牛流行热病毒G1抗原表位基因在大肠杆菌中的表达、纯化及抗原性鉴定

Expression,purification and antigenic characterization of the Epitope-G1 gene of Bovine ephemeral fever virus in Escherichia coli

摘要:

从包含牛流行热病毒G蛋白基因的质粒pMD-G中克隆G1抗原表位区基因，与表达载体pGEX-4T-1连接，成功构建重组质粒pGEX-G₁。重组质粒转化BL21（DE3），以IPTG进行诱导，并确定了最佳表达条件的IPTG浓度为0.1mmol/L、反应温度为16℃、诱导时间为18h。可溶性表达的目的蛋白经Glutathione Sepharose TM^4B介质纯化，纯度达80%；以包涵体形式存在的重组蛋白以2%的脱氧胆酸钠洗涤、0.5%的N-十二烷基肌氨酸钠溶解、透析复性、Glutathione Sepharose TM^4B纯化后，纯度达85%以上。Western blot试验表明纯化的目的蛋白有良好的反应原性。经间接ELISA检测，测得牛流行热病毒12份阳性血清的OD490值平均为1.813±0.231，12份阴性血清的OD490值平均为0.359±0.032，差异极显著（P<0.01）。将重组蛋白作为抗原免疫兔子，试验兔均产生了高滴度的抗体，证实该蛋白有免疫原性。将目的蛋白作为包被抗原，测得8份狂犬病病毒阳性血清的OD490值平均为0.324±0.031，与所测12份阴性血清的OD490值接近，说明不存在交叉反应。以上结果均证实纯化后的重组蛋白有良好的生物学活性和特异性，可作为包被抗原，开发ELISA试剂盒。

Abstract:

The epitope-G₁ gene, cloned from the pMD-G plasmid including G protein gene of bovine ephemeral fever virus (BEFV), was subcloned into expression vector pGEX-4T-1 to construct pGEX-G₁ recombinant plasmid successfully. The pGEX-G₁ was transformed into E.coli BL21(DE3) to be induced with IPTG. The optimal expression conditions for G₁ gene were obtained, which included reaction temperature 16℃, induction time 18h and IPTG concentration 0.1mmol/L. The soluble target protein was purified with Glutathione Sepharose TM^4B and the purity reached 80%. The inclusion body washed with 2% deoxycholic acid sodium salt and dissolved with 0.5% N-lauroyl sarcosine sodium was recovered by the way of dialysis, then the protein was purified with Glutathione Sepharose TM^4B and its purity was above 85%. The protein purified had nicer reaction activity by analysis of Western blot. The target protein was used as coating antigen to detect the sera against BEFV by an indirect ELISA. The 12 positive sera to BEFV were detected and the average of OD₄₉₀ was 1.813±0.231, while the average of OD₄₉₀ from 12 negative sera was 0.359±0.032, and the distinction was very remarkable (P<0.01). All the rabbits inoculated with the target protein had produced high titer of antibodies, which indicated that the target protein had immunological activity. The average of OD₄₉₀ detecting the 8 positive sera to rabies virus (RV) with the target protein purified was 0.324±0.031 which closed the datum obtained from the negative sera to BEFV, and it showed no cross-reaction between the sera to RV and BEFV. All the results above indicated that the target protein expressed had nicer biological activity and specificity, so the protein could be used as coating antigen to develop ELISA Kit for diagnosing BEF.