【摘要】 为了验证重组抗菌肽Ranalexin的抑菌活性,提取牛蛙皮肤组织总RNA,经RT-PCR扩增抗菌肽基因ranalexin,克隆测序后,将该基因插入pGEX-3X融合表达载体,进行原核表达,通过改变IPTG剂量、培养液pH值、诱导表达温度和诱导时间优化表达条件,并采用谷胱甘肽氧化/还原系统对重组蛋白进行复性,用琼脂扩散法测定其体外抑菌活性.结果显示,扩增获得的牛蛙皮肤抗菌肽基因ranalexin大小为201bp,其序列与GenBank公布序列同源性达99.57%;正确构建了pGEX-3X-Ranalexin表达载体,最佳表达条件为IPTG终浓度1.0mmol?L-1、诱导时间5～6h;表达产物的分子质量约为34kDa,并以包涵体的形式表达,经复性纯化后该重组蛋白对金黄色葡萄球菌具有明显的体外抑菌活性.说明牛蛙皮肤抗菌肽基因ranalexin的原核表达载体构建成功,并获得了具有抑菌活性的重组抗菌肽GST-Ranalexin.更多还原

【Abstract】 In order to verify antibacterial activity of recombinant antibacterial peptide Ranalexin,the total RNA was extracted from the skin of Rana catesbiana and the ranalexin gene was amplified by RT-PCR. After DNA sequencing,the gene was cloned into fusion expression vector pGEX-3X and the fusion protein of GST-Ranalexin was produced under inducement with IPTG. This expression condition was optimized under inducement with different dosages of IPTG,pH,temperature and time. Then the expressed protein was renatured by the systems of GSH and GSSG and its antibacterial activity was determined in vitro by agar diffusion method.The results indicate that the ranalexin DNA fragment at the length of 201 bp was amplified and the nucleotide homology with the sequence on GenBank was 99.57%,the recombinant plasmid pGEX-3X-Ranalexin was constructed correctly and the optimal expression condition was the IPTG final concentration of 1.0 mmol·L-1,the induction time of 5-6 h,the expressed recombinant protein existed in the form of inclusion bodies,with molecular mass of about 34 kDa. After refolding,the purified recombinant protein exhibited the antibacterial activity to Staphylococcus aureus in vitro.It is indicated that the prokaryotic expression vector for antimicrobial peptide ranalexin gene from the skin of Rana catesbiana was successfully constructed,and the recombinant antimicrobial peptide Ranalexin was prepared.更多还原