文献类型：期刊文献

中文题名：巴马香猪Toll样受体4基因cDNA的克隆及生物信息学分析

英文题名：Cloning and Bioinformatics Analysis of TLR4 cDNA in Bama Miniature Pig

作者：巨向红[1];徐汉进[2];雍艳红[1];安立龙[2];效梅[1];许英梅[2]

机构：[1]广东海洋大学动物医学系;[2]广东海洋大学动物科学系

年份：2010

卷号：18

期号：3

起止页码：185

中文期刊名：中国实验动物学报

外文期刊名：Acta Laboratorium Animalis Scientia Sinica

收录：CSTPCD、、CSCD2011_2012、CSCD

基金：广东海洋大学博士启动基金(编号:0712107)

语种：中文

中文关键词：猪;Toll样受体;生物信息学分析

外文关键词：Pig; Toll-like receptor; Bioinformatics analysis

中文摘要：目的研究TLR4在猪自然免疫中的作用及机制,为抗病育种及免疫佐剂的开发提供依据。方法利用NCBI公布的TLR4基因序列设计引物,RT-PCR技术克隆巴马香猪TLR4基因。结果所得基因序列提交GenBank,登录号:GQ304754。经序列分析,发现巴马香猪TLR4基因开放阅读框长2526 bp,编码785个氨基酸,该蛋白等电点为6.58,分子量为96.4×103;与普通猪比对发现巴马香猪TLR4基因有5个碱基发生突变;与小鼠、狗、鸡、牛、羊和人的同源性分别为71.9%、81.5%、54.2%、86.4%、85.5%和81.9%;TLR4膜外区蛋白为背侧多个α螺旋和内侧多个β折叠平行交替排列构成一个弯曲状螺旋结构;N末端存在信号肽,且可能在23～24位氨基酸处存在裂解位点;胞外区有13个明显的LRR,分别位于第53～74、77～100、101～124、149～173、174～192、201～225、372～393、398～429、446～469、470～494、495～518、519～541、543～566位氨基酸区;膜外区含8个N连接的糖基化位点。结论本研究成功克隆巴马香猪TLR4基因,为进一步研究该基因的功能和蛋白质的特性奠定了基础。

外文摘要：Objective Bama Miniature pig is originated in the Chengguan area of Bama County and Yiwei area of Dongtian County in Guangxi Zhuang Autonomous Region of China.The form of this breed is the results of long time inbreeding,so the heredity and production performance is very stabilized,and are used as an experiment animal model normally.Toll-like receptor 4（TLR4） is an important member of Toll-like receptor family,it mainly responses to lipopolysaccharide（LPS） and plays a vital role in the host immune system.In order to explore the molecular mechanism of TLR4 participating in immunosuppression induced by heat stress in Bama Miniature Pigs.The present study is conducted to clone the TLR4 gene cDNA and make bioinformatics analysis of the protein after translated into amino acids.Methods Total 12 bama miniature pigs aged approximately 3-4 months were used in this study.One milliliter blood was collected into vacutainers by anterior vena cava puncture.Lymphocytes were isolated from 1 mL of whole blood by density gradient centrifugation using LTS-1077 lymphocyte separation solution.TLR4 gene was cloned from peripheral blood mononuclear cells by reverse transcription polymerase chain reaction（RT-PCR）.The Identical alignment of the amino acid sequence was compared with other animals by DNAman software.Homology modeling to prediction the three-dimensional structure models of TLR4 extracellular region was conducted by ESyPred3D Web Server 1.0 software.The N-terminal signal peptide structure was predicted by Signal P3.0 software.The transmembrane region structure was predicted by TMHMM Server v.2.0 software.The conserved domain region was predicted by SMART service software and the N-glycosylation site by NetGlyc1.0 Server software.Results The results showed that Toll-like receptor 4 gene consisted of 2358 base pairs.The gene sequence was submitted to GenBank（gi： GQ304754）.The TLR4 gene coded 785 amino acids.The molecular weight of TLR4 protein was 96.4 kD and the isoelectric point was 6.58.This protein had a transmembrane region（589-611） and a signal peptide in N-terminal,and might be a schizolysis site in 23-24 amino acids.5 bases of TLR4 gene in Bama miniature pig were mutated compared with that of common pig,but the structure did not change significantly.The Bama miniature pig TLR4 gene sequence had a high homology compared with that of mice（73.4%）,dog（82.4%）,chicken（59.5%）,cattle（85.3%）,sheep（84.6%） and man（82.4%）.The structure of extracellular region of TLR4 protein showed a forniciform helix structure,and consisted of a lot of α-helix in inside and β-sheet in outside of the arc and they arranged parallely and alternately.There were thirteen leucine-rich repeats located at 53-74,77-100,101-124,149-173,174-192,201-225,372-393,398-429,446-469,470-494,495-518,519-541 and 543-566 amino acid site,respectively,and there were also 8 N-linked glycosylation sites out of the membrane region.Conclusion TLR4 gene of Bama miniature pigs has been successfully cloned and the possible structure and function are pretested by bioinformatic analysis in the present study.