Prokaryotic expression, purification of prM of JEV and preparation of monoclonal antibody.
- Author:
Bei-fang NING
1
;
Huai-min ZHU
;
Xiao-jun ZHOU
;
Yi CAO
;
Ai-guo ZHOU
Author Information
- Publication Type:Journal Article
- MeSH: Animals; Antibodies, Monoclonal; analysis; immunology; isolation & purification; Antibody Specificity; BALB 3T3 Cells; Cell Line; Cloning, Molecular; Electrophoresis, Polyacrylamide Gel; Encephalitis Virus, Japanese; genetics; immunology; Epitopes; immunology; Escherichia coli; genetics; Mice; Plasmids; genetics; metabolism; Prokaryotic Cells; metabolism; Sequence Analysis, DNA; Viral Proteins; biosynthesis; genetics; immunology; isolation & purification
- From: Chinese Journal of Experimental and Clinical Virology 2008;22(1):65-67
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo prepare monoclonal antibody (mAb) against prM epitope.
METHODSThe gene encoding prM was isolated using RT-PCR from brain of JEV infected mouse and cloned into prokaryotic expression vector pET-32a. Recombinant plasmid was transformed into E.coli BL21/DE3/LysS, then the transformed cells were expressed with the induction of IPTG. The expression and purification of the prM protein was analyzed by SDS-PAGE. The BALB/c mice were immunized with purified prM protein. Hybridoma cell lines secreting monoclonal antibodies against prM were established after cell fusion of mouse splenic cell and P3-X63-Ag8.653 cells. The specificity of mAb was identified by ELISA, Western Blot and Immunohistochemistry assay.
RESULTSmAb against prM epitope of JEV was prepared successfully.
CONCLUSIONThe obtained prM specific mAb was valuable for the prevention and dignosis of Japanese encephalitis.
