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

Construction and virulence evaluation of the virB1-89K gene knockout mutant of type IV-like secretion system of Streptococcus suis serotype 2

摘要:

摘要:【目的】构建高致病性2 型猪链球菌IV 型样分泌系统virB1-89K基因的敲除株和互补株，研究virB1-89K基因缺失对细菌毒力的影响。【方法】通过同源重组技术敲除virB1-89K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virB1-89K敲除株中，构建互补株。比较野生株05 ZYH33、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△virB1-89K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05 ZYH33的IV样分泌 系统的重要组分，与其高致病性密切相关。

关键词:关键词:2 型猪链球菌，Ⅳ型样分泌系统，virB1 基因，基因敲除

Abstract:

Abstract:［Objective］To construct the virB1-89K gene knockout mutant and its complementary strain of Streptococcus suis serotype 2 (SS2) highly virulent strain 05ZYH33 and evaluate the role of virB1-89K 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 ΔvirB1-89K．The virB1-89K gene and its upstream promoter were cloned into the E．coli-S．suis shuttle vector pSET1，and the recombinant plasmid was electrotransformed into the ΔvirB1-89K 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 ΔvirB1-89K and its complementary strain CvirB1-89K were successfully constructed． No significant differences in biological characteristics were found among the three strains． However，the virulence of the ΔvirB1-89K mutant was reduced to 30% of the wildtype level and functional complementation of virB1-89K restored its pathogenicity．[Conclusion] The virB1-89K gene plays an important role in the pathogenesis of S．suis 2 infection．

Key words:Keywords: Streptococcus suis serotype 2，type IV-like secretion system，virB1 gene，knockout