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目的 :利用基因工程技术制备全分子人源抗c-Met抗体,分析该抗体免疫学特性,并观察该全分子抗体对鼻咽癌细胞增殖、迁移和侵袭的影响。方法:设计引物扩增抗c-Met Fab抗体的重轻链可变区序列,将其分别克隆到真核表达载体p FUSECHIg-h G1和p FUSE-CLIg-hκ中。转染真核细胞,表达产物用Protein A柱纯化。通过ELISA、Western blot和免疫荧光等方法鉴定该抗体的免疫学特性。CCK-8检测抗体对人鼻咽癌细胞增殖的抑制作用,划痕实验、Transwell侵袭实验分析抗体对人鼻咽癌细胞迁移和侵袭的影响。结果:成功重组全分子人源抗c-Met抗体的表达载体,获得的全分子抗体保留了与抗原的特异性结合活性。CCK-8实验中结果显示全分子抗体能抑制c-Met阳性鼻咽癌细胞CNE2和HONE1的增殖。细胞划痕和Transwell侵袭实验结果显示全分子抗体能抑制c-Met阳性细胞CNE2和HONE1的迁移和侵袭能力。结论:本研究成功制备人源全分子抗c-Met抗体,该抗体有明显的中和活性,为探索鼻咽癌分子靶向治疗奠定了基础。
OBJECTIVE: To prepare a full-scale human anti-c-Met antibody using genetic engineering technique and analyze the immunological characteristics of this antibody. The effect of this all-molecule antibody on the proliferation, migration and invasion of nasopharyngeal carcinoma cells was also observed. Methods: The heavy and light chain variable regions of anti-c-Met Fab antibody were designed and cloned into eukaryotic expression vector p FUSECHIg-h G1 and p FUSE-CLIg-hκ respectively. The eukaryotic cells were transfected and the expression product was purified on a Protein A column. The immunological characteristics of the antibody were identified by ELISA, Western blot and immunofluorescence. The inhibitory effect of CCK-8 on the proliferation of human nasopharyngeal carcinoma cells, scratch test and Transwell invasion assay were used to analyze the effect of antibody on the migration and invasion of human nasopharyngeal carcinoma cells. RESULTS: The expression vector of recombinant human full-fledged human anti-c-Met antibody was successfully obtained. The obtained whole molecule antibody retained the specific binding activity with the antigen. CCK-8 results showed that all-molecule antibodies can inhibit the proliferation of CNE2 and HONE1 in c-Met positive nasopharyngeal carcinoma cells. Cell scratch and Transwell invasion assay results showed that all-antibody can inhibit the migration and invasion of c-Met positive cells CNE2 and HONE1. Conclusion: The human full-fledged anti-c-Met antibody was successfully prepared in this study. The antibody has obvious neutralization activity, which lays the foundation for the exploration of molecular targeted therapy for nasopharyngeal carcinoma.