【作者】 李江；

【导师】 马永平；

【作者基本信息】 重庆医科大学 ， 生物化学与分子生物学， 2011， 硕士

【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。更多还原

【Abstract】 Enterovirus type 71(EV71) is one of the major etiological agents of hand-foot-mouth disease (HFMD), which often causes severe neurological corqplications. Vp1 is the most important antigenic genes of EV71, it’s product as a king of outer capsid protein takes important role in the infection of the host cell. So we choose vp1 as the target gene for an oral vaccine. LTB, a non-toxic subunit, is reported has powerful immunogen and adjuvant, so we expressed it with VP1 as intramolecular adjuvant in this study.Bifidobacterium exist in human intestinal tract normaly. It is an important beneficial bacterium, which has an important regulating function to the microenvironment of human intestinal tract and a special protection to the intestinal tract of an infant. Bifidobacteria can adhere to the gut. So we try to develop it as a gastrointestinal tract administers live vaccine system carrying EV71 VP1 and LTB of ETEC.ObjectiveIn order to develop HFMD vaccine preventing EV71 infection, the recombined expression vector for the vp1-ltb fusion gene was constructed and expressed both in E.coli BL21 (DE3) and Bifidobacterium. The immunicity of the recombinant Bifidobacterium was analyzed with ELISA. MethodsEV71 Vp1 gene was synthesised with 14 pairs of primers and the ltb gene fragment of ETEC (44815) plasmid DNA was amplified by PCR. Two fragments were linked by overlap PCR and the product was identified by sequencing analysis and inserted into the expression vector pBEX. The constructed recombinant plasmid pBEX-VP1-LTB was transformed into E.coli BL21(DE3). The expression product of VP1-LTB was analyzed by SDS-PAGE and Western blotting. The mice were immunized with the expressed product. The IgG and IgA levels were determined in sera, faces and intestines mucus to identify the immunogenicity of the expressed product. Then the recombined plasmid was transformed into Bifidobacterium by electroporation. The antigenicity of target protein was identified by Western blot. The specific serum IgG and fecal IgA was detected with ELISA.ResultsThe vp1 gene was 891bp and its sequence was consistent with that respected. Both PCR and restriction enzyme analysis showed that recombinant plasmid pBEX-VP1-LTB contained the target fusion gene. The expression product with a relative molecular mass of 73kD existed mostly in the form of inclusion body. Western-blot showed that the protein has a reactionogenicity with antibody of vp1-positive sera of rabbits.The IgG and IgA levels of mice immunized with VP1-LTB were significantly higher than those of control mice. The recombinant plasmids were successfully transformed into Bifidobacterium by electroporation. Western-blot showed that the protein has a reactionogenicity with antibody of vp1-positive sera of rabbits. Specific IgG in blood serum and IgA in intestinal tract of SD rat were found with ELISA.ConclusionThe recombinant expression vector for the vp1-ltb fusion gene was successfully constructed with overlap PCR technique. This recombinant expression vector was expressed both in E.coli BL21 (DE3) and Bifidobacterium. The recombinant protein showed good antigenicity and immunogenicity. This work paved the way for developing intramolecular adjuvant enginered vaccine against HFMD infected by EV71. The recombinant plasmid pBEX-VP1-LTB can express the target VP1-LTB with a reactionogenicity. The recombinant Bifidobacterium expressing VP1-LTB can induce specific membrana mucosa immune response which laid the foundation for the next research of an oral vaccine of EV71 with the Bifidobacterium expression system.更多还原