摘要
目的 用噬菌体表面展示技术 ,直接从抗血小板表面蛋白全套单链抗体噬菌体展示文库中筛选能与糖蛋白 (GP)Ⅱb/Ⅲa复合物高亲合力结合的单链抗体克隆 ,为开发有自主知识产权的抗栓药物奠定基础。方法 从活化血小板免疫的小鼠脾淋巴细胞中提取总RNA ,反转录成cDNA后 ,用抗体可变区混合引物扩增全套轻、重链可变区基因 ,经重叠延伸反应 ,装配成单链抗体 (ScFv)基因 ,将其克隆到噬菌体载体pHEN1中 ,构建单链抗体噬菌体抗体库。对抗体库进行亲合筛选后 ,ELISA法鉴定抗GPⅡb/Ⅲa复合物单链抗体。结果 经过 4轮“吸附———竞争性洗脱———扩增”的富集过程 ,phage -ELISA得到一个ELISA活性较高的能与GPⅡb/Ⅲa结合的克隆。序列测定符合抗体可变区结构特点。结论 成功构建了全套抗血小板表面蛋白单链抗体噬菌体展示文库 。
Objective The purpose of this study was to obtain the high-affinity single-chain Fv antibody against platelet membrane glycoprotein(GP) Ⅱb/Ⅲa from anti-activity platelet phage display library. Methods The heavy-chain and light-chain variable region gene(VH and VL)repertoire of immunoglobulin were amplified from the speen cells mRNA by RT-PCR and joined by a DNA linker encoding peptide as a single-chain Fv(ScFv) fragment with overlap extension. These fragments were cloned into the phagemid pHEN1 and the phage display library was constructed. The affinity selection and ELISA were adopted for identification of specific phage antibody to GPⅡb/Ⅲa. Results After through 4 rounds of panning, the high affinity ScFv gene was obtained, the sequence was comfined with mouse antibody. Conclusion Phage display library of repertoire single chain antibody of anti activity platelet was successfully constructed, and gene of single chain antibody which have binding ability to activity platelet was screened.
出处
《苏州大学学报（医学版）》
CAS
2003年第2期142-146,共5页
Suzhou University Journal of Medical Science
