鼠防御素基因Crp4在大肠杆菌中的表达及活性检测

通过基因工程技术,以鼠防御素基因Crp4为研究对象,从鼠回肠末端克隆了鼠防御素Crp4基因的成熟编码序列(约为102 bp),将其与另外2个鼠防御素基因的氨基酸序列进行比对,发现同源性分别高达97.8%和97.1%。将该序列克隆到原核表达载体pET-28a中,构建了重组质粒pET-Crp4,转化DE3感受态并用IPTG进行诱导表达。SDS-PAGE和Wersiernblot结果表明,该基因在pET-28a中成功表达。体外抑菌试验证实,重组Crp4蛋白对金黄色葡萄球菌、李斯特菌、大肠杆菌等具有一定的抑菌活性。

Prokaryotic Expression of Mouse Defensin Crp4 in Escherichia coli and Its Activity Detection

Using genetic engineering technique,mouse defensin Crp4 with its length 102 bp is cloned from mouse ileum.Through sequence analysis of Crp4 with other two mouse defensins reported,we found their homogeneity is up to 97.8% and 97.1%.Then the target DNA fragment was cloned into pET-28a vector,and the recombinant vectors were transformed into E.coli DE3,so the expression was induced based on the optimal values of the IPTG concentration incubation temperature and induction time determined in the previous section.The expressed proteins were analyzed by result of SDS-PAGE and Wertern blot.The recombinant Crp4 fusion proteins possess the antimicrobial activity to staphylococcus aureus,E.coli in the assay of drug susceptibility by the method of agar diffusion.So we can further study biological characteristics of defensin and its medical value.