摘要
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 produce a full molecule human anti-c-Met antibody with genetic engineering, and analyze the biological activity of the antibody and observe its influence on proliferation, migration and invasion of nasopharyngeal carcinoma cells. Methods：We designed a primer for amplifying the variable region of anti-c-Met Fab antibody. VH and VL genes were cloned into pFUSE-CHIg-hG1 and pFUSE-CLIg-hκ expression vectors, respectively, and were both transfected into eukaryotic cells. The expression product was purified using protein A column. The identification and immunological characteristics of antibody were analyzed by enzyme-linked immune sorbent assay （ELISA）, Western blotting assay and immunofluorescence assay. The inhibition function of the antibody in nasopharyngeal carcinoma cells was analyzed by CCK-8. The effects of the antibody on the invasion and migration of nasopharyngeal carcinoma cells was determined by wound-healing assay and Transwell invasion assay. Results：The results demonstrated that the full molecule human anti-c-Met antibody was successfully produced, and the antibody effectively recognized c-Met protein. In nasopharyngeal carcinoma cell experiment, CCK8 assay demonstrated that the antibody played a significant role in proliferation of CNE2 and HONE1. Wound-healing assay and Transwell invasion assay demonstrated that the antibody could suppressed the migration and invasion of CNE2 and HONE1. Conclusion：In this study, full molecule human anti-c-Met antibody was established successfully and has obvious neutralizing effect. The antibody is expected to become a candidate molecule for molecular targeted therapy of nasopharyngeal carcinoma.
出处
《南京医科大学学报（自然科学版）》
CAS
CSCD
北大核心
2016年第6期687-692,共6页
Journal of Nanjing Medical University(Natural Sciences)
基金
江苏省科技厅临床医学科技专项计划(BL2013038)
国家自然科学基金(81502673)
关键词
鼻咽癌
C-MET
人源抗体
nasopharyngeal carcinoma
c-Met
full molecule human antibody
