Sperm-specific
antigens are attractive candidates for the development of a
contraceptive vaccine. Using the subtractive
cDNA hybridization technology, the present study was conducted to obtain a human sperm-specific
antigen. The 32P-labeled single stranded
cDNA of human testis, subtracted with
poly(A)+ RNA of human peripheral white blood cells, was used to screen the human testis
cDNA-ZAP II library. The putative positive clones were further screened for binding with the solubilized human oocyte zona pellucida preparation (HZP). After screening 10(7) colonies, one positive clone, designated
contraceptive vaccinogen (CV), was obtained. It had an insert of approximately 1.3 kb, that was cloned and sequenced. The sense strand was identified by using the in vitro transcription and translation procedures, and the full-length sequence was obtained by using the 5' rapid amplification of 5' -
cDNA ends (5'-RACE) procedure. The full-length CV
cDNA has an ORF of 312
amino acids (aa) with the first ATG Met
start codon at
nucleotide (nt) 35 and the stop
codon TAA, at nt 959. The translated
protein has a calculated molecular mass of 35.3 kD and four potential N-linked glycosylation and several phosphorylation sites. Hydropathy plot generated from the deduced aa sequence showed it to be a membrane-anchored
peptide. Extensive computer search in the database did not find any homology of existing sequences with CV both for nt and aa. Northern blot analysis indicated the human testis-specific expression of CV
antigen. The coding region of CV
cDNA was subcloned into pET22b(+) vector and expressed. The expressed recombinant (r)CV
protein had a molecular size of approximately 44 kD, and it specifically reacted with the ZP3 component of HZP. Rabbit rCV
antibodies recognized the rCV, and a cognate
antigen of approximately 64 kD in the human sperm extract. The
antibodies showed binding with the live and
methanol-fixed human sperm, and significantly (P < 0.001) inhibited human sperm penetration of
zona-free hamster oocytes, as well as human sperm binding to human oocyte zona pellucida. These findings indicate that the testis/sperm- specific CV
antigen has a role in human sperm function and may find clinical applications in the
contraceptive vaccine development and in the specific diagnosis and treatment of
male infertility.