肺炎克雷伯菌调控蛋白RcsAB的构建表达及活性鉴定

Construction Expression and Activity Identification of Regulatory Protein RcsAB in Klebsiella pneumoniae

为了探讨转录调控子Rcs AB对靶基因的转录调控作用,构建肺炎克雷伯菌RcsA和RcsB的重组质粒,之后诱导蛋白表达,提取纯化后测定其活性。PCR得到rcsA、rcsB片段,分别将两DNA片段克隆至表达载体pMAL-C5X、pET28a,构建重组质粒。再将重组质粒导入E. coli BL21(DE3)菌株中,经IPTG诱导后收集菌体,超声破碎。破碎后的上清过柱、透析纯化,得到高纯度的蛋白,通过EMSA进行蛋白活性鉴定。RcsA,RcsB蛋白成功表达纯化,并能够与靶基因结合,初步证明蛋白具有生物学活性。成功制备有生物学活性的RcsA、RcsB蛋白,为进一步研究RcsAB蛋白复合物特异的生物学功能提供物质基础。

To investigate the transcriptional regulation of transcriptional regulator RcsAB for target gene, this study constructed the recombinant plasmid of RcsA and RcsB in Klebsiella pneumoniae, and induced the protein expression, then detected its activity after extraction and purification. rcsA and rcsB fragments were amplified by PCR, of which the DNA fragments were cloned into expression vectors pMAL-C5 X and pET28 a, respectively. And we constructed recombinant plasmids, which were transformed into E. coli BL21(DE3) strain, and the strains were collected and broken by ultrasonic waves after induced by IPTG. After fragmentation, the supernatants were purified by column and dialysis, and high purity proteins were obtained. The protein activity was identified by EMSA. RcsA and RcsB proteins were successfully expressed, purified and bound to a target gene which therefore prove the biologically activity of proteins. RcsA and RcsB proteins which have the biologically activities were successfully synthesized, which might laythe foundation for further studying the specific biological functions of RcsAB protein complexes.