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目的真核表达人类免疫缺陷病毒1型(HIV-1)CRF01_AE重组型gp120蛋白的V1/V2结构域,制备其单克隆抗体并鉴定其抗原反应性。方法构建pTriEx-3-V1/V2CNE55真核表达载体,在HEK293F细胞中表达V1/V2-His融合蛋白,纯化后作为抗原免疫BALB/c小鼠,取免疫后的小鼠脾细胞与Sp2/0骨髓瘤细胞融合,ELISA筛选分泌抗V1/V2重组蛋白单克隆抗体的阳性杂交瘤,对单克隆抗体的特异性、型别以及效价进行鉴定。结果制备并获得一株稳定分泌抗HIV-1 AE亚型V1/V2结构域单克隆抗体的杂交瘤细胞株,ELISA测定腹水效价高达1∶81 000,单克隆抗体类型属于IgG1/κ型。Western blot结果表明该单克隆抗体同样能够识别不同亚型的HIV gp120蛋白。结论成功制备出抗HIV-1 AE重组型gp120蛋白V1/V2结构域的单克隆抗体。
Objective To elicit the V1 / V2 domain of human immunodeficiency virus type 1 (HIV-1) CRF01_AE recombinant gp120 protein and prepare its monoclonal antibody and identify its antigenic reactivity. Methods The eukaryotic expression vector pTriEx-3-V1 / V2CNE55 was constructed and the V1 / V2-His fusion protein was expressed in HEK293F cells. The purified fusion protein was used as antigen to immunize BALB / c mice. The spleen cells were incubated with Sp2 / 0 Myeloma cells were fused. Positive hybridomas secreting anti-V1 / V2 recombinant protein monoclonal antibodies were screened by ELISA, and the specificity, type and potency of the monoclonal antibodies were identified. Results A hybridoma cell line secreting anti-HIV-1 AE subtype V1 / V2 domain monoclonal antibody was prepared and obtained. The titer of ascites was as high as 1:81 000 by ELISA and the type of monoclonal antibody was IgG1 / κ. Western blot results show that the monoclonal antibody can also recognize different subtypes of HIV gp120 protein. Conclusion Monoclonal antibodies against HIV-1 AE recombinant gp120 protein V1 / V2 domain were successfully prepared.