hUNC93B1基因的克隆、表达纯化及抗体制备（医学论文）

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1、hUNC93B1基因的克隆.表达纯化及抗体制备作者：蔡宇红张铁柱毛云英【Abstract]AIM:ToconstructtheprokaryoticexpressionvectorcontaininghUNC93Blgene,theninducetheexpressionofthisgeneandpurifythefusionprotein,andatlastusingtargetproteintomakethespecificandhightiterantibody.METHODS:Thespecificprimerso

2、fhUNC93geneweredesignedandthegenewasamplifiedbyPCR・ThePCRproductwasthenligatedintopMD18Tvector.A仕ersequenceanalysis,itwassubclonedintopRSETAwhichcouldexpressthetargetproteinasa(His)6taggedfusion.ThebacterialstrainBL21(DE3)wasusedasthehostcelltoinducetheexpressiono

3、fthefusionproteinbyIPTG・ThepurifiedhUNC93BlfusionproteinwaseasilyachievedonaNi2NTAaffinitycolumnbyvirtueofits(His)6tag.Rabbitswereimmunedwiththeexpressedproteinandimmunologicaladjuvantforspecificantibodypreparation.RESULTS:An1808bplengthfragmentwasamplifiedbyPCR.T

4、hesequencewascompletelymatchedwithhUNC93BlgenesequenceregisteredinGenbank;hUNC93BlpRSETAvectorwassuccessfullyconstructed・AfterthefusionproteinwastransfectedintoE.coil,aMr72000newproteinwasgotbyIPTGinduction.AspecificandhightiterantihUNC93Blserumin1：64000wasgaineda

5、tlast.CONCLUSION:hUNC93BlgenehasbeensuccessfullyclonedandhUNC93BlfusionproteinhasbeencorrectlyexpressedinE.coilandpurifiedbyNi2NTAagarosebeads・Aspecificandhight让erantibodyhasbeenprepared・AllthismakesitpossibletodofurtherinvestigationontherelationshipbetweenhUNC93B

6、landheartfailure・【Keywords]hUNC93Blgene;cardiovasculardiseases;geneexpression;proteinpurification【摘要】目的：构建人hUNC93El基因的原核表达载体,诱导其表达并纯化该蛋白，制备特异性高效价抗体.方法：设计针对hUNC93Bl基因的特异性引物，通过PCR的方法扩增目的基因，克隆入pMD18T载体，测序全部正确后亚克隆入原核表达载体pRSETA.将构建的载体导入大肠杆菌BL21中，用IPTG诱导表达.经SDSPAGE电泳鉴定后

7、用操柱纯化融合蛋白.与免疫佐剂联用，免疫家兔制备特异性抗体.结果：PCR扩增得到长度为1808bp的片断，结果与GenBank中人hUNC93Bl的基因序列完全一致，成功构建了原核表达载体hUNC93BlpRSETA.Bl血清.导入大肠杆菌后经诱导得到一条约Mr72000的新蛋白条带.免疫家兔后获得了1:64000的高效价特异性抗人hUNC93结论：成功地克隆了人hUNC93El基因，使其编码的蛋白质在原核细胞中顺利表达，并纯化出该种蛋白•制备了特异性抗体，为研究hUNC93El基因与人类心血管疾病的关系提供依据.【关键词

8、】hUNC93Bl基因；心血管疾病；基因表达;蛋白纯化0引言hUNC93Bl基因最早是由Kashuba等人于2002年最早发现并克隆的，该基因定位于llql3染色体上，由11个外显子组成，cDNA全长2282bp,编码597个氨基酸，表达的蛋白质Mr66600,GenBank登陆号为：NM_030930