边界病病毒E2蛋白质的原核表达及间接ELISA检测方法的建立  

Prokaryotic expression of border disease virus E2 and establishment of an indirect ELISA for serum antibody detection

作　　者：毛立[1] 刘霞[1,2] 李文良[1] 杨蕾蕾[1] 张纹纹[1] 魏建忠[2] 江杰元[1] 

机构地区：[1]江苏省农业科学院兽医研究所/农业部兽用生物制品工程技术重点实验室/国家兽用生物制品工程技术研究中心,江苏南京210014 [2]安徽农业大学动物科技学院,安徽合肥230036

出　　处：《江苏农业学报》2015年第1期93-99,共7页Jiangsu Journal of Agricultural Sciences

基　　金：江苏省农业科技自主创新基金项目[CX(14)2090]

摘　　要：边界病病毒(Border disease virus,BDV)是导致绵羊和山羊繁殖障碍的重要病原。为了建立检测BDV特异抗体的ELISA方法,本研究将BDV基因克隆入原核表达载体p ET-32a(+),并构建重组表达菌,经SDS-PAGE和Western blot鉴定目的蛋白质。以纯化的重组表达蛋白质为抗原,建立间接ELISA(r E2-ELISA)检测方法。通过反应条件优化,确定抗原包被浓度4.0μg/ml;封闭液为20 g/L BSA;血清最佳稀释度为1∶50,孵育1 h;二抗最佳稀释倍数为1∶30 000,作用45 min;以TMB为底物显色10 min。用该ELISA方法检测瘟病毒属的CSFV、BVDV阳性血清,结果均为阴性。对187份山羊血清样品进行临床检测,与Svanova试剂盒检测结果阳性符合率为76.25%,总符合率为74.87%;上述检测方法同时与Western blot鉴定结果进行比较,结果显示,Western blot结果与r E2-ELISA检测结果的符合率为79.55%,高于与Svanova试剂盒检测的符合率(72.73%)。表明建立的间接ELISA检测方法可适用于临床BDV血清样品的抗体检测。Border disease virus（ BDV） is main pathogen causing reproductive manifestations of sheep and goat. To develop an ELISA for detection of the specific antibody of BDV,the antigenic region of BDV E2 protein was amplified by RT-PCR,and cloned into p ET-32a（ ＋）. The positive plasmid was transformed to BL21 and the recombinant bacteria were obtained. The recombinant protein was identified by SDSPAGE and Western blot. The indirect ELISA was developed successfully through the optimization of reaction system which involved coated antigen of 4. 0 μg / ml,sealing buffer of 20 g / L BSA,serum sample at 1∶ 50 dilution and incubation for 1 h at 37 ℃,HRP conjugated rabbit anti-pig Ig G at 1∶ 30 000 dilution and the reaction time of 45 min at 37 ℃,and colour development at room temperature for 10 min. Coating antigen had no cross reaction with the antibodies against BVDV and CSFV. The r E2-ELISA showed a BDV positive coincidence rate of 76. 25% and a total coincidence rate of 74. 87% with Svanova ELISA kit. Western blot identification revealed a coincidicen rate of 79. 55% in 44 serum sample,higher than that identified by Svanova ELISA kit. It was indicated that the r E2-ELISA was suitable for large-scale epidemiological investigation for BDV infection.

关 键 词：边界病病毒 E2蛋白质 间接ELISA 

分 类 号：Q939.4[生物学—微生物学]