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目的构建日本血吸虫PP2C-1蛋白磷酸酶表达载体,并进行体外表达和酶活性分析。方法从日本血吸虫基因组数据库中找到一个可能编码PP2C-1蛋白磷酸酶的基因Sj-PP2C-1,设计引物并通过PCR技术扩增得到该基因,将其成熟编码区与pET28a表达载体连接后转化大肠埃希菌并用IPTG诱导表达,对表达产物的磷酸酶活性进行分析。结果成功构建了Sj-PP2C-1蛋白磷酸酶表达载体,其编码区能够在大肠埃希菌中表达分子质量单位为40ku的目的蛋白;采用V2460系统进行检测,Sj-PP2C-1具有PP2C磷酸酶活性。结论 Sj-PP2C-1基因原核表达产物具有蛋白磷酸酶活性,为研究PP2C类蛋白磷酸酶的功能打下了基础。
Objective To construct the PP2C-1 protein phosphatase expression vector of Schistosoma japonicum and analyze its expression in vitro and enzyme activity. Methods Sj-PP2C-1, a gene encoding PP2C-1 protein phosphatase, was found from the genome of Schistosoma japonicum. The gene was designed and amplified by PCR. The mature coding region was ligated with pET28a expression vector and transformed into the large intestine The Escherichia coli was induced by IPTG and the phosphatase activity of the expressed product was analyzed. Results The Sj-PP2C-1 protein phosphatase expression vector was successfully constructed. The coding region of the Sj-PP2C-1 protein was successfully expressed in Escherichia coli with a molecular weight of 40 ku. The Sj-PP2C-1 protein was detected by the V2460 system. active. Conclusion The prokaryotic expression product of Sj-PP2C-1 gene has the activity of protein phosphatase, which lays the foundation for studying the function of PP2C protein phosphatase.