猪伪狂犬病病毒gB蛋白B细胞表位解析及其阻断ELISA抗体检测方法的建立

发布时间：2018-06-28 12:08

本文选题：猪伪狂犬病病毒 + 单克隆抗体　；参考：《南京农业大学》2015年硕士论文

【摘要】：伪狂犬病(Pseudorabies,PR)是一种由伪狂犬病病毒(Pseudorabies virus,PRV)引起的猪、牛、羊等多种哺乳动物的重要传染病,给全球养猪业带来巨大的经济损失。目前,疫苗免疫成为防控伪狂犬病的主要手段。但是,2011年以来,我国许多规模化猪场再次出现PRV大流行,给我国养猪业带来严重经济损失。本研究从我国发病猪场分离得到1株PRV强毒株,采用大肠杆菌表达系统表达获得的PRV超强毒株ZJ01 gB和gE重组蛋白,制备获得4株抗PRV单克隆抗体,阐明了 gB蛋白一个抗原表位,采用重组gB蛋白和辣根过氧化物酶标记的gB蛋白单抗,成功建立了检测PRV抗体的阻断ELISA方法,为该病防控奠定了重要基础。具体研究内容包括:1.江苏省猪伪狂犬病病毒强毒株分离与致病性研究从临床发病猪群分离获得一株PRVNJ毒株,gC和gD基因推导的氨基酸序列与先前报道的毒株相比分别有7个与2个氨基酸的插入,基因进化树结果显示,该分离株位于相对独立的分支,与亚洲分离株亲缘关系较近。100日龄健康猪攻毒比较结果显示,PRVNJ株滴鼻接种组猪出现明显临床症状,攻毒后7d内全部死亡,组织病理变化明显,而传统毒株LA接种组仅表现体温变化与轻微呼吸道症状,组织病理变化轻微,表明PRV NJ分离毒株毒力明显增强。2.猪伪狂犬病病毒gB和gE蛋白单克隆抗体的制备与鉴定本研究采用大肠杆菌表达系统表达获得PRV超强毒株ZJ01 gB和gE重组蛋白,免疫BALB/c小鼠,通过细胞融合、克隆和间接ELISA方法筛选,制备获得4株能稳定分泌抗PRV单克隆抗体的杂交瘤细胞,细胞培养上清ELISA抗体效价为1:1600～6400,腹水抗体效价达1:4×105;连续培养20代,抗体效价基本一致。抗gB单抗B1B6和B3D7属于IgG2b,抗gE单抗E1B11和E5C10属于IgG1,轻链均为1κ型。Western blot和IFA结果显示,4株单抗均能与PRV流行毒株ZJ01、HZ、SD、NJ和LA发生特异性反应,B1B6和B3D7还能与PRV gE基因缺失疫苗毒株Bartha-K61发生反应,但E1B11和E5C10b不能与Bartha-K61反应,为PRV功能蛋白和抗体鉴别诊断试剂盒研制奠定了重要基础。3.猪伪狂犬病毒ZJ01毒株gB蛋白单克隆抗体的B}0胞表位鉴定为了对已获得的2株抗PRV gB蛋白的单克隆抗体针对的抗原表位进行定位,本研究利用原核表达系统截断表达了 7个重组gB蛋白。通过SDS-PAGE和Western blot鉴定目的蛋白成功表达后,再通过Western blot对2株抗PRV gB蛋白的单克隆抗体所识别的抗原表位进行定位。结果显示,抗gB单抗B1B6和B3D7识别相同的抗原表位E(104)YGDLDART(112),与不同来源的PRV毒株进行序列比对分析,结果发现该表位在所参考毒株中高度保守。本研究为gB蛋白功能分析研究和PRV诊断方法的建立奠定了基础。4.猪伪狂犬病病毒阻断ELISA抗体r检测方法的建立与应用本研究采用猪伪狂犬病毒(PRV)ZJ01毒株重组gB蛋白和HRP标记单克隆抗体,建立检测PRV gB抗体的阻断ELISA方法,其优化的反应条件为:抗原包被浓度0.5μg·mL-1,待检血清1:1稀释,作用时间37℃1h,酶标单抗稀释度为1:15000,作用时间为37℃ 0.5h.血清样品阻断率PI≥28.67%时判为阳性,PI≤18.27%时判为阴性,18.27%PI28.67%时判为可疑。该方法与PRRSV、PCV2、CSFV和FMDV抗体阳性血清无交叉反应性。批间和批内变异系数均小于10%。敏感性、特异性分别为80.9%和96.4%。与IDEXX PRV gB抗体检测试剂盒符合率为85.1%。535份临床猪血清样品检测结果显示,PRV抗体阳性率达61.87%。
[Abstract]:Pseudorabies (PR) is an important infectious disease of pigs, cattle, sheep and other mammals caused by Pseudorabies virus (PRV). It has brought huge economic losses to the global pig industry. At present, vaccine immunity has become the main means to prevent and control pseudorabies. But since 2011, many large-scale pig farms in our country have been re established. The PRV pandemic has brought serious economic loss to the pig industry in China. 1 PRV strains were isolated from the swine farm of our country. The superstrong PRV strain ZJ01 gB and gE recombinant protein expressed in the Escherichia coli expression system was used to prepare 4 monoclonal anti PRV monoclonal antibodies, and the epitope of gB protein was clarified, and the recombinant gB was used as a recombinant gB. The gB protein monoclonal antibody labeled with protein and horseradish peroxidase successfully established the blocking ELISA method for detecting PRV antibody and laid an important foundation for the prevention and control of the disease. 1. the specific research contents include: isolation and pathogenicity of the virulent strain of porcine pseudorabies virus in Jiangsu Province, a PRVNJ strain was isolated from the clinical swine, gC and gD genes were pushed. 7 and 2 amino acids were inserted in the amino acid sequence compared with the previously reported strains. The results of gene evolution tree showed that the isolate was located in a relatively independent branch. Compared with the Asian isolates, the results showed that the swine inoculation group of PRVNJ strains had obvious clinical symptoms and 7 after attack of.100 day old pigs. 7 D all died and the pathological changes were obvious, while the traditional LA inoculation group showed only the temperature change and the mild respiratory symptoms, and the histopathological changes were slight. It showed that the virulence of the PRV NJ isolates significantly enhanced the preparation and identification of the monoclonal antibodies of the.2. porcine pseudorabies virus gB and gE protein. PRV superstrong strain ZJ01 gB and gE recombinant protein were obtained, and BALB/c mice were immunized by cell fusion, cloning and indirect ELISA screening. 4 hybridoma cells capable of stable secretion of anti PRV monoclonal antibodies were prepared. The titer of the cell culture supernatant ELISA antibody was 1:1600 to 6400 and the antibody titer of ascites reached 1:4 * 105; the antibody titer was continuously cultured for 20 generations. The anti gB monoclonal antibody B1B6 and B3D7 belong to IgG2b, and the anti gE monoclonal antibody E1B11 and E5C10 belong to IgG1, and the light chains are all 1 kappa.Western blot and IFA results. It can react with Bartha-K61 and establish an important basis for the development of the PRV functional protein and antibody differential diagnostic kit. The B}0 cell epitopes of the gB protein monoclonal antibody of the.3. porcine pseudorabies virus ZJ01 strain are identified in order to locate the antigen epitopes against the obtained 2 monoclonal antibodies against PRV gB protein. This study uses the prokaryotic expression system. 7 recombinant gB proteins were truncated. After the target protein was successfully expressed by SDS-PAGE and Western blot, the antigen epitopes identified by 2 monoclonal antibodies against PRV gB protein were identified by Western blot. The results showed that the antigen epitope E (104) of the anti gB McAb B1B6 and B3D7 identified (104), 112, and different sources. Sequence alignment analysis showed that the epitope was highly conserved in the reference strain. This study laid the foundation for the establishment and application of the gB protein function analysis and the establishment of the PRV diagnosis method for the.4. porcine pseudorabies virus blocking the ELISA antibody r detection method. The study adopted the recombinant gB eggs of the porcine pseudorabies virus (PRV) ZJ01 strain. White and HRP labeled monoclonal antibodies were used to establish a blocking ELISA method for detecting PRV gB antibodies. The optimal reaction conditions were: the antigen inclusion concentration was 0.5 mu g. ML-1, the serum 1:1 was diluted, the action time was 37 1H, the dilution of the enzyme labeled monoclonal antibody was 1:15000, the action time was 37 C 0.5H. serum sample blocking rate PI > 28.67% and PI was less than 18.27% 18.27%PI28.67% was found to be doubtful. The method had no cross reactivity with PRRSV, PCV2, CSFV and FMDV antibody positive serum. The coefficient of variation between batch and batch was less than 10%. sensitivity, the specificity was 80.9% and the coincidence rate of 96.4%. and IDEXX PRV gB antibody detection kit was shown in 85.1%.535 portion of clinical pig serum samples. The positive rate of antibody is 61.87%.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

【相似文献】