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利用mRNA提取试剂盒直接从健康人外周血白细胞中提取mRNA ,逆转录为cDNA ,合成引物扩增人抗体分子IgG1亚型Fc基因 ,克隆到pGEMT Vector中并测序。合成引物扩增人抗体分子轻、重链信号肽序列 ,分别克隆并测序。将轻链信号肽和轻链 (kappa)VL CL基因进行重组形成轻链全分子基因 ,再将其克隆到哺乳细胞表达载体pcDNA3.1中。将重链信号肽、重链 (gamma)VH CH1和Fc基因进行重组形成重链全分子基因 ,再将其克隆到哺乳细胞表达载体 pCI DHFR1中。将轻、重链全分子表达载体同时转染中国仓鼠卵细胞 (CHO细胞 ) ,以药物G418和氨甲喋呤 (MTX)筛选细胞克隆 ,检测细胞克隆的培养上清中抗HBsAg抗体活性 ,结果筛到数株较高表达的克隆 ,当MTX药物浓度增加到 1× 10 -7mol/L时 ,平均每 10 6个细胞培养上清抗HBsAg最高表达量可达 8μg/d。回收细胞培养上清 ,以ProteinL亲和层析柱纯化重组抗体。该抗体在非还原SDS PAGE中表现为一条高分子量 (>10 0kD)的蛋白质带 ,在还原SDS PAGE中表现为一条约 5 0kD的带和一条约 2 5kD的带 ,Western印迹分析结果显示 ,该蛋白质的全分子和重链分子可以和羊抗人Fc抗血清发生特异性免疫反应。
MRNA was extracted directly from peripheral blood leukocytes of healthy people by mRNA extraction kit and reverse transcribed into cDNA. The Fc gene of human IgG1 subtype was amplified by PCR and cloned into pGEMT Vector and sequenced. Synthetic primers amplified human antibody molecules light and heavy chain signal peptide sequences were cloned and sequenced. The light chain signal peptide and light chain (kappa) VL CL gene were recombined to form a light chain full molecule gene, and then cloned into the mammalian expression vector pcDNA3.1. The heavy chain signal peptide, gamma VH CH1 and Fc genes were recombined to form a heavy chain full molecule gene, and then cloned into the mammalian expression vector pCI DHFR1. The full-length light and heavy chain expression vectors were transfected into Chinese hamster ovary cells (CHO cells) simultaneously. The cell clones were screened with drugs G418 and methotrexate (MTX), and the anti-HBsAg activity of the cell culture supernatants was detected. When the concentration of MTX drug was increased to 1 × 10 -7 mol / L, the highest expression level of anti-HBsAg per 10 6 cell culture supernatants was up to 8 μg / d. The cell culture supernatant was recovered and the recombinant antibody was purified with Protein L affinity chromatography. The antibody appeared as a high molecular weight (> 100 kD) protein band in non-reducing SDS PAGE and showed a band of about 50 kD and a band of about 25 kD in reducing SDS PAGE. Western blot analysis showed that the Protein whole and heavy chain molecules can specifically immunoreact with goat anti-human Fc antisera.