摘要
目的制备胃癌单抗 MGd1的单链可变区片段 (single chain variable fragm ent,Sc Fv) ,为胃癌体内诊疗研究提供候选靶向载体分子。方法从 MGd1杂交瘤分离 m RNA,RT- PCR分别扩增抗体重、轻链可变区基因 (VH和 VL DNA) ,二者经 linker DNA连接形成 Sc Fv DNA。将 Sc Fv DNA与载体 p CANTAB5 E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 Sc Fv。以高表达 MGd1结合抗原的细胞株 KATO 对重组噬菌体抗体 Sc Fv进行两轮筛选后 ,随机挑取克隆经 EL ISA筛选 MGd1Sc Fv单克隆 ,并对其结合抗原的能力进行鉴定。结果 VH、VL 和 Sc Fv DNA分别约为 340、32 0和 75 0 bp。经两轮亲和筛选后 ,在随机筛检的 30个克隆中得到 12个噬菌体呈现型 MGd1Sc Fv单克隆 ,其中结合抗原能力强的克隆有 5个。结论用噬菌体呈现技术成功地获得了单抗 MGd1的 Sc Fv,为拓展该抗体的应用范围奠定了基础。
ObjectiveTo provide possible tumor targeting vehicle for in vivo study on diagnosis and treatment of gastric carcinoma by generating single chain variable fragments (ScFv) of monoclonal antibody MGd1 recognizing the carcinoma. Methods mRNA was isolated from the hybridoma cell line producing monoclonal antibody MGd1. Heavy and light chain genes were amplified separately and assembled into ScFv DNAs with a specially constructed linker DNA by polymerase chain reaction. 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 displays ScFv fragments on the surface of the filamentous phage M13. After two rounds of panning with gastric carcinoma cell line KATOⅢ highly expressing MGd1 binding antigen, the phage clones displaying ScFv of the antibody were selected by ELISA from the enriched phages. The affinity of the positive phage clones was assayed by competition ELISA. Results The VH,VL and ScFv DNAs were about 340, 320 and 750 bp respectively. 12 phage clones displaying ScFv of MGd1 were selec ted from 30 enriched phage clones. 5 of the 12 phage clones could strongly compete with the original antibody MGd1 for binding to the antigen highly expressed on KATOⅢ cells. Conclusion [WT5”,6BZ]The phage displayed ScFv of monoclonal antibody MGd1 is successfully produced by phage display technology, which may pave a way for broadening the application range of the antibody. [
出处
《免疫学杂志》
CAS
CSCD
北大核心
2001年第3期165-168,共4页
Immunological Journal
基金
国家杰出青年基金! (395 2 5 0 2 0 )
国家"86 3"课题! (10 2 - 10 - 0 1- 0 6 )
国家自然科学基金! (3990 0 0 6 8)资助项目
关键词
胃癌
单克隆抗体
单链可变区片段
噬菌体呈现
抗MGd1
SCFV
gastric carcinoma
monoclonal antibody
antibody single chain variable fragment
phage display technology
