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Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1α Promoter Using Gibson Assembly
Published on: February 9, 2015
Construction of plasmids expressing Sars-CoV encoding proteins and their effects on transcription of hfgl2
Hongwu Wang1, Meifang Han, Huaning Yao
1Department of Infectious Disease, Huazhong University of Science and Technology, Wuhan, 430030, China. hongwuwang@126.com
Abstract:
SARS coronavirus (SARS-CoV) is the etiologic agent of severe acute respiratory syndrome. The aim of this study was to construct Sars-CoV membrane (M), nucleocapsid (N) and spike 2 (S2) gene eukaryotic expression plasmids, and identify their expression in vitro. Gene fragments encoding N protein, M protein and S2 protein of SARS-CoV were amplified by PCR using cDNA obtained from lung samples of SARS patients as template, and subcloned into pcDNA3.1 vector to form eukaryotic expression plasmids. SARS-CoV protein eukaryotic expression plasmids were transfected respectively into CHO cells. Immunohistochemistry was employed to detect the expression of the structural proteins of SARS-CoV in transfected cells. SARS-CoV protein eukaryotic expression plasmids were successfully constructed by identification with digestion of restriction enzymes and sequencing. M, N and S2 proteins of SARS-CoV were detected in the cytoplasm of transfected CHO cells. It was concluded that these recombinant eukaryotic expression plasmids were constructed successfully, and SARS-CoV encoding proteins could activate transcription and expression of hfgl2 gene.
