结核分枝杆菌Rv2629基因产物的亚细胞定位和穿梭质粒转化耻垢分枝杆菌

Subcellular location of Rv2629 in M.tuberculosis and its transformed version into M.smegmatis using the pMV261 plasmid

目的研究结核分枝杆菌利福平耐药相关蛋白Rv2629在细胞内的亚定位及其与药敏的相关性。方法采用差速离心进行细胞组分分离及蛋白质印迹法(Western blot)检测初步判定蛋白亚细胞定位;采用pMV261转化耻垢分枝杆菌,BACTEC MGIT960测定转化菌株的利福平耐受性。结果Rv2629蛋白主要定位于结核分枝杆菌的细胞壁和细胞膜,重组有Rv2629突变位点191C质粒的耻垢分枝杆菌对利福平的最低抑菌浓度(MIC)为160mg/L,相应的携带有野生型基因191A的宿主菌MIC为20mg/L。结论Rv2629基因191A/C突变同利福平耐药相关。

Objective To detect the subcellular location of Rv2629 in M. tuberculosis, and to test the drug susceptibility of M. smegmatis following the introduction of Rv2629 using the pMV261 plasmid.Methods Different components of M. tuberculosis were separated by differential velocity centrifugation, and subcellular location of Rv2629 was examined by Western blot. The pMV261 plasmid was used to transformM, smegmatis so that it contained the Rv2629 gene. Rifampicin （RIF） resistance was assayed using a BACTEC MGIT 960 instrument. Results Rv2629 was found mainly in the cell wall and cell membrane of M. tuberculosis. The minimum inhibitory concentration （MIC） of RIF for the transformed M. smegmatis containing the mutated Rv2629 gene （strain 191C） was 160 mg/L, while the MIC for the wild type Rv2629 gene （strain 191A） was 20 mg/L, similar to the parental strain. Conclusion These results indicate that the 191A/C mutation of Rv2629 gene is associated with RIF resistance.