金黄色葡萄球菌核酸酶在大肠杆菌中的表达及其活性分析

Expression and nuclease activity analysis of staphylococcus nuclease in E.coli

目的:构建金黄色葡萄球菌核酸酶(SN)基因的原核表达载体,研究其在大肠杆菌中的表达,并制备兔抗SN抗体。方法:从质粒pPLCSN中扩增SN基因片段,插入表达载体pLEX后转化大肠杆菌GI724,以色氨酸诱导表达。以表达的重组蛋白免疫日本大耳白兔制备抗SN抗体,用Westernblot分析兔抗SN抗体的特异性。结果:重组表达载体pLEXSN在大肠杆菌中获得表达,表达的可溶性蛋白占菌体蛋白总量的37%,具有良好的生物学活性。制备的兔抗SN抗体能够特异识别SN。结论:成功地在大肠杆菌中表达具有生物学活性的SN,并制备了兔抗SN抗体,为进一步探讨其作为抗病毒因子应用于病毒性疾病的治疗奠定了基础。

AIM: To express staphylococcus nuclease (SN) in E.coli and prepare rabbit antisera against SN. METHODS: The SN gene was amplified by high-fidelity PCR from plasmid pPLC-SN and then subcloned into expression vector pLEX to obtain the recombinant plasmid pLEX-SN. The expression of recombinant protein was induced by tryptophan. The expressed SN was used to immunize a rabbit to prepare specific antibody. RESULTS: SDS-PAGE analysis showed that the relative molecular mass (M_r) of the expressed SN was about 17 000 and the expressed SN accounted for about 37% of total bacterial proteins. The prepared antisera were specific to react with recombinant SN. CONCLUSION: Expression vector of SN has been successfully constructed and rabbit antibody against SN was prepared. These results lay the foundation for developing SN as antiviral protein.