Cloning And Sequence Analysis Of α And β Chain Of DR Locus From Hebao Pigs

Hebao pig is one of Chinese swine breeds with fine quality, and evolved more primarily, therefore it should have important scientific research value. DR locus belongs to the important functional genes of swine major histocompatibility complex (MHC) class II, also named as swine leukocyte antigen (SLA) class Ⅱ, including a chain (DRA) and β chain (DRB), which are the marker of molecular evolution. In order to study the SLA-DR locus of Hebao pig, RT-PCR was used to amplify SLA-DRA using primers of DRAF1/DRAR1 and to amplify SLA-DRB using primers of DRBF1/DRBR1 from the spleen tissues of Hebao pigs, respectively. The PCR products were inserted into pMD18-T vector and then transformed to Escherichia coli JM109. The positive clones were selected by digestion with the EcoR I and Hind III. Finally, all sequences were compared, analyzed and phylogenetic-tree constructed by biological software DANMAN and Mega 5.0 software. The results showed that three SLA-DRA-HB01-03 alleles and three SLA-DRB-HB01-03 alleles were cloned successfully. By sequencing, it was shown that the length of DRA was 779 bp, which coded 252 amino acids. After submission, the three SLA-DRA-HB01-03 alleles were qualified accession number of LC002668, LC002669 and LC002670, respectively. For β chain of SLA-DRB-HB01-03 alleles, the length was 836 bp, coded for 266 amino acids. After submission, the three SLA-DRB-HB01-03 were qualified accession number of LC20542, LC20543 and LC20544, respectively. By comparing the amino acids of SLA-DRA-HB01-03 alleles with other SLA-DRA alleles, it was shown that the mutated amino acids were few. For β chain of SLA-DRB-HB01-03 alleles, the polymorphism was clear with lots of mutated amino acids. The homology between SLA-DRA-HB01-03 and other SLA-DRA alleles was among 98.7% and 99.6%, while for SLA-DRB-HB01-03, the homology to other alleles was among 90.3% and 99.8%. For the phylogenetic tree, the SLA-DRA-HB01-03 clustered into one branch, but close to other DRA alleles in evolution. For β chain of SLA-DRB-HB01-03 alleles, they were clustered into one branch and far away from other alleles, and they were more primarily evolved.In this research, the a chain and β chain of DR locus were cloned from Hebao pigs successfully, and it was concluded that the a chain of SLA-DRA-HB01-03 alleles was conservative, while the β chain of SLA-DRB-HB01-03 was more polymorphic and displays more molelular characteristics in evolution. The research will lay a base to study the structure and function of SLA-DR locus in future.