依赖利福平结核分枝杆菌的分子生物学研究  

Molecular biological on rifampim-depending M.Tuberculosis straims isolated from patients

作　　者：钟敏[1] 温博海[2] 陈荣[2] 陈炜[2] 王易伟[1] 王安容[1] 钟明[1] 

机构地区：[1]重庆市肺科医院检验科,400020 [2]第三军医大学微生物学教研室

出　　处：《中华检验医学杂志》2003年第1期42-45,共4页Chinese Journal of Laboratory Medicine

基　　金：am重庆市医学重点项目 (No .99 -10 0 2 );重庆市科委攻关项目 ( 2 0 0 0 - 6 30 9)

摘　　要：目的　从分子生物学的角度研究依赖利福平结核分枝杆菌 (依R菌 )。方法　采用DNA自动测序仪 ,对菌株rpoB基因片段 ( 319bp)的序列进行分析 ;采用随机扩增DNA聚丙酰胺凝胶电泳 (RAPD PAG)指纹分型法 ,对菌株的DNA指纹进行分型 ;采用SDS聚丙酰胺凝胶蛋白电泳法 ,对菌株的菌体蛋白进行分析。结果　 30株依R菌rpoB基因的突变率为 96 7% ( 2 9/ 30 ) ,37株耐利福平结核分枝杆菌 (耐R菌 )为 81 1% ( 30 / 37) (P <0 0 1) ,依R菌和耐R菌的突变高发位点均为 5 31位( 37/ 5 9,6 3 2 % ;)密码子、突变 ,其次为 5 16位 ( 13/ 5 9,2 2 8% )和 5 2 6位 ( 7/ 5 9,12 3% ) ;依R菌和耐R菌两组菌株的DNA指纹结果可分为 5种类型 ,其中以Ⅱ型 ( 31/ 6 7,46 2 % )、Ⅰ型 ( 2 0 / 6 7,2 9 9% )和Ⅲ型 ( 13/ 6 7,19 4% )为主 ;3种主要类型的指纹在两组菌株中的分布无明显特殊性 ;依R菌在含R管和对照管中的菌体蛋白电泳图谱有 76 7% ( 2 3/ 30 )的不同 ,其在含R管中菌体蛋白表达丰富 ,且在分子量 310 0 0～ 43 0 0 0间有一宽带 ;37株耐R菌其两管的菌体蛋白电泳图谱各自均相同。结论　依R菌和耐R菌的基因型密切相关 ;依R菌的蛋白表达可依赖于R。Objective To study the molecular biology of rifampin-depending M. Tuberculosis. Methods The seguence (a 319-bp DNA fragment) of rpoB gene were analyzed by automated DNA sequencing machine. (2) The fingerprints of genomic DNA were obtained by random amplified polymorphic DNA (RAPD) fingerprinting. (3)The protein electrophoresis of bacterium by SDS-polyacrylamide gel (SDS-PAG).(4) The cases of pulmonary tuberculosis by rifampin-depending strains were retrospectively analyzed. Results (1) rpoB gene sequenced: The point mutationrate of rifampin-depending strainswas 96.7%(29/30) and that of rifampin-residtant strains 81.1%(30/37), P<0.01, the most positions of the mutation were at codon 531(37/59), followed by 516(13/59) and 526(7/59).(2)DNA fingerprints:Five types of DNA fingerprints were determined; the main types were Ⅱ, Ⅰ and Ⅲ and they were 46.2%(31/67), 29.9%(20/67)and19.4% (13/67), respectively. The 3 main types distribution in rifampin-depending and rifampin-resistant strains were not signicantly different. (3)Protein electrophoresis showed The inequality of protein expression (76.7%, 23/30) of rifampin-depending strains in the media containing rifampin and control stains in L-J media, but both had a wrde band of between 31 000 and 43 000 in the media containing rifampin. Conclusions (1)Our results reveal that there is a strong correlation(the Genotype) between rifampin-depending and rifampin-resistant of M. Tuberculosis. (2)The protein expression of rifampin-depending strains dependon. rifampin.

关 键 词：利福平 结核分枝杆菌 分子生物学 结核病 基因 

分 类 号：R346[医药卫生—基础医学]