鼠衣原体毒力基因的筛选和基因差异型菌株的建立

Screening of virulence gene and establishment of isogenic Chlamydia muridarum clones with different genotype

摘要:

【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。

Abstract:

[Objective] To find difference in whole genome sequence between Chlamydia muridarum standard strain and attenuated strain, screening virulence gene and establish the corresponding isogenic organisms with different genotype, and it may lay a foundation for analyzing pathogenic mechanism. [Methods] C. muridarum strain Nigg G0 and attenuated strain G28 were sequenced by deep sequcencing. virulence-associated genes were identified by whole genome sequencing and alignment. A plaque-forming assay was used to isolate many plaques from both CM.G0 and CM.G28 population organisms. Based on PCR sequencing results from virulence-associated gene, individual clones with different genotype were selected. [Results] Whole genome sequencing and alignment revealed that TC0412, TC0237 and TC0668 gene have much difference between CM.G0 and CM.G28. We had obtained 111 plaque samples by plaque-forming assay, and 56 individual clones from CM.G0 and CM.G28 were selected by PCR identification of TC0412, TC0237 and TC0668. Some clones were matched in three pairs (B3, D1, E1) by TC0412 proteiform, and each pair contained the matched organisms which lack of mutation or carrying 100% mutation of TC0237 and TC0668 gene. [Conclusion] The results showed that TC0412, TC0237 and TC0668 gene may be associated with pathogenicity of C. muridarum. The screened clones which have been matched in pair can be used for next function research of virulence gene.