摘要
目的 制备胃癌单抗MGb1的噬菌体呈现型单链可变区片段 (ScFv) ,为获得用于胃癌体内诊疗研究的靶向载体分子奠定基础。方法 从杂交瘤细胞分离mRNA ,扩增抗体重、轻链可变区(VH和VL)cDNA ,经linkerDNA连接形成ScFvDNA。将ScFvDNA与pCANTAB5E的连接产物转化于大肠杆菌TG1,经M13KO7感染后 ,获得重组噬菌体抗体ScFv。经亲和筛选和ELISA检测获得呈现MGb1ScFv的噬菌体单克隆。结果 VH、VL和ScFvDNA分别约为 340bp、32 0bp和 75 0bp。经筛选得到 17个呈现MGb1ScFv的噬菌体单克隆 ,其中结合抗原能力强的克隆有 4个。结论 获得了单抗MGb1的噬菌体呈现ScFv ,为拓展抗体的应用范围创造了条件。
Objective To lay a foundation for obtaining a tumor targeting vehicle for in vivo study on diagnosis and treatment of gastric carcinoma by generating single chain variable fragments(ScFv) of monoclonal antibody MGb1 directed against the cancer. Methods mRNA was isolated from MGb1 producing mouse hybridoma cell line, and the variable regions of heavy and light chain cDNAs were amplified separately and assembled into ScFv DNAs with a specially constructed linker DNA by PCR. The ScFv DNAs were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into competent E.coli TG1. The transformed cells were infected with M13KO7 helper phage to yield recombinant phage,which disply ScFv fragments as a fusion with gene 3 protein on the tips of the phage M13. After two rounds of panning with gastric carcinoma cell line KATOⅢ highly expressing MGb1 binding antigen, the phage clones displayed ScFv fragments of the antibody were selected by enzyme linked immunosorbent assay(ELISA) from the enriched phages. The affinity of the positive phage clones was detected by competition ELISA. Results The VH,VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. 17 phage clones displayed ScFv of MGb1 were selected from 40 enriched phage clones. 4 out of the 17 phage clones could strongly compete with the original hybridoma antibody MGb1 for binding to the antigen expressed on KATOⅢ cells. Conclusion The phage displayed ScFv fragments of monoclonal antibody MGb1 are successfully produced by phage antibody technology, which may be useful to widen the range of application of the antibody.
出处
《中华内科杂志》
CAS
CSCD
北大核心
2000年第9期585-587,共3页
Chinese Journal of Internal Medicine
基金
国家杰出青年基金! (395 2 5 0 2 0 )
国家"86 3"课题!资助项目 (10 2 10 0 1 0 6 )
