Abstract The Toll like receptor family (TLRs) is an important type of pattern recognition receptor (PRRs), which has been widely studied in recent years. TLRs activates cascade reaction in the process of signaling pathway by identifying the invasion of pathogenic microorganisms, inducing tissue or organism to produce cytokines and inflammatory factors, which plays an extremely important role in the host against pathogen infection. The purpose of this study was to explore the correlation between the overexpression of goat (Capra hircus) TLR4 gene in mouse macrophages and the expression of TLR2 gene. In this experiment, the whole length of CDS of goat TLR4 gene was cloned by reverse transcription PCR (RT-PCR) amplification combined with T cloning method. The pEGFP-N1-TLR4 eukaryotic expression vector was transiently transfected to mouse macrophages (Ana-1) with lipofectamine3000TM after identification the eukaryotic expression vector by connection, transformation and double enzyme digestion. The total RNA was extracted after transfection, and TLR4 and TLR2 gene mRNA in transfected cell Ana-1 were detected by qRT-PCR and the differential expression was analyzed by the software of SPSS 18.0. Results showed that the TLR4 gene CDS region was 2 537 bp, 3 mutation sites (T426C, C1254T, G1515A) in the CDS region of TLR4 gene were found, and 3 mutations did not cause changes in amino acids, which belonged to synonymous mutations. The eukaryotic expression vector of pEGFP-N1-TLR4 were successfully transfected and expressed in the mouse macrophages, the qRT-PCR test showed that the expression of TLR4 and TLR2 was positive group＞negative group＞control group (blank), in control group, there was no significant difference in mRNA expression between TLR4 and TLR2 gene (P＞0.05), while mRNA expression in TLR4 positive group was significantly higher than that in TLR2 positive group (0.01＜P＜0.05). The expression level of TLR2 gene mRNA in the positive group was also significantly higher than that in the blank group (0.01＜P＜0.05). The results showed that the overexpression of TLR4 could promote the TLR2 gene. This study provide a basis for further study of the interaction between TLR4 and TLR2 genes and their intracellular expression mechanism.
XU Wei,LUO Wei-Xing,FENG Jun-Qi, et al. Expression of Goat (Capra hircus) TLR4 Gene in Mouse (Mus musculus) Macrophages and Its Correlation with the Expression of TLR2 Gene[J]. , 2018, 26(11): 1900-1908.
[1]陈富强，张小丽，李发等.基因在小尾寒羊乳房炎乳腺组织中的表达[J].农业生物技术学报, 2016, 24(03):349-356 [2]翟春媛.2010. 猪TLR4基因的突变与功能分析[D]. 东北农业大学，导师：刘娣，pp.6-7.（Di C Y. 2010. The Mutation of TLR4 Gene in Pig and Function Analysis[D]. Northeast Agricultural University. Supervisor: Liu D, pp.6-7.） [3]吕伟丽.2014.甘肃地区小尾寒羊TLR4、TLR9基因多态性与呼吸系统疾病的相关性分析[D].甘肃农业大学, 导师:马小军, pp.25-29.（Lv W L. 2014. The Correlation Research between Polymorphism of Toll-like Receptor 4 Gene and Respiratory System Disease[D]. Gansu Agricultural University, Supervisor: Ma X J, pp25-29.） [4]马腾壑，许尚忠，王兴平，等.奶牛基因遗传变异与乳房炎体细胞评分的相关研究[J].畜牧兽医学报, 2007, 38(04):332-336 [5]王会敏.2017.中国美利奴羊TLR4、TLR2基因多态性及其与布鲁氏菌病的相关性分析[D].石河子大学, 导师:高剑峰, pp.28-31.（Wang H M.2017. Analysis of correlation between the Polymorphisms of TLR4、TLR2 genes with brucellosis in Chinese Merino sheep[D]. Shihezi University, Supervisor: Gao J F, pp28-31.） [6]于鑫, 王月秋, 李明恒等.Toll受体4在脂多糖诱导人牙周膜成纤维细胞表达细胞核因子-κB受体活化因子配基中的作用.[J[ 325-328[J].华西口腔医学杂志, 2012, 30(03):325-328 [7]钟晓.2017.TLR4基因多态性与大动脉粥样硬化型脑梗死的相关性研究[D]. 青岛大学，导师: 孙丽，pp.17-18.(Zhong X. 2017. Association between TLR4 gene polymorphisms and the Large-artery Atherosclerosis Stroke[D].Qiingdao University. Supervisor:Sun L, pp.17-18. [8]Albiger B, Dhalberg S, Henriques- normark B, et al.Role of the innate immune system inhost defence against bacterical infectious: Focus on the Toll- like receptors[J].Journal of Internal Medicine, 2007, 261(06):511-528 [9]Candia L, Marquez J, Hernandez C, et al.Toll-like receptor-2 expression is upregulated in antigen-presenting cells from patients with psoriatic arthritis: a pathogenic role for innate immunity[J].JRheumatol, 2007, 34(2):374-379 [10]Goldammer T, Zerbe H, M olenaar A, et al.Masitfs increases mammary m RNA abundance of 3-defensin 5,Toll-like receptor 2 (TLR2),and TLR4 but not TLR9 in cattle[J].Clinical and Diagnostic Laboratory Immunology, 2004, 11(1):174-185 [11]Hashimoto C, Hudson K L, Anderson K V.The Toll gene of Drosophila,required for dorsalventral embryonic polarity,appears to encode a transmembrane protein1988,52:269-279.[J].Cell, 1988, 52(02):269-279 [12]Lien E, Ingalls R R.Toll-like receptors[J].Critical care medicine, 2002, 30(1):S1-S11 [13]Medzihtov R, PrestonHurlburt P, Janeway CAJr.A human homologue of the DrosoPihla Toll Proetin signal saetivation of adaPtive immunity,388:394-397.[J].Nature, 1999, 388(6640):394-397 [14]Monica Molteni, Sabrina Gemma, and Carlo Rossetti.The Role of Toll-Like Receptor 4 in Infectious and Noninfectious Inflammation[J].Mediators of Inflammation, 2016, 2016(07):1-9 [15]Proenca M, Juliana Oliveira G, Cristina A, et al.TLR2 and TLR4 polymorphisms influence mRNA and protein expression in colorectal cancer., 2015 Jul 7; 21(25):30–7741.[J].world J.Gastroenterol, 2015, 21(25):7730-7741 [16]Sarah S M, Tamilselvan S, Kamatchiammal S, et al.Expression of Toll-like receptors 2 and 4 in gingivitis and chronic periodontitis[J].Indian J Dent Res, 2006, 17(3):114-116 [17]Takeda K, Kaisho T, Akira S.Toll-like receptors 2003, 335-376[J].Annu Rev Immuno1, 2003, 21(1):335-376 [18]West XZ, Malinin NL, Merkulova AA, Tischenko M, et al.Oxidative stress induce angiogenesis by activating TLR2 with novel endogenous ligands.[J].Nature, 2010, 467(7318):972-976 [19]Yu L Wang L., Chen S. .Endogenous toll-like receptor ligands and their biological significance. [J].Journal of Cellular and Molecular Medicine., 2010, 14(11):2592-2603 [20]Zhong Jia, Colincino MS, Lin Xinyi, et al.Cardiac Autonomic Dysfunction: Particulate Air Pollution Effects Are Modulated by Epigenetic Immunoregulation of Toll-like Receptor 2 and Dietary Flavonoid Intake.[J].Journal of the American Heart Association, 2015, 4(1):1420-1423 [21]Zhang Z, Bashiruddin J B, Doel C, et al.Cytokine and Toll-like receptor m RNAs in the nasal-associated lymphoid tissues of cattle during foot-and-mouth disease virus infection[J].J Comp Pathol, 2006, 134(1):56-62
