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1、分類號S852.1學(xué)校代碼10129UDC577學(xué)號08201059蒙古綿羊GhrelincDNA的克隆和原核表達ThecDNACloningandProkaryoticExpressionofMongoliaSheepGhrelin申請人：梁建榮學(xué)科門類：農(nóng)學(xué)學(xué)科專業(yè)：基礎(chǔ)獸醫(yī)學(xué)研究方向：動物組織胚胎與發(fā)育生物學(xué)指導(dǎo)教師：曹貴方教授論文提交日期：二〇一一年五月摘要Ghrelin是Kojima等1999年發(fā)現(xiàn)的生長激素促分泌素受體(GHSR)的第一個內(nèi)源性配體，Ghrelin除調(diào)節(jié)生長激素分泌外，還有增加食欲，調(diào)節(jié)能量平衡，促進胃酸分泌等作用
2、。根據(jù)Genbank中檢索羊preproGhrelinmRNA序列，設(shè)計了蒙古綿羊Ghrelin原核表達引物。通過RT-PCR技術(shù)擴增GhrelincDNA，將其與pMD-19TVector連接，轉(zhuǎn)入大腸桿菌經(jīng)鑒定正確后，回收目的片段與原核表達載體pET-32a(+)相連接，并轉(zhuǎn)化BL21(DE3)大腸桿菌。經(jīng)IPTG誘導(dǎo)后進行SDS-PAGE和Western-blot分析。結(jié)果顯示克隆的蒙古綿羊GhrelincDNA序列與已發(fā)表序列有兩個堿基的差異，該堿基的變化并不影響氨基酸序列，目的蛋白主要以可溶性形式存在，Western-blot初步證
3、實了所獲的融合蛋白是特異的，結(jié)果表明成功構(gòu)建了蒙古綿羊Ghrelin基因的原核表達載體，并在大腸桿菌中獲得高效表達，為進一步研究蒙古綿羊Ghrelin基因的功能提供參考。關(guān)鍵詞：Ghrelin；基因克?。辉吮磉_；蒙古綿羊ThecDNACloningandProkaryoticExpressionofMongoliaSheepGhrelinAbstractGhrelinisthefirstendogenousligandofgrowthhormonesecretogogue(GHSR),itisfoundinratstomachin1999b
4、yKojima.GhrelincannotonlyregulatethesecretionofGH,butalsostimulateappetite，regulateenergybalanceandstimulategastricacidsecretion.AccordingtothesequenceofpreproghrelinmRNA，apairofprimeroftheexpressionsystemofprokaryoticwasdesigned.GhrelingenewasamplifiedbyRT-PCRandthePCRprod
5、uctwereclonedintothepMD-19TVector,subclonedintotheexpressionvectorpET-32a(+)andtransformedintohostEscherichiacolistrainBL2l(DE3)forexpression.ComparingwiththesequencesofsheepGhrelinreportedinGenbank,thereweretwonucleotidedifferences,butitdidnotaffecttheaminoacidsequence.The
6、expressionproductwasobservedwithsolubleprotein.Western-blotshowedthattherecombinantproteinwasrecognizedbyhis-antibodyspecifically.ThenucleotidesequenceisolatedfromtherecombinantplasmidpET-32a-Ghrelinwasthesameasexpected;theGhrelinproteinwasexpressedsuccessfullyinEscherichia
7、coli,andprovidedabasisforfurtherinvestigationofGhrelinfunction.Keywords:Ghrelin;cloninggene;prokaryoticexpression;MongoliaSheepDirectedby:Prof.CaoGuifangApplicantforMasterdegree:LiangJianrong(HistologyandDevelopmentalbiology)(CollegeofAnimalScience,InnerMongoliaAgricultural
8、University,huhhot010018,China)目錄1引言···············································
