Prokaryotic expression, purification and antigenicity identification of mouse prostate stem cell antigen.
- Author:
Jinkai DONG
1
;
Jin LUO
;
Jinqi YAN
;
Liang ZHANG
;
Jiangping GAO
;
Jiyun YU
Author Information
- Publication Type:Journal Article
- MeSH: Animals; Antigens, Neoplasm; genetics; immunology; isolation & purification; Cloning, Molecular; Escherichia coli; metabolism; GPI-Linked Proteins; genetics; immunology; isolation & purification; Genetic Vectors; Male; Mice; Neoplasm Proteins; genetics; immunology; isolation & purification; Plasmids; Prostate; cytology
- From: Journal of Southern Medical University 2012;32(4):502-506
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo amplify mouse prostate stem cell antigen (mPSCA) gene and construct a recombinant plasmid to obtain mPSCA protein and identify its antigenicity.
METHODSThe gene of mPSCA was amplified by RT-PCR from mouse prostate cancer cell line RM-1 with the signal peptide sequence removed. The PCR product was cloned into pET-42a prokaryotic expression vector to construct the recombinant plasmid pET-42a-mPSCA, which was transformed into BL21 (DE3) for mPSCA expression. The fusion protein was purified and identified by SDS-PAGE and Western blotting. The antigenicity of the purified protein was characterized by ELISA.
RESULTSThe mPSCA gene was obtained with an identical sequence to that retrieved in GenBank. The prokaryotic expression vector for mPSCA was successfully constructed as confirmed by enzyme digestion and DNA sequencing. Both Western blotting and ELISA demonstrated the antigenicity of the purified mPSCA protein.
CONCLUSIONThe purified mPSCA obtained possesses good antigenicity, which will facilitate further study of immunotherapy for prostate cancer targeting PSCA.
