| 灰葡萄孢BcKMO基因的原核表达分析 |
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| 引用本文： | 王敏,刘媛媛,周帆,姜婷婷,郑旭,张靖,时翠平,邢继红,董金皋.灰葡萄孢BcKMO基因的原核表达分析[J].华北农学报,2017,32(1). |
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| 作者姓名： | 王敏 刘媛媛 周帆 姜婷婷 郑旭 张靖 时翠平 邢继红 董金皋 |
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| 作者单位： | 河北农业大学,真菌毒素与植物分子病理学实验室,河北保定071001 |
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| 基金项目： | 河北省高等学校科学技术研究项目 |
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| 摘 要： | 为构建灰葡萄孢犬尿氨酸单加氧酶(Bc KMO)基因的原核表达载体并进行高效表达,获得纯化的Bc KMO蛋白。以灰葡萄孢野生型BC22为试材,通过反转录PCR扩增Bc KMO基因,回收Bc KMO基因片段克隆到p MD19-T载体中,测序正确后,酶切p MD19-T-Bc KMO和带有GST标签蛋白的p GEX4T-1质粒,将目的片段进行连接,构建Bc KMO基因的原核表达载体p GEX4T-1-Bc KMO-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Bc KMO蛋白,大小约71 k Da。SDS-PAGE分析表明,该蛋白在0.2 mmol/L IPTG诱导12 h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Bc KMO基因的体外诱导表达成功。
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| 关 键 词： | 灰葡萄孢 BcKMO 原核表达 纯化 PCR |
Prokaryotic Expression Analysis of BcKMO Gene from Botrytis cinerea |
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| WANG Min,LIU Yuanyuan,ZHOU Fan,JIANG Tingting,ZHENG Xu,ZHANG Jing,SHI Cuiping,XING Jihong,DONG Jingao.Prokaryotic Expression Analysis of BcKMO Gene from Botrytis cinerea[J].Acta Agriculturae Boreali-Sinica,2017,32(1). |
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| Authors: | WANG Min LIU Yuanyuan ZHOU Fan JIANG Tingting ZHENG Xu ZHANG Jing SHI Cuiping XING Jihong DONG Jingao |
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| Abstract: | 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. |
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| Keywords: | Botrytis cinerea BcKMO Prokaryotic expressin Purification PCR |
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