猪流行性腹泻病毒E基因真核表达载体的构建及融合蛋白的表达

Construction of Eukaryotic Expression Vector of Porcine Epidemic Diarrhea Virus E Gene and Expression of Fusion Protein

E蛋白是猪流行性腹泻病毒的小膜蛋白,为了研究E蛋白在病毒侵染宿主细胞过程中的作用,构建了PEDV E基因的真核表达载体。通过RT-PCR技术扩增E基因片段,经双酶切连接至pEGFP-N1载体,酶切鉴定及测序结果证实PEDV E基因成功克隆到pEGFP-N1中,并将重组质粒命名为pEGFP-N1-E。将pEGFP-N1-E转染He La,利用Western Blot和免疫荧光技术检测pEGFP-N1-E的表达以及定位情况,结果显示pEGFP-N1-E在He La细胞中成功表达,并且在胞质胞核均匀分布。结果表明:pEGFP-N1-E已被成功构建,并且在He La细胞中正确表达。为后续研究关于E基因的功能及其在抗病毒中所发挥的作用等方面奠定了研究基础。

E protein encoded envelop protein of the porcine epidemic diarrhea virus. In order to study the role of E protein in the process of infecting host cells, the eukaryotic expression vector of PEDV E gene was constructed. E gene fragment was amplified by RT-PCR and ligated into pEGFP-N1 after double enzyme digestion. The results of restriction enzyme digestion analysis and sequence showed that PEDV E gene was successfully cloned into pEGFP-N1, and the recombinant plasmid was named as pEGFP- N1-E. Then pEGFP-N1-E was transfected into HeLa cell, Western Blot and immunofluorescence were used to detect its expression and location. The results showed that pEGFP-N1-E was successfully expressed in HeLa cells, and distributed in the cytoplasm and nucleus. These results indicated that pEGFP-N1-E had been constructed successfully, and expressed in HeLa cells correctly. This study might provide the foundation for further research on the function of E gene and its role in antiviral effect.