Recombinant human brain myelin basic protein and its antibody.
- Author:
Ji LIU
1
;
Jianye CHEN
;
Ruohan WANG
;
Junjie CHEN
Author Information
1. School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041.
- Publication Type:Journal Article
- MeSH:
Animals;
Antibody Formation;
Autoantibodies;
Brain;
immunology;
metabolism;
Cloning, Molecular;
DNA, Complementary;
Escherichia coli;
genetics;
metabolism;
Gene Expression;
Humans;
Myelin Basic Protein;
biosynthesis;
genetics;
immunology;
Rabbits;
Recombinant Fusion Proteins;
biosynthesis;
immunology
- From:
Journal of Biomedical Engineering
2003;20(1):64-67
- CountryChina
- Language:Chinese
-
Abstract:
We constructed the expression vector by inserting 21.5 KDa MBP human brain full-length cDNA coding sequence digested with restriction enzyme EcoR I and Sal I into downstream of pGEX-5T expression vector. The recombinant vector p5TMP was transformed into E. coli and the positive clonies were selected and incubated in LB medium induced by IPTG (isopropyl- -D-thiogalactoside). A new polypeptide band with apparent molecular weight 42 KDa was detected in transformed cell lysates by SDS-PAGE. Western blotting analysis confirmed that this fusion protein reacted specifically with antibodies to MBP, the expression level of MBP was about 414.6 mg/L medium estimated by immuno-dot blot, ELISA and absorbance scanning. Newzealand rabbits were immunized by subcutaneous injection of the purified recombinant MBP. The titer was obtained at 1:16 after 5 injections. The specificity of the antibody to MBP was confirmed by immuno-blot and Western blotting.
