噬菌体抗体库的构建及抗肝癌抗体的筛选

Cloning of Anti-liver Cancer Monoclonal Antibodies from Phage Antibody Library

目的 构建噬菌体抗体库及筛选抗人肝癌特异性噬菌体抗体。 方法 以ＲＴ－ＰＣＲ法从经Ｂｅｌ７４０４细胞免疫的ＢＡＬＢ／Ｃ小鼠脾淋巴细胞扩增免疫球蛋白的Ｆｄ 段及к链基因，克隆到表达载体ｐＣＯＭＢ３Ｈ－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Ｂｅｌ７４０４细胞为抗原对噬菌体抗体库进行４轮“吸附－洗脱－扩增”的亲和筛选，挑取部分克隆检测与抗原的结合活性。 结果 建成容量为２×１０６ＣＦＵ 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２４５ 倍，含Ｆａｂ 基因的克隆比率也由２６％ 增到８３％ ，挑取的１０ 个克隆中有８ 个与Ｂｅｌ７４０４细胞有结合活性。 结论 噬菌体抗体库技术系高效筛选体系。

To construct a phage antibody library and select specific phage antibodies against human liver cancer from the library \ Methods\ RT PCR was used to amplified immunnoglobulin heavy chain Fd genes and κ chain genes from spleen lymphocytes of BALB/C mice immunized with human liver cancer cell line Bel\-\{7404\} \ These genes were recombined with vector pCOMB3H SS and Fab fragment were displayed on filamentous phage to construct a phage antibody library \ After four rounds of selection against Bel\-\{7404\} cell,some clone were isolated for detect the binding capacity \ Results\ A phage antibody library with a repertoire of 2×10\+6 CFU was constructed \ From the 1st round to the 4th round of the panning,the number of the eluted phages had increased 245 times and the rate of the clones containing Fab genes was raised from 25% to 83% \ Eight of the ten clones exhibited Bel\-\{7404\} cell binding capacity All these indicated that the specific phage antibodies had been enriched during the panning \ Conclusion\ The technology of phage antibody library was a high selective system,which could serve as a new useful method in the production of McAbs toward human cancer \;