Cloning,Prokaryotic Expression And Polymorphism Study Of SLA-DOB Gene

Major histocompatibility complex (MHC) antigen was such gene cluster that linked with certain chromosome encoding a group of cell membrance glycoprotein that correlated with immune response. Its function was not only confined in rejection of transplantation but also correlated with immune response for extra-antigen. It was studied widely in human, mouse, sheep, cattle and dog in recent years due to MHC gene's very important roles in organ transplantation and immune response. SLA-DOB gene was an important function gene that participated antigen presentation of SLA II molecule. But until now no reports have been observed. In this study, 137 samples(individuals) from six breeds of Bama mini pigs, Wuzhishan mini pigs, Diannan mini pigs, Hezuo pigs, Ganzi Tibet pig, Hebao pigs and organizes of another 1 Bama mini pig were used to study SLA-DOB gene using methods of bioinformatics, RT-PCR, prokaryotic expression, SSCP-PCR and sequencing. In order to mine mechanism of SLA-DOB gene that was influencing immune response in pigs and provide some tissue expression, polymorphism identification of this gene were described in this paper. The major resuts are follows:1. EST database of pig was screening by information-probe of cDNA sequence of HLA-DOB.Then cDNA sequence of SLA-DOB1 gene was cloned, 897bp. Obtained CDS sequence was analyzed with open reading frame by ORF finder server of NCBI. The results was displayed that 277 amino acids was predicted in ORF. Specificity primer was designed between start codon and termination codon. Then the clone results (GenBank accession: DQ333334)was consistent with sequence of electronic clone by RT-PCR and sequence analysis. Sequence of SLA-DOB1 was cloned that was high homology with the other species by multiple sequences alignment and phylogeny evolution analysis. Basic character, structure and function of SLA-DOB1 protein was predicted by bioinfomatics. The results showed that SLA-DOB1 molecule was a precursor protein of MHC II β with signal peptide, which have a transmembrane structure near to termination of N. A comparison of the nucleotide sequences showed that the SLA-DOB1 gene was highest homology to Mus musculus(71%), cattle(77%),...