Human Lyc4 Cloning Cum Lyc4, Lyc3 Cdv-1r Relations Of Reproductive Research

Using the nucleotide sequence of the human lysozyme as a query for a BLAST search at the EST database of GenBank, we got a series of homologous EST and assemble them as a contig. By screening of human testis A cDNA library, a length 831 bp cDNA sequence containing an open reading frame was obtained and submitted to GenBank(accession number AF 326749). This novel human gene LYC4 encodes a 148 amino acid protein with a signal peptide. The mature LYC4 sequence identity to human lysozyme is 38%. The eight conserved cysteine and other properties of LYC4 attribute it to a member of c type lysozyme /α-lactalbumin superfamily. Comparing to the other members, LYC4 and LYC1, LYC2, LYC3, LYC5 form a group in the unrooted phylogenetic tree analysed by ClustalW program, suggesting a human lysozyme-like gene family exist.LYC4 was expressed specifically in human testis by northern blotting analysis. In situ hybridization experiments showed that the LYC4 mRNA was intensively expressed in the caput epididymis and weakly detected in the corpus and cauda epididymis, being restricted to the epithelial cells, whereas, no obvious hybridization signal was found in the seminiferous tubles of the testis.Immunohistochemistry with antibody to purified recombinant LYC4 revealed high-level expression of LYC4 in the human caput epididymis and moderate-level in the corpus and cauda epididymis. In addition to epithelial cells, immunohistochemical staining is also presented in the lumen of the epididymis. A band at 14kDa was detected in human seminal plasma and also was found in the protein extraction from mouse caput, corpus and cauda epididymis respectively by Western blot analysis.By fused with his-tag, recombinant LYC4 was expressed and purified. A bactericidal activity to Gram-positive M. luteus and B.subtilis was demonstrated by LYC4, but failed to kill Gram-negative E.coli. Furthermore, LYC4 revealed an antimicrobial activity to some particular bacteria collected from the clinical microbes in women vaginas. The experiment of HEK293T cell transfection also showed the LYC4 has an antimicrobial activity. We suggest that LYC4 contribute to epididymal innate immunity and protect the sperm against attack by microorganisms in the male and female reproductive tracts.The antisera of LYC1, LYC2, LYC3, LYC4 and LYC5 were raised by synthesis peptides and recombinant proteins. A significant decrease in the number of sperm bound or penetrate to zona-free hamster eggs were observed in the presence of antiserum to LYC3, LYC4 and LYC5, comparing to their preimmune serum respectively, the values of two-tails student's t-test were P<0.005, P<0.05 and P<0.05. However, LYC1 and LYC2 demonstrated negative results by above hamster egg penetration test (HEPT), suggesting that LYC3, LYC4, LYC5 are...