Cloning, expression and purification of human nuclear autoantigenic sperm protein (hNASP) and preparation of its polyclonal antibody.
- Author:
Min WANG
1
;
Jian-Li SHI
;
Guo-Yan CHENG
;
Yan-Qin HU
;
Chun-Meng LIU
;
Chen XU
Author Information
- Publication Type:Journal Article
- MeSH: Adult; Animals; Antibody Formation; Autoantigens; biosynthesis; genetics; immunology; Cloning, Molecular; Humans; Male; Nuclear Proteins; biosynthesis; genetics; immunology; Rabbits; Recombinant Fusion Proteins; biosynthesis; immunology; Reverse Transcriptase Polymerase Chain Reaction
- From: National Journal of Andrology 2006;12(10):867-871
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo acquire the purified human nuclear autoantigenic sperm protein (hNASP) and its polyclonal antibody for investigating the possible functions of hNASP involved in fertilization.
METHODSThe coding sequence of hNASP gene was amplified from human testis RNA with specific primers, and the PCR product was cloned first into pMD-18T and then into pET-28a ( + ) after restriction digestion with BamH I and Hind III. The fusion protein was expressed in E. coli BL21 (DE3) after induction with IPTG. The recombinant protein NASP was purified from the supernatant with Ni2 -NTA His-bind resin under native conditions.
RESULTSThe results of DNA sequencing and SDS-PAGE analysis showed the protein to be what we had hoped to acquire. ELISA showed that we had acquired rabbit anti-hNASP serum with high titer.
CONCLUSIONHigh purity recombinant hNASP protein could be obtained with the above-mentioned prokaryotic expression method, and so could the rabbit anti-hNASP serum with high titer and high specificity.
