哈萨克绵羊DRB1基因外显子1和4多态性与布鲁氏菌病的相关性研究

doi:10.16431/j.cnki.1671-7236.2020.12.006

摘要/Abstract

摘要： 试验旨在对哈萨克绵羊DRB1基因外显子1和4多态性与布鲁氏菌病的相关性进行研究。使用虎红平板凝集试验（RBPT）对试羊的血清进行血清学检测，参考GenBank中绵羊MHC ClassⅡ区DRB1基因序列（登录号：NC_040271.1），对其外显子1和4片段设计引物，采用PCR-SSCP和DNA测序技术对230只哈萨克绵羊的DRB1基因进行多态性检测，分析其多态位点与哈萨克绵羊布鲁氏菌易感性之间的关系。RBPT检测发现66只哈萨克绵羊为布鲁氏菌感染阳性，阳性检出率为28.7%。外显子1片段存在一个SNP位点（F1-G22A），测序确定两种基因型（GG、GA），优势等位基因和基因型分别为G、GG，F1-G22A多态位点的易感基因型为GA。卡方检验表明，哈萨克绵羊DRB1基因F1-G22A多态位点与布鲁氏菌易感性的相关性不显著（P>0.05）。通过生物信息学在线软件分析得出，F1-G22A多态位点导致了RNA二级结构的改变和最小自由能的降低，引起了蛋白质二级结构的改变。DRB1基因外显子4片段未发现SNPs。由此得出，哈萨克绵羊DRB1基因F1-G22A多态位点与布鲁氏菌易感性可能存在一定的相关性。

关键词: 哈萨克绵羊; DRB1基因; PCR-SSCP; SNP; 布鲁氏菌病

Abstract: The purpose of this experiment was to study the correlation between exon 1 and 4 polymorphisms of DRB1 gene and brucellosis in Kazakh sheep.Using RBPT serological tests to try sheep serum,reference in GenBank sheep MHC Class Ⅱ area DRB1 gene sequences (Accession No.:NC_040271.1),the exon 1 and 4 pieces designed primers,using PCR-SSCP and DNA sequencing technology to 230 Kazak sheep DRB1 gene polymorphism detection,analyze its polymorphism loci and the relationship between the Kazak sheep Brucella susceptibility.The results showed that 66 Kazakh sheep were positive for Brucella in RBPT test,and the positive detection rate was 28.7%.There was one SNP locus (F1-G22A) in exon 1 fragment,and sequencing determined two genotypes (GG and GA),the dominant allele and genotype were G and GG respectively,and the susceptibility genotype of the polymorphisms of F1-G22A was GA.Chi-square test showed that there was no significant correlation between the polymorphisms of DRB1 gene F1-G22A and Brucella susceptibility in Kazakh sheep (P>0.05).According to the analysis of bioinformatics online software,the F1-G22A polymorphic sites lead to the change of RNA secondary structure and the decrease of minimum free energy,and lead to the change of protein secondary structure.No SNPs were found in DRB1 exon 4 fragment.Therefore,there might be a certain correlation between the polymorphisms of DRB1 gene F1-G22A and Brucella susceptibility in Kazakh sheep.

Key words: Kazakh sheep; DRB1 genes; PCR-SSCP; SNP; brucellosis

中图分类号:

S813.3

马雪珍, 徐杰, 高剑峰, 李刚. 哈萨克绵羊DRB1基因外显子1和4多态性与布鲁氏菌病的相关性研究[J]. 中国畜牧兽医, 2020, 47(12): 3844-3851.

MA Xuezhen, XU Jie, GAO Jianfeng, LI Gang. The Correlation Between Polymorphisms of Exon1 and Exon4 of DRB1 Gene and Brucellosis in Kazakh Sheep[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(12): 3844-3851.

参考文献

[1] LI T,TONG Z,HUANG M,et al.Brucella melitensis M5-90Δbp26 as a potential live vaccine that allows for the distinction between natural infection and immunization[J].Canadian Journal of Microbiology,2017,63(8):719-729.
[2] 鲁友铭,李俊萱,吴胜昔,等.布鲁氏菌BP26蛋白的原核表达及鉴定[J].重庆理工大学学报(自然科学),2019,33(10):185-190. LU Y M,LI J X,WU S X,et al.Prokaryotic expression and identification of Brucella BP26 protein[J].Journal of Chongqing University of Technology(Natural Science),2019(10):185-190.(in Chinese)
[3] CORBEL M J.Brucellosis:An overview[J].Emerging Infectious Diseases,1997,3(2):213.
[4] SCHURIN G G,SRIRANGANATHAN N,CORBEL M J.Brucellosis vaccines:Past,present and future[J].Veterinary Microbiology,2002,90(1-4):479-496.
[5] 孙丽媛.吉林省布鲁氏菌流行株种属鉴定及其耐药分子机制的研究[D].长春:吉林大学,2011. SUN L Y.Identification of Brucella isolates and the molecular resistance mechanism from Jilin regions[D].Changchun:Jilin University,2011.(in Chinese)
[6] 张广俊.羊布鲁氏菌病的危害与防治[J].四川畜牧兽医,2013,40(1):57-58. ZHANG G J.Harm and prevention of brucellosis of sheep[J].Sichuan Animal & Veterinary Sciences,2013,40(1):57-58.(in Chinese)
[7] 刘文进,何成武,宁多武,等.新疆畜间布鲁氏菌病免疫评估与防控模式研究[J].家畜生态学报,2019,40(10):67-71. LIU W J,HE C W,NING D W,et al.Study on immunization evaluation and control mode of livestock brucellosis in Xinjiang[J].Journal of Domestic Animal Ecology,2019,40(10):67-71.(in Chinese)
[8] 陈月娥,李建华,苟亚峰,等.绵羊和人主要组织相容性复合体DRB1基因外显子2多态性及生物信息学分析[J].中国畜牧兽医,2014,41(4):66-70. CHEN Y E,LI J H,GOU Y F,et al.Polymorphisms and bio-informatics analysis of Ovis aries and Homo sapiens DRB1 genes exon2[J].China Animal Husbandry & Veterinary Medicine,2014,41(4):66-70.(in Chinese)
[9] 徐飞,成述儒,刘秀,等.藏绵羊MHC-DRB1基因第3外显子的PCR-SSCP检测及其序列分析[J].农业生物技术学报,2011,19(5):873-880. XU F,CHENG S R,LIU X,et al.PCR-SSCP detection and DNA sequence analysis of MHC-DRB1 gene exon 3 in Tibetan sheep[J].Journal of Agricultural Biotechnology,2011,19(5):873-880.(in Chinese)
[10] 黄兰,徐伟,封竣淇,等.贵州白山羊DRA和DRB1基因的表达差异分析[J].基因组学与应用生物学,2016,35(8):1931-1934. HUNG L,XU W,FANG J Q,et al.The expression differences analysis of DRA,DRB1 genes about Guizhou White goat[J].Genomics and Applied Biology,2016,35(8):1931-1934.(in Chinese)
[11] 徐飞,成述儒,罗玉柱.绵羊DRB1基因生物信息学分析[J].生物技术通报,2011,1:113-118. XU F,CHENG S R,LUO Y Z.Bioinformatics analysis of sheep DRB1 gene[J].Biotechnolog Bulletin,2011,1:113-118.(in Chinese)
[12] 成述儒,王华,樊庆山,等.甘肃高山细毛羊DRB1基因内含子2微卫星多态性与生长速度的关联分析[J].浙江农业学报,2016,28(12):2021-2027. CHENG S R,WANG H,FAN Q S,et al.Correlation analysis of DRB1 gene intron 2 microsatellite polymorphism and growth rate on Gansu Alpine Merino[J].Acta Agriculturae Zhejiangensis,2016,28(12):2021-2027.(in Chinese)
[13] LI W X,XIA J,ZHOU X,et al.Association of HLA alleles (A,B,DRB1) and HIV-1 infection in the Han population of Hubei,China[J].Journal of Huazhong University of Science and Technology (Medical Sciences),2017,37(1):131-139.
[14] SHAWKATOVÁ I,JAVOR J,PARNICKA Z,et al.Analysis of ICAM1 gene polymorphism in Slovak multiple sclerosis patients[J]. Folia Microbiologica,2017,62(4):287-293.
[15] 王元元,严国,罗成,等.哈萨克羊DRB1基因外显子3多态性及生物信息学分析[J].中国畜牧兽医,2018,45(3):721-729. WANG Y Y,YAN G,LUO C,et al.Polymorphism and bioinformatics analysis of DRB1 gene exon 3 in Kazakh sheep[J]. China Animal Husbandry & Veterinary Medicine, 2018,45(3):721-729.(in Chinese)
[16] 陈月娥.中国美利奴羊MHC-DRB1 exon2单倍型构建及与布鲁氏菌病易感性关联性[D].石河子:石河子大学,2014. CHEN Y E.Haplotype for the MHC-DRB1 gene of Chinese Merino sheep and association with brucellosis susceptibility[D].Shihezi:Shihezi University,2014.(in Chinese)
[17] 王文文,许万云,胡梦薇,等.中国美利奴羊OLA-DQB1基因exon 2遗传多态性与布鲁氏菌病易感性的关联分析[J].中国畜牧兽医,2015,42(6):1495-1503. WANG W W,XU W Y,HU M W,et al The correlation analysis between Chinese Merino sheep OLA-DQB1 gene exon 2 genetic polymorphism and brucellosis susceptibility[J].China Animal Hus-bandry & Veterinary Medicine,2015,42(6):1495-1503.(in Chinese)
[18] 冯华.改良试管凝集试验和虎红平板凝集试验检测羊布鲁氏菌病结果比较[J].中国草食动物科学,2017,37(4):69-70. FENG H.Comparison of improved tube agglutination test and tiger red plate agglutination test in the detection of brucellosis[J].China Herbivore Science,2017,37(4):69-70.(in Chinese)
[19] CHEN Y,XU W,CHEN C,et al.Variations in MHC-DRB1 exon2 and associations with brucellosis susceptibility in Chinese Merino sheep[J].BioRxiv,2016:038539.
[20] BENTZEN A K,HADRUP S R.Evolution of MHC-based technologies used for detection of antigen-responsive T-cells[J].Cancer Immunology,Immuno-therapy,2017,66(5):657-666.
[21] KNITTEL D,GADZINSKI A,HUA S,et al.Heparan sulfates targeting increases MHC class Ⅰ-and MHC class Ⅱ-restricted antigen presentation and CD8⁺ T-cell response[J].Vaccine,2016,34(27):3093-3101.
[22] ALI A O A,STEAR A,FAIRLIE-CLARKE K,et al.The genetic architecture of the MHC class Ⅱ region in British Texel sheep[J].Immunogenetics,2017,69(3):157-163.
[23] SHEN H,HAN G,JIA B,et al.MHC-DRB1/DQB1 gene polymorphism and its association with resistance/susceptibility to cystic echinococcosis in Chinese Merino sheep[J].Journal of Parasitology Research,2014,2014:272601.
[24] LI R Y,HUI W Q,JIA B,et al.The relationship between MHC-DRB1 gene second exon polymorphism and hydatidosis resistance of Chinese Merino (Sinkiang Junken type),Kazakh and Duolang sheep[J].Parasite,2011,18(2):163-169.
[25] GRUSZCZYSKA J,BROKOWSKA K,CHARON K M,et al.Restriction fragment length polymorphism of exon 2 Ovar-DRB1 gene in Polish Heath sheep and Polish Lowland sheep[J].Journal of Applied Genetics,2005,46(3):311-314.
[26] 李洁.甘肃高山细毛羊微卫星亲子鉴定及DRB1基因第3外显子多态性研究[D].兰州:甘肃农业大学,2013. LI J.Paternity test with microsatellite and DRB1 exon 3 polymorphism in Gansu Alpine Merion[D].Lanzhou:Gansu Agricultural University,2013.(in Chinese)