哈萨克羊DQB2基因外显子2多态性及遗传变异分析

doi:10.16431/j.cnki.1671-7236.2017.12.022

摘要/Abstract

摘要：

试验旨在研究DQB2基因外显子2多态性与哈萨克羊布鲁氏菌病易感性的相关性。通过PCR-SSCP技术对146只布鲁氏菌阴性哈萨克羊血液样本和28只布鲁氏菌阳性哈萨克羊血液样本中的白细胞表面抗原DQB2基因外显子2的多态性进行研究，挑取不同的等位基因进行克隆测序，经卡方检验分析每个SNP位点的基因频率、基因型频率及其多态性与布鲁氏菌病易感性的相关性，应用生物信息学软件分析与哈萨克羊布鲁氏菌病易感性相关的不同等位基因的mRNA二级结构及蛋白质的二级结构、三级结构和抗原表位。结果发现，在270 bp的外显子2序列中共检测到33个SNPs，其中C9G、A180G位点的基因频率在病例组和对照组中的分布具有极显著性差异（P<0.01），其基因型频率在病例组和对照组中存在显著差异（P<0.05）；A13T、C133G位点的基因频率在病例组和对照组中存在显著差异（P<0.05）；进一步分析发现，A180G突变位点的最小自由能最低，其mRNA二级结构最稳定；A13T和C133G 2个突变位点均引起mRNA二级结构及蛋白质二级结构、三级结构和抗原表位的改变。本试验结果表明DQB2基因外显子2多态性与哈萨克羊布鲁氏菌病易感性呈显著相关。

关键词: DQB2基因; PCR-SSCP; SNPs; 布鲁氏菌病; 易感性; 生物信息学

Abstract:

This study was aimed to investigate the association between the polymorphism of DQB2 gene exon 2 and the susceptibility to Kazakh sheep brucellosis. The DQB2 gene exon 2 of Kazakh sheep lymphocyte antigen was amplified by PCR-SSCP method from 146 healthy and 28 infected with Brucella Kazakh sheep, and the single nucleotide polymorphisms (SNP) was analyzed, then the different alleles were selected for cloning and sequencing. In order to analyze its correlation with brucellosis susceptibility, the differences in gene frequency and genotype frequency of each SNP locus were analyzed by Chi-square test. Bioinformatics softwares were used to analyze the secondary structure of mRNA, the secondary structure, tertiary structure and epitope of protein. The sequencing result showed that 33 SNPs were detected in 270 bp DNA sequence, the gene frequencies of C9G and A180G were extremely significantly different in case group and control group (P<0.01), and its genotype frequencies presented significantly difference (P<0.05). Similarly, A13T and C133G loci were significant difference in case group and control group (P<0.05). Further analysis result showed that the minimum free energy of the A180G mutation site was the lowest and its mRNA secondary structure was the most stable; Both A13T and C133G mutation sites caused the changes of mRNA secondary structure, protein secondary, tertiary structure and antigenic epitope of protein, respectively. The results showed that the polymorphism of DQB2 gene exon 2 might be significantly correlated with brucellosis susceptibility in Kazakh sheep.

Key words: DQB2 gene; PCR-SSCP; SNPs; brucellosis; susceptibility; bioinformatics

中图分类号:

S813.3

王元元, 严国, 罗成, 齐江姣, 周光普, 谭君, 高剑峰. 哈萨克羊DQB2基因外显子2多态性及遗传变异分析[J]. 《中国畜牧兽医》, 2017, 44(12): 3543-3553.

WANG Yuan-yuan, YAN Guo, LUO Cheng, QI Jiang-jiao, ZHOU Guang-pu, TAN Jun, GAO Jian-feng. Polymorphisms and Its Genetic Variation Analysis of DQB2 Gene Exon 2 in Kazakh Sheep[J]. , 2017, 44(12): 3543-3553.

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