摘要目的 通过建立新型人源化抗HIV-1 P24单克隆抗体制备技术平台,研制1～2种抗HIV-1 P24抗体.方法 使用连接P24的磁珠分选特异性分泌P24抗体的B细胞,有限稀释后,提取总mRNA,采用逆转录和巢式PCR扩增免疫球蛋白重链和轻链基因,经测序鉴定后克隆到真核表达载体Cloning vector AbVec-hIgG1、Cloning vector AbVec-hIgKappa、Cloning vector AbVec-hIgLambda中;通过瞬时转染293T细胞得到重组抗体;使用proteinA亲和层析纯化抗体.结果 成功筛选到5对抗HIV-1 P24的人源单克隆抗体基因.结论 本研究初步成功地建立了人源化HIV-1 P24单克隆抗体的制备及纯化方法,为HIV早期诊断以及筛选其他人源化单克隆抗体奠定了基础.

abstractsObjective We have developed an a new and efficient humanized anti-HIV-1 P24 monoclonal antibody preparation platform to develop 1-2 anti-HIV-1 P24 antibodies.Methods Specific secretory P24 antibody B cells were sorted by connecting P24 resin,after limited dilution,the total mRNA was extracted then immunoglobulin heavy and light chain genes were amplified by reverse transcription and nested PCR.Then the sequence was cloned into the eukaryotic expression vector,Cloning vector AbVechIgG1,Cloning vector AbVec-hIgKappa,Cloning vector AbVec-hIg Lambda,after the sequencing.We obtained the recombinant antibody by the instantaneous transmission of 293T cells to obtain recombinant antibody.Then the HIV-1 P24 antibody was purified by protein A affinity chromatography.Results We have successfully screened five groups of humanized anti-HIV-1 P24 monoclonal antibody genes.Conclusion We initially and successfully established the humanized anti-HIV-1 P24 monoclonal antibody preparation and purification methods.