抗前列腺特异抗原重链可变区单域抗体基因的构建及表达_医学论文.docVIP

抗前列腺特异抗原重链可变区单域抗体基因的构建及表达_医学论文 抗前列腺特异抗原重链可变区单域抗体基因的构建及表达_医学论文 作者：袁建林　王智　武国军　郝晓柯 【关键词】 前列腺特异抗原 　　关键词: 前列腺特异抗原单域抗体克隆表达 　　摘 要:目的 扩增出抗前列腺特异抗原（PSA）单克隆抗体（MAb）的重链可变区（VH）单域抗体基因，并在大肠杆菌中表达. 方法 从分泌抗PSA mAb的杂交瘤细胞系E4 B7 中提取总RNA，经RT-PCR扩增VH 单域抗体基因，将其克隆到融合蛋白表达载体pGEX-4T-1中进行表达. 结果 VH 单域抗体基因全长363bp，含起始码和终止码.将其克隆到pGEX-4T-1内，转化大肠杆菌DH5α，获得高效表达，表达量占全菌总蛋白质的38%，以包涵体形式存在.经初步纯化和复性后，用谷胱甘肽（GST）亲和色谱纯化，再经凝血酶水解获得抗PSA VH 单域抗体.竞争结合抑制实验证明，该表达产物具有前列腺癌细胞亲和活性. 结论 构建成功抗PSA的VH 单域抗体，为进一步临床应用奠定基础. 　　Keywords:prostate-specific antigensingle domain antibodycloningexpression 　　Abstract:AIM To amplify the VH single domain gene of an-ti-prostate specific antigen（PSA）monoclonal antibody（mAb），and express it in E.coli.METHODS Total RNA was extracted from the hybridoma cell line E4 B7 ，which se-creted McAb against PSA，and subjected to reverse tran-scription.The VH single domain antibody gene was amplified by PCR，cloned into the prokaryotic fusion protein expres-sion vector pGEX-4T-1and expressed in E.coli.RESULTS The VH single domain antibody gene consisted of363bp，which was cloned into an expression vector pGEX-4T-1and transformed into E.coli DH5α.Clones harboring plasmid pGEX-VH produced a40kDa of fusion protein in E.coli at a level of38%of the total cellular protein.After primary pu-rification and renaturation，the fusion protein was further pu-rified by GST-affinity chromatography.The fusion protein was digested with thrombin and the VH single domain anti-body was separated，the binding activity of anti-PSA VH sin-gle domain antibody was confirmed by competitive binding in-hibition assay in vitro.CONCLUSION Anti-PSA single do-main antibody has been successfully constructed for the use in clinical studies. 　　0 引言 　　前列腺特异抗原（Prostate specific antigen）是一种灵敏度较高的前列腺癌肿瘤标志物，131 I标记抗PSA单克隆抗体用于前列腺癌放射免疫显像，有较高的灵敏度和特异性［1］ .针对鼠源性单克隆抗体对人体存在着免疫原性的问题，自20世纪80年代以来基因工程抗体的研究愈来愈受到重视，其中重链可变区（VH ）单域抗体由于只有一个功能区，保留了较好的抗原结合能力，在制备和应用上有其优点.我们应用RT-PCR方法从抗PSA单克隆抗体的杂交瘤细胞系E4 B7 中扩增出VH 单域抗体基因，在大肠杆菌中获得了高效表达，并进行亲和活性测定. 　　1 材料和方法