Analysis Of Sequence And Its Function On Three Swine Leutocyte Antigen Gene In Inbreeding Line Of Wuzhishan Pig Population

To detect the alleles of SLA classâ…¡DRA, DRB gene in inbreeding line of Wuzhishan pig(WZSP) group. In the research, the method of PCR-SSCP,direct sequence of PCR and cloning sequence of four pairs SLA classâ… 3　primers were used , and a novel SLA class â…¡DRB allele was cloned and analysis by RT-PCR. The results indicate that: (1) In inbreeding line of WZSP group, SLA-DRA, DRB have respectively two alleles and found a novel DRB allele. The exon2 of SLA-DRA that one base transversion of G/T is highly conserved, at the level of nucleotide sequences and amino acid sequences, its homology reached 99%. While that of SLA-DRB possess polymorphism, the homology of nucleotide sequence and amino acid sequence is respectively beyond 85%, 70%. (2) Ten novel alleles of SLA class â… 3 gene were detected in this study, but they had a few nucleotide sequence differences. (3) Alignments of amino acid sequences of a total of all the 23 different alleles so far identified in the SLA class â… 3 gene including 9 new alleles identified in this study. In the a1 domain of SLA classâ… 3 molecules, multiple amino acid allelic differences were clustered at amino acid positions 50-85, while amino acid residues with extensive variation on the β-strand and a-helix in the a₂ domain were clustered at amino acid positions 98-119 and 146-172, respectively. The side chain of five residues-98,100,102,117 and 119-on the β-strand were suggested to correlate with the portion pointing into the binding site for processed antigen of the HLA class â… molecules. There residues (158,158 and 170) on the a-helix of the a₂ domain in the SLA class â… molecules, at positions 146-172, with extensive variation are also located in the recognition site for processed foreign antigen of the HLA class â… molecules. Furthermore, three amino acid residues of WZSP at positions 105,112 and 118, required for binding of human CD8 to the HLA class â… molecules, were well conserved at the identical position. (4) A novel DRB Alleles was thus obtained, which had 801 nucleotides with an complete open reading frame (ORF). Moreover, a phylogenetic analysis of the sequence revealed that SLA-DRB of the swine strains were more similar to HLA-DRB1*09012, *120101 than to H-2-E B; The human CD4-binding sites of HLA-DRB molecule and their counterparts in swine were further analyzed. These data indicated that the sites are completely conserved among the related molecules in porcine and in human, which might provide an understanding of how to the immune system evolved. Moreover, Construction of a phylogenetic tree using the nucleotide sequences of those SLAâ…¡DRB and SLAâ… 3 alleles by the neighbor-joining method on the basis of Tamura-Nei distances of the MEGA3 program indicated that, in inbreeding line of WZSP, there were itself special genetic resource. Conclusion: Success to detect alleles of SLA-DRA, DRB1 in inbreeding WZSP group, providing firm basis on the genetype of WZSP SLA-DR and its gene function.