新疆地区牛分枝杆菌29个毒力基因的突变分析

为了解我国新疆地区牛分枝杆菌的毒力基因变异情况并初步评估其毒力变化,利用聚合酶链式反应(PCR)检测临床分离的66株牛分枝杆菌的29个毒力基因,经Sanger测序后与标准株AF2122/97和本实验室保存的两株牛分枝杆菌(M.bovis C68004和M.bovis N)进行序列比对,对基因突变情况进行统计,分析基因突变对其编码蛋白功能和结构的影响。结果表明:1)临床分离株与本实验室保存的高毒力菌株M.bovis N毒力基因突变位点具有很高的相似性,在检测的29个毒力基因中,50%以上临床分离株与M.bovis N共有25个基因序列一致;2)临床分离株广泛存在的Mb2958、Mb2982C和Mb1553C基因突变PROVEAN评分均低于-2.5,可能改变其编码的酶催化活性,进而影响细胞壁脂类合成;3)Mb0607、Mb0609、Mb0606、Mb2979C和Mb1214基因突变分别造成哺乳动物细胞入侵蛋白、甲基转移酶和酰基转移酶蛋白二级结构改变,或许对细菌的胞内生存产生影响。综上,本研究筛选出8个关键毒力基因,这些基因的突变可能在新疆地区牛分枝杆菌流行菌株的毒力变化中发挥重要作用;同时,本研究揭示了关键毒力基因的突变特征,为进一步研究牛分枝杆菌遗传多样性及进化规律奠定了基础。

Mutation analysis of 29 virulence genes of Mycobacterium bovis in Xinjiang

The purpose of this study was to understand the virulence of Mycobacterium bovis(M. bovis) in Xinjiang. A total of 29 virulence genes of 66 clinical isolates of M. bovis were detected by polymerase chain reaction assay(PCR). After Sanger sequencing, the sequences were compared with M. bovis AF2122/97, M. bovis C68004 and M. bovis N. The effects of gene mutations on protein function and structure were analyzed. The results showed that: 1)The isolates tested in this study possessed virulence gene sequences, which shared high similarity to those in highly virulent M. bovis strain N. Among the 29 virulence genes detected, more than 50% of the clinical isolated strains had 25 genes that were consistent with M. bovis N. 2)The PROVEAN score of Mb2958, Mb2982C and Mb1553C gene mutations in clinical isolates were lower than -2. 5, which might change the catalytic activity of the enzyme encoded, thus affected the biosynthesis of cell wall lipids. 3)Mutations of Mb0607, Mb0609, Mb0606, Mb2979C and Mb1214 genes cause the secondary structural changes of mammalian cell entry protein, methyltransferase and acyltransferase proteins, which might affect the intracellular survival of M. bovis. In summary, the mutations of 8 virulence genes might play an important role in the virulence changes of epidemic strains in Xinjiang. This study provides a preliminary reference for further research of molecular characteristics of M. bovis and provides a solid basis for further evolutionary analysis of M. bovis.