重组人Leptin的表达、纯化及免疫学与生物学活性鉴定

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1、重组人Leptin的表达.纯化及免疫学与生物学活性鉴定张珏，王晓辉，冯泽国(解放军总医院麻醉手术中心北京100853)【摘耍】目的：为了获得重组人Leptin(rh-leptin)蛋白高表达的菌株及大量具有生物活性的rh-leptin蛋白,构建Leptin重组表达载体,转化感受态细菌进行表达,对rh-leptin蛋白进行复性及纯化，并鉴定其免疫学及生物学活性。方法：提取脂肪组织RNA,RT-PCR方法获得用PCR方法扩增人Leptin的编码区，使产物与pGEM-Teasy载体相连，转染DH5«o测序后，选出阳性克隆质粒扩增。将酶切后的leptin片段与pET-28a(+)

2、载体连接，获得leptin的表达质粒。将重组表达质粒转入BL21(DE3)，测序结果显示Leptin编码区没有突变产生，用TPTG诱导leptin重组蛋白表达。表达产物进行SDS-PAGE电泳&Western印迹杂交进行免疫学分析。并通过Km小鼠减肥实验及胃肠推进率测定实验检验其生物学活性。结果：在包涵体蛋白22kDa处有明显的新增蛋白带，其含量超过总蛋白的50%；经Western-blot证实为重组的人leptin蛋口。大量表达后，经蛋口复性和纯化，通过Km小鼠减肥实验及胃肠推进率测定实验证实重组表达的人Leptin蛋白具有生物学活性。结论：成功构建了rh—leptin

3、的高表达菌株，建立了rh—leptin的纯化与复性方法，经验证表达产物具有免疫学及生物学活性，可供进一步基础及临床研究应用。【关键词】rh—leptin；克隆；表达；融合蛋白；大肠杆菌；复性；蛋白纯化；生物活性Expression,PurificationandIdentificationforImmunologicalandBiologicalActivityofRecombinedHumanLeptin[Abstract]Objective:Toobtaineffectivemethodofhighrecombinedhumanleptin(rh-leptin)exp

4、ressioninE.coliandthemethodofpurifyingandrenaturingtherh-leptionprotein,thentoobtainalargeamountofrh-leptinproteinwithimmunologicalandbiologicalactivityforfurtherresearch..Methods:TheadiposeLeptinCDswasobtainedbyreversetranscription-polymerasechainreaction(RT-PCR)andclonedbyPCR,andtheprod

5、uctwasconnectedwithpGEM-TeasyandtransformedintoDH5a•AfterDNAsequencing,thepositivecolonieswerepickedoutandamplified.ThecleavedleptinfragmentwasclonedintopET-28a(+).Then,therecombinedplasmidofleptinwastransformedintoBL21(DE3).DNAsequencingresultshowedthatnomutationwasfoundinrh-leptin'sCDS.

6、AfterIPTGinduction,thecelllysatewasdirectlyusedforSDS-PAGE.Afterpurificationandrenaturation,theimmunologicalactivityofrh-leptinwascheckedbyWestern-blot.ItsbiologicalactivitywascheckedbyreducingtheweightandfoodintakeandinhibitingthegastrointestinalmotilityofKmmice・Results:Therewasanewbando

7、fprotein(>50%totalprotein)around22KDaonSDS-PAGE.ThebandwasprovedbyWestern-blottoberh-leptin,whichwashighlyexpressedintheBL21(DE3)/pET-28a(+)system,andtherh-leptinhasimmunologicalactivity.Itsbiologicalactivitywasconfirmedbyreducingtheweightandfoodintakeandinhibitingt