CN112175928B - 一种源于沙门氏菌噬菌体基因编码的蛋白在作为革兰氏阴性菌裂解酶中的应用 - Google Patents
一种源于沙门氏菌噬菌体基因编码的蛋白在作为革兰氏阴性菌裂解酶中的应用 Download PDFInfo
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Abstract
本发明属于生物技术领域,具体涉及一种源于沙门氏菌噬菌体基因编码的蛋白在作为裂解酶中的应用,所述的蛋白的氨基酸序列为SEQ ID NO.2所示,编码所述蛋白的基因的核苷酸序列为SEQ ID NO.1所示。本发明首次发现SEQ ID NO.2所示的蛋白具备裂解酶的作用,且能自内裂解大肠杆菌等革兰氏阴性细菌,该裂解酶的裂解效应高,对多种不同的大肠杆菌以及沙门氏菌均具备裂解效果。
Description
技术领域
本发明属于生物技术领域,具体涉及一种源于沙门氏菌噬菌体基因编码的蛋白在作为裂解酶中的应用。
背景技术
噬菌体编码产生的裂解酶在噬菌体侵染细菌的后期产生,裂解酶通过作用于细菌细胞壁的肽聚糖层达到裂菌的效果。目前,国际上对于阳性菌裂解酶的研究较多,由于其不存在细胞壁外膜,可以通过将外源加入的裂解酶直接作用于细菌,通过裂解细菌细胞壁达到裂菌、杀菌的效果。然而对于革兰氏阴性菌裂解酶,其存在细胞壁外膜,对于外源添加的裂解酶具有高度抗性,目前研究中多使用透膜剂与裂解酶联用,研究发现存在少部分裂解酶具有独立裂解阴性菌的能力,这被推测与裂解酶自身的带电荷性质有关。大多数噬菌体裂解酶具有双结构域的特点,即N端的催化结构域与C端的结合结构域,结合结构域负责与宿主细菌细胞壁上的特异性位点相结合,催化结构域则能够特异性的切断肽聚糖上的化学键,从而达到裂解细菌的效果。此外,还有部分具有其它结构特点的裂解酶,比如部分革兰氏阴性菌裂解酶的催化域处于C端,结合域位于N端;一些裂解酶还具有多个结合结构域或不同催化结构域串联的结构。
随着测序技术、大规模病毒宏基因组学和培养技术的发展,数据库中完整的噬菌体基因组序列越来越多,截至2019年9月,其基因组量超过8000,继续研究新噬菌体基因组有利于我们进一步理解噬菌体基因组的多样性及噬菌体与细菌之间的相互作用机制。噬菌体中还有许多功能蛋白需要挖掘,可以通过ORF预测、功能预测、毒力因子或耐药因子预测,为噬菌体后续在食品、医学及农业等领域的安全应用提供科学依据,此外,可以在其功能基因组学分析的基础上对噬菌体蛋白的开发、噬菌体的进化及裂解机制等进行研究。然而,目前通过软件对裂解酶功能进行预测仍有较高的不准确性,如对于预测具有裂解功能的裂解酶,经实验验证具有裂解活菌能力的仅占5%左右。
噬菌体裂解酶因其高效的杀菌性、种属特异性和安全性,使其在医疗、农业、食品安全等领域具有广阔的应用前景,目前,革兰氏阳性菌裂解酶已被应用于食品安全领域,成功消除食源性病原菌。然而对于革兰氏阴性菌裂解酶,由于其外膜的制约,导致其相关的应用实例较少。裂解酶通过自内裂解革兰氏阴性菌形成的菌蜕细胞,可用于菌蜕疫苗或活性物质递送载体的研究,然而并非所有裂解酶都具有良好的自内裂解效果,因此部分裂解酶不具备制作革兰氏阴性菌菌蜕细胞的能力。
菌蜕是来自革兰氏阴性细菌的细胞外膜,其不含所有的细胞质成分,但具有完整的细胞形态,包括所有的细胞表面结构。因此菌蜕可用作疫苗、药物或活性物质的递送载体等。与传统的疫苗相比,菌蜕作为疫苗具有靶向性、固有佐剂特性和裂解过程不产生变性从而保留所有抗原决定簇等优势。目前,菌蜕细胞已经在大肠杆菌、金黄色葡萄球菌、铜绿假单胞菌、肺炎克雷伯氏菌等多种菌株中构建成功。
发明内容
一种源于沙门氏菌噬菌体基因编码的蛋白在作为裂解酶中的应用,所述的蛋白序列为SEQ ID NO.2所示,编码其核苷酸序列为SEQ ID NO.1所示。
为了达到上述目的,本发明采取以下技术措施:
一种源于沙门氏菌噬菌体基因编码的蛋白在作为裂解酶中的应用,所述的蛋白序列为SEQ ID NO.2所示,所述的应用包括利用该蛋白直接制备成裂解酶,该裂解酶可直接通过添加诱导剂裂解细胞,无需添加任何其他材料,可用于蛋白表达中的细胞破碎;本发明的应用还包括利用上述编码该蛋白的基因制备成菌蜕(致病性大肠杆菌及沙门氏菌),制备的菌蜕细胞用作疫苗制备的抗原物质,还可作为药物、异源抗原、核酸等的递送载体,其由于缺乏内含物而更加安全。
与现有技术相比,本发明具有以下优点:
1.本发明首次发现SEQ ID NO.2所示的蛋白具备裂解酶的作用,且能自内裂解大肠杆菌与沙门氏菌等革兰氏阴性细菌;
2.本发明提供的裂解酶的裂解效应高,对多种不同的革兰氏阴性菌均具备裂解效果。
附图说明
图1为插入基因ORF-40的PCR扩增产物。
图2为重组质粒转化子的菌液PCR验证;
其中,A为大肠杆菌BL21(DE3)-pBAD 24-ORF 40菌液PCR验证;B为大肠杆菌BL21(AI)-pBAD 24-ORF 40菌液PCR验证;C为大肠杆菌BL21(DE3)pLysS-pBAD 24-ORF 40菌液PCR验证;M:Marker Marker DL2000;泳道1-3:同一种菌挑取了3个转化子做验证。图3为自诱导工程型大肠杆菌裂解曲线;
其中,A为E.coli trans5α-pBAD 24-ORF 40的裂解曲线、B为E.coli BL21(DE3)-pBAD24-ORF 40的裂解曲线、C为E.coli BL21(AI)-pBAD 24-ORF 40的裂解曲线、D为E.coliBL21(DE3)plySs-pBAD 24-ORF 40的裂解曲线。
图4为氯霉素抗性重组载体构建和转化子的菌液PCR验证。
图5为含有重组溶菌质粒pBAD 24-ORF 40的沙门氏菌和大肠杆菌的裂解曲线。
具体实施方式
下列实施例旨在进一步举例说明,而不是限制本发明。本领域技术人员可以理解到,在不背离本发明的精神和原则的前提下,对本发明的任何平行改变和改动都将落入本发明的保护范围内,本发明所述的技术方案,如未特别说明,均为本领域的常规方案;所述试剂或材料,如未特别说明,均来源于商业渠道。
实施例1:
构建工程菌株及自裂解曲线的测定
1.裂解酶基因克隆及pBAD24-ORF 40载体构建
以ORF 40(SEQ ID NO.1所示,对应的氨基酸序列为SEQ ID NO.2所示)的碱基序列为模板设计插入基因的上下游引物ORF 40-F和ORF 40-R,以质粒pBAD 24的序列为模板设计质粒线性化的上下游引物pBAD24-F和pBAD24-R,插入基因的引物ORF 40-F和ORF 40-R与质粒pBAD 24的引物pBAD24-F和pBAD24-R具有同源序列,由上海生工公司合成。
orf 40-f:ttgggctagcaggaggaattcaccatgaagtcgcgtcgaa ga aga
orf 40-r:caaaacagccaagcttgcacgttattattgcaccggtttt gaca
pbad24-f:taacgtgcaagcttggctgttttg
pbad24-r:ggtgaattcctcctgctagcccaa
PCR扩增目的基因ORF40的程序如下:以1μL基因为模板,上下游引物ORF 40-F和ORF 40-R各加入100ng进行PCR扩增,PCR条件为:(1)94℃预变性10min;(2)94℃变性30sec,55℃退火30sec,72℃延伸30sec,30个循环;(3)72℃延伸5min。
载体pBAD24的线性化PCR扩增程序如下:以1μL基因为模板,上下游引物pBAD24-F和pBAD24-R各加入100ng进行PCR扩增,PCR条件为:(1)98℃预变性30sec;(2)98℃变性10sec,60℃退火30sec,72℃延伸2min 30sec,25个循环;(3)72℃延伸5min。
利用同源重组原理构建重组质粒pBAD24-ORF 40:将1μL插入基因的PCR产物和1μL质粒线性化的PCR产物转入到50μL感受态E.coli trans5α中,以ORF 40-F与ORF40-R为引物对转化子进行PCR验证,可扩增得到全长的ORF 40基因,如图1所示,图中M为标准分子量DL2000,1~5为扩增条带,与目标大小576bp一致。
2.构建大肠工程菌株自诱导裂解系统
将步骤1中构建好的重组质粒分别转化到E.coli trans5α、E.coli BL21(DE3)、E.coli BL21(DE3)pLysS和E.coli BL21(AI)中,挑取正确转化子并进行验证,验证结果如图2所示,将验证正确的E.coli trans5α-pBAD 24-ORF 40、E.coli BL21(DE3)-pBAD 24-ORF 40、E.coli BL21(AI)-pBAD 24-ORF 40和E.coli BL21(DE3)pLysS-pBAD 24-ORF 40的单克隆接种于5mL对应抗性的LB培养基中,于37℃、180rpm培养8h,取100μL菌液转接到10mL对应抗性的LB培养基中,于37℃、180rpm培养至OD600=0.6,向E.coli trans5α-pBAD 24-ORF 40、E.coli BL21(DE3)-pBAD 24-ORF 40和E.coli BL21(DE3)pLysS-pBAD 24-ORF 40的菌液中分别加入1mol/L IPTG诱导剂,向E.coli BL21(AI)-pBAD 24-ORF 40的菌液中加入1mol/L IPTG诱导剂和20%阿拉伯糖,分别于37℃、180rpm进行诱导表达,未诱导的菌株作为对照,测量其菌液的OD600值,绘制裂解曲线(图3)。
如图3所示,与对照相比,裂解酶基因(ORF 40)在工程大肠杆菌trans5α、BL21(DE3)、BL21(DE3)plysS、BL21(AI)中表达时,其OD值会下降,证明裂解酶的自内裂解的能力有利于工程大肠杆菌的裂解,其有利于细菌内容物的释放,即重组蛋白的释放。
因此,可构建含有ORF 40基因的重组质粒,将该重组质粒和表达异源蛋白的重组质粒都转入大肠杆菌中(二者诱导条件不同),先对于异源蛋白进行诱导表达,然后加入IPTG和阿拉伯糖对自内裂解酶进行诱导表达,根据SAR-endolysin(自内裂解酶)的裂解能力,可以在异源蛋白表达体系中替代物理破碎方法,温和裂解表达菌株;此外,还可与物理方法联合使用,从而避免传统物理破碎方法带来的弊端(如传统物理破碎法强度较高,可能导致细胞过度破碎,这不利于可溶性蛋白的提取)。
根据结果显示,对于不同的工程菌株,裂解酶的裂解效果不一,大肠杆菌trans5α、BL21(AI),BL21(DE3)、pLysSBL21(AI)分别在8h、8h、3.5h、3.5h菌液OD600降至最低,即该时刻裂解效率最大。
实施例2:
构建大肠杆菌菌蜕细胞
1.氯霉素抗性pBAD 24-ORF 40重组质粒的鉴定
制作大肠杆菌X7213感受态细胞,将已构建的重组质粒pBAD24-ORF 40转化至X7213感受态细胞中。
以ORF 40-F和ORF 40-R为引物对转化子进行PCR验证,取PCR产物进行凝胶核酸电泳,显示条带与目标条带大小一致,如图4中A所示。
2.接合转移至鼠伤寒沙门氏菌和致病性大肠杆菌
以鼠伤寒沙门氏菌ATCC 13311和大肠杆菌ATCC 700728为受体菌,携带有氯霉素抗性的重组质粒pBAD 24-ORF 40的大肠杆菌X7213为供体菌,通过接合转移构建菌蜕菌株。在转化平板上挑取转化子接种培养,使用菌液为模板,ORF 40-F和ORF 40-R为引物进行PCR验证,验证结果如图4中B,4中C所示,结果显示,pBAD 24-ORF 40重组质粒已成功转入鼠伤寒沙门氏菌ATCC 13311和大肠杆菌ATCC 700728中,获得重组菌株鼠伤寒沙门氏菌ATCC13311-pBAD 24-ORF 40和大肠杆菌ATCC 700728-pBAD 24-ORF 40。
3.菌蜕细胞裂解曲线的绘制
挑取验证正确的鼠伤寒沙门氏菌ATCC 13311-pBAD 24-ORF 40和大肠杆菌ATCC700728-pBAD 24-ORF 40单克隆接种于5mL LB+Cm培养基中,于37℃、180rpm培养8h,按1:100的比例转接至10mL LB+Cm培养基中,37℃、180rpm培养至OD600为0.6左右,分别加入终浓度为0.2%的阿拉伯糖诱导剂,于37℃、180rpm进行诱导表达,未诱导的菌株作为对照,测量其菌液的OD600值,每30min取样绘制裂解曲线,裂解后需要进行离心获得菌蜕细胞,6000rpm离心15min收集裂解完成的菌体,加入PBS清洗3次后,冻干,储存于-80℃。
裂解结果如图5所示,具体数据如下:
本发明制得的菌蜕细胞的作用与其他菌蜕细胞相似,本实施例中制得的细菌菌蜕可用作高效的DNA疫苗载体,在人类医药中,基因治疗应用主要集中于DNA传递到靶细胞引起强烈有效的免疫反应,这在疫苗和肿瘤治疗新策略的研究中至关重要。带负电荷的DNA可通过菌蜕表面的跨膜孔道结合于带正电荷的细菌内膜上,且细菌菌蜕表面具备完善的配体,与细胞表面的受体结合,可靶向性的将DNA疫苗导入受体细胞中。
当其用于DNA疫苗载体时,可参考Wen J,Yang Y,Zhao G,et al.Salmonellatyphi Ty21a bacterial ghost vector augments HIV-1gp140 DNA vaccine-inducedperipheral and mucosal antibody responses via TLR4 pathway[J].Vaccine,2012,30(39):5733-5739.,具体如下:
用装有HIVgp140 DNA疫苗的Ty21a沙门氏菌菌蜕转染鼠巨噬细胞RAW264.7,发现菌蜕能够被有效的摄取。与裸露DNA疫苗免疫鼠相比,用装有HIVgp140 DNA疫苗的沙门氏菌菌蜕免疫小鼠后能够引起强烈的全身和局部抗gp140抗体的反应。
将DNA载入菌蜕中
通过在含有HIVgp140 DNA的缓冲液中(100mM NaCl,10mM sodium acetate,10mMHEPES,pH 7)悬浮沙门氏菌细菌菌蜕,将DNA转入沙门氏菌菌蜕细胞中(在使用细菌菌蜕装载荧光蛋白表达质粒时,结果显示转染率达50%以上)。
将菌蜕接种到巨噬细胞
将含有DNA的沙门氏菌菌蜕接种到巨噬细胞中,菌蜕与巨噬细胞比例为1:10~1:100时巨噬细胞生长最好,在接入后的12h、24h、36h、48h收集上清液用于细胞因子测量。
小鼠免疫实验在小鼠皮下接种100μg沙门氏菌菌蜕DNA疫苗、20μg裸DNA或PBS,每隔两周接种相同的疫苗,在基线时间和最终免疫后10天收集小鼠血清,储存在-20℃,通过ELISA法分别测量抗体数量。
序列表
<110> 华中农业大学
<120> 一种源于沙门氏菌噬菌体基因编码的蛋白在作为革兰氏阴性菌裂解酶中的应用
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gatggttcac ctaccaaaat atatactgct gctcgtaaac aaaactggac tgaaatgtgt 480
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aaggaaaaag cgctatgtct aaaaccggtg caataa 576
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Claims (2)
1.一种源于沙门氏菌噬菌体基因编码的蛋白在自内裂解大肠杆菌(Escherichia coli)或肠炎沙门氏菌肠炎亚种鼠伤寒血清型(Salmonella enterica subsp. enterica serovar Typhimurium)中的应用,所述的蛋白的氨基酸序列为SEQ ID NO.2所示。
2.根据权利要求1所述的应用,所述的蛋白在制备大肠杆菌(Escherichia coli)或肠炎沙门氏菌肠炎亚种鼠伤寒血清型(Salmonella enterica subsp. enterica serovar Typhimurium)菌蜕细胞中的应用。
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