PRRSV M蛋白基因片段的原核表达及间接ELISA检测方法的初步建立  

Establishment of an indirect ELISA using the prokaryotically-expressed M protein for the detection of antibodies against PRRSV

作　　者：罗忠永 王印[1] 杨泽晓[1] 

机构地区：[1]四川农业大学动物医学院动物检疫实验室,四川成都611130

出　　处：《中国兽医科学》2017年第1期95-99,共5页Chinese Veterinary Science

基　　金："十二五"国家科技支撑计划项目(2013BAD12B04)

摘　　要：利用PCR方法获得PRRSV M基因,使用原核表达系统p ET-28a(+)-BL21(DE3)诱导表达M蛋白。用表达的M蛋白建立检测PRRSV抗体的间接ELISA。通过特异性试验、阻断试验和重复性试验考查基于重组M蛋白建立的ELISA(M-ELISA)的实用性,同时使用M-ELISA和IDEXX Herd Chek ELISA for PRRSV试剂盒检测收集的400份临床血清样品。结果,用PCR扩增到了PRRSV M基因,获得了重组M蛋白,建立了用于检测PRRSV抗体的间接ELISA(M-ELISA);该M-ELISA具有良好的特异性和重复性。400份临床血清的检测结果与IDEXX试剂盒的检测结果的符合率为95.3%。上述结果表明,本研究基于表达的重组M蛋白,成功建立了检测RRSV抗体的间接ELISA。In this study,the segment of M gene from porcine reproductive and respiratory syndrome virus（PRRSV） was amplified by PCR using a pair of specific primers designed according to the conserva- tive region of M gene from many PRRSV strains. The M gene fragment was expressed using the prokaryotic expression system pEr-28a（＋） -Ecoli BL21（DE3） induced with IPTG. The good immunoreactivity of the expressed M protein to PRRSV antibodies was proved by Western-blot analysis. An indirect M-ELISA assay for the detection of PRRSV antibodies in swine serum was established using the expressed protein M. rhe M-ELISA kit reacted to positive serum against PRRSV but did not react to negative serum against PRRSV, positive sera against CSFV,PRV PPV and PCV. The kit did not react to positive serum against PRRSVwhen the serum was mixed with protein M before the ELISA test. The detection results of the using same posi- tive serum sample against PRRSV showed no obvious difference when the sample was detected with differ- ent M-ELISA kits made in this study. 400 clinical serum samples were detected with the M-ELISA kit and the IDEXX HerdChek ELISA for PRRSV kit,and the detection results of the M-LISA kit showed a consisten- cy of 95.3% compared to the IDEXX HerdChek ELISA. In brief, the M gene from PRRSV was expressed and an indirect ELISA assay was established using the expressed recombinant protein M.

关 键 词：猪繁殖与呼吸障碍综合征病毒 M蛋白基因 原核表达 N-ti-ELISA 

分 类 号：S852.65[农业科学—基础兽医学]