长爪沙鼠TLR4基因片段克隆及其在脏器中的表达和分布特征  

Cloning of TLR4 Gene Fragment and Its Expression and Distribution Characteristic in Organs of Mongolian Gerbil(Meriones unguiculatus)

作　　者：朱乐欣[1] 蒋小武[1] 刘月环[2] 罗廉佳 李剑[1] 吴旧生[1] 

机构地区：[1]浙江大学动物科学学院,杭州310058 [2]浙江省医学科学院,杭州310013

出　　处：《农业生物技术学报》2016年第10期1544-1551,共8页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.1502023)

摘　　要：Toll样受体(Toll-like receptors,TLRs)是参与调节先天免疫的一类具有高度保守性的蛋白受体。为克隆长爪沙鼠(Meriones unguiculatus)TLR4基因片段,分析其在内脏组织中的分布特征,本研究通过分析比较大鼠(Rattus norvegicus)、小鼠(Mus musculus)与仓鼠(Mesocricetus auratus)TLR4基因序列,选择保守区设计引物,扩增长爪沙鼠TLR4基因片段;将所得片段连接到克隆载体上,测序并使用DNASTAR Meglign软件分析该序列与其他不同种属动物TLR4基因序列同源性;采用半定量PCR与免疫组织化学技术分析TLR4在正常长爪沙鼠脏器组织中的表达情况。结果显示,本研究成功克隆了长爪沙鼠TLR4基因片段(Gen Bank登录号:KX137123),BLAST分析比对发现,与其他鼠类的TLR4基因同源性在87.4%以上,与仓鼠的同源性较高(89.1%),与人(Homo sapiens)、猪(Sus scrofa)的TLR4同源性较低,分别为69.5%和73.1%,提示长爪沙鼠与仓鼠的亲缘关系较近,与人和猪的亲缘关系较远。半定量PCR分析结果显示,TLR4基因在长爪沙鼠心、肝、脾、肺和肾中呈差异表达,以肺脏中表达量最高,肝脏中表达量最低。免疫组化结果表明,在长爪沙鼠的肺脏和脾脏组织中可见较多TLR4阳性反应产物,在肺脏中阳性反应产物主要见于肺泡支气管上皮细胞;在脾脏中阳性反应产物主要见于脾小结周围的边缘区,淋巴小结内也可见少量分布;肾脏中偶见阳性反应产物分布,心脏和肝脏上均未检出,这与转录水平结果较一致。研究结果为探究长爪沙鼠先天免疫及其作为良好的动物感染模型提供了理论依据。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 m RNA and protein in heart, liver, spleen, lung and kidney of normal MongolianGerbil, respectively. Results demonstrated that TLR4 gene fragment of Mongolian Gerbil（Gen Bank 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） showed69.5% 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 f

关 键 词：长爪沙鼠 Toll样受体4基因(TLR4) 克隆 组织分布 

分 类 号：S852[农业科学—基础兽医学]