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【目的】探讨鼠衣原体(Chlamydia muridarum,Cm)标准株与减毒株全基因组序列中存在的差异,筛选毒力基因并建立不同基因型的单克隆菌株,为后续致病机制的研究奠定基础。【方法】将Cm标准株G0和减毒株G28以高通量测序法进行全基因组测序,通过全基因组序列比对分析并筛选潜在的毒力相关基因;经空斑形成实验(Plaque assay)在混合菌G0和G28中大量挑取空斑,以毒力靶基因的PCR测序鉴定从G0和G28中筛选含有不同毒力基因型的单克隆菌株。【结果】全基因测序结果显示Cm G0与G28的TC0412、TC0237和TC0668基因明显不同。通过挑取空斑初步筛选了111个空斑样品,通过3个毒力基因的PCR测序鉴定最终获得了G0和G28来源的56个单克隆菌株,并根据TC0412蛋白型分成B3、D1和E1三组,每组中含有TC0237和TC0668基因差异性菌株。【结论】Cm的致病能力可能与TC0412、TC0237和TC0668基因有关,筛选获得的单克隆菌株通过分组匹配后可用于后续的毒力基因功能研究。
【Objective】 To investigate the differences in the genome-wide sequences of the standard and attenuated strains of Chlamydia muridarum (Cm), to screen virulence genes and to establish monoclonal strains with different genotypes, which lays the foundation for further study on pathogenic mechanism. 【Method】 Whole-genome sequencing of Cm standard strain G0 and attenuated strain G28 was carried out by high-throughput sequencing, and the potential virulence-related genes were screened through genome-wide sequence alignment. The plaque assay Plaques were screened from mixed bacteria G0 and G28. The PCR products of virulent target genes were used to screen out the G0 and G28 monoclonal strains containing different virulence genotypes. 【Result】 The results of whole genome sequencing showed that TC0412, TC0237 and TC0668 genes of Cm G0 and G28 were significantly different. A total of 111 plaque samples were screened by plaque selection. By PCR sequencing of 3 virulence genes, 56 monoclonal strains derived from G0 and G28 were finally obtained and divided into B3, D1 and E1 tris Groups, each containing TC0237 and TC0668 gene differential strains. 【Conclusion】 The pathogenicity of Cm may be related to the gene TC0412, TC0237 and TC0668. The monoclonal strains screened by the screening can be used for the subsequent study of virulence gene function after grouping.