人线粒体色氨酰-trna合成酶的克隆、表达与纯化

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1、中南林业科技大学硕士学位论文人线粒体色氮酰砸暇A合成酶的克隆、表达与纯化A-bstractAmiIloacyl删AsyntkI切∞s(a出S)are恤脚insearli刚yeIne玛edin她progressionof饼ganiceVolution．AaRSc觚catalysis锄inoacidsbilldingtocognatet舭，andalsobeinvolvedinmeproce骆ofg胁e郴sionandregulati呱rRNAsynthellsis锄dI龇～即licin舀印optosisacC0们ing

2、t0rec翎tlyresearches．The州ectiVeofmisstudyist00btajnmehum觚mitochondria衄噼0ph觚yl—tRNAS)，rImetaSe(hmtTIp_RS)，趾ddetemi鹏itsl【ineticpammetersbytllehum孤mitochondfiatRNA1twhichw弱traIlscriptediIlVi们．Itpr州idesab弱isofthe咖dy0ntIles仃uctI鹏，fImction锄dtlleSpecies—specifici锣ofthe

3、amiIloilcyl—t】卧泼s)，lllhetaseh删唧RS．ThetotalI龇～w勰ex觚lcted蠡的mbrcastcarcinomacells(MDA小m_435S)，andthecodingsequenceofh删唧RSwim‘)utsignalpeptidesw弱舯plifiedwithrcV粥e的nscripti啷l(RD·PCR．1hcPcRproductw舔firstclonedint0舭pMDl8一T'tllen，pⅧ18-hmt唧RSandpET24a(+)werebo也dige洲wi也f

4、e矧“鲫唧eMIand砌Rl，锄dligatedt0cons伽lctt11eexpressionpl硒midwhichnamedpET24a(+)-hmt唧RS．111epositivecloncswclreid哪ified觚d蚓ectcdbyantibioticsrcsistancegelleScI优ning，molecularweight，nle纳gmentaf衙digesting觚dcolonyPCR．pET24a(+)-h傩唧RS、Ⅳ舔tra璐f0册edintoBL2l—C0(1伽【Plus(DE3)划L．1

5、hcexpressionconditionswereoptimizI耐bychar培ingt11elPTGconcentration，illductiontimeandtempc粕：turc．ThereSultsindicatedtIlattllehmtT巾RSexpre踮ionquantit)，w弱tIlemostwhenultimacollc删ionofIPTGw硒O．1mmol／I。alldinduced4hat30℃，觚dtllewei出sizeis38Kdaconformited州tll舭t11eory．T

6、hehmtTrpRSwastIlenp嘶6edwimNi2+-]帆AHis-bindresiIl，孤dhighspecificit)，His一噼dhIIItl却RSprmeinw舔obtained．1khum孤mitochon“at对蛆。巾wascons咖ctedby似ost印sPCR锄dn孤sc—ptedinvi仃o．矧itwas拄yptoph彻yla．刚byhmt唧RS．1he‰is1．08岬ol／L，aIldk嘲is0．02s-1，雠specificac“V畸0f也ep嘶fiedsol曲leproteinis4

7、411．18U／mg．Inconclusion，nleprokaryoticeXpressionVectorf．ork7唧RSissuccess剐lyc0潞tmcted觚dhigllspecifjc时His-协ggedhmt呻RSpr．oteinwasobtained．Andthee硒eIlciencyofaminoacylationwasmeas叫edbytRNArrD．Keywords：Mitochondria；Tryptophanyl．tRNASynthetase；tI讯AT叩；clone；expn晦sion；

8、puri矗cati佃H中南林业科技大学硕士学位论文人线粒体色氨酰糠NA合成酶的克隆、表达与纯化缩略词注：文中用氨基酸三字符号加RS来表示某种氨基酸特异的酶Hl中南林业科技大学硕士学位论文人线粒体色氮酰堰NA合成酶的克隆、表达与纯化目录摘要⋯⋯⋯⋯⋯⋯．⋯⋯⋯⋯⋯⋯⋯⋯⋯．⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．．】【Abs仃