ABSTRACT
<p><b>AIM</b>To investigate the possible functions of human sperm membrane protein (hSMP-1) in the process of fertilization.</p><p><b>METHODS</b>A 576-bp cDNA fragment of HSD-1 gene coding for the extracellular domain of hSMP-1 was cloned and expressed. The localization of this protein on human and mouse sperm was determined by indirect immunofluorescent staining by using anti-recombinant hSMP-1 (anti-rhSMP-1) antibodies. Sperm acrosome reaction and sperm-zona pellucida (ZP) binding assay were carried out in 10-week-old BALB/c mice.</p><p><b>RESULTS</b>Recombinant hSMP-1 was successfully cloned and expressed. The expression of the native protein was limited on the acrosome of human and mouse sperm. Treatment of anti-rhSMP-1 antibodies significantly decreased the average number of sperms bound to each egg. Meanwhile, the percentage of acrosome reaction was decreased in comparison to pre-immune control after treatment with anti-rhSMP-1 (P < 0.05).</p><p><b>CONCLUSION</b>The results suggest that anti-rhSMP-1 antibody inhibited mouse acrosome reaction and sperm-ZP binding.</p>
Subject(s)
Animals , Female , Humans , Male , Mice , Acrosome Reaction , Allergy and Immunology , Physiology , Antibodies , Pharmacology , Antigens, Surface , Genetics , Cloning, Molecular , DNA Primers , Escherichia coli , Genetics , Fertilization , Fluorescent Antibody Technique, Indirect , Membrane Proteins , Genetics , Mice, Inbred BALB C , Recombinant Proteins , Metabolism , Reverse Transcriptase Polymerase Chain Reaction , Zona Pellucida , Allergy and Immunology , PhysiologyABSTRACT
<p><b>OBJECTIVE</b>To acquire the purified human nuclear autoantigenic sperm protein (hNASP) and its polyclonal antibody for investigating the possible functions of hNASP involved in fertilization.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>