带信号肽人血管内皮生长因子基因VEGF_(121)及VEGF_(165)载体的克隆

Cloning of expression vector for VEGF_(121) and VEGF_(165)gen es encoding human vascular endothelial growth factor

目的 克隆及构建带信号肽人血管内皮生长因子基因VEGF121及VEGF165表达载体。方法 对正常引产胎儿肺组织的总RNA进行逆转录-聚合酶链反应（RT-PCR），克隆入T载体，测定扩增片段序列，进而克隆入表达质粒pcDNA3.1^-,酶切鉴定重组子。结果 用一对引物（5′引物为-21-7bp,3′引物为554-576bp)可扩增出两个条带，短条带为VEGF121（487bp)，长条带经筛选可得到两个片段，一为正常的VEGF165（619bp)，另一个为新的VEGFmRNA“异常”剪切片段。测序结果显示，此“异常”剪切片段扩增长度为639bp，同样为VEGF165全长核苷酸席子邓列，但在VEGF165的第3种和4外显子之间插入了长度为20bp的片段，经序列检索发现20bp的插入序殓为滞留的第3内含子终末序列，含内含子剪切信号。结论 克隆出带信号肽人血管内皮生长因子基因VEGF121及VEGF165表达载体。同时在正常引产胎儿的肺组织中可能发现了一种新的VEGFmRNA可变剪切形式。

Objective To cloneandconstructtheexpressionvectorfor humanvascularendothelialgrowthfactor(VEGF)genes.Methods TotalRNAswere extractedfrom humanlungtissueof a4-month-oldfetusand subjectedto reverse transcriptase-polymerasechainreaction(RT-PCR)withtheamplifiedproductsclonedintopMD 18-T vector.Sequenceanalysis wasperformedbeforetheamplifiedproductswereclonedintotheexpressionplasmidpcDNA3.1 - ,therecombinantof which wasverifiedby endonucleasedigestion.Results AfterRT-PCRusinga pairof primers(sense-21/7bpandantisense554/576bp),twobandswereidentified.Theband(487bp)shorterinlengthwasconfirmedas VEGF 121 (withthefulllengthof VEGF 121 being444bp)whilethelongerband(619bp)was normalVEGF 165 (withthefulllengthof VEGF 165 being576bp).Interestingly,anotherslightlylongerVEGF 165 nucleotidesequencewasidentifiedby sequencinganalysis,whichfeaturedan unique20bp insertionpreciselybetweenexon3andexon4fromthefirstATGof humanVEGF 165 cDNA.The20bp insert wasidentifiedas theretainingintron3terminalnucleotidescontainingthesplicingsignal,whichcausedframeshiftmutation inthereadingframeandcouldprobablygiveriseto a shortpolypeptidesconsistingof only97aminoacidresidualsdueto the earlyappearanceof stopcodeUAGin themiddleof exon4.Conclusion Wehavesuccessfullyconstructedtheexpression vectorforVEGF 121 andVEGF 165 genes,anda newpossiblealternativesplicingisoformof VEGFis identifiedinnormalfetus whosemolecularmechanismandphysiologicalfunctionneedsfurtherinvestigation.