[Cloning, expression and purification of human nuclear autoantigenic sperm protein (hNASP) and preparation of its polyclonal antibody].
OBJECTIVE
To acquire the purified human nuclear autoantigenic sperm protein (hNASP) and its polyclonal antibody for investigating the possible functions of hNASP involved in fertilization.


METHODS
The 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.


RESULTS
The 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.


CONCLUSIONS
High 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.
Wang
Min
M
Department of Histology and Embryology, Shanghai Jiaotong University School of Medicine/Shanghai Key Laboratory for Reproductive Medicine, Shanghai 200025, China.
Shi
Jian-Li
JL
Cheng
Guo-Yan
GY
Hu
Yan-Qin
YQ
Liu
Chun-Meng
CM
Xu
Chen
C
D000328:Adult; D000818:Animals; D000917:Antibody Formation; D001324:Autoantigens / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D003001:Cloning, Molecular; D006801:Humans; D008297:Male; D009687:Nuclear Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction
D001324:Autoantigens; C489390:NASP protein, human; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins