A versatile cloning vector facilitates target gene expression in prokaryotic and eukaryotic cells
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A versatile cloning vector facilitates target gene expression in prokaryotic and eukaryotic cells
Military Medical ResearchVol. 26, Issue 4, Pages: 204-212(2011)
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CLC:R346
Published:2011
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[1].A versatile cloning vector facilitates target geneexpression in prokaryotic and eukaryotic cells[J].Journal of Medical Colleges of PLA,2011,26(04):204-212.
DOI:
[1].A versatile cloning vector facilitates target geneexpression in prokaryotic and eukaryotic cells[J].Journal of Medical Colleges of PLA,2011,26(04):204-212.DOI:
A versatile cloning vector facilitates target gene expression in prokaryotic and eukaryotic cells
摘要
Abstract
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.
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