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.
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[2] Zhao W，Wang J，Shen J，et al. Mutations in VEGFA are associated with congenital left ventricular outflow tract obstruction[J]. Biochem Biophys res Commun， 2010， 396( 2) : 483 － 488. [2] McBride KL，Pignatelli r，Lewin M， et al. Inheritance analysis of congenital left ventricular outflow tract obstruction malformations:Segregation，multiplex relative risk，and heritability[J]. Am J Med Genet A，2005，134A( 2) : 180 － 186. [3] Aboulhosn J，Child JS. Left ventricular outflow obstruction:subaortic stenosis，bicuspid aortic valve，supravalvar aortic stenosis，and coarctation of the aorta[J]. Circulation，2006，114( 22) : 2412 － 2422. [4] Wessels MW，Berger rM，Frohn-Mulder IM，et al. Autosomal dominant inheritance of left ventricular outflow tract obstruction [J]. Am J Med Genet A， 2005， 134A( 2) : 171 － 179. [5] Kerstjens-Frederikse WS，Du Marchie Sarvaas GJ，ruiter JS，etal. Left ventricular outflow tract obstruction: should cardiac screening be offered to first-degree relatives? [J]. Heart，2011，97( 15) : 1228 － 1232. [6] McBride KL，riley MF，Zender GA， et al. NOTCH1 mutations in individuals with left ventricular outflow tract malformations reduce ligand-induced signaling[J]. Hum Mol Genet，2008，17( 18) :2886 － 2893. [7] 李宝珍，李素梅，施卫明，等. 利用重叠PCr 技术对OsAMT112 进行定点突变[J]. 生物技术通报，2009 ( 9 ) :69 － 72. [8] Tee MK， Jaffe rB. A precursor form of vascular endothelial growth factor arises by initiation from an upstream in-frame CUG codon[J].Biochem J，2001，359( Pt 1) : 219 － 226. [9] Huez I，Bornes S，Bresson D，et al. New vascular endothelial growth factor isoform generated by internal ribosome entry sitedriven CUG translation initiation[J]. Mol Endocrinol，2001，15( 12) : 2197 － 2210. [10] Meiron M，Anunu r，Scheinman EJ， et al. New isoforms of VEGF are translated from alternative initiation CUG codons located in its5' UTr[J]. Biochem Biophys res Commun，2001，282 ( 4 ) :1053 － 1060 -
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