中国美利奴羊TLR2基因多态性及其与布鲁氏菌病的相关性分析

doi:10.16431/j.cnki.1671-7236.2018.05.003

摘要/Abstract

摘要：

试验旨在研究Toll样受体2（Toll-like receptor，TLR2）基因的多态性及其与中国美利奴羊布鲁氏菌病易感性的相关性。利用生物信息学方法对NCBI上公布的绵羊TLR2基因序列进行比对，选出多态位点丰富的片段进行扩增，运用PCR-SSCP的方法对206个中国美利奴布鲁氏菌病阴性样本和80个中国美利奴羊布鲁氏菌病阳性样本进行TLR2基因的多态性检测，然后对不同等位基因的PCR产物进行测序，确定该基因的多态性位点，经卡方检验分析每个SNP位点的等位基因频率、基因型频率及其多态性与布鲁氏菌病易感性的相关性，利用生物信息学软件分析RNA二级结构及蛋白质的二级结构。结果表明，在279 bp的序列中共检测到3个SNPs，分别为：C1731T、G1737C和G1749T，均未引起对应氨基酸的改变，属于无义突变。这些位点在病例组和对照组之间的等位基因频率及基因型频率均不存在显著差异（P>0.05）。各突变位点均能引起RNA二级结构和最小自由能的改变，而蛋白质的二级结构均未改变。由此得出，中国美利奴羊TLR2基因的3个SNPs位点（C1731T、G1737C和G1749T）与中国美利奴羊布鲁氏菌病易感性无相关性。

关键词: 中国美利奴羊; TLR2基因; PCR-SSCP; 多态性; 布鲁氏菌病

Abstract:

This experiment was aimed to study the correlation between Toll-like receptor 2 (TLR2) gene polymorphisms and susceptibility to brucellosis of China Merino sheep.Bioinformatics methods were used for comparing the TLR2 gene sequences of sheep which were published on NCBI,and selected abundant fragments of polymorphic loci for amplification.The method of PCR-SSCP was used to detect the polymorphism of TLR2 gene from 206 brucellosis negative samples and 80 brucellosis positive samples of Chinese Merino sheep,the PCR products of different alleles were sequenced to determine the polymorphic loci of TLR2 gene.The correlation between allele frequency,genotype frequency and polymorphism of each SNP and susceptibility to brucellosis were analyzed by Chi square test.Bioinformatics analysis software was used to analyze the secondary structures of RNA and protein.The results showed that 3 SNPs (C1731T,G1737C and G1749T) were detected in the sequence of 279 bp,which didn't lead to the change of corresponding amino acids,and belonged to nonsense mutation.There was no significant difference in allele frequency and genotype frequency among the cases and controls (P>0.05).The secondary structures and the minimum free energy of RNA were changed at each mutation site,but the secondary structures of protein was not changed.In conclution,there was no correlation between 3 SNPs (C1731T,G1737C and G1749T) of TLR2 gene and brucellosis susceptibility of Chinese Merino sheep.

Key words: China Merino sheep; TLR2 gene; PCR-SSCP; polymorphism; brucellosis

中图分类号:

Q813.3

罗成, 王元元, 王会敏, 谭君, 周光普, 高剑峰. 中国美利奴羊TLR2基因多态性及其与布鲁氏菌病的相关性分析[J]. 《中国畜牧兽医》, 2018, 45(5): 1145-1152.

LUO Cheng, WANG Yuanyuan, WANG Huimin, TAN Jun, ZHOU Guangpu, GAO Jianfeng. Polymorphism of TLR2 Gene in Chinese Merino Sheep and Its Correlation Analysis with Brucellosis[J]. , 2018, 45(5): 1145-1152.

参考文献

[1] 古丽努尔·马哈提,玛尔孜亚·亚森,努力古丽·塔力甫汗,等.中国美利奴羊周岁母羊羊毛纤维直径及其变异分析[J].草食家畜,2017,2:27-31. GURINUR M,MARZIYA Y,NULIGURI T,et al.The diameter and variation analysis of Chinese Merino wool yearling ewe[J].Grass-feeding Livestock,2017,2:27-31.(in Chinese)
[2] 王科.新疆发展现代畜牧业的优势及影响因素分析[J].饲料工业,2015,36(15):60-64. WANG K.Research on developing modern animal husbandry of the advantages and influence factors in Xinjiang[J].Feed Industry, 2015,36(15):60-64.(in Chinese)
[3] GAROFOLO G, GIANNATALE E D, PLATONE I,et al.Origins and global context of Brucella abortus in Italy[J].BMC Microbiology,2017,17(1):2-7.
[4] IMRAN H,TUMER G,YUKSEL M,et al.Brucellosis as an aetiology of septic arthritis[J].Tropical Doctor,2014,44(1):48-49.
[5] SOHRAB N,ABDOLREZA K,MANOOCHEHR R,et al.Association of interleukin-2 gene variants (positions +114 and -384) and susceptibility to brucellosis in Iranian population[J].AIMS Molecular Science,2017,4(1):103-109.
[6] DING J,ZHU L,PENG Y,et al.Rough Brucella strain RM57 is attenuated and confers protection against Brucella melitensis[J].Microbial Pathogenesis,2017,3(107):270-275.
[7] MEDZHITOV R,PRESTON H P,JANEWAY C.A human homologue of the Drosophila Toll protein signals activation of adaptive immunity[J].Nature,1997,388(6640):394-397.
[8] ZINNGREBE J,WALCZAK H.TLRs go linear-on the ubiquitin edge[J].Trends in Molecular Medicine,2017,23(4):296-309.
[9] YU S Y,NIE Y C,KNOWLES B,et al.TLR sorting by Rab11 endosomes maintains intestinal epithelial-microbial homeostasis[J].EMBO JOURNAL,2014,33(17):1882-1895.
[10] KORNRRV K V,ATRETKHANY Y S,DRUTSKAYA M S,et al.TLR-signaling and proinflammatory cytokines as drivers of tumorigenesis[J].Cytokine,2017,89:127-135.
[11] SIOROWSSKI K,BROKOS B,ECHEVERRIA V,et al.RAGE-TLR crosstalk sustains chronic inflammation in neurodegeneration[J].Molecular Neurobiology,2017,Epub ahead of print.
[12] BRENCICOVA E,JAGGER A L,EVANS H G,et al.Interleukin-10 and prostaglandin E2 have complementary but distinct suppressive effects on Toll-like receptor-mediated dendritic cell activation in ovarian carcinoma[J].PLoS One,2017,12(4):1-24.
[13] TÖRÖK H P,BELLON V,KONRAD A,et al.Functional Toll-like receptor (TLR)2 polymorphisms in the susceptibility to inflammatory bowel disease[J].PLoS One,2017,12(4):1-14.
[14] WU H C,JIANG K F,ZHANG T,et al.Hydroxytyrosol exerts an anti-inflammatory effect by suppressing Toll-like receptor 2 and TLR2 downstream pathways in Staphylococcus aureus-induced mastitis in mice[J].Journal of Functional Foods,2017,6(35):595-604.
[15] ELSHABRAWY H A,ESSANI A E,SZEKANECZ Z,et al.TLRs,future potential therapeutic targets for RA[J].Autoimmunity Reviews,2017,16(2):103-113.
[16] 盛金良,陈创夫,杨霞,等.绵羊Toll样受体家族在肺泡巨噬细胞的分布及脂多糖(LPS)刺激对TLR2、TLR4表达的影响[J].华北农学报,2010,25(1):30-35. SHENG J L,CHEN C F,YANG X,et al.Distribution of sheep Toll like receptor family in alveolar macrophages and the effect of lipopolysaccharide (LPS) stimulation on the expression of TLR2 and TLR4[J].Acta Agriculturae Boreali-Sinic,2010,25(1):30-35.(in Chinese)
[17] 杜润慈,刘建华,冉多良,等.TLRs介导的信号通路在马链球菌感染RAW264.7细胞中的作用[J].中国畜牧兽医,2016,43(5):1270-1276. DU R C,LIU J H,RAN D L,et al.Effect of TLRs medinated signal pathway on Streptococcus equi infection in RAW264.7 cells[J].China Animal Husbandry & Veterinary Medicine,2016,43(5):1270-1276.(in Chinese)
[18] LAURA C,VANCRAEYNESTE H,DIDIER L R,et al.Role of TLR1,TLR2 and TLR6 in the modulation of intestinal inflammation and Candida albicans elimination[J].Gut Pathogens,2017,9(1):9.
[19] ABDELHABIB S,NARASIMHA R P,ABDULLAH A A,et al.Association between TLR-9 polymorphisms and colon cancer susceptibility in Saudi Arabian female patients[J].Onco Targets and Therapy,2017,10:1-11.
[20] GAO G J,XU J.Important biology events and pathways in Brucella infection and implicateons for novel antibiotic drug targets[J].Critical Reviews in Eukaryotic Gene Expression,2013,23(1):65-76.
[21] ANKITAL J,MAYANK R,CHAKARAVARTI S,et al.Brucella phage lysate bacterin induces elevated TLRs and cytokines response in murine model[J].Journal of Pure & Applied Microbiology,2016,10(3):2063-2070.
[22] 张欣,张小丽,宋晓育,等.甘肃高山细毛羊TLR2基因遗传多态性及其与乳房炎相关性[J].江苏农业学报,2015,1(6):1357-1361. ZHANG X,ZHANG X L,SUN X Y,et al.Genetic polymorphism of TLR2 gene and its correlation with mastitis in Gansu fine-wool sheep[J].Jiangsu Journal of Agrricultural Sciences,2015,1(6):1357-1361.(in Chinese)
[23] 曹智.新型表示模式下的DNA序列和RNA二级结构分析方法研究[D].长沙:湖南大学,2010. CAO Z.The research on analysis methods of DNA sequences and RNA secondary structures on representation models[D].Changsha:Hunan University,2010.(in Chinese)