We found a match
Your institution may have access to this item. Find your institution then sign in to continue.
- Title
Molecular Cloning, Expression of Testicular Transcript Abundant in Germ Cells and Immunobiological Effects of the Recombinant Protein.
- Authors
Verma, Saguna; Mohapatra, Bhagyalaxmi; Jagadish, Nirmala; Selvi, Ramasamy; Roy, Pragyan; Rana, Ritu; Lakshmi, Kavitha; Suri, Anil
- Abstract
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 <em>hsap</em> cDNA clone was obtained, sequenced and subjected to tissue specificities studies by Northern blotting. The cell type-specific expression was done using <em>in situ</em> RNA hybridization studies. To obtain recombinant HSAP (r-HSAP), <em>hsap</em> cDNA was cloned in pET 22b(+) expression vector. r-HSAP was expressed as polyhistidine fusion protein in <em>Escherichia coli</em> and purified. Rabbits were immunized with the purified r-HSAP, which led to generation of antibodies. In order to evaluate <em>in vitro</em> 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...
- Subjects
ANTIGEN-antibody reactions; RECOMBINANT proteins; INFERTILITY; IMMUNOGLOBULINS; SPERMATOZOA
- Publication
American Journal of Reproductive Immunology, 2004, Vol 52, Issue 2, p164
- ISSN
1046-7408
- Publication type
Article
- DOI
10.1111/j.1600-0897.2004.00188.x