哈氏弧菌(Vibrio harveyi)lexA基因的克隆与原核表达

Cloning and Prokaryotic Expression of lexA Gene of Vibrio harveyi

LexA是调节SOS反应的阻遏蛋白,而且与细菌耐药性的形成密切相关。本研究以水产养殖经济动物的条件致病菌哈氏弧菌ZJ0603基因组DNA为模板,设计同源引物克隆出SOS基因lexA,并对lexA基因进行了生物信息学分析;构建了lexA基因的原核表达载体PGEX-lexA,在大肠杆菌BL21（DE3）中诱导表达成功,还对LexA蛋白表达的诱导温度、时间、IPTG浓度等条件进行了优化。结果表明,lexA基因的开放阅读框为621 bp,编码206个氨基酸,LexA蛋白理论分子量为22.68 kD;重组蛋白表达的最优条件为37℃、0.04 mmol/L IPTG诱导6 h。

LexA is a repressor protein that regulates the SOS response, and it is closely related to the formation of bacterial resistance. In this paper, a template was designed to amplify and clone the SOS gene lexA basing on the lexA genomic DNA of Vibrio harveyi ZJ0603, which is the conditional pathogen of the aquatic economic animals. The bioinformatics analysis on lexA was performed as well. The prokaryotic expression vector PGEX-IexA oflexA gene was constructed, and the expression of LexA was successfully induced in E. coli BL21 （DE3）. The induction temperature, time and IPTG concentration were optimized to express LexA as well. The results showed that the open reading frame oflexA gene was 621 bp, encoding 206 amino acids, and the molecular weight of LexA protein was 22.68 kD. The optimal conditions for the expression of recombinant protein were 0.04 mmol/L inducing IPTG for 6 hours at 37℃.