猪伪狂犬病毒gD蛋白的截短表达与PPA-ELISA抗体检测方法的建立  

Development of PPA-ELISA for Detecting Antibodies to Pseudo Rabies Virus Using Recombinant Truncated Glycoprotein gD Expressed in E.coli

作　　者：祖立闯 沈志强[1,2] 郭广君 王金良[2] 苗立中[2] 董林[2] 吕素芳 

机构地区：[1]山东绿都生物科技有限公司,山东滨州256600 [2]山东省滨州畜牧兽医研究院,山东滨州256600

出　　处：《家畜生态学报》2014年第1期54-60,共7页Journal of Domestic Animal Ecology

基　　金：山东省自然科学基金(ZR2012CQ012);滨州市应用技术研究与开发专项(200706)

摘　　要：为建立一种快速的猪伪狂犬病毒抗体检测方法,本研究参照已发表的PRV SA株基因组序列,PCR扩增了长约1 070bp的gD基因片段,将目的片段定向克隆到pET30a原核表达载体,转化BL21表达菌,经IPTG诱导后获得了以包涵体形式表达的重组gD蛋白。重组蛋白纯化后,经免疫印迹检测证明具有良好的抗原性和特异性。以该蛋白作为包被抗原,以辣根过氧化物酶标记葡萄球菌A蛋白为二抗,建立了检测猪伪狂犬病毒抗体的PPA-ELISA检测方法。该方法与其他7种常见猪病病毒(CSFV、PPV、PRRSV、JEV、PCV-2、PEDV、TGEV)的阳性血清不发生交叉反应;批内重复性试验的变异系数小于5%,批间重复性试验的变异系数小于10%;与IDEXX gD-ELISA试剂盒相比较,符合率、敏感性和特异性分别为92.0%、95.1%和88.1%。本研究建立的PRV gD-PPA-ELISA抗体检测方法具有良好的重复性、敏感性和特异性,为PRV的免疫猪群抗体监测、快速诊断和PRV流行病学调查提供一种快速、简便的血清学诊断方法。To develop a method to detect the antibody of pseudo rabies virus, a fragment of about 1070 bp long was amplified by PCR technique after analyzing the published PRY genome sequence, the target fragment was directionally cloned into pET30a vector, the recombinant protein gD was expressed in inclusion body after induced by IPTG. Western blot showed that the purified recombinant protein retained better antigenicity and specificity. An indirect ELISA was successfully developed to detect anti-PRY antibody using the purified recombinant gD protein as coating antigen and horseradish peroxidase-staphylococcal protein A （HRP-SPA） as the second antibody. The PRY gD-PPA击LISA showed no cross-reaction with the positive sera of other seven diseases（CSFV、PPV、PRRSV,JEV、PCV-2、PEDV、TGEV）. Coefficient of variability percent （C. V %） of intro-batch and inter-batch duplicative tests was less than 5 % and 10 %,respectively. Comparing with the IDEXX ELISA Kit, the concordance was 92.0 %,the sensitivity was 95.1 %,and the specificity was 88. 1%. Therefore, this PRY gD-PPA-ELlSA has good specificity, sensi tivity and repeatability, which could be used as a simple and rapid serology detection method for monitoring of anti-PRY antibody and epidemiologic survey of PRY.

关 键 词：猪伪狂犬病毒 gD蛋白 截短表达 PPA—ELISA 检测 

分 类 号：S811.6[农业科学—畜牧学]