Objective: To facilitate manipulation of gene expression in different host cells, we used pEGFP-N1 as backbone to construct a versatile vector that can drive foreign gene expression in prokaryotic and eukaryotic cells...Objective: To facilitate manipulation of gene expression in different host cells, we used pEGFP-N1 as backbone to construct a versatile vector that can drive foreign gene expression in prokaryotic and eukaryotic cells. Methods: A cloning and expression vector, pEGFP-N1-lac, was constructed by inserting the prokaryotic lac promoter of pUC19 into the eukaryotic expression vector, pEGFP-N1, between the eukaryotic PCMV promoter and enhanced green fluorescent protein (EGFP) open reading frames. To assess the function of pEGFP-N1-lac, the nucleotide sequence encoding the hepatitis C virus (HCV) core protein was cloned into the multiple cloning sites. Western blotting analysis was used to detect the expression of the HCV core protein in Escherichia coli DH5α and HepG2 cells. Results: Restriction enzymedigestion and sequence analysis indicated that pEGFP-N1-lac was successfully constructed and the HCV core gene was cloned into this vector. The Western blotting results showed that pEGFP-N1-lac promoted expression of HCV core gene in prokaryotic E. coli DH5α and eukaryotic HepG2 cells. Conclusion: The pEGFP-N1-lac vector has been successfully constructed and functions in both prokaryotic and eukaryotic cells. The EGFP reporter can be used as an insert-inactivation marker for clone selection or as an expression tag. This vector can be used for cloning and expression of genes in both prokaryotic and eukaryotic cells, making gene cloning, expression and functional studies convenient as well as time- and labor-efficient.展开更多
基金Supported by the National High Technology Research and Development Program of China (863 Program, 2009AA02Z111)the National Natural Science Foundation of China (30872223)the Funds of the State Key Laboratory of Pathogen and Biosecurity
文摘Objective: To facilitate manipulation of gene expression in different host cells, we used pEGFP-N1 as backbone to construct a versatile vector that can drive foreign gene expression in prokaryotic and eukaryotic cells. Methods: A cloning and expression vector, pEGFP-N1-lac, was constructed by inserting the prokaryotic lac promoter of pUC19 into the eukaryotic expression vector, pEGFP-N1, between the eukaryotic PCMV promoter and enhanced green fluorescent protein (EGFP) open reading frames. To assess the function of pEGFP-N1-lac, the nucleotide sequence encoding the hepatitis C virus (HCV) core protein was cloned into the multiple cloning sites. Western blotting analysis was used to detect the expression of the HCV core protein in Escherichia coli DH5α and HepG2 cells. Results: Restriction enzymedigestion and sequence analysis indicated that pEGFP-N1-lac was successfully constructed and the HCV core gene was cloned into this vector. The Western blotting results showed that pEGFP-N1-lac promoted expression of HCV core gene in prokaryotic E. coli DH5α and eukaryotic HepG2 cells. Conclusion: The pEGFP-N1-lac vector has been successfully constructed and functions in both prokaryotic and eukaryotic cells. The EGFP reporter can be used as an insert-inactivation marker for clone selection or as an expression tag. This vector can be used for cloning and expression of genes in both prokaryotic and eukaryotic cells, making gene cloning, expression and functional studies convenient as well as time- and labor-efficient.
