【作者】 高慎阳；

【导师】 李一经；

【作者基本信息】 东北农业大学 ， 预防兽医学， 2006， 硕士

【摘要】 M蛋白是猪流行性腹泻病毒(PEDV )的主要的结构蛋白,与Genbank上的其它毒株相比较发现M基因具有很高的保守性,所以重组M蛋白可作为理想的检测抗原用于临床PEDV感染的检测。本实验为使M蛋白在原核细胞中得以表达,对M基因设计了几个大小不同的片断,同时采用不同的载体系统,探索了M基因的表达效果,即将猪流行性腹泻病毒的M基因及编码M蛋白膜外区M基因片断(M’)分别亚克隆到原核表达载体pJLA605和pGEX-6p-1中,构建了重组质粒pJLA605-M、pGEX-6p-M-GST、pGEX-6p-M和pGEX-6p-M’,转化大肠杆菌后,探索了M蛋白在N端和C端不带有谷胱苷肽巯基转移酶(GST)、N端和C端均带有GST、只在N端带有GST以及只含有M蛋白的膜外区M’蛋白的表达情况。SDS-PAGE分析结果表明,N端和C端带有GST的pGEX-6p-M-GST和只含有膜外区M’基因的重组质粒pGEX-6p-M’获得了高效表达,其表达产物分别以融合蛋白GST-M和GST-M’形式存在,经AlpharImager2200软件分析,表达量分别为20%和45%。对重组融合的M’蛋白进行包涵体提取及凝胶过滤层析纯化,得到纯度达90%以上的重组蛋白。分别用抗CV777株PEDV M蛋白的单克隆抗体和纯化PEDV病毒粒子对重组M’蛋白及由其制备的多克隆抗血清进行了鉴定,Western blotting及ELISA检测结果均表明重组M’蛋白有很好免疫原性与反应原性,因此重组M’蛋白与天然PEDV M蛋白有相同的生物学活性。总之,本实验报道了PEDV中国地方流行毒株LJB/03重组M蛋白的表达,同时采用不同的载体系统,探索了M基因表达效果,获得了完整M蛋白和M蛋白的膜外区,并对重组M’蛋白进行了初步纯化与鉴定,实验所得各种数据,为以后该蛋白体外表达、分离、纯化以及诊断试剂的研制提供了具有参考价值的资料,并为PEDV M蛋白的结构和功能研究奠定了重要的物质基础。更多还原

【Abstract】 The membrane protein (M protein) is the main structure protein of PEDV and the sequence analysis suggests that the M gene of PEDV is surprisingly conventional, hence the recombinant of M protein may be useful as an effective diagnositic antigen for detecting PEDV infection. In this work, the whole M gene of PEDV and its truncated fragment M’gene which encodes the exterior of the viral envelope, were subcloned into pJLA605 and pGEX-6p-1, respectively. The recombinant plasmids pJLA605-M, pGEX-6p-M, pGEX-6p-M’and pGEX-6p-M-GST were constructed and transformed into E.coli for expression. SDS-PAGE analysis showed recombinant M protein and truncated M’protein were highly expressed by pGEX-6p-M-GST and pGEX-6p-M’, and the expression products as fusion protein GST-M and GST-M’account reached 20% and 45% in the total bacteria protein, respectively, with the analysis of software AlpharImager2200. The crude inclusion bodies (IBs) extracts of recombinant M’protein were partially purified by gel filtration chromatographer and then identified by Western blotting and InELISA, using a monoclonal antibody against PEDV M protein of CV777 strain and the polyclonal serum antibodies generating by recombinant M’protein. The result showed that the antigenicity and reactivity of the purified protein were extremely closed to the natural biology activity of PEDV, therefore, directly to be used as an rapidly feasible clinical diagnostic antigen reagent, the purified recombinant M’protein was qualified.Conclusively, the prokaryotic expression of recombinant M protein of local isolated LJB/03 strain PEDV were first reported in China. The whole M protein and truncated M’protein were highly expressed by using different expression vector systems, and the recombinant M’protein were primarily purified and identified. These data are useful for the development of a new diagnostic reagent for detecting PEDV infection in China, and helpful for the further study on M protein’s structure and characterization更多还原