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【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virB1-89 K基因缺失对细菌毒力的影响。【方法】通过同源重组技术敲除virB1-89 K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△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05ZYH33的IV样分泌系统的重要组分,与其高致病性密切相关。
【Objective】 To construct the knockout and complementation strains of the virB1-89K gene of the highly pathogenic Streptococcus suis type 2 Streptococcus suis serotype 2 and to study the influence of virB1-89 K deletion on bacterial virulence. 【Method】 The virB1-89 K gene was knocked out by homologous recombination technique. The knockout strains were screened by multiplex PCR and sequenced. The virB1-89K gene was cloned into shuttle plasmid pSET1 and transformed into virB1-89K knockout strain to construct a complementary strain. The differences of the basic biological characteristics between the wild-type strain 05ZYH33, the mutant strain △ virB1-89K and the complementary strain CvirB1-89K were compared. The effect of virB1-89K gene knockout on the virulence of the bacteria was analyzed in mice. 【Result】 The mutant strain △ virB1-89K and the complementary strain CvirB1-89K were successfully constructed. The knockout strain was reduced to 30% of the wild strain without any significant change in the basic biological traits, and the complementation strain recovered its toxicity. 【Conclusion】 The virB1-89K gene is an important component of the IV-like secretion system of the highly pathogenic strain 05ZYH33 of Streptococcus suis type 2, which is closely related to its high pathogenicity.