小反刍兽疫病毒H蛋白的表达及其B细胞线性表位筛选

发布时间：2018-01-28 08:46

本文关键词： 小反刍兽疫小反刍兽疫病毒 H基因 H蛋白线性B细胞表位筛选　出处：《安徽农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：小反刍兽疫(PPR)是由小反刍兽疫病毒(PPRV)感染山羊、绵羊等家养和野生小反刍动物的一种烈性、接触性、传染性疫病。2007年,在我国西藏地区首次暴发小反刍兽疫。目前,我国已有20个省市相继发现了感染病例。探索有效的快速诊断和防治相结合的防治措施对于及时应对随时可能发生的小反刍兽疫疫情具有重要的意义。本研究利用蛋白质免疫印迹方法,以免疫的新西兰大白兔血清为一抗,通过覆盖H基因的重叠16肽片段与血清的特异性反应,鉴定小反刍兽疫病毒H蛋白的线性B细胞表位。本文主要进行了以下研究:1.PPRV H基因原核表达载体的构建以密码子优化的Peste des petits ruminants virus strain China/Tibet/Geg/07-30(FJ905304)的H基因为模版,扩增H基因片段,构建了1个克隆载体pJET1.2/blunt-PPRV-H和3个表达载体pET28a-PPRV-H,pET30a-PPRV-H和pET32a-PPRV-H。2.His-H蛋白的表达与纯化对重组表达载体pET30a-PPRV-H的诱导表达条件进行优化,确定浓度为1mmol/L IPTG,诱导5 h为最佳诱导表达条件。在此条件下,H蛋白主要以包涵体形式存在。通过尿素溶液洗涤包涵体,去除杂蛋白,纯化和浓缩目的蛋白,浓缩后的蛋白浓度最高为2 mg/mL。3.免疫血清的制备以纯化后的重组H蛋白免疫新西兰大白兔(0.5 mg/只),分别在14 d、28d、42 d加强免疫一次,56 d颈动脉采血收集血清。通过间接ELISA方法测定血清的滴度,1号,2号和3号兔子免疫血清的滴度分别达到1∶25600,1∶12800,1∶6400。从而说明获得的抗血清可以特异性识别重组表达的His-H蛋白。4.H蛋白B细胞线性表位的筛选设计并合成覆盖H基因16肽的基因序列,构建表达载体pXXGST-3-P76-(1F~75F),将重组质粒转化大肠杆菌BL21。以兔抗H蛋白抗血清作为一抗,利用蛋白质免疫印迹来筛选重组表达的16肽片段,从而确定H蛋白的线性B细胞表位,结果共筛选出30个阳性的16肽。
[Abstract]:Ruminant ruminant disease (PPRV) is a strong, contagious, infectious disease of small ruminants infected by small ruminant virus (PPRV) in goats, sheep and other domestic and wild ruminants. 2007. The first outbreak of small ruminant epidemic in Tibet, China. 20 provinces and cities in China have found infected cases one after another. It is of great significance to explore effective prevention and treatment measures combining rapid diagnosis and prevention and treatment for the possible small ruminant epidemic situation at any time. Western blotting was used. The immunized New Zealand white rabbit serum was used as the first antibody, and the specific reaction between the 16 peptide fragment and the serum was obtained by covering the H gene. The linear B cell epitopes of ruminant ruminant virus H protein were identified. In this paper, the following studies were carried out: 1. Construction of a prokaryotic expression vector of PPRVH gene with codon optimized Peste des. Petits ruminants virus strain China / beta / Gegr / 07-30. FJ905304) the H base because of the template. The H gene fragment was amplified and a clone vector pJET1.2/blunt-PPRV-H and three expression vectors pET28a-PPRV-H were constructed. Expression and purification of pET30a-PPRV-H and pET32a-PPRV-H.2.His-H protein to induce expression of Recombinant expression Vector pET30a-PPRV-H. To optimize. The optimal expression conditions were determined to be 1 mmol / L IPTG and induced for 5 h. Under these conditions, the protein mainly existed in the form of inclusion body, and the inclusion body was washed by urea solution. Remove miscellaneous proteins, purify and concentrate target proteins. The concentration of concentrated protein was up to 2 mg / mL 路3.The purified recombinant H protein was used to immunize New Zealand white rabbits with 0.5 mg / L for 14 days and 28 days respectively. The serum was collected from carotid artery for 56 days after 42 days of enhanced immunization. The titer of serum was determined by indirect ELISA method. The titers of 2 and 3 rabbits were 1: 25600 and 1: 12800, respectively. 1:. The results showed that the obtained antiserum could specifically identify the recombinant His-H protein .4.4. the screening of linear epitopes of B cells and synthesis of the gene sequence covering the 16 peptide of H gene. To construct the expression vector pXXGST-3-P76-P76-P76-P76-P76-P76-P76-P76-P76-P76-F7 / 75FN, the recombinant plasmid was transformed into Escherichia coli BL21. Western blot was used to screen the recombinant 16 peptide fragments, and then the linear B cell epitopes of H protein were determined. A total of 30 positive 16 peptides were screened out.
【学位授予单位】：安徽农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

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