难治性分化型甲状腺癌患者抗VEGF抗体Fab片段筛选鉴定的实验研究  

Experimental Study on the Screening and Identification of Anti-VEGF Fab Fragments in Patients with Refractory Differentiated Thyroid Cancer

作　　者：吴飞飞[1] 张承宇 卢朝秀 段涛 申虎威[1] WU Feifei;ZHANG Chengyu;LU Chaoxiu(Department of Endocrinology, Peace Hospital Affiliated to Changzhi Medical College)

机构地区：[1]长治医学院附属和平医院内分泌科,046000

出　　处：《长治医学院学报》2022年第1期7-11,共5页Journal of Changzhi Medical College

基　　金：长治医学院科研启动基金项目(QDZ201639)。

摘　　要：目的:制备难治性分化型甲状腺癌患者的抗血管内皮生长因子(VEGF)抗体的Fab片段,并验证其抗VEGF活性。方法:利用含高滴度VEGF抗体的难治性分化型甲状腺癌患者的外周血淋巴细胞构建一个噬菌体文库。采用核苷酸测序和免疫印迹法筛选特异性抗VEGF抗体的Fab片段,再使用细胞增殖法检测Fab片段的抗肿瘤活性。结果:从噬菌体库中筛选并分析6种Fab片段,均与VEGF具有结合活性,其中2种抗体Fab6(OD值为0.72±0.02)、Fab16(OD值为0.56±0.02)与VEGF有较高的结合活性,能够在体外阻断人类脐静脉内皮细胞(HUVEC)中VEGF的表达。对6个克隆的重链可变区和轻链可变区序列分析表明,它们的重链可变区(VH)序列与抗原表位直接相互作用的互补决定区(CDRS)有12个显著差异。Fab2、Fab6、Fab16三个克隆片段均表现出抑制活性,差异有统计学意义。结论:噬菌体展示技术可用于筛选人源性VEGF单克隆抗体,为研究人源性VEGF单克隆抗体对难治性分化型甲状腺癌的治疗作用奠定基础。Objective:The Fab fragments of the anti-vascular endothelial growth factor(Vascular growth factor,VEGF)in patients with refractory differentiated thyroid cancer antibodies were prepared and verified for their anti-VEGF activity.Methods:Peripheral blood lymphocytes from a refractory differentiated thyroid carcinoma patient who has high titer of VEGF antibody were utilized to construct a phage library.The Fab fragments with specific anti-VEGF antibodies were screened by nucleotide sequencing and immune blot analysis,and then the anti-tumor activity of the Fab fragments was detected using the cell proliferation assay.Results:Six Fab fragments were screened and analyzed from the phage library,all of which showed binding activity to VEGF,including two antibodies Fab6(OD value 0.72±0.02)and Fab16(OD value 0.56±0.02)showed high binding activity to VEGF,and could block the expression of VEGF in human umbilical vein endothelial cells(HUVEC)in vitro.The sequences of heavy chain variable region and light chain variable region of 6 clones showed 12 significant differences in complementary determination regions(CDRS)of direct interaction between heavy chain variable region(VH)sequences and antigen epitopes.Three cloned fragments,Fab2,Fab6 and Fab16,all showed inhibitory activity,and the difference was statistically significant.Conclusion:Phage display technology can be used to screen human VEGF monoclonal antibody,which lays a foundation for studying the therapeutic effect of human VEGF monoclonal antibody on refractory differentiated thyroid cancer.

关 键 词：难治性分化型甲状腺癌 人类基因工程抗体 噬菌体展示 

分 类 号：R736.1[医药卫生—肿瘤]