基于双歧杆菌表达系统的人轮状病毒口服重组活疫苗研究

发布时间：2018-01-05 05:33

本文关键词：基于双歧杆菌表达系统的人轮状病毒口服重组活疫苗研究　出处：《重庆医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：轮状病毒是引起婴幼儿腹泻的首要病毒性病原体,全球每年超过一亿儿童感染该病毒并导致近百万例的死亡。Vp4是轮状病毒主要的抗原基因之一,其产物外衣壳蛋白在感染宿主细胞时起重要作用。因此,我们选择vp4作为研制相关疫苗的目标基因。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。因此,用双歧杆菌作为表达系统的口服轮状病毒疫苗有其独特的安全性和经济的方便的优势。目的:构建表达轮状病毒抗原基因vp4的原核表达载体并在大肠杆菌中进行初步表达鉴定;制备轮状病毒vp4基因工程双歧杆菌,验证重组载体pBES-vp4在双歧杆菌中的表达情况并初步分析重组工程菌的免疫原性。方法:(1)PCR扩增vp4基因,双酶切目的基因片断和经本室构建的表达载体pBES并以T4连接酶连接,先转入大肠杆菌BL21(DE3)进行表达并做SDS-PAGE和Western-blot分析;(2)电转法将重组质粒转入双歧杆菌,SDS-PAGE和Western-blot分析其表达情况;(3)重组双歧杆菌口服免疫SD大鼠,ELISA检测机体特异的血清IgG和肠道IgA抗体。结果:(1)酶切和测序结果说明目的基因被成功克隆进经改造的表达载体;PCR和限制酶切鉴定说明重组载体pBES-vp4被成功转化进双歧杆菌;(2)经大肠杆菌和BL21(DE3)双歧杆菌表达均可见约87kD的目的蛋白;Western-blot分析结果表明该蛋白具有与轮状病毒抗体的反应原性;(3)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:该研究表明重组载体pBES-vp4可以在大肠杆菌和BL21(DE3)双歧杆菌中表达。该轮状病毒vp4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轮状病毒重组亚单位口服活疫苗奠定了基础。
[Abstract]:Rotavirus is the leading viral pathogen causing infantile diarrhea. More than 100 million children are infected with rotavirus every year and nearly one million deaths. Vp4 is one of the major antigen genes of rotavirus. Capsid proteins play an important role in the infection of host cells. Therefore, we select vp4 as the target gene for the development of related vaccines. Bifidobacterium is an important and beneficial bacteria in the human intestinal tract, which has an important regulatory function to the human intestinal microenvironment, and has a unique protective effect on the intestinal tract of infants. Oral rotavirus vaccine using Bifidobacterium as expression system has its unique advantages of safety and economic convenience. Objective: to construct the prokaryotic expression vector of rotavirus antigen gene vp4 and express it in Escherichia coli. Rotavirus vp4 gene engineering bifidobacterium was prepared to verify the expression of recombinant vector pBES-vp4 in Bifidobacterium and to analyze the immunogenicity of recombinant engineering strain. Methods the vp4 gene was amplified by PCR, the target gene fragment was digested by double enzyme and the expression vector pBES was constructed by our laboratory and ligated with T4 ligase. E. coli BL21 (DE3) was transferred to E. coli for SDS-PAGE and Western-blot analysis. (2) the recombinant plasmid was transformed into Bifidobacterium by SDS-PAGE and Western-blot. The serum IgG and intestinal IgA antibody were detected by Elisa in SD rats immunized with recombinant Bifidobacterium. Results the result of digestion and sequencing showed that the target gene was successfully cloned into the modified expression vector. PCR and restriction enzyme digestion showed that the recombinant vector pBES-vp4 was successfully transformed into Bifidobacterium. (2) about 87kD target protein was expressed by E. coli and BL21DE3) Bifidobacterium. The results of Western-blot analysis showed that the protein could react with rotavirus antibody. Detection of specific IgG and IgA antibodies in SD rats immunized with recombinant Bifidobacterium Conclusion: the recombinant vector pBES-vp4 can be used in Escherichia coli and BL21DDE3. The rotavirus vp4 gene engineering bifidobacterium can induce the specific mucosal immune response of SD rats. It lays a foundation for the further development of rotavirus recombinant subunit oral live vaccine 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

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