人乳头瘤病毒16型L1蛋白表达及其单克隆抗体杂交瘤细胞株的建立

目的表达并纯化重组人乳头瘤病毒16型（HPV16）L1蛋白，建立分泌抗HPV16 L1单克隆抗体（mAb）的杂交瘤细胞株。方法以基因工程表达HPV16L1蛋白。用Ni—NTA技术进行纯化后免疫BALB／c小鼠，取脾细胞与SP2／0骨髓瘤细胞融合，经含次黄嘌呤、氨基蝶呤、胸腺嘧啶（HAT）的选择培养基及有限稀释法进行克隆化，用间接ELISA法筛选阳性杂交瘤细胞，对获得的杂交瘤细胞株染色体进行计数．同时测定细胞培养上清mAb的效价。结果筛选出2株可分泌抗HPV16 L1 mAb的杂交瘤细胞，命名为AE3和AG7，染色体数分别为130条和98条。2株杂交瘤细胞AE3和AG7培养上清的效价分别为1：5120和1：80。结论成功建立了2株可分泌抗HPV 16 L1 mAb的杂交瘤细胞，为进一步研制HPV感染检测试剂盒提供实验基础。

Expression of human papillomavirus type 16 L1 and construction of hybridoma cell strain of human papillomavirus type 16 L1 monoclonal antibody

Objective To construct hybridoma cell strain that secretes monoclonal antibodies (mAb) of human papillomavirus type 16 (HPV16) L1 antigen and to express and recombine HPV16 L1. Methods Protein of HPV 16 L1 gene was expressed by gene engineering bacterium and the protein was purified by Ni-NTA. BALB/c mice were immunized with purified HPV 16 L1 protein. The fusion cells were obtained from splenocytes of immunized mice and myeloma SP2/0 cells. Monoclonal hybridoma cell lines were obtained by limiting dilution, HAT selective culture. The mAbs was screened by ELISA and the chromosome number of hybridoma cell was counted. Agarose gel double diffusion test was used to detect the titer of mAbs in the supernatant of the culture medium. Results Two hybridoma cell strains AE3 and AG7 were selected, which secreted HPV 16 L1 mAbs, name as AE3 and AG7, classified into IgG1. The chromosome number of AE3 and AG7 was 130 and 98, respectively. The titer of AE3 and AG7 supernatant was 1:5 120 and 1:80, respectively. Conclusion Two hybridoma cell lines which have ability of secreting mAbs against HPV 16 L1 antigen have been established successfully, which lays the foundation for developing HPV detecting kit.