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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 prokaryotic express β-carbonic anhydrase (β-CA), a potential drug target of Schistosoma japonicum, and determine its catalytic activity. Methods According to the conserved sequence of β-CA, the β-CA sequence was identified in the genome of Schistosoma japonicum. After the nucleic acid sequence was completely synthesized, it was introduced into the prokaryotic expression system for fusion expression. The expressed proteins were identified by SDS-PAGE and Western blotting, then purified by Ni affinity chromatography and the carbonic anhydrase activity was determined. Results The DNA sequence Sjp_0056790.1 had a conserved sequence of β-CAs. The DNA sequence was successfully introduced into the recombinant expression vector p ET-32a (+) - Sja CA. The results of SDS-PAGE and Western blotting showed that the fusion protein had a molecular weight of about 38 kDa; carbonic anhydrase activity assay results show that the protein has carbonic anhydrase activity, and this activity is inhibited by acetazolamide. Conclusion Sjp_0056790.1 encodes β-CA of Schistosoma japonicum, and the β-CA of Schistosoma japonicum with catalytic activity was successfully expressed in this study, which laid the foundation for subsequent drug screening.