分枝杆菌利福平耐药基因rpoB高通量突变文库的构建  

Construction of a High-Throughput Mutation Library of Mycobacterium Rifampin Resistance Gene rpoB

作　　者：陈勇[1] 冯思源 徐霖[2] 田国宝[2] CHEN Yong;FENG Si-yuan;XU lin;TIAN Guo-bao(School of Laboratory Medicine,Chengdu Medical College,Chengdu 610500,China;Department of Microbiologyand Immunology,Zhongshan School of Medicine,Sun Yat-sen University,Guangzhou 510080,China)

机构地区：[1]成都医学院医学检验系,四川成都610500 [2]中山大学中山医学院免疫学与微生物学系,广东广州510080

出　　处：《中山大学学报（医学科学版）》2023年第4期634-641,共8页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：国家自然科学基金(81830103,82061128001)。

摘　　要：【目的】建立利福平耐药基因rpoB高通量突变文库。【方法】通过同源重组和L5 attB噬菌体整合位点交换构建耻垢分枝杆菌ΔrpoB attB::rpoB菌株;基于L5 attB质粒交换系统和抗性选择培养基,挑取48个克隆以验证质粒替换的效率;针对利福平耐药决定区(RRDR)区域内以每3个碱基为单位设计简并性引物,通过PCR扩增获得该区域81个碱基的全覆盖式突变文库;文库片段与载体进行无缝克隆转化到大肠杆菌中形成大肠杆菌突变文库;基于质粒交换原理,将突变质粒库转化至耻垢分枝杆菌ΔrpoB attB::rpoB菌株中,替换原有L5 attB位点质粒,形成耻垢分枝杆菌突变文库;通过基因组提取、建库和高通量测序明确文库的基因型种类。【结果】与野生型rpoB基因(5600 bp)相比,敲除株的扩增片段为2200 bp,证明耻垢分枝杆菌ΔrpoB attB::rpoB条件性敲除株构建成功;质粒替换的成功率为100%;大肠杆菌文库和耻垢分枝杆菌文库中单氨基酸突变类型均为540种,大肠杆菌文库多点突变5301种,耻垢分枝杆菌文库多点突变853种,大肠杆菌文库和耻垢分枝杆菌文库相关系数为0.84。【结论】开发了一种针对分枝杆菌必需基因rpoB突变体文库的构建策略,成功建立了利福平耐药基因rpoB的突变文库。【Objective】To establish a mutation library of rifampicin resistance gene rpoB.【Methods】TheΔrpoB attB::rpoB strain of Mycobacterium smegmatis(M.smegmatis)be constructed by homologous recombination and L5 attB phage integration site exchange.Based on the L5 attB plasmid exchange system and resistance selection medium,48 clones are selected to verify plasmid replacement efficiency.Degenerate primers are designed every 3 bases in the rifampicin resis⁃tance determining region(RRDR),and a full-coverage mutation library of 81 bases in RRDR region is obtained by PCR amplification.The library fragments are seamlessly cloned into the vector and transformed into Escherichia coli(E.coli)to form an E.coli mutation library.Based on the principle of plasmid exchange,the mutant plasmid library is transformed into the M.smegmatis strainΔrpoB attB::rpoB,and the original L5 attB site plasmid is replaced to form the M.smegmatis mu⁃tant library.The genotype of the library are determined by genome extraction,library construction and high-throughput se⁃quencing.【Results】Compared with the wild-type rpoB gene(5600 bp),the amplified fragment of the rpoB knockout strain is 2200 bp,which proved that theΔrpoB attB::rpoB conditional knockout strain of M.smegmatis is successfully constructed.The success rate of plasmid replacement is 100%.There were 540 kinds of single amino acid mutations in both E.coli library and M.smegmatis library,5301 kinds of multi-point mutations in E.coli library,and 853 kinds of multi-point mutations in M.smegmatis library.The correlation coefficient between E.coli library and M.smegmatis library is 0.84.【Conclusions】We have developed a strategy to construct a library of mutants targeting the essential mycobacterial gene rpoB,and successfully established a mutant library of rifampicin resistance gene rpoB.

关 键 词：分枝杆菌 利福平耐药 突变文库 RPOB 

分 类 号：R378[医药卫生—病原生物学]