蒙古绵羊Ghrelin cDNA的克隆和原核表达.pdf

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1、分类号S852.1学校代码10129UDC577学号08201059蒙古绵羊GhrelincDNA的克隆和原核表达ThecDNACloningandProkaryoticExpressionofMongoliaSheepGhrelin申请人：梁建荣学科门类：农学学科专业：基础兽医学研究方向：动物组织胚胎与发育生物学指导教师：曹贵方教授论文提交日期：二〇一一年五月摘要Ghrelin是Kojima等1999年发现的生长激素促分泌素受体(GHSR)的第一个内源性配体，Ghrelin除调节生长激素分泌外，还有增加食欲，调节能量平衡，促进胃酸分泌等作用

2、。根据Genbank中检索羊preproGhrelinmRNA序列，设计了蒙古绵羊Ghrelin原核表达引物。通过RT-PCR技术扩增GhrelincDNA，将其与pMD-19TVector连接，转入大肠杆菌经鉴定正确后，回收目的片段与原核表达载体pET-32a(+)相连接，并转化BL21(DE3)大肠杆菌。经IPTG诱导后进行SDS-PAGE和Western-blot分析。结果显示克隆的蒙古绵羊GhrelincDNA序列与已发表序列有两个碱基的差异，该碱基的变化并不影响氨基酸序列，目的蛋白主要以可溶性形式存在，Western-blot初步证

3、实了所获的融合蛋白是特异的，结果表明成功构建了蒙古绵羊Ghrelin基因的原核表达载体，并在大肠杆菌中获得高效表达，为进一步研究蒙古绵羊Ghrelin基因的功能提供参考。关键词：Ghrelin；基因克隆；原核表达；蒙古绵羊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)目录1引言···············································