用RT-PCR方法扩增出传染性支气管炎病毒(IBV )H120S1基因468 bp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18 ku左右大小的目的蛋白,IPTG最佳诱导浓度为0 .2 mmol/L ,最适诱导时间为4 h ,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 induc-tion .To obtain the purified recombinant protein ,the concentration of IPTG and the induction time were op-timized .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 .
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