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 107 colonies, one positive clone, designated contraceptive vaccinogen (CV), was obtained. It had an insert of ∼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 ∼44 kD, and it specifically reacted with the ZP3 component of HZP. Rabbit rCV antibodies recognized the rCV, and a cognate antigen of ∼64 kD in the human sperm extract. The antibodies showed binding with the live and methanol-fixed human sperm, and significantly (P
