Construction And Relationship With Brucellosis Susceptibility Of Haplotypes Of MHC-DQB2 From Sheep

Object: SNPs in MHC-DQB2 exon2 and exon3 of case and control Kazak sheep were analyzed through the experiment,and then SHEsis software was used to analyze the haplotype of SNPs.Expect there are significant differences in the two groups between individual SNPs loci and haplotype combinations,as genetic markers associated with brucellosis susceptibility,for the future development of Brucella molecular marker assisted selection provides certain reference for research,in order has a foundation to speed up the breeding new breeds of sheep with brucellosis resistance.Methods: 1.For the detection of Brucella in 100 Kazakh sheep with Bengal plate agglutination test of positive group and negative group respectively using PCR-SSCP technology to detect MHC-DQB2 Exon2,exon3 SNPs,was cloned and sequenced with different genotype,then using sequence alignment software to find the Exon2 and the exon3 SNPs loci,through statistical analysis,there were significant differences between positive group and negative group,obtained SNPs loci associated with the susceptibility of brucellosis.2.All of the SNPs loci through the detected test coincides with the minimum allele frequency and the balance of Hardy-Weinberg,select qualified SNPs loci,and then the SNPs haplotype analysis was performed using SHEsis software,has been associated with brucellosis susceptibility haplotype combination..Results: 1.Both exon2 and exon3 have abundant SNPs and SAPs loci in Kazak sheep MHC-DQB2,and the SNPs and SAPs loci of the Exon2 and the amino acid missense mutation loci are more abundant than exon3.2.Using the results of PCR-SSCP and sequencing analysis,44 SNPs loci were detected in MHC-DQB2 exon2 and 22 SNPs loci were detected in MHC-DQB2 Exon3 gene in Kazak sheep.3.SAP analysis of MHC-DQB2 gene in sheep was obtained,exon 2 encoding 90 amino acids,of which 34 are SAPs loci;in 34 SAPs loci,3 silent mutations,the remaining 31 are missense mutations.Exon 3 encodes a total of 93 amino acids,of which there are 20 SAPs loci.Of the 20 SAPs loci,there were 6 missense mutations and the rest were silent mutation.4.Through correlation analysis of two exons of MHC-DQB2 gene SNP and brucellosis,Exon2 found that only 9C/G allele frequency showed significant difference in the two groups;through the analysis of the genotype frequencies of Exon2 SNPs loci,117G/T,131C/G,9G/C,157C/A,180G/A,5 loci were significantly different between cases group and normal group(P < 0.05).There are significant differences in the distribution of 155T/C loci exon3 alleles in two groups of samples(P<0.05);the exon3 genotype of each polymorphism analysis found no significant difference in the distribution of each genotype in two groups of samples.5.The Kazak sheep MHC-DQB2 exon 2 in line with the conditions of the 18 SNPs loci by linkage disequilibrium analysis,obtained the exon 2 of 13 linkage domain,named Block1-Block13,which has 9 Block(Block2,Block3,Block4,Block6,Block7,Block8,Block10,Block12,Block13)a strong linkage disequilibrium.For MHC-DQB2 exon 3 linkage disequilibrium analysis,obtained the exon 3 of 8 linkage domain,named Block1-Block8,which has 4 Block(Block1,Block2,Block5,Block8)is a strong linkage disequilibrium.6.The SHSsis haplotype analysis revealed that 33 SNP loci of Kazak sheep MHC-DQB2 exon 2 can be constructed 17 haplotypes,including Hap12,Hap13,Hap14,Hap15,Hap16,Hap17 were much higher than the normal frequency group frequency,reached a significant level.21 haplotypes can be constructed by 18 SNP loci of exon3,Hap4,Hap6 in case group frequency is far lower than the normal group frequency reached significant level.Conclusion: 1.The Exon2 and exon3 of the MHC-DQB2 gene of Kazak sheep are rich in SNPs and SAPs,and the SNPs and SAPs sites of the exon2 and the amino acid missense mutation sites are more abundant than exon3.2.it is presumed that 9G/C loci of Kazakhstan sheep MHC-DQB2 gene Exon2 associated with the susceptibility of Brucella,Some genotypes of 9G/C、117G/T、131C/G、157C/A、180G/A loci are related to susceptibility of brucellosis,in other words,they to be infected more likely with specific genotypes in these loci of individual type.Preliminary analysis showed that the 155T/C of MHC-DQB2 exon3 were associated with susceptibility to brucellosis.3.The gene locus of the 9 strong linkage disequilibrium Block of MHC-DQB2 gene exon2 and the 4 strong linkage disequilibrium of exon3 in Kazak sheep may be a genetic linkage.4.it is presumed,they are more likely to suffer from brucellosis,with Hap12,Hap13,Hap14,Hap15,Hap16,Hap17 of the 6 haplotype combinations of individual for the Kazak sheep MHC-DQB2 exon2.they are more susceptibility to brucellosis with Hap9 individual and they are more resistant to brucellosis with Hap4,Hap6 individual for the Kazak sheep MHC-DQB2 exon3.