新耐药基因HA117多克隆抗体的制备与鉴定

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1、新耐药基因HA117多克隆抗体的制备与鉴定陈建飞，金先庆，丁雄辉，孙艳辉，赵利华，郭振华，王士奇(400014重庆，重庆医科大学附属儿童医院儿童发育疾病研究省部共建教育部重点实验室)［摘要」目的制备HA117多克隆抗体。方法以HA117表达质粒pAdTrack-HAll7为模板，PCR扩増HA117基因，扩增产物转入BI.21(DE3)感受态细胞，进行HA117蛋白诱导表达，采用SDS-PAGE检测表达产物。纯化后免疫新西兰大口兔，制备多克隆抗体。通过间接ELISA检测其效价。将转染携带HA117

2、重组腺病毒的HEK293细胞为实验组，未转染的IIEK293细胞为对照组,用细胞免疫荧光和Westernblot方法检测其过表达的HA117蛋白,验证制备的IIA117多克隆抗体的特界性。结果成功获得I1A117多克隆抗体，间接EL1SA&检测显示抗1IA117血清效价为1:729000。细胞免疫荧光和Westernblot检测示制备的HA117多克隆抗体可以特异性识别HA117抗原蛋白。结论获得了效价和特杲性较高的HA117多克隆抗体。［关键词］HA117；多克隆抗体；抗体制备；鉴定［中图法分类

3、号］［文献标志码］［基金项目］国家自然科学基金面上项冃(39970768)［通信作者］金先庆，电话：(023)63638834,E-mail：etzhl@163.comPreparationandidentiHcationofpolyclonalantibodyofnovelmultidrugresistancegeneHA117CHENJianfei,JINXianqing,DINGXionghui,SUNYanhui,ZHAOLihua,GUOZhenhua,WANGShiqi(KeyLabo

4、ratoryofDevelopmentalDiseasesinChildhood,ChongqingMedicalUniversity,MinistryofEducation)(问题1:单位英文信息请参照本刊写法完善。)［Abstract］Objective:PreparingpolyclonalantibodyofI1A117inordertoexplorethedistributioncharacter!stiesandexpressionlevelofHA117intumorandnornm

5、ltissues・Methods:pAdTrack-HAl17plasmidwasusedasthetemplatetoamplifythetotalcDNAofHA117usingPCR.TheproductsofPCRweretransferredintoBL21(DE3)competentcellstoinducetheproteinexpressionofHA117andidentifiedbySDS-PAGE.NewZealandrabbitswereimmunedwith1IA117p

6、roteinafterbeenpurifiedinordertogetthepolyclonalantibodyofHA117・IndirectELISAwasusedtodetectthetiterofthepolyclonalantibodyofIIA117・AdenovirusvectorencodingHA117genewastransductedintoHEK293・ImmunofluorescenceandWesternblotwereusedtodetectthespecificit

7、yofpolyclonalantibodyofIIA117.Results:PolyclonalantibodyofHA117wasgotsuccessfully・Theanli-HA117scrumtiterwas1:729000showedbyindirectELISA.ThepolycloncantibodyofHA117couldidentifythe1IA117proteinintrcinsducted11EK293specificallyshowedbyimmunofluorescen

8、eeandWesternbolt・Conclusion:TheacquireofthepolyclonalantibodywithahightiterandhighspecificityofI1A117providedausefulandstrongtoolforexploringthedistributioncharacteristicsandexpressionlevelofHA117intumorandnormaltissue,providedagoodwayforfurth