中文摘要：

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。

英文摘要：

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.