论文部分内容阅读
为制备抗丙型肝炎病毒核心蛋白的抗独特型单链可变区抗体 (抗 IdscFv) ,采用噬菌体表面展示技术 ,将抗 HCV核心蛋白的单克隆抗体固相包被于Nunc板 ,从噬菌体单链可变区抗体库中经过 5轮“吸附 洗脱 扩增”筛选过程 ,获得可与HCV核心蛋白单克隆抗体结合的抗独特型人源单链可变区抗体的噬菌体集落。随机挑选 6 0个克隆 ,利用酶联免疫吸附法 (ELISA)、交叉反应和竞争抑制实验 ,对其进行免疫学检测和鉴定。获得与HCV核心蛋白单克隆抗体结合活性较强的抗 IdscFv阳性克隆 ,并对HCV核心蛋白特异性抗 IdscFv的编码序列进行测定分析。结果经过 5轮“吸附 洗脱 扩增”筛选 ,在随机挑选的 6 0个克隆中 ,有 2 0株克隆ELISA的吸光度(A4 5 0nm)值较高 ,这些噬菌体上清与牛血清白蛋白 (BSA)进行交叉反应后 ,确定了其中有 6株交叉反应较弱 ,结合 2次ELISA重复实验的A值及竞争抑制实验结果 ,最后确定 1株阳性克隆 ,提取质粒 ,进行DNA序列测定 ,DNA大小为 76 8bp。本实验结果提示用噬菌体抗体库技术能够成功地获得抗 HCV核心蛋白的抗 IdscFv ,为开展用抗 IdscFv防治慢性丙型肝炎的研究创造了条件
To prepare an anti-idiotypic single chain variable region antibody against Hepatitis C virus core protein (anti-IdscFv), the anti-HCV core protein monoclonal antibody was coated on the Nunc plate by phage surface display technique, After five rounds of “adsorption elution amplification” screening in the variable region antibody library, phage colonies of anti-idiotypic human single-chain variable region antibodies that bind to the HCV core protein monoclonal antibody were obtained. Sixty clones were selected at random, and their immunological detection and identification were carried out by enzyme-linked immunosorbent assay (ELISA), cross-reaction and competition inhibition experiments. The anti-IdscFv positive clone with strong binding activity to HCV core protein monoclonal antibody was obtained, and the coding sequence of HCV core protein specific anti-IdscFv was assayed. Results After 5 rounds of “adsorption and elution amplification”, 20 out of 60 random clones were selected and the absorbance (A450 nm) of 20 clones was higher. These phage supernatants were incubated with bovine serum albumin BSA) were cross-reacted to determine the cross-reaction was weak among them, combined with two ELISA repeat A values and competitive inhibition test results, and finally identified a positive clone, plasmid extraction, DNA sequencing, DNA size For 76 8bp. The results of this experiment suggest that anti-IdscFv anti-HCV core protein can be successfully obtained by the phage antibody library technology, which provides the conditions for the research on anti-IdscFv prevention and treatment of chronic hepatitis C