Construction And Characterization Of Eukaryotic Expression System Of Chicken MHC Ⅰα Gene

Major histocompatibility complex (MHC) plays an important role in the immune response. Chicken BF gene encoding the mammalian homological MHC Class I molecules, which are primarily responsible for the presentation of endogenous antigen peptide recognized by CD8+T cell. Chicken B-F2gene encoding a chain of MHC I becomes a candidate marker genes for disease resistance. MHC has a unique effect in the field of immunogenetics, population genetics and breeding for disease resistance due to its outstanding characteristics and important function. The more research have been carried out about MHC molecular structure and function of human, mouse and other mammals, While a little of imformation about chicken MHC molecules is reported, especially the MHC Class I molecule. To further study of chicken MHC class I molecules in this study a P815cell strain stable expressing chicken MHC I a gene was built.First, a pair of primers was designed based on the reported chicken MHC I a gene sequences (GenBank accession number:AY989897) and pEGFP-Nl multiple cloning sites sequence. mRNA was extracted from chicken peripheral blood mononuclear cells as the template, then the MHC I a gene fragment was amplificated by RT-PCR The DNA fragment was inserted into vector pEGFP-Nl. After the transfection and selection culture with kanamycin a recombinant eukaryotic plasmid pEGFP-Nl-MHC I a was achieved. This plasmid was further identificated with sequencing, by endonucleases, Ecor I and Sal I double restriction and PCR.Secondly, the pEGFP-Nl-MHC I a was transfected into COS7. After culture of24h the samples were observed with fluorescence microscope for the expression of fluorescence. The fluorescence was mainly located in the endomembrane system. Then alignment of the sequence was carried out and a phylogenetic tree was constructed among chicken, quail, duck, mouse, human and cattle. The results showed that MHC I a chains of birds and mammals belong to different branches of the phylogenetic distance, the quail and chicken were evolutionarily recent, followed a duck, mouse, human and cattle compared to a class. Comparison of prediction protein structure of chicken, duck, mouse and human MHC I a, revealed that they all have a high similarity of structure. Their similar function was assumed. The results of prediction of intracellular localization showed chicken MHC I a molecule were distributed in the endomembrane system.Finally, pEGFP-Nl-MHC I a was transfected into P815cell strain by Lipofection, in which the working concentration of G418was600[J],g/ml. And then after Characterization a P815(cMHC la) cell strain was built, which could stably express cMHC I a gene, which distributed in the endomembrane system, and the specific DNA segment was amplificated from the cell DNA by PCR. All these results suggest that the MHC I a molecules locate in the cell endomembrane system.In this study, the recombinant plasmid pEGFP-N1-MHC I a and the P815(cMHC I a) cell strain were successfully constructed. The P815(cMHC I a) could act as a tumor cell model to study of function of chicken MHC I molecule.