【摘要】 目的制备2009H1N1流感病毒神经氨酸酶(NA)重组蛋白,为建立H1N1快速检测方法和神经氨酸酶抑制剂筛选模型奠定基础。方法采用MDCK细胞方法分离2009H1N1流感病毒,提取病毒RNA,RT-PCR生成NA基因,构建原核表达载体PET-102/D-TOPO-NA,IPTG诱导表达重组蛋白,SDS-PAGE凝胶电泳、WersternBlotting鉴定重组蛋白。结果成功分离2009H1N1流感病毒,NA蛋白119、152、275、292位氨基酸分别为Glu、Arg、His和Cys。SDS-PAGE凝胶电泳蛋白分子相对分子质量约为64000,与预期一致。WesternBlotting证实该蛋白具有神经氨酸酶抗原活性。结论 IPTG诱导原核表达神经氨酸蛋白的最适浓度为0.1mmol/L。实验得到的蛋白尚需进一步纯化。更多还原

【Abstract】 Objective To isolate the virus of 2009 H1N1 and clone its neuraminidase(NA) gene in order to study the structure and function of NA.Methods The virus was isolated from the sample of the patient infected with 2009 H1N1 influenza by MDCK cell culture.The NA gene was amplified by RT-PCR and then cloned into the prokaryotic expression vector PET-102/D-TOPO for the production of recombinant NA.The expressed products were separated on SDS-PAGE gel and its antigenicity was identified by Western Blotting.Results 2009 H1N1 influenza virus were successfully isolated from the sample using MDCK cell culture method.The NA amino acids at positions 119,152,275 and 292 are Glu,Arg,His and Cys,respectively.NA protein was successfully expressed in E.coli BL21(DE3).The results of SDS-PAGE showed the recombinant NA protein was about 64 kDa,consisting with the expected size of the protein molecule.Antigen activity of the expressed NA protein was confirmed by Western Blotting.Conclusion The isolate is sensitive to oseltamivir.The optimal IPTG concentration to induce prokaryotic expression of NA is 0.1 mmol/L.The protein needs purification before being further used.更多还原