日本血吸虫潜在药物靶点β-碳酸酐酶的表达与鉴定  

Cloning and characterization of β-carbonic anhydrase,a potential drug target of Schistosoma japonicum

作　　者：张聪慧[1] 朱淮民[1] 

机构地区：[1]第二军医大学病原生物学教研室,上海200433

出　　处：《中国血吸虫病防治杂志》2016年第2期161-166,共6页Chinese Journal of Schistosomiasis Control

基　　金：国家科技重大专项(2012ZX10004220);卫生行业科研专项(201202019)

摘　　要：目的原核表达日本血吸虫(Schistosoma japonicum)潜在的药物靶点β-碳酸酐酶(β-CA)并测定其催化活性。方法根据β-CA保守序列,在日本血吸虫基因组中鉴定β-CA序列,核酸序列全合成后将其导入原核表达系统进行融合表达。SDS-PAGE及Western blotting鉴定表达蛋白后,采用Ni亲和层析方法纯化目的蛋白,并测定碳酸酐酶活性。结果c DNA序列Sjp_0056790.1具有β-CAs的保守序列;成功将该核酸序列导入重组表达载体p ET-32a(+)-Sja CA中;SDSPAGE和Western blotting检测结果显示融合蛋白分子量大小约为38 k Da;碳酸酐酶活性测定结果显示该蛋白具有碳酸酐酶活性,且该活性受乙酰唑胺抑制。结论 Sjp_0056790.1编码日本血吸虫β-CA,本研究成功表达了具有催化活性的日本血吸虫的β-CA,为后续的药物筛选奠定了基础。Objective To express the beta carbonic anhydrase（β-CA）of Schistosoma japonicum,and analyze its catalytic activity.Methods The c DNA and amino sequence which may encode β-CA of S.japonicum were obtained by the bioinformatics-method,and then the c DNA sequence was cloned into prokaryotic expression vector p ET-32a（＋）and expressed.After examining by SDS-PAGE and Western blotting,the recombinant protein was purified by Ni-affinity chromatography and the catalytic activity was determined.Results The sequence Sjp_0056790.1 took on the conservative position of β-CAs.The PCR and restriction enzyme digestion confirmed the construction of recombinant plasmid p ET-32a（＋）-Sja CA.SDS-PAGE and Western blotting analyses showed that the molecular weight of recombinant protein was about 38 k Da as expected,and it could be recognized by anti-His tag antibody.The catalytic activity determining revealed that the recombinant protein Sja CA owned the carbonic anhydrase activity.Conclusion Sjp_0056790.1 encodes the β-CA of S.japonicum,and the β-CA with catalytic activity is successfully expressed,so it lays a foundation for the subsequent research of pharmacological inhibition,providing theoretic basis for searching and developing a new feasible anti-schistosome drug.

关 键 词：日本血吸虫 β-碳酸酐酶(β-CA) 药物靶点 表达 活性测定 

分 类 号：R383.24[医药卫生—医学寄生虫学]