摘要
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.
作者
郑亮
邹德华
程丽鑫
李兴志
张雅婷
沈玉江
武雪宁
徐嘉欣
王显赫
张华
曹宏伟
Zheng Liang;Zou Dehua;Cheng Lixin;Li Xingzhi;Zhang Yating;Shen Yujiang;Wu Xuening;Xu Jiaxin;Wang Xianhe;Zhang Hua;Cao Hongwei(College of Life Science and Technology,Heilongjiang Bayi Agricultural University,Daqing 163319)
出处
《黑龙江八一农垦大学学报》
2018年第4期81-85,共5页
journal of heilongjiang bayi agricultural university
基金
国家自然科学基金(31570159)
黑龙江省教育厅科学技术研究面上项目(12541578)
黑龙江八一农垦大学博士科研启动基金(XDB2015-16
XDB2015-18)
黑龙江八一农垦大学青年创新人才项目(CXRC2016-12)
黑龙江八一农垦大学研究生创新科研项目(YJSCX2017-Y60
YJSCX2017-Y66)
关键词
PEDV
E基因
载体构建
蛋白表达
porcine epidemic diarrhea virus
E gene
vector construction
protein expression