Abstract：Toll-like receptors (TLRs) are highly conservative protein regulators participating in innate immune response. This test aimed to clone and analyse TLR4 gene fragment and distribution in organs of experimental animal Mongolian Gerbil (Meriones unguiculatus). Conservative region of TLR4 gene which was compared among rat (Rattus norvegicus), mouse (Mus musculus) and hamster (Mesocricetus auratus) was chosen to design the primers, and Mongolian Gerbil TLR4 was cloned. After sequencing, the sequences were further analyzed and compared homology with DNASTAR Meglign software between Mongolian Gerbil and other different species of animals. Semi-quantitative PCR and immunohistochemical assay were used to determine the expression levels of TLR4 mRNA and protein in heart, liver, spleen, lung and kidney of normal Mongolian Gerbil, respectively. Results demonstrated that TLR4 gene fragment of Mongolian Gerbil (GenBank accession No. KX137123) was successfully cloned and showed more than 87.4% homologous with other mouse genus, and the highest for 89.1% with hamster. Homology with human (Homo sapiens) and swine (Sus scrofa) showed 69.5% and 73.1%, respectively. These results showed that the kinship of TLR4 gene might be relatively closer between Mongolian Gerbil and hamster, and farther with human and swine. Semi-quantitative PCR showed that TLR4 differentially expressed in 5 different tissues, and the expression was the highest in lung but the lowest in liver. Immunohistochemical assay further confirmed the semi-quantitative PCR result in protein level. Positive reaction products mainly distributed in lung and spleen in Mongolian Gerbil. High abundance of TLR4 proteins distributed mainly in alveolar bronchial epithelial cells of lung. TLR4 was located mainly in marginal zone around splenic nodule and with little on nodule region of spleen. Sporadic TLR4 proteins could be found in kidney. But no observant reaction products was detected in heart and liver definitely. This study will be a pioneer test for further research on innate immunity of Mongolian Gerbil which performs excellent infection model for some specific pathogens.
[1]陈逢, 刘玉兰, 李权, 等.信号通路关键基因在断奶仔猪不同组织中的表达[J].中国畜牧杂志, 2013, 49(17):53-57
[2]陈富强, 张小丽, 李发弟, 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(3):349-356
[3]侯巍, 岳华, 汤承.荧光定量-检测雏鸡感染强毒后胸腺和法氏囊中和 的表达[J].中国预防兽医学报, 2010, 32(4):254-258
[4]李厚达, 赵继勋, 尹松林, 等.2003. 实验动物学[M].北京:中国农业出版社 pp. 280-282. (Li H D, Zhao J X, Yin S L. 2003. Laboratory Animal Science[M]. Chinese agriculture press, Beijing, China, pp. 280-282.)
[5]李长龙, 杜小燕, 陈振文.长爪沙鼠资源开发利用进展[J].中国实验动物学报, 2014, 22(6):106-109
[6]林宝山, 兰道亮, 黄偲, 等.牦牛和基因 在不同组织中的表达分布研究[J].中国预防兽医学报, 2014, 36(11):901-903
[7]肖丽, 戴方伟, 宋晓明, 等.长爪沙鼠在病原感染研究中的应用[J].中国比较医学杂志, 2013, 23(7):61-67
[8]赵一萍, 黄金龙, 白东义, 等.蒙古马不同组织器官、、和 转录水平研究[J].中国兽医学报, 2012, 32(10):1542-1546
[9]赵文杰, 宋志琦, 程广宇, 等.长爪沙鼠正常泌尿器官的组织学观察[J].中国比较医学杂志, 2014, 24(12):24-26
[10]Akira S, Takeda K, Kasha T.Toll-like receptors: critical proteins linking innate and acquired immunity[J].Nature Immunology, 2001, 2(8):675-680
[11]Asahina, Y, Yoshioka N, Kano R, et al.Full-length coda cloning of Toll-like receptor 4 in dogs and cats[J].Veterinary immunology and immunopathology, 2003, 96(3-4):159-167
[12]Akira S, Takeda K.Toll-like receptor signaling[J].Nature reviews Immunology, 2004, 4(7):499-511
[13]Alvarez B, Revile C, Chamorro S, et al.Molecular cloning,characterization and tissue expression of porcine Toll-like receptor 4[J].Developmental and comparative immunology, 2006, 30(4):345-355
[14]Abbas L, Revolt M N.Aminoglycoside ototoxicity and hair cell ablation in the adult gerbil: A simple model to study hair cell loss and regeneration[J].Hearing research, 2015, 325:12-26
[15]Creagh E M, O'Neill L A.TLRs,NLRs and RLRs: a trinity of pathogen sensors that co-operate in innate immunity[J].Trends in immunology, 2006, 8(27):352-357
[16]Chen C H, Zibiao Z, Ming C, et al.Expression pattern of Toll-like receptors (TLRs) in different organs and effects of lipopolysaccharide on the expression of TLR2 and 4 in reproductive organs of female rabbit[J].Developmental and comparative immunology, 2014, 46(2):341-348
[17]Chrzeszczyk, D, Konopka, T, Zeta, M.Polymorphisms of Toll-Like Receptor 4 as a Risk Factor for Periodontitis: Meta-Analysis[J].Advances in clinical and experimental medicine: official organ Wroclaw Medical University, 2015, 24(6):1059-1070
[18]Cheng, L, Chen, Q, Zhu, W, et al.Toll-Like Receptors 4 and 5 Cooperatively Initiate the Innate Immune Responses to Uropathogenic Ecoli Infection in Mouse Epididymal Epithelial Cells[J].Biology of reproduction, 2016, 94(3):1-22
[19]Derda M A, Wojtkowiak-Giera A, Kolasa-Wolosiuk, et al.Acanthamoeba infection lungs of mice expressed by toll-like receptors (TLR2 and TLR4)[J]., 2016, 165:30-34
[20]Fang X, Liu X, Ming C, et al.Breed-linked polymorphisms of porcine toll-like receptor 2 (TLR2) and TLR4 and the primary investigation on their relationship with prevention against Mycoplasma pneumonia and bacterial LPS challenge[J].Immunogenetics, 2013, 65(11):829-834
[21]Iqbal M, Philbin V J, Smith A L.Expression patterns of chicken Toll-like receptor mRNA in tissues,immune cell subsets and cell lines[J].Veterinary immunology and immunopathology, 2005, 104(1-2):117-127
[22]Lee J C, Won M H.Neuroprotection of antioxidant enzymes against transient global cerebral ischemia in gerbils[J].Anatomy & cell biology, 2005, 47(3):149-156
[23]Isobe H, Nishiyama A, Takano T, et al.Reduction of Overall Helicobacter pylori Colonization Levels in the Stomach of Mongolian Gerbil by Lactobacillus johnsonii La1 (LC1) and Its in Vitro Activities against Hpylori Motility and Adherence[J].Bioscience Biotechnology and Biochemistry, 2012, 76(4):850-852
[24]Kuchinka J, Nowak E, Szczurkowski A, et al.Arteries supplying the base of the brain in the Mongolian gerbil (Meriones unguiculatus)[J].Polish journal of veterinary sciences, 2008, 11(4):295-299
[25]Kawai T, and Akira S.Toll-like receptors and their crosstalk with other innate receptors in infection and immunity[J].Immunity, 2011, 34(5):637-650
[26]Menzies M, Ingham A.Identification and expression of Toll-like receptors 1-10 in selected bovine and ovine tissues[J].Veterinary immunology and immunopathology, 2006, 109(1-2):23-30
[27]Miller, S I, Ernst, R K, Bader, M W.LPS,TLR4 and infectious disease diversity Nature reviews[J].Nature reviews. Microbiology, 2005, 3(1):36-46
[28]Nishimura M, Naito S.Tissue-Specific mRNA Expression Profiles of Human Toll-like Receptors and Related Genes[J].Biological & Pharmaceutical Bulletin, 2005, 28(5):886-892
[29]Schmausser B M, Andrulis S, Endrich H K, et al.Toll-like receptors TLR4,TLR5 and TLR9 on gastric carcinoma cells: an implication for interaction with Helicobacter pylori[J].International journal of medical microbiology, 2005, 295(3):179-185
[30]Shi H, Kokoeva M V, Inouye K, et al.TLR4 links innate immunity and fatty acid-induced insulin resistance[J].The Journal of clinical investigation, 2006, 116(11):3015-3025
[31]Slotwinski R, Slotwinska S, Kedziora S, et al.Innate immunity signaling pathways: links between immunonutrition and responses to sepsis[J].Archivum immunologiae et therapiae experimentalis, 2011, 59(2):139-150
[32]Thirugnanam S, Pandiaraja P, Ramaswamy K, et al.Brugia malayi: comparison of protective immune responses induced by Bm-alt-2 DNA,recombinant Bm-ALT-2 protein and prime-boost vaccine regimens in a jird model[J].Experimental parasitology, 2007, 116(4):483-491
[33]Vahanan B M, Raj G D, Pawar R M, et al.Expression profile of toll like receptors in a range of water buffalo tissues (Bubalus bubalis)[J].Veterinary immunology and immunopathology, 2008, 126(1-2):149-155
[34]Yin Y W, Wang Q, Sun Q Q, et al.Toll-like receptor 4 gene Asp299Gly and Thr399Ile polymorphisms in type 2 diabetes mellitus: a meta-analysis of 15,059 subjects[J].Diabetes research and clinical practice, 2015, 107(3):338-347
[35]Zarember K A, Godowski P J.Tissue expression of human Toll-like receptors and differential regulation of Toll-like receptor mRNAs in leukocytes in response to microbe,their products,and cytokines[J].Nature reviews. Immunology, 2002, 168(2):554-561
[36]Zhang S, Moss S.Rodent models of Helicobacter infection, inflammation, and disease[J].Methods in molecular biology, 2012, 921:89-98
