[Confirmation and expression analysis of three predicted genes in sheep MHC region]

Previous DNA sequencing of BAC clones covering entire ovine MHC (OLA) region resulted in identification of approximately 130 functional genes in the region, of which 8 were predicted by computer software to be exclusively existed in sheep, but not in any other species known to date. In the present study, we successfully identified and cloned cDNA sequence of OaN2, OaN5, and OaN6 from representative sheep tissues, confirmed their existence in reality. The sequences obtained experimentally exactly identical to those predicted previously. The length of cDNA fragments for OaN2, OaN5, and OaN6 was 270 bp, 309 bp, and 205 bp, respectively, with GenBank accession number assigned as JF330782 (OaN2), JF330783 (OaN5), and JF330784 (OaN6). Northern analyses indicated that the mRNA transcripts of OaN2 were mainly seen in ovine mesenteric lymph nodes and spleen, while OaN5 was observed in only in mesenteric lymph nodes. In contrast, OaN6 transcripts were detected in all tissues except for liver and heart. Western blot showed that OaN2 protein expression level was detected in mesenteric lymph nodes, spleen, and liver, essentially consistent with that of mRNA transcripts. Immunohistochemistry analysis showed that OaN2 protein was highly expressed in ovine mesenteric lymph nodes, moderately expressed in, and not expressed in heart, liver, and pancreas, consistent with the results of Western blotting. The cloning and expression analysis of 3 novel genes provide a basis for revealing their specificities and would be helpful to further study of their expression profile and their potential functions.