新城疫病毒HN基因的截短表达及间接ELISA抗体检测方法的建立与应用  

Development and Application of Indirect ELISA for Detecting Antibodies Against Newcastle Disease Virus with Recombinant Truncated HN Protein Expressed in E.coli

作　　者：田莉莉[1] 李丽[1] 

机构地区：[1]辽宁医学院畜牧兽医学院,辽宁锦州121000

出　　处：《中国家禽》2012年第6期29-32,共4页China Poultry

摘　　要：为建立一种快速的新城疫病毒(NDV)抗体检测方法,参照已发表的NDV基因组序列,设计合成1对特异性引物,PCR扩增了长约1044bp的HN基因片段。将目的片段定向克隆到pGEX-6p-1原核表达载体,酶切及测序鉴定均正确后,转化BL21表达菌,经IPTG诱导得到了以包涵体形式表达的重组蛋白。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。以该蛋白作为诊断抗原,建立了检测新城疫病毒抗体的间接ELISA诊断方法。该方法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%;与血凝抑制试验(HI)相比较,符合率、敏感性和特异性分别为92.0%、91.0%和93.6%。To develop a method to detect the antibody against Newcastle disease virus,a fragment of about 1 044bp long was obtained by RT-PCR technique with specific primers based on published NDV genome sequence. Then the target fragment was directionally cloned into pGEX-6p-1 vector. After identifying with digesting and sequencing,the recombinant plasmid was transformed into E.coli BL21 （DE3）. The recombinant protein HN was expressed in inclusion body form in E.coli after induction with IPTG. Western blotting showed that the purified recombinant protein retain better antigenicity and specificity. An indirect ELISA was successfully developed to detect anti-NDV antibody using the purified recombinant HN protein as coating antigen. Coefficient of variability percent （C.V.%） of intro-batch and inter-batch duplicative tests was less than 5% and 10%, respectively. Comparing with the HI,the concordance was 92.0%,the sensitivity was 91.0%,and the specificity was 93.6%.

关 键 词：新城疫病毒 HN蛋白 原核表达 间接ELISA 

分 类 号：S858.315.3[农业科学—临床兽医学]