野生型及突变型人血管内皮生长因子A真核表达载体的构建与鉴定
Construction and identification of eukaryotic expression vectors of human wild-type and mutant-type vascular endothelial growth factor A gene
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摘要: 目的:构建人野生型和c.454CT突变型血管内皮生长因子A(VEGFA)真核表达载体。方法:采用反转录聚合酶链反应扩增人VEGFA基因,用限制性核酸内切酶BglⅡ和SalⅠ双酶切后连接真核表达载体pEGFP-N1,构建野生型VEGFA真核表达载体(野生型pEGFP-VEGFA),通过双酶切和测序进行鉴定。采用定点诱变PCR技术构建c.454CT突变型人VEGFA真核表达载体(突变型pEGFP-VEGFA),同样通过双酶切和测序进行鉴定。结果:野生型和突变型pEGFP-VEGFA被双酶切为4 697 bp和1 251 bp两条条带。测序结果证实野生型pEGFP-VEGFA VEGFA序列与GenBank公布的VEGFA mRNA序列完全一致,突变型pEGFP-VEGFA除第454位碱基C被T替代以外,其余序列与野生型完全一致。结论:成功构建了野生型和突变型pEGFP-VEGFA,为下一步研究VEGFA基因功能提供参考。Abstract: 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.