猪链球菌2型ABC转运蛋白gene0910敲除突变体的构建及活性分析

Construction and virulence evaluation of gene knock-out mutant of an ABC transporter gene0910 of Streptococcus suis Type 2

目的:构建猪链球菌2型(Streptococcus suis type 2)强毒株05ZYH3389K毒力岛上的ABC转运蛋白gene0910敲除突变体,并初步分析其活性,为进一步研究猪链球菌假想毒力因子在致病中的作用提供实验基础。方法:以猪链球菌2型05ZYH33基因组为模板,扩增gene0910两侧各约500bp左右的片段为上下游同源臂,以pSET1质粒为模板,扩增氯霉素抗性基因Cm为中间片段,采用重叠PCR方法搭建三个片段,并克隆到自杀载体pSET4S上,构建基因敲除的载体。电转化05ZYH33感受态细胞,经30℃双交换和40℃质粒丢失,最后点板法筛选出基因敲除突变体△0910。对突变株和野生株的生物学活性及小鼠的致病性进行了初步比较。结果:PCR分析和测序结果均显示gene0910完全被氯霉素抗性基因Cm所替代,基因敲除突变体构建成功。结论:突变株的生物学活性和对小鼠的致病性与野生株相比差异不显著。

Objective：To construct gene knock-out mutant of an ABC transporter gene0910 which is located on candidate pathogenicity island 89 K of Streptococcus suis type 2（SS2） virulent strain 05ZYH33 and evaluate its biological activity in order to provide an experimental basis for studying the role of SS2＇putative virulence factors in pathogenesis.Methods：Genomic DNA of Streptococcus suis type 2 05ZYH33 and pSET1 plasmid as template were used to amplify the 500 bp fragments of an ABC transporter gene0910 and the chromosomal（Cm） resistance cassette,respectively.By overlap extension PCR method.recombinant gene knock-out vector was constructed consisting of Cm cassette with flanking homology regions to the target gene.The plasmid pSET4S-0910 was transformed into wild strain 05ZYH33,by 30℃ double-crossover and 40℃ plasmid loss,the gene knock-out mutant strain △0910 was screened.Biological characteristics and virulence of mutant strain △0910 in mice were compared with wild-type strain 05ZYH33.Results：PCR analysis and sequencing confirmed that the coding gene0910 was replaced completely by Cm cassette.Conclusions：There were no significant differences in biological characteristics and virulence in mice between mutant strain △0910 and wild-type strain 05ZYH33.