结核分枝杆菌异烟肼耐药基因(katG)调控蛋白的表达  

Cloning and expression of the genes coding for Mycobacterium tuberculosis antigen katG

作　　者：袁薇[1] 陈旭 赵兵兵 魏川川[1] 许士池 刘英[1] YUAN Wei;CHEN Xu;ZHAO Bing-bing;WEI Chuan-chuan;XU Shi-chi;LIU Ying(Institute of Tuberculosis Control,Guizhou Center for Disease Control and Prevention,Guiyang550004,China;不详)

机构地区：[1]贵州省疾病预防控制中心结核病防治研究所,贵阳550004 [2]贵州中医药大学附属二院

出　　处：《医学动物防制》2021年第4期374-377,共4页Journal of Medical Pest Control

基　　金：贵州省科技计划项目(黔科合支撑[2018]2762,黔科合基础[2019]1186号)。

摘　　要：目的katG基因表达异常对一线抗痨药物异烟肼的耐药性意义重大,克隆并表达结核分枝杆菌katG基因的调控蛋白—furA、katG、senX3和regX3,有助于探讨其耐药机制。方法以结核分枝杆菌标准菌株H37Rv基因组DNA序列为模板,设计furA、katG、senX3和regX3四种基因的特异性引物,分别进行PCR扩增,获得目的基因,通过克隆载体pMD19-T构建重组质粒,转化E coli DH5α,经DNA测序并与Gen Bank进行比对分析验证,质粒酶切后再亚克隆到表达载体pET42a(+),转化BL21(DE3)中,IPTG诱导重组目的蛋白表达,SDS-PAGE电泳分析重组蛋白大小,Western Blot鉴定表达情况。结果PCR扩增出furA、katG、senX3和regX3基因,成功构建重组质粒pET42a(+)-furA、pET42a(+)-katG、pET42a(+)-senX3、pET42a(+)-regX3,0.5 mmol/L IPTG诱导表达重组蛋白,经SDS-PAGE电泳和Western Blot验证,furA重组蛋白在23 KD、regX3重组蛋白在28 KD、senX3重组蛋白在46 KD和katG重组蛋白在80 KD处分别出现蛋白条带,重组蛋白主要以可溶形式表达。结论应用分子生物学技术成功构建furA、katG、senX3和regX3基因原核表达系统,并获得相应重组蛋白,为进一步研究其结构和功能,探讨以katG为核心的异烟肼耐药机制奠定基础。Objective Abnormal katG expression is significant for resistance to the first-line anti-TB drug isoniazid,to clone and express the genes furA,katG,senX3 and regX3 coding for Mycobacterium tuberculosis,obtain the recombinant protein,could help to explore the mechanism of drug resistance.Methods The genes furA,katG,senX3 and regX3 were amplified from Mycobacterium tuberculosis H37 Rv genomic DNA by polymerase chain reaction(PCR),and cloned into plasmid pMD19-T.The DNA was sequenced and compared with Gen Bank for verification.By restricted enzymes digestion,the plasmid pET42 a(+)-furA,pET42 a(+)-katG,pET42 a(+)-senX3 and pET42 a(+)-regX3 were transformed into E coli DH5α,extraction plasmid,the plasmid pET42 a(+)-furA,pET42 a(+)-katG,pET42 a(+)-senX3 and pET42 a(+)-regX3 were transformed into E coli BL21(DE3)and induced by IPTG;the furA,katG,senX3 and regX3 fusion protein expressed was analyzed by SDS-PAGE and Western Blot.Results The furA,katG,regX3 and senX3 were amplified by PCR,recombinant plasmids pMD19-T-furA,pMD19-T-katG,pMD19-T-senX3 and pMD19-T-regX3 were consistent with those reported by Gen Bank.The proteins were expressed in E coli BL21(DE3)as soluble protein.SDS-PAGE and Western Blot showed that the Mr of these gene product were 23 KD,80 KD,46 KD and 28 KD.Conclusion The furA,katG,senX3 and regX3 proteins were successfully obtained by molecular biological technique and obtained the corresponding,laying the foundation for further study of their structure and function and exploring the mechanism of isoniazid resistance with katG as the core,which provide an experimental basis for further studies of the structure of the genes.

关 键 词：结核分枝杆菌 基因克隆 KATG基因 二元信号转导系统 蛋白表达 耐药 

分 类 号：R378.911[医药卫生—病原生物学]