Construction and identification of eukaryotic expression vectors of human wild-type and mutant-type vascular endothelial growth factor A gene

ObjectiveTo construct the eukaryotic expression vectors of human wild-type and c.454C T mutant-type vascular endothelial growth factor A( VEGFA) gene. Methods: Human VEGFA mrNA was amplified by reverse transcriptase-polymerase chainreaction( rT-PCr) ， and rT-PCr product was digested by restrictive endonucleases BglⅡ and SalⅠ.The wild type recombinant vector( wild-type pEGFP-VEGFA) was constructed by connecting enzyme digestion product and eukaryotic expression vector pEGFP-N1and identified by BglⅡ and SalⅠ double-enzyme digestion and sequence analysis.The mutant-type recombinant vector ( mutant-type pEGFP-VEGFA) was constructed by overlap extension PCr method and also identified by the above methods.Results: Wild-type and mutant-type pEGFP-VEGFA were all digested into two bands of 4 697 and 1 251 bp， representing the pPEGFP-N1 empty plasmid and VEGFA gene， respectively.Sequencing results showed that the sequence of wild-type pEGFP-VEGFA was identical to GenBank VEGFA accession number NM_001025366.The sequence of mutant-type pEGFP-VEGFA was identical to that of wild-type pEGFP-VEGFA， except that base C was replaced by T at position 454 in the mrNA sequence of the VEGFA gene. Conclusions: The eukaryotic expression vectors of human wild-type and mutant-type VEGFA gene have been successfully constructed，which lays a foundation for the biological function research of VEGFA gene in the future.