全人源抗c-Met Fab抗體的篩選及鑒定
發(fā)布時(shí)間：2018-12-12 22:28
【摘要】： c-Met是由原癌基因Met編碼的酪氨酸激酶受體,位于細(xì)胞膜上,包含1個(gè)50kDa的α亞基和1個(gè)150kDa的β亞基,兩者經(jīng)二硫鍵連接形成異二聚體。c-Met是肝細(xì)胞生長(zhǎng)因子(hepatocyte growth factor/scatterfactor,HGF/SF)在體內(nèi)的唯一受體,兩者結(jié)合并隨之激活后可促進(jìn)腫瘤細(xì)胞的增殖、遷徙和新血管生成,最終導(dǎo)致惡性腫瘤的侵襲和轉(zhuǎn)移。目前HGF/SF和c-Met多種拮抗劑的研究已在體外和動(dòng)物實(shí)驗(yàn)中證明可有效地抑制腫瘤的發(fā)生、侵襲和轉(zhuǎn)移,HGF/SF-c-Met已經(jīng)成為腫瘤診斷和生物治療的新靶點(diǎn)。鼠源抗HGF/SF和c-Met單克隆抗體和/或多克隆抗體已在體外和動(dòng)物實(shí)驗(yàn)中顯示出高親和力和中和活性,但是鼠源性單克隆抗體引起的人抗鼠抗體反應(yīng)(human antimouse antibody,HAMA)限制了鼠源抗體在臨床中的應(yīng)用。這就推動(dòng)了人源化單克隆抗體的研究。與鼠源性單克隆抗體相比,人源化單克隆抗體免疫源性明顯減弱,并且血清半衰期顯著延長(zhǎng),促進(jìn)了單克隆抗體在臨床中的應(yīng)用。本文利用固相篩選方法從大容量人源天然Fab抗體庫中篩選抗c-Met Fab抗體,并對(duì)其與人肝癌細(xì)胞的結(jié)合活性進(jìn)行鑒定,為研制用于肝癌生物治療的靶向藥物,提供候選分子。 材料與方法 1.以Met-Fc融合蛋白包板,擴(kuò)增本實(shí)驗(yàn)室制備的大容量人源天然Fab抗體庫(庫容為1.2×10~9)。將噬菌體抗體加入Met-Fc包被的ELISA板,進(jìn)行6輪洗脫、富集,從第6輪篩選后得到的細(xì)菌集落中隨機(jī)挑取60個(gè)克隆,分別用Met-Fc、IgG包板,用Phage ELISA方法鑒定它們的免疫學(xué)特性。 2.可溶性表達(dá):用符合條件(Met-Fc(+)、IgG(-))的細(xì)菌克隆質(zhì)粒轉(zhuǎn)染Top 10F',經(jīng)IPTG誘導(dǎo)表達(dá)抗c-Met Fab抗體。誘導(dǎo)表達(dá)上清通過Protein L親和層析法純化。 3.用Western blotting、IP、FACS、免疫熒光等方法鑒定抗c-Met Fab抗體與c-Met表達(dá)陽性細(xì)胞表面c-Met分子的結(jié)合活性。 結(jié)果 1.用Met(c-28)兔抗Met多克隆抗體鑒定SMMC7721、BEL7402兩株肝癌細(xì)胞,結(jié)果顯示c-Met均表達(dá)于肝癌細(xì)胞表面。 2.經(jīng)過6輪Met-Fc固相篩選的噬菌體感染XL I-Blue大腸桿菌,隨機(jī)挑取60個(gè)克隆表達(dá),進(jìn)行Phage ELISA鑒定。其中54個(gè)克隆與Met-Fc呈陽性反應(yīng),陽性率為90%。54個(gè)克隆中與Fc呈陰性反應(yīng)者有4個(gè)克隆(AM1-14、AM2-9、AM2-18、AM2-26),BstOI酶切鑒定顯示為同一克隆,測(cè)序結(jié)果顯示為人Fab片段。 3.陽性克隆經(jīng)IPTG誘導(dǎo)表達(dá),誘導(dǎo)表達(dá)上清經(jīng)Protein L親和層析純化得到抗c-Met Fab抗體,經(jīng)SDS-PAGE電泳,在相對(duì)分子量26、34kDa處各出現(xiàn)一條條帶,經(jīng)Western blotting檢測(cè)為人Fab片段。 4.IP、FACS、免疫熒光結(jié)果顯示AM2-26可與SMMC7721、BEL7402細(xì)胞表面c-Met分子結(jié)合。 結(jié)論 從大容量天然人源Fab抗體庫中篩選的AM2-26能夠與肝癌細(xì)胞表面c-Met分子特異性結(jié)合,為研制用于肝癌生物治療的靶向藥物,提供了候選分子。
[Abstract]:C-Met, a tyrosine kinase receptor encoded by proto-oncogene Met, is located on the cell membrane and contains a 偽 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.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392
本文編號(hào)：2375341
[Abstract]:C-Met, a tyrosine kinase receptor encoded by proto-oncogene Met, is located on the cell membrane and contains a 偽 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.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392
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