牛传染性鼻气管炎病毒gE蛋白原核表达及间接ELISA检测方法研究

Prokayotic Expression of Infectious Bovine Rhinotracheitis Virus gE Protein and Indirect ELISA

本研究以牛传染性鼻气管炎gE基因的原核表达产物为抗原建立检测IBRV抗体间接ELISA检测方法。通过DANStar比对分析牛传染性鼻气管炎病毒gE蛋白序列,通过亲和层析对重组表达产物进行纯化,经Western blot检测证明重组表达产物能被IBRV标准性鼻气管炎gE蛋白长度为500 bp的特异性肽序列,随后构建了pCold-TF-gE原核表达体系,并用Ni-IDA进行纯化。以纯化鉴定后的表达产物为抗原(1 μg/mL)包被酶标板制备多克隆抗体血清,并建立检测牛传染性鼻气管炎血清抗体间接gE-ELISA方法。结果显示:阳性OD_(450)值为0.369,组内组间重复性小于5%,敏感性强,与口蹄疫等阳性血清均无交叉反应且特异性达95%。利用建立的gE-ELISA方法,同时和IDEXX(IBR)抗体检测试剂盒对200份临床阳性血清进行检测比较,符合率均达到95%。该方法特异敏感高效,可用于牛传染性鼻气管炎血清抗体的检测。

In this study,an indirect ELISA assay for detection of Infectious bovine rhinotracheitis virus(IBRV)antibodies was developed using the prokaryotic expression product of the bovine infectious rhinotracheitis gE gene as an antigen.The gE sequence of IBRV was analyzed by DANStar.The specific peptide sequence of the gE protein was a length of 500 bp.Subsequently,the pCold-TF-gE plasmid was constructed and Ni was used in prokaryotic expression system.The expressed recombinant gE protein was purified by NTA affinity chromatography and recognized by IBRV standard positive serum in Western blot.The purified gE protein was used as a coating antigen at 1 μg/mL to develop an indirect gE-ELISA method for detecting serum antibodies against IBRV.The results showed that the positive OD_(450) value was 0.369 and the repeatability between the groups was less than 5%.There was no cross-reaction with other positive serum samples such as foot-and-mouth disease.The gE-ELISA method and IDEXX(IBR)antibody detection kit were used to test 200 clinical positive serum samples and the coincidence rate reached 95%.Therefore,the gE-ELISA method developed here was specific,sensitive and efficient for use in detecting serum antibodies against IBRV.