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1、·1268·江苏医药2008年12月第34卷第12期JiangsuMedJ,December2008,Vol34,No.12·论著·人胰升血糖素样肽1突变体基因的克隆及在原核细胞的表达朱清秦红兵顾锦华朱红艳【摘要】目的构建人胰升血糖素样肽1(hGLP21)突变体基因的原核表达载体,并在大肠杆菌8BL21中进行表达。方法用生物合成方法获得hGLP21突变体基因Val2hGLP21,将其克隆于载体8pGEX24T21中,构建pGEX24T21/Val2hGLP21融合表达载体,转化大肠杆菌BL21,酶切电泳以及测序鉴定阳性克隆,用SDS2PAGE和蛋白免疫印迹法检
2、测其重组蛋白诱导表达情况。结果重组质粒8pGEX24T21/Val2hGLP21经双酶切电泳鉴定与测序分析证实构建成功,SDS2PAGE与蛋白免疫印迹8分析证实其成功诱导表达重组融合蛋白。结论成功地克隆了hGLP21突变体基因Val2hGLP21,并在大肠杆菌中进行表达,为下一步获得含突变体基因的重组hGLP21蛋白及其生物学功能研究奠定良好基础。【关键词】人胰升血糖素样肽1;原核表达;突变体【中图分类号】Q78【文献标识码】A【文章编号】025323685(2008)1221268203Cloningofhumanglucagon2likepeptide21
3、mutantgeneanditsexpressioninE.colicellsZHUQing,QINHongbing,GUJinhua,etal.DepartmentofPharmacology,MedicalCollegeofNantongUniversity,Nantong226001,CHINA【Abstract】ObjectiveToconstructtheprokaryoticexpressionvectorofhumanglucagon2likepeptide21(hGLP21)mutantgene,andexpressitinE.colicell
4、s.MethodsThemutantgeneofhGLP281(Val2hGLP21)wasobtainedbybiosynthesistechnique.Themutantgenewasclonedintothevector8pGEX24T21toconstructtheexpressionvectorofpGEX24T21/Val2hGLP21.TherecombinantplasmidwastransformedintoE.colicells(BL21),andthenthepositiveclonewasidentifiedbyelectrophore
5、sisafterenzymedigestionandDNAsequencing.TheexpressionoffusionproteininducedbyIPTGwasanalyzedbySDS2PAGEandWesternblot.ResultsElectrophoreticanalysisandDNAsequencing8confirmedthattherecombinantplasmidpGEX24T21/Val2hGLP21hadbeenconstructedsuccessfully,andtheexpressionofrecombinantfusio
6、nproteininE.colicellswasdisplayedbySDS2PAGEand8testifiedbyWesternblotanalysis.ConclusionThemutantgeneVal2hGLP21hasbeensuccessfullyclonedandexpressedinE.colicells,whichmayprovidethefoundationforfurtherobtainingrecombinanthGLP21mutantandexperimentalstudiesonitsbiologicalfunction.【Keyw
7、ords】Humanglucagon2likepeptide21;Prokaryoticexpression;Mutant[JiangsuMedJ,December2008,34(12):126821270.]人胰升血糖素样肽1(hGLP21)是由肠道L细失去生物活性,限制了其临床应用。因此,开发抵抗胞分泌的肠促胰岛素,它最重要的生理作用是葡萄DPP2IV降解的GLP21长效类似物,对于糖尿病的[1,2][7]糖依赖的胰岛素刺激作用,几乎不引起低血糖。防治具有重要意义。本研究选择hGLP21(7～8有学者研究发现,其促胰岛素分泌作用是通过促进37)第8位的丙氨
8、酸(Ala)作为突变位点,生物合成[3
