本文關(guān)鍵詞：基于雙歧桿菌表達(dá)系統(tǒng)的人輪狀病毒口服重組活疫苗研究　出處：《重慶醫(yī)科大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 輪狀病毒 雙歧桿菌 疫苗

【摘要】：輪狀病毒是引起嬰幼兒腹瀉的首要病毒性病原體,全球每年超過(guò)一億兒童感染該病毒并導(dǎo)致近百萬(wàn)例的死亡。Vp4是輪狀病毒主要的抗原基因之一,其產(chǎn)物外衣殼蛋白在感染宿主細(xì)胞時(shí)起重要作用。因此,我們選擇vp4作為研制相關(guān)疫苗的目標(biāo)基因。 雙歧桿菌正常存在于人類腸道內(nèi),是一種重要的有益的生菌,對(duì)人體腸道微環(huán)境有重要的調(diào)節(jié)功能,對(duì)嬰幼兒腸道有獨(dú)特的保護(hù)作用。因此,用雙歧桿菌作為表達(dá)系統(tǒng)的口服輪狀病毒疫苗有其獨(dú)特的安全性和經(jīng)濟(jì)的方便的優(yōu)勢(shì)。 目的:構(gòu)建表達(dá)輪狀病毒抗原基因vp4的原核表達(dá)載體并在大腸桿菌中進(jìn)行初步表達(dá)鑒定;制備輪狀病毒vp4基因工程雙歧桿菌,驗(yàn)證重組載體pBES-vp4在雙歧桿菌中的表達(dá)情況并初步分析重組工程菌的免疫原性。 方法:(1)PCR擴(kuò)增vp4基因,雙酶切目的基因片斷和經(jīng)本室構(gòu)建的表達(dá)載體pBES并以T4連接酶連接,先轉(zhuǎn)入大腸桿菌BL21(DE3)進(jìn)行表達(dá)并做SDS-PAGE和Western-blot分析;(2)電轉(zhuǎn)法將重組質(zhì)粒轉(zhuǎn)入雙歧桿菌,SDS-PAGE和Western-blot分析其表達(dá)情況;(3)重組雙歧桿菌口服免疫SD大鼠,ELISA檢測(cè)機(jī)體特異的血清IgG和腸道IgA抗體。 結(jié)果:(1)酶切和測(cè)序結(jié)果說(shuō)明目的基因被成功克隆進(jìn)經(jīng)改造的表達(dá)載體;PCR和限制酶切鑒定說(shuō)明重組載體pBES-vp4被成功轉(zhuǎn)化進(jìn)雙歧桿菌;(2)經(jīng)大腸桿菌和BL21(DE3)雙歧桿菌表達(dá)均可見(jiàn)約87kD的目的蛋白;Western-blot分析結(jié)果表明該蛋白具有與輪狀病毒抗體的反應(yīng)原性;(3)ELISA檢測(cè)表明重組雙歧桿菌免疫后的SD大鼠體內(nèi)產(chǎn)生了特性的IgG和IgA抗體 結(jié)論:該研究表明重組載體pBES-vp4可以在大腸桿菌和BL21(DE3)雙歧桿菌中表達(dá)。該輪狀病毒vp4基因工程雙歧桿菌可誘發(fā)SD大鼠特異的粘膜免疫反應(yīng),為進(jìn)一步研制基于雙歧桿菌表達(dá)系統(tǒng)的輪狀病毒重組亞單位口服活疫苗奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392

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