全人源抗c-Met Fab抗体的筛选及鉴定
发布时间：2018-12-12 22:28
【摘要】： c-Met是由原癌基因Met编码的酪氨酸激酶受体,位于细胞膜上,包含1个50kDa的α亚基和1个150kDa的β亚基,两者经二硫键连接形成异二聚体。c-Met是肝细胞生长因子(hepatocyte growth factor/scatterfactor,HGF/SF)在体内的唯一受体,两者结合并随之激活后可促进肿瘤细胞的增殖、迁徙和新血管生成,最终导致恶性肿瘤的侵袭和转移。目前HGF/SF和c-Met多种拮抗剂的研究已在体外和动物实验中证明可有效地抑制肿瘤的发生、侵袭和转移,HGF/SF-c-Met已经成为肿瘤诊断和生物治疗的新靶点。鼠源抗HGF/SF和c-Met单克隆抗体和/或多克隆抗体已在体外和动物实验中显示出高亲和力和中和活性,但是鼠源性单克隆抗体引起的人抗鼠抗体反应(human antimouse antibody,HAMA)限制了鼠源抗体在临床中的应用。这就推动了人源化单克隆抗体的研究。与鼠源性单克隆抗体相比,人源化单克隆抗体免疫源性明显减弱,并且血清半衰期显著延长,促进了单克隆抗体在临床中的应用。本文利用固相筛选方法从大容量人源天然Fab抗体库中筛选抗c-Met Fab抗体,并对其与人肝癌细胞的结合活性进行鉴定,为研制用于肝癌生物治疗的靶向药物,提供候选分子。 材料与方法 1.以Met-Fc融合蛋白包板,扩增本实验室制备的大容量人源天然Fab抗体库(库容为1.2×10~9)。将噬菌体抗体加入Met-Fc包被的ELISA板,进行6轮洗脱、富集,从第6轮筛选后得到的细菌集落中随机挑取60个克隆,分别用Met-Fc、IgG包板,用Phage ELISA方法鉴定它们的免疫学特性。 2.可溶性表达:用符合条件(Met-Fc(+)、IgG(-))的细菌克隆质粒转染Top 10F',经IPTG诱导表达抗c-Met Fab抗体。诱导表达上清通过Protein L亲和层析法纯化。 3.用Western blotting、IP、FACS、免疫荧光等方法鉴定抗c-Met Fab抗体与c-Met表达阳性细胞表面c-Met分子的结合活性。 结果 1.用Met(c-28)兔抗Met多克隆抗体鉴定SMMC7721、BEL7402两株肝癌细胞,结果显示c-Met均表达于肝癌细胞表面。 2.经过6轮Met-Fc固相筛选的噬菌体感染XL I-Blue大肠杆菌,随机挑取60个克隆表达,进行Phage ELISA鉴定。其中54个克隆与Met-Fc呈阳性反应,阳性率为90%。54个克隆中与Fc呈阴性反应者有4个克隆(AM1-14、AM2-9、AM2-18、AM2-26),BstOI酶切鉴定显示为同一克隆,测序结果显示为人Fab片段。 3.阳性克隆经IPTG诱导表达,诱导表达上清经Protein L亲和层析纯化得到抗c-Met Fab抗体,经SDS-PAGE电泳,在相对分子量26、34kDa处各出现一条条带,经Western blotting检测为人Fab片段。 4.IP、FACS、免疫荧光结果显示AM2-26可与SMMC7721、BEL7402细胞表面c-Met分子结合。 结论 从大容量天然人源Fab抗体库中筛选的AM2-26能够与肝癌细胞表面c-Met分子特异性结合,为研制用于肝癌生物治疗的靶向药物,提供了候选分子。
[Abstract]:C-Met, a tyrosine kinase receptor encoded by proto-oncogene Met, is located on the cell membrane and contains a 伪 subunit of 50kDa and a 尾 subunit of 150kDa. C-Met is the only receptor of hepatocyte growth factor (hepatocyte growth factor/scatterfactor,HGF/SF) in vivo, which can promote the proliferation, migration and angiogenesis of tumor cells. Finally, it leads to the invasion and metastasis of malignant tumor. At present, many antagonists of HGF/SF and c-Met have been proved to be effective in inhibiting tumor occurrence, invasion and metastasis in vitro and animal experiments. HGF/SF-c-Met has become a new target for tumor diagnosis and biotherapy. Mouse monoclonal antibodies against HGF/SF and c-Met and / or polyclonal antibodies have shown high affinity and neutralization activity in vitro and in animal experiments, but mouse monoclonal antibodies induce human anti-mouse antibody response to (human antimouse antibody,. HAMA limits the clinical application of murine antibodies. This promotes the study of humanized monoclonal antibodies. Compared with murine monoclonal antibody, the immunogenicity of humanized monoclonal antibody was obviously weakened, and the half-life of serum was significantly prolonged, which promoted the application of monoclonal antibody in clinic. In this paper, a solid phase screening method was used to screen the anti c-Met Fab antibody from a large human natural Fab antibody library, and its binding activity with human hepatoma cells was identified, which provides candidate molecules for the preparation of targeted drugs for the biotherapy of liver cancer. Materials and methods 1. A large human natural Fab antibody library (1.2 脳 10 ~ (9) was amplified by using Met-Fc fusion protein envelope. The phage antibody was added to the ELISA plate coated with Met-Fc for 6 rounds of elution and enrichment. 60 clones were randomly selected from the bacterial colonies obtained after the sixth round of screening, and the Met-Fc,IgG plates were used respectively. Their immunological properties were identified by Phage ELISA method. 2. Soluble expression: Top 10 FN was transfected with bacterial clone plasmid (Met-Fc (), IgG (-), and the anti c-Met Fab antibody was induced by IPTG. The supernatant was purified by Protein L affinity chromatography. 3. Western blotting,IP,FACS, immunofluorescence assay was used to identify the binding activity of anti c-Met Fab antibody to c-Met molecules on the surface of c-Met positive cells. Result 1. Met (c-28) rabbit anti Met polyclonal antibody was used to identify two SMMC7721,BEL7402 hepatoma cells. The results showed that c-Met was expressed on the surface of HCC cells. 2. Six rounds of Met-Fc solid phase screening phage infected XL I-Blue Escherichia coli, 60 clones were randomly selected and identified by Phage ELISA. Among them, 54 clones were positive for Met-Fc, and the positive rate was 90.54 clones. 4 clones were negative for Fc. The results of sequencing showed a human Fab fragment. 3. The positive clones were induced to express by IPTG and the supernatants were purified by Protein L affinity chromatography. After SDS-PAGE electrophoresis, one band appeared at the relative molecular weight of 264kDa, and the human Fab fragment was detected by Western blotting. 4. The immunofluorescence results showed that AM2-26 could bind to c-Met molecules on the surface of SMMC7721,BEL7402 cells. Conclusion the AM2-26 screened from large capacity natural human Fab antibody library can specifically bind to c-Met molecules on the surface of hepatoma cells, which provides candidate molecules for the development of targeted drugs for liver cancer biotherapy.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392
本文编号：2375341
[Abstract]:C-Met, a tyrosine kinase receptor encoded by proto-oncogene Met, is located on the cell membrane and contains a 伪 subunit of 50kDa and a 尾 subunit of 150kDa. C-Met is the only receptor of hepatocyte growth factor (hepatocyte growth factor/scatterfactor,HGF/SF) in vivo, which can promote the proliferation, migration and angiogenesis of tumor cells. Finally, it leads to the invasion and metastasis of malignant tumor. At present, many antagonists of HGF/SF and c-Met have been proved to be effective in inhibiting tumor occurrence, invasion and metastasis in vitro and animal experiments. HGF/SF-c-Met has become a new target for tumor diagnosis and biotherapy. Mouse monoclonal antibodies against HGF/SF and c-Met and / or polyclonal antibodies have shown high affinity and neutralization activity in vitro and in animal experiments, but mouse monoclonal antibodies induce human anti-mouse antibody response to (human antimouse antibody,. HAMA limits the clinical application of murine antibodies. This promotes the study of humanized monoclonal antibodies. Compared with murine monoclonal antibody, the immunogenicity of humanized monoclonal antibody was obviously weakened, and the half-life of serum was significantly prolonged, which promoted the application of monoclonal antibody in clinic. In this paper, a solid phase screening method was used to screen the anti c-Met Fab antibody from a large human natural Fab antibody library, and its binding activity with human hepatoma cells was identified, which provides candidate molecules for the preparation of targeted drugs for the biotherapy of liver cancer. Materials and methods 1. A large human natural Fab antibody library (1.2 脳 10 ~ (9) was amplified by using Met-Fc fusion protein envelope. The phage antibody was added to the ELISA plate coated with Met-Fc for 6 rounds of elution and enrichment. 60 clones were randomly selected from the bacterial colonies obtained after the sixth round of screening, and the Met-Fc,IgG plates were used respectively. Their immunological properties were identified by Phage ELISA method. 2. Soluble expression: Top 10 FN was transfected with bacterial clone plasmid (Met-Fc (), IgG (-), and the anti c-Met Fab antibody was induced by IPTG. The supernatant was purified by Protein L affinity chromatography. 3. Western blotting,IP,FACS, immunofluorescence assay was used to identify the binding activity of anti c-Met Fab antibody to c-Met molecules on the surface of c-Met positive cells. Result 1. Met (c-28) rabbit anti Met polyclonal antibody was used to identify two SMMC7721,BEL7402 hepatoma cells. The results showed that c-Met was expressed on the surface of HCC cells. 2. Six rounds of Met-Fc solid phase screening phage infected XL I-Blue Escherichia coli, 60 clones were randomly selected and identified by Phage ELISA. Among them, 54 clones were positive for Met-Fc, and the positive rate was 90.54 clones. 4 clones were negative for Fc. The results of sequencing showed a human Fab fragment. 3. The positive clones were induced to express by IPTG and the supernatants were purified by Protein L affinity chromatography. After SDS-PAGE electrophoresis, one band appeared at the relative molecular weight of 264kDa, and the human Fab fragment was detected by Western blotting. 4. The immunofluorescence results showed that AM2-26 could bind to c-Met molecules on the surface of SMMC7721,BEL7402 cells. Conclusion the AM2-26 screened from large capacity natural human Fab antibody library can specifically bind to c-Met molecules on the surface of hepatoma cells, which provides candidate molecules for the development of targeted drugs for liver cancer biotherapy.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392
【参考文献】
相关期刊论文 前7条
1 曹卡加;范乔阳;刘奕龙;黄荣;尹传忠;马国胜;刘子群;万德森;曾益新;;广州市2000～2002年恶性肿瘤的发病率与死亡率分析[J];癌症;2008年03期
2 汤琪云;王学浩;;肝癌患者肝移植术前、术后化疗16例分析[J];南京医科大学学报(自然科学版);2006年04期
3 万佳艺;孙慧;焦永军;朱晓娟;朱进;刘政;冯振卿;;人源天然Fab噬菌体抗体库的构建及抗c-Met抗体的筛选、鉴定[J];南京医科大学学报(自然科学版);2008年06期
4 张兰;戴龙;谭东;;厦门市2004-2005年度恶性肿瘤监测结果分析[J];实用预防医学;2007年05期
5 George F. VANDE WOUDE;HGF/SF-Met signaling in tumor progression[J];Cell Research;2005年01期
6 张思维;陈万青;孔灵芝;李连弟;鲁凤珠;李光琳;孟佳;赵平;;中国部分市县1998～2002年恶性肿瘤的发病与死亡[J];中国肿瘤;2006年07期
7 罗荣城,尤长宣;肿瘤生物治疗新进展[J];中国新药杂志;2005年02期
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