人胰高血糖素样肽.docx

人胰高血糖素样肽【摘要】目的：对人胰高血糖素样肽-1进行基因突变，构建GST融合蛋白原核表达质粒pGEX-4T/hGLP-1，并在大肠杆菌中诱导表达。方法：选择大肠杆菌偏爱密码子，将hGLP-1第2位的丙氨酸定点突变为缬氨酸，直接生物合成4个寡聚核苷酸片段并进行基因拼接，形成完整的hGLP-1突变体基因hGLP-1，并连接到载体pGEX-4T-1，转化大肠杆菌BL21，BamHI与XhoI双酶切电泳鉴定与序列测定，然后诱导表达其融合蛋白。结果：经酶切电泳鉴定与测序分析，证实所合成的突变体基因hGLP-1已克隆到pGEX-4T-1中，经SDS-PAGE分析可以诱导表达出融合蛋白。结论：成功地构建了GST融合蛋白原核表达质粒pGEX-4T/hGLP-1，为进一步获得重组hGLP-1蛋白奠定基础，为产业化规模制备hGLP-1突变体提供实验依据。【关键词】人胰高血糖素样肽-1；融合蛋白；基因突变；克隆；糖尿病ConstructionofexpressionvectorofhumanGlucagon-likepeptide-1mutantgene[Abstract]Objective:ToobtainthemutantgeneofhumanGlucagon-likepeptide-1(hGLP-1),constructtheexpressionvectorofpGEX-4T/hGLP-1,andexpresstheGSTfusionproteinincells.Methods:Withthepreferentialcodonofbeingselected,fouroligonucleotidefragmentsweredirectlybiosynthesized,thenweresplicedtoformacompletemutantgeneofhGLP-1whichchangedthecodonforthesecondaminoacidofhGLP-1(7~37)-alanine(Ala8)intovaline(Val8)bymeansofsite-directedmutagenesis,soitnamedhGLP-1.ThemutantgenewasinsertedintothevectorpGEX-4T-1withBamHIandXhoIrestrictionrecombinantplasmidofpGEX-4T/hGLP-1wastransformedintoE.colicells(BL21),thenidentifiedbyelectrophoresisafterenzymedigestionandtestifiedbyDNAsequencing,furtherinducedtoexpressthefusionproteinbyIPTG.Results:TheresultsofelectrophoreticanalysisandDNAsequencingconfirmedthatthesyntheticmutantgene(Val8)hGLP-1hadbeenclonedintothepGEX-4T-1successfully,andinducedexpressionoffusionproteinbySDS-PAGEanalysis.Conclusion:TheGSTfusionproteinexpressionplasmidofpGEX-4T/(Val8)hGLP-1wassuccessfullyconstructed，whichlaidthefoundationforfurtherobtainingrecombinanthGLP-1mutantforexperimentalorclinicalstudiesandprovidedthetechnicalroutesofindustrialization.[Keywords]HumanGlucagon-likepeptide-1;Fusionprotein;Genemutation;Cloning;Diabetesmellitus人胰高血糖素样肽－1是由人胰高血糖素基因编码、并由肠道L细胞分泌的一种肽类激素，它是随着食物在肠道的消化吸收过程而分泌的，其功能由hGLP-1受体(hGLP-1R)介导，是目前能诱导胰岛素分泌的最强物质之一，还能以同样的强度抑制胰高血糖素的分泌，它促胰岛素分泌的作用具有葡萄糖依赖性，随着葡萄糖浓度降低，其促胰岛素分泌作用逐渐减弱，因而hGLP-1降糖作用是自限性的，不会增加2型糖尿病患者发生低血糖的危险性，这是hGLP-1优于磺酰脲类降糖药及胰岛素最显着的特点[1,2]。另外研究发现GLP-1具有改善胰腺B细胞及胰岛功能、增加胰岛素敏感性、抑制胃排空