汉坦病毒SEO型S基因的克隆及在大肠杆菌中的高效表达

Cloning of S Gene of Hantavirus Type SEO and Its High Expression in E.coli

目的构建汉坦病毒SEO型S基因原核表达载体。方法应用RT-PCR方法扩增汉坦病毒SEO型的YZG-Changchun株S基因,克隆至pMD18-T载体中,经酶切鉴定及PCR分析后,定向克隆入原核表达载体pET-28a中,转化E.coliRosetta,经IPTG诱导表达,SDS-PAGE和Western blot分析外源蛋白的表达情况。结果表达载体经双酶切和测序证明构建正确。IPTG浓度为1.0 mmol/L,诱导4.5 h时,S基因在原核细胞中得到了高效表达,表达的蛋白占菌体蛋白总量的37%,纯化后的蛋白具有良好的抗原活性。结论已成功构建了汉坦病毒SEO型S基因原核表达载体,并得到高效表达。

Objective To construct a prokaryotic expression vector of S gene of Hantavirus type SEO. Methods Amplify the S gene of YZG-Changchun strain of Hantaan virus type SEO by RT-PCR and clone into vector pMD18-T. Identify the recombinant plasmid by restriction analysis and PCR, and transfoma the target gene to E. coli Rosetta for expression under induction of IPTG. Identify the expressed product by SDS-PAGE and Western blot.Results Restriction analysis and sequencing proved that the expression vector was constructed correctly. After induction with 1.0 mmol/L IPTG for 4.5 h,S gene was highly expressed. The expressed product contained 37% of total somatic protein and showed specific reaction with mouse IgG against Hantaan virus type SEO. Conclusion The prokaryotic expression vector of S gene of Hantaan virus type SEO was successfully constructed, and the target gene was highly expressed.