CN101096680A - A DNA vaccine eukaryotic expression vector and its application in the preparation of gene vaccine - Google Patents
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Abstract
本发明公开了一种DNA疫苗真核表达载体及其应用。其目的是提供一种DNA疫苗真核表达载体与其在制备抗肿瘤等基因疫苗中的应用。所述DNA疫苗真核表达载体是在pVAX1载体骨架中添加内部核糖体进入位点编码序列得到的重组pVAX1真核表达载体。本发明的基因疫苗有望对乳腺癌、胰腺癌、结直肠癌和前列腺癌等多种恶性实体瘤发挥广谱治疗作用,特别是以上述真核表达载体构建的抗肿瘤基因疫苗具有较好的体液免疫和细胞免疫效果,对肿瘤生长具有显著的抑制作用,将在肿瘤的治疗及其药物开发领域发挥重要作用,应用前景广阔。The invention discloses a DNA vaccine eukaryotic expression vector and its application. Its purpose is to provide a DNA vaccine eukaryotic expression vector and its application in preparing anti-tumor isogenic vaccines. The DNA vaccine eukaryotic expression vector is a recombinant pVAX1 eukaryotic expression vector obtained by adding an internal ribosome entry site coding sequence to the pVAX1 vector backbone. The gene vaccine of the present invention is expected to play a broad-spectrum therapeutic effect on various malignant solid tumors such as breast cancer, pancreatic cancer, colorectal cancer and prostate cancer, especially the anti-tumor gene vaccine constructed with the above-mentioned eukaryotic expression vector has better humoral The effect of immunity and cellular immunity has a significant inhibitory effect on tumor growth, and will play an important role in the field of tumor treatment and drug development, with broad application prospects.
Description
技术领域technical field
本发明涉及载体及其应用,特别是涉及一种DNA疫苗真核表达载体与其在制备抗肿瘤等基因疫苗中的应用。The invention relates to a vector and its application, in particular to a DNA vaccine eukaryotic expression vector and its application in preparing an anti-tumor isogenic vaccine.
背景技术Background technique
当前,恶性肿瘤已经成为威胁人类健康的最重要杀手之一,因此,加强恶性肿瘤的防治研究具有重大意义。目前,手术切除、化疗和放疗仍然是治疗恶性肿瘤的主要治疗方法。传统的手术治疗方法虽可使患者的生存率有所提高,但是术后5年的复发率依然在半数以上,这是因为手术虽然能够有效地切除肉眼所见的瘤块,但无法保证去除所有的肿瘤细胞,也未能改变肿瘤发生的内环境;而化疗和放疗除了有严重的毒副作用外,许多肿瘤如肾癌等对其不敏感或产生抗性。肿瘤的转移和复发是影响病人预后的重要因素,更是恶性肿瘤发生和演进中最危险的阶段,目前,上述传统治疗方法在克服肿瘤复发和转移方面存在很大的局限性,成为肿瘤治疗的瓶颈。近年来逐步发展的免疫基因治疗手段,因其具有特异性高、针对性强和副作用小的特点,已经成为恶性肿瘤治疗的重要发展方向。At present, malignant tumors have become one of the most important killers that threaten human health. Therefore, it is of great significance to strengthen the research on the prevention and treatment of malignant tumors. Currently, surgical resection, chemotherapy, and radiotherapy are still the main treatments for malignant tumors. Although the traditional surgical treatment method can improve the survival rate of patients, the recurrence rate is still more than half after 5 years after surgery. In addition to severe toxic side effects of chemotherapy and radiotherapy, many tumors such as kidney cancer are insensitive or resistant to it. Tumor metastasis and recurrence are important factors affecting the prognosis of patients, and they are also the most dangerous stage in the occurrence and evolution of malignant tumors. At present, the above-mentioned traditional treatment methods have great limitations in overcoming tumor recurrence and metastasis, and have become the mainstay of tumor treatment. bottleneck. Immunogene therapy, which has been gradually developed in recent years, has become an important development direction for the treatment of malignant tumors because of its high specificity, strong pertinence and small side effects.
DNA疫苗也称基因疫苗或核酸疫苗,是20世纪90年代发展起来的一种新型疫苗,它是指将外源抗原基因克隆到真核表达载体上,然后再将该重组的质粒DNA免疫动物。外源抗原基因能够在宿主体内表达,并刺激其产生体液免疫和细胞免疫。许多动物模型和人体实验都证明,DNA疫苗可以有效地激发机体的体液免疫应答和细胞免疫应答,被誉为第三次疫苗革命。DNA vaccine, also known as gene vaccine or nucleic acid vaccine, is a new type of vaccine developed in the 1990s. It refers to cloning foreign antigen genes into eukaryotic expression vectors, and then immunizing animals with the recombinant plasmid DNA. Exogenous antigen genes can be expressed in the host and stimulate humoral and cellular immunity. Many animal models and human experiments have proved that DNA vaccines can effectively stimulate the body's humoral immune response and cellular immune response, known as the third vaccine revolution.
DNA疫苗具有许多突出的优点:①DNA质粒表达的抗原接近天然构象,抗原性强;②激发机体全面的免疫应答,其保守抗原的保护性免疫应答对不同亚型的病原体有交叉抵御作用;③制备简单,成本低廉,易进行规模化生产,且运输、保存方便;④使用安全,没有感染病原的危险,DNA疫苗仅仅是病原体某种抗原的基因片段,而不是整个病原体的基因,且利用质粒作载体,不涉及感染性因子;⑤免疫具有持续性,一次接种可获得长期免疫力,避免了灭活疫苗、重组亚单位疫苗等需多次加强免疫的繁琐;⑥能联合免疫,即可将编码不同抗原的基因构建在同一个质粒中或将含不同抗原基因的多种质粒联合应用,制备多价DNA疫苗;⑦DNA疫苗既有预防作用,也有治疗作用;⑧接种途径多样化,既能采用注射方式接种(包括皮内、皮下、肌注、基因枪注射技术),亦能采用口服或喷雾接种方式。DNA vaccines have many outstanding advantages: ①The antigen expressed by the DNA plasmid is close to the natural conformation and has strong antigenicity; ②It stimulates a comprehensive immune response in the body, and the protective immune response of its conserved antigen has cross-resistance to different subtypes of pathogens; ③Preparation Simple, low cost, easy to carry out large-scale production, and convenient to transport and store; ④ safe to use, no risk of infection of pathogens, DNA vaccine is only a gene fragment of a certain antigen of the pathogen, not the gene of the whole pathogen, and uses the plasmid as The carrier does not involve infectious agents; ⑤ Immunity is persistent, and long-term immunity can be obtained by one vaccination, avoiding the cumbersomeness of repeated booster immunizations such as inactivated vaccines and recombinant subunit vaccines; The genes of different antigens are constructed in the same plasmid or multiple plasmids containing different antigen genes are used in combination to prepare multivalent DNA vaccines; ⑦ DNA vaccines have both preventive and therapeutic effects; Inoculation methods (including intradermal, subcutaneous, intramuscular injection, gene gun injection techniques), oral or spray inoculation can also be used.
Survivin属于凋亡抑制蛋白(IAP)家族成员,研究显示,Survivin是迄今为止发现的最强的凋亡抑制因子,也是IAP家族成员中最小的一个。人Survivin基因定位于染色体(17q25),包含4个外显子和3个内含子,编码一种分子量为16.5kD的胞质蛋白,含有一个杆状病毒重复(BIR)结构功能区,羧基末端缺乏锌指结构而代之以α螺旋结构。其mRNA在体内经过不同的剪切方式可产生三种异构体:Survivin、Survivin-2B和Survivin-EX3。Survivin is a member of the inhibitor of apoptosis protein (IAP) family. Studies have shown that Survivin is the strongest inhibitor of apoptosis discovered so far, and it is also the smallest member of the IAP family. The human Survivin gene is located on the chromosome (17q25), contains 4 exons and 3 introns, encodes a cytoplasmic protein with a molecular weight of 16.5kD, contains a baculovirus repeat (BIR) structural functional region, and has a carboxy-terminal Lack of zinc finger structure and replaced by α-helical structure. Its mRNA can produce three isoforms in vivo through different splicing modes: Survivin, Survivin-2B and Survivin-EX3.
Survivin在胚胎组织及恶性肿瘤中普遍表达,如胃癌、结直肠癌、乳腺癌和肺癌等;但在分化成熟的组织及癌旁正常组织中无表达。这一特点使得Survivin成为肿瘤靶向治疗的重要靶点,具有良好的特异性及安全性。研究证实,靶向Survivin治疗可以促进肿瘤细胞凋亡并抑制其增殖,而对正常组织几乎无不良影响,这一显著优势使Survivin逐渐成为抗肿瘤免疫治疗研究中的一大热点。Survivin is commonly expressed in embryonic tissues and malignant tumors, such as gastric cancer, colorectal cancer, breast cancer and lung cancer, etc., but not in differentiated mature tissues and normal adjacent tissues. This feature makes Survivin an important target for tumor targeted therapy, with good specificity and safety. Studies have confirmed that targeted Survivin therapy can promote tumor cell apoptosis and inhibit its proliferation, but has almost no adverse effects on normal tissues. This remarkable advantage makes Survivin gradually become a hot spot in the research of anti-tumor immunotherapy.
Rong Xiang等将鼠全长Survivin和分泌型趋化因子CCL21的基因连接至pBudCE4.1真核表达载体上,制备成DNA疫苗,在C57BL/6J小鼠体内的免疫实验结果显示,该DNA疫苗发引发CD8+CTL反应,对肺癌细胞具有很好的杀伤效果[Rong X,Noriko M,Yunping L,et al.A DNA vaccine targeting survivin combines apoptosiswith suppression of angiogenesis in lung tumor eradication.Cancer Research.2005,65(2):553-561]。Marc Schmitz等人证实凋亡抑制蛋白Survivin的自氨基端第5-14位氨基酸(TLPPAWQPFL)和第95-104位氨基酸(ELTLGEFLKL)可在体外有效地激发特异性CD8+CTL杀伤肿瘤细胞(Marc S,Petra D,Bernd W,et al.Generationof Survivin-specific CD8+T Effector Cells by Dendritic Cells Pulsed withProtein or Selected Peptides.Cancer research,2000,60(17):4845-4849)。Kerstin Otto等人又用这段短肽在IV期黑色素瘤患者体内进行了功能实验,ELISPOT检测结果表明,该短肽可以激发强烈的T细胞免疫反应(Kerstin O,Mads HA,AndreasE,et al.Lack of toxicity of therapy-induced T cell responses against theuniversal tumour antigen surviving.Vaccine,2005,23(7):884-889)。YoshihikoHirohashi等研制的抗肿瘤多肽疫苗是以人Survivin-2B的自氨基端第80-88位氨基酸(AYACNTSTL)为靶点,这段短肽是HLA-A24限制性T淋巴细胞表位,可被CD8+细胞毒T淋巴细胞特异性识别,实验结果证明,以这段短肽为靶抗原制备的疫苗对结肠癌细胞具有很好的杀伤抑制作用,目前该疫苗已经进入II期临床试验阶段(Yoshihiko H,Toshihiko T,Akiko M,et al.An HLA-A24-restricted Cytotoxic T LymphocyteEpitope of a Tumor-associated Protein,Survivin.Clinical Cancer Research,2002,8(6):1731-1739)。Rong Xiang et al. connected the gene of mouse full-length Survivin and secreted chemokine CCL21 to the pBudCE4.1 eukaryotic expression vector to prepare a DNA vaccine. The results of immunization experiments in C57BL/6J mice showed that the DNA vaccine developed Initiate CD8+CTL response and have a good killing effect on lung cancer cells [Rong X, Noriko M, Yunping L, et al. A DNA vaccine targeting survivin combines apoptosis with suppression of angiogenesis in lung tumor eradication. Cancer Research. 2): 553-561]. Marc Schmitz et al confirmed that the 5-14th amino acid (TLPPAWQPFL) and the 95th-104th amino acid (ELTLGEFLKL) of the apoptosis inhibitor protein Survivin can effectively stimulate specific CD8+ CTL to kill tumor cells in vitro (Marc S , Petra D, Bernd W, et al. Generation of Survivin-specific CD8+T Effector Cells by Dendritic Cells Pulsed with Protein or Selected Peptides. Cancer research, 2000, 60(17): 4845-4849). Kerstin Otto et al. used this short peptide to conduct functional experiments in patients with stage IV melanoma. ELISPOT test results showed that the short peptide can stimulate a strong T cell immune response (Kerstin O, Mads HA, Andreas E, et al. Lack of toxicity of therapy-induced T cell responses against the universal tumor antigen surviving. Vaccine, 2005, 23(7): 884-889). The anti-tumor peptide vaccine developed by Yoshihiko Hirohashi et al. is based on the 80th-88th amino acid (AYACNTSTL) from the amino terminal of human Survivin-2B. This short peptide is an HLA-A24 restricted T lymphocyte epitope, which can be detected by CD8 Cytotoxic T lymphocytes are specifically recognized. Experimental results have proved that the vaccine prepared with this short peptide as the target antigen has a good killing and inhibitory effect on colon cancer cells. At present, the vaccine has entered the phase II clinical trial stage (Yoshihiko H, Toshihiko T, Akiko M, et al. An HLA-A24-restricted Cytotoxic T Lymphocyte Epitope of a Tumor-associated Protein, Survivin. Clinical Cancer Research, 2002, 8(6): 1731-1739).
人绒毛膜促性激素(human chorionic gonadotropin,hCG)是由胚胎合体滋养层细胞所分泌的糖蛋白激素,正常hCG由α链和β链亚单位以非共价键连接构成二聚体。几乎所有的恶性肿瘤细胞均能表达异位hCG。hCGβ是多种肿瘤的特征性蛋白,多种组织的肿瘤细胞均选择性分泌单链hCGβ或hCGβ的核心片段,研究表明,该蛋白与恶性肿瘤转移特性、恶性化程度以及肿瘤微环境和免疫耐受的形成等均有一定关系,具体表现为:hCG的糖基序列大多集中在hCGβ的梭基末端多肽(hCGp-CTP)区域,肿瘤细胞表面表达的hCGβ链越多,糖基化程度越高,肿瘤细胞表面所带的负电荷就越多,而肿瘤细胞表面带负电荷可促进肿瘤细胞与血管内皮细胞的粘附、刺激有关粘附分子和趋化因子的生成,从而使肿瘤细胞越容易转移。同时,由于强负电荷的存在,可以抑制NK、T细胞、巨噬细胞等免疫细胞的活化,导致免疫细胞的活性降低,甚至无能,造成肿瘤的免疫耐受。Human chorionic gonadotropin (hCG) is a glycoprotein hormone secreted by embryonic syncytiotrophoblast cells. Normal hCG consists of α-chain and β-chain subunits connected by non-covalent bonds to form a dimer. Almost all malignant tumor cells can express ectopic hCG. hCGβ is a characteristic protein of various tumors. Tumor cells of various tissues selectively secrete single-chain hCGβ or the core fragment of hCGβ. The formation of receptors has a certain relationship, the specific performance is: the glycosyl sequence of hCG is mostly concentrated in the shuttle base terminal polypeptide (hCGp-CTP) region of hCGβ, the more hCGβ chains expressed on the surface of tumor cells, the higher the degree of glycosylation , the more negative charges on the surface of tumor cells, and the negative charges on the surface of tumor cells can promote the adhesion between tumor cells and vascular endothelial cells, stimulate the production of relevant adhesion molecules and chemokines, thus making it easier for tumor cells to transfer. At the same time, due to the existence of strong negative charges, it can inhibit the activation of immune cells such as NK cells, T cells, and macrophages, resulting in a decrease in the activity of immune cells, or even incompetence, resulting in immune tolerance of tumors.
由于hCGβ亚基与促黄体激素(LH)有较高的同源性,因此以hCGβ亚基为免疫原产生的抗体会与LH产生交叉反应。只有hCGβ羧基端自109-145位氨基酸残基组成的37肽(hCGβ-CTP37)是hCG分子所特有的,并存在hCG的特异抗原表位,其诱导产生的抗体才具有更好的抗原特异性。Since the hCGβ subunit has high homology with luteinizing hormone (LH), the antibody produced using the hCGβ subunit as an immunogen will cross-react with LH. Only the 37 peptide (hCGβ-CTP37) composed of amino acid residues 109-145 at the carboxy-terminal of hCGβ is unique to hCG molecules and has a specific antigenic epitope of hCG, so the antibodies induced by it have better antigen specificity .
以异位hCGβ亚基为靶抗原激发的体液和细胞免疫反应可有效地攻击肿瘤细胞,已成为肿瘤生物治疗新的研究热点。国外利用该靶点设计的胰腺癌肿瘤疫苗Avicine已经结束II期临床试验,结直肠癌疫苗已进入III期临床试验的关键时期,显示该靶点对多种恶性肿瘤术后复发和转移具免疫保护功能,具有良好的应用前景。Humoral and cellular immune responses stimulated by ectopic hCGβ subunits as target antigens can effectively attack tumor cells, which has become a new research hotspot in tumor biotherapy. Avicine, a pancreatic cancer vaccine designed using this target abroad, has completed phase II clinical trials, and colorectal cancer vaccines have entered the critical period of phase III clinical trials, showing that this target has immune protection against postoperative recurrence and metastasis of various malignant tumors function, has a good application prospect.
hCGβ链的核心片段-CTP37,是多种肿瘤的特征性蛋白,与恶性肿瘤转移、恶性化程度以及肿瘤微环境和免疫耐受的形成等均有一定关系。研究表明,以CTP37为靶抗原激发的体液和细胞免疫反应,可有效地攻击肿瘤细胞,现已成为肿瘤生物治疗的另一研究热点。The core fragment of the hCGβ chain - CTP37, is a characteristic protein of various tumors, and has a certain relationship with the metastasis of malignant tumors, the degree of malignancy, and the formation of tumor microenvironment and immune tolerance. Studies have shown that the humoral and cellular immune responses stimulated by CTP37 as the target antigen can effectively attack tumor cells, and it has become another research hotspot in tumor biotherapy.
Li-Zhen He等将hCGβ的全基因连接在抗DC细胞抗体B11重链的3’端构成融合基因,再将其插入真核表达载体pMMV4中构成DNA疫苗,体外实验证明该疫苗可激发产生强烈的T细胞免疫反应杀死肿瘤细胞(He LZ,Ramakrishna V,Connolly JE,etal.A novel human cancer vaccine elicits cellular responses to thetumor-associated antigen,human chorionic gonadotropin beta[J].Clin CancerRes,2004,10(6):1920-7.)。Moulton等将含有hCGβ的COOH-末端37个氨基酸残基(CTP37)的短肽与白喉类毒素相偶连制备成蛋白质疫苗,可激发机体产生抗CTP37的抗体抑制肿瘤生长,现在该疫苗治疗结直肠癌的实验已经进入III期临床试验的关键时期(Moulton HM,Yoshihars PH,Mason DH,et al.Active specificimmunotherapy with a beta-human chorionic gonadotropin peptide vaccine inpatients with metastatic colorectal cancer:antibody response is associatedwith improved survival[J].Clin Cancer Res,2002,8(7):2044-51)。He Yi等于近期发表的一篇论文中介绍了他们研制的一种新型的以CTP37为靶点的核酸疫苗,是将CTP37与分枝杆菌热休克蛋白HSP65相连,并将这段融合基因插入真核表达载体构成核酸疫苗,在小鼠体内进行了抑瘤效果的实验。结果发现该核酸疫苗可诱发机体产生高滴度的亲和性抗体,体内和体外实验结果均表明该疫苗可有效抑制乳腺癌细胞的生长,为肿瘤的免疫治疗提供了一条新思路(Yi H,Rong Y,Yankai Z,et al.Improved efficacy of DNA vaccination against breast cancer by boosting withthe repeat beta-hCG C-terminal peptide carried by mycobacterial heat-shockprotein HSP65[J].Vaccine,2006,24(14):2575-84.)。Li-Zhen He et al. connected the whole gene of hCGβ to the 3' end of the heavy chain of the anti-DC cell antibody B11 to form a fusion gene, and then inserted it into the eukaryotic expression vector pMMV4 to form a DNA vaccine. In vitro experiments proved that the vaccine can induce strong T cell immune response to kill tumor cells (He LZ, Ramakrishna V, Connolly JE, et al. A novel human cancer vaccine elicits cellular responses to the tumor-associated antigen, human chorionic gonadotropin beta[J]. Clin CancerRes, 2004, 10( 6): 1920-7.). Moulton et al. coupled a short peptide containing 37 amino acid residues (CTP37) at the COOH-terminus of hCGβ to diphtheria toxoid to prepare a protein vaccine, which can stimulate the body to produce antibodies against CTP37 and inhibit tumor growth. Now the vaccine treats colorectal cancer Cancer experiments have entered a critical period of phase III clinical trials (Moulton HM, Yoshihars PH, Mason DH, et al. Active specific immunotherapy with a beta-human chorionic gonadotropin peptide vaccine patients with metastatic colorectal cancer: antibody response is associated with ]. Clin Cancer Res, 2002, 8(7): 2044-51). In a recent paper published by He Yi et al., they introduced a new type of nucleic acid vaccine targeting CTP37, which is to link CTP37 with mycobacterial heat shock protein HSP65 and insert this fusion gene into eukaryotic cells. The expression vector constitutes a nucleic acid vaccine, and the tumor-suppressing effect experiment was carried out in mice. It was found that the nucleic acid vaccine can induce the body to produce high-titer affinity antibodies, and both in vivo and in vitro experiments showed that the vaccine can effectively inhibit the growth of breast cancer cells, providing a new idea for tumor immunotherapy (Yi H, Rong Y, Yankai Z, et al.Improved efficacy of DNA vaccination against breast cancer by boosting with the repeat beta-hCG C-terminal peptide carried by mycobacterial heat-shockprotein HSP65[J]. Vaccine, 2006, 257-14: 84.).
基因治疗的关键环节除了正确选取最特异、高效、广泛的靶抗原之外,还包括合理选择适当的真核表达载体,这也是核酸疫苗研制的前提条件。pVAX1载体是被美国药品及食物鉴定委员会承认的用于疫苗临床使用的真核表达载体,它具有卡那霉素抗性,非常适合于人体应用。In addition to the correct selection of the most specific, efficient and broad target antigens, the key link of gene therapy also includes the rational selection of appropriate eukaryotic expression vectors, which is also a prerequisite for the development of nucleic acid vaccines. The pVAX1 vector is a eukaryotic expression vector recognized by the Drug and Food Certification Committee of the United States for the clinical use of vaccines. It has kanamycin resistance and is very suitable for human applications.
当前,DNA疫苗研究领域的一个关键性问题是如何提高疫苗的免疫保护力,这一问题关系到DNA疫苗最终能否转化为产品。以往人们为了构建多价DNA疫苗及增强DNA疫苗的免疫原性,多构建双启动子表达载体、重组表达融合蛋白或通过携带不同抗原基因和佐剂基因的质粒共同免疫,虽然双启动子载体可用于两个基因的协同表达,但启动子之间的相互干扰会造成转录效果的不一致,减弱甚至丧失某些基因的表达(Tahara H,Zitvogel L,Storkus WJ,et al.Effective eradication of establishedmurine tumors with IL-12 gene therapy using a polycistronic retroviralvector[J].J Immunol,1995,154(12):6466-74.);此外,多载体共免疫的效率也较低,常常不能取得令人满意的效果。采用IRES元件构建双顺反子表达载体,可允许两个目的基因同时有效表达(Wagstaff M,Lilley C,Smith J,et al.Gene transferusing a disabled herpes virus vector containing the EMCV IRES allows multiplegene expression in vitro and in vivo.Gene Ther.1998,5(11):1566-70.),是近年来多基因表达的重要方法。At present, a key issue in the field of DNA vaccine research is how to improve the immune protection of vaccines, which is related to whether DNA vaccines can eventually be transformed into products. In the past, in order to construct multivalent DNA vaccines and enhance the immunogenicity of DNA vaccines, people often constructed dual-promoter expression vectors, recombinantly expressed fusion proteins, or co-immunized with plasmids carrying different antigen genes and adjuvant genes. Although dual-promoter vectors can be used for Co-expression of two genes, but mutual interference between promoters will cause inconsistencies in transcriptional effects, weaken or even lose the expression of some genes (Tahara H, Zitvogel L, Storkus WJ, et al.Effective eradication of established murine tumors with IL -12 gene therapy using a polycistronic retroviral vector [J]. J Immunol, 1995, 154 (12): 6466-74.); In addition, the efficiency of multi-vector co-immunization is also low, and often cannot achieve satisfactory results. Using IRES elements to construct bicistronic expression vectors can allow two target genes to be effectively expressed simultaneously (Wagstaff M, Lilley C, Smith J, et al. Gene transferring a disabled herpes virus vector containing the EMCV IRES allows multiple gene expression in vitro and in vivo.Gene Ther.1998, 5(11):1566-70.), is an important method for multi-gene expression in recent years.
内部核糖体进入位点(internal ribosome entry site,IRES)是最早发现于动物病毒基因组5’非编码区的一段DNA序列,它具有不依赖于5’帽子结构的翻译起始功能,可以使来源于同一mRNA的2个开放读码框架正确表达,是一种双顺反子表达元件。在两个蛋白编码序列之间插入IRES,在上游启动子的控制下,转录后为同一条mRNA;翻译时,IRES上游基因翻译起始遵循一般的真核生物基因的翻译起始规律(即帽结构依赖方式),而IRES下游基因则通过IRES引导核糖体进入,启始基因的翻译,从而实现两个基因的同时表达。由于两个基因翻译自同一条mRNA,表达出的蛋白在时间和空间上接近,尤适用于研究相互作用的两个蛋白质或通过报告基因追踪待检测蛋白的转录及表达,也可作为多表位DNA疫苗的表达载体。The internal ribosome entry site (IRES) is a DNA sequence that was first discovered in the 5' non-coding region of the animal virus genome. It has a translation initiation function independent of the 5' cap structure and can enable The two open reading frames of the same mRNA are correctly expressed, which is a bicistronic expression element. Insert IRES between two protein coding sequences, and under the control of the upstream promoter, it will be the same mRNA after transcription; during translation, the translation initiation of the upstream gene of IRES follows the general translation initiation rule of eukaryotic genes (ie cap structure-dependent manner), while the IRES downstream gene guides the entry of ribosomes through IRES and initiates the translation of the gene, thereby realizing the simultaneous expression of the two genes. Since the two genes are translated from the same mRNA, the expressed proteins are close in time and space, which is especially suitable for studying two interacting proteins or tracking the transcription and expression of the protein to be detected through the reporter gene, and can also be used as a multi-epitope Expression vectors for DNA vaccines.
正常情况下,机体对自身抗原具有耐受性,不产生免疫应答。利用异种同源基因作为抗原,通过生物种与种之间基因的同源性,设法诱导机体产生交叉免疫反应,有利于打破机体的自身免疫耐受,能够使疫苗有效地诱导体液和细胞免疫保护反应,高效发挥抗肿瘤转移和复发的作用。Under normal circumstances, the body is tolerant to self-antigens and does not generate an immune response. Using heterologous genes as antigens, through the homology of genes between species, try to induce cross-immune reactions in the body, which is conducive to breaking the body's autoimmune tolerance and enabling the vaccine to effectively induce humoral and cellular immune protection Response, effectively play the role of anti-tumor metastasis and recurrence.
异种同源抗原是打破肿瘤免疫耐受的有效策略。研究发现,生物在由低级物种向高级物种进化演变过程中,不同种属的同一基因表现为一定的同源性。可利用异种同源细胞或分子诱导肿瘤细胞的自体免疫反应达到抗肿瘤的目的。通过利用生物种与种之间基因的同源性,设法诱导机体产生交叉免疫反应(cross-reaction),可能会打破自身免疫耐受(Ciesielski MJ,Apfel L,Barone TA,et al.Antitumor effects ofa xenogeneic surviyin bone marrow derived dendritic cell vaccine againstmurine GL261 gliomas.Cancer Immunol Immunother,2006,55(12),1491-503)。研究表明,使用与肿瘤抗原(HER-2/neu)高度同源的蛋白质,可以诱导出比使用肿瘤抗原本身大得多的机体免疫反应。国内魏于全院士等发现用异种血管内皮生长因子极其受体作为疫苗免疫小鼠,可诱导针对肿瘤新生血管的自身免疫反应,从而可抑制肿瘤生长,在动物体内取得了较好的抑瘤效果(Wei YQ,Huang MJ,Yang L,et al.Immunogene therapy of tumors with vaccine based on Xenopus homologous vascularendothelial growth factor as a model antigen.[J]Proc Natl Acad Sci USA.2001 Sep 25;98(20):11545-50)。目前,以异种抗原作为免疫原研制抗肿瘤疫苗已经成为重要的设计策略,国内外在多种疫苗设计上均应用此策略,并取得了令人满意的免疫保护效果。Xenologous antigens are an effective strategy to break tumor immune tolerance. Studies have found that during the evolution of organisms from low-level species to high-level species, the same gene of different species shows a certain degree of homology. The autoimmune response of tumor cells can be induced by heterologous cells or molecules to achieve the purpose of anti-tumor. By using the homology of genes between species, try to induce the body to produce cross-reaction (cross-reaction), which may break autoimmune tolerance (Ciesielski MJ, Apfel L, Barone TA, et al.Antitumor effects ofa xenogeneic surviyin bone marrow derived dendritic cell vaccine against murine GL261 gliomas. Cancer Immunol Immunother, 2006, 55(12), 1491-503). Studies have shown that the use of proteins highly homologous to tumor antigens (HER-2/neu) can induce a much larger immune response than the use of tumor antigens themselves. Domestic academician Wei Yuquan found that immunizing mice with xenogeneic vascular endothelial growth factor and its receptor as a vaccine can induce an autoimmune response against tumor neovascularization, thereby inhibiting tumor growth, and achieved a good tumor inhibitory effect in animals (Wei YQ, Huang MJ, Yang L, et al.Immunogene therapy of tumors with vaccine based on Xenopus homologous vascularendothelial growth factor as a model antigen.[J]Proc Natl Acad Sci USA.2001
肿瘤免疫基因治疗的主要目的是使机体产生具有抗肿瘤特异性和细胞毒活性的T淋巴细胞,从而达到特异性攻击肿瘤的作用。T细胞的活化需双信号刺激:第一信号由T细胞抗原受体(TCR)与抗原提呈细胞(APC)上的抗原肽-MHC分子复合物结合提供,第二信号由APC上的共刺激分子与T细胞上的相应配体结合提供。只有双信号共同刺激才能有效激活特异性的T细胞,缺乏第二信号造成T细胞克隆凋亡,使肿瘤细胞产生免疫逃避。APC上的共刺激分子主要是一些粘附分子,其中,B7分子是当今的研究热点。The main purpose of tumor immune gene therapy is to make the body produce T lymphocytes with anti-tumor specificity and cytotoxic activity, so as to achieve the effect of specifically attacking tumors. The activation of T cells requires dual-signal stimulation: the first signal is provided by the combination of T cell antigen receptor (TCR) and the antigen peptide-MHC molecule complex on the antigen-presenting cell (APC), and the second signal is provided by costimulation on the APC The molecule is provided in conjunction with the corresponding ligand on the T cell. Only dual-signal co-stimulation can effectively activate specific T cells, and the lack of the second signal will cause T cell clone apoptosis and make tumor cells immune evasion. The co-stimulatory molecules on APC are mainly some adhesion molecules, among which, the B7 molecule is the current research hotspot.
B7.1是APC上的主要共刺激分子,它与配体CD28分子的相互作用是T细胞激活的第二信号。B7.1与其配体CD28结合后,可促进T细胞增殖、阻止T细胞凋亡、产生T细胞趋化因子(Herold K,Lu J,Rulifson T,et al.Regulation of C-Cchemokineproduction by murine T cells by CD28/B7 costimulation[J].J Immunol,1997,159(9):4150-53.);上调IL-2Rα、β和γ,增加IL-2 mRNA的转录,增强细胞因子分泌。上调CD40L的表达和CTLA 24的mRNA水平;还可增强T细胞对超抗原的敏感性,介导CTL对靶细胞的效应功能;介导T-B细胞间的粘附,促进体液免疫应答等。B7.1主要共刺激CD8+T细胞向CTL分化CD4+T细胞向Th1细胞分化,这在增强和维持有效免疫应答中起到关键作用。同时,B7.1还具有较强的共刺激GM-CSF产生的作用(Boussiotis V,Freeman G,Gribben J,et al.The role of B7.1/B7.2:CD28/CTLA-4 pathways in the prevention of anergy,induction of productiveimmunity and down-regulation of the immune response[J].Immunol Rev,1996,153:5-26.)。B7.1 is the main co-stimulatory molecule on APC, and its interaction with ligand CD28 molecule is the second signal of T cell activation. After binding with its ligand CD28, B7.1 can promote T cell proliferation, prevent T cell apoptosis, and produce T cell chemokines (Herold K, Lu J, Rulifson T, et al. Regulation of C-Cchemokineproduction by murine T cells by CD28/B7 costimulation[J]. J Immunol, 1997, 159(9): 4150-53.); up-regulate IL-2Rα, β and γ, increase the transcription of IL-2 mRNA, and enhance cytokine secretion. Up-regulate the expression of CD40L and the mRNA level of CTLA 24; it can also enhance the sensitivity of T cells to superantigens, mediate the effector function of CTLs on target cells; mediate the adhesion between T-B cells, and promote humoral immune responses, etc. B7.1 mainly co-stimulates the differentiation of CD8+T cells into CTLs and the differentiation of CD4+T cells into Th1 cells, which plays a key role in enhancing and maintaining effective immune responses. At the same time, B7.1 also has a strong co-stimulatory effect on the production of GM-CSF (Boussiotis V, Freeman G, Gribben J, et al. The role of B7.1/B7.2: CD28/CTLA-4 pathways in the prevention of energy, induction of productive immunity and down-regulation of the immune response [J]. Immunol Rev, 1996, 153: 5-26.).
粒细胞巨噬细胞集落刺激因子(granulocyte macrophage colony stimulatingfactor,GM-CSF)主要由T细胞和巨噬细胞产生,是一种具有多种生物活性的细胞因子,可以促进DC等APC分化、成熟和活化及上调CD86的表达以激发对肿瘤的免疫应答(Kass E,Parker J,Schlom,et al.Comparat ive studies of the effects ofrecombiant GM-CSF and GM-CSF administered via a poxvirus to enhance theconcentration of antigen presenting cells in regional lymphnodes[J].Cytokine,2000,12(7):960-71.);在体外具有促进髓样祖细胞增殖,增强中性粒细胞、单核巨噬细胞、嗜酸性粒细胞对肿瘤细胞的吞噬作用和ADCC效应等活性(Yu J,Burwick J,Dranoff G,et al.Gene therapy for metastatic brain tumors byvaccination with granulocyte-macrophage colony-stimulating factor transducedtumors cells[J].Human gene therpy,1997,8(9):1065-72.);促进Th、Tc、NK在肿瘤部位浸润,从而杀伤肿瘤(Nakazaki Y,Tani K,Lin ZT,et al.Vaccine effectof granulocyte-macrophage colony-stimulating factor or CD80 gene-transducedmurine hematopoietic tumor cells and their cooperative enhancement ofanti-tumor immunity[J].Gene Ther,1998,5(10):1355-62.)。大量文献表明,GM-CSF具有很好的协同抗肿瘤作用(Armstrong C,Botella R,Galloway T,et al.Anti-tumor effects of granulocyte macrophage colony-stimulating factorproduction by melanoma cells[J].Cancer Res,1996,56(9):2191-98;WakimotoH,Abe J,Tsunoda R,et al.Intensified anti-tumor immunity by a cancer vaccinethat produces granulocyte-macrophage colony stimulating factor plusinterleukin 4[J].Cancer Res,1996,56(8):1828-33.)。Granulocyte macrophage colony stimulating factor (GM-CSF) is mainly produced by T cells and macrophages. It is a cytokine with various biological activities, which can promote the differentiation, maturation and activation of DC and other APCs. and upregulate the expression of CD86 to stimulate the immune response to tumors (Kass E, Parker J, Schlom, et al.Comparative studies of the effects of recombinant GM-CSF and GM-CSF administered via a poxvirus to enhance the concentration of antigen presenting cells in regional lymphnodes[J]. Cytokine, 2000, 12(7): 960-71.); in vitro, it can promote the proliferation of myeloid progenitor cells, and enhance the effect of neutrophils, mononuclear macrophages and eosinophils on tumor cells Phagocytosis and ADCC effects and other activities (Yu J, Burwick J, Dranoff G, et al. Gene therapy for metastatic brain tumors byvaccination with granulocyte-macrophage colony-stimulating factor transduced tumors cells[J]. Human gene therapy, 1997, 8( 9): 1065-72.); Promote the infiltration of Th, Tc, and NK in the tumor site, thereby killing the tumor (Nakazaki Y, Tani K, Lin ZT, et al. Vaccine effect of granulocyte-macrophage colony-stimulating factor or CD80 gene-transduced murine hematopoietic tumor cells and their cooperative enhancement of anti-tumor immunity [J]. Gene Ther, 1998, 5(10): 1355-62.). A large number of literatures show that GM-CSF has a good synergistic anti-tumor effect (Armstrong C, Botella R, Galloway T, et al. Anti-tumor effects of granulocyte macrophage colony-stimulating factorproduction by melanoma cells[J]. Cancer Res, 1996 , 56(9): 2191-98; WakimotoH, Abe J, Tsunoda R, et al. Intensified anti-tumor immunity by a cancer vaccine that produces granulocyte-macrophage colony stimulating factor plus interleukin 4[J]. Cancer Res, 1996, 5 8): 1828-33.).
仅靠肿瘤特异性抗原难以有效激发机体抗肿瘤免疫,尚需B7.1共刺激分子与其配体等相互作用参与激活T细胞。许多研究也已证实多数肿瘤细胞不表达B7.1共刺激分子,从而逃避了机体免疫监视,以至肿瘤逃避免疫监视而在体内无限制生长(杨旭伟,陈元仲,林振兴.人B7.1基因cDNA的克隆及其逆转录病毒表达载体构建[J].福建医科大学学报,2001,35(2):109-111.)。而GM-CSF具有激活树突细胞等专职抗原递呈细胞作用,使抗原特异的辅助T淋巴细胞和抗肿瘤效应细胞增生,在体内能激活较强的抗肿瘤免疫反应。若将两者联合应用,即可启动针对肿瘤抗原的免疫,又可促进辅助T淋巴细胞和抗肿瘤免疫效应细胞增生,产生协同的抗肿瘤效应。目前已有研究证实,将GM-CSF与B7.1分子联合应用会提高疫苗对肿瘤细胞的杀伤作用。Sumimoto等人的研究结果表明,B7.1基因和GM-CSF基因共转染的肺癌细胞株LLC制备的瘤苗(LLC/GM+B7),诱发的CTL细胞杀伤活性明显高于单独转染B7.1或GM-CSF基因的LLC细胞(Sumimoto H,Tani K,Nakazaki Y,et al.GM-CSF and B7-1(CD80)co-stimulatory signals co-operate in the induction of effective anti-tumorimmunity in syngeneic mice.Int J Cancer.1997,73(4):556-61.)。Nakazaki等人的研究结果表明,胸腺淋巴瘤细胞/GM-CSF与胸腺淋巴瘤细胞/B7.1瘤苗联合使用具有协同抗肿瘤免疫效果,其机制可能是GM-CSF基因转导的瘤苗主要是通过APC介导的间接途径影响Th细胞进而激活CTL;B7.1基因转录的瘤苗是通过直接途径影响CTL,而将两者联合使用可诱发系统性抗肿瘤免疫反应,导致肿瘤部分根除(Nakazaki Y,Tani K,L in Z,et al.Vaccine effect of granulocyte macrophagecolony stimulating factor or CD80 gene transduced murine hematopoietic tumorcells and their cooperative enhancement of anti-tumor immunity[J].Gene Ther,1998,5(10):1355-62.)。It is difficult to effectively stimulate the body's anti-tumor immunity only by tumor-specific antigens, and the interaction between B7.1 co-stimulatory molecules and their ligands is still required to participate in the activation of T cells. Many studies have also confirmed that most tumor cells do not express B7.1 co-stimulatory molecules, thereby evading the body's immune surveillance, so that tumors escape immune surveillance and grow indefinitely in the body (Yang Xuwei, Chen Yuanzhong, Lin Zhenxing. Human B7.1 gene cDNA Cloning and construction of retroviral expression vector [J]. Journal of Fujian Medical University, 2001, 35(2): 109-111.). GM-CSF has the function of activating professional antigen-presenting cells such as dendritic cells, making antigen-specific helper T lymphocytes and anti-tumor effector cells proliferate, and can activate a strong anti-tumor immune response in vivo. If the two are used in combination, immunity against tumor antigens can be initiated, and the proliferation of helper T lymphocytes and anti-tumor immune effector cells can be promoted, resulting in a synergistic anti-tumor effect. At present, studies have confirmed that the combined application of GM-CSF and B7.1 molecules can improve the killing effect of vaccines on tumor cells. The research results of Sumimoto et al. showed that the tumor vaccine (LLC/GM+B7) prepared by the lung cancer cell line LLC co-transfected with B7.1 gene and GM-CSF gene, the killing activity of CTL cells induced was significantly higher than that transfected with B7 alone. .1 or GM-CSF gene LLC cells (Sumimoto H, Tani K, Nakazaki Y, et al. GM-CSF and B7-1(CD80) co-stimulatory signals co-operate in the induction of effective anti-tumorimmunity in syngeneic mice. Int J Cancer. 1997, 73(4): 556-61.). The research results of Nakazaki et al. showed that the combined use of thymic lymphoma cells/GM-CSF and thymic lymphoma cells/B7.1 tumor vaccine had a synergistic anti-tumor immune effect, and the mechanism may be the main mechanism of tumor vaccine transduced by GM-CSF gene. The indirect pathway mediated by APC affects Th cells and then activates CTL; the tumor vaccine transcribed by B7.1 gene affects CTL through a direct pathway, and the combined use of the two can induce a systemic anti-tumor immune response, resulting in partial tumor eradication ( Nakazaki Y, Tani K, L in Z, et al. Vaccine effect of granulocyte macrophagecolony stimulating factor or CD80 gene transduced murine hematopoietic tumor cells and their cooperative enhancement of anti-tumor immunity[J].
LDH法测定细胞CTL效应的原理如图27所示:乳酸脱氢酶(LDH)是活细胞胞浆中的内含酶之一,当效应细胞和靶细胞共同孵育后,靶细胞的细胞膜受损,膜的通透性发生改变使胞浆中的LDH释放到胞外。取含有LDH的培养上清,经酶促反应使底物(tetrazolium salt)转化成一种紫红色的甲肷类化合物(formazan),在490nm处有很高的吸收峰,测定其A490值就能间接反映出效应细胞对靶细胞的杀伤状况。The principle of the LDH method for measuring the CTL effect of cells is shown in Figure 27: Lactate dehydrogenase (LDH) is one of the endogenous enzymes in the cytoplasm of living cells. When the effector cells and target cells are co-incubated, the cell membrane of the target cells is damaged. , the permeability of the membrane changes so that the LDH in the cytoplasm is released to the outside of the cell. Take the culture supernatant containing LDH, and convert the substrate (tetrazolium salt) into a purple-red formazan compound (formazan) through an enzymatic reaction, which has a high absorption peak at 490nm, and can be measured indirectly by measuring its A490 value. It reflects the killing status of effector cells on target cells.
发明内容Contents of the invention
本发明的目的是提供一种可用于构建基因疫苗且可同时启动两种基因共表达的DNA疫苗真核表达载体。The purpose of the present invention is to provide a DNA vaccine eukaryotic expression vector that can be used to construct a gene vaccine and can simultaneously promote the co-expression of two genes.
为解决上述技术问题,本发明采取以下技术方案:一种DNA疫苗真核表达载体,是在pVAX1载体骨架中含有内部核糖体进入位点(internal ribosome entry site,IRES)编码序列的重组真核表达载体。In order to solve the above-mentioned technical problems, the present invention adopts the following technical solutions: a DNA vaccine eukaryotic expression vector is a recombinant eukaryotic expression vector containing an internal ribosome entry site (internal ribosome entry site, IRES) coding sequence in the pVAX1 vector backbone. carrier.
由于所述内部核糖体进入位点的上、下游均可以启动基因表达,因此为方便外源基因的插入,在上述DNA疫苗真核表达载体中的内部核糖体进入位点编码序列的两端还可再添加一种或多种限制性内切酶识别位点,所述限制性内切酶的选择是广泛的,包括EcoR V、BamH I、Cla I、BstB I、Pvu I和Psi I、BssH II、Swa I、Pac I、Nsi I、Sma I、Xho I、Not I、Bst XI、Nhe I和Sal I等。Since the upstream and downstream of the internal ribosome entry site can initiate gene expression, in order to facilitate the insertion of foreign genes, the two ends of the internal ribosome entry site coding sequence in the above-mentioned DNA vaccine eukaryotic expression vector are also One or more restriction enzyme recognition sites can be added, the choice of restriction enzymes is extensive, including EcoR V, BamH I, Cla I, BstBI, Pvu I and Psi I, BssH II, Swa I, Pac I, Nsi I, Sma I, Xho I, Not I, Bst XI, Nhe I and Sal I, etc.
具体来讲,在所述内部核糖体进入位点编码序列的5’端添加限制性内切酶EcoRV、Cla I、BstB I、Pvu I和Psi I识别位点,在其3’端添加限制性内切酶BssH II、Swa I、Pac I、Nsi I和Xho I识别位点的DNA疫苗真核表达载体为pVAX1-IRES。Specifically, restriction endonucleases EcoRV, Cla I, BstBI, Pvu I and Psi I recognition sites were added at the 5' end of the internal ribosome entry site coding sequence, and restriction enzymes were added at its 3' end. The DNA vaccine eukaryotic expression vector of endonuclease BssH II, Swa I, Pac I, Nsi I and Xho I recognition sites is pVAX1-IRES.
所述DNA疫苗真核表达载体可按照基因工程领域的常规方法构建。The DNA vaccine eukaryotic expression vector can be constructed according to conventional methods in the field of genetic engineering.
所述DNA疫苗真核表达载体在制备基因疫苗中的应用也属于本发明的保护范围,所述基因疫苗包括针对多种疾病的基因疫苗,如抗肿瘤、抗流感、抗艾滋病、抗病毒性肝炎、抗结核、抗狂犬病或抗疟疾等基因疫苗。The application of the eukaryotic expression vector of the DNA vaccine in the preparation of the gene vaccine also belongs to the scope of protection of the present invention, and the gene vaccine includes the gene vaccine against various diseases, such as anti-tumor, anti-influenza, anti-AIDS, anti-viral hepatitis , anti-tuberculosis, anti-rabies or anti-malarial and other genetic vaccines.
本发明另一目的在于提供所述DNA疫苗真核表达载体的一种应用,即提供一种高效广谱实体肿瘤治疗性基因疫苗。Another object of the present invention is to provide an application of the DNA vaccine eukaryotic expression vector, that is, to provide a high-efficiency broad-spectrum therapeutic gene vaccine for solid tumors.
其中,以本发明的DNA疫苗真核表达载体为出发载体构建的抗肿瘤基因疫苗,其活性成分是在本发明的DNA疫苗真核表达载体的载体骨架中核糖体进入位点编码序列的上游含有Survivin-2B自氨基端第5-28位和第80-121位氨基酸残基编码序列构成的融合基因,以及人和猴的绒毛膜促性腺激素β链的核心片段CTP37区域编码序列构成的融合基因的重组真核表达载体。Among them, the anti-tumor gene vaccine constructed with the DNA vaccine eukaryotic expression vector of the present invention as the starting vector, its active ingredient is contained in the upstream of the ribosome entry site coding sequence in the vector backbone of the DNA vaccine eukaryotic expression vector of the present invention Survivin-2B is a fusion gene composed of the coding sequence of amino acid residues 5-28 and 80-121 at the amino terminal, and a fusion gene composed of the coding sequence of the core fragment CTP37 region of the human and monkey chorionic gonadotropin β chain recombinant eukaryotic expression vector.
所述Survivin-2B的自氨基端第5-28位氨基酸残基的编码序列和自氨基端第80-121位氨基酸残基的编码序列可通过连接臂“nG”连接,其中,n可以为K(赖氨酸)、R(精氨酸)、C(半胱氨酸)、Q(谷氨酰胺)G(甘氨酸)或N(天冬酰胺)。所述由连接臂“nG”连接的Survivin-2B自氨基端第5-28位和第80-121位氨基酸残基编码序列的融合基因的核苷酸序列可如序列表中序列1所示。The coding sequence of amino acid residues 5-28 from the amino terminal of Survivin-2B and the coding sequence of amino acid residues 80-121 from the amino terminal can be connected by a connecting arm "nG", wherein n can be K (lysine), R (arginine), C (cysteine), Q (glutamine), G (glycine), or N (asparagine). The nucleotide sequence of the fusion gene of Survivin-2B linked by the linker "nG" from the 5th-28th and 80th-121st amino acid residue coding sequence of the amino terminal can be shown as
所述Survivin-2B的自氨基端第5-28位氨基酸残基编码序列位于Survivin-2B的自氨基端第80-121位氨基酸残基编码序列的的3’或5’端均可。The Survivin-2B coding sequence of amino acid residues 5-28 from the amino terminal can be located at the 3' or 5' end of the coding sequence of amino acid residues 80-121 from the amino terminal of Survivin-2B.
所述人和猴的绒毛膜促性腺激素β链的核心片段CTP37区域编码序列也可通过连接臂“nG”连接,其中,n为K、R、C、Q、G或N。所述由连接臂“NG”连接的人和猴的绒毛膜促性腺激素β链的核心片段CTP37区域编码序列的融合基因的核苷酸序列可如序列表中序列2所示。The human and monkey chorionic gonadotropin β-chain core fragment CTP37 region coding sequences can also be connected by linking arm "nG", wherein n is K, R, C, Q, G or N. The nucleotide sequence of the fusion gene of the coding sequence of the core fragment CTP37 region of human chorionic gonadotropin β chain connected by the connecting arm "NG" can be shown as
所述人的绒毛膜促性腺激素β链的核心片段CTP37区域编码序列可位于猴的绒毛膜促性腺激素β链的核心片段CTP37区域编码序列的3’或5’端均可。The human chorionic gonadotropin beta chain core fragment CTP37 region coding sequence can be located at the 3' or 5' end of the monkey chorionic gonadotropin beta chain core fragment CTP37 region coding sequence.
此外,所述Survivin-2B自氨基端第5-28位和第80-121位氨基酸残基编码序列构成的融合基因位于人和猴绒毛膜促性腺激素β链的CTP37区域编码序列构成的融合基因的3’或5’端均可。In addition, the Survivin-2B fusion gene composed of the coding sequence of amino acid residues 5-28 and 80-121 at the amino terminal is located in the fusion gene composed of the coding sequence of the CTP37 region of the beta chain of human and monkey chorionic gonadotropin Either the 3' or 5' end can be used.
为引导目的基因在真核细胞内获得有效表达和分泌,所述抗肿瘤基因疫苗的真核表达载体的载体骨架中内部核糖体进入位点编码序列的上游还可连接有人Igκ链前导信号肽(sig)编码序列和/或人IgG-Fc段编码序列和/或糖基磷脂酰肌醇(GPI)锚定信号肽编码序列。In order to guide the target gene to obtain effective expression and secretion in eukaryotic cells, the upstream of the internal ribosome entry site coding sequence in the vector backbone of the eukaryotic expression vector of the anti-tumor gene vaccine can also be connected with a human Igκ chain leading signal peptide ( sig) coding sequence and/or human IgG-Fc segment coding sequence and/or glycosylphosphatidylinositol (GPI) anchor signal peptide coding sequence.
此外,为进一步增强所述抗肿瘤基因疫苗激活免疫应答的能力,在所述抗肿瘤基因疫苗的真核表达载体的载体骨架中内部核糖体进入位点编码序列的下游还可连接有人GM-CSF和/或B7.1共刺激分子编码序列,以及白细胞介素、干扰素和/或肿瘤坏死因子等免疫激活细胞因子的编码序列。In addition, in order to further enhance the ability of the anti-tumor gene vaccine to activate the immune response, the downstream of the internal ribosome entry site coding sequence in the vector backbone of the eukaryotic expression vector of the anti-tumor gene vaccine can also be connected with human GM-CSF And/or B7.1 co-stimulatory molecule coding sequence, and the coding sequence of immune activation cytokines such as interleukin, interferon and/or tumor necrosis factor.
具体来讲,在本发明的DNA疫苗真核表达载体的载体骨架中内部核糖体进入位点编码序列的上游含有Survivin-2B自氨基端第5-28位和第80-121位氨基酸残基编码序列构成的融合基因,以及人和猴的绒毛膜促性腺激素β链的核心片段CTP37区域编码序列构成的融合基因的重组真核表达载体可为pVAX1-2PAG。Specifically, in the vector backbone of the DNA vaccine eukaryotic expression vector of the present invention, the upstream of the internal ribosome entry site coding sequence contains Survivin-2B from amino terminal 5-28 and 80-121 amino acid residues The recombinant eukaryotic expression vector of the fusion gene composed of the fusion gene composed of sequence and the coding sequence of the core fragment CTP37 region of human and monkey chorionic gonadotropin β chain can be pVAX1-2PAG.
在本发明的DNA疫苗真核表达载体的载体骨架中在所述内部核糖体进入位点编码序列的上游依次连接有人Igκ链前导信号肽编码序列、Survivin-2B自氨基端第5-28位和第80-121位氨基酸残基编码序列构成的融合基因、人和猴的绒毛膜促性腺激素β链的核心片段CTP37区域编码序列构成的融合基因,以及人IgG-Fc段编码序列和糖基磷脂酰肌醇锚定信号肽编码序列的重组真核表达载体可为pVAX1-2PAG-Fc-GPI。In the vector backbone of the DNA vaccine eukaryotic expression vector of the present invention, the human Igκ chain leader signal peptide coding sequence, Survivin-2B from the 5th to 28th positions of the amino terminal and the upstream of the internal ribosome entry site coding sequence are sequentially connected The fusion gene composed of the coding sequence of amino acid residues 80-121, the fusion gene composed of the coding sequence of the CTP37 region of the core fragment of human and monkey chorionic gonadotropin β chain, and the coding sequence of human IgG-Fc segment and glycosylphospholipid The recombinant eukaryotic expression vector of the acyl inositol anchor signal peptide coding sequence can be pVAX1-2PAG-Fc-GPI.
在本发明的DNA疫苗真核表达载体的载体骨架中在所述内部核糖体进入位点编码序列的上游依次连接有人Igκ链前导信号肽编码序列、Survivin-2B自氨基端第5-28位和第80-121位氨基酸残基编码序列构成的融合基因(抗原基因)、人和猴绒毛膜促性腺激素β链的核心片段CTP37区域编码序列构成的融合基因(抗原基因)、人IgG-Fc段编码序列和糖基磷脂酰肌醇锚定信号肽编码序列(所述人Igκ链前导信号肽编码序列、人IgG-Fc段编码序列和糖基磷脂酰肌醇锚定信号肽编码序列与所述抗原基因融合表达),在所述内部核糖体进入位点编码序列的下游连接有人GM-CSF和B7.1共刺激分子编码序列的重组真核表达载体可为pVAX1-sig-2PAG-FC-GPI-GM/B7(简称pVAX1-2PFcGB)。In the vector backbone of the DNA vaccine eukaryotic expression vector of the present invention, the human Igκ chain leader signal peptide coding sequence, Survivin-2B from the 5th to 28th positions of the amino terminal and the upstream of the internal ribosome entry site coding sequence are sequentially connected Fusion gene (antigen gene) composed of coding sequence of amino acid residues 80-121, fusion gene (antigen gene) composed of coding sequence of core fragment CTP37 region of human and monkey chorionic gonadotropin β chain, human IgG-Fc segment Coding sequence and glycosyl phosphatidylinositol anchor signal peptide coding sequence (the human Igκ chain leading signal peptide coding sequence, human IgG-Fc fragment coding sequence and glycosyl phosphatidylinositol anchor signal peptide coding sequence are the same as the antigen gene fusion expression), the recombinant eukaryotic expression vector that connects human GM-CSF and B7.1 co-stimulatory molecule coding sequence downstream of the internal ribosome entry site coding sequence can be pVAX1-sig-2PAG-FC-GPI -GM/B7 (referred to as pVAX1-2PFcGB).
需要的时候,在上述疫苗的制备中还可以加入一种或多种药学上可接受的载体。所述载体包括药学领域常规的稀释剂、吸收促进剂或表面活性剂等。When necessary, one or more pharmaceutically acceptable carriers can also be added in the preparation of the above vaccine. The carrier includes conventional diluents, absorption enhancers or surfactants in the field of pharmacy.
上述疫苗的用量一般为0.01-200μg/kg体重/天,可以一次或多次使用,疗程一般为20-40天,剂量和疗程都可根据实际情况进行调整。The dosage of the above-mentioned vaccines is generally 0.01-200 μg/kg body weight/day, and can be used once or multiple times. The course of treatment is generally 20-40 days, and the dose and course of treatment can be adjusted according to the actual situation.
为提高疗效,本发明的药物还可以与抗生素、免疫刺激剂等进行组合治疗。In order to improve the curative effect, the medicine of the present invention can also be combined with antibiotics, immunostimulants, etc. for treatment.
本发明提供了一种DNA疫苗真核表达载体。该载体是针对DNA疫苗在恶性肿瘤治疗中展现出来的优势,在真核表达载体pVAX1的基础之上,引入了双顺反子表达元件-IRES序列,并在IRES序列的两端插入了多种稀有酶切位点,从而构建成功的一种新型DNA疫苗载体pVAX1-IRES。并在此NA疫苗载体pVAX1-IRES的基础上构建了一种多靶点复合抗肿瘤基因疫苗,该抗肿瘤基因疫苗是在pVAX1-IRES中IRES编码序列的上游含有人Igκ链前导信号肽(sig)、人Survivin-2B主要T细胞表位区域、人和猴绒毛膜促性腺激素β链的核心片段CTP37区域、人IgG-Fc段编码序列和糖基磷脂酰肌醇(GPI)锚定肽的多基因融合序列,在pVAX1-IRES中IRES编码序列的IRES下游含有人GM-CSF和B7.1融合基因的重组真核表达载体pVAX1-sig-2PAG-FC-GPI-GM/B7(简称pVAX1-2PFcGB)。转染有该质粒的293T细胞的流式细胞仪和免疫荧光检测结果表明,pVAX1-2PFcGB在293T细胞中获得很好的表达,此外,本发明的抗肿瘤基因疫苗还具有较好的体液免疫和细胞免疫效果,同时对肿瘤生长具有显著的抑制作用。以本发明的真核表达载体制备的基因疫苗有望对乳腺癌、胰腺癌、结直肠癌和前列腺癌等多种恶性实体瘤发挥广谱治疗作用,在肿瘤的治疗及其药物开发领域发挥重要作用,应用前景广阔。The invention provides a DNA vaccine eukaryotic expression vector. The vector aims at the advantages of DNA vaccines in the treatment of malignant tumors. On the basis of the eukaryotic expression vector pVAX1, a bicistronic expression element-IRES sequence is introduced, and a variety of Rare enzyme cutting sites, thus successfully constructing a new type of DNA vaccine vector pVAX1-IRES. And on the basis of this NA vaccine vector pVAX1-IRES, a multi-target composite anti-tumor gene vaccine was constructed. The anti-tumor gene vaccine contained the human Igκ chain leading signal peptide (sig ), human Survivin-2B main T cell epitope region, human and monkey chorionic gonadotropin β-chain core fragment CTP37 region, human IgG-Fc segment coding sequence and glycosylphosphatidylinositol (GPI) anchor peptide Multi-gene fusion sequence, the recombinant eukaryotic expression vector pVAX1-sig-2PAG-FC-GPI-GM/B7 containing human GM-CSF and B7.1 fusion gene in the IRES downstream of the IRES coding sequence in pVAX1-IRES (abbreviated as pVAX1- 2PFcGB). The results of flow cytometry and immunofluorescence detection of 293T cells transfected with the plasmid show that pVAX1-2PFcGB is well expressed in 293T cells. In addition, the anti-tumor gene vaccine of the present invention also has better humoral immunity and Cellular immune effect, at the same time has a significant inhibitory effect on tumor growth. The gene vaccine prepared with the eukaryotic expression vector of the present invention is expected to play a broad-spectrum therapeutic effect on various malignant solid tumors such as breast cancer, pancreatic cancer, colorectal cancer and prostate cancer, and play an important role in the field of tumor treatment and drug development , the application prospect is broad.
下面结合具体实施例对本发明做进一步详细说明。The present invention will be described in further detail below in conjunction with specific embodiments.
附图说明Description of drawings
图1为PCR扩增的IRES编码序列的1.2%琼脂糖凝胶电泳检测结果Figure 1 is the 1.2% agarose gel electrophoresis detection result of the IRES coding sequence amplified by PCR
图2为携带IRES编码序列的重组载体pSimple18-EcoRV/BAP-IRES的EcoR V和Xho I双酶切鉴定结果Figure 2 is the EcoR V and Xho I double enzyme digestion identification results of the recombinant vector pSimple18-EcoRV/BAP-IRES carrying the IRES coding sequence
图3为pSimple18-EcoRV/BAP-IRES和真核表达载体pVAX1的EcoR V和Xho I双酶切产物的1.2%琼脂糖凝胶电泳检测结果Figure 3 is the 1.2% agarose gel electrophoresis detection results of pSimple18-EcoRV/BAP-IRES and the EcoR V and Xho I double digestion products of the eukaryotic expression vector pVAX1
图4为真核DNA疫苗载体pVAX1-IRES的EcoR V和Xho I双酶切产物的1.2%琼脂糖凝胶电泳检测结果Fig. 4 is the 1.2% agarose gel electrophoresis detection result of the EcoR V of eukaryotic DNA vaccine vector pVAX1-IRES and the Xho I double digestion product
图5A为PCR扩增的DsRed1基因片段的1.2%琼脂糖凝胶电泳检测结果Figure 5A is the detection result of 1.2% agarose gel electrophoresis of the DsRed1 gene fragment amplified by PCR
图5B为PCR扩增的EGFP基因片段的1.2%琼脂糖凝胶电泳检测结果Figure 5B is the 1.2% agarose gel electrophoresis detection result of the EGFP gene fragment amplified by PCR
图6A为携带DsRed1基因的重组载体pGEM-T Easy-DsRed1的BamH I和Pvu I双酶切产物的1.2%琼脂糖凝胶电泳检测结果Figure 6A is the result of 1.2% agarose gel electrophoresis detection of the BamH I and Pvu I double digestion products of the recombinant vector pGEM-T Easy-DsRed1 carrying the DsRed1 gene
图6B为携带EGFP基因的重组载体pGEM-T Easy-EGFP的BssH II和Nsi I双酶切产物的1.2%琼脂糖凝胶电泳检测结果Figure 6B is the 1.2% agarose gel electrophoresis detection result of the BssH II and Nsi I double digestion products of the recombinant vector pGEM-T Easy-EGFP carrying the EGFP gene
图7为pGEM-T Easy-DsRed1、pGEM-T Easy-EGFP和pVAX1-IRES双酶切产物的1.2%琼脂糖凝胶电泳检测结果Figure 7 shows the results of 1.2% agarose gel electrophoresis detection of pGEM-T Easy-DsRed1, pGEM-T Easy-EGFP and pVAX1-IRES double digestion products
图8A为携带DsRed1基因的真核表达载体pVAX1-IRES-DsRed1的BamH I和Pvu I双酶切产物的1.2%琼脂糖凝胶电泳检测结果Figure 8A is the 1.2% agarose gel electrophoresis detection result of the BamH I and Pvu I double digestion products of the eukaryotic expression vector pVAX1-IRES-DsRed1 carrying the DsRed1 gene
图8B为携带EGFP基因的真核表达载体pVAX1-IRES-EGFP的Nsi I和BssH II双酶切产物的1.2%琼脂糖凝胶电泳检测结果Figure 8B is the 1.2% agarose gel electrophoresis detection result of the Nsi I and BssH II double enzyme digestion products of the eukaryotic expression vector pVAX1-IRES-EGFP carrying the EGFP gene
图8C为携带DsRed1和EGFP基因的真核表达载体pVAX1-DsRed1-IRES-EGFP的BamH I和Xho I双酶切产物的1.2%琼脂糖凝胶电泳检测结果Figure 8C is the 1.2% agarose gel electrophoresis detection result of the BamH I and Xho I double digestion product of the eukaryotic expression vector pVAX1-DsRed1-IRES-EGFP carrying DsRed1 and EGFP genes
图9为各种重组质粒在293T细胞内表达情况的激光扫描共聚焦显微镜检测结果Figure 9 shows the results of laser scanning confocal microscope detection of the expression of various recombinant plasmids in 293T cells
图10为各种重组质粒在293T细胞内瞬时表达情况的流式细胞仪检测结果Figure 10 is the flow cytometry detection result of the transient expression of various recombinant plasmids in 293T cells
图11为抗原复合物2PAG融合基因的结构式意图Figure 11 is a schematic diagram of the structural formula of the antigen complex 2PAG fusion gene
图12为PCR扩增的Sur-SC和SC-hmCTP及抗原复合物2PAG融合基因的1%琼脂糖凝胶电泳检测结果Figure 12 is the detection result of 1% agarose gel electrophoresis of Sur-SC and SC-hmCTP and antigen complex 2PAG fusion gene amplified by PCR
图13为PCR扩增的人GM-CSF因子和B7.1融合基因的1%琼脂糖凝胶电泳检测结果Figure 13 is the 1% agarose gel electrophoresis detection result of the human GM-CSF factor and B7.1 fusion gene amplified by PCR
图14为含有抗原复合物2PAG融合基因的真核表达质粒pCI-2PAG-Fc-GPI的菌落PCR及酶切鉴定结果Figure 14 is the colony PCR and enzyme digestion identification results of the eukaryotic expression plasmid pCI-2PAG-Fc-GPI containing the antigen complex 2PAG fusion gene
图15为真核表达质粒pVAX-sig-2P-FC-GPI-IRES的酶切鉴定结果Fig. 15 is the enzyme digestion identification result of the eukaryotic expression plasmid pVAX-sig-2P-FC-GPI-IRES
图16为真核表达质粒pVAX1-IRES-GM/B7的酶切鉴定结果Fig. 16 is the enzyme digestion identification result of the eukaryotic expression plasmid pVAX1-IRES-GM/B7
图17为pVAX1-sig-2PAG-Fc-GPI-GM/B7(pVAX1-2PFcGB)的酶切鉴定结果Figure 17 shows the enzyme digestion and identification results of pVAX1-sig-2PAG-Fc-GPI-GM/B7 (pVAX1-2PFcGB)
图18为pVAX1-2PFcGB在293T转染细胞内的瞬时表达效率的流式细胞仪检测结果Figure 18 is the result of flow cytometry detection of the transient expression efficiency of pVAX1-2PFcGB in 293T transfected cells
图19为pVAX1-2PFcGB在293T转染细胞内的瞬时表达效率的免疫荧光检测结果Figure 19 shows the results of immunofluorescence detection of the transient expression efficiency of pVAX1-2PFcGB in 293T transfected cells
图20为Balb/c小鼠的免疫方案Figure 20 is the immunization scheme of Balb/c mice
图21为各组免疫小鼠皮下移植瘤的平均成瘤时间的统计结果Figure 21 is the statistical result of the average tumor formation time of the subcutaneous transplanted tumors in each group of immunized mice
图22为各组免疫小鼠体内移植瘤的生长曲线Figure 22 is the growth curve of transplanted tumors in each group of immunized mice
图23为各组免疫小鼠皮下移植瘤攻击30天后离体肿瘤体积的比较结果Figure 23 is the comparison result of the tumor volume in vitro after 30 days of challenge with subcutaneous xenograft tumors of immunized mice in each group
图24为皮下移植瘤攻击35天后各组免疫小鼠体内的平均瘤重统计结果Figure 24 is the statistical result of the average tumor weight in each group of immunized mice after 35 days of subcutaneous transplantation challenge
图25为皮下移植瘤攻击35天后各免疫组的肿瘤抑制率的统计结果Figure 25 is the statistical result of the tumor inhibition rate of each immune group after 35 days of challenge with subcutaneous transplanted tumors
图26为2D组、E组和F组免疫小鼠血清抗体效价的ELISA检测结果Figure 26 is the ELISA detection result of serum antibody titer of immunized mice in 2D group, E group and F group
图27为LDH法测定细胞CTL效应的原理示意图Figure 27 is a schematic diagram of the principle of the LDH method for measuring the effect of cell CTL
图28为各免疫组小鼠脾细胞的杀伤活性检测结果Figure 28 is the detection result of the killing activity of mouse splenocytes in each immune group
图29为pVAX1-2PFcGB、pVAX1-2PAG、pVAX1-2PAG-Fc-GPI的结构示意图Figure 29 is a schematic diagram of the structure of pVAX1-2PFcGB, pVAX1-2PAG, pVAX1-2PAG-Fc-GPI
具体实施方式Detailed ways
下述实施例中所用方法如无特别说明均为常规方法,具体步骤可参见:《Molecular Cloning:A Laboratory Manual》(Sambrook,J.,Russell,David W.,Molecular Cloning:A Laboratory Manual,3rd edition,2001,NY,Cold SpringHarbor)。所用引物及DNA序列如无特别说明均由北京三博远志生物技术有限责任公司合成。所有百分比浓度如无特别说明均为体积/体积(V/V)或质量/体积(W/V)百分比浓度。The methods used in the following examples are conventional methods unless otherwise specified, and the specific steps can be found in: "Molecular Cloning: A Laboratory Manual" (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3 rd edition, 2001, NY, Cold Spring Harbor). The primers and DNA sequences used were synthesized by Beijing Sanbo Yuanzhi Biotechnology Co., Ltd. unless otherwise specified. All percentage concentrations are volume/volume (V/V) or mass/volume (W/V) percentage concentrations unless otherwise specified.
实施例1、DNA疫苗载体pVAX1-IRES的构建
一、RES编码序列的PCR扩增与纯化1. PCR amplification and purification of RES coding sequence
首先根据pIRES-neo载体中的IRES编码序列(IRES序列的位置为1295-1880bp)设计其PCR扩增引物,并在用于PCR扩增IRES编码序列的上游引物IRES-F中添加了限制性内切酶EcoR V、Cla I、BstB I、Pvu I和Psi I识别位点,在用于PCR扩增IRES编码序列的下游引物IRES-R中添加了限制性内切酶BssH II、Swa I、Pac I、Nsi I和Xho I识别位点,具体引物序列如下:IRES-F(上游引物):5′-CCGATATCGATTCGAACGATCGTTATAACTAGGGCGGCCAATTCCGCC-3′IRES-R(下游引物):5′-GCCTCGAGCGCGCATTTAAATTAATTAATGCATTGGCTGCAGGAATTATCCCG-3′。引物合成后,用灭菌蒸馏水配制成20μmol/L的使用液,分装,-20℃保存备用。First, design its PCR amplification primers according to the IRES coding sequence in the pIRES-neo vector (the position of the IRES sequence is 1295-1880bp), and add a restriction internal to the upstream primer IRES-F for PCR amplification of the IRES coding sequence Dicer EcoR V, Cla I, BstB I, Pvu I, and Psi I recognition sites, restriction endonucleases BssH II, Swa I, Pac added to the downstream primer IRES-R for PCR amplification of the IRES coding sequence I, Nsi I and Xho I recognition sites, specific primer sequences are as follows: IRES-F (upstream primer): 5'-CCGATATCGATTCGAACGATCGTTATAACTAGGGCGGCCAATTCCGCC-3'IRES-R (downstream primer): 5'-GCCTCGAGCGCGCATTTAAATTAATTAATGCATTGGCTGCAGGAATTATCCCG-3'. After the primers are synthesized, use sterilized distilled water to prepare a 20 μmol/L use solution, aliquot them, and store them at -20°C for later use.
然后,以质粒pIRES-neo(Clontech公司)为模板,在引物IRES-F和IRES-R的引导下,在Perkin Elmer公司的GeneAmp PCR System2400型PCR仪上PCR扩增IRES的编码序列,50μlPCR反应体系为:5×PrimeSTARTM缓冲液(含Mg2+)10μl,dNTPs(2.5mmol/L)4μl,上游引物(20μmol/L)1μl,下游引物(20μmol/L)1μl,PrimeSTARTM HS DNA Polymerase(2.5U/μl,TaKaRa公司)0.5μl,模板(pIRES-neo质粒稀释100倍)1μl,灭菌蒸馏水32.5μl。PCR反应条件为:先95℃ 3min;然后94℃ 45s,60℃ 45s,72℃ 1min 30s,共循环30次;最后72℃延伸7min,4℃保存。反应结束后,取5μl PCR扩增产物进行1.2%琼脂糖凝胶电泳检测,检测结果如图1所示(泳道M为DL 2000 DNA Marker(购自TaKaRa公司),泳道1为:IRES编码序列的PCR扩增产物),经扩增获得了682bp的DNA条带,与预期大小一致。用博大泰克公司的PCR产物胶回收试剂盒并参照试剂盒说明书回收并纯化该目的条带。Then, using the plasmid pIRES-neo (Clontech Company) as a template, under the guidance of primers IRES-F and IRES-R, PCR amplified the coding sequence of IRES on the GeneAmp PCR System2400 type PCR instrument of Perkin Elmer Company, 50 μl PCR reaction system For: 5×PrimeSTAR TM buffer (containing Mg 2+ ) 10μl, dNTPs (2.5mmol/L) 4μl, upstream primer (20μmol/L) 1μl, downstream primer (20μmol/L) 1μl, PrimeSTAR TM HS DNA Polymerase (2.5 U/μl, TaKaRa Company) 0.5 μl, template (pIRES-neo plasmid diluted 100 times) 1 μl, sterilized distilled water 32.5 μl. The PCR reaction conditions were as follows: first 95°C for 3min; then 94°C for 45s, 60°C for 45s, 72°C for 1min and 30s, a total of 30 cycles; finally 72°C for 7min, and 4°C for storage. After the reaction was finished, 5 μl of the PCR amplification product was taken for 1.2% agarose gel electrophoresis detection, and the detection results were as shown in Figure 1 (swimming lane M is
二、携带IRES编码序列的pSimple18-EcoRV/BAP重组载体的构建与鉴定2. Construction and identification of pSimple18-EcoRV/BAP recombinant vector carrying IRES coding sequence
1、大肠杆菌DH5α感受态细胞的制备(CaCl2法)1. Preparation of Escherichia coli DH5α competent cells (CaCl 2 method)
a.取-70℃保存的大肠杆菌DH5α菌种,将其划线接种于LB琼脂平板上,在37℃温箱中培养过夜(12-24小时);a. Take the Escherichia coli DH5α strain stored at -70°C, inoculate it on the LB agar plate, and cultivate it overnight (12-24 hours) in a 37°C incubator;
b.挑取湿润光滑,边缘整齐的大肠杆菌DH5α单个菌落接种至3mL LB液体培养基中,在37℃、200rpm下振荡培养过夜;b. Pick a single colony of Escherichia coli DH5α that is moist, smooth, and with neat edges and inoculate it into 3 mL of LB liquid medium, and cultivate overnight at 37°C and 200 rpm;
c.将活化的菌种按1%接种于50mL LB液体培养基中,37℃振荡培养2-4h,使细菌生长达到对数生长期,即OD600nm=0.3-0.6;c. Inoculate the activated bacteria at 1% in 50mL LB liquid medium, shake and culture at 37°C for 2-4 hours, so that the bacteria grow to the logarithmic growth phase, that is, OD 600nm =0.3-0.6;
d.在无菌条件下将菌液转移至无菌、预冷的聚丙烯管中,冰浴10min后4℃、8000rpm离心10min,使菌体完全沉淀;d. Transfer the bacterial solution to a sterile, pre-cooled polypropylene tube under aseptic conditions, and then centrifuge at 4°C and 8000rpm for 10 minutes after ice bathing for 10 minutes to completely precipitate the bacteria;
e.弃上清,用10mL预冷的0.1mol/L CaCl2重悬菌体沉淀,冰浴20min后,4℃、6000rpm离心10min;e. Discard the supernatant, resuspend the bacterial pellet with 10 mL of pre-cooled 0.1 mol/L CaCl 2 , and then centrifuge at 4°C and 6000 rpm for 10 min after ice bathing for 20 min;
f.弃上清,用2mL预冷的0.1mol/L CaCl2重悬菌体沉淀,冰浴3.5h后,加入15%甘油,分装,-70℃保存备用。f. Discard the supernatant, resuspend the bacterial pellet with 2 mL of pre-cooled 0.1 mol/L CaCl 2 , and ice-bath for 3.5 hours, add 15% glycerol, aliquot, and store at -70°C for later use.
2、PCR扩增的IRES编码序列的5’末端磷酸化2. Phosphorylation of the 5' end of the PCR-amplified IRES coding sequence
用TaKaRa公司的DNA Kination Kit高效磷酸化试剂盒并参照试剂盒说明书对步骤一扩增的IRES编码序列进行5’末端磷酸化,具体步骤如下:Use the DNA Kination Kit high-efficiency phosphorylation kit from TaKaRa Company and refer to the kit instructions to phosphorylate the 5' end of the IRES coding sequence amplified in
a.在微量离心管中配制下列反应液,全量为50μl;a. Prepare the following reaction solution in a microcentrifuge tube, the total volume is 50 μl;
T4 Polynucleotide KinaseT 4 Polynucleotide Kinase
2μl2 μl
(10U/μl)(10U/μl)
ATP(10mM) 5μlATP(10mM) 5μl
10×Reaction缓冲液 5μl10×Reaction buffer 5μl
PCR纯化产物 4μlPCR purification product 4μl
ddH2O 34μlddH 2 O 34 μl
总体积 50μlTotal volume 50μl
b.37℃反应30分钟;b. React at 37°C for 30 minutes;
c.70℃加热10分钟,使酶失活;c. Heat at 70°C for 10 minutes to inactivate the enzyme;
d.加入50μl的ddH2O,补充体积至100μl;d. Add 50 μl of ddH 2 O to make up the volume to 100 μl;
e.加入10μl(1/10量)的3M CH3COONa(pH5.2);e. Add 10 μl (1/10 amount) of 3M CH 3 COONa (pH5.2);
f.加入4μl的DNAmate溶液,均匀混合;f. Add 4 μl of DNAmate solution and mix evenly;
g.加入250μl的-20℃预冷的无水乙醇,充分混匀;g. Add 250 μl of -20°C pre-cooled absolute ethanol and mix thoroughly;
h.4℃、12000rpm离心10分钟,回收沉淀,用70%冷乙醇清洗沉淀后,室温干燥10分钟;h. Centrifuge at 4°C and 12000rpm for 10 minutes, recover the precipitate, wash the precipitate with 70% cold ethanol, and dry at room temperature for 10 minutes;
6.将沉淀溶解于20μl的ddH2O中。6. Dissolve the pellet in 20 μl of ddH2O .
3、5’末端磷酸化后的IRES编码序列与pSimple18-EcoRV/BAP载体的连接3. Ligation of the phosphorylated IRES coding sequence at the 5' end to the pSimple18-EcoRV/BAP vector
用TaKaRa公司的Ligation Mix高效DNA连接酶将步骤2经5’末端磷酸化后的IRES编码序列与载体pSimple18-EcoRV/BAP(TaKaRa公司)进行连接,连接体系如下:Ligate the IRES coding sequence phosphorylated at the 5' end of
pSimple18-EcoRV/BAP载体 1μlpSimple18-EcoRV/
5’末端磷酸化后的IRES编码序列 4μlIRES coding sequence phosphorylated at the 5'
Ligation Mix 5μlLigation Mix 5μl
总体积 10μlTotal volume 10μl
连接条件为:16℃连接1h。The connection conditions are: 16°C connection for 1h.
4、将连接产物转化大肠杆菌DH5α感受态细胞4. Transform the ligation product into Escherichia coli DH5α competent cells
取步骤1制备的新鲜大肠杆菌感受态细胞DH5α 200μl,置于冰浴中,加入步骤3的连接产物10μl,轻轻混匀后冰浴30min,立即转移到42℃水浴中热休克90s,再冰浴静置2min,加入0.5mL不加抗生素的LB液体培养基,37℃水浴15min后,37℃摇床轻摇45min;随后,取200μl转化菌体,均匀涂布于含有氨苄青霉素(100μg/mL)的LB平板培养基上,在超净工作台吹干约10min,37℃倒置培养12-16h。然后,从LB抗性平板中挑取长出的单一菌落5个,分别接种于含氨苄青霉素(100μg/mL)的LB液体培养基中(5mL/管),37℃摇床培养过夜。Take 200 μl of fresh Escherichia coli competent cells DH5α prepared in
5、提取质粒5. Extract plasmid
将步骤4)获得的转化有携带IRES编码序列的pSimple18-EcoRV/BAP的重组质粒菌液过夜培养,用博大泰克公司的碱裂解法试剂盒并参照试剂盒说明书提取质粒。The recombinant plasmid bacteria liquid transformed with pSimple18-EcoRV/BAP carrying the IRES coding sequence obtained in step 4) was cultured overnight, and the plasmid was extracted with the alkaline lysis kit of Biotech and referring to the kit instructions.
6、酶切鉴定及测序6. Enzyme digestion identification and sequencing
对步骤5用碱裂解法试剂盒提取的质粒用限制性内切酶EcoR V和Xho I进行双酶切鉴定,酶切体系如下:The plasmid extracted with the alkaline lysis kit in
10×NEBuffer3 2μl10×NEBuffer3 2μl
100×BSA 0.2μl100×BSA 0.2μl
重组质粒质粒DNA 5μlRecombinant plasmid plasmid DNA 5μl
Xho I 1μlXho I 1μl
EcoR V 1μlEcoR V 1μl
灭菌双蒸水 10.8μlSterilized double distilled water 10.8μl
总体积 20μlTotal volume 20μl
酶切条件为:37℃水浴酶切过夜。对双酶切产物进行1.2%琼脂糖凝胶电泳鉴定,鉴定结果如图2所示(泳道M为DL 2000 DNA Marker,泳道1为:EcoR V和Xho I双酶切产物),经酶切获得了2692bp和682bp的DNA条带,与预期结果一致,表明IRES编码序列已成功连入pSimple18-EcoRV/BAP载体中。然后,将经酶切鉴定正确的阳性克隆菌液送北京三博远志生物技术有限责任公司进行测序,测序结果表明获得了序列正确的携带IRES编码序列的pSimple18-EcoRV/BAP重组载体,将其命名为pSimple18-EcoRV/BAP-IRES。The enzyme digestion conditions are: 37°C water bath enzyme digestion overnight. The double digestion products were identified by 1.2% agarose gel electrophoresis, and the identification results are shown in Figure 2 (lane M is
三、真核DNA疫苗载体pVAX1-IRES的构建与鉴定3. Construction and identification of eukaryotic DNA vaccine vector pVAX1-IRES
1、酶切1. Enzyme digestion
将步骤二经测序正确的阳性重组质粒pSimple18-EcoRV/BAP-IRES和真核表达载体pVAX1(invitrogen公司)分别用限制性内切酶EcoR V和Xho I进行双酶切,酶切条件为37℃水浴过夜,酶切体系如下:The positive recombinant plasmid pSimple18-EcoRV/BAP-IRES and the eukaryotic expression vector pVAX1 (invitrogen company) that were correctly sequenced in
2、酶切产物的回收、连接和转化2. Recovery, ligation and transformation of enzyme digestion products
对步骤1的双酶切产物进行1.2%琼脂糖凝胶电泳检测,检测结果如图3所示(泳道M为DL 2000 DNA Marker,泳道1为:pVAX1的EcoR V和Xho I双酶切产物,泳道2为pSimple18-EcoRV/BAP-IRES的EcoR V和Xho I双酶切产物),pVAX1经酶切获得了大小约为3000bp的DNA条带,pSimple18-EcoRV/BAP-IRES经酶切获得了682bp的DNA条带,与预期结果一致。用北京博大泰克生物基因技术有限责任公司的“PCR片断快速胶回收试剂盒”并参照试剂盒说明书回收并纯化经酶切过夜的重组质粒pSimple18-EcoRV/BAP-IRES中的IRES编码序列的DNA片段和真核表达载体pVAX1。将纯化后的目的基因IRES和线性化载体pVAX1进行连接,16℃连接1h,连接体系如下:Perform 1.2% agarose gel electrophoresis on the double-digestion product of
目的基因IRES 5μlTarget gene IRES 5μl
线性化pVAX1载体 5μl
Ligation Mix 10μlLigation Mix 10μl
总体积 20μl。
然后,用与步骤二相同的方法将连接产物转化大肠杆菌DH5α感受态细胞。Then, the ligation product was transformed into Escherichia coli DH5α competent cells by the same method as
3、阳性重组质粒的筛选与鉴定3. Screening and identification of positive recombinant plasmids
挑取在含卡那霉素(50μg/mL)LB抗性平板上长出的单菌落6个(编号1-6),分别接种于含卡那霉素(50μg/mL)的LB液体培养基中(5mL/管),37℃摇床培养过夜,提质粒,用限制性内切酶EcoR V和Xho I进行双酶切鉴定,酶切体系如下:
然后,对双酶切产物进行1.2%琼脂糖凝胶电泳检测,检测结果如图4所示(泳道M为DL 2000 DNA Marker,泳道1为:pVAX1空载体,泳道2为IRES的DNA片段,泳道3-8为挑选的1-6号单克隆质粒的EcoR V和Xho I双酶切产物),阳性重组质粒经双酶切可获得3000bp和682bp的DNA条带,其中4,5,7,8号单克隆为阳性克隆,与预期结果一致,证明获得了序列及插入位置均正确的含有IRES编码序列的pVAX1重组载体,命名为pVAX1-IRES,即本发明的真核DNA疫苗载体。Then, carry out 1.2% agarose gel electrophoresis detection to the double-digestion product, and detection result is as shown in Figure 4 (swimming lane M is
实施例2、真核DNA疫苗载体pVAX1-IRES的表达功能验证Example 2, Verification of the expression function of the eukaryotic DNA vaccine vector pVAX1-IRES
为了检验实施例1构建的真核DNA疫苗载体pVAX1-IRES是否具有同时启动两种基因进行共表达的功能,首先从红色荧光蛋白表达载体pDsRed1-N1(Clontech公司)中扩增出红色荧光蛋白基因DsRed1(EF 517319),从增强型绿色荧光蛋白表达载体pEGFP-N1(Clontech公司)中扩增出增强型绿色荧光蛋白基因EGFP(EF 587314),然后将扩增出的DsRed1基因片段和EGFP基因片段分别插入pVAX1-IRES载体中IRES编码序列的上游和下游,得到重组表达质粒pVAX1-IRES-DsRed1、pVAX1-IRES-EGFP、pVAX1-DsRed1-IRES-EGFP。将这三个重组真核表达质粒瞬时转染人胚胎肾细胞293T,通过流式细胞仪和激光扫描共聚焦显微镜分析DsRed1、EGFP的表达情况,从而检验所构建的DNA疫苗载体pVAX1-IRES是否具有同时启动两种基因进行共表达的功能,In order to check whether the eukaryotic DNA vaccine vector pVAX1-IRES constructed in Example 1 has the function of co-expressing two genes at the same time, the red fluorescent protein gene was first amplified from the red fluorescent protein expression vector pDsRed1-N1 (Clontech Company) DsRed1 (EF 517319), the enhanced green fluorescent protein gene EGFP (EF 587314) was amplified from the enhanced green fluorescent protein expression vector pEGFP-N1 (Clontech Company), and then the amplified DsRed1 gene fragment and EGFP gene fragment The upstream and downstream of the IRES coding sequence were respectively inserted into the pVAX1-IRES vector to obtain recombinant expression plasmids pVAX1-IRES-DsRed1, pVAX1-IRES-EGFP, and pVAX1-DsRed1-IRES-EGFP. These three recombinant eukaryotic expression plasmids were transiently transfected into 293T human embryonic kidney cells, and the expression of DsRed1 and EGFP were analyzed by flow cytometry and laser scanning confocal microscopy, so as to test whether the constructed DNA vaccine vector pVAX1-IRES had Simultaneously activate the function of co-expression of two genes,
具体实验方法如下:The specific experimental method is as follows:
6.PCR扩增DsRed1基因和EGFP基因片段6. PCR amplification of DsRed1 gene and EGFP gene fragments
6.引物设计6. Primer Design
根据红色荧光蛋白基因DsRed1(EF 517319)和增强型绿色荧光蛋白EGFP基因(EF587314)设计PCR扩增引物,引物序列如下:PCR amplification primers were designed according to the red fluorescent protein gene DsRed1 (EF 517319) and the enhanced green fluorescent protein EGFP gene (EF587314). The primer sequences are as follows:
F-DsRed1(上游引物):5’-CCG GGATCCACCGGTCGCCACCATGGTGC-3’(带下划线碱基为限制性内切酶BamH I识别位点)F-DsRed1 (upstream primer): 5'-CCG GGATCC ACCGGTCGCCACCATGGTGC-3' (underlined base is the recognition site of restriction endonuclease BamH I)
R-DsRed1(下游引物):5’-CCG CGATCGCTACAGGAACAGGTGG-3’(带下划线碱基为限制性内切酶Pvu I识别位点);R-DsRed1 (downstream primer): 5'-CCG CGATCG CTACAGGAACAGGTGG-3' (underlined base is the recognition site of restriction endonuclease Pvu I);
F-EGFP(上游引物):5’-CCG ATGCATCGCCACCATGGTGAGC-3’(带下划线碱基为限制性内切酶Nsi I识别位点)F-EGFP (upstream primer): 5'-CCG ATGCAT CGCCACCATGGTGAGC-3' (underlined bases are restriction endonuclease Nsi I recognition sites)
R-EGFP(下游引物):5’-CCG GCGCGCTTACTTGTACAGCTCG-3’(带下划线碱基为限制性内切酶BssH II识别位点)。R-EGFP (downstream primer): 5'-CCG GCGCGC TTACTTGTACAGCTCG-3' (underlined base is the recognition site of restriction endonuclease BssH II).
引物合成、纯化后用灭菌蒸馏水配制成20μmol/L的使用液,分装,-20℃保存备用。After the primers were synthesized and purified, they were prepared into a 20 μmol/L use solution with sterilized distilled water, aliquoted, and stored at -20°C for later use.
2、DsRed1基因片段的PCR扩增及其回收、纯化2. PCR amplification of DsRed1 gene fragment and its recovery and purification
以质粒pDsRed1-N1(Clontech公司)为模板,在引物F-DsRed1和R-DsRed1的引导下,在Perkin Elmer公司的GeneAmp PCR System 2400型PCR仪上PCR扩增DsRed1基因序列,50μl PCR反应体系为:10×Ex Taq缓冲液5μl,dNTPs(2.5mmol/L)4μl,上游引物(20μmol/L)1μl,下游引物(20μmol/L)1μl,TaKaRa ExTaq(5U/μl,TaKaRa公司)1μl,模板(pDsRed1-N1质粒稀释100倍)1μl,灭菌蒸馏水37μl。PCR反应条件为:先95℃ 5min;然后94℃ 45s,62℃ 45s,72℃ 2min,共循环35次;最后72℃延伸10min。反应结束后,取5μl PCR扩增产物进行1.2%琼脂糖凝胶电泳检测,检测结果如图5A所示(泳道M为DL 2000 DNA Marker,泳道1为:DsRed1基因的PCR扩增产物),经扩增获得了723bp的DNA条带,与预期大小一致。用博大泰克公司的PCR产物胶回收试剂盒并参照试剂盒说明书回收并纯化该目的条带。Using the plasmid pDsRed1-N1 (Clontech Company) as a template, under the guidance of primers F-DsRed1 and R-DsRed1, the DsRed1 gene sequence was amplified by PCR on the GeneAmp PCR System 2400 PCR instrument of Perkin Elmer Company, and the 50 μl PCR reaction system was : 5 μl of 10×Ex Taq buffer, 4 μl of dNTPs (2.5 mmol/L), 1 μl of upstream primer (20 μmol/L), 1 μl of downstream primer (20 μmol/L), 1 μl of TaKaRa ExTaq (5U/μl, TaKaRa Company), template ( pDsRed1-N1 plasmid diluted 100 times) 1 μl, 37 μl of sterilized distilled water. The PCR reaction conditions were as follows: first 95°C for 5min; then 94°C for 45s, 62°C for 45s, 72°C for 2min, a total of 35 cycles; finally, 72°C for 10min. After the reaction, 5 μl of the PCR amplification product was taken for 1.2% agarose gel electrophoresis detection, and the detection result was as shown in Figure 5A (swimming lane M is
3、EGFP基因片段的PCR扩增及其回收、纯化3. PCR amplification of EGFP gene fragment and its recovery and purification
以质粒pEGFP-N1(Clontech公司)为模板,在引物F-EGFP和R-EGFP的引导下,PCR扩增EGFP基因序列,反应体系及反应条件与步骤2相同。反应结束后,取5μl PCR扩增产物进行1.2%琼脂糖凝胶电泳检测,检测结果如图5B所示(泳道M为DL 2000DNA Marker,泳道1为:EGFP基因的PCR扩增产物),经扩增获得了746bp的DNA条带,与预期大小一致。用博大泰克公司的PCR产物胶回收试剂盒并参照试剂盒说明书回收并纯化该目的条带。Using the plasmid pEGFP-N1 (Clontech Company) as a template, under the guidance of primers F-EGFP and R-EGFP, the EGFP gene sequence was amplified by PCR, and the reaction system and reaction conditions were the same as in
二、pGEM-T Easy-DsRed1、pGEM-T Easy-EGFP重组克隆载体的构建2. Construction of pGEM-T Easy-DsRed1 and pGEM-T Easy-EGFP recombinant cloning vectors
1、将纯化后的PCR扩增产物与pGEM-T Easy载体连接及连接产物的转化1. Ligate the purified PCR amplification product to the pGEM-T Easy vector and transform the ligation product
将步骤一扩增并经纯化的DsRed1基因和EGFP基因分别连接入大肠杆菌克隆载体pGEM-T Easy(购自TaKaRa公司)中,16℃连接过夜,连接体系如下:The amplified and purified DsRed1 gene and EGFP gene in
PCR扩增的目的基因纯化产物 6.0μlPCR amplified target gene purification product 6.0μl
pGEM-T Easy 2.0μlpGEM-T Easy 2.0μl
10×T4 DNA Ligase buffer 1.0μl10×T 4 DNA Ligase buffer 1.0μl
T4DNA Ligase(12μ/μl) 1.0μlT 4 DNA Ligase (12μ/μl) 1.0μl
总体积 10μl。The total volume is 10 μl.
反应结束后,用与实施例1步骤二中相同的方法将两个基因的连接产物转化大肠杆菌DH5α感受态细胞。After the reaction, the ligation product of the two genes was transformed into Escherichia coli DH5α competent cells by the same method as in
2、阳性重组质粒的筛选与鉴定2. Screening and identification of positive recombinant plasmids
挑取在含氨苄青霉素(100μg/mL)LB抗性平板上长出的分别转化DsRed1基因和EGFP基因的单菌落各6个(均编号1-6),分别接种于含氨苄青霉素(Amp+)(100μg/mL)的LB液体培养基中(5mL/管),37℃摇床培养过夜,提质粒,对携带DsRed1基因的质粒(命名为pGEM-T Easy-DsRed1)用限制性内切酶BamH I和Pvu I进行双酶切鉴定,酶切体系如下:
对携带EGFP基因的质粒(命名为pGEM-T Easy-EGFP)用限制性内切酶BssH II和NsiI进行双酶切鉴定,酶切体系如下:The plasmid carrying the EGFP gene (named pGEM-T Easy-EGFP) was identified by double enzyme digestion with restriction endonucleases BssH II and NsiI. The enzyme digestion system is as follows:
酶切反应条件均为37℃水浴酶切过夜。然后,对双酶切产物进行1.2%琼脂糖凝胶电泳鉴定,其中,携带DsRed1基因的质粒pGEM-T Easy-DsRed1的酶切鉴定结果如图6A所示(泳道M为DL 2000 DNA Marker,泳道1-6为挑选的1-6号单克隆质粒的BamH I和Pvu I双酶切产物,泳道7为DsRed1基因片段),阳性重组质粒经双酶切可获得3015bp和717bp的DNA条带,其中1,3,4,5号单克隆为阳性克隆,与预期结果一致,最后将经酶切鉴定正确的阳性克隆菌液送北京三博远志生物技术有限责任公司进行测序,测序结果表明获得了序列及插入位置均正确的含有DsRed1基因的重组大肠杆菌克隆载体pGEM-T Easy-DsRed1;携带EGFP基因的质粒pGEM-T Easy-EGFP的酶切鉴定结果如图6B所示(泳道M为DL 2000 DNA Marker,泳道1-6为挑选的1-6号单克隆质粒的BssH II和Nsi I双酶切产物),阳性重组质粒经双酶切可获得3015bp和740bp的DNA条带,其中1,2,4,5,6号单克隆为阳性克隆,与预期结果一致,最后将经酶切鉴定正确的阳性克隆菌液送北京三博远志生物技术有限责任公司进行测序,测序结果表明获得了序列及插入位置均正确的含有EGFP基因的重组大肠杆菌克隆载体pGEM-T Easy-EGFP。The enzyme digestion reaction conditions were 37°C water bath enzyme digestion overnight. Then, carry out 1.2% agarose gel electrophoresis identification to the product of double enzyme digestion, wherein, the enzyme digestion identification result of the plasmid pGEM-T Easy-DsRed1 carrying DsRed1 gene is shown in Figure 6A (swimming lane M is
三、重组载体pVAX1-IRES-DsRed1、pVAX1-IRES-EGFP和pVAX1-DsRed1-IRES-EGFP的构建3. Construction of recombinant vectors pVAX1-IRES-DsRed1, pVAX1-IRES-EGFP and pVAX1-DsRed1-IRES-EGFP
1、pVAX1-IRES-DsRed1的构建1. Construction of pVAX1-IRES-DsRed1
1)酶切1) enzyme digestion
将步骤二获得的携带DsRed1基因的阳性重组质粒pGEM-T Easy-DsRed1和实施例1构建的真核表达载体pVAX1-IRES用限制性内切酶BamH I和Pvu I进行双酶切,37℃水浴过夜,酶切体系如下:The positive recombinant plasmid pGEM-T Easy-DsRed1 carrying the DsRed1 gene obtained in
然后,对双酶切产物进行1.2%琼脂糖凝胶电泳鉴定,酶切鉴定结果如图7所示(泳道M为DL 2000 DNA Marker,泳道2为携带DsRed1基因的阳性重组质粒pGEM-TEasy-DsRed1的BamH I和Pvu I双酶切产物,泳道4为pVAX1-IRES的BamH I和PvuI双酶切产物),pGEM-T Easy-DsRed1经双酶切获得717bp的DNA条带,pVAX1-IRES经双酶切可获得大小约为3600bp的DNA条带,与预期结果相符。Then, carry out 1.2% agarose gel electrophoresis identification to the double-enzyme digestion product, the enzyme digestion identification result is shown in Figure 7 (swimming lane M is
2)酶切产物的回收、连接和转化2) Recovery, ligation and transformation of enzyme-cleaved products
用博大泰克公司的PCR产物胶回收试剂盒并参照试剂盒说明书回收并纯化717bp的DsRed1基因和3600bp的线性化质粒pVAX1-IRES的条带,然后将纯化后的目的基因DsRed1和线性化载体pVAX1-IRES进行连接,16℃连接1h,连接体系如下:Recover and purify the 717bp DsRed1 gene and the 3600bp linearized plasmid pVAX1-IRES band using the PCR product gel recovery kit from Biotech and referring to the kit instructions, and then combine the purified target gene DsRed1 and the linearized vector pVAX1- IRES for connection, 16°C for 1h, the connection system is as follows:
DsRed1基因 5μlDsRed1 gene 5μl
pVAX1-IRES载体 5μlpVAX1-
Ligation Mix 10μlLigation Mix 10μl
总体积 20μl。
然后,用与与实施例1步骤二中相同的方法将连接产物转化大肠杆菌DH5α感受态细胞。Then, the ligation product was transformed into Escherichia coli DH5α competent cells by the same method as in
3)阳性重组质粒的筛选与鉴定3) Screening and identification of positive recombinant plasmids
挑取在含卡那霉素(50μg/mL)LB抗性平板上长出的单菌落8个(编号1-8),分别接种于含卡那霉素(50μg/mL)的LB液体培养基中(5mL/管),37℃摇床培养过夜,提质粒,用限制性内切酶BamH I和Pvu I进行双酶切鉴定,酶切体系如下:
酶切鉴定结果如图8A所示(泳道M为DL 2000 DNA Marker,泳道1-8为挑选的1-8号单克隆质粒的BamH I和Pvu I双酶切产物),阳性重组质粒经双酶切可获得3600bp和717bp的DNA条带,其中1,2,4号单克隆为阳性克隆,与预期结果一致,最后将经酶切鉴定正确的阳性克隆菌液送北京三博远志生物技术有限责任公司进行测序,测序结果表明获得了序列及插入位置均正确的含有DsRed1基因的重组真核表达载体,命名为pVAX1-IRES-DsRed1。The results of enzyme digestion identification are shown in Figure 8A (lane M is
2、pVAX1-IRES-EGFP的构建2. Construction of pVAX1-IRES-EGFP
1)酶切1) enzyme digestion
将步骤二获得的携带EGFP基因的阳性重组质粒pGEM-T Easy-EGFP和实施例1构建的真核表达载体pVAX1-IRES用限制性内切酶Nsi I和BssH II进行双酶切,37℃水浴过夜,酶切体系如下:The positive recombinant plasmid pGEM-T Easy-EGFP carrying the EGFP gene obtained in
然后,对双酶切产物进行1.2%琼脂糖凝胶电泳鉴定,酶切鉴定结果如图7所示(泳道M为DL 2000 DNA Marker,泳道1为携带EGFP基因的阳性重组质粒pGEM-T Easy-EGFP的Nsi I和BssH II双酶切产物,泳道3为pVAX1-IRES的Nsi I和BssH II双酶切产物),pGEM-T Easy-EGFP经双酶切可获得740bp的DNA条带,pVAX1-IRES经双酶切可获得3600bp的DNA条带,与预期结果相符。Then, carry out 1.2% agarose gel electrophoresis identification to the product of double enzyme digestion, the result of enzyme digestion identification is shown in Figure 7 (swimming lane M is
2)酶切产物的回收、连接和转化2) Recovery, ligation and transformation of enzyme-cleaved products
用博大泰克公司的PCR产物胶回收试剂盒并参照试剂盒说明书回收并纯化740bp的EGFP基因和3600bp的线性化质粒pVAX1-IRES的条带,然后将纯化后的目的基因EGFP和线性化载体pVAX1-IRES进行连接,16℃连接1h,连接体系如下:Recover and purify the 740bp EGFP gene and the 3600bp linearized plasmid pVAX1-IRES band using the PCR product gel recovery kit from Biotech and referring to the kit instructions, and then combine the purified target gene EGFP and the linearized vector pVAX1- IRES for connection, 16°C for 1h, the connection system is as follows:
EGFP基因 5μl
pVAX1-IRES载体 5μlpVAX1-IRES vector 5μl
Ligation Mix 10μlLigation Mix 10μl
总体积 20μl。
然后,用与与实施例1步骤二中相同的方法将连接产物转化大肠杆菌DH5α感受态细胞。Then, the ligation product was transformed into Escherichia coli DH5α competent cells by the same method as in
3)阳性重组质粒的筛选与鉴定3) Screening and identification of positive recombinant plasmids
挑取在含卡那霉素(50μg/mL)LB抗性平板上长出的单菌落3个(编号1-3),分别接种于含卡那霉素(50μg/mL)的LB液体培养基中(5mL/管),37℃摇床培养过夜,提质粒,用限制性内切酶Nsi I和BssH II进行双酶切鉴定,酶切体系如下:
酶切鉴定结果如图8B所示(泳道M为DL 2000 DNA Marker,泳道1-3为挑选的1-3号单克隆质粒的Nsi I和BssH II双酶切产物),阳性重组质粒经双酶切可获得3600bp和740bp的DNA条带,其中3号单克隆为阳性克隆,与预期结果一致,最后将经酶切鉴定正确的阳性克隆菌液送北京三博远志生物技术有限责任公司进行测序,测序结果表明获得了序列及插入位置均正确的含有EGFP基因的重组真核表达载体,命名为pVAX1-IRES-EGFP。The results of enzyme digestion identification are shown in Figure 8B (lane M is
3、pVAX1-DsRed1-IRES-EGFP的构建3. Construction of pVAX1-DsRed1-IRES-EGFP
1)酶切1) enzyme digestion
将步骤1获得的携带DsRed1基因的真核表达载体pVAX1-IRES-DsRed1用限制性内切酶Nsi I和BssH II进行双酶切,37℃水浴过夜,酶切体系如下:The eukaryotic expression vector pVAX1-IRES-DsRed1 obtained in
然后,对双酶切产物进行1.2%琼脂糖凝胶电泳鉴定,经双酶切可获得大小约为4300bp的DNA条带,与预期结果相符。Then, 1.2% agarose gel electrophoresis was performed on the double-enzyme digestion product to identify a DNA band with a size of about 4300 bp, which was consistent with the expected result.
2)酶切产物的回收、连接和转化2) Recovery, ligation and transformation of enzyme-cleaved products
用博大泰克公司的PCR产物胶回收试剂盒并参照试剂盒说明书回收并纯化大小约为4300bp的线性化质粒pVAX1-IRES-DsRed1的条带,然后将纯化后的EGFP基因和线性化质粒pVAX1-IRES-DsRed1进行连接,16℃连接1h,连接体系如下:Recover and purify the linearized plasmid pVAX1-IRES-DsRed1 band with a size of about 4300bp using the PCR product gel recovery kit from Biotech and referring to the kit instructions, and then combine the purified EGFP gene and the linearized plasmid pVAX1-IRES -DsRed1 for connection, 16°C for 1h, the connection system is as follows:
EGFP基因 5μl
pVAX1-IRES-DsRed1 5μlpVAX1-IRES-
Ligation Mix 10μlLigation Mix 10μl
总体积 20μl。The total volume is 20 μl.
然后,用与与实施例1步骤二中相同的方法将连接产物转化大肠杆菌DH5α感受态细胞。Then, the ligation product was transformed into Escherichia coli DH5α competent cells by the same method as in
3)阳性重组质粒的筛选与鉴定3) Screening and identification of positive recombinant plasmids
挑取在含卡那霉素(50μg/mL)LB抗性平板上长出的单菌落3个(编号1-3),分别接种于含卡那霉素(50μg/mL)的LB液体培养基中(5mL/管),37℃摇床培养过夜,提质粒,用限制性内切酶Xho I和BamH I进行双酶切鉴定,酶切体系如下:
酶切鉴定结果如图8C所示(泳道M为DL 2000 DNA Marker,泳道1为挑选的单克隆质粒,泳道2为挑选的单克隆质粒的Xho I和BamH I双酶切产物),阳性重组质粒经双酶切可获得3600bp和2100bp左右的DNA条带,与预期结果一致,最后将经酶切鉴定正确的阳性克隆菌液送北京三博远志生物技术有限责任公司进行测序,测序结果表明获得了序列及插入位置均正确的含有DsRed1基因和EGFP基因的重组真核表达载体,命名为pVAX1-DsRed1-IRES-EGFP。The results of enzyme digestion identification are shown in Figure 8C (lane M is
4)大量提质粒4) Extract a large number of plasmids
将鉴定正确的分别转染有质粒pVAX1-IRES-DsRed1、pVAX1-IRES-EGFP及pVAX1-DsRed1-IRES-EGFP的重组克隆菌液过夜培养,按照Promega公司WizardPlusMinipreps DNA Purification system并参照产品说明书提取质粒。Cultivate the correctly identified recombinant clones transfected with plasmids pVAX1-IRES-DsRed1, pVAX1-IRES-EGFP, and pVAX1-DsRed1-IRES-EGFP overnight, and extract them according to Promega's Wizard® Plus Minipreps DNA Purification system and refer to the product manual. plasmid.
四、重组真核表达质粒瞬时转染293T细胞及表达水平检测4. Transient transfection of recombinant eukaryotic expression plasmids into 293T cells and detection of expression levels
1、转染293T细胞1. Transfection of 293T cells
采用脂质体法将步骤三构建的三种真核表达载体pVAX1-IRES-DsRed1、pVAX1-IRES-EGFP及pVAX1-DsRed1-IRES-EGFP分别转染人胚肾293T细胞,具体方法为:将293T细胞(购自协和医科大学细胞中心)培养于含10%胎牛血清的RPMI1640培养基(Gibco BRL公司)中,取对数生长期的293T细胞,用0.25%胰酶(或EDTA)常规消化后接种入6孔培养板中,于37℃、50mL/L CO2孵育箱中培养24h(细胞生长密度为80-90%),然后将阳性对照质粒pEGFP-N1、pDsRed1-N1和鉴定正确的重组质粒pVAX1-IRES-DsRed1、pVAX1-IRES-EGFP和pVAX1-DsRed1-IRES-EGFP分别在Invitrogen公司的转染试剂LipofectamineTM2000的介导下转染293T细胞,具体操作参照试剂盒说明书,空白对照组只加入等量的空白脂质体。The three eukaryotic expression vectors pVAX1-IRES-DsRed1, pVAX1-IRES-EGFP and pVAX1-DsRed1-IRES-EGFP constructed in
2、激光扫描共聚焦显微镜检测各种重组质粒在293T细胞内的表达情况2. Detection of expression of various recombinant plasmids in 293T cells by laser scanning confocal microscope
用Bio-Rad公司的Radiance 2100TM激光扫描共聚焦显微镜检测步骤1的各种重组质粒在293T转染细胞内的表达情况,检测方便包括以下步骤:Use the Radiance 2100TM laser scanning confocal microscope of Bio-Rad to detect the expression of various recombinant plasmids in
a.转染48h后用0.25%的胰酶消化293T转染细胞,轻轻吹打制备成单细胞悬液;a. 48 hours after transfection, digest the 293T transfected cells with 0.25% trypsin, and gently pipette to prepare a single cell suspension;
b.用冰浴预冷的含有2%新生小牛血清的PBS清洗细胞3次;b. Wash the
c.用1%多聚甲醛重悬细胞,调整细胞浓度,将细胞悬液滴到载玻片上,封片剂封存;c. Resuspend the cells with 1% paraformaldehyde, adjust the cell concentration, drop the cell suspension onto the glass slide, and seal it with the mounting medium;
d.用激光扫描共聚焦显微镜检测各种重组质粒的表达情况。d. Detect the expression of various recombinant plasmids with a laser scanning confocal microscope.
瞬时转染48小时后的293T细胞的激光扫描共聚焦显微镜检测结果如图9所示(a:pDsRed1-N1;b:pEGFP-N1;c:pVAX1-IRES-DsRed1;d:pVAX1-IRES-EGFP;e-g:pVAX1-DsRed1-IRES-EGFP;h:空白对照),转染阳性对照质粒pDsRed1-N1和pEGFP-N1的293T细胞均有红色或绿色的荧光呈现(a,b),证明转染体系建立成功;转染重组质粒pVAX1-IRES-DsRed1、pVAX1-IRES-EGFP的293T细胞也发出红色或绿色的荧光(c,d),证明连入的DsRed1、EGFP基因均可以在已构建的pVAX1-IRES载体中正常表达;分别在575nm或518nm两种波长激发下观察转染重组质粒pVAX1-DsRed1-IRES-EGFP的293T细胞,可见红色或绿色荧光呈现(e,f),当用575nm和518nm两种波长共同激发时,可见某些293T细胞呈黄色(g),证明连入pVAX1-IRES载体中IRES编码序列上游的DsRed1基因和下游的EGFP基因不仅可以分别在293T细胞中表达,而且可以在同一个细胞中通过IRES的作用进行共表达,证明本发明的DNA疫苗载体pVAX1-IRES具有同时启动两种基因进行共表达的功能。The laser scanning confocal microscopy results of 293T cells after transient transfection for 48 hours are shown in Figure 9 (a: pDsRed1-N1; b: pEGFP-N1; c: pVAX1-IRES-DsRed1; d: pVAX1-IRES-EGFP ; e-g: pVAX1-DsRed1-IRES-EGFP; h: blank control), 293T cells transfected with positive control plasmids pDsRed1-N1 and pEGFP-N1 all had red or green fluorescence (a, b), proving the transfection system The establishment was successful; 293T cells transfected with recombinant plasmids pVAX1-IRES-DsRed1 and pVAX1-IRES-EGFP also emitted red or green fluorescence (c, d), proving that the inserted DsRed1 and EGFP genes could be expressed in the constructed pVAX1- It is normally expressed in the IRES vector; the 293T cells transfected with the recombinant plasmid pVAX1-DsRed1-IRES-EGFP were observed under the excitation of two wavelengths of 575nm and 518nm, and red or green fluorescence was seen (e, f). When excited by different wavelengths together, some 293T cells can be seen to be yellow (g), which proves that the upstream DsRed1 gene and the downstream EGFP gene connected to the IRES coding sequence in the pVAX1-IRES vector can not only be expressed in 293T cells respectively, but also can be expressed in the same The co-expression is carried out through the action of IRES in one cell, which proves that the DNA vaccine vector pVAX1-IRES of the present invention has the function of simultaneously promoting the co-expression of two genes.
3、流式细胞仪测定各种重组质粒的瞬时表达效率3. Determination of the transient expression efficiency of various recombinant plasmids by flow cytometry
用BD公司的FACSCalibur流式细胞仪测定步骤1瞬时转染48小时后各种重组质粒在293T转染细胞内的瞬时表达效率,具体方法如下:The FACSCalibur flow cytometer of BD Company was used to measure the transient expression efficiency of various recombinant plasmids in 293T transfected cells after 48 hours of transient transfection in
a.转染48h后用0.25%的胰酶消化293T转染细胞,轻轻吹打制备成单细胞悬液;a. 48 hours after transfection, digest the 293T transfected cells with 0.25% trypsin, and gently pipette to prepare a single cell suspension;
b.用冰浴预冷的含有2%新生小牛血清的PBS清洗细胞3次;b. Wash the
c.用0.5mL 1%多聚甲醛重悬细胞,最后用流式细胞仪测定各种重组质粒在293T转染细胞内的表达情况。c. Resuspend the cells with 0.5
瞬时转染48小时后的各种重组质粒在293T细胞内的瞬时表达效率的流式细胞仪检测结果如图10所示(A:空白对照;B:pDsRed1-N1;C:pEGFP-N1;D:pVAX1-IRES-DsRed1;E:pVAX1-IRES-EGFP;F:pVAX1-DsRed1-IRES-EGFP(UL表示up-left,左上角,575nm,反应的是只单独表达红色荧光蛋白的细胞;LR即low-right,右下角,518nm,反应的是只单独表达绿色荧光蛋白的细胞;UR即up-right,右上角,575nm+518nm,双表达即同一个细胞中既表达红色荧光蛋白又表达绿色荧光蛋白;LL即low-left,左下角,表示的是双阴的细胞),可见质粒pVAX1-DsRed1-IRES-EGFP中DsRed1基因的表达情况为(10.24%+18.86%=29.10%),EGFP基因的表达情况(5.24%+18.86%=24.10%),DsRed1基因和EGFP基因同时表达的情况(18.86%)。表明连入pVAX1-IRES载体中IRES编码序列上游的DsRed1基因和下游的EGFP基因不仅可以分别在293T细胞中表达,而且可以在同一个细胞中通过IRES的作用进行共表达,进一步证明本发明的DNA疫苗载体pVAX1-IRES具有同时启动两种基因进行共表达的功能。The flow cytometric detection results of the transient expression efficiency of various recombinant plasmids in 293T cells 48 hours after transient transfection are shown in Figure 10 (A: blank control; B: pDsRed1-N1; C: pEGFP-N1; D : pVAX1-IRES-DsRed1; E: pVAX1-IRES-EGFP; F: pVAX1-DsRed1-IRES-EGFP (UL means up-left, the upper left corner, 575nm, it reflects the cells that only express red fluorescent protein alone; LR means low-right, lower right corner, 518nm, reflects cells that only express green fluorescent protein alone; UR is up-right, upper right corner, 575nm+518nm, double expression means that both red fluorescent protein and green fluorescent protein are expressed in the same cell protein; LL is low-left, the lower left corner represents double negative cells), it can be seen that the expression of the DsRed1 gene in the plasmid pVAX1-DsRed1-IRES-EGFP is (10.24%+18.86%=29.10%), and the expression of the EGFP gene Expression situation (5.24%+18.86%=24.10%), the situation (18.86%) of DsRed1 gene and EGFP gene expression simultaneously.It shows that the DsRed1 gene and the downstream EGFP gene connected in the IRES coding sequence upstream in the pVAX1-IRES vector can not only It is expressed in 293T cells, and can be co-expressed in the same cell through the action of IRES, which further proves that the DNA vaccine vector pVAX1-IRES of the present invention has the function of simultaneously promoting the co-expression of two genes.
实施例3、多靶点复合抗原抗肿瘤基因疫苗的构建及真核表达Example 3, Construction and eukaryotic expression of multi-target compound antigen anti-tumor gene vaccine
一、抗原复合物2PAG融合基因的设计1. Design of antigen complex 2PAG fusion gene
Survivin主要激发细胞免疫反应,Survivin的mRNA在体内经过不同的剪切方式可产生三种异构体:Survivin、Survivin-2B和Survivin-EX3,其中以Survivin-2B的自氨基端第80-88位氨基酸残基为特异性靶点的抗肿瘤疫苗已经进入II期临床试验阶段。经大量文献调研发现,Survivin-2B的T细胞抗原表位主要集中在自氨基端第5-28和第80-121位氨基酸残基之间,因此选取这两个区域作为本发明多靶点复合抗原抗肿瘤基因疫苗的靶点之一。从Genebank中查找人Survivin 2B(NM-001012271)中相对应于上述两个区域的碱基序列(134-205,359-513),再通过连接臂“-nG-,其中n可以为K(赖氨酸)、R(精氨酸)、C(半胱氨酸)、Q(谷氨酰胺)、G(甘氨酸)或N(天冬酰胺),本实施例n为N(天冬酰胺)”编码序列将两段DNA序列连接从而构成Sur融合基因,其核苷酸序列如序列表中的序列1所示,全长273bp,编码91个氨基酸残基。Survivin mainly stimulates cellular immune response. Survivin mRNA can produce three isoforms through different cutting methods in vivo: Survivin, Survivin-2B and Survivin-EX3, among which the 80th-88th amino terminal of Survivin-2B Anti-tumor vaccines with amino acid residues as specific targets have entered phase II clinical trials. A large number of literature investigations found that the T cell antigen epitope of Survivin-2B is mainly concentrated between amino acid residues 5-28 and 80-121 from the amino terminal, so these two regions were selected as the multi-target complex of the present invention. Antigen is one of the targets of anti-tumor gene vaccine. Find the base sequence (134-205, 359-513) corresponding to the above two regions in human Survivin 2B (NM-001012271) from Genebank, and then through the linker "-nG-, wherein n can be K (Lai amino acid), R (arginine), C (cysteine), Q (glutamine), G (glycine) or N (asparagine), in this embodiment n is N (asparagine)" The coding sequence connects the two DNA sequences to form the Sur fusion gene. Its nucleotide sequence is shown as
CTP37是位于hCGβ链羧基端第109-145位氨基酸残基的37肽,存在hCG的特异抗原表位。将人和猴的CTP37联合使用构建复合靶抗原,方法为:从Genebank中查找人和猴的hCGβ-CTP37的编码序列[人:NM_033043;猴:NM_001032928(同源性比较结果,猴:人=76%;猴:小鼠=100%),通过连接臂-nG-(n可以为K、R、C、Q、G或N,本实施例n为N)将两个物种的CTP37编码序列相连接构成融合基因hmCTP,其核苷酸序列如序列表中的序列2所示,全长为228bp,编码76个氨基酸残基。CTP37 is a 37-peptide located at amino acid residues 109-145 at the carboxy-terminal of the hCGβ chain, and has a specific antigenic epitope of hCG. Combine human and monkey CTP37 to construct a composite target antigen. The method is: search for the coding sequence of human and monkey hCGβ-CTP37 from Genebank [human: NM_033043; monkey: NM_001032928 (homology comparison results, monkey: human = 76 %; monkey: mouse=100%), the CTP37 coding sequences of two species are connected by connecting arm-nG-(n can be K, R, C, Q, G or N, n is N in this embodiment) The fusion gene hmCTP is constituted, its nucleotide sequence is shown as
将融合基因Sur的3’端和融合基因hmCTP的5’端相连接即得到抗原复合物2PAG融合基因,其结构式意图如图11所示。The antigen complex 2PAG fusion gene was obtained by connecting the 3' end of the fusion gene Sur with the 5' end of the fusion gene hmCTP, and its structural formula is shown in Figure 11.
二、抗原复合物2PAG融合基因的PCR扩增2. PCR amplification of antigen complex 2PAG fusion gene
1、引物设计1. Primer design
根据Sur和hmCTP基因的核苷酸序列设计两对引物(带下划线碱基为限制性内切酶识别位点),其中Sur-F1的5’端含有限制性内切酶Xho I识别位点,hmCTP-R1的5’端含有限制性内切酶EcoR I识别位点,Sur-SC-R1和Sur-SC-F1有20bp的互补区域,具体引物序列如下:According to the nucleotide sequences of Sur and hmCTP genes, two pairs of primers were designed (underlined bases are restriction endonuclease recognition sites), wherein the 5' end of Sur-F1 contains restriction endonuclease Xho I recognition sites, The 5' end of hmCTP-R1 contains a restriction endonuclease EcoR I recognition site, Sur-SC-R1 and Sur-SC-F1 have a 20bp complementary region, and the specific primer sequences are as follows:
Sur-F1:5’-CCCGGCTCGAGACATTGCCCCCTG-3’Sur-F1: 5'-CCCGGCTCGAGACATTGCCCCCTG-3'
Sur-SC-R1:5’-CATCACAGGTCTTCTTATTGTTGG-3’Sur-SC-R1: 5'-CATCACAGGTCTTTCTTATTGTTGG-3'
Sur-SC-F1:5’-CAATAAGAAGACCTGTGATGACCCCCG-3’Sur-SC-F1: 5'-CAATAAGAAGACCTGTGATGACCCCCG-3'
hmHCG-R1:5’-GGCCCGAATTCTTGTGGAAGGAAAGGG-3’。hmHCG-R1: 5'-GGCCCGAATTCTTGTGGAAGGAAAGGG-3'.
2、抗原复合物2PAG融合基因的PCR扩增2. PCR amplification of antigen complex 2PAG fusion gene
首先,由北京擎天生物技术有限公司合成上述两条融合基因Sur和hmCTP,并分别连接至T-easy载体pGEM-T Easy(TaKaRa公司)中,将含有融合基因Sur的重组载体命名为Sur-T-easy,将含有融合基因hmCTP的重组载体命名为hmCTP-T-easy。然后,分别以含有合成基因的质粒Sur-T-easy和hmCTP-T-easy为模板,PCR扩增出融合基因Sur-SC和SC-hmCTP。其中,Sur-SC的3’端和SC-hmCTP的5’端含有20bp的互补序列。PCR扩增体系为:10×PCR缓冲液5μl,dNTPs Mixture(2.5mM)5μl,引物(20pM)Sur-F1和Sur-SC-R1(或Sur-SC-F1和hmCTP-R1)各1μl,模板Sur-T-easy(或hmCTP-T-easy)1μl,Taq DNA聚合酶(5U/μl)1μl,加去离子水至50μl。PCR反应条件为:先94℃预变性5min;然后94℃变性30s,58℃退火30s,72℃延伸45s,共5个循环;最后72℃继续延伸10min。反应结束后,取5μl PCR扩增产物进行1%琼脂糖凝胶电泳检测,检测结果如图12所示(泳道M为DL 2000 DNA Marker,泳道1为:融合基因Sur-SC,泳道2为:融合基因SC-hmCTP),经扩增获得了273bp和228bp的DNA条带,与预期大小一致。用博大泰克公司的PCR产物胶回收试剂盒并参照试剂盒说明书回收并纯化融合基因Sur-SC(273bp)和融合基因SC-hmCTP(228bp)的目的条带。再以PCR扩增的融合基因Sur-SC和SC-hmCTP稀释50倍后各取1μl共同为模板,用搭桥PCR的方法扩增抗原复合物2PAG融合基因,在引物Sur-F1和hmCTP-R1的引导下进行PCR扩增,反应结束后,取5μl PCR扩增产物进行1%琼脂糖凝胶电泳检测,检测结果如图12所示(泳道M为DL 2000 DNA Marker(购自TaKaRa公司),泳道3为PCR扩增的抗原复合物2PAG融合基因),经扩增获得了523bp的DNA条带,与预期大小一致。用博大泰克公司的PCR产物胶回收试剂盒并参照试剂盒说明书回收并纯化该抗原复合物2PAG融合基因的目的条带。First, the above two fusion genes Sur and hmCTP were synthesized by Beijing Qingtian Biotechnology Co., Ltd., and connected to the T-easy vector pGEM-T Easy (TaKaRa Company) respectively, and the recombinant vector containing the fusion gene Sur was named Sur- T-easy, the recombinant vector containing the fusion gene hmCTP was named hmCTP-T-easy. Then, using the plasmids Sur-T-easy and hmCTP-T-easy containing the synthetic genes as templates, fusion genes Sur-SC and SC-hmCTP were amplified by PCR. Among them, the 3' end of Sur-SC and the 5' end of SC-hmCTP contain a 20bp complementary sequence. The PCR amplification system is: 5 μl of 10×PCR buffer, 5 μl of dNTPs Mixture (2.5mM), 1 μl of each primer (20pM) Sur-F1 and Sur-SC-R1 (or Sur-SC-F1 and hmCTP-R1), template Sur-T-easy (or hmCTP-T-easy) 1μl, Taq DNA polymerase (5U/μl) 1μl, add deionized water to 50μl. The PCR reaction conditions were as follows: pre-denaturation at 94°C for 5 min; then denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 45 s, a total of 5 cycles; finally, extension at 72°C for 10 min. After the reaction, 5 μl of the PCR amplification product was taken for 1% agarose gel electrophoresis detection, and the detection results are shown in Figure 12 (lane M is
三、人GM-CSF和B7.1融合基因的获得3. Acquisition of human GM-CSF and B7.1 fusion gene
按GeneBank上公开的人GM-CSF(NM-000758)和B7.1(NM-005191)的编码序列设计其PCR扩增引物序列,序列如下:According to the coding sequences of human GM-CSF (NM-000758) and B7.1 (NM-005191) published on GeneBank, the PCR amplification primer sequences are designed as follows:
GMF1(上游引物):5’-CCG ATGCATATGTGGCTGCAGAGCCTG-3’(带下划线碱基为限制性内切酶Nsi I识别位点)GMF1 (upstream primer): 5'-CCG ATGCAT ATGTGGCTGCAGAGCCTG-3' (underlined bases are restriction endonuclease Nsi I recognition sites)
GMR1(下游引物):5’-GCCGCCGCGTCGACCCCTGCCGCCCTCCTGGACTGGCTCCCAGC-3’;GMR1 (downstream primer): 5'-GCCGCCGCGTCGACCCCTGCCGCCCTCCTGGACTGGCTCCCAGC-3';
B7 F1(上游引物):5’-GGCGGCAGGGGTCGACGCGGCGGCGGTCTTTCTCACTTCTGTTC-3’B7 F1 (upstream primer): 5'-GGCGGCAGGGGTCGACGCGGCGGCGGTCTTTCTCACTTCTGTTC-3'
B7 R1(下游引物):5’-CCG GCGCGCTTATACAGGGCGTACACTTTCCC-3’(带下划线碱基为限制性内切酶BSSH II识别位点)。B7 R1 (downstream primer): 5'-CCG GCGCGC TTATACAGGGCGTACACTTTCCC-3' (bases underlined are recognition sites for restriction endonuclease BSSH II).
首先,由北京擎天生物技术有限公司合成人GM-CSF和B7.1的编码序列,并分别连接至pGEM-T Easy载体(购自TaKaRa公司)中,将含有人GM-CSF编码序列的重组载体命名为pGEM-TEasy-GM-CSF,将含有B7.1编码序列的重组载体命名为pGEM-TEasy-B7.1。然后,分别以含有合成基因的质粒pGEM-TEasy-GM-CSF和pGEM-TEasy-B7.1为模板,在GMF1和GMR1的引导下PCR扩增GM-CSF基因全长序列,在B7 F1和B7 R1的引导下PCR扩增B7.1基因全长序列,然后将各自的PCR产物稀释50倍后各取1μl共同作为模板,在引物GMF1和B7 R1的引导下PCR扩增GM-CSF和B7.1融合基因。具体反应体系和反应条件参见步骤二。反应结束后,取5μl PCR扩增产物进行1%琼脂糖凝胶电泳检测,检测结果如图13所示(泳道M为DL 2000 DNA Marker,泳道1为:人GM-CSF基因,泳道2为:B7.1基因,泳道3为PCR扩增的人GM-CSF因子和B7.1融合基因),经扩增获得了1245bp的DNA条带,与预期大小一致。用博大泰克公司的PCR产物胶回收试剂盒并参照试剂盒说明书回收并纯化该人GM-CSF和B7.1融合基因的目的条带。人GM-CSF基因和B7.1基因通过8肽编码序列连接,表达后可以在体内普遍存在的Furin蛋白酶作用下使人GM-CSF因子和B7.1分子分离,从而不影响它们的空间构象,以利于发挥各自的功能。First, the coding sequences of human GM-CSF and B7.1 were synthesized by Beijing Qingtian Biotechnology Co., Ltd., and respectively ligated into the pGEM-T Easy vector (purchased from TaKaRa Company), and the recombinant human GM-CSF coding sequence The vector was named pGEM-TEasy-GM-CSF, and the recombinant vector containing the B7.1 coding sequence was named pGEM-TEasy-B7.1. Then, using the plasmids pGEM-TEasy-GM-CSF and pGEM-TEasy-B7.1 containing synthetic genes as templates, the full-length sequence of the GM-CSF gene was amplified by PCR under the guidance of GMF1 and GMR1. Under the guidance of R1, the full-length sequence of the B7.1 gene was amplified by PCR, and then 1 μl of each PCR product was diluted 50 times and used as a template, and GM-CSF and B7.1 were amplified by PCR under the guidance of primers GMF1 and B7 R1. 1 fusion gene. For specific reaction system and reaction conditions, refer to step 2. After the reaction, 5 μl of PCR amplification products were taken for 1% agarose gel electrophoresis detection, and the detection results were as shown in Figure 13 (swimming lane M is
四、DNA疫苗pVAX1-2PFcGB的构建4. Construction of DNA vaccine pVAX1-2PFcGB
1、含有抗原复合物2PAG融合基因的真核表达质粒pCI-2PAG-Fc-GPI的构建1. Construction of eukaryotic expression plasmid pCI-2PAG-Fc-GPI containing antigen complex 2PAG fusion gene
将步骤二获得的抗原复合物2PAG融合基因用限制性内切酶Xho I和EcoR I进行双酶切后与经相同酶双酶切的含有人Igk链前导信号肽(sig)、人Ig G-Fc和糖基磷脂酰肌醇(GPI)锚定信号肽基因序列的真核表达载体pCI-GPI(在Promega公司的真核表达质粒pCI-neo+的基础上构建而成,构建方法为:在pCI-neo+原多克隆位点处的限制性内切酶Nhe I和Xba I之间插入通用序列构建而成,该通用序列的基因顺序为5’-Nhe I-人Igk链前导信号肽(sig)编码序列-Xho I-EcoR V-EcoR I-人IgG Fc段编码序列-糖基化磷脂酰肌醇(GPI)锚定信号肽的编码序列-Xba I-3’,外源免疫分子基因可以在Nhe I-EcoR I之间选择合适的酶切位点插入。pCI-GPI除含有人IgG Fc段编码序列和糖基化磷脂酰肌醇(GPI)锚定信号肽的编码序列外,还含有pCI载体的主要骨架结构,如CMV早期启动子、嵌合内含子、Neo筛选标记、SV40增强子和氨苄抗性基因Ampr+等)连接,将连接产物转化至大肠杆菌DH5α感受态细胞,筛选阳性克隆,用引物Sur-F1和hmCTP-R1进行菌落PCR鉴定,然后提质粒,用限制性内切酶Nhe I和Not I进行双酶切鉴定,PCR及酶切鉴定结果如图14所示(泳道M:DL 2000 DNA Marker;泳道1:2PAG融合基因的Xho I+EcoR I酶切产物;泳道2:pCI-GPI的Xho I+EcoR I酶切产物;泳道3:菌落PCR鉴定结果;泳道4:pCI-neo-2PAG-Fc-GPI质粒;泳道5:pCI-neo-2PAG-Fc-GPI的Nhe I+Not I酶切产物),阳性克隆质粒经酶切可获得5472bp和2473bp的DNA条带,将鉴定正确的阳性克隆菌株由北京三博生物有限公司进行测序,测序结果表明获得序列及插入位置均正确含有抗原复合物2PAG融合基因的真核表达质粒,命名为pCI-2PAG-Fc-GPI。The antigen complex 2PAG fusion gene obtained in step 2 was double-digested with restriction endonucleases Xho I and EcoR I, and then double-digested with the human Igk chain leading signal peptide (sig), human IgG- The eukaryotic expression vector pCI-GPI with Fc and glycosylphosphatidylinositol (GPI) anchoring signal peptide gene sequence (constructed on the basis of Promega's eukaryotic expression plasmid pCI-neo+, the construction method is: in pCI -neo+ The restriction endonuclease Nhe I and Xba I at the original multiple cloning site are constructed by inserting a universal sequence, and the gene sequence of the universal sequence is 5'-Nhe I-human Igk chain leader signal peptide (sig) Coding sequence-Xho I-EcoR V-EcoR I-human IgG Fc segment coding sequence-glycosylated phosphatidylinositol (GPI) anchoring signal peptide coding sequence-Xba I-3', exogenous immune molecule gene can be found at Select a suitable restriction site for insertion between Nhe I-EcoR I. In addition to the coding sequence of the human IgG Fc segment and the coding sequence of the glycosylated phosphatidylinositol (GPI) anchor signal peptide, pCI-GPI also contains pCI The main skeleton structure of the vector, such as CMV early promoter, chimeric intron, Neo selection marker, SV40 enhancer and ampicillin resistance gene Amp r+ , etc.) is connected, and the connection product is transformed into E. coli DH5α competent cells, and the screening is positive Cloning, carrying out colony PCR identification with primers Sur-F1 and hmCTP-R1, then extracting the plasmid, and carrying out double enzyme digestion identification with restriction endonucleases Nhe I and Not I, PCR and enzyme digestion identification results are shown in Figure 14 (swimming lane M: DL 2000 DNA Marker; lane 1: Xho I+EcoR I digestion product of 2PAG fusion gene; lane 2: Xho I+EcoR I digestion product of pCI-GPI; lane 3: colony PCR identification result; lane 4: pCI-neo-2PAG-Fc-GPI plasmid; Lane 5: Nhe I+Not I digestion product of pCI-neo-2PAG-Fc-GPI), the positive clone plasmid can be digested to obtain DNA bands of 5472bp and 2473bp, The correctly identified positive cloned strains were sequenced by Beijing Sanbo Biological Co., Ltd. The sequencing results showed that the sequence and insertion position were correct and contained the eukaryotic expression plasmid of the antigen complex 2PAG fusion gene, which was named pCI-2PAG-Fc-GPI.
2、真核表达质粒pVAX-sig-2P-FC-GPI-IRES的构建2. Construction of eukaryotic expression plasmid pVAX-sig-2P-FC-GPI-IRES
将测序正确的阳性重组质粒pCI-2PAG-Fc-GPI先用限制性内切酶Not I进行单酶切并经纯化后,用Klenow酶(TaKaRa公司)并参照试剂盒说明书补平单酶切后产生的粘性末端,然后将纯化后的产物再用限制性内切酶Nhe I进行单酶切,随后切胶回收含有2473bp线性化的sig-2PAG-FC-GPI的目的片段(5’端为Nhe I酶切后产生的粘性末端,3’端为Klenow补平后产生的平端)。然后,将实施例1构建的真核表达载体pVAX1-IRES用限制性内切酶Nhe I和EcoR V进行双酶切和纯化后与sig-2PAG-Fc-GPI进行连接,将连接产物转化至大肠杆菌DH5α感受态细胞,筛选阳性克隆,用引物Sur-F1和hmCTP-R1进行菌落PCR鉴定,然后提质粒,提质粒,用限制性内切酶Nhe I和Cla I进行双酶切鉴定,菌落PCR及酶切鉴定结果如图15所示(泳道M:DL 2000 DNAMarker;泳道1:sig-2PAG-Fc-GPI/Not I→Klenow→Nhe I;泳道2:pVAX1-IRES的NheI+EcoR V酶切产物;泳道3:菌落PCR鉴定结果;泳道4:pVAX1-sig-2PAG-Fc-GPI-IRES质粒;泳道5:pVAX1-sig-2PAG-Fc-GPI-IRES的Nhe I+Cla I酶切产物),阳性克隆质粒经酶切可获得3600bp和2473bp的DNA条带,将鉴定正确的阳性克隆菌株由北京三博生物有限公司进行测序,测序结果表明获得序列及插入位置均正确含有sig-2PAG-FC-GPI DNA片段的真核表达质粒,命名为pVAX-sig-2P-FC-GPI-IRES。The correctly sequenced positive recombinant plasmid pCI-2PAG-Fc-GPI was digested with restriction endonuclease Not I and purified, and then filled with Klenow enzyme (TaKaRa Company) and referred to the kit instructions. The resulting cohesive ends, then the purified product is then single-digested with restriction endonuclease Nhe I, followed by gel cutting to recover the target fragment containing 2473bp linearized sig-2PAG-FC-GPI (the 5' end is Nhe The sticky end produced after digestion with I enzyme, the 3' end is the blunt end produced after Klenow filling). Then, the eukaryotic expression vector pVAX1-IRES constructed in Example 1 was double digested and purified with restriction endonucleases Nhe I and EcoR V, then ligated with sig-2PAG-Fc-GPI, and the ligated product was transformed into the large intestine Bacillus DH5α competent cells, screening positive clones, using primers Sur-F1 and hmCTP-R1 for colony PCR identification, then extracting plasmids, extracting plasmids, using restriction endonucleases Nhe I and Cla I for double digestion identification, colony PCR The results of enzyme digestion and identification are shown in Figure 15 (lane M:
3、pVAX1-IRES-GM/B7的构建3. Construction of pVAX1-IRES-GM/B7
将步骤三扩增的GM-CSF和B7.1融合基因用限制性内切酶Nsi I和BSSH II进行双酶切后与经同样酶双酶切的载体pVAX1-IRES进行连接,将连接产物转化至大肠杆菌DH5α感受态细胞,筛选阳性克隆,提质粒,用限制性内切酶Nsi I和BSSHII进行双酶切鉴定,酶切鉴定结果如图16所示(泳道M:DL 2000 DNA Marker;泳道1:pVAX1-IRES-GM/B7质粒;泳道2:GM-CSF和B7.1融合基因;泳道3:pVAX1-IRES-GM/B7质粒的Nsi I+BSSHII酶切产物),阳性克隆质粒经酶切可获得3600bp和1245bp的DNA条带,将鉴定正确的阳性克隆菌株由北京三博生物有限公司进行测序,测序结果表明获得序列及插入位置均正确含有GM-CSF和B7.1融合基因的真核表达质粒,命名为pVAX1-IRES-GM/B7。The GM-CSF and B7.1 fusion gene amplified in
4、DNA疫苗pVAX1-2PFcGB的获得4. Acquisition of DNA vaccine pVAX1-2PFcGB
将步骤2鉴定正确后的阳性重组质粒pVAX1-sig-2PAG-Fc-GPI-IRES用限制性内切酶Nhe I和Cla I进行双酶切后,对酶切产物进行1%琼脂糖凝胶电泳检测,回收并纯化2473bp的含有sig-2PAG-FC-GPI的目的片段,然后再与经Nhe I和Cla I双酶切的真核表达质粒pVAX1-IRES-GM/B7进行连接,将连接产物转化至大肠杆菌DH5α感受态细胞,筛选阳性克隆,提质粒,用限制性内切酶Nsi I和BSSH II进行双酶切鉴定,酶切鉴定结果如图17所示(泳道M:DL 2000 DNA Marker;泳道1:sig-2PAG-Fc-GPI的NheI+Cla I酶切产物;泳道2:pVAX1-IRES-GM/B7的Nhe I+Cla I酶切产物;泳道3:PCR鉴定;泳道4:pVAX1-sig-2PAG-Fc-GPI-GM/B7质粒;泳道5:pVAX1-sig-2PAG-Fc-GPI-IRES的Nhe I+Cla I酶切产物),阳性克隆质粒经酶切可获得大小约为2000bp的DNA条带,将鉴定正确的阳性克隆菌株由北京三博生物有限公司进行测序,测序结果表明获得序列及插入位置均正确的真核表达质粒,即本发明的多靶点复合抗原抗肿瘤基因疫苗,命名为pVAX1-sig-2PAG-Fc-GPI-GM/B7(简称pVAX1-2PFcGB)。After the positive recombinant plasmid pVAX1-sig-2PAG-Fc-GPI-IRES identified correctly in
五、多靶点复合抗原抗肿瘤基因疫苗的真核表达5. Eukaryotic expression of multi-target compound antigen anti-tumor gene vaccine
1、DNA疫苗pVAX1-2PFcGB的真核表达1. Eukaryotic expression of DNA vaccine pVAX1-2PFcGB
采用与实施例2中相同的脂质体法将步骤四构建的DNA疫苗pVAX1-2PFcGB转染人胚肾293T细胞,同时设立三个实验组和空白对照组,空白对照组只加入等量的空白脂质体,分别用于流式细胞仪检测和免疫荧光检测。Using the same liposome method as in Example 2, the DNA vaccine pVAX1-2PFcGB constructed in
2、流式细胞仪检测2. Flow cytometry detection
采用与实施例2中相同的方法用BD公司的FACSCalibur流式细胞仪测定步骤1瞬时转染48小时后pVAX1-2PFcGB在293T转染细胞内的瞬时表达效率,在pVAX1-2PFcGB中,2PAG融合基因的羧基端融合了人IgG Fc段基因,可作为检测融合蛋白表达的标签,因此分别用FITC标记的兔抗人IgG(购自北京中杉金桥技术有限公司)、PE标记的兔抗人B7的抗体(EBioScience公司)与转染48小时后的细胞进行孵育,用以检测插入片段2PAG-Fc-GPI和载体中GM-CSF/B7.1融合基因在293T细胞中的表达情况,流式细胞仪检测结果如图18所示(A:空白对照组(FITC);B:实验组(FITC);C:空白对照组(PE);D:实验组(PE)),转染48小时后,293T细胞中插入片段2PAG-Fc-GPI的阳性表达率为44.46%,平均荧光强度为334.17;载体中GM-CSF/B7.1的阳性表达率为52.26%,平均荧光强度为497.86,表明连接入pVAX1-IRES载体中IRES编码序列上游的2PAG-Fc-GPI基因片段和下游的GM-CSF/B7.1融合基因均获得表达。The same method as in Example 2 was used to measure the transient expression efficiency of pVAX1-2PFcGB in 293T transfected cells after 48 hours of transient transfection in
3、免疫荧光检测3. Immunofluorescence detection
采用与实施例2中相同的方法处理细胞,滴片后在荧光显微镜下直接进行免疫荧光检测。分别用FITC标记的兔抗人IgG、PE标记的兔抗人B7的抗体与转染48小时后的细胞进行孵育,用以检测插入片段2PAG-Fc-GPI和载体中GM-CSF/B7.1融合基因在293T细胞中的表达情况,免疫荧光检测结果如图19所示(A:经FITC-IgG抗体孵育的293T细胞;B:经PE-B7抗体孵育的293T细胞),经荧光显微镜观测发现,瞬时转染pVAX1-2PFcGB 48h后的293T细胞均有荧光显示,且主要分布于细胞膜上(图6),而未转染重组质粒pVAX1-2PFcGB的293T细胞检测结果均为阴性,证明插入片段2PAG-Fc-GPI和载体中GM-CSF/B7.1融合基因均可以有效表达。The cells were treated with the same method as in Example 2, and immunofluorescence detection was directly performed under a fluorescence microscope after dropping the slides. Incubate the cells 48 hours after transfection with FITC-labeled rabbit anti-human IgG and PE-labeled rabbit anti-human B7 antibodies to detect the insert fragment 2PAG-Fc-GPI and GM-CSF/B7.1 in the vector The expression of the fusion gene in 293T cells, the results of immunofluorescence detection are shown in Figure 19 (A: 293T cells incubated with FITC-IgG antibody; B: 293T cells incubated with PE-B7 antibody), and observed by fluorescence microscope , the 293T cells transiently transfected with pVAX1-2PFcGB for 48 hours showed fluorescence, and were mainly distributed on the cell membrane (Figure 6), while the test results of 293T cells not transfected with the recombinant plasmid pVAX1-2PFcGB were all negative, proving that the inserted fragment 2PAG - Both Fc-GPI and GM-CSF/B7.1 fusion gene in the vector can be effectively expressed.
实施例4、多靶点复合抗原抗肿瘤基因疫苗pVAX1-2PFcGB的抑瘤活性检测Example 4. Detection of anti-tumor activity of multi-target composite antigen anti-tumor gene vaccine pVAX1-2PFcGB
用下述方法检测本发明多靶点复合抗原抗肿瘤基因疫苗pVAX1-2PFcGB的抑瘤活性,同时以实施例3构建的质粒pVAX1-2PAG、pVAX1-2PAG-Fc-GPI(结构示意图见图29)和pVAX1-IRES-GM/B7为对照,检测方法如下:The following method was used to detect the anti-tumor activity of the multi-target compound antigen anti-tumor gene vaccine pVAX1-2PFcGB of the present invention, and the plasmids pVAX1-2PAG and pVAX1-2PAG-Fc-GPI constructed in Example 3 were used simultaneously (see Figure 29 for a schematic diagram of the structure) Compared with pVAX1-IRES-GM/B7, the detection method is as follows:
一、多靶点复合抗原抗肿瘤基因疫苗pVAX1-2PFcGB免疫小鼠1. Mice immunized with multi-target complex antigen anti-tumor gene vaccine pVAX1-2PFcGB
1、免疫用质粒DNA的大量提取与纯化1. Mass extraction and purification of plasmid DNA for immunization
用北京博大泰克生物基因技术有限责任公司“B型超纯质粒大量快速提取试剂盒”并参照试剂盒说明书大量制备免疫用质粒DNA,具体方法包括以下步骤:Use the "B-Type Ultrapure Plasmid Mass Rapid Extraction Kit" of Beijing Broadtech Biogene Technology Co., Ltd. and refer to the kit instructions to prepare a large amount of plasmid DNA for immunization. The specific method includes the following steps:
a.将200mL携带pVAX1-2PFcG的大肠杆菌DH5α重组菌37℃培养12小时后收集菌体,加入7mL溶液1,振荡至彻底悬浮;a. Collect 200 mL of Escherichia coli DH5α recombinant bacteria carrying pVAX1-2PFcG at 37°C for 12 hours, collect the bacteria, add 7 mL of
b.加入10.5mL溶液2,轻轻颠倒混匀,使菌体充分裂解,随后冰上放置5分钟;b. Add 10.5mL of
c.加入10.5mL溶液3,轻轻颠倒混匀,室温放置10分钟,4℃、12,000rpm离心15min;c. Add 10.5mL of
d.将上清与0.6倍体积的异丙醇混匀后,4℃、10,000rpm离心20min;d. After mixing the supernatant with 0.6 times the volume of isopropanol, centrifuge at 4°C and 10,000rpm for 20min;
e.收集沉淀,溶于2mL双蒸水中并加入80μl 10mg/mL RNase A,37℃保温30分钟;e. Collect the precipitate, dissolve it in 2mL double-distilled water, add 80μl 10mg/mL RNase A, and incubate at 37°C for 30 minutes;
f.加入等体积的结合缓冲液,混匀后移入吸附柱,室温、12,000rpm离心1min;f. Add an equal volume of binding buffer, mix well, transfer to the adsorption column, and centrifuge at 12,000rpm for 1min at room temperature;
g.倒掉废液,加入500μl去内毒素缓冲液,室温、2,000rpm离心1min,重复1次后,再次于室温、12,000rpm离心3min;尽量去除去内毒素缓冲液;g. Pour off the waste liquid, add 500 μl of endotoxin-removing buffer, centrifuge at room temperature and 2,000 rpm for 1 min, repeat once, then centrifuge again at room temperature and 12,000 rpm for 3 min; remove endotoxin-removing buffer as much as possible;
h.倒掉废液,加入800μl漂洗缓冲液,静置1分钟后12,000rpm离心1min,重复2次后,再次12,000rpm离心2min,尽量去除漂洗缓冲液;h. Pour off the waste liquid, add 800 μl of washing buffer, let stand for 1 minute, then centrifuge at 12,000 rpm for 1 min, repeat twice, then centrifuge again at 12,000 rpm for 2 min to remove the washing buffer as much as possible;
I.将吸附柱放入一个干净的离心管中,在吸附膜的中央加入50μl无菌水,室温下静置2-5min,12,000rpm离心2min,收集洗脱液,-20℃贮存备用。I. Put the adsorption column into a clean centrifuge tube, add 50 μl sterile water to the center of the adsorption membrane, let it stand at room temperature for 2-5 minutes, centrifuge at 12,000 rpm for 2 minutes, collect the eluate, and store it at -20°C for later use.
收集洗脱液即为提纯的质粒DNA,加入10×PBS使质粒DNA溶于PBS溶液。紫外分光光度法测定DNA含量和纯度,结果DNA含量约为1μg/μl,纯度达90%,-20℃保存。The collected eluate is the purified plasmid DNA, and 10×PBS is added to dissolve the plasmid DNA in the PBS solution. The DNA content and purity were measured by ultraviolet spectrophotometry, and the result was that the DNA content was about 1 μg/μl and the purity was 90%, and it was stored at -20°C.
2、动物分组2. Animal grouping
将6周龄、体重18-20g的雌性Balb/c小鼠(购自军事医学科学院实验动物中心)随机分为6组,每组9只,A组为PBS对照组,B组为pVAX1空载体组,C组为pVAX1-IRES-GM/B7组,D组为pVAX1-2PAG组,E组为pVAX1-2PAG-Fc-GPI组,F组为pVAX1-2PFcGB组(基因疫苗pVAX1-2PFcGB)。Female Balb/c mice aged 6 weeks and weighing 18-20 g (purchased from the Experimental Animal Center of the Academy of Military Medical Sciences) were randomly divided into 6 groups, 9 mice in each group, group A was the PBS control group, and group B was the pVAX1 empty vector Group C is the pVAX1-IRES-GM/B7 group, Group D is the pVAX1-2PAG group, E group is the pVAX1-2PAG-Fc-GPI group, and F group is the pVAX1-2PFcGB group (gene vaccine pVAX1-2PFcGB).
3、皮下移植瘤攻击3. Attack of subcutaneous tumor transplantation
收获对数生长期的小鼠乳腺癌细胞EMT6(购自中国科学院上海细胞资源中心),制备单细胞悬液,用PBS洗2次,调整细胞浓度至2×106个/mL,于-d5天在小鼠背部右侧皮下接种100μl细胞悬液即2×105个/只。Harvest the mouse breast cancer cells EMT6 in the logarithmic growth phase (purchased from the Shanghai Cell Resource Center of the Chinese Academy of Sciences), prepare a single cell suspension, wash twice with PBS, adjust the cell concentration to 2×10 6 cells/mL, and store on -d5 On the first day, 100 μl of cell suspension was subcutaneously inoculated on the right side of the back of the mouse, that is, 2×10 5 cells/mouse.
4、Balb/c小鼠的免疫4. Immunization of Balb/c mice
免疫方案如图20所示,Balb/c小鼠经皮下移植瘤攻击5天后,开始接种疫苗。免疫方式采用股四头肌肌肉注射,A组注射100μl PBS,其余各组分别注射相应的DNA质粒100μg/只,分别于d0、d5、d15和d20共免疫4次。每次免疫前,首先注射100μl 0.25%布比卡因预处理接种部位,72h后在相同部位接种疫苗。于末次免疫后第5天(d25)检测小鼠体内抗体的产生情况并收集每组2只小鼠的脾细胞,准备CTL杀伤实验;d30天断颈处死每组其余各只小鼠并摘取肿瘤组织称重。The immunization scheme is shown in Figure 20. Balb/c mice were vaccinated 5 days after subcutaneously transplanted tumors were challenged. The method of immunization was intramuscular injection of quadriceps femoris. Group A was injected with 100 μl PBS, and the other groups were injected with corresponding
二、免疫效果检测2. Detection of immune effect
1、疫苗免疫保护效果观察和检测1. Observation and detection of immune protection effect of vaccines
1)免疫小鼠的成瘤时间1) Tumor formation time of immunized mice
首先给Balb/c小鼠皮下注射接种2×105个EMT6细胞,接种细胞后5d,开始给各组小鼠股四头肌分别注射相应的质粒DNA或PBS,观察并记录各组小鼠的肿瘤结节形成时间(可扪及,长径约2-3mm)。First, Balb/c mice were subcutaneously inoculated with 2× 105 EMT6 cells, and 5 days after the inoculation of cells, the corresponding plasmid DNA or PBS was injected into the quadriceps muscles of the mice in each group, and the mice in each group were observed and recorded. Tumor nodule formation time (palpable, about 2-3 mm in diameter).
各组免疫小鼠皮下移植瘤的成瘤时间统计结果如表1和图21所示,可以看出,各免疫组的肿瘤结节形成时间存在不同程度的差别。A组的成瘤时间最早,约6天左右;B组、C组的成瘤时间相差不多,无显著性差别(P>0.05)。含有抗原复合物2PAG的D、E和F组小鼠成瘤时间均晚于对照组,其中D组小鼠的成瘤时间较早,约8天左右;E组次之,而F组小鼠的成瘤时间最晚,直到12天左右才可以检测到肿瘤结节的形成。E组和F组与对照组相比,小鼠的成瘤时间差异显著(P<0.05)。The statistical results of the tumor formation time of subcutaneous transplanted tumors in each group of immunized mice are shown in Table 1 and Figure 21. It can be seen that the tumor nodule formation time of each immunization group is different to varying degrees. The time of tumor formation in group A was the earliest, about 6 days; the time of tumor formation in group B and group C was similar without significant difference (P>0.05). The tumor formation time of the mice in groups D, E and F containing the antigen complex 2PAG was later than that of the control group, and the tumor formation time of the mice in the D group was earlier, about 8 days; the E group followed, and the mice in the F group The tumor formation time of the tumor was the latest, and the formation of tumor nodules could not be detected until about 12 days. Compared with the control group, the tumor formation time of the mice in groups E and F was significantly different (P<0.05).
表1各组免疫小鼠皮下移植瘤的成瘤时间Table 1 Tumor formation time of subcutaneous transplanted tumors in each group of immunized mice
注:※与对照组比较,该组小鼠成瘤潜伏期延长(P<0.05)Note: ※ Compared with the control group, the latent period of tumor formation in this group was prolonged (P<0.05)
2)免疫小鼠体内移植瘤生长情况检测2) Detection of transplanted tumor growth in immunized mice
预先选定各免疫组中的7只小鼠,皮下注射接种2×105个/只EMT6细胞后第5天开始接种疫苗。从小鼠皮下移植瘤攻击后开始至处死小鼠的35天内,每2天用游标卡尺测量一次肿瘤的垂直长径和垂直短径,并通过如下公式计算每组小鼠体内肿瘤的平均体积,绘制其肿瘤生长曲线,对肿瘤生长曲线进行线性回归并对其斜率做t检验,小鼠体内肿瘤的平均体积的计算公式如下:Seven mice in each immunization group were pre-selected, and 2×10 5 EMT6 cells per mouse were subcutaneously injected and vaccinated on
免疫小鼠体内移植瘤的生长曲线如图22所示,pVAX1空载体组(B组)和不含复合抗原2PAG的pVAX1-IRES-GM/B7组(C组)小鼠体内的内移植生长情况与PBS对照组之间无显著差别(P>0.05);经疫苗治疗的D、E和F组内的小鼠,其无瘤潜伏期均比对照组延长,肿瘤的生长均比对照组缓慢(P<0.05)。其中,E组和F组与PBS对照组相比,差异极其显著(P<0.01)。F组内小鼠的无瘤潜伏期最长,在d22前其肿瘤的生长速度最为缓慢,与E组相比有显著性差异(P<0.05),但当d22之后差异不显著(P>0.05),这可能与第二次与第三次免疫之间时间的延长有关,未能及时抑制住肿瘤的生长。The growth curves of transplanted tumors in immunized mice are shown in Figure 22. The growth of transplanted tumors in pVAX1 empty vector group (group B) and pVAX1-IRES-GM/B7 group (group C) without complex antigen 2PAG There was no significant difference with the PBS control group (P>0.05); the tumor-free incubation period of mice in groups D, E and F treated with the vaccine was longer than that of the control group, and the growth of tumors was slower than that of the control group (P <0.05). Among them, the difference between E group and F group and PBS control group was extremely significant (P<0.01). The mice in group F had the longest tumor-free latency period, and the tumor growth rate was the slowest before d22, which was significantly different from group E (P<0.05), but the difference was not significant after d22 (P>0.05) , which may be related to the prolongation of the time between the second and third immunizations, which failed to inhibit the growth of tumors in time.
3)皮下移植瘤攻击后35天小鼠的存活情况3) Survival of mice 35 days after subcutaneous transplantation challenge
皮下注射接种2×105个/只EMT6细胞后35天内,各组小鼠(7只/组)的存活情况如表2所示,PBS对照组和pVAX1空载体组均有1只小鼠自然死亡,其余各组的7只小鼠均存活,表明本发明的基因疫苗具有较高的安全性。Within 35 days after subcutaneous injection of 2×10 5 EMT6 cells, the survival of mice in each group (7 mice/group) is shown in Table 2. One mouse in the PBS control group and the pVAX1 empty vector group had natural died, and all the other 7 mice in each group survived, showing that the genetic vaccine of the present invention has higher safety.
表2皮下移植瘤攻击后35天各组免疫小鼠的存活情况Table 2 Survival of immunized mice in each group 35 days after subcutaneous transplantation challenge
4)免疫小鼠对皮下移植瘤的抑制作用4) Inhibitory effect of immunized mice on subcutaneous transplanted tumors
皮下注射接种2×105个/只EMT6细胞后35天断颈处死各组内的存活小鼠,摘取肿瘤组织,并拍照。称量各组瘤重后,根据公式计算各组疫苗对肿瘤的抑制率,肿瘤抑制率的计算公式如下:The surviving mice in each group were sacrificed by neck dislocation 35 days after inoculation of 2×10 5 EMT6 cells subcutaneously, and the tumor tissues were removed and photographed. After weighing the tumor weight of each group, the tumor inhibition rate of each group of vaccines was calculated according to the formula, and the calculation formula of the tumor inhibition rate was as follows:
皮下接种EMT6细胞并经疫苗免疫30天后,各免疫组小鼠的离体肿瘤体积比较结果如图23所示(A:PBS空白对照组;B:pVAX1组;C:pVAX1-IRES组;D:pVAX1-2PAG组;E:pVAX1-2PAG-Fc-GPI组;F:pVAX1-2PFcGB组),皮下移植瘤攻击35天后各组小鼠的平均瘤重如图24所示,各免疫组的肿瘤抑制率的统计结果如表3和图25所示,结果显示,A组、B组和C组的平均瘤重相近,没有显著性差异(P>0.05);与对照组相比,D组、E组和F组的平均瘤重均明显降低,表明本发明含复合抗原基因2PAG的基因疫苗具有较高的肿瘤抑制率(P<0.01);其中F组的平均瘤重最小,具有最高的肿瘤抑制率,与对照组的差异极其显著(P<0.001)。After 30 days of subcutaneous inoculation of EMT6 cells and immunization with the vaccine, the comparison results of the tumor volumes in vitro of the mice in each immunized group are shown in Figure 23 (A: PBS blank control group; B: pVAX1 group; C: pVAX1-IRES group; D: pVAX1-2PAG group; E: pVAX1-2PAG-Fc-GPI group; F: pVAX1-2PFcGB group), the average tumor weight of mice in each group after 35 days of subcutaneous transplantation challenge is shown in Figure 24, and the tumor inhibition of each immune group The statistical results of the rate are shown in Table 3 and Figure 25. The results showed that the average tumor weights of Group A, Group B and Group C were similar without significant difference (P>0.05); compared with the control group, Group D, Group E The average tumor weights of group F and group F all decreased significantly, indicating that the gene vaccine containing the compound antigen gene 2PAG of the present invention has a higher tumor inhibition rate (P<0.01); wherein the average tumor weight of group F is the smallest, and has the highest tumor inhibition rate. The rate was significantly different from that of the control group (P<0.001).
表3各组免疫小鼠对肿瘤的抑制作用Table 3 Inhibitory effect of each group of immunized mice on tumor
注:※与对照组相比,该组小鼠对肿瘤的抑制作用非常显著(P<0.01);※※P<0.001。Note: ※ Compared with the control group, this group of mice had a very significant inhibitory effect on the tumor (P<0.01); ※※ P<0.001.
2、血清抗体的ELISA检测2. ELISA detection of serum antibodies
末次免疫后第5天(d25),取D、E和F组免疫小鼠的尾静脉血,室温放置3h后3000rpm离心10min,取上层血清,用Sur融合蛋白包板,用ELISA法检测抗体滴度,同时设立免疫前鼠血清的阴性对照组,检测方法包括以下步骤:On the 5th day after the last immunization (d25), the tail vein blood of the immunized mice in groups D, E and F was collected, left at room temperature for 3 hours, centrifuged at 3000rpm for 10 minutes, and the upper layer of serum was collected, coated with Sur fusion protein, and detected by ELISA. Degree, set up the negative control group of mouse serum before immunization simultaneously, detection method comprises the following steps:
a.包被:蛋白用包被液稀释至终浓度为3μg/mL,包被ELISA板,4℃过夜;a. Coating: Dilute the protein with coating solution to a final concentration of 3 μg/mL, coat the ELISA plate, and leave overnight at 4°C;
b.洗涤:甩弃ELISA板孔中包被液,PBST洗涤1次;b. Washing: discard the coating solution in the wells of the ELISA plate, and wash once with PBST;
c.封闭:2%酪蛋白室温封闭4h,弃去封闭液,拍干ELISA板;c. Blocking: block with 2% casein at room temperature for 4 hours, discard the blocking solution, and pat dry the ELISA plate;
d.检测:免疫鼠血清用PBS倍比稀释后加入各孔,100ul/孔,37℃、30min;d. Detection: the immune mouse serum was diluted with PBS and added to each well, 100ul/well, 37°C, 30min;
e.标记:PBST洗涤后加入HRP标记的羊抗鼠IgG(购自北京中杉金桥技术有限公司),37℃反应20min;e. Labeling: After washing with PBST, add HRP-labeled goat anti-mouse IgG (purchased from Beijing Zhongshan Jinqiao Technology Co., Ltd.), and react at 37°C for 20 minutes;
f.显色:PBST洗涤后,TMB显色10min,2M硫酸终止反应。用SPECTRAIII酶联仪检测450nm的OD值。结果判定:待测样品的OD值(P)与阴性对照的OD值(N)之比大于或等于2.1(P/N≥2.1)为阳性,显示阳性结果的最大抗体稀释度为免疫小鼠血清的抗体效价。f. Color development: After washing with PBST, develop color with TMB for 10 min, and stop the reaction with 2M sulfuric acid. The OD value at 450nm was detected with a SPECTRAIII enzyme-coupled instrument. Result judgment: the ratio of the OD value (P) of the sample to be tested to the OD value (N) of the negative control is greater than or equal to 2.1 (P/N≥2.1) is positive, and the maximum antibody dilution that shows a positive result is the immune mouse serum antibody titer.
D、E和F组免疫小鼠血清的抗体效价的ELISA检测结果如图26所示,D、E和F组免疫小鼠血清中均有特异性抗体出现,D组的血清抗体效价为1∶3200,E组和F组相同,均为1∶6400。上述检测结果表明本发明的基因疫苗具有较好的免疫原性。The ELISA detection results of the antibody titers of the immunized mice serum of the D, E and F groups are shown in Figure 26, all have specific antibodies in the immunized mice serum of the D, E and F groups, and the serum antibody titers of the D group are 1:3200, group E and group F are the same, both are 1:6400. The above test results show that the genetic vaccine of the present invention has better immunogenicity.
3、免疫小鼠CTL杀伤活性的测定3. Determination of CTL killing activity in immunized mice
测定免疫小鼠CTL杀伤活性,具体方法包括以下步骤:Measuring the CTL killing activity of immunized mice, the specific method comprises the following steps:
1)免疫小鼠脾脏单个核细胞悬液的制备1) Preparation of spleen mononuclear cell suspension of immunized mice
末次免疫后第5天(d25),断颈处死各组中剩余的两只小鼠,在无菌条件下取小鼠的脾脏制备脾细胞悬液,具体操作如下:On the 5th day (d25) after the last immunization, the remaining two mice in each group were killed by neck dislocation, and the spleen of the mice was taken under aseptic conditions to prepare a spleen cell suspension. The specific operations were as follows:
a.打开超净工作台的紫外灯照射30分钟,取小鼠拉颈处死,自来水冲洗后浸泡于75%乙醇中5min,随即放入超净台内小鼠解剖板上左侧卧位;a. Turn on the ultra-clean workbench for 30 minutes of ultraviolet light irradiation, take the mouse to pull the neck to kill, rinse with tap water, soak in 75% ethanol for 5 minutes, and then put it in the left side lying position on the mouse dissection board in the ultra-clean workbench;
b.用75%乙醇消毒小鼠腹部,无菌手术开腹腔取出脾脏,去除脂肪和结缔组织,剪成(5-7段)放于200目不锈钢网筛,将网筛置于预先放入2-3mL无血清RPMI-1640培养液的平皿中,用研磨棒挤压出脾细胞,尽量挤压干净;b. Disinfect the abdomen of the mouse with 75% ethanol, open the abdominal cavity to take out the spleen by aseptic operation, remove fat and connective tissue, cut into (5-7 sections) and put them on a 200-mesh stainless steel mesh screen, and put the mesh screen in 2 - Squeeze out splenocytes with a grinding rod in a plate of 3mL serum-free RPMI-1640 culture medium, and squeeze them as clean as possible;
c.用适量无血清RPMI-1640培养液冲洗钢网,冰浴中静置3-5min,取上清部分移入离心管中,补加培养液到10mL;c. Rinse the stencil with an appropriate amount of serum-free RPMI-1640 culture medium, let it stand in an ice bath for 3-5 minutes, take the supernatant and transfer it to a centrifuge tube, and add culture medium to 10 mL;
d.准备两只透明刻度离心管,分别加入4mL淋巴细胞分离液,将脾细胞悬液缓慢加在分离液上方,1500rpm离心20min;d. Prepare two transparent graduated centrifuge tubes, add 4mL lymphocyte separation medium respectively, slowly add spleen cell suspension on top of the separation medium, and centrifuge at 1500rpm for 20min;
e.离心后取白膜,加入10mL培养基洗2次(1000rpm离心10min);e. Take buffy membrane after centrifugation, add 10mL culture medium and wash twice (centrifuge at 1000rpm for 10min);
f.最后将细胞重悬于淋巴细胞培养基中(不完全培养基中加入20%FCS)。用淋巴细胞计数液对细胞进行计数,一般来讲,脾细胞悬液中可以收获108的细胞,经淋巴细胞分离液分离后可获得107的细胞,调整细胞浓度至1×106/mL,加入维持淋巴细胞生长的细胞因子PHA-P 2μg/mL和IL-2 20IU/mL,放入CO2孵箱中培养。f. Finally, the cells were resuspended in the lymphocyte culture medium (20% FCS was added to the incomplete medium). Count the cells with lymphocyte counting solution. Generally speaking, 108 cells can be harvested from the spleen cell suspension, and 107 cells can be obtained after separation by lymphocyte separation medium. Adjust the cell concentration to 1×10 6 /mL. Add cytokines PHA-P 2μg/mL and IL-2 20IU/mL to maintain the growth of lymphocytes, and culture them in a CO 2 incubator.
2)效应细胞的制备2) Preparation of effector cells
取免疫后小鼠的脾单个核细胞悬液,加PHA-P 2μg/mL和IL-2 10IU/mL维持淋巴细胞生长,为检测特异性杀伤效应,加入常规方法制备的重组Sur原核蛋白,工作浓度25μg/mL,与细胞共刺激,CO2孵箱中培养3天后用作效应细胞。Take the splenic mononuclear cell suspension of the immunized mice, add PHA-
3)靶细胞的制备3) Preparation of target cells
将小鼠乳腺癌细胞EMT6在CO2孵箱中培养备用。Mouse breast cancer cells EMT6 were cultured in a CO 2 incubator for later use.
4)乳酸脱氢酶(LDH)-释放法测杀伤活性4) Lactate dehydrogenase (LDH)-release method to measure killing activity
A、反应体系的建立A. The establishment of the reaction system
效靶细胞共孵育杀伤过程,在96孔圆底细胞培养板中进行,整个反应体系要设置严格的对照孔和试验孔,每组设3个复孔,均为200μl体系,包括以下内容:The process of co-incubating and killing effective target cells is carried out in a 96-well round-bottomed cell culture plate. The entire reaction system should be set up with strict control wells and test wells. Each group should set up 3 duplicate wells, all of which are 200μl systems, including the following:
a.不同浓度效应细胞LDH自然释放孔(由效应细胞与无血清RPMI-1640培养基组成):收集效应细胞,调整细胞浓度倍比稀释,每孔50μl;a. Different concentrations of effector cells LDH natural release wells (composed of effector cells and serum-free RPMI-1640 medium): collect effector cells, adjust the cell concentration and dilute, 50 μl per well;
b.靶细胞LDH自然释放孔(靶细胞与无血清培养基组成):每孔加入靶细胞(1×104/mL)50μl,用无血清培养基补足至100μl;b. Target cell LDH natural release well (composed of target cells and serum-free medium): add 50 μl of target cells (1×10 4 /mL) to each well, and make up to 100 μl with serum-free medium;
c.靶细胞最大释放孔(由靶细胞、无血清RPMI-1640培养基和随后加入的10×裂解液组成):每孔加入靶细胞(1×104/mL)50μl,用无血清培养基补足100μl;c. Target cell maximum release well (composed of target cells, serum-free RPMI-1640 medium and 10× lysate added later): add 50 μl of target cells (1×10 4 /mL) to each well, and use serum-free medium Make up 100μl;
d.培养基背景值孔:加入100μl无血清RPMI-1640培养基,用于校正培养基中LDH的自然释放;d. Medium background value well: add 100 μl serum-free RPMI-1640 medium to correct the natural release of LDH in the medium;
e.体积校正孔:加入100μl无血清RPMI-1640培养基和10×裂解液(Promega公司的CytoTox 96@ Non-Radioactive Cytotoxicity Assay试剂盒提供),校正10μl裂解液引起的体积误差;e. Volume correction well: Add 100 μl serum-free RPMI-1640 medium and 10× lysate (Promega’s CytoTox 96@ Non-Radioactive Cytotoxicity Assay kit), and correct the volume error caused by 10 μl lysate;
f.实验孔:每孔加入靶细胞(5×104/mL)50μl,加入倍比稀释好的效应细胞50μl,使效靶比为60∶1;30∶1;15∶1;7.5∶1。f. Experimental wells: add 50 μl of target cells (5×10 4 /mL) to each well, and add 50 μl of effector cells after doubling dilution, so that the effect-target ratio is 60:1; 30:1; 15:1; 7.5:1 .
所有反应设置完成后,于室温300rpm离心4min。After all the reaction settings were completed, centrifuge at 300 rpm for 4 min at room temperature.
B、细胞培养和收获上清B. Cell culture and harvest supernatant
将96孔培养板放入CO2孵箱中培养4小时,培养结束前45min,在靶细胞最大释放孔中,加入10×裂解液,继续培养并在显微镜下观察最大释放孔中靶细胞的裂解情况,如果裂解不完全可补加入10×裂解液。结束培养后,在室温下250rpm离心4min。Put the 96-well culture plate into the CO2 incubator and cultivate it for 4 hours. 45 minutes before the end of the culture, add 10× lysate to the maximum release hole of the target cells, continue the culture and observe the lysis of the target cells in the maximum release hole under the microscope In some cases, if the lysis is not complete, add 10× Lysis Buffer. After finishing the culture, centrifuge at 250 rpm for 4 min at room temperature.
C、LDH测定C. LDH determination
用Promega公司的CTL活性检测试剂盒CytoTox 96@ Non-RadioactiveCytotoxicity Assay并参照试剂盒说明书测定各免疫组小鼠脾细胞的杀伤活性,方法为:准备另一块96孔酶联板,从96孔圆底培养板中取上清50μl移入酶联板中相对应位置,每孔中加入50μl底物液,盖好盖室温避光反应30min后,再加入50μl终止液,490nm处读取吸光值。Use Promega's CTL activity detection kit CytoTox 96@ Non-Radioactive Cytotoxicity Assay and refer to the kit instructions to measure the killing activity of mouse splenocytes in each immune group. Take 50 μl of the supernatant from the culture plate and transfer it to the corresponding position in the enzyme-linked plate, add 50 μl of substrate solution to each well, cover the well and react in the dark at room temperature for 30 minutes, then add 50 μl of stop solution, and read the absorbance at 490 nm.
D、计算实验结果D. Calculate the experimental results
先求出各组实验数据所设复孔的平均值,然后计算各组数据的纠正值,即实验组和自然释放组减去培养基背景释放值;最大释放组减去体积纠正释放值,应用各组实验数据的纠正值,按如下公式计算:First calculate the average value of the multiple wells set for each group of experimental data, and then calculate the corrected value of each group of data, that is, the experimental group and the natural release group minus the background release value of the medium; the maximum release group minus the volume corrected release value, apply The correction value of each group of experimental data is calculated according to the following formula:
各免疫组小鼠脾细胞的杀伤率统计结果如表4和图28所示,实验结果显示,D、E和F组免疫小鼠体内均产生了不同程度的细胞免疫反应,随着效靶比的增加,CTL特异性杀伤率也逐渐上升,与对照组具有显著性差异(P<0.05),其中F组的CTL杀伤率最高,在效靶比为60∶1、30∶1和15∶1时均与对照组具有极其显著性差异(P<0.01);D组和E组的CTL杀伤率基本相同。Table 4 and Figure 28 show the statistical results of the killing rate of splenocytes in each immunized group of mice. The experimental results show that the immunized mice in groups D, E and F all produced different degrees of cellular immune responses. The specific killing rate of CTL also gradually increased, which was significantly different from that of the control group (P<0.05). Among them, the killing rate of CTL in group F was the highest, and the effective target ratio was 60:1, 30:1 and 15:1. There was an extremely significant difference (P<0.01) between the time average and the control group; the CTL killing rates of the D group and the E group were basically the same.
表4不同效靶比的各免疫组小鼠脾细胞的杀伤活性The killing activity of mouse splenocytes in each immunization group of different effect target ratios of table 4
注:※与对照组相比,该组小鼠对肿瘤的抑制作用非常显著(P<0.05);※※P<0.01。Note: ※ Compared with the control group, the mice in this group had a very significant inhibitory effect on the tumor (P<0.05); ※※ P<0.01.
序列表sequence listing
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CN103068985A (en) * | 2010-06-24 | 2013-04-24 | 美国政府(由卫生和人类服务部、疾病控制和预防中心的部长所代表) | Pan-lyssavirus vaccines against rabies |
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CN105400798A (en) * | 2008-11-17 | 2016-03-16 | Vgx药品有限责任公司 | Antigens that elicit immune response against flavivirus and methods of using same |
CN103068985A (en) * | 2010-06-24 | 2013-04-24 | 美国政府(由卫生和人类服务部、疾病控制和预防中心的部长所代表) | Pan-lyssavirus vaccines against rabies |
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CN107281475A (en) * | 2017-06-15 | 2017-10-24 | 杭州贝罗康生物技术有限公司 | A kind of gene vaccine for preventing and treating tumour and its production and use |
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