Molecular Cloning, Expression of Testicular Transcript Abundant in Germ Cells and Immunobiological Effects of the Recombinant Protein
Article first published online: 26 JUL 2004
American Journal of Reproductive Immunology
Volume 52, Issue 2, pages 164–173, August 2004
How to Cite
Verma, S., Mohapatra, B., Jagadish, N., Selvi, R., Roy, P., Rana, R., Lakshmi, K. and Suri, A. (2004), Molecular Cloning, Expression of Testicular Transcript Abundant in Germ Cells and Immunobiological Effects of the Recombinant Protein. American Journal of Reproductive Immunology, 52: 164–173. doi: 10.1111/j.1600-0897.2004.00188.x
- Issue published online: 26 JUL 2004
- Article first published online: 26 JUL 2004
- Submitted November 11, 2003; revised March 29, 2004; accepted May 3, 2004.
- flow cytometry;
- indirect immunofluorescence;
- in situ RNA hybridization;
- recombinant protein
Problem: It has been well documented that antisperm antibodies can be causative factors for infertility. In this report we have identified a protein on human sperm referred as human sperm-associated protein (HSAP) using serum of an immunoinfertile woman; it is thus a sperm-specific protein – a candidate molecule for control of fertility.
Method of study: An immunoinfertile woman serum showing head–head sperm agglutination and acrosomal localization, reacted with human sperm protein of apparent molecular weight of 48 kDa on Western blot. Anti-48 kDa antiserum was raised in rabbit by eluting 48 kDa protein and was used to screen the human testis cDNA expression library. A putative positive hsap cDNA clone was obtained, sequenced and subjected to tissue specificities studies by Northern blotting. The cell type-specific expression was done using in situ RNA hybridization studies. To obtain recombinant HSAP (r-HSAP), hsap cDNA was cloned in pET 22b(+) expression vector. r-HSAP was expressed as polyhistidine fusion protein in Escherichia coli and purified. Rabbits were immunized with the purified r-HSAP, which led to generation of antibodies. In order to evaluate in vitro immunocontraceptive potential, the anti-r-HSAP antibodies were characterized by agglutination assay, zona-free hamster egg penetration assay, indirect immunofluorescence (IIF) assay, and by flow cytometry analysis.
Results: We have cloned a human testis gene encoding a protein (HSAP) of 328 amino acids. Antibodies against the purified recombinant protein specifically recognized approximately 40 kDa r-HSAP, and a cognate 48 kDa protein band in human sperm extract in Western blot procedure. The anti-r-HSAP antibodies localized acrosomal compartment, inhibited sperm binding/attachment in zona-free hamster penetration assay and revealed surface binding with human live sperm by flow cytometry. The cDNA sequence has been submitted to EMBL and has been given the accession number Y16676.
Conclusion: This study has put in evidence that novel sperm-specific r-HSAP has role in sperm function and may have application in the development of a contraceptive vaccine. The availability of the recombinant protein will facilitate studies on the assessment of its potential as a contraceptive immunogen.