58型人乳头瘤病毒E7蛋白单克隆抗体的制备及鉴定

Preparation and identification of monoclonal antibody against E7 oncoprotein from human papillomavirus type 58

为获得抗58型人乳头瘤病毒(HPV58)E7蛋白的单克隆抗体,乳糖诱导重组工程菌表达HPV58 E7融合蛋白,以纯化后的该重组蛋白为免疫原,经细胞融合和有限稀释后,得到6株分泌抗HPV58 E7单克隆抗体(McAb)的工程细胞株,并采用体内接种方法及亲和柱层析纯化技术制备6株抗体.酶联免疫吸附测定(ELISA)、Western-blot实验和细胞免疫荧光检测结果表明,所得抗体均为IgG2a类,κ型,抗体效价均可达10-5,并且能够与原核及真核系统表达的HPV58 E7蛋白发生特异性抗原-抗体反应.该抗体的成功制备为后续HPV58 E7致癌生物学研究及相关肿瘤诊断试剂的开发奠定基础.

To obtain monoclonal antibodies （McAbs） against E7 oncoprotein of human papillomavirus type 58 （HPV -58 ） ,lactose induction was applied to express GST -HPV58E7 fusion protein in recombinant bacterial strain. Using simply GST affinity chromatographic separation,the target protein was purified and used as a novel antigen to produce antibodies. After mouse immunization, somatic hybridization, cloned culture, positive hybrido- ma scanning, ascites preparation, rProtein, and affinity purification, 6 strains of purified and high titer anti - HPV58E7 McAbs were prepared. Further studies used ELISA, Western -blot and immunofluorescence, and proved these obtained antibodies could specifically react with HPV -58 E7 which was expressed either in pro- karyotic or eukaryotic cells. Moreover, another indirect ELISA also revealed that these 6 strains of McAbs were IgG2a K type antibodies and bound with the same epitope in entire sequence of HPV -58 E7 oncoprotein. In conclusion, the prepared McAbs provides the basis for tumor biology research and the development of related di- agnostic reagents which are targeting HPV -58 ET, and these studies are very important to effective screening, di- agnosis, prevention and treatment of cervical cancer caused by HR - HPV detect in our country.