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【目的】明确肺炎链球菌荚膜多糖合成操纵子的启动子序列出现的点突变313713 T→C是否可导致细菌荚膜缺失。【方法】Western blot检测肺炎链球菌突变株SPY1(NC_008533.1 313713 T→C)荚膜多糖含量;实时定量荧光PCR分析荚膜多糖合成基因cps2A、cps2B、cps2C以及cps2D的表达量;构建重组质粒pEVP3-cps promoterD39和pEVP3-cps promoterSPY1,分别转化D39和SPY1菌株,通过β-半乳糖苷酶活性检测来验证转入的启动子序列对细菌荚膜合成的影响,并通过电镜观察荚膜结构和荚膜肿胀试验进一步验证。【结果】肺炎链球菌SPY1的荚膜多糖含量较野生型显著下降,其相关基因cps2A、cps2B、cps2C以及cps2D的表达量较野生型D39均显著降低;与D39-pEVP3-cps promoterD39对比,D39-pEVP3-cps promoterSPY1的β-半乳糖苷酶活性下降了76%,与SPY1-pEVP3-cps promoterD39相比,SPY1-pEVP3-cps promoterSPY1的β-半乳糖苷酶活性下降了约79%;电镜结果显示,重组SPY1-pEVP3-cps promoterD39荚膜可恢复至野生型水平,并且重组D39-pEVP3-cps promoterSPY1(NC_008533.1 313713 T→C)荚膜肿胀试验呈阴性。【结论】荚膜多糖合成操纵子的启动子序列点突变313713 T→C可导致荚膜多糖合成基因表达显著下调,从而引起菌株SPY1的荚膜显著减少甚至缺失。
【Objective】 To determine whether point mutation 313713 T → C in the promoter sequence of S. pneumoniae capsular polysaccharide synthesis operon can result in bacterial capsular deletion. 【Methods】 The content of capsular polysaccharide of Streptococcus pneumoniae mutant strain SPY1 (NC_008533.1 313713 T → C) was detected by Western blot. The expression of capsular polysaccharide synthesis genes cps2A, cps2B, cps2C and cps2D was analyzed by real-time quantitative PCR. The recombinant plasmid pEVP3-cps promoterD39 and pEVP3-cps promoterSPY1 were transformed into D39 and SPY1 strains respectively. The effect of the promoter sequence introduced into the capsid was verified by β-galactosidase activity assay. Capsule swelling test further validation. Results Compared with wild-type D39-pEVP3-cps promoter D39, the expression of capsular polysaccharide of Streptococcus pneumoniae SPY1 was significantly lower than that of wild-type D39-pEVP3-cps promoter D39, and the expressions of cps2A, cps2B, cps2C and cps2D were significantly decreased The β-galactosidase activity of pYP3-cps promoterSPY1 decreased by 76%, and the β-galactosidase activity of SPY1-pEVP3-cps promoterSPY1 decreased by about 79% compared with that of SPY1-pEVP3-cps promoterD39. The result of electron microscopy , The recombinant SPY1-pEVP3-cps promoterD39 capsular returned to the wild-type level, and the recombinant capsid swelling test of D39-pEVP3-cps promoterSPY1 (NC_008533.1 313713 T → C) was negative. 【Conclusion】 The point mutation 313713 T → C in the promoter region of capsular polysaccharide synthesis operon can result in significant down-regulation of the gene expression of capsular polysaccharide synthesis, resulting in significant reduction or even loss of the capsular strain of strain SPY1.