日本血吸虫T2核酸酶的原核表达及活性分析

目的 构建日本血吸虫T2核酸酶表达载体,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与曼氏血吸虫虫卵抗原Omega-1同源性最高的蛋白AY814845,设计引物并通过PCR技术扩增得到该基因,将其成熟编码区连入pET32a表达载体,转化大肠杆菌并用IPTG诱导其表达,最后对表达产物的核酸酶活性进行分析。结果 成功的构建了日本血吸虫T2核酸酶的表达载体,其编码区能够在大肠杆菌中表达,且表达产物具有一定的核酸酶活性。结论 从体外扩增了日本血吸虫T2核酸酶AY814845,明确其表达产物具有核酸酶活性,为今后深入研究该蛋白的功能打下了基础。

Expression and activity analysis of a kind of T2 Rnase from Schistosoma japonicum

To construct the prokaryotic expression plasmid containing T2 Rnase gene of Schistosorna japonicum and ana- lyse the Rnase activity of recombinant protein, the gene fragment containing the mature ORF of AY814845, which shows the highest homologous identity to Omega 1 from S. mansoni, was amplified by PCR and then cloned into pET32a. The recombi- nant plasmid was confirmed by PCR and restriction enzyme analysis. The correct plasmid was named as pET32a-AY814845 and then transformed into Escherichia coli Rosseta （DE3） and finally induced with isopropyl-~-D-thiogalactopyranosid （IPTG）. The expressed protein products were analyzed by SDS-PAGE and identified by Rnase activity research. In this study, the re- sults showed that T2 Rnase gene AY814845 of S. japonicum could be successfully expressed in E. coli and the recombinant protein showed Rnase activity. The conclusion suggests that the recombinant ,plasmid might be used for further study on the function of AY814845 in adjusting the immune response.