论文标题:牛病毒性腹泻病毒p80基因的分段表达及其抗原性分析
Expression of the p80 Fragments of BVDV and Antigenicity Analysis
论文作者
论文导师 王君伟,论文学位 硕士,论文专业 预防兽医学
论文单位 东北农业大学,点击次数 134,论文页数 57页File Size3021K
2006-04-25论文网 http://www.lw23.com/lunwen_5870777/
BVDV;p80 gene;expression;purification;antigenicity analysis;indirect ELISA
牛病毒性腹泻病毒(Bovine viral diarrhea virus，BVDV)是黄病毒科(Flaviridae)、瘟病毒属(Pestivirus)成员，引起牛病毒性腹泻-黏膜病(BVD-MD)，症状主要表现为腹泻，急、慢性黏膜病，持续性感染与免疫耐受，免疫抑制，母畜流产、死胎和畸胎等。该病主要发生于牛，还可感染猪、羊、鹿、骆驼及其它野生反刍动物。牛病毒性腹泻黏膜病呈世界性分布，我国目前已有二十个省市自治区存在本病。由于我国对BVDV的危害性缺乏足够重视，目前尚缺少相应的疫苗，也没有完善的牛病毒性腹泻的诊断、监测技术，面对该病在我国有逐步扩大的趋势，开展BVD／MD的诊断和检测技术的研究迫在眉睫。BVDV的非结构蛋白p80在瘟病毒属中非常保守，是一种免疫优势蛋白，在自然感染或者弱毒活疫苗免疫后的动物中都可以产生针对p80蛋白的抗体，具有重要的诊断价值。 本研究将p80基因分成三段，参照GenBank上发表的BVDV VEDEVAC株基因序列(序列号为AJ585412)设计三对引物，应用PCR方法扩增出p80的三段基因片段，分别命名为p80A基因片段(939bp)、p80B基因片段(570bp)和p80C基因片段(672bp)。分别克隆至pMD-18-T载体，构建重组质粒pMD18-T-p80A、pMD18-T-p80B、pMD18-T-p80C。测序结果表明p80A、p80B、p80C基因片段与VEDEVAC株基因序列(序列号为AJ585412)的同源性分别为99.15％、98.95％和99.85％。推导氨基酸的同源性分别为99.04％、98.42％和100％。 利用DNA重组技术，将BVDV p80A、p80B和p80C基因片段分别亚克隆至原核表达载体pGEX-6P-1上，重组质粒分别命名为pGEX-6P-p80A、pGEX-6P-p80B和pGEX-6P-p80C。将上述重组质粒转化至工程菌Rosetta(DE3)pLyss和BL21(DE3)pLysS中，在IPTG诱导下重组质粒pGEX-6P-p80A、pGEX-6P-p80B和pGEX-6P-p80C均得到成功表达。Western blowing分析表达的三个融合蛋白，结果显示，只有p80B基因片段(289-477aa)表达的融合蛋白能与牛病毒性腹泻的阳性血清反应，表明p80蛋白的免疫优势区域集中在289-477aa区域，位于p80蛋白的解旋酶区域。 蛋白的可溶性分析显示，p80B基因片段蛋白表达产物主要以包涵体形式存在。对包涵体进行提取及切胶纯化，用纯化蛋白作抗原，初步建立间接ELISA方法，结果显示，p80B基因片段表达的融合蛋白能用作ELISA的诊断抗原，用于检测BVD抗体。本实验为进一步开发诊断p80抗体的试剂盒研究工作奠定了基础；同时该方法可以作为与基因工程亚单位疫苗匹配的鉴别诊断方法，能有效的区分动物的疫苗免疫和自然感染，为BVDV的净化提供技术手段。
Bovine viral diarrhea virus (BVDV) is the member of the pestivirus genus, family Flaviviridae, mainly caused bovine viral diarrhea-mucosal disease. Bovine infected by BVDV showed diarrhea, acute and chronic mucosal diaease, persistent infection and immunotolerance, immunosupression, pregnant cow abortion, dead fetus and abnormal fetus. BVDV is a worldwide spreaded diseases, and control of BVDV infection is economically important to the Europe and America cattle industry. BVDV infection has spreaded in twenty province in China, but attention has not been directed towards BVDV infection, so far neither BVDV vaccine applied nor adoption of measures to control of BVDV. It has shown that BVDV infection has expand in China, so it is of great interest to study the pathogen from the molecular biology. Among the non-structural proteins, the NS3(p80) protein is highly immunogenic and highly conserved, so it has important diagnostic significance. So gaining p80 gene and its expressing product is key to the preparation of the specific diagnosis antigen.In this study, three pair of primers were designed to amplify p80A, p80B and p80C gene fragments by PCR according to the published sequence of BVDV VEDEVAC (AJ585412). p80A, p80B and p80C gene fragments were cloned into the vector pMD18-T and the recombinant were named pMD18-T-p80A, pMD18-T-p80B, pMD18-T-p80C. Sequencing and homologous analysis showed that the homology of nucleotide sequence of p80A, p80B and p80C between VEDEVAC were 99.15%, 98.95% and 99.85% respectively. The deduced amino acid sequences were 99.04%, 98.42% and 100% correspondingly.By recombinant DNA techniques, p80A, p80B and p80C gene fragments of BVDV VEDEVAC strain were subcloned into prokaryotic expression vector pGEX-6P-1 respectively to construct recombinant expression vector of pGEX-6P-p80A, pGEX-6P-p80B and pGEX-6P-p80C. Then the recombinant expression plasmid above all were transformed into engineering bacteria of E.coli Rosetta(DE3)pLysS and BL21(DE3) pLysS respectively, recombinant expression plasmid of pGEX-6P-p80A, pGEX-6P-p80B and pGEX-6P-p80C successfully expressed after induced with IPTG. Only fragment p80B(289-477aa)reacted with BVDV positive serum in Western blotting and demonstrated that an immunodominant region of the p80 protein was defined by 289-477aa.This region is contained in domain of BVDV p80 helicase.The p80B recombinant fusion protein was analysised with SDS-PAGE after ultrasonication,
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