抗组氨酰-ｔＲＮＡ合成酶单克隆抗体制备及初步鉴定

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1、抗组氨酰-tRNA合成酶单克隆抗体制备及初步鉴定【摘要】制备均一性和特异性高的抗组氨酰-tRNA合成酶抗体，为研究皮肌炎和多肌炎的检测提供方法，为制备检测试剂盒提供原料。【方法】采用常规融合和间接ELISA检测法，获取杂交瘤细胞株；ProteinGSepharose4FF和SephadexG50结合对含有单克隆抗体的腹水进行纯化；SDS-PAGA电泳技术进行单克隆抗体的鉴定。【结果】筛选获得了2株稳定分泌阳性抗体的杂交瘤细胞株，分别命名为W001和W002；杂交瘤细胞克隆后，100%的检测孔保持了分泌抗组氨酰-tRNA合成酶抗体的能力；腹水纯化达到了较理想的

2、效果；该2株单克隆抗体能特异性针对组氨酰-tRNA合成酶，并能跟天然的蛋白结合。【结论】所制备的均一性和特异性高的抗组氨酰-tRNA合成酶抗体，可直接用于在ELISA、免疫印迹、免疫组化学等研究方面，达到了试验的目的。【关键词】组氨酰-tRNA合成酶抗组氨酰-tRNA合成酶单克隆抗体鉴定Abstract：[Objective]Toproduceanti~histidyl-tRNAsynthetase,whichenjoyhighdegreeofhomogeneityanddistinction,toprovidethetestmethodsforthestu

3、dyofthediseasesofdermatomy0sitisandpolymyositis・Besides,toproducematerialsforthetest1iquid・【Methods]Withtheregularcombinedmethodsavai1ab1eandindirectELISAtoobtainhybridomacell1ines；topurifytheasciteswithmonoclonalant■1bodyinthecombineduseofproteinGSepharose4FFandsephadexG50；toident

4、ifymonoc1ona1antibodywiththeelectrophoresistechnique.【Results]Twohybridomace111ines,namedW001andWO02,havebeenfoundintheresearch,whichcansecreteanti~histidyl-tRNAsynthetasemonoclonalantibodystab1y.Afterthecellsofhybridomabeingcloned,100%checkingholeskeepthecapabi1ityofsecretinganti-

5、histidy1-tRNAsynthetasemonoc1ona1antibody・AndtheascitesofthemonoclonalantibodyhasbeenpurifiedbyProteinGSepharose4FFandSephadexG50,inwhichthepurificationwassatisfying・Inaddition,thetwomonoclonalantibodiesshowedtheirabi1itytocomposemold,andtointegratewithnaturala1bumen.【Conclusion】Th

6、eanti-histidy1-tRNAsynthetasemonoc1ona1antibodiesinthisstudyarehomogeneousanddistinctive,whichcanbeuseddirect1yintheresearchofELISA,WesternBlotandImmunohistochemistryetc・Obviously,theaimofthisresearchhasbeenachieved・Keywords：histidyl-tRNAsynthetase；anti-histidyl-tRNAsynthetase；mono

7、clonalantibody；identification抗组氨酰-tRNA合成酶抗体是一类结缔组织病中的多肌炎(po1ymyositis)和皮肌炎(dermatomyositis,PM/DM)的标志性自身抗体。PM/DM指横纹肌弥漫性炎症疾患，主要累及对称性的近端肢带肌，颈和咽部肌肉无力和萎缩，严重者导致心肌、呼吸肌群受累，引起死亡。只出现肌痛肌无力现象者称多肌炎；在多肌炎的基础上，出现典型皮疹者称皮肌炎。PM/DM是自身免疫性疾病，临床检验发现25%〜30%的患者含抗组氨酰-tRNA合成酶抗体[1](临床上又称抗Jo-1抗体)，而其他抗合成酶抗体在PM/

8、DM中的阳性率仅为1%〜3%,其中PM的阳性率为30