CD59-linker-CD2融合基因真核表达系统的构建 .pdfVIP

CD59-linker-CD2融合基因真核表达系统的构建

钟丹丹;高美华;李伟伟;张蓓

【摘要】目的CD59和CD2都是重要的淋巴细胞膜表面分子,参与T细胞信号转

导.文中构建CD59和CD2融合基因真核表达系统,探讨CD59向T细胞传递信号

的机制及CD59与CD2的相互作用.方法在CD59基因和CD2基因间加入一段

柔性链接头(linker)基因序列,构建CD59-linker-CD2融合基因真核表达系统.利用

RT-PCR法从人T细胞白血病细胞系Jurkat细胞总RNA中扩增CD59和CD2基

因,分别构建CD59-T和CD2-T重组克隆载体,经限制性核酸内切酶酶切后与

pIRES质粒进行重组,构建pIRES-CD59-linker-CD2融合基因真核表达系统.结果

测序证实CD59和CD2基因扩增成功;经限制性核酸内切酶酶切、PCR和测序鉴

定表明携带CD59-linker-CD2融合基因的重组质粒构建成功.结论构建CD59-

linker-CD2真核表达系统为在真核细胞中表达CD59与CD2并进一步探讨其在T

细胞信号转导中的作用提供了实验材料.%ObjectiveBothCD59andCD2are

importantmoleculesonthesurfaceoflymphocytesandinvolvedinTcell

signaltranscluction.Thepurposeofthisstudywastoconstructa

eukaryoticexpressionvectoroftheCD59andCD2fusiongenetofacilitate

theinvestigationofthemechanismofsignaltransductionfromCD59toT

cellsandtheinteractionbetweenCD59andCD2.MethodsWe

constructedaeukaryoticexpressionvectoroftheCD59-linker-CD2fusion

genebylinkingtheCD59andCD2geneswithaflexiblechain(linker).The

CD59andCD2geneswereclonedbyRT-PCRfromthetotalRNAofJurkat

TcellstoestablishCD59-TandCD2Tcloningvectors,respectively,whichin

turnwereclonedintotheplasmidpIRESfollowingrestriction

endonucleasedigestiontoconstructaeukaryoticvectorcontainingthe

CD59-linker-CD2fusiongene.ResultsDNAsequencingconfirmedthe

successfulamplificationoftheCD59andCD2genes.Restriction

endonucleasedigestion,PCRandDNAsequencinganalysisprovedthat

theeukaryoticexpressionvectorcontainingtheCD59-linker-CD2fusion

genewasconstructedcorrectly.ConclusionThesuccessfulconstruction

oftheeukaryoticexpressionvectorcontainingtheCD59-linker-CD2fusion

genehaspreparedtheexperimentalgroundforfurtherstudyofthe

expressionsofCD59andCD2ineuk