我国结核分枝杆菌现代北京基因型菌株Rv3303c~Rv3304的多态性及生理意义研究  

Polymorphism of Rv3303c−Rv3304 of Mycobacterium tuberculosis modern Beijing genotype strains and physiological significance in China

作　　者：南晓甜 李马超[1] 刘海灿[1] 王蔚 赵秀芹[1] 万康林[1] 赵丽丽[1] Nan Xiaotian;Li Machao;Liu Haican;Wang Wei;Zhao Xiuqin;Wan Kanglin;Zhao Lili(National Institute for Communicable Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China)

机构地区：[1]中国疾病预防控制中心传染病预防控制所,北京102206

出　　处：《疾病监测》2024年第5期592-596,共5页Disease Surveillance

基　　金：传染病重大专项课题(No.2018ZX10302302)。

摘　　要：目的分析我国141株结核分枝杆菌现代北京基因型菌株Rv3303c~Rv3304基因间隔区的多态性,探讨Rv3303c~Rv3304基因间隔区的生理意义。方法通过聚合酶链式反应(PCR)及测序技术分析141株结核分枝杆菌现代北京基因型菌株中Rv3303c~Rv3304基因间隔区序列,构建Rv3303c~Rv3304基因间隔区的重组质粒,将重组质粒及空载质粒电转入耻垢分枝杆菌mc2155中,β-半乳糖苷酶(报告基因lacZ表达产物)活性试验检测报告基因lacZ的表达水平;使用SPSS 25.0软件进行单因素方差分析。结果141株现代北京基因型菌株中Rv3303c~Rv3304基因间隔区存在376 bp、260 bp和144 bp 3种不同长度序列,分别含有4、2和0个拷贝重复序列,以2个拷贝重复序列为主,占比约95.74%;基于这3种不同拷贝重复序列,构建重组质粒pSD5B-334、pSD5B-332、pSD5B-330,并将重组质粒及空载质粒转入耻垢分枝杆菌中;β-半乳糖苷酶活性检测结果显示,与菌株MSMEG-pSD5B相比,重组菌株MSMEG-334、MSMEG-332、MSMEG-330的β-半乳糖苷酶活性均上升,菌株间的β半乳糖苷酶活性的差异有统计学意义(P<0.001)。结论我国结核分枝杆菌现代北京基因型菌株Rv3303c~Rv3304基因间隔区具有多态性,通过模式菌株验证发现其能显著影响报告基因lacZ的转录。Objective To analyze the polymorphism of gene spacer region of Rv3303c−Rv3304 of 141 strains of Mycobacterium tuberculosis in China and discuss the physiological significance of Rv3303c−Rv3304 gene spacer.Methods The sequence of Rv3303c−Rv3304 gene spacer in 141 strains of M.tuberculosis modern Beijing genotype was analyzed by polymerase chain reaction(PCR)and sequencing,and the recombinant plasmid of Rv3303c−Rv3304 gene spacer was constructed,and the recombinant plasmid and empty plasmid were electroporated into Mycobacterium smegmatis mc2155.βgalactosidase(lacZ gene expression product)activity assay was used to detect the expression level of reporter gene lacZ.Software SPSS 25.0 was used to conduct a one-way analysis of variance.Results There were 376 bp,260 bp and 144 bp sequences in the spacer region of the Rv3303c−Rv3304 gene of 141 strains of the modern Beijing genotype,which contained 4,2 and 0 copy repeats,respectively.Two copy repeats were the predominant sequence,accounting for 95.74%.Based on these three different copy repeats,recombinant plasmids pSD5B-334,pSD5B-332,and pSD5B-330 were constructed,and the recombinant plasmids and empty plasmids were transferred into Mycobacterium smegmatis.The results ofβ-galactosidase activity detection showed that theβ-galactosidase activity of recombinant strains MSMEG-334,MSMEG-332 and MSMEG-330 increased compared with strains MSMEG-pSD5B,and the differences ofβ-galactosidase activity among strains were significant(P<0.001).Conclusion In China,the gene spacer region of Rv3303c−Rv3304 of the modern Beijing genotype strain of M.tuberculosis,has polymorphism,which can significantly affect the transcription of reporter gene lacZ indicated by model strain verification.

关 键 词：结核分枝杆菌 现代北京基因型菌株 Rv3303c~Rv3304基因间隔区 Β-半乳糖苷酶活性 

分 类 号：R211[医药卫生—中医学] R521