基于测序技术的海南地方猪TLR2基因多态性分析
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摘要
为了解海南地方猪TLR2基因的多态性,本研究采用PCR法从五指山猪、临高猪和屯昌猪3种海南地方猪的血液中克隆Toll样受体2(TLR2)基因,并进行测序及序列分析。结果显示,3种海南地方猪TLR2基因的扩增长度均为2 649 bp,编码区长2 358 bp。3种猪TLR2基因序列的种内比对结果显示,五指山猪种内有7个核苷酸位点存在多态性,2处位于编码区;屯昌猪种内有5个核苷酸位点存在多态性,均在编码区外;临高猪种内有4个核苷酸位点存在多态性,1处位于编码区。种间比对结果显示3种猪有12个核苷酸位点存在多态性,其中3处为错义突变,导致氨基酸改变。同源性分析结果显示,3种猪的TLR2基因序列高度保守,同源性在99.6%～99.9%之间。蛋白质结构预测分析显示,海南猪TLR2基因在876、1454位点呈现的AG突变均引起了其蛋白质二级结构和三级结构的改变,提示可能存在TLR2功能的变化。本研究填补了海南地方猪TLR2基因多态性空白,为深入研究TLR2基因多态性与猪疫病易感性的关系提供基础研究资料。
To understand the polymorphism of the TLR2 gene in native Hainan pig breeds, the gene from the blood samples of the Wuzhishan pigs, the Lingao pigs and the Tunchang pigs was cloned by PCR and sequenced, and was analyzed using bioinformatics. The results showed that the TLR2 DNA sequence in native Hainan pig breeds was 2649 bp and its CDS was 2358 bp. The intra-specific alignment of the TLR2 gene sequences of the three pigs showed that there were seven nucleotide polymorphisms in the Wuzhishan pigs(two of which were located in the coding region), five nucleotide polymorphisms in the Tunchang pigs(all of which were outside the coding region) and four nucleotide polymorphisms in the Lingao pigs(of which was located in the coding region). The results of inter-specific comparison showed that there were 12 nucleotide polymorphisms in three pig breeds, three of the polymorphisms were missense mutations resulting in changes in amino acid. The results of homologous analysis showed that the TLR2 gene sequences of the three pig breeds were highly conservative, with homology ranging from 99.6% to 99.9%. Prediction and analysis of protein structure showed that AG mutation at the sites of 876 and 1454 of the TLR2 gene in native Hainan pigs caused changes in the secondary and tertiary structure of the protein, suggesting possible changes in the function of TLR2. This study provided reference for further research on the relationship between polymorphism of the TLR2 gene and the epidemic susceptibility of pigs.
To understand the polymorphism of the TLR2 gene in native Hainan pig breeds, the gene from the blood samples of the Wuzhishan pigs, the Lingao pigs and the Tunchang pigs was cloned by PCR and sequenced, and was analyzed using bioinformatics. The results showed that the TLR2 DNA sequence in native Hainan pig breeds was 2649 bp and its CDS was 2358 bp. The intra-specific alignment of the TLR2 gene sequences of the three pigs showed that there were seven nucleotide polymorphisms in the Wuzhishan pigs(two of which were located in the coding region), five nucleotide polymorphisms in the Tunchang pigs(all of which were outside the coding region) and four nucleotide polymorphisms in the Lingao pigs(of which was located in the coding region). The results of inter-specific comparison showed that there were 12 nucleotide polymorphisms in three pig breeds, three of the polymorphisms were missense mutations resulting in changes in amino acid. The results of homologous analysis showed that the TLR2 gene sequences of the three pig breeds were highly conservative, with homology ranging from 99.6% to 99.9%. Prediction and analysis of protein structure showed that AG mutation at the sites of 876 and 1454 of the TLR2 gene in native Hainan pigs caused changes in the secondary and tertiary structure of the protein, suggesting possible changes in the function of TLR2. This study provided reference for further research on the relationship between polymorphism of the TLR2 gene and the epidemic susceptibility of pigs.
引文
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[15] Muneta Y,Minagawa Y,Kusumoto M,et al.Development of allele-specific primer PCR for a swine TLR2 SNP and comparison of the frequency among several pig breeds of Japan and the Czech Republic[J].J Vet Med Sci,2012,74(5):553-559.
[2] Cao Z,Zheng M,Lv H,et al.Tissue expression of Toll-like receptors 2,3,4 and 7 in swine in response to the Shimen strain of classical swine fever virus[J].Mol Med Rep,2018,17(5):7122-7130.
[3] Nemati M,Larussa T,Khorramdelazad H,et al.Toll-like receptor 2:An important immunomodulatory molecule during Helicobacter pylori infection[J].Life Sci,2017,178:17-29.
[4] Mukherjee S,Karmakar S,Babu S P.TLR2 and TLR4 mediated host immune responses in major infectious diseases:a review[J].Braz J Infect Dis.,2016,20(2):193-204.
[5] Choi J Y,Kim B G.Toll-like Receptor 2:A Novel Therapeutic Target for Ischemic White Matter Injury and Oligodendrocyte Death[J].Exp Neurobiol,2017,26(4):186-194.
[6] Ahmadishoar S,Kariminik A.Toll-like receptor 2 and its roles in immune responses against Legionella pneumophila[J].Life Sci,2017,188:158-162.
[7] Knowlton A A.Paying for the Tolls:the high cost of the innate immune system for the cardiac myocyte[J].Adv Exp Med Biol,2017,1003:17-34.
[8] Darfou-Oduro K A,Megens H J,Roca A,et al.Evidence for adaptation of porcine Toll-like receptors[J].Immunogenetics,2016,68(3):179-189.
[9] Pattabiraman G,Panchal R,Medvedev AE.The R753Q polymorphism in Toll-like receptor 2 (TLR2) attenuates innate immune responses to mycobacteria and impairs MyD88 adapter recruitment to TLR2[J].J Biol Chem,2017,292(25):10685-10695.
[10] Guo Y,Fukud T,Nakamura S,et al.Interaction between Leptospi-ral Lipopolysaccharide and Toll-like Receptor 2 in Pig Fibroblast Cell Line,and Inhibitory Effect of Antibody against Leptospiral Lipopolysaccharide on Interaction[J].Asian-Australas J Anim Sci,2015,28(2):273-279.
[11] Wu S,Huang W,Wang D,et al.Evaluation of TLR2,TLR4,and TOLLIP polymorphisms for their role in tuberculosis susceptibility[J].APMIS,2018,126(6):501-508.
[12] Colombo R,Boccia S,Ricciardi W,et al.TLR2 polymorphism and susceptibility to vulvovaginal colonisation by Candida species[J].J Obstet Gynaecol,2017,37(3):404-405.
[13] 何小兵,房永祥,贾怀杰,等.Toll 样受体 2、4 对病毒囊膜蛋白的识别[J].免疫学杂志,2011,27(10):902-909.
[14] Shinkai H,Suzuki R,Akiba M,et al.Porcine Toll-like receptors:recognition of Salmonella enterica serovar Choleraesuis and influence of polymorphisms[J].Mol Immunol,2011,48(9-10):1114-1120.
[15] Muneta Y,Minagawa Y,Kusumoto M,et al.Development of allele-specific primer PCR for a swine TLR2 SNP and comparison of the frequency among several pig breeds of Japan and the Czech Republic[J].J Vet Med Sci,2012,74(5):553-559.