[Recombinant human brain myelin basic protein and its antibody].
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.
Liu
Ji
J
School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041.
Chen
Jianye
J
Wang
Ruohan
R
Chen
Junjie
J
D000818:Animals; D000917:Antibody Formation; D001323:Autoantibodies; D001921:Brain / Q000276:immunology / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D004676:Myelin Basic Protein / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000276:immunology
D001323:Autoantibodies; D018076:DNA, Complementary; D004676:Myelin Basic Protein; D011993:Recombinant Fusion Proteins