牛传染性鼻气管炎病毒gB基因的截短表达与间接ELISA诊断方法的建立

An Indirect ELISA for Detection of Antibody against Infectious Bovine Rhinotracheitis Virus Using Recombinant Truncated Glycoprotein B Expressed in E.coli

经DNAstar分析,据牛传染性鼻气管炎病毒(IBRV)全基因序列设计合成gB基因的特异性引物,以构建的pGM-T-gB为模板,PCR扩增IBRV gB蛋白192-266位氨基酸的基因序列。将目的片段克隆,酶切鉴定并测序鉴定后,定向插入pET32a表达载体中,转化表达菌BL21。SDS-PAGE电泳分析表明,诱导表达产物以包涵体和可溶形式存在,大小约为29.2 ku,与预测值相符。亲和纯化后重组蛋白浓度为2.000 mg/mL,纯度为79.2%。间接ELISA检测证明,表达蛋白具有抗原性。以该蛋白作为诊断抗原,建立了间接ELISA诊断方法。确定的抗原包被浓度为50μg/mL,血清的最佳稀释度为1∶40,阳性标准定为S/P>0.395。交叉试验表明对牛流热(BEV)、牛肺疫(CBPP)、牛病毒性腹泻(BVD)、牛结核(BTB)、牛副结核(PBB)以及牛赤羽病(BAD)等其它6种疾病阳性血清不发生交叉反应。与中和试验相比较,特异性、敏感性和符合率分别为83.3%、90.9%和88.9%;与进口法国IBRV全病毒ELISA抗体诊断试剂盒比较,特异性、敏感性和符合率分别为92%、92.7%和92.5%。上述结果表明该诊断方法具有良好的特异性和敏感性,显示了良好的应用前景。

The gene, which codes the amid acid residency from 192th to 266th of Infectious Bovine Rhinotracheitis Virus（IBRV） glycoprotein B（gB）, was amplified by PCR technique with specific primers after analyzing glycoprotein B with biosoftware DNAstar. Then the amplified product was cloned into T-vector. After identifying with double-enzyme cut and sequencing for the accuracy of the product of amplification, the expected band on the agarose gals was recovered and directional inserted into pET32a vector. The recombinant vector was transformed into BL21. SDSPAGE analysis showed that the fusion protein of 29.2 ku as expected was in solution forms partially. The recombinant protein was purified by affinity chromatography, the concentration of purified protein was 2.000 mg/mL and the purity was 79.2%. The recombinant protein was checked by the indirect enzyme-link assay, which showed that it retains antigenicity of the nature protein. An indirect enzyme linked-assay（antigen concentration： 50 μg/mL; serum dilution： 1 ： 40） was developed with the recombinant protein as a diagnosis antigen, the positive criterion was S/P〉0. 395. There was no cross reaction with positive sura of other six diseases, including BEF （Bovine ephemeral fever）, CBPP（Contagious bovine pleuropneumonia）, BVD-MD（Bovine viral diarrhea-mucosal disease）, BTB（Bovine tuberculosis）, BPB （Bovine paratuberculosis） and BAD （Bovine Akabane disese）. Comparing with the Virus Neutralization testing, the speciality was 83.3% ; the sensitivity was 90.9% ; the accordance rate was 88. 9 %. Comparing with the IBR ELISA kit from France, the speciality was 92% ; the sensitivity was 92.7% ; the accordance rate was 92.5 %. The assay was confirmed to be specific and sensitive by the results above, suggesting a good application prospect.