人源性噬菌体抗体库的构建及抗amylin抗体的初步筛选

Construction of human phage antibody library and screening for human monoclonal antibodies of amylin

目的:构建人源性噬菌体抗体库,从中筛选胰淀素(amylin)单克隆抗体(mAb),测定其特异性及抗原结合活性。方法:从正常健康人的外周血淋巴细胞中提取总RNA,用RT-PCR方法扩增人免疫球蛋白Fd段和轻链基因,构建噬菌体抗体库。酶切和PCR鉴定后,阳性克隆进行DNA测序分析。用人amylin抗原对抗体库进行筛选富集,将得到的阳性克隆进行Phage-ELISA鉴定,结果进行统计学分析。结果:最终构建的抗体库库容约为0.8×108,酶切鉴定显示有插入片段,抗体库重组率为70%。阳性克隆进行DNA测序证实所获基因为人免疫球蛋白可变区基因。以amylin抗原进行3轮筛选,抗体库得到特异性富集。阳性克隆进行Phage-ELISA检测证实有良好的抗胰淀素抗原的特异性。结论:成功构建了一个人源性噬菌体抗体库,为从中获得人源抗amylin的mAb奠定了实验基础。

AIM： To screen monoclonal antibodies to amylin from a constructed human phage antibody library and identify their antigenic specificity and combining activities. METHODS： The heavy chain Fd fragment and light chain of human immunoglobulin genes were amplified from peripheral blood lymphocytes of healthy donors using RT-PCR, and then inserted into phagemid pComb3XSS to generate a human phage antibody library. The insertion of light chain or heavy chain Fd genes were identified by PCR after the digestion of Sac I, Xba I, Xho I and Spe I. One of positive clones was analyzed by DNA sequencing. The spe- cific anti-amylin clones were screened from antibody library against human amylin antigens and then the positive clones were determined by Phage-ELISA analysis. RESULTS： A Fab phage antibody library with 0. 8 x 108 members was constructed with the efficacy of about 70%. DNA sequence analysis indicated VH gene belonged to VH3 gene family and Vx gene belonged to the Vx gene family. Using human amylin as panning antigen, specific antJ-amylin Fab antibodies were enriched by screening the library for three times. Phage-ELISA assay showed the positive clones had very good specificity to amylin antigen. CONCLUSION： The successful construction of a phage antibody library and the identification of antJ-amylin Fab antibodies provide a basis for further study and preparation of human anti-amylin antibodies.