副溶血性弧菌vtrB基因敲除菌株的构建及其在抵抗胆汁中的作用

Construction of vtrB gene knockout strain of Vibrio parahaemolyticus and its role in bile resistance

摘要:

构建副溶血性弧菌转录调节因子VtrB （V.parahaemolyticus T3SS2 regulator B）敲除菌株和回补菌株，探讨VtrB在副溶血性弧菌抵抗胆汁中的作用。根据GenBank数据库中副溶血性弧菌vtrB基因上下游序列设计引物，通过融合PCR将vtrB基因上下游同源臂融合并克隆入自杀质粒pDS132中，将重组自杀质粒转入大肠杆菌S17-1 λpir中，再接合转移至副溶血性弧菌中进行同源重组，通过10%蔗糖筛选获得vtrB基因突变株ΔvtrB。将pBAD33-vtrB重组质粒电转入ΔvtrB突变株中获得回补菌株C-ΔvtrB。研究副溶血性弧菌野生型、突变株和回补株在2%脱氧胆酸盐（DOC）处理后的存活率。基于同源重组原理，经vtrB基因的PCR扩增和转录水平检测结果表明，成功获得副溶血性弧菌vtrB基因缺失突变株ΔvtrB和回补株C-ΔvtrB。与野生型和回补菌株C-ΔvtrB相比，ΔvtrB突变株在2% DOC处理20、40和60 min后的存活率分别为17%、4%和1%，vtrB基因的失活使得副溶血性弧菌对胆汁抵抗能力极显著降低（P<0.001），表现出存活能力的缺陷。由此表明，转录调节因子VtrB有助于副溶血性弧菌对胆汁的抵抗。

Abstract:

Vibrio parahaemolyticus transcriptional regulator vtrB gene knockout and complementary strains were constructed, and the role for VtrB in bile resistance was also explored. Two pairs' primers were designed according to the vtrB gene in GenBank. The upstream and downstream flanking DNA fragments of vtrB gene were fused by PCR, and then cloned into the suicide plasmid pDS132. The recombinant suicide vector was transferred from E. coli S17-1 λpir to V. parahaemolyticus for homologous recombination. The vtrB gene mutants were screened by 10% sucrose. Complement strain C-ΔvtrB was constructed by transferring pBAD33-vtrB into ΔvtrB mutant strain. The survival rates of wild type, ΔvtrB mutant and C-ΔvtrB strains cultured in media containing 2% deoxycholate (DOC) were determined. Based on the principle of homologous recombination, the PCR amplification and transcription level detection results of the vtrB gene showed that V. parahaemolyticus vtrB gene mutant ΔvtrB and its complementary strain C-ΔvtrB were successfully constructed. The survival rates of the ΔvtrB strain was 17%, 4% and 1% in the presence of 2% DOC for 20 min, 40 min and 60 min, respectively. This was a significantly lower level of survival than that of the wild type and C-ΔvtrB strains (P<0.001). The results indicated that the VtrB is helpful to the bile resistance of V. parahaemolyticus.