| Objective: Comparative analysis for DRA gene sequence and the exon2 sequence on sheep genome MHC segment;Build recombination carrier p YD1-DRA of saccharomyces cerevisiae surface display;Amplify exon2 sequence of OLA-DRA gene on risk and normal sheep individual using DNA pooling to get the polymorphism loci by analyzing polymorphism; mutate into specific enzyme sites by fast fixed-point gene mutation on the both ends of exon2 sequence of OLA-DRA gene;Build saccharomyces cerevisiae surface display library on DRA gene;Detect whether DRA gene stable expression on saccharomyces cerevisiae cell surface or not, and explore feasible way of the preliminary screening of combined brucella antigen peptide with OLA-DRA protein. Lay a foundation for disease resistance breeding of sheep and further study the disease-resistant mechanism of MHC, and provide certain reference basis and theory support, so as to accelerate the process of molecular genetic disease resistance breeding.Methods: 1. Analyze m RNA sequence of MHCâ…¡DRA gene on cattle and sheep by using BLAST to find sequence of MHCâ…¡DRA gene and the exon sequence on cattle and sheep, and seek SNPS loci of cattle and sheep through the SNP databases in Gen Bank. Analyze the SNP loci of exon of MHCâ…¡DRA gene on cow by using DNAMAN6.0 software, and obtain the exon sequence with relatively rich polymorphism of MHCâ…¡DRA gene on cattle, and get exon2 sequence of MHCâ…¡DRA gene on sheep with higher similarity to those exons on cattle by using DNAStar Seq Man software. 2. This experiment detects brucella positive serum of 240 sheep blood samples by using brucella tiger red plate agglutination diagnostic kit and tude agglutination test, then tests SNP loci of exon2 sequence on OLA-DRA gene by using PCR-SSCP technology, and tests again SNPs of exon2 sequence on OLA-DRA gene in 600 brucella masculine and feminine individuals by using direct sequencing method of DNA pooling PCR products, and cloning exon2 containing SNP loci and sequencing, and then compare and analyze polymorphic loci by using molecular biology software. 3. Build OLA-DRA gene yeast surface display library by inserting into the restriction enzyme sites through site-directed mutation method, and experiment with protein induced expression and cell immunofluorescence, and inspect whether OLA-DRA gene display on the yeast cell surface or not by using fluorescence microscope.Results: 1. Obtain MHCâ…¡DRA gene sequence on sheep with high homology with cattle through bioinformatics analysis and gene cloning sequencing, and release from NCBI with KR422362 for Gen Bank login, and get its exon2 sequence with polymorphism by comparative analysis. 2. Get 27.5% detection rate of brucella positive individuals eventually after reviewing the positive individuals in the detection of a total of 240 sheep containing 174 negative individuals and 66 positive individuals by using brucella tiger red plate agglutination test and tude agglutination test to detectbrucella infections masculine or feminine individuals of sheep. 3. Find exon2 sequence of DRA gene on the sheep with polymorphism, and 143 bp of exon2 sequence base mutation changed from G to A so that amino acid from Arg into His by detecting exon2 sequence of MHCâ…¡DRA gene on the 240 sheep individuals with the method of PCR-SSCP technology. 4. Insert the specific restriction enzyme sites successfully through site-directed mutation method, and build yeast surface display library of OLA-DRA gene eventually, and appear green fluorescence under the fluorescent microscope after inducing protein expression and cellular immunofluorescence, and display OLA-DRA protein on cell surface in yeast successfully.Conclusion: 1. This research finds exon2 to own polymorphism by analyzing exon2 sequence polymorphism of OLA-DRA gene with the method of PCR-SSCP technology and sequencing the PCR products directly and comparative alignment analysis. 2. This paper gets 3 polymorphic loci including exon2 46 th base C/G, 120 th bases G/A and 143 th bases G/A by analyzing polymorphism loci of OLA-DRA gene and comparing the exon2 sequence with the length of 246 bp, which is consistent with the immune gene encoding product diversity. 3. This study builds OLA-DRA gene yeast surface display library which is inspired to green fluorescence under the fluorescent microscope after induction expression and cell immunofluorescence, which shows that expression protein of OLA-DRA gene is displayed in yeast cell surface, and proves that this method has certain feasibility and scientific innovation. |
