抗HIV-1 gp120单克隆抗体的制备及鉴定

Preparation and identification of anti-HIV-1 gp120 monoclonal antibody

目的制备抗HIV-1 gp120单克隆抗体,鉴定其特性,为进一步研制HIV治疗性抗体提供贮备。方法将HIV-1 gp120基因片段连接到经酶切的原核表达载体PEGX-4T-2中,利用大肠杆菌XL1-blue表达GST-HIV蛋白。表达的蛋白纯化后免疫BALB/c小鼠,取免疫小鼠脾细胞与SP2/0骨髓瘤细胞融合,间接ELISA筛选阳性克隆。重组免疫印迹分析(RIBA)和Western blot检测抗体特异性。结果构建的重组质粒的外源基因片段经测序与预期HIV-1 gp120抗原基因片段大小一致。纯化蛋白SDS-PAGE可见1条相对分子质量(Mr)32 000的表达蛋白条带。获得6株稳定分泌单克隆抗体的杂交瘤细胞株,其细胞培养上清效价为10-3,小鼠腹水效价为10-5,抗体亚型均为IgG1。RIBA结果显示6株单抗皆只与HIV-1 gp120抗原反应。Western blot结果显示在Mr32 000处可见1条单一目的条带。结论获得6株稳定分泌抗HIV-1 gp120单克隆抗体的杂交瘤细胞株,为进一步研制抗HIV治疗性抗体奠定了基础。

Purpose To Prepare anti- HIV-1 gpl20 monoclonal antibodies and to identify the specificity of antibodies in order to provide technique for preparing HIV remedial antibodies. Methods The gene fragment of HIV-1 gp120 was connected to PEGX-4T-2 prokaryotic expressing vector. The vector was cut by enzyme. GST- HIV protein was expressed by E. coli XLl-blue. The BALB/e mice were immunized with purified GST-HIV antigen, and then the fusion of mice spleen cell and myeloma cell SP2/0 was executed as the routine cell-fu- sion technique. Positive cells were screened by Indirect ELISA. Immuno-blotting assay and Western blot identi- fied the specificity of antibodies. Results External gene section from the recombinant plasmid by sequencing showed the same size of HIV-1 gpl20 gene sequences tained after purified protein SDS-PAGE electrophoresis An external expressed protein band of 32 KD was ob- It indicated that six strains of hybridoma cells secreting special monoclone antibodies had been obtained. ELISA resu]ts showed that six strains monoclonal antibodies only reacted to HIV-1 gpl20 antigen. Western blot results showed that a band with molecular weight 32 kDa was obtained, which could interact with HIV-1 gp120 monoclonal antibody. Conclusion Six strains of hybrido- ma cells secreting special monoclone antibodies had been obtained, The prepared monoclonal antibodies have established a basis for HIV remedial antibody.