CN110551187B - 化学合成的h7n9禽流感病毒na蛋白胞外区抗原片段及制备方法和应用 - Google Patents
化学合成的h7n9禽流感病毒na蛋白胞外区抗原片段及制备方法和应用 Download PDFInfo
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Abstract
本发明提供一种化学合成的H7N9禽流感病毒NA蛋白胞外区抗原片段及制备方法和应用,涉及基因工程技术领域。该方法通过生物信息学分析,筛选出H7N9禽流感病毒NA蛋白胞外区抗原片段,基于E.coli原核生物偏爱密码子进行密码子优化,化学合成抗原表位的全新基因序列;然后利用基因工程技术,构建具有编码NA胞外区域的DNA表达载体,将该载体转化到感受态细胞中,从而得到以包涵体形式表达的重组tN9;对该包涵体进行超声处理得到重新折叠的重组tN9;通过SDS‑PAGE收集并分析正确折叠的tN9,进行蛋白亲和纯化和质谱分析。本发明制备的重组蛋白可用于H7N9禽流感感染NA抗体的检测及单抗和多抗制备,为深入探索NA的蛋白结构及功能、H7N9禽流感病毒致病机制和建立快速检测方法奠定基础。
Description
技术领域
本发明涉及基因生物工程技术领域,具体涉及H7N9禽流感病毒NA蛋白胞外区抗原片段及制备方法和应用。
背景技术
2013年3月中国首次报道人感染新型H7N9禽流感病毒病例,该病能引起严重甚至致命呼吸道疾病。研究证实H7N9禽流感病毒在多次大流行期间,感染家禽和人类且对小鼠、雪貂和鸡群的致病性增强。因此,美国疾病预防和控制中心结合流感风险评估结果,将H7N9亚型禽流感病毒列为高风险病原。流行病学调查表明:大部分患者具有活禽接触史,意味着活禽市场和H7N9流感爆发存在着紧密联系。目前,H7N9病毒无法通过呼吸道、母婴、血液等方式实现人人之间传播,但它具有与人类感染相关的基因组成,在少量非人源高致病H7N9毒株中,发现神经氨酸酶抑抗性和哺乳动物适应性突变。此外,H7N9禽流感病毒人人传播突变动力学模型分析表明,将来H7N9病毒可以获得人人传播的特征,因此,对H7N9禽流感病毒的研究工作亟需深入开展,以避免给人类健康带来威胁。
NA是流感病毒主要囊膜纤突之一,天然NA蛋白有4个相同的单体组成四聚体,每种单体全长约470个氨基酸,并有胞内区、跨膜区、茎部区及头部区4个区域组成。近来研究成果表明,NA除了在病毒释放中发挥作用,还有助于病毒与细胞糖蛋白唾液酸基团结合,补充血凝素(Hemagglutinin,HA)的受体结合功能,提高NA酶活性,并促进病毒感染。无论是自然突变还是通过遗传修饰获得的NA活性位点突变体,其框架和催化残基都可以在不同程度上改变病毒复制能力、感染性和对抗病毒抑制剂的敏感性。总之,NA在病毒吸附、侵入、释放以及维持与HA的功能平衡等方面起着重要的多功能作用。
流感病毒基因易发生抗原漂移、抗原转换,从而可能导致流感爆发,NA基因是流感病毒基因组突变率较高的基因。在一些H7N9临床分离株中发现了与耐药性相关的NA氨基酸突变位点NA-R292K,数据表明H7N9分离株NA的R292K取代,对奥司他韦和帕拉米韦产生高度耐药性,对扎那米韦产生部分耐药性。而且,耐药突变位点的H7N9病毒株与未产生突变的病毒株相比,其在人体的病毒复制能力、对小鼠的毒力和在豚鼠间的传播能力并没有下降。
目前,流感病毒疫苗的有效成分主要基于HA蛋白,研究表明NA可能也具备良好的免疫原性。有研究人员制备了基于NA的重组病毒样颗粒疫苗,免疫N1 NA VLPs的雪貂产生高滴度的血清NA抑制抗体,可以保护雪貂免受致死剂量的病毒侵袭,这为基于NA疫苗的开发提供了支持。目前,尚无关于N9 NA作为疫苗标靶的报道,试推测N9蛋白可能同样存在病毒特异性抗原表位,诱导产生保护性抗体。总之,NA在病毒侵入、释放、小分子药物作用标靶和免疫原性方面发挥多重重要功效。因此,深入研究NA在流感感染和复制周期中的复杂作用,特别是考虑到H7N9病毒HA和NA糖蛋白如何各自发挥作用,实现功能平衡以维持病毒适应性等方面具有重要意义。然而目前尚无针对N9蛋白的特异性抗体,一定程度上限制了对N9蛋白的深入研究。
发明内容
本发明的主要目的是:提供一种经过纯化的NA胞外区蛋白,为后续单克隆抗体制备、开展NA蛋白结构和功能研究、建立H7N9禽流感病毒血清学检测方法和进行H7N9病毒对人和动物的感染机制以及新型疫苗的开发提供条件。
本发明的第一个目的,提供一种重组H7N9禽流感病毒NA蛋白原核表达载体。
本发明的第二个目的,提供一种重组表达宿主细胞。
本发明的第三个目的,提供一种禽流感病毒NA蛋白胞外区抗原基因。
本发明的第四个目的,提供所述的H7N9禽流感病毒NA胞外区重组蛋白。
本发明的第五个目的,提供了重组质粒pET28b-tN9的构建方法。
本发明的第六个目的,提供了一种重组蛋白纯化方法。
本发明的第七个目的,提供了所述的重组质粒pET28b-tN9、重组宿主细胞,重组蛋白在建立N9亚型禽流感病毒血清学检测方法中的应用。
为了解决上述技术问题,本发明所采用的技术方案为:
一种化学合成的编码H7N9禽流感病毒NA蛋白胞外区的抗原片段,其特征在于,该抗原片段序列全长为1322bp,所述抗原片段的核苷酸序列SEQ ID NO:1如下:
5’CC ATG GGC CAC CTG AAA CCG GGT TGC AAC TGC AGC CAC AGC CAG CCG GAAACC ACC AAC ACC AGC CAA ACC ATC ATT AAC AAC TAC TAT AAC GAG ACC AAC ATC ACCAAC ATT CAG ATG GAG GAA CGT ACC AGC CGT AAC TTC AAC AAC CTG ACC AAA GGT CTGTGC ACC ATC AAC AGC TGG CAC ATT TAC GGC AAG GAC AAC GCG GTG CGT ATC GGC GAGAGC AGC GAT GTG CTG GTT ACC CGT GAA CCG TAC GTT AGC TGC GAC CCG GAT GAG TGCCGT TTT TAT GCG CTG AGC CAG GGT ACC ACC ATC CGT GGC AAA CAC AGC AAC GGT ACCATT CAC GAC CGT AGC CAA TAC CGT GCG CTG ATT AGC TGG CCG CTG AGC AGC CCG CCGACC GTG TAT AAC AGC CGT GTT GAA TGC ATT GGC TGG AGC AGC ACC AGC TGC CAC GATGGC AAG AGC CGT ATG AGC ATC TGC ATT AGC GGT CCG AAC AAC AAC GCG AGC GCG GTGGTT TGG TAC AAC CGT CGT CCG GTG GCG GAG ATC AAC ACC TGG GCG CGT AAC ATT CTGCGT ACC CAG GAG AGC GAA TGC GTG TGC CAC AAC GGT GTT TGC CCG GTG GTT TTT ACCGAT GGT AGC GCG ACC GGT CCG GCG GAT ACC CGT ATC TAC TAC TTC AAA GAG GGT AAAATC CTG AAG TGG GAA AGC CTG ACC GGT ACC GCG AAA CAC ATC GAG GAA TGC AGC TGCTAC GGT GAA CGT ACC GGC ATT ACC TGC ACC TGC CGT GAC AAC TGG CAG GGT AGC AACCGT CCG GTG ATC CAA ATT GAT CCG GTT GCG ATG ACC CAC ACC AGC CAA TAT ATC TGCAGC CCG GTG CTG ACC GAC AAC CCG CGT CCG AAC GAT CCG AAC ATT GGC AAA TGC AACGAC CCG TAC CCG GGT AAC AAC AAC AAC GGT GTT AAG GGC TTC AGC TAT CTG GAT GGCGCG AAC ACC TGG CTG GGT CGT ACC ATC AGC ACC GCG AGC CGT AGC GGC TAC GAA ATGCTG AAA GTG CCG AAC GCG CTG ACC GAC GATCGT AGC AAG CCG ATC CAG GGT CAA ACCATT GTTCTG AAC GCG GAC TGG AGC GGT TAC AGCGGC AGC TTC ATG GAC TAT TGG GCG GAGGGC GAT TGC TAC CGT GCG TGC TTT TAT GTT GAG CTG ATC CGT GGT CGT CCG AAA GAAGAC AAA GTG TGG TGG ACC AGC AAC AGC ATT GTT AGC ATG TGC AGC AGC ACCGAA TTCCTG GGC CAA TGG AAC TGG CCG GAT GGT GCG AAG ATC GAG TAT TTT CTG CTC GAG CACCAC CAC CAC CACCAC 3’。
一种制备所述的编码H7N9禽流感病毒NA蛋白胞外区的抗原片段的方法,包括H7N9禽流感病毒NA蛋白胞外区基因片段重组质粒的构建、表达融合蛋白工程菌的筛选、表达产物的处理,具体步骤如下:
(1)H7N9禽流感病毒NA蛋白胞外区基因片段重组质粒的构建:
用Nco I和Xho I内切酶双酶切质粒表达载体pET28b和权利要求1所述的抗原片段,经核酸电泳凝胶回收后,用T4 DNA连接酶连接,使所述的抗原片段的基因插入到载体pET28b内的NocI和XhoI位点之间,与载体上的起始密码子翻译框架一致,表达融合蛋白,融合蛋白全长包括440个氨基酸,融合蛋白的N端融合了载体上的2个氨基酸,C端包含Leu Glu和6个组氨酸His标签,表达的融合蛋白的氨基酸序列SEQ ID NO:2如下:
Met Gly His Leu Lys Pro Gly Cys Asn Cys Ser His Ser Gln Pro Glu
Thr Thr Asn Thr Ser Gln Thr Ile Ile Asn Asn Tyr Tyr Asn Glu Thr
Asn Ile Thr Asn Ile Gln Met Glu Glu Arg Thr Ser Arg Asn Phe Asn
Asn Leu Thr Lys Gly Leu Cys Thr Ile Asn Ser Trp His Ile Tyr Gly
Lys Asp Asn Ala Val Arg Ile Gly Glu Ser Ser Asp Val Leu Val Thr
Arg Glu Pro Tyr Val Ser Cys Asp Pro Asp Glu Cys Arg Phe Tyr Ala
Leu Ser Gln Gly Thr Thr Ile Arg Gly Lys His Ser Asn Gly Thr Ile
His Asp Arg Ser Gln Tyr Arg Ala Leu Ile Ser Trp Pro Leu Ser Ser
Pro Pro Thr Val Tyr Asn Ser Arg Val Glu Cys Ile Gly Trp Ser Ser
Thr Ser Cys His Asp Gly Lys Ser Arg Met Ser Ile Cys Ile Ser Gly
Pro Asn Asn Asn Ala Ser Ala Val Val Trp Tyr Asn Arg Arg Pro Val
Ala Glu Ile Asn Thr Trp Ala Arg Asn Ile Leu Arg Thr Gln Glu Ser
Glu Cys Val Cys His Asn Gly Val Cys Pro Val Val Phe Thr Asp Gly
Ser Ala Thr Gly Pro Ala Asp Thr Arg Ile Tyr Tyr Phe Lys Glu Gly
Lys Ile Leu Lys Trp Glu Ser Leu Thr Gly Thr Ala Lys His Ile Glu
Glu Cys Ser Cys Tyr Gly Glu Arg Thr Gly Ile Thr Cys Thr Cys Arg
Asp Asn Trp Gln Gly Ser Asn Arg Pro Val Ile Gln Ile Asp Pro Val
Ala Met Thr His Thr Ser Gln Tyr Ile Cys Ser Pro Val Leu Thr Asp
Asn Pro Arg Pro Asn Asp Pro Asn Ile Gly Lys Cys Asn Asp Pro Tyr
Pro Gly Asn Asn Asn Asn Gly Val Lys Gly Phe Ser Tyr Leu Asp Gly
Ala Asn Thr Trp Leu Gly Arg Thr Ile Ser Thr Ala Ser Arg Ser Gly
Tyr Glu Met Leu Lys Val Pro Asn Ala Leu Thr Asp Asp Arg Ser Lys
Pro Ile Gln Gly Gln Thr Ile Val Leu Asn Ala Asp Trp Ser Gly Tyr
Ser Gly Ser Phe Met Asp Tyr Trp Ala Glu Gly Asp Cys Tyr Arg Ala
Cys Phe Tyr Val Glu Leu Ile Arg Gly Arg Pro Lys Glu Asp Lys Val
Trp Trp Thr Ser Asn Ser Ile Val Ser Met Cys Ser Ser Thr Glu Phe
Leu Gly Gln Trp Asn Trp Pro Asp Gly Ala Lys Ile Glu Tyr Phe Leu
Leu Glu His His His His His His;
(2)表达融合蛋白工程菌的筛选鉴定:
将重组质粒pET28b-tN9分别转化E.coli BL21(DE3)、Rosetta、Arctic Express(DE3)感受态细胞,挑取重组单菌落分别于含50μg/ml卡那霉素的LB液体培养基中培养,37℃过夜培养,次日随机挑取转化菌落和含质粒pET28的对照菌,提取质粒,用限制性内切酶NcoI和XhoI对质粒双酶切,用1.0g/mL的Agarose凝胶检测双酶切产物,将含有重组质粒的阳性转化子接种至含卡那霉素50μg/mL的LB培养基内,于30℃、37℃、16℃分别振荡培养至OD值为0.5,加IPTG诱导剂至其终浓度为0.2mmol/L,继续振荡培养诱导5h,离心收集菌体,进行超声破碎,经SDS-PAGE检测,证实重组蛋白在E.coli BL21(DE3)、Rosetta(DE3)、Arctic Express(DE3)均可表达;将表达产物进行质谱分析,以确认表达产物为目标产物;
(3)表达包涵体产物的处理:
将E.coli BL21(DE3)诱导表达融合蛋白的工程菌离心,收获培养物,菌体重悬于洗脱液内,洗脱液的组成为0.1M PBS PH7.2~7.4、10mmol/L EDTA、0.5%Triton-X100,离心收集沉淀,以洗脱液重悬菌体2~3次,超声破菌30min,然后重新溶解在含8M尿素的buffer A缓冲液中,得到解离状态的重组NA蛋白胞外区抗原的包涵体;将所述包涵体稀释在含氯化镁、4M尿素的buffer B缓冲液中,得到第一复性液;将所述第一复性液稀释在含有Triton X-100、2M尿素的buffer C缓冲液中,4℃透析12h,得到第二复性液;将所述第二复性液稀释在含精氨酸、1M尿素的buffer D缓冲液中,4℃条件透析12h,得到第三复性液,将所述第三复性液4℃条件透析12h,得到最终复性液;
将所述最终复性液利用Ni柱分离,用不同咪唑浓度的洗脱液分次洗脱目的蛋白,即得到纯化的NA蛋白胞外区抗原片段。
所述不同咪唑浓度的洗脱液分次洗脱目的蛋白时洗脱液的浓度分别为10mMoL/L、100mMoL/L、150mMoL/L和250mMoL/L。
所述的H7N9禽流感病毒NA蛋白胞外区抗原片段在H7N9禽流感病毒血清学检测中的应用。
所述的H7N9禽流感病毒NA蛋白胞外区抗原片段在对人和动物的感染机制以及疫苗中的应用。
所述的H7N9禽流感病毒NA蛋白胞外区抗原片段在H7N9禽流感病毒NA抗体的检测及单克隆抗体的制备中的应用。
所述的H7N9禽流感病毒NA蛋白胞外区抗原片段在H7N9禽流感病毒NA检测中试剂盒的应用。
本发明的优点:
1.本发明通过化学合成H7N9禽流感病毒NA蛋白胞外区抗原全新基因片段,利用基因工程技术,制备新型重组蛋白。通过计算机分析,筛选出含强H7N9禽流感病毒NA蛋白胞外区抗原片段,基于原核生物密码子偏爱性,化学合成新的基因序列,利用基因工程技术进行体外表达和纯化,制备的蛋白可用于H7N9禽流感病毒NA抗体的检测及单克隆抗体的制备等方面。
2.本发明根据生物信息学的分析,筛选出的NA胞外区片段氨基酸序列,选择E.coli偏爱的密码子进行化学合成编码基因序列,该基因适宜在E.coli内的高效表达。
3.本发明构建的重组菌,通过HIS标签易于后续大量纯化、制备该蛋白。
4.本发明表达的tN9蛋白具有良好的免疫原性,能诱导兔子产生高水平免疫反应,所制备的特异性多克隆抗体具有较高的效价。
5.本发明为开展NA蛋白结构和功能研究、建立H7N9禽流感病毒血清学检测方法,进一步探讨NA蛋白特性、H7N9病毒对人和动物的感染机制以及新型疫苗的开发提供了理论基础。
6.本发明提供一种禽流感病毒NA蛋白胞外区片段的表达、纯化和鉴定方法,以期为N9亚型禽流感病毒血清学检测方法的建立奠定基础。
附图说明
图1为H7N9禽流感病毒NA蛋白胞外区抗原片段二级结构分析图;
Alpha helix,缩写为h:α螺旋;Extended strand,缩写为e:β折叠;Beta turn,缩写为t:β转角;Random coil,缩写为c:无规则卷曲。
图2为NA蛋白截短前(A、C)后(B、D)三级结构比对图。
图3为tN9基因序列密码子优化前后比对图。
图4为重组菌落PCR后凝胶电泳图,泳道1:阳性重组菌落;泳道2:阴性重组菌落;M:DL2000Marker。
图5为SDS-PAGE检测重组tN9蛋白的表达图;
M:蛋白Marker;1-7:分别为Rosetta重组菌诱导前、30℃诱导后、30℃诱导后超声破碎上清、30℃诱导后超声破碎沉淀、37℃诱导后、37℃诱导后超声破碎上清、37℃诱导后超声破碎沉淀;8、16、17分别为Arctic Express(DE3)重组菌诱导后、诱导后超声破碎上清、诱导后超声破碎沉淀;9-15:分别为BL21(DE3)重组菌诱导前、30℃诱导后、30℃诱导后超声破碎上清、30℃诱导后超声破碎沉淀、37℃诱导后、37℃诱导后超声破碎上清、37℃诱导后超声破碎沉淀。
图6为SDS-PAGE检测tN9重组蛋白亲和纯化产物图;
图中,1:流穿液;2:10mMoL/L的咪唑洗脱液;3-5:100mMoL/L的咪唑洗脱液;6:150mMoL/L的咪唑洗脱液;10:BSA标准品;11:纯化后的目标蛋白;M:蛋白Marker。
图7为质谱分析tN9蛋白数据库的搜索结果图。
图8为纯化后抗体SDS-PAGE分析图;M:蛋白质分子质量标准;1:纯化后浓缩抗体。
图9为Western blotting鉴定tN9多克隆抗体免疫反应性;
图中,1:重组蛋白;2:pet28a载体诱导后;M:蛋白Marker;3:人用H7N9灭活病毒原液(上海株)。
图10为ELISA检测tN9蛋白多克隆抗体效价。
具体实施方式
下面通过实例和附图对本发明进一步阐述、说明。
原核表达载体pET28b(+)、E.coli DH5α和E.coli BL21(DE3)感受态细胞由新乡学院医学院医学检验技术中心实验室保存;E.coli Rosetta和E.coli Arctic Express(DE3)感受态细胞购自南京钟鼎生物公司。
Fast Digest限制性内切酶Nco I和Xho I购自Thermo Scientific公司;质粒小量制备试剂盒和Solution I购自TAKARA公司;DNA凝胶回收试剂盒购自OMEGA BIO-TEK公司;Ni Sepharose 6FF、Sepharose 4B纯化填料购自GE公司;HRP标记山羊抗兔IgG和DAB显色液购自武汉博士德生物工程有限公司;TMB显色液购自上海碧云天生物技术有限公司。
优化后的基因片段由南京金斯瑞生物科技有限公司合成。
实施例1新型H7N9禽流感病毒NA蛋白胞外区片段的生物学信息分析
采用EXPASY系统的Protparam程序,分析新型H7N9禽流感病毒(安徽株)NA蛋白胞外区片段(truncated N9,tN9)理化特征(https://web.expasy.org/protparam/)。
分析后得知,tN9分子式为C2154 H3283 N623 O674S27,分子质量单位为49.6Ku,理论等电点为6.56,半衰期为30h(mammalian)、>20h(yeast)、>10h(Escherichia coli),不稳定指数43.49,为不稳定蛋白。
使用Protscale程序Kyte&Doolittle算法分析其疏水性(https://web.expasy.org/protscale/)。
构建的疏水图谱显示,多肽链第270位的Asp具有最高的分值1.389,疏水性最强,第292位的Pro具有最低的分值-2.978,亲水性最强,整体来看,该基因编码蛋白为亲水性蛋白。
使用SOPMA软件(https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html)预测二级结构。
结果表明,α螺旋(Alpha helix,h)占7.95%;β折叠(Extended strand,e)占31.82%;β转角(Beta turn,t)占7.73%;无规则卷曲(Random coil,c)占52.50%,主要以无规则卷曲、β折叠为主(详见图1)。使用PHYRE2 protein fold recognition serve(http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index)预测全长NA蛋白和NA蛋白胞外区片段三级结构。
其PDB文件用VMD(Visual Molecular Dynamics)1.9.3软件进行可视化分析。
将截短前后NA蛋白序列提交至PHYRE2服务器自动建模,构建了NA蛋白截断前后的三维结构(见图2A、图2B)。
将PDB文件用VMD软件进行处理,并标记出HIS原子位置,比对分析显示截短前后蛋白质茎部区和头部区结构无显著差异,删除胞内区和跨膜区不会影响NA蛋白的免疫原性,HIS tag可以用于后续蛋白纯化(见图2C、图2D)。
根据生物信息学分析结果,以GenScript Rare Codon Analysis工具对NA胞外区核苷酸序列(1290bp)进行基因密码子优化设计。
确认目标序列合框后由南京金斯瑞公司进行合成。密码子偏好性是影响蛋白表达的重要因素之一。
经在线稀有密码子分析工具进行密码子优化分析,结果表明tN9编码基因密码子适应指数(codon adaptation index,CAI)为0.59,GC含量为43.64%,密码子使用频率分布系数CFD为13%。
根据大肠杆菌密码子偏好性,对tN9序列稀有密码子进行同义替换后,CAI指数为0.91,达到理想值范围(0.8~1.0),GC含量为58.55%,符合理想值区间(30%~70%),有利于后续蛋白的高效表达。
使用序列处理在线工具包(SMS,http://www.bio-soft.net/sms/index.html)对优化前后的序列进行比对,稀有密码子被替换为原核生物偏爱密码子,结果见图3。
实施例2目标基因重组质粒的构建
目标基因合成时,在5’端添加Nco I酶切位点(CCATGG),在3’端删除TAA终止密码子,添加Xho I酶切位点(CTCGAG),合成后克隆至PUC57载体,得到的抗原片段SEQ ID NO:1。
用Nco I和Xho I内切酶双酶切质粒表达载体pET28b和上述抗原片段SEQ ID NO:1,经核酸电泳凝胶回收后,用T4 DNA连接酶连接,使所述抗原片段的基因插入到载体pET28b内的Nco I和Xho I位点之间,与载体上的起始密码子翻译框架一致,表达融合蛋白,融合蛋白全长包括440个氨基酸,融合蛋白的N端融合了载体上的2个氨基酸,C端包含LeuGlu和6个组氨酸His标签,表达的融合蛋白的氨基酸序列如SEQ ID NO:2。
以载体通用引物为扩增引物,以重组单菌落为模板,进行PCR鉴定,PCR反应程序见表1。
表1 PCR反应程序
PCR反应结束后,在1%的琼脂糖中进行核酸凝胶电泳,结果如图4所示。提取阳性克隆送至武汉金开瑞生物工程有限公司进行测序验证,测序正确的表达质粒命名为pET28b-tN9。
实施例3表达融合蛋白工程菌的筛选
将重组质粒pET28b-tN9分别转化E.coli BL21(DE3)、Rosetta、Arctic Express(DE3)感受态细胞,挑取重组单菌落分别于含50μg/ml卡那霉素的LB液体培养基中培养,37℃过夜培养,次日随机挑取转化菌落和含质粒pET28的对照菌,提取质粒,用限制性内切酶NcoI和XhoI对质粒双酶切,用1.0g/mL的Agarose凝胶检测双酶切产物,将含有重组质粒的阳性转化子接种至含卡那霉素50μg/mL的LB培养基内,于30℃、37℃、16℃分别振荡培养至OD值为0.5,加IPTG诱导剂至其终浓度为0.2mmol/L,继续振荡培养诱导5h,离心收集菌体,进行超声破碎,经SDS-PAGE检测,证实重组蛋白在E.coli BL21(DE3)、Rosetta(DE3)、Arctic Express(DE3)均可表达(见图5)。
(3)表达包涵体产物的处理
将E.coli BL21(DE3)诱导表达融合蛋白的工程菌离心,收获培养物,菌体重悬于洗脱液内,洗脱液的组成为0.1M PBS PH7.2~7.4、10mmol/L EDTA、0.5%Triton-X100,离心收集沉淀,以洗脱液重悬菌体2~3次,超声破菌30min,然后重新溶解在含8M尿素的buffer A缓冲液中,得到解离状态的重组NA蛋白胞外区抗原的包涵体;将所述包涵体稀释在含氯化镁、4M尿素的buffer B缓冲液中,得到第一复性液;将所述第一复性液稀释在含有Triton X-100、2M尿素的buffer C缓冲液中,4℃透析12h,得到第二复性液;将所述第二复性液稀释在含精氨酸、1M尿素的buffer D缓冲液中,4℃条件透析12h,得到第三复性液,将所述第三复性液4℃条件透析12h,得到最终复性液。具体各缓冲液配方见表2。
表2各缓冲液组成
实施例4目的蛋白的亲和纯化和质谱分析
将最终复性液利用Ni柱分离,用不同咪唑浓度的洗脱液分次洗脱目的蛋白,即得到纯化的NA蛋白胞外区抗原片段。
具体为:用镍柱亲和层析法纯化目标蛋白,并用10mMoL/L、100mMoL/L、150mMoL/L和250mMoL/L的咪唑梯度进行洗脱,收集洗脱液,进行SDS-PAGE鉴定。
将纯化后的蛋白浓缩,并以SDS-PAGE电泳检测蛋白纯度,图6A表明蛋白纯度不低于85%。
将电泳后目标条带切割,送至生工生物工程(上海)股份有限公司进行串联飞行时间质谱(Maldi-tof-tof)检测,以进一步验证目标蛋白。
混合梯度洗脱样品,以BSA标准品为对照,SDS-PAGE分析其纯度不低于85%。切胶后目标条带(见图6B),进行串联飞行时间质谱分析,经数据库搜索后,得到的蛋白质匹配得分为455,分子质量49524u,等电点(PI)6.56,匹配度21%(见图7)。
实施例5动物免疫
将验证正确的目的蛋白与等体积的弗氏完全佐剂充分混合后,以多点皮下方式免疫新西兰大白兔(400μg/只)。
免疫前耳缘静脉采血分离血清,用作阴性对照。
每2周免疫1次,之后用相同剂量的重组蛋白与弗氏不完全佐剂配伍,第四次免疫一周后采血大量制备抗血清蛋白,并准备纯化。具体免疫程序见表3;
表3免疫程序
针次 | 时间(天数) | 剂量 |
第1针 | 0 | 400μg抗原+等体积FCA |
第2针 | 14 | 400μg抗原+等体积FICA |
第3针 | 28 | 400μg抗原+等体积FICA |
第4针 | 35 | 400μg抗原+等体积FICA |
实施例6多抗纯化
取重组蛋白(抗血清蛋白)至少3mg,用PBS 4℃过夜透析,再将透析袋置于溶肽Buffer中继续透析4-6h,取出备用;
按每毫升琼脂糖凝胶Sepharose 4B(琼脂糖凝胶)偶联3mg蛋白的量,称取所需的Sepharose 4B总量,用1mM HCl溶胀15min后装入纯化柱内;
将Sepharose 4B胶取出,平均分配于2ml EP管中,瞬时离心后吸出上清液;
加入多肽溶液或透析好的蛋白溶液,混匀;用封口膜封口后固定在旋转培养器上反应,4℃过夜;
将偶联后的蛋白溶液胶装入纯化柱中;
用10-20倍胶体积的溶肽液过柱;
用Tris-HCl封闭2h;用10-20倍胶体积的Tris-HCl过柱;
用10-20倍1×PBS过柱平衡,平衡好的亲和柱于4℃保存;
将所得抗血清与PBS等量混合后缓慢上样,待抗体结合后用甘氨酸缓冲液洗脱,然后在PBS中4℃透析过夜,得到多克隆抗体,进行后续多克隆抗体的检测。
实施例7抗体纯度鉴定
纯化后抗体通过SDS-PAGE电泳,考马斯亮蓝染色观察纯化抗体的纯度在85%以上,见图8。
实施例8 Western blotting法鉴定多抗特异性
采用Western blotting方法检测抗体的特异性。
将纯化的重组蛋白、pet28a空载体诱导后产物和人用H7N9灭活病毒原液(上海株),经SDS-PAGE电泳后电转至PVDF膜,以1%的BSA于37℃封闭1h,PBST洗涤3次,每次5min;
用制备的多克隆抗体(1:3000稀释)作为一抗,室温孵育1h,PBST洗涤3次,每次5min;
加入1:1500稀释的山羊抗兔HRP-IgG,室温孵育1h,PBST洗涤3次,每次5min,DAB显色并拍照留用。
Western blotting检测结果见图9,图9泳道1和3,50KD左右位置各出现一条蛋白条带(tN9分子量49.6KD,全长NA分子量51.8KD),表明制备的抗体能与经IPTG诱导的重组蛋白和天然新型H7N9禽流感病毒(上海株)发生特异性反应,具有良好的反应原性。
实施例9间接ELISA法测定多克隆抗体的效价
将重组蛋白用包被液稀释成3μg/mL,每孔100μL,4℃包被。次日弃去包被液,洗板3次,每孔加入200μL封闭液,37℃恒温孵育1h,用于ELISA检测。
制备的抗体按1:500倍比稀释,HRP标记的山羊抗兔IgG稀释度为1:2000,显色液为TMB,用酶标仪测定各孔吸光度(A450)值。
实验设阴性血清对照,样品A450值≥阴性对照A450值的2.1倍,判为阳性。
将tN9蛋白免疫兔子制备的抗体进行1:500倍比稀释,间接ELISA法测定抗体效价,结果显示效价为1:256000(见图10)。
序列表
<110> 新乡学院
<120> 化学合成的H7N9禽流感病毒NA蛋白胞外区抗原片段及制备方法和应用
<130> PCR扩增
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1322
<212> DNA
<213> 人工序列()
<400> 1
ccatgggcca cctgaaaccg ggttgcaact gcagccacag ccagccggaa accaccaaca 60
ccagccaaac catcattaac aactactata acgagaccaa catcaccaac attcagatgg 120
aggaacgtac cagccgtaac ttcaacaacc tgaccaaagg tctgtgcacc atcaacagct 180
ggcacattta cggcaaggac aacgcggtgc gtatcggcga gagcagcgat gtgctggtta 240
cccgtgaacc gtacgttagc tgcgacccgg atgagtgccg tttttatgcg ctgagccagg 300
gtaccaccat ccgtggcaaa cacagcaacg gtaccattca cgaccgtagc caataccgtg 360
cgctgattag ctggccgctg agcagcccgc cgaccgtgta taacagccgt gttgaatgca 420
ttggctggag cagcaccagc tgccacgatg gcaagagccg tatgagcatc tgcattagcg 480
gtccgaacaa caacgcgagc gcggtggttt ggtacaaccg tcgtccggtg gcggagatca 540
acacctgggc gcgtaacatt ctgcgtaccc aggagagcga atgcgtgtgc cacaacggtg 600
tttgcccggt ggtttttacc gatggtagcg cgaccggtcc ggcggatacc cgtatctact 660
acttcaaaga gggtaaaatc ctgaagtggg aaagcctgac cggtaccgcg aaacacatcg 720
aggaatgcag ctgctacggt gaacgtaccg gcattacctg cacctgccgt gacaactggc 780
agggtagcaa ccgtccggtg atccaaattg atccggttgc gatgacccac accagccaat 840
atatctgcag cccggtgctg accgacaacc cgcgtccgaa cgatccgaac attggcaaat 900
gcaacgaccc gtacccgggt aacaacaaca acggtgttaa gggcttcagc tatctggatg 960
gcgcgaacac ctggctgggt cgtaccatca gcaccgcgag ccgtagcggc tacgaaatgc 1020
tgaaagtgcc gaacgcgctg accgacgatc gtagcaagcc gatccagggt caaaccattg 1080
ttctgaacgc ggactggagc ggttacagcg gcagcttcat ggactattgg gcggagggcg 1140
attgctaccg tgcgtgcttt tatgttgagc tgatccgtgg tcgtccgaaa gaagacaaag 1200
tgtggtggac cagcaacagc attgttagca tgtgcagcag caccgaattc ctgggccaat 1260
ggaactggcc ggatggtgcg aagatcgagt attttctgct cgagcaccac caccaccacc 1320
ac 1322
<210> 2
<211> 440
<212> PRT
<213> 人工序列()
<400> 2
Met Gly His Leu Lys Pro Gly Cys Asn Cys Ser His Ser Gln Pro Glu
1 5 10 15
Thr Thr Asn Thr Ser Gln Thr Ile Ile Asn Asn Tyr Tyr Asn Glu Thr
20 25 30
Asn Ile Thr Asn Ile Gln Met Glu Glu Arg Thr Ser Arg Asn Phe Asn
35 40 45
Asn Leu Thr Lys Gly Leu Cys Thr Ile Asn Ser Trp His Ile Tyr Gly
50 55 60
Lys Asp Asn Ala Val Arg Ile Gly Glu Ser Ser Asp Val Leu Val Thr
65 70 75 80
Arg Glu Pro Tyr Val Ser Cys Asp Pro Asp Glu Cys Arg Phe Tyr Ala
85 90 95
Leu Ser Gln Gly Thr Thr Ile Arg Gly Lys His Ser Asn Gly Thr Ile
100 105 110
His Asp Arg Ser Gln Tyr Arg Ala Leu Ile Ser Trp Pro Leu Ser Ser
115 120 125
Pro Pro Thr Val Tyr Asn Ser Arg Val Glu Cys Ile Gly Trp Ser Ser
130 135 140
Thr Ser Cys His Asp Gly Lys Ser Arg Met Ser Ile Cys Ile Ser Gly
145 150 155 160
Pro Asn Asn Asn Ala Ser Ala Val Val Trp Tyr Asn Arg Arg Pro Val
165 170 175
Ala Glu Ile Asn Thr Trp Ala Arg Asn Ile Leu Arg Thr Gln Glu Ser
180 185 190
Glu Cys Val Cys His Asn Gly Val Cys Pro Val Val Phe Thr Asp Gly
195 200 205
Ser Ala Thr Gly Pro Ala Asp Thr Arg Ile Tyr Tyr Phe Lys Glu Gly
210 215 220
Lys Ile Leu Lys Trp Glu Ser Leu Thr Gly Thr Ala Lys His Ile Glu
225 230 235 240
Glu Cys Ser Cys Tyr Gly Glu Arg Thr Gly Ile Thr Cys Thr Cys Arg
245 250 255
Asp Asn Trp Gln Gly Ser Asn Arg Pro Val Ile Gln Ile Asp Pro Val
260 265 270
Ala Met Thr His Thr Ser Gln Tyr Ile Cys Ser Pro Val Leu Thr Asp
275 280 285
Asn Pro Arg Pro Asn Asp Pro Asn Ile Gly Lys Cys Asn Asp Pro Tyr
290 295 300
Pro Gly Asn Asn Asn Asn Gly Val Lys Gly Phe Ser Tyr Leu Asp Gly
305 310 315 320
Ala Asn Thr Trp Leu Gly Arg Thr Ile Ser Thr Ala Ser Arg Ser Gly
325 330 335
Tyr Glu Met Leu Lys Val Pro Asn Ala Leu Thr Asp Asp Arg Ser Lys
340 345 350
Pro Ile Gln Gly Gln Thr Ile Val Leu Asn Ala Asp Trp Ser Gly Tyr
355 360 365
Ser Gly Ser Phe Met Asp Tyr Trp Ala Glu Gly Asp Cys Tyr Arg Ala
370 375 380
Cys Phe Tyr Val Glu Leu Ile Arg Gly Arg Pro Lys Glu Asp Lys Val
385 390 395 400
Trp Trp Thr Ser Asn Ser Ile Val Ser Met Cys Ser Ser Thr Glu Phe
405 410 415
Leu Gly Gln Trp Asn Trp Pro Asp Gly Ala Lys Ile Glu Tyr Phe Leu
420 425 430
Leu Glu His His His His His His
435 440
Claims (7)
1.一种化学合成的编码H7N9禽流感病毒NA蛋白胞外区的抗原片段的核苷酸序列,其特征在于,该抗原片段的核苷酸序列全长为1322bp,所述抗原片段的核苷酸序列SEQ ID NO:1如下:
5’CC ATG GGC CAC CTG AAA CCG GGT TGC AAC TGC AGC CAC AGC CAG CCG GAA ACCACC AAC ACC AGC CAA ACC ATC ATT AAC AAC TAC TAT AAC GAG ACC AAC ATC ACC AACATT CAG ATG GAG GAA CGT ACC AGC CGT AAC TTC AAC AAC CTG ACC AAA GGT CTG TGCACC ATC AAC AGC TGG CAC ATT TAC GGC AAG GAC AAC GCG GTG CGT ATC GGC GAG AGCAGC GAT GTG CTG GTT ACC CGT GAA CCG TAC GTT AGC TGC GAC CCG GAT GAG TGC CGTTTT TAT GCG CTG AGC CAG GGT ACC ACC ATC CGT GGC AAA CAC AGC AAC GGT ACC ATTCAC GAC CGT AGC CAA TAC CGT GCG CTG ATT AGC TGG CCG CTG AGC AGC CCG CCG ACCGTG TAT AAC AGC CGT GTT GAA TGC ATT GGC TGG AGC AGC ACC AGC TGC CAC GAT GGCAAG AGC CGT ATG AGC ATC TGC ATT AGC GGT CCG AAC AAC AAC GCG AGC GCG GTG GTTTGG TAC AAC CGT CGT CCG GTG GCG GAG ATC AAC ACC TGG GCG CGT AAC ATT CTG CGTACC CAG GAG AGC GAA TGC GTG TGC CAC AAC GGT GTT TGC CCG GTG GTT TTT ACC GATGGT AGC GCG ACC GGT CCG GCG GAT ACC CGT ATC TAC TAC TTC AAA GAG GGT AAA ATCCTG AAG TGG GAA AGC CTG ACC GGT ACC GCG AAA CAC ATC GAG GAA TGC AGC TGC TACGGT GAA CGT ACC GGC ATT ACC TGC ACC TGC CGT GAC AAC TGG CAG GGT AGC AAC CGTCCG GTG ATC CAA ATT GAT CCG GTT GCG ATG ACC CAC ACC AGC CAA TAT ATC TGC AGCCCG GTG CTG ACC GAC AAC CCG CGT CCG AAC GAT CCG AAC ATT GGC AAA TGC AAC GACCCG TAC CCG GGT AAC AAC AAC AAC GGT GTT AAG GGC TTC AGC TAT CTG GAT GGC GCGAAC ACC TGG CTG GGT CGT ACC ATC AGC ACC GCG AGC CGT AGC GGC TAC GAA ATG CTGAAA GTG CCG AAC GCG CTG ACC GAC GATCGT AGC AAG CCG ATC CAG GGT CAA ACC ATTGTTCTG AAC GCG GAC TGG AGC GGT TAC AGCGGC AGC TTC ATG GAC TAT TGG GCG GAG GGCGAT TGC TAC CGT GCG TGC TTT TAT GTT GAG CTG ATC CGT GGT CGT CCG AAA GAA GACAAA GTG TGG TGG ACC AGC AAC AGC ATT GTT AGC ATG TGC AGC AGC ACCGAA TTC CTGGGC CAA TGG AAC TGG CCG GAT GGT GCG AAG ATC GAG TAT TTT CTG CTC GAG CAC CACCAC CAC CACCAC 3’。
2.一种制备权利要求1所述的编码H7N9禽流感病毒NA蛋白胞外区的抗原片段的核苷酸序列的方法,其特征在于,包括H7N9禽流感病毒NA蛋白胞外区基因片段重组质粒的构建、表达融合蛋白工程菌的筛选、表达产物的处理,具体步骤如下:
(1)H7N9禽流感病毒NA蛋白胞外区基因片段重组质粒的构建:
用Nco I和Xho I内切酶双酶切质粒表达载体pET28b和权利要求1所述的抗原片段,经核酸电泳凝胶回收后,用T4 DNA连接酶连接,使所述的抗原片段的基因插入到载体pET28b内的NocI和XhoI位点之间,与载体上的起始密码子翻译框架一致,表达融合蛋白,融合蛋白全长包括440个氨基酸,融合蛋白的N端融合了载体上的2个氨基酸,C端包含Leu Glu和6个组氨酸His标签,表达的融合蛋白的氨基酸序列SEQ ID NO:2如下:
Met Gly His Leu Lys Pro Gly Cys Asn Cys Ser His Ser Gln Pro Glu
Thr Thr Asn Thr Ser Gln ThrIle Ile Asn Asn Tyr Tyr Asn Glu Thr
Asn Ile Thr Asn Ile Gln Met Glu Glu Arg Thr Ser Arg Asn Phe Asn
Asn Leu Thr Lys Gly Leu Cys Thr Ile Asn Ser Trp His Ile Tyr Gly
Lys Asp Asn Ala Val Arg Ile Gly Glu Ser Ser Asp Val Leu Val Thr
Arg Glu Pro Tyr Val Ser Cys Asp Pro Asp Glu Cys Arg Phe Tyr Ala
Leu Ser Gln Gly Thr ThrIle Arg Gly Lys His Ser Asn Gly ThrIle
His Asp Arg Ser Gln Tyr Arg Ala Leu Ile Ser Trp Pro Leu Ser Ser
Pro Pro Thr Val Tyr Asn Ser Arg Val Glu Cys Ile Gly Trp Ser Ser
Thr Ser Cys His Asp Gly Lys Ser Arg Met Ser Ile Cys Ile Ser Gly
Pro Asn Asn Asn Ala Ser Ala Val Val Trp Tyr Asn Arg Arg Pro Val
Ala Glu Ile Asn Thr Trp Ala Arg Asn Ile Leu Arg Thr Gln Glu Ser
Glu Cys Val Cys His Asn Gly Val Cys Pro Val Val Phe Thr Asp Gly
Ser Ala Thr Gly Pro Ala Asp Thr Arg Ile Tyr Tyr Phe Lys Glu Gly
Lys Ile Leu Lys Trp Glu Ser Leu Thr Gly Thr Ala Lys His Ile Glu
Glu Cys Ser Cys Tyr Gly Glu Arg Thr Gly Ile Thr Cys Thr Cys Arg
Asp Asn Trp Gln Gly Ser Asn Arg Pro Val Ile Gln Ile Asp Pro Val
Ala Met Thr His Thr Ser Gln Tyr Ile Cys Ser Pro Val Leu Thr Asp
Asn Pro Arg Pro Asn Asp Pro Asn Ile Gly Lys Cys Asn Asp Pro Tyr
Pro Gly Asn Asn Asn Asn Gly Val Lys Gly Phe Ser Tyr Leu Asp Gly
Ala Asn Thr Trp Leu Gly Arg Thr Ile Ser Thr Ala Ser Arg Ser Gly
Tyr Glu Met Leu Lys Val Pro Asn Ala Leu Thr Asp Asp Arg Ser Lys
Pro Ile Gln Gly Gln ThrIle Val Leu Asn Ala Asp Trp Ser Gly Tyr
Ser Gly Ser Phe Met Asp Tyr Trp Ala Glu Gly Asp Cys Tyr Arg Ala
Cys Phe Tyr Val Glu Leu Ile Arg Gly Arg Pro Lys Glu Asp Lys Val
Trp Trp Thr Ser Asn SerIle Val Ser Met Cys Ser Ser Thr Glu Phe
Leu Gly Gln Trp Asn Trp Pro Asp Gly Ala Lys Ile Glu Tyr Phe Leu
Leu Glu His His His His His His;
(2)表达融合蛋白工程菌的筛选鉴定:
将重组质粒pET28b-tN9分别转化E.coli BL21(DE3)、Rosetta、Arctic Express(DE3)感受态细胞,挑取重组单菌落分别于含50μg/ml卡那霉素的LB液体培养基中培养,37℃过夜培养,次日随机挑取转化菌落和含质粒pET28的对照菌,提取质粒,用限制性内切酶NcoI和XhoI对质粒双酶切,用1.0g/mL的Agarose凝胶检测双酶切产物,将含有重组质粒的阳性转化子接种至含卡那霉素50μg/mL的LB培养基内,于30℃、37℃、16℃分别振荡培养至OD值为0.5,加IPTG诱导剂至其终浓度为0.2mmol/L,继续振荡培养诱导5h,离心收集菌体,进行超声破碎,经SDS-PAGE检测,证实重组蛋白在E.coli BL21(DE3)、Rosetta(DE3)、ArcticExpress(DE3)均可表达;将表达产物进行质谱分析,以确认表达产物为目标产物;
(3)表达包涵体产物的处理:
将E.coli BL21(DE3)诱导表达融合蛋白的工程菌离心,收获培养物,菌体重悬于洗脱液内,洗脱液的组成为0.1M PBS PH7.2~7.4、10mmol/L EDTA、0.5%Triton-X100,离心收集沉淀,以洗脱液重悬菌体2~3次,超声破菌30min,然后重新溶解在含8M尿素的buffer A缓冲液中,得到解离状态的重组NA蛋白胞外区抗原的包涵体;将所述包涵体稀释在含氯化镁、4M尿素的buffer B缓冲液中,得到第一复性液;将所述第一复性液稀释在含有TritonX-100、2M尿素的buffer C缓冲液中,4℃透析12h,得到第二复性液;将所述第二复性液稀释在含精氨酸、1M尿素的buffer D缓冲液中,4℃条件透析12h,得到第三复性液,将所述第三复性液4℃条件透析12h,得到最终复性液;
将所述最终复性液利用Ni柱分离,用不同咪唑浓度的洗脱液分次洗脱目的蛋白,即得到纯化的NA蛋白胞外区抗原片段。
3.根据权利要求2所述的H7N9禽流感病毒NA蛋白胞外区抗原片段的核苷酸序列的方法,其特征在于,所述不同咪唑浓度的洗脱液分次洗脱目的蛋白时洗脱液的浓度分别为10mmol/L、100mmol/L、150mmol/L和250mmol/L。
4.一种权利要求1所述的H7N9禽流感病毒NA蛋白胞外区抗原片段的核苷酸序列在以非诊断目的的H7N9禽流感病毒血清学检测中的应用。
5.一种权利要求1所述的H7N9禽流感病毒NA蛋白胞外区抗原片段的核苷酸序列在对人和动物的感染机制研究以及疫苗制备中的应用。
6.一种权利要求1所述的H7N9禽流感病毒NA蛋白胞外区抗原片段的核苷酸序列在以非诊断目的的H7N9禽流感病毒NA抗体的检测及单克隆抗体的制备中的应用。
7.一种权利要求1所述的H7N9禽流感病毒NA蛋白胞外区抗原片段的核苷酸序列在制备H7N9禽流感病毒NA检测试剂盒中的应用。
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