灰葡萄孢BcKMO基因的原核表达分析

摘要

The aim of this study is prokaryotic expression analysis of BcKMO gene from Botrytis cinerea and obtain the purified BcKMO protein.The BcKMO gene was amplified by RT-PCR technology using the cDNA of the Botrytis cinerea wild type BC22,cloned into the pMD19-T vector and sequenced.The results of sequencing showed that the BcKMO gene sequence was right.The pMD19-T-BcKMO and pGEX4T-1 plasmids were digested using restriction enzyme.The BcKMO gene segments were collected and cloned into the pGEX4T-1 vector.The results of restriction enzyme digestion and sequencing showed that the vector pGEX4T-1-BcKMO-GST was successfully constructed.The vector pGEX4T-1-BcKMO-GST was transformed into E.coli BL21 strain.The results of IPTG inducement indicated that the pGEX4T-1-BcKMO-GST was successfully expressed in E.coli BL21 strain,with the molecular weight 71 kDa.The optimal conditions of the prokaryotic expression of BcKMO were determined as 0.2 mmol/L IPTG treatment 12 h.Western Blot results showed that the GST antibody could specifically bound to purified pGEX4T-1-BcKMO-GST fusion protein,suggesting that the expression of BcKMO gene was successfully in vitro.%为构建灰葡萄孢犬尿氨酸单加氧酶(BcKMO)基因的原核表达载体并进行高效表达,获得纯化的BcKMO蛋白.以灰葡萄孢野生型BC22为试材,通过反转录PCR扩增BcKMO基因,回收BcKMO基因片段克隆到pMD19-T载体中,测序正确后,酶切pMD19-T-BcKMO和带有GST标签蛋白的pGEX4T-1质粒,将目的片段进行连接,构建BcKMO基因的原核表达载体pGEX4T-1-BcKMO-GST.经酶切和测序鉴定正确后,将构建好的原核载体转化大肠杆菌BL21.经IPTG诱导,在大肠杆菌BL21菌株中成功表达了与GST标签蛋白融合的BcKMO蛋白,大小约71 kDa.SDS-PAGE分析表明,该蛋白在0.2 mmol/L IPTG诱导12h时高效表达;Western Blot结果发现目的蛋白能与GST特异性抗体起特异性反应,表明BcKMO基因的体外诱导表达成功.