摘要
囊膜蛋白(E蛋白)是新型冠状病毒(Severe Acute Respiratory Syndrome Coronavirus-2,SARS-CoV-2)的小包膜蛋白,在调控冠状病毒复制方面有重要作用。为了更有利地研究SARS-CoV-2 E蛋白的生物学功能,研究成功构建了SARS-CoV-2 E基因的真核表达载体。首先,通过PCR技术扩增SARS-CoV-2 E基因,并将pCMV-Myc载体进行双酶切。随后,利用同源重组将SARS-CoV-2 E基因连接至pCMV-Myc载体。经酶切和测序鉴定发现,SARS-CoV-2 E基因成功克隆至pCMV-Myc载体中,并将重组质粒命名为pCMV-Myc-SARS-CoV-2-E。将重组质粒pCMV-Myc-SARS-CoV-2-E转染至HEK-293T细胞,利用Western Blot和间接免疫荧光技术检测重组质粒的表达以及定位情况。结果表明,重组质粒pCMV-Myc-SARS-CoV-2-E在HEK-293T细胞中成功表达,并且发现重组蛋白主要分布于胞质,少量存在于胞核,为进一步研究SARS-CoV-2 E蛋白的功能奠定基础。
SARS-CoV-2 E protein was the envelope protein of SARS-CoV-2(Severe Acute Respiratory Syndrome Coronavirus-2),which played an important role in viral replication.In order to study the biological function of SARS-CoV-2 E protein,eukaryotic expression vector of SARS-CoV-2 E gene was successfully constructed.The E gene fragment was amplified by PCR and linked to the pCMV-Myc vector by double enzyme digestion.Then,the E gene of SARS-CoV-2 was linked to pCMV-Myc vector by homologous recombination.The results of enzyme digestion confirmed that the SARS-CoV-2 E gene was successfully cloned into the pCMV-Myc vector and the recombinant plasmid was named pCMV-Myc-SARS-CoV-2-E.The recombinant plasmid was transfected into HEK-293T cells and the expression and localization of pCMV-MyC-SARS-CoV-2-E were detected by Western Blot and indirect immunofluorescence.The results showed that pCMV-Myc-SARS-CoV-2-E was successfully expressed in HEK-293T cells and found that the recombinant protein was mainly distributed in the cytoplasm,and a small amount exists in the nucleus.It laid a foundation for further study on the function of SARS-CoV-2 E protein.
作者
于雯
郑亮
王宏宇
高鑫誉
吴志军
曹宏伟
张华
Yu Wen;Zheng Liang;Wang Hongyu;Gao Xinyu;Wu Zhijun;Cao Hongwei;Zhang Hua(College of Life Science and Technology,Heilongjiang Bayi Agricultural University,Daqing163319;College of Animal Science and Technology,Heilongjiang Bayi Agricultural University;Biotechnology Center,Heilongjiang Bayi Agricultural University)
出处
《黑龙江八一农垦大学学报》
2022年第5期85-91,共7页
journal of heilongjiang bayi agricultural university
基金
黑龙江八一农垦大学青年创新人才项目(CXRC2016-12)。
