MOLECULAR-CLONING AND SEQUENCING OF CDNA-ENCODING FOR A NOVEL TESTIS-SPECIFIC ANTIGEN

cDNA encoding for a sperm antigen, designated NZ-1, was cloned and seq uenced from murine testis cDNA-lambda gt11 expression library using an tibodies to human sperm surface antigens belonging to 14-18 kD molecul ar region. These sperm antigens are involved in zona pellucida binding and have tyrosine phyosphorylation activity. Computer generated trans lation analysis of 1395-bp cDNA yielded an open reading frame (ORF) of 152 aa with first ATG, Met start codon at nt 32 and the stop codon TG A at nt 487. The translated protein has a calculated molecular weight of 17.9 kD and a potential tyrosine phosphorylation site at aa 46-54, besides at least two O-linked glycosylation sites. The hydropathy plot generated from the deduced aa sequence indicated it to be a membrane- anchored peptide with a hydrophobic NH2-terminus that is characteristi c of a signal peptide, Extensive computer search in the GenBank, NBRF, and Swiss sequence banks, indicating it to be a novel protein. Northe rn blot analysis indicated testis-specific expression of NZ-1 antigen. The NZ-1 cDNA was subcloned into pGEX-1 lambda T vector and expressed in glutathione-S-transferase gene fusion system to obtain the recombi nant protein. The recombinant protein specifically reacted with the or iginal antibodies raised against the native 14-18 kD sperm proteins. T hese findings suggest that the sperm-specific recombinant NZ-1 may fin d applications in the development of a contraceptive vaccine, and in s tudying tile normal and abnormal sperm function and the signal transdu ction mechanism. (C) 1997 Wiley-Liss, Inc.