血管内皮生长因子基因3'UTR 不同基因型载体的构建与鉴定

目的:构建含SNP位点的血管内皮生长因子(VEGF)基因3’UTR的荧光素酶报告基因载体,为进一步揭示VEGF基因3’UTR的单核苷酸多态性(SNP)影响肺癌发病风险的分子机制奠定基础。方法:以rs3025039和rs3025040两个位点均为C纯合子的非癌症病人血液DNA为模板,扩增出两位点为C/C单体型、长度为1448 bp的VEGF基因3’UTR目的片段,测序验证后将其克隆至pMIR-REPORT荧光素酶报告基因载体上,得到重组质粒pMIR-C/C。同时,我们以pMIR-C/C为模板定点突变两个SNP位点,得到具有T/T单体型的重组质粒pMIR-T/T。将各重组质粒转化大肠杆菌DH10B,筛选阳性克隆后提取质粒进行双酶切鉴定及DNA测序鉴定。结果:单菌落质粒测序验证显示带有C/C单体型的VEGF基因3’UTR重组质粒pMIR-C/C构建成功;经两次定点突变,成功将pMIR-C/C质粒转变为pMIR-T/T,经测序验证未引入任何其他突变。同时生物信息学预测还显示rs3025040位点位于miR-199a/b与VEGF基因mRNA的结合位置,其改变可以影响miRNA与mRNA的结合效率。结论:本研究成功构建了含有两个连锁SNP的VEGF基因3’UTR的荧光素酶报告基因载体,为今后VEGF基因3’UTR的功能研究奠定基础。

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.