牛传染性鼻气管炎病毒gG iELISA抗体检测方法的建立

Establishment of gG iELISA to detect antibody of bovine herpes virus 1

为了建立有效鉴别牛传染性鼻气管炎病毒（BoHV-1）gg-／tk-基因缺失疫苗人工免疫和BoHV-1野毒株自然感染的鉴别诊断方法，以BoHV-1gG蛋白为抗原建立了间接ELISA（iELISA）抗体检测方法。根据编码BoHV1gG蛋白的基因序列设计特异性扩增引物，以BoHV1基因组DNA为模板扩增gg基因截短片段，克隆至原核表达载体pGEX-6p-1并进行IPTG诱导表达。SDS-PAGE和Westernblot分析表明，gg基因在大肠杆菌BL21（DE3）中呈可溶性及包涵体2种形式表达，纯化的gG蛋白具有良好的反应原性。将可溶性gG蛋白纯化后作为包被抗原，建立了BoHV-1gGiELISA抗体检测方法，并优化了各反应条件。使用该方法与商业化试剂盒BoHV-1gE和gB阻断ELIsA进行比较检测，并与其他5种牛常见病原体的阳性血清进行交叉反应检测，同时进行了该方法的重复性试验、保存期试验及消长规律试验；最后使用该方法对临床1031份牛血清进行了检测，血清流行率为83．71%,4（95%CI：81．30％～85．90％）。结果显示，该方法的阴、阳性血清检测的临界值（S／P值）为0．398，与进口试剂盒比较显示该方法具有良好的诊断敏感性和特异性，且本试剂盒可在1：50倍样本稀释下进行检测，而商业化试剂盒是1：1倍样本稀释检测。此外，该方法分析特异性良好，与其他非相关病原体的阳性血清无交叉反应；检测重复性良好；在4℃条件下可保存6个月。该方法在人工感染BoHV～1野毒株和人工免疫BoHv-1gg-／tk-疫苗株20d后可以明显区分疫苗株免疫和野毒株感染。综上所述，本研究成功建立了BoHV-1 gG iELISA抗体检测方法，敏感性和特异性良好，既可以用于有效鉴别疫苗免疫和自然感染，也可作为BoHV-1的血清学诊断方法。

In order to establish an effective differential diagnosis technique to differentiate the Bo- HV-1 natural infection from the animals vaccinated BoHV-1 gg-/tk- gene-deleted vaccine,there- fore the gG protein was made as an antigen to establish BoHV-1 gO iELISA method. Specific primers were designed to amplify the truncated fragment of gg gene according to BoHV-1 gg gene sequences and the truncated gg gene was cloned into the pGEX-6p-1 vector and expressed by the induction of IPTG. Sodium docecyl sufate-polyacrylamide gel （SDS-PAGE） and Western blot assay showed that gg gene was expressed as both soluble form and inclusion body by the transformed E. coli BL21 strain （DE3）. Afterwards,the soluble form gG protein was purified and selected asthe coating antigen to establish BoHV-1 gG iELISA. The working condition of gG iELISA was op- timized. The cutoff values was determined and diagnostic sensitivity, specificity and agreement rate of the gG iELISA were analyzed by using commercial gE and gB blocking ELISA kits as the refer- ence. Cross-reactivity assay of gG iELISA was completed by detecting five positive sera against common bovine pathogens. This study also completed the repeatability and shelf life test. Finally,gG iELISA was applied to detect 1 031 clinical serum samples. The results showed that the cutoff expressed as S/P value of this gG iELISA was 0. 398. The diagnostic sensitivity,specificity and a- greement rate of the gG iELISA were very good. In addition,the samples were diluted 1 - 50 for gG iELISA,while the samples were diluted 1 ： 1 with the commercial kits. Cross-reactivity assay showed that gG iELISA was specific to BoHV-1. In additon, it has a nice repeatability. The gG iELISA could be stable at 4℃ for at least 6 months by doing the high temperature destruction as-say. The gG iELISA could differentiate the natural infection from vaccinated animals （DIVA） after 20 d of infection or vaccination. Finally the gG iELISA was applied to detect 1 031 clinical serum samples whose seroprevalence was 83.71% （95%CI： 81. 30%-85.90%）. In conclusion, this paper established BoHV-1 gG iELISA method with good sensitivity and specificity, and it can not only differentiate the natural infection from vaccinated animals with BoHV-1 gg/tk , but also detect infection of BoHV-1.