EV71病毒抗原蛋白VP1基因重组载体疫苗研究

发布时间：2018-10-12 09:29

【摘要】：肠道病毒7l型(EV71)是导致手足口病的主要病原体之一,常引起多种与神经系统相关的严重疾病。vp1是EV71病毒主要的抗原基因,其编码的外衣壳蛋白在感染宿主细胞时起重要作用。因此,我们选择vp1作为研制EV71疫苗的目标基因。LTB有很强的免疫原性和佐剂活性且无毒。本研究中选用LTB作为分子内粘膜免疫佐剂。双歧杆菌正常存在于人类肠道内,是一种重要的有益的生菌,对人体肠道微环境有重要的调节功能,对婴幼儿肠道有独特的保护作用。双歧杆菌是人类肠道的自然宿主且可以粘附于肠道上皮细胞。因此,本研究的目的是将双歧杆菌发展成一个表达EV71 VP1蛋白和ETEC LTB蛋白的双价口服活疫苗的抗原表达系统。目的构建携带EV71病毒vp1基因和ltb基因的融合表达载体并在大肠杆菌和双歧杆菌中进行表达,鉴定其抗原性和免疫原性。为制备预防EV71感染的口服疫苗奠定基础。方法用重叠延伸PCR(overlap extension PCR)法获得人肠道病毒71型vp1片段与大肠杆菌不耐热肠毒素B亚单位(ltb)的融合基因。设计14对引物通过重叠延伸PCR技术合成vp1基因,以ETEC(44815)质粒DNA为模板,PCR扩增ltb基因,将vp1基因与ltb基因连接并测序,连接产物插入原核表达质粒pBEX,构建重组质粒pBEX-VP1-LTB,转化E.coli B1221(DE3)进行表达,SDS-PAGE及Western blotting分析其表达。将表达VP1-LTB的大肠杆菌超声破碎后离心提取包涵体制备抗原,经口灌喂免疫小鼠,检测小鼠血清中抗VP1的IgG、IgA,粪便sIgA和肠粘液sIgA,鉴定其免疫原性。电转化法将重组质粒pBEX-VP1-LTB转入双歧杆菌,Western-blot分析其抗原性。口服免疫SD大鼠,采集血清和粪便样品,ELISA检测机体特异的血清IgG和肠道IgA抗体。结果合成的vp1基因全长891bp,测序结果与预期相符,重组表达质粒pBEX-VP1-LTB经PCR及双酶切鉴定,表明构建正确;目的蛋白在E.coli BL21(DE3)中获得了表达,Western-blot分析结果表明该蛋白具有与EV71 VP1抗体结合的抗原性,免疫后小鼠血清抗VP1的IgG、IgA以及粪便sIgA和肠粘液sIgA明显高于PBS和GST对照组。电转法成功将重组质粒转入双歧杆菌, Western-blot分析结果表明该蛋白具有与EV71病毒抗体结合的抗原性。ELISA检测表明重组双歧杆菌免疫后的SD大鼠体内产生了特性的IgG和IgA抗体。结论应用重叠延伸PCR技术成功构建了EV71 VP1-LTB融合表达质粒,并在E.coli BL21(DE3)和双歧杆菌中获得有生物学功能的VP1表达产物,该蛋白具有良好的抗原性和免疫原性,为研制EV71分子内佐剂疫苗打下了基础。该研究表明重组载体pBEX-VP1-LTB可以在双歧杆菌中表达。该重组VP1-LTB基因工程双歧杆菌可诱发SD大鼠特异的粘膜免疫反应,为进一步研制基于双歧杆菌表达系统的手足口病EV71重组亚单位口服活疫苗奠定了基础。
[Abstract]:Enterovirus 7l (EV71) is one of the major pathogens leading to HFMD, which often causes many serious diseases related to the nervous system. Vp1 is the main antigen gene of EV71 virus, and its coat shell protein plays an important role in the infection of host cells. Therefore, we selected vp1 as the target gene for the development of EV71 vaccine. LTB has strong immunogenicity, adjuvant activity and non-toxicity. In this study, LTB was selected as intramolecular mucosal immune adjuvant. Bifidobacterium is an important and beneficial bifidobacterium that exists in human intestinal tract and has important regulation function to human intestinal microenvironment and unique protective effect to infant intestinal tract. Bifidobacterium is the natural host of the human gut and adheres to intestinal epithelial cells. Therefore, the aim of this study was to develop a bivalent oral live vaccine expressing EV71 VP1 protein and ETEC LTB protein into an antigen expression system of Bifidobacterium. Objective to construct a fusion expression vector carrying vp1 gene and ltb gene of EV71 virus and express them in Escherichia coli and Bifidobacterium to identify their antigenicity and immunogenicity. It lays the foundation for the preparation of oral vaccine to prevent EV71 infection. Methods the fusion gene of human enterovirus 71 vp1 fragment and Escherichia coli heat-labile enterotoxin B subunit (ltb) was obtained by overlapping extension PCR (overlap extension PCR) method. A total of 14 pairs of primers were designed to synthesize vp1 gene by overlapping extension PCR technique. The ltb gene was amplified by PCR using ETEC (44815) plasmid DNA as template. Vp1 gene was linked to ltb gene and sequenced. The recombinant plasmid pBEX-VP1-LTB, was transformed into E.coli B1221 (DE3) and expressed by SDS-PAGE and Western blotting. The inclusion bodies of E. coli expressing VP1-LTB were extracted by centrifugation after ultrasonic crushing. The mice were immunized by oral administration. The immunogenicity of IgG,IgA, stool sIgA and intestinal mucus sIgA, against VP1 in the serum of mice was detected. The recombinant plasmid pBEX-VP1-LTB was transformed into Bifidobacterium by electroporation and its antigenicity was analyzed by Western-blot. SD rats were immunized orally. Serum and fecal samples were collected. Serum IgG and intestinal IgA antibody were detected by ELISA. Results the total length of vp1 gene was 891bp.The result of sequencing was consistent with the expectation. The recombinant expression plasmid pBEX-VP1-LTB was identified by PCR and double enzyme digestion, which showed that the construction was correct. Objective the protein was expressed in E.coli BL21 (DE3). The results of Western-blot analysis showed that the protein was antigenicity to EV71 VP1 antibody. The serum anti-VP1 IgG,IgA, stool sIgA and intestinal mucus sIgA were significantly higher in immunized mice than those in PBS and GST control groups. The recombinant plasmid was successfully transferred into Bifidobacterium by electroporation. Western-blot analysis showed that the protein had the antigenicity of binding to EV71 virus antibody. ELISA detection showed that the SD rats immunized with recombinant bifidobacterium produced characteristic IgG and IgA antibodies. Conclusion the fusion expression plasmid of EV71 VP1-LTB was successfully constructed by using overlapping extension PCR technique, and the bifidobacterium (E.coli BL21) and bifidobacterium were used to obtain the bifidobacterium VP1 expression product. The protein has good antigenicity and immunogenicity. It lays a foundation for the development of EV71 intramolecular adjuvant vaccine. This study showed that the recombinant vector pBEX-VP1-LTB could be expressed in Bifidobacterium. The recombinant VP1-LTB gene engineering bifidobacterium can induce specific mucosal immune response in SD rats, which lays a foundation for the further development of oral live vaccine for EV71 recombinant subunit of HFMD based on Bifidobacterium expression system.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

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