抗肝癌噬菌体单链抗体二聚体的表达、纯化及初步鉴定
发布时间：2019-02-16 05:01
【摘要】： 目的:将我室构建的抗肝癌噬菌体单链抗体二聚体(scFv-BDM2)在大肠杆菌可溶性表达、纯化,并对其亲和力与特异性行初步鉴定,为下一步构建~(99m)Tc标记的scFv-BDM2及scFv-BDM2-发热磁性纳米颗粒的临床应用奠定基础。 方法:1.将抗肝癌scFv-BDM2由E.coli TGl转入E.coli HB2151中,IPTG诱导可溶性表达,制备大量scFv-BDM2;利用HiTrap Anti-E Tag亲和层析柱对表达的二聚体进行纯化。 2.将纯化后的scFv-BDM2进行聚丙烯酰胺凝胶电泳,以Bradford法测定其浓度,酶联免疫吸附实验法(ELISA)检测其生物活性和特异性;采用非竞争酶免疫技术测定上述纯化的scFv-BDM2亲和力常数,并比较其大小变化。 结果:1.将抗肝癌scFv DM二聚体在E.coli HB2151大量表达并纯化后经聚丙烯酰胺凝胶电泳分析,可见一清晰蛋白条带;经分子相对迁移率初步计算估计,目的条带的分子量约为60KD,纯化后的二聚体浓度约为16μg/ml。 2.采用非竞争酶免疫法测定上述纯化的单链抗体二聚体的活性效价和亲和力常数分别为1:64、3.735×10~7mol/L~(-1),而scFv DM的亲和力常数为2.44×10~7mol/L(-1),二聚体的亲和力为scFv DM的1.53倍。以ELISA法对所获阳性克隆的特异性分析发现,抗肝癌scFv-BDM2均与三种肝癌细胞结合,而与正常肝细胞、胃癌细胞和膀胱癌细胞不结合。 结论:经大肠杆菌表达及亲和层析纯化后获得一定浓度的scFv DM二聚体,其亲和力较前提高,和抗肝癌噬菌体单链抗体同样具有良好的抗原结合特异性。
[Abstract]:Objective: to express and purify anti-hepatoma phage single-chain antibody dimer (scFv-BDM2) in Escherichia coli, and to identify its affinity and specificity. It lays a foundation for the clinical application of ~ (99m) Tc labeled scFv-BDM2 and scFv-BDM2- febrile magnetic nanoparticles. Methods: 1. The anti-hepatoma scFv-BDM2 was transferred from E.coli TGl to E.coli HB2151, and the soluble expression was induced by IPTG. A large number of scFv-BDM2; was purified by HiTrap Anti-E Tag affinity chromatography. 2. The purified scFv-BDM2 was detected by polyacrylamide gel electrophoresis and its concentration was determined by Bradford method. The bioactivity and specificity of the purified scFv-BDM2 were detected by Elisa (ELISA). The affinity constants of the purified scFv-BDM2 were determined by non-competitive enzyme immunoassay and the changes of its size were compared. Results: 1. The anti-hepatoma scFv DM dimer was expressed in E.coli HB2151 and purified by polyacrylamide gel electrophoresis, and a clear protein band was found. The molecular weight of the target band is about 60 KD and the concentration of the purified dimer is about 16 渭 g / ml. 2. The activity titers and affinity constants of the purified scFv dimer were 1: 64-3.735 脳 10 ~ (7) mol / L ~ (-1) and 2.44 脳 10~7mol/L (-1) by non-competitive enzyme immunoassay, respectively. The affinity of dimer was 1.53 times of that of scFv DM. The specific analysis of the positive clones by ELISA method showed that anti-HCC scFv-BDM2 was bound to three kinds of hepatoma cells, but not to normal hepatocytes, gastric cancer cells and bladder cancer cells. Conclusion: a certain concentration of scFv DM dimer was obtained after expression of Escherichia coli and purification by affinity chromatography. The affinity of scFv DM dimer was improved compared with that before, and it had good antigen-binding specificity as well as anti-hepatoma phage scFv.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392;R735.7
[Abstract]:Objective: to express and purify anti-hepatoma phage single-chain antibody dimer (scFv-BDM2) in Escherichia coli, and to identify its affinity and specificity. It lays a foundation for the clinical application of ~ (99m) Tc labeled scFv-BDM2 and scFv-BDM2- febrile magnetic nanoparticles. Methods: 1. The anti-hepatoma scFv-BDM2 was transferred from E.coli TGl to E.coli HB2151, and the soluble expression was induced by IPTG. A large number of scFv-BDM2; was purified by HiTrap Anti-E Tag affinity chromatography. 2. The purified scFv-BDM2 was detected by polyacrylamide gel electrophoresis and its concentration was determined by Bradford method. The bioactivity and specificity of the purified scFv-BDM2 were detected by Elisa (ELISA). The affinity constants of the purified scFv-BDM2 were determined by non-competitive enzyme immunoassay and the changes of its size were compared. Results: 1. The anti-hepatoma scFv DM dimer was expressed in E.coli HB2151 and purified by polyacrylamide gel electrophoresis, and a clear protein band was found. The molecular weight of the target band is about 60 KD and the concentration of the purified dimer is about 16 渭 g / ml. 2. The activity titers and affinity constants of the purified scFv dimer were 1: 64-3.735 脳 10 ~ (7) mol / L ~ (-1) and 2.44 脳 10~7mol/L (-1) by non-competitive enzyme immunoassay, respectively. The affinity of dimer was 1.53 times of that of scFv DM. The specific analysis of the positive clones by ELISA method showed that anti-HCC scFv-BDM2 was bound to three kinds of hepatoma cells, but not to normal hepatocytes, gastric cancer cells and bladder cancer cells. Conclusion: a certain concentration of scFv DM dimer was obtained after expression of Escherichia coli and purification by affinity chromatography. The affinity of scFv DM dimer was improved compared with that before, and it had good antigen-binding specificity as well as anti-hepatoma phage scFv.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392;R735.7
【参考文献】
相关期刊论文 前4条
1 孙志伟,赵君,王双,俞炜源,刘彦仿;抗肝癌人源化Diabody在大肠杆菌中的可溶性表达及活性鉴定[J];世界华人消化杂志;2004年11期
2 叶刚;杨冬华;汤绍辉;周e，
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