高致病性2型猪链球菌Ⅳ型样分泌系统virB1-89K基因的敲除及其对毒力的影响

摘要

[Objective] To construct the virB1-S9K gene knockout mutant and its complementary strain of Streptococcus suis serotype 2 ( SS2 ) highly virulent strain 05ZYH33 and evaluate the role of virB1-S9K in the pathogenesis of SS2.[Methods] The virB1-89K gene was knocked out by homologous recombination, then multiple-PCR and sequence analysis were used to identify the knockout strain △virB/-89K.The virB1 -89 K gene and its upstream promoter were cloned into the E.coli-S.suis shuttle vector pSETl , and the recombinant plasmid was electrotransformed into the △virBi-89K mutant to generate the complementary strain CvirB1-89K.The effects of virB1-89K deletion on the basic biological characteristics and virulence of SS2 were then determined in this study.[Results] The isogenic mutant △virB1-89K and its complementary strain △virB1-89K were successfully constructed.No significant differences in biological characteristics were found among the three strains.However, the virulence of the △virBl -89K.mutant was reduced to 30% of the wild-type level and functional complementation of virBl-89K restored its pathogenicity.[Conclusion] The virB1-89K gene plays an important role in the pathogenesis of S.suis 2 infection.%[目的]构建高致病性2型猪链球菌Ⅳ型样分泌系统vir B1-89K基因的敲除株和互补株,研究vir B1-89K基因缺失对细菌毒力的影响.[方法]通过同源重组技术敲除vir B1-89K基因,多重PCR筛选敲除株并测序鉴定.再将virB1-89K基因克隆到穿梭质粒pSET1后转入vir B1-89K敲除株中,构建互补株.比较野生株05ZYH33、突变株△virB1-89K和互补株CvirB1-89K三者基本生物学特性的差异,小鼠实验分析virB1-89K基因敲除后对细菌毒力的影响.[结果]成功构建突变株△vir B1-89K和互补株CvirB1-89K,在基本生物学性状无明显改变的情况下,敲除株的毒性降低到野生株的30％,互补株可恢复其毒性.[结论]virB1-89K基因作为2型猪链球菌高致病性菌株05ZYH33的Ⅳ样分泌系统的重要组分,与其高致病性密切相关.