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目的:研制具有型特异性和构象依赖性的人乳头瘤病毒16主衣壳L1蛋白(HPV16 L1)单克隆抗体(mAb)。方法:利用昆虫-杆状病毒系统表达有生物活性的HPV16 L1蛋白。非变性条件下纯化HPV16 L1蛋白,免疫6周龄雌性BALB/c小鼠。末次加强免疫后取其脾细胞与骨髓瘤Sp2/0细胞融合,应用间接ELISA筛选阳性克隆。非变性Western blot鉴定mAb的型特异性和构象依赖性,细胞免疫组化确定抗体的型特异性。间接ELISA法测定杂交瘤分泌上清及腹水中抗体效价。mAb亚类试剂盒鉴定mAb亚类。结果:获得1株稳定分泌具有型特异性和构象依赖性的HPV16 L1抗体的杂交瘤细胞株,命名为2E3。2E3杂交瘤细胞株细胞培养上清中抗体效价为1∶800;小鼠腹水中抗体效价为1∶12800。亚类鉴定结果为IgG1κ。Western blot及细胞免疫组化分析结果显示,2E3mAb具有型特异性和构象依赖性,只与非变性的HPV16L1蛋白发生反应,不与HPV18L1、HPV58L1、HPV11L1产生交叉反应。结论:成功制备了型特异性HPV16 L1 mAb,为进一步研究HPV16 L1的抗原表位奠定了基础。
OBJECTIVE: To develop a monoclonal antibody (mAb) of human papillomavirus 16 capsid L1 protein (HPV16 L1) with specificity and conformation dependence. Methods: The biologically active HPV16 L1 protein was expressed using an insect-baculovirus system. HPV16 L1 protein was purified under non-denaturing conditions and 6-week-old female BALB / c mice were immunized. After the last booster immunization, the spleen cells were fused with myeloma Sp2 / 0 cells, and the positive clones were screened by indirect ELISA. The nonspecific Western blot was used to identify the type-specific and conformation-dependent mAbs, and the cellular immunohistochemistry was used to determine the antibody-type specificity. Indirect ELISA for the determination of antibody titer in hybridoma secreted supernatant and ascites fluid. mAb subclass kit to identify mAb subclasses. Results: One hybridoma cell line stably secreting HPV16 L1 antibody with specificity and conformation dependence was obtained. The antibody titer of the 2E3.2E3 hybridoma cell line was 1: 800. The mouse ascites fluid Middle antibody titer is 1: 12800. The subclass was identified as IgG1κ. The results of Western blot and immunohistochemistry showed that 2E3 mAb was type-specific and conformation-dependent, only reacts with non-degenerate HPV16 L1 protein and did not cross-react with HPV18 L1, HPV58 L1 and HPV11 L1. Conclusion: The type-specific HPV16 L1 mAb was successfully prepared, which lays the foundation for further study on the epitope of HPV16 L1.