H1亚型猪流感病毒抗体间接ELISA检测方法的建立及其应用  

Indirect ELISA for detection of antibodies against swine influenza virus (H1N1)

作　　者：高蕾 刘思当[1] 肖一红[1] 刘为民[2] 刘文军[3] 孙蕾[3] 

机构地区：[1]山东农业大学动物医学院,泰安271018 [2]佛山科学技术学院,佛山5282313 [3]中国科学院微生物研究所中国科学院病原微生物与免疫学重点实验室,北京100101

出　　处：《生物工程学报》2011年第5期805-811,共7页Chinese Journal of Biotechnology

基　　金：山东省自主创新成果转化重大专项(No.2008ZHZX1A1101);中国科学院知识创新工程重要方向项目(Nos.KSCX2-YW-R-158;KSCX2-EW-Q-14);国家科技支撑计划(No.2010BAD04B01-7);中国科学院病原微生物与免疫学重点实验室开放基金(No.2009CASPMI-005)资助~~

摘　　要：为建立H1亚型猪流感病毒抗体检测方法,扩增了H1N1亚型猪流感病毒流行株的血凝素基因HA1部分,构建原核表达载体pET30a-HA1,并转化大肠杆菌BL21表达重组蛋白。对重组蛋白包涵体进行变性、复性和Ni-NTA亲和层析纯化。以纯化后的蛋白作包被抗原,建立间接ELISA检测方法。利用该检测方法检测了2008？2009年采集的猪血清785份,阳性率为15.54%,不同省份的阳性率存在差异（8%~47%）。以IDEXX相关试剂盒检测结果作为参照,该方法的诊断特异性达到91%,诊断敏感性达到95%。In order to detect antibody against swine influenza virus（H1N1）,HA1region of hemagglutinin gene in epidemic swine influenza virus（H1N1） strain was amplified and subcloned into prokaryotic expression vector pET30a.Then recombinant HA1 protein was expressed by Escherichia coli BL21.The purified recombinant HA1 protein was obtained after the treatment of denaturing,refolding and affinity chromatography with immobilized nickel chelating NTA（Ni-NTA）.An indirect enzyme-linked 806 ISSN1000-3061 CN11-1998/Q Chin J Biotech May 25,2011 Vol.27 No.5 immunosorbent assay（ELISA） method was established using the purified protein as antigen.Then 785 swine serum samples collected during 2008-2009 were detected by this method,and the positive ratio was 15.54%.There were diversities among provinces（8%-47%）.The diagnostic specificity and diagnostic sensitivity of this method arrived at 91% and 95% respectively,using the results of IDEXX ELISA kit as reference.

关 键 词：H1亚型猪流感病毒 血凝素 间接ELISA 

分 类 号：S858.28[农业科学—临床兽医学]