人源全分子抗c-Met抗体的制备及对鼻咽癌细胞生物学特性的影响  

Preparation of full molecule human anti-c-Met antibody and its effects on nasopharyngeal carcinoma cells

作　　者：白璐月 刘金荣 唐奇[3] 熊四平[3] 陈雅[3] 杜银娟 周荧[1] 朱进[4] 陈仁杰[1] 

机构地区：[1]南京医科大学第二附属医院耳鼻咽喉科,江苏南京210011 [2]南京医科大学附属南京妇幼保健院妇产科,江苏南京210004 [3]南京医科大学卫生部抗体技术重点实验室,江苏南京211166 [4]南京军区军事医学研究所,江苏南京210002

出　　处：《南京医科大学学报（自然科学版）》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)

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.

关 键 词：鼻咽癌 C-MET 人源抗体 

分 类 号：R739.63[医药卫生—肿瘤]