牛传染性鼻气管炎病毒gG蛋白的表达及gG-ELISA的建立

Infectious Bovine Rhinotracheitis Viral gG Expression and gG-ELISA Development

以牛传染性鼻气管炎病毒（IBRV）基因组DNA为模板,PCR扩增gG基因,克隆到T载体pMD18-T,经限制性酶切分析及序列测定鉴定正确后,进一步亚克隆至原核表达载体pGEX-KG。SDS-PAGE和Western blot分析表明,该基因在大肠杆菌BL21（DE3）中呈可溶性及包涵体两种形式表达,重组蛋白质具有免疫学活性。包涵体蛋白经提纯、变性、复性后,作为包被抗原建立了gG-ELISA诊断方法。应用该方法与进口试剂盒（IDEXX）平行检测380份血清样品,两种方法的符合率为92%（351/380）。对6份病毒分离阳性血清的检测均为阳性,对非相关病原的阳性血清及中监所的阴性血清检测均为阴性,表明所建立的IBRVgG抗体的ELISA检测具有良好的灵敏度与特异性。对1248份奶牛血清样本进行了检测,进口牛群的IBRV抗体阳性率平均为21.7%,湖北本地中国黑白花奶牛的抗体阳性率在不同牧场之间变化很大,范围为0.0%-41.5%。

Taking the genome DNA of Infectious Bovine Rhinotracheitis Virus（IBRV）as the template,the gG gene was amplified with PCR and cloned into the T cloning vector pMD18-T.After being identified by restriction digestion and DNA sequencing,the insert was subcloned into the expression vector pGEX-KG.Sodium docecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE）and Western blot assay showed that this gene was expressed as both soluble form and inclusion body by the transformed E.coli BL21 strain（DE3）.The fusion protein was purified and used as the coating antigen to develop the indirect Enzyme-Linked Immunosorbent Assay（ELISA）.Comparison between this gG-ELISA and commercial IBRV gB-ELISA Kit（IDEXX）was made in the detection of 380 cow serum samples.The results demonstrated an agreement of 92%.By using this novel gG-ELISA,1248 cow serum samples were tested and the average positive rate of IBRV antibodies for imported cows is 21.7%,while the positive rate ranged greatly from 0.0%～41.5% for Hubei local Chinese Black and White Dairy Cows.