论文部分内容阅读
目的 探讨过氧化氢酶编码基因 (KatG)基因点突变和结核分枝杆菌异烟肼 (INH)耐药性之间的关系 ,筛选相关位点用于筛查临床INH耐药株基因型。方法 采用三步法聚合酶链反应(PCR)对临床耐异烟肼菌株中发现的 6个位点进行定点诱变 ,用含诱变基因的 pET2 4b质粒转化大肠埃希菌BL2 1菌株 ,在异丙基硫代 β D半乳糖苷 (IPTG)诱导下表达 ,使用镍亲和柱和Q 琼脂糖快速流动凝胶纯化各KatG诱变蛋白并测定其过氧化氢酶和过氧化物酶活性。结果 完成KatG基因 6个位点的诱变及相应诱变蛋白的表达和纯化 ,过氧化氢酶、过氧化物酶活性测定发现 ,3个诱变蛋白[KatG(W32 1G)、KatG(R4 18Q)和KatG(A4 5 6S) ]基本失活 ,其余 3个诱变蛋白的活性亦有不同程度下降。结论 所引入的 6个突变均可造成KatG蛋白过氧化氢 过氧化物酶活性下降 ,与结核分枝杆菌INH耐药有关 ,可用于筛查临床INH耐药株基因型
Objective To investigate the relationship between point mutation of catalase (KatG) gene and resistance to isoniazid (INH) in Mycobacterium tuberculosis and screen the relevant sites for the screening of clinical INH resistant strains. Methods Six sites were identified in clinical iso-isoniazid-resistant strains by three-step polymerase chain reaction (PCR), and the Escherichia coli BL21 strain was transformed with the pET2 4b plasmid containing the mutagenesis gene. Isopropylthiogalactopyranoside (IPTG), each KatG mutagenesis protein was purified using a nickel affinity column and Q Sepharose fast-flow gel and its catalase and peroxidase activities were determined. Results The mutagenesis of KatG gene at 6 loci and the expression and purification of the corresponding mutagenized proteins were completed. The activities of catalase and peroxidase were determined. Three mutagenic proteins [KatG (W32 1G), KatG (R4 18Q ) And KatG (A4 5 6S)] were basically inactivated, and the activity of the other three mutagenic proteins also decreased to some extent. Conclusion All of the 6 mutations introduced can cause the decrease of catalase activity of KatG protein, which is related to the resistance of Mycobacterium tuberculosis INH and could be used to screen the genotype of INH resistant strains