Construction and Identification of Eukaryotic Expression Vectors Including SNPs in VEGF gene 3'UTR

Objective: To construct luciferase reporter gene vectors containing vascular endothelial growth factor(VEGF) gene 3'UTR with two SNP sites and to detect microRNA binding stability in different genotype VEGF gene 3'UTR.Methods: Non-cancer pa-tients' genomic DNA with C/C homozygote in the two linked SNP sites(rs3025039 and rs3025040) was used as a PCR template to amplify 1448bp 3'UTR of VEGF gene.After the PCR product was cloned into the pMIR-REPORT luciferase miRNA expression report vector,this pMIR-C/C vector was used as PCR template to construct the pMIR-T/T by site-specific mutagenesis.Both pMIR-C/C and pMIR-T/T vectors were transferred into E.coli DH10B and verified by double-enzyme digestions and DNA sequencing.Results: Two recombinant plasmids were successfully constructed and the sequences of them were the same as expected.Furthermore,the bioinformatics analysis suggested that rs3025040 T allele may increase miR-199 a/b regulation of VEGF gene expression.Conclusions: Luciferase reporter vec-tors pMIR-REPORT-VEGF 3'UTR containing two SNPs homozygotes in VEGF gene 3'UTR were successfully constructed and these constructions of the recombinant plasmids will be further studied in related SNP function analysis in lung cells.