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目的 构建抗人肝癌的噬菌体抗体库并对该库进行初步筛选。方法 以 RT- PCR法从经 Bel740 4细胞免疫的 BAL B/ C小鼠脾淋巴细胞扩增免疫球蛋白的 Fd段及 k链基因 ,克隆到表达载体 p COMB3H- SS中并将抗体 Fab段表达于单链噬菌体表面 ,构建噬菌体抗体库 ;以 Bel740 4细胞为抗原对所建噬菌体抗体库进行 4轮“吸附 -洗脱 -扩增”的亲和筛选。结果 建成了容量为 2× 10 6 cfu的噬菌体抗体库 ,在亲和筛选过程中 ,噬菌体收获率逐轮得到提高 ,第 4轮为第一轮的 2 46倍 ,含 Fab基因的克隆比率也由 2 5 %增到 83% ,表明特异性噬菌体抗体得到了富集。结论 噬菌体抗体库技术系高效筛选体系 ,为肿瘤单抗的制备提供了有效的新途径
Objective To construct a phage antibody library against human hepatocellular carcinoma (HCC) and preliminary screen the library. Methods Fd and k chain genes of immunoglobulin were amplified from splenic lymphocytes of BALB / c mice immunized with Bel740 4 cells by RT-PCR and cloned into the expression vector p COMB3H-SS and the Fab fragment was expressed Phage antibody library was constructed on the surface of single-stranded phage, and 4 rounds of “adsorption-elution-amplification” affinity screening with the Bel740 4 cell antigen was performed. Results A phage antibody library with a capacity of 2 × 10 6 cfu was constructed. During the affinity screening process, the phage harvesting rate was improved by rounds, the fourth round was 246 times of the first round, and the cloning rate of the Fab gene was also increased from 25% to 83%, indicating that specific phage antibodies are enriched. Conclusion The phage antibody library technology is an efficient screening system that provides an effective new approach for the preparation of tumor monoclonal antibodies