Construction of Eukaryotic Expression Plasmids for Erns and Npro Proteins in BVDV
Objective The experiment studies aimded to construct recombinant expression vectors for the structural protein(glycosylated envelope protein Erns)and non structural protein(Npro)of bovine viral diarrhea virus(BVDV),and to express Erns and Npro using a mammalian eukaryotic expression system.Methods The primers Npro-F/R and Erns-F/R were designed referring to the nucleotide sequence of the BVDV reference strain published in GenBank.The cDNA of BVDV virus was used for PCR amplification.The recovered and purified Npro and Erns target genes and the eukaryotic expression vector pCMV-Myc were digested with double enzyme cleavage.The agarose gel electrophoresis was used for the recovery and purification.The harvested and purified products were linked with T4 DNA ligase and transformed into BL-21 competent cells,and the eukaryotic expression plasmid was constructed.The recombinant plasmids were named pCMV-Myc-Npro and pCMV-Myc-Erns.After the double enzyme digestion and sequencing identification were correct,HEK-293 was transfected.The expression of Npro and Erns was detected in the cells by PCR and Western blotting.Results The pCMV-Myc-Npro and pCMV-Myc-Erns eukaryotic expression plasmids was successfully constructed,and Npro and Erns were successfully expressed in HEK293 cells.Conclusion The successful expression of Npro and Erns proteins will provide theoretical references for exploring protein biological functions,studying viral infection mechanisms,and developing subunit vaccines.
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