sprE基因敲除粪肠球菌突变株的构建和功能的初步研究

目的构建丝氨酸蛋白酶同源基因(serine protease gene,sprE)敲除粪肠球菌突变株,来研究sprE基因的功能。方法用pTX4577质粒构建粪肠球菌sprE基因重组自杀质粒pCQ001,通过体内同源重组,筛选获得sprE基因的突变株,体外研究不同温度和氧化条件对突变株生长能力的影响。结果经同源重组,利用卡那霉素抗性筛选,PCR、脉冲场电泳和southern blot进行鉴定获得基因突变株#sprE,突变株在40℃的生长能力以及在氧化条件下的存活率均明显降低。结论sprE基因敲除粪肠球菌突变株#sprE构建成功,为进一步研究其功能打下基础。

Construction of the sprE gene knock-out mutant of Enterococcus faecalis and its preliminary functional study

The knock-out mutant on the serine protease homeobox gene(sprE) of Enterococcus faecalis was constructed in the present study in order to investigate its function,in which recombinant suicide vector pCQ001 of E.faecalis was firstly constructed by means of plasmid pTX4577,and then the isogenic sprE-deficient mutant was screened by allelic replacement.Moreover,in order to investigate the growth ability,mutant strains were compared with wide-type strain in vitro.It was shown that the mutant with sprE gene mutation could be obtained after kanamycin-screening and identification by PCR,pulsed field gel electrophoresis(PFGE) and Southern blotting. The experimental evidence indicated that the growth ability under 40℃ and the survival rate under oxidative stress of the mutant strain was remarkably reduced in comparison with those of the wide-type strain.