Construction of plasmids expressing Sars-CoV encoding proteins and their effects on transcription of hfgl2 prothrombinase.
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.
Wang
Hongwu
H
Department of Infectious Disease, Huazhong University of Science and Technology, Wuhan, 430030, China. hongwuwang@126.com
Han
Meifang
M
Yao
Huaning
H
Wang
Zhanhui
Z
Xi
Dong
D
Yan
Weiming
W
Hou
Jinlin
J
Luo
Xiaoping
X
Ning
Qin
Q
D000818:Animals; D016466:CHO Cells; D000086862:Coronavirus M Proteins; D000086462:Coronavirus Nucleocapsid Proteins; D006224:Cricetinae; D003412:Cricetulus; D005340:Fibrinogen / Q000096:biosynthesis / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics; D019590:Nucleocapsid Proteins / Q000096:biosynthesis* / Q000235:genetics; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics* / Q000378:metabolism; D045169:Severe Acute Respiratory Syndrome / Q000821:virology; D064370:Spike Glycoprotein, Coronavirus; D013925:Thromboplastin / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics; D014162:Transfection; D014759:Viral Envelope Proteins / Q000096:biosynthesis / Q000235:genetics; D014763:Viral Matrix Proteins / Q000096:biosynthesis / Q000235:genetics
D000086862:Coronavirus M Proteins; D000086462:Coronavirus Nucleocapsid Proteins; C095202:FGL2 protein, human; C000706237:M protein, SARS-CoV; D008562:Membrane Glycoproteins; D019590:Nucleocapsid Proteins; D011994:Recombinant Proteins; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; D014763:Viral Matrix Proteins; C578557:spike glycoprotein, SARS-CoV; D005340:Fibrinogen; D013925:Thromboplastin