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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。
OBJECTIVE: To construct human phage antibody library from which amylin monoclonal antibody (mAb) was screened for its specificity and antigen binding activity. Methods: Total RNA was extracted from peripheral blood lymphocytes of healthy volunteers. The Fd segment and light chain of human immunoglobulin were amplified by RT-PCR and the phage antibody library was constructed. After digestion and PCR identification, the positive clones were subjected to DNA sequencing analysis. The human amylin antigen was used to screen and enrich the antibody library. The positive clones obtained were identified by Phage-ELISA and the results were statistically analyzed. Results: The final antibody library construction capacity of about 0.8 × 108, restriction enzyme digestion showed inserts, antibody library recombination rate of 70%. Positive clones were subjected to DNA sequencing to confirm that the gene was a human immunoglobulin variable region gene. Three rounds of screening with amylin antigen resulted in specific enrichment of the antibody library. Phage-ELISA assays of positive clones confirmed the specificity of the anti-amylin antigen. Conclusion: A human phage antibody library was successfully constructed, which laid the foundation for obtaining human anti-amylin mAb.