The Construction Of Ovis Aries MHC ? DRB1 Gene Yeast Surface Display Library And Screening Of Brucella M5 Strain Epitope

Object: Detection of polymorphisms of DRB1 gene exon 3 in positive and negative Kazakh sheep individual for Brucella using PCR-SSCP techniques to obtain polymorphic loci;Construct yeast surface display recombinant expression vector p YD1-DRB1;insert into specific restriction sites with Site-directed mutagenesis of exon 2 of sheep DRB1 gene;Build yeast surface display library on DRB1 gene;Detecting the expression of DRB1 gene on the surface of yeast cells;Detection of peripheral blood leukocytes is luminescing when the mice were immunized with pmc221-GFP-M5 and and explore a feasible way to screen brucella antigen peptide library.Lay a foundation for breeding of sheep and for the control mechanism of brucellosis,and to provides a certain theoretical basis.Methods: 1.Brucella positive serum of 180 Kazakh sheep samples were detected using brucella rose bengal plate agglutination test kit.The polymorphism in exon 3 of DRB1 gene in Kazakh sheep was analyzed by direct sequencing method combined with PCR-SSCP technique.2.Construct construct a yeast polymorphism display library of sheep DRB1 gene with inserting into restriction sites at the end of exon 2 of DRB1 gene,then induced with galactose and detect the expression of the target protein by cellular immunofluorescence.3.The mice were infected with brucella pmc221-GFP-M5,the peripheral blood leukocytes were isolated by density gradient centrifugation.Whether the leukocytes had green fluorescence was observed by using confocal laser scanning.Results: 1.To detect the infection rate of brucella by rose bengal plate agglutination test,146 negative samples and 34 positive samples were detected in 180 Kazakh sheep with a positive detection rate of 18.89%.2.To detectn polymorphism in exon 3 of DRB1 gene in Kazakh sheep by PCR-SSCP combined with direct sequencing,the result showed that the 7 SNPs sites(T10C,C119 T,G215C,A238 G,T245G,G256 A,and C259T)of DRB1 gene exon 3 had no correlation with brucellosis susceptibility in Kazakh sheep.3.Through the point mutation,a specific restriction enzyme site was successfully introduced to construct the yeast polymorphic surface display library of sheep DRB1 gene,induced by galactose and detected by cellular immunofluorescence.The results showed that the sheep DRB1 gene was successfully expressed on the yeast surface and had correct spatial conformation.4.Using the laser confocal microscope to observe the white blood cells obtained by density gradient centrifugation,The white blood cells obtained by density gradient centrifugation were observed using laser confocal microscope and green fluorescence appeared in the field of vision.Conclusion: 1.The polymorphism of exon 3 of MHC-DRB1 gene in Kazakh sheep was detected and showed that the exon 3 sequence of DRB1 gene in Kazakh sheep had low polymorphism.2.The yeast polymorphism display library of sheep DRB1 gene was successfully constructed,then induced by galactose,which is inspired green fluorescence appeared under confocal laser scanning,indicates that DRB1 protein was successfully expressed on the surface of yeast cells,indicated this method can be applied to the eukaryotic expression of sheep MHC class II genes.3.Detection of green fluorescence appeared in white blood cells using laser confocal after the density gradient centrifugation,which shows that the pmc221-GFP-M5 Brucella can successfully invaded mononuclear macrophages and make them luminesced.