截短的传染性支气管炎病毒S1基因原核表达与分析

Prokaryotic Expression and Analysis of Truncated IBV S1 Gene

用RT-PCR方法扩增出传染性支气管炎病毒(IBV)H120S1基因468bp的目的片段,将该片段定向克隆到pQE-30原核表达载体中,并转化到大肠埃希菌BL21(DE3)感受态细胞中;经IPTG诱导进行表达,分别对IPTG的浓度和诱导时间进行优化,对表达的重组蛋白进行纯化,用Western blot验证该蛋白的活性。结果显示,成功构建了pQE-30-S1重组菌,经IPTG诱导,得到18ku左右大小的目的蛋白,IPTG最佳诱导浓度为0.2mmol/L,最适诱导时间为4h,该蛋白主要以包涵体形式存在,Western blot显示获得的重组蛋白能够与IBV多克隆抗体特异性反应。

The target fragment of 468 bp of IBV H120 S1 gene was amplified by RT-PCR.The fragment was cloned into pQE-30 prokaryotic expression vector and the recombinant plasmids were transformed into competent BL21（DE3）Escherichia coli,followed by recombinant protein expression using IPTG induction.To obtain the purified recombinant protein,the concentration of IPTG and the induction time were optimized.The Western blot was utilized to verify the activity of the protein.The results showed that pQE-30-S1 recombinant strain was successfully constructed,and the target protein with the size of about 18 ku was expressed by IPTG induction.The optimal induction concentration was 0.2 mmol/L and the optimum induction time was 4 h.The protein mainly existed in inclusion bodies,and Western blot showed that the recombinant protein could react specifically with IBV polyclonal antibodies.