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目的 测定脓胸 (CF)病人铜绿假单胞菌分离株外毒素A(ETA)的ADP核糖转移酶(ADPRT)活性及其酶活区 (Ⅲ区 )序列的变化 ,为研究ETA在CF病人慢性肺部感染致病机理中的作用提供试验基础和理论依据。方法 采用PCR技术 ,以铜绿假单胞菌PA SD株的染色体DNA为模板 ,扩增ETAⅢ区基因。将产物克隆于pGEMR TEasy载体 ,鉴定后测序。用DNASTAR软件将测定的序列与GenBank中获得的其它CF分离株序列进行比较。同时平行测定PA SD株与标准株PA10 3的ADPRT活性。结果 扩增出目的基因片段 ,长度为 6 5 8bp ,获得了重组质粒 (PA SD ETA)。测序结果表明 ,PA SD株与所比较菌株的氨基酸的同源性在 94 .4 %～ 98.1%之间 ,其中与标准株的同源性为98.1%。第 6 0 8位发生了相对于所比较的全部分离株的突变 ,第 5 15位发生Ser到Gly的突变 ,同时ADPRT活性比标准株PA10 3低。结论 本研究进一步阐明第 5 15位氨基酸与ADPRT活性的关系 ,从而为研究ETA在CF病人慢性肺部感染致病机理中的作用提供理论依据。
OBJECTIVE: To determine the changes of ADPRT activity and its active region (Ⅲ region) sequence in exotoxin A (ETA) isolated from patients with empyema (CF) The pathogenesis of pulmonary infection in the role of providing experimental basis and theoretical basis. Methods The chromosomal DNA of Pseudomonas aeruginosa PA SD strain was used as a template to amplify the ETA Ⅲ region gene by PCR. The product was cloned into pGEMR TEasy vector and sequenced. The determined sequences were compared with other CF isolates obtained in GenBank using DNASTAR software. ADPRT activity of the PA SD strain and the standard strain PA10 3 were measured in parallel. Results The target gene fragment was amplified with a length of 658 bp and a recombinant plasmid (PA SD ETA) was obtained. The sequencing results showed that the amino acid homology between PA SD strain and the tested strains was between 94.4% ~ 98.1%, and the homology with the standard strain was 98.1%. The mutation at position 608 relative to all the isolates was mutated, a Ser to GIy mutation at position 5-15, and a lower ADPRT activity than the standard strain PA103. Conclusion This study further elucidates the relationship between amino acid number 5 15 and ADPRT activity, which will provide a theoretical basis for studying the role of ETA in the pathogenesis of chronic pulmonary infection in CF patients.