Construction of the Eukaryotic Expression Vector of SARS-CoV-2 Nsp1 Gene and Effects on Host Protein Translation
Non-structural protein 1(Nsp1)was the virulence determinant of the new coronavirus(Severe acute respiratory syndrome coronavirus-2,SARS-CoV-2),which played an important role in regulating coronavirus replication.In order to study the biological function of SARS-CoV-2 Nsp1 protein more effectively,the eukaryotic expression vector of SARS-CoV-2 Nsp1 gene was successfully constructed and verified its function of inhibiting translation of host and foreign proteins.The SARS-CoV-2 Nsp1 gene was amplified by PCR technology and then connected to the pEGFP-N1 vector using homologous recombination technology.The SARS-CoV-2 Nsp1 gene was successfully cloned into the pEGFP-N1 vector by double-enzyme digestion and sequencing results.The recombinant plasmid pEGFP-N1-SARS-CoV-2-Nsp1 was transfected into HEK-293T cells and HeLa cells.After labeling with puromycin,the expression of the recombinant plasmid and puromycin were detected by Western Blot and Coomassie brilliant blue staining.The results showed that the recombinant plasmid pEGFP-N1-SARS-CoV-2-Nsp1 was successfully expressed in HEK-293T cells and HeLa cells,and it was verified that it inhibited the translation of host proteins and foreign proteins transferred into cells.It laid the foundation for a more in-depth study of the function of the SARS-CoV-2 Nsp1 protein.
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