Expression and identification of the soluble monoclonal antibody ScFv fragment
- VernacularTitle:一种可溶性单抗ScFv片段的表达及鉴定
- Author:
Xiaolin ZHONG
;
Huiguang GAO
;
Qing JI
;
Gang HUANG
;
- Publication Type:Journal Article
- Keywords:
colorectal carcinoma;
monoclonal antibody;
single chain variable fragments;
phage
- From:Journal of Third Military Medical University
2003;0(08):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To express soluble single chain variable fragments (ScFv) of monoclonal antibody MC3 recognizing colorectal carcinomas in E. coli HB2151 and to purify the soluble ScFv and identify its antigen binding activities to find new target vectors for the diagnosis and therapy of colorectal carcinomas. Methods The phage clones displaying ScFv fragment of the monoclonal antibody MC3 were used to infect E. coli HB2151 to express soluble antibodies. The soluble ScFvs were identified by Dot blot and Western blot and their antigen binding activities were determined by ELISA. The VH and VL DNAs of the ScFv DNA derived were sequenced based on the dideoxy method. Results The soluble MC3 ScFvs were expressed successfully. The expression products with a proximate MW of 32?10 3 were mainly secreted into the periplasm. The soluble ScFv containing periplasmatic extracts derived from three clones could inhibit the binding of MC3 with its antigen, and the inhibition rates were 41.19%, 36.89% and 33.77% respectively. The sequences of the VH and VL DNAs of the MC3 ScFv showed that the variable antibody genes belonged to the IgG1 subgroup and ? type. Conclusion Generation of E. coli HB2151 expressed ScFv of monoclonal antibody MC3 paves the way for further use of the antibody.
