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目的制备细粒棘球绦虫胰岛素受体(EgIR)蛋白的多克隆抗体,为进一步研究EgIR蛋白的功能提供检测抗体。方法利用Gamier-Robson、Chou-Fasman、DNAMAN等软件预测EgIR蛋白抗原表位,进行引物序列设计。将预测的EgIR基因部分序列克隆至原核表达载体pGEX-4T-1,转化E.coli ROSETTA(DE3)株,IPTG诱导蛋白表达,表达产物EgIR蛋白经谷胱甘肽琼脂糖柱纯化后注射免疫家兔10d,收集免疫血清,采用ELISA法测定抗体效价,采用Western blot检测制备的多克隆抗体与EgIR蛋白的结合特性。结果构建的原核表达载体转化DE3后经0.8mmol/L IPTG诱导,成功表达约55ku的EgIR重组蛋白;目的蛋白纯化后的浓度为3mg/ml,纯度为85%;免疫家兔后收集的血清经ELISA法检测,抗体效价>1∶51 200;Western blot检测该抗体能与10、5、1ng/ml的EgIR重组蛋白特异结合,该抗体1∶1 000稀释后仍可检测到原头蚴、囊泡蛋白样品中的胰岛素受体蛋白。结论成功制备兔抗EgIR亚基多克隆抗体,为EgIR蛋白的功能和药物靶标研究奠定了基础。
Objective To prepare a polyclonal antibody against the echinococcus granulosus insulin receptor (EgIR) protein and to provide a test antibody for further study on the function of EgIR protein. Methods Gamier-Robson, Chou-Fasman, DNAMAN and other software were used to predict the epitope of EgIR protein and the primer sequence was designed. The partial sequence of predicted EgIR gene was cloned into prokaryotic expression vector pGEX-4T-1 and transformed into E.coli ROSETTA (DE3) strain. IPTG induced protein expression, EgRNA protein was purified by glutathione sepharose column and immunized Rabbit 10d, immune serum was collected, the antibody titers were measured by ELISA, and the binding characteristics of the prepared polyclonal antibody and EgIR protein were detected by Western blot. Results The prokaryotic expression vector was transformed into DE3 and induced by IPTG at 0.8 mmol / L. The recombinant protein was about 55 ku of EgIR recombinant protein. The purity of purified recombinant protein was 3 mg / ml and the purity was 85% ELISA method, antibody titer> 1:51 200; Western blot detection of this antibody with 10,5,1 ng / ml EgIR recombinant protein specific binding of the antibody 1: 1 000 dilution of the original cercariae can still be detected, Insulin receptor protein in vesicle proteins. Conclusion The successful preparation of rabbit anti-EgIR subunit polyclonal antibody laid the foundation for the study of the function and drug target of EgIR protein.