苏钟猪TLR4多态性及编码区C1027A功能分析

doi:10.3864/j.issn.0578-1752.2012.06.020

中国农业科学 ›› 2012, Vol. 45 ›› Issue (6): 1206-1214.doi: 10.3864/j.issn.0578-1752.2012.06.020

苏钟猪TLR4多态性及编码区C1027A功能分析

刘筱, 方晓敏, 邹晓龙, 赵芳, 任守文

1.江苏省农业科学院畜牧研究所，南京210014
2.南京农业大学动物科学院，南京210095

收稿日期:2011-03-04 出版日期:2012-03-15 发布日期:2011-07-04
通讯作者: 通信作者任守文，Tel：025-84390349；E-mail：renshouwen@ yahoo.com.cn；通信作者方晓敏，Tel：025-84391941；E-mail：Fxmw2000@yahoo.com.cn
作者简介:刘筱，E-mail：xiao-xiao678@163.com
基金资助:
家生猪产业技术体系（nycytx-009）、江苏省农业科技自主创新基金项目（CX（10）121）、转基因生物新品种培育重大专项（2009ZX08009- 153B）、江苏省农科院学科带头人优秀后备人才项目（6510818）、中波合作项目

Polymorphism of TLR4 and Function Analysis of C1027A in Suzhong Pig

LIU Xiao, FANG Xiao-Min, ZOU Xiao-Long, ZHAO Fang, REN Shou-Wen

1.江苏省农业科学院畜牧研究所，南京210014
2.南京农业大学动物科学院，南京210095

Received:2011-03-04 Online:2012-03-15 Published:2011-07-04

摘要/Abstract

摘要： 【目的】研究苏钟猪TLR4的多态性及其编码区第1 027 bp处突变对TLR4蛋白功能的影响。【方法】采用PCR-SSCP的方法检测TLR4在苏钟猪中的多态性并利用real-time PCR方法检测不同基因型（CC型和AC型）的猪肺泡巨噬细胞经脂多糖（lipopolysaccharide，LPS）诱导后，TLR4及促炎症因子TNF-α、IL-1β表达量的变化情况，探讨该处突变对TLR4识别LPS的影响。【结果】①共检测到3个突变：G962A、C1027A、G960A，其中前两个为有义突变，且C1027A突变可引起编码氨基酸性质的改变；②LPS能快速诱导猪肺泡巨噬细胞中TLR4及促炎症因子TNF-α和IL-1β表达水平上调且TLR4编码区C1027A不同基因型（CC型和AC型）的猪肺泡巨噬细胞（pulmonary alveolar macrophages，PAMs）对LPS的敏感性及反应强度存在显著性差异。【结论】苏钟猪TLR4编码区的序列相对保守、多态含量较低，群体遗传变异程度较小。TLR4编码区C1027A突变能影响TLR4识别LPS的能力，等位基因C为苏钟猪抗革兰阴性菌感染的优势基因。

关键词: 苏钟猪, TLR4, PCR-SSCP, LPS, TNF-&alpha, IL-1&beta

Abstract: As a significant pattern-recognition receptor, toll-like receptor (TLR) is essential for the natural immunity. Its polymorphism has a profound influence on responses to a wide range of pathogens and is associated with resistance and susceptibility to diseases.【Objective】This study speculated on the polymorphisms of TLR4 and function analysis of the mutation (C1027A) in its coding sequence in Suzhong pig. 【Method】 PCR-SSCP was used to search the mutations of TLR4 in 127 samples of Suzhong pigs. The expression levels of TLR4 and proinflammatory cytokines TNF-α and IL-1β were detected by real-time PCR in porcine alveolar macrophages (PAMs) that were induced by LPS. And the PAMs were grouped by their different genotypes (CC and AC) in 1027 bp of coding sequence (CDS) to discuss whether this mutation of TLR4 influenced its sensitivity to LPS. 【Result】 Three SNPs were found, including G962A, C1027A and G960A, and the first two of them were nonsynonymous SNPs and C1027A changed the character of amino acid. LPS stimulation markedly increased TLR4 and proinflammatory cytokines TNF-α and IL-1β mRNA expression. And PAMs had different sensitivities and response to LPS between genotypes CC and AC of C1027A. 【Conclusion】The coding sequence of TLR4 was relatively conservative in Suzhong pig and this population had low polymorphism and genetic variation. The mutation in C1027A can affect the binding ability to LPS. So, allele C in 1027 bp of TLR4 is the resistance allele to Gram-negative bacterial infections in Suzhong pig.

Key words: Suzhong pig, TLR4, PCR-SSCP, LPS, TNF-α, IL-1β

刘筱, 方晓敏, 邹晓龙, 赵芳, 任守文. 苏钟猪TLR4多态性及编码区C1027A功能分析[J]. 中国农业科学, 2012, 45(6): 1206-1214.

LIU Xiao, FANG Xiao-Min, ZOU Xiao-Long, ZHAO Fang, REN Shou-Wen. Polymorphism of TLR4 and Function Analysis of C1027A in Suzhong Pig[J]. Scientia Agricultura Sinica, 2012, 45(6): 1206-1214.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2012.06.020

https://www.chinaagrisci.com/CN/Y2012/V45/I6/1206

参考文献

[1]Akira S. Mammalian toll-like receptors. Current Opinion Immunology, 2003, 15(2): 5-11.

[2]Trinchieri G, Sher A. Cooperation of toll-like receptor signals in innate immune defence. Nature Reviews Immunology, 2007, 7: 179-190.

[3]Akira S, Takeda K, Kaisho T. Toll-like receptors: critical proteins linking innate and acquired immunity. Nature Immunology, 2001, 2: 675-680.

[4]Muneta Y, Uenishi H, Kikuma R, Yoshihara K, Shimoji Y, Yamamoto R, Hamashima N, Yokomizo Y, Mori Y. Porcine TLR2 and TLR6: identification and their involvement in mycoplasma hyopneumoniae infection. Journal Interferon and Cytokine Research, 2003, 23: 583-590.

[5]Yang I A, Barton S J, Rorke S, Cakebread J A, Keith T P, Clough J B, Holgate S T, Holloway J W. Toll-like receptor 4 polymorphism and severity of atopy in asthmatics. Genes and Immunity, 2004, 5: 41-45.

[6]Uenishi H, Shinkai H. Porcine toll-like receptors: the front line of pathogen monitoring and possible implications for disease resistance. Developmental and Comparative Immunology, 2009, 33: 353-361.

[7]Lorenz E, Mira J P, Frees K L, Schwartz D A. Relevance of mutations in the TLR4 receptor in patients with gram-negative septic shock. Archives of Internal Medicine, 2002, 162(9): 1028-1032.

[8]Poltorak A, He X, Smirnova I, Liu M Y, Van Huffel C, Du X, Birdwell D, Alejos E, Silva M, Galanos C, Freudenberg M, Ricciardi- Castagnoli P, Layton B, Beutler B. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science, 1998, 282(5396): 2085-2088.

[9]王伯瑶, 黄宁, 吴琦. 炎症反应toll信号传导通路. 中国病理生理杂志, 2000, 16 (6): 567-571.

Wang B Y, Huang N, Wu Q. Toll receptor signaling pathway in inflammation, Chinese Journal of Pathophysiology, 2000, 16(6): 567-571. (in Chinese)

[10]Shinkai H, Tanaka M, Morozumi T, Eguchi-Ogawa T, Okumura N, Muneta Y, Awata T, Uenishi H. Biased distribution of single nucleotide polymorphisms(SNPs) in porcine toll-like receptor 1(TLR1), TLR2, TLR4, TLR5, and TLR6 genes. Immunogenetics, 2006, 58(4): 324-330.

[11]Basta S, Carrasco C P, Knoetig S M, Rigden R C, Gerber H, Summerfield A, McCullough K C. Porcine alveolar macrophages: poor accessory or effective suppressor cells for T-lymphocytes. Veterinary Immunology and Immunopathology, 2000, 77(3/4): 177-190.

[12]Moue M, Tohno M, Shimazu T, Kido T, Aso H, Saito T, Kitazawa H. Toll-like receptor 4 and cytokine expression involved in functional immune response in an originally established porcine intestinal epitheliocyte cell line. Biochimica et Biophysica Acta, 2008, 1780(2): 134-144.

[13]Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods, 2001, 25: 402-408.

[14]Thomas A V, Broers A D, Vandegaart H F, Desmecht D J. Genomic structure, promoter analysis and expression of the porcine(Sus scrofa) TLR4 gene. Molecular Immunology, 2006, 43(6): 653-659.

[15]Álvarez B, Revilla C, Chamorro S, López-Fraga M, Alonso F, Domínguez J, Ezquerra A. Molecular cloning, characterization and tissue expression of porcine toll-like receptor 4. Developmental and Comparative Immunology, 2006, 30(4): 345-355.

[16]Foster S L, Hargreaves D C, Medzhitov R. Gene-specific control of inflammation by TLR-induced chromatin modifications. Nature, 2007, 447: 972-978.

[17]Koay M A, Gao X, Washington M K, Parman K S, Sadikot R T, Blackwell T S, Christman J W. Macrophages are necessary for maximal nuclear factor-kappa B activation in response to endotoxin. American Journal of Respiratory Cell and Molecular Biology, 2002, 26(5): 572-578.

[18]Matsuguchi T, Musikacharoen T, Ogawa T, Yoshikai Y. Gene expressions of toll-like receptor 2, but not toll-like receptor 4, is induced by LPS and inflammatory cytokines in mouse macrophages. Journal of Immunology, 2000, 165(10): 5767-5772.

[19]盛金良, 陈创夫, 杨霞, 王远志, 张辉. 绵羊toll样受体家族在肺泡巨噬细胞的分布及脂多糖(LPS)刺激对TLR2、TLR4表达的影响. 华北农学报, 2010, 25(1): 30-35.

Sheng J L, Cheng C F. Yang X, Wang Y Z, Zhang H. Distribution of toll-like receptors(TLRs) and TLR2, TLR4 expression during LPS stimulus sheep alveolar macrophage. Acta Agriculturae Boreali-Sinica, 2010, 25(1): 30-35. (in Chinese)

[20]Beutler B. Tlr4: central component of the sole mammalian LPS sensor. Current Opinion in Immunology, 2000, 12(1): 20-26.

[21]Bihl F, Salez L, Beaubier M, Torres D, Larivière L, Laroche L, Benedetto A, Martel D, Lapointe J M, Ryffel B, Malo D. Overexpression of toll-like receptor 4 amplifies the host response to lipopolysaccharide and provides a survival advantage in transgenic mice. Journal of Immunology, 2003, 170: 6141-6150.

[22]Kim G Y, Roh S I, Park S K, Ahn S C, Oh Y H, Lee J D, Park Y M. Alleviation of experimental septic shock in mice by acidic polysaccharide isolated from the medicinal mushroom phellinus linteus. Biological and Pharmaceutical Bulletin, 2003, 26(10): 1418-1423.

苏钟猪TLR4多态性及编码区C1027A功能分析

Polymorphism of TLR4 and Function Analysis of C1027A in Suzhong Pig

PDF

赞

可视化

被引次数

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

Metrics

本文评价

推荐阅读 0

[1]	陈志,张逸,路钦越,郭佳禾,梁艳,张明怡星,杨章平. 茶树油对LPS诱导的奶牛乳腺炎的作用及其机制[J]. 中国农业科学, 2021, 54(14): 3124-3133.
[2]	应诗家，戴子淳，郭佳佳，施振旦. LPS对鹅等级卵泡基质层TLR家族基因表达的影响[J]. 中国农业科学, 2017, 50(6): 1147-1156.
[3]	陈志, 罗卫星, 刘若余, 蔡惠芬, 承杰, 宋桃伟, 张依裕. 贵州山羊品种RERG基因多态性与生长性状的相关性[J]. 中国农业科学, 2013, 46(9): 1915-1922.
[4]	郭凡溪, 刘腾飞, 耿智霞, 蒋凡, 余祖功. 复方甘草酸单胺可溶性粉对恩诺沙星联合LPS 致鸡肝损伤的免疫调节[J]. 中国农业科学, 2013, 46(12): 2576-2583.
[5]	吴井生, 王金玉. FSHR基因第10外显子多态性及其与小梅山猪产仔数的相关性[J]. 中国农业科学, 2012, 45(13): 2728-2736.
[6]	刘重旭1;王凭青1;张宝云1;储明星2;邓腊梅1;谭颖1;樊奇1. 贵州白山羊和古蔺马羊脂联素基因多态性及其与繁殖性能的关联研究[J]. 中国农业科学, 2011, 44(9): 1916-1922.
[7]	王洪梅,张利博,侯明海,王长法,王玲玲,孙涛,何洪彬,仲跻峰 . 牛Nramp1基因启动子的克隆及其活性分析[J]. 中国农业科学, 2011, 44(5): 1022-1028 .
[8]	成述儒, 罗玉柱, 胡江, 王继卿, 刘秀. 藏绵羊DQA1基因多态性分析[J]. 中国农业科学, 2011, 44(10): 2146-2153.
[9]	陈仁金,杨章平,毛永江,陈莹,常玲玲,吴海涛,冀德君,李云龙,张亚琴 . 中国荷斯坦牛LYZ基因多态性及其与乳房炎的关联分析[J]. 中国农业科学, 2010, 43(23): 4936-4941 .
[10]	陈仁金,杨章平,毛永江,陈莹,常玲玲,吴海涛,施雪奎,周里根,王建勇,尹召华,梁祥焕 . 中国荷斯坦牛CXCR1基因遗传多态性与体细胞评分的关联分析[J]. 中国农业科学, 2010, 43(18): 3848-3856 .
[11]	毛永江,陈仁金,陈莹,常玲玲,施雪奎,杨章平,施健,黄回华 . 南方地区中国荷斯坦牛DGAT1基因多态性与泌乳性状的关联分析[J]. 中国农业科学, 2010, 43(14): 2990-2995 .
[12]	张龙,朱方俊,王彦,兰茜,吕荣平,刘益平,朱庆 . 鸡FATP1基因多态性与屠体性状的相关分析[J]. 中国农业科学, 2009, 42(9): 3272-3278 .
[13]	刘秀,胡江,罗玉柱 . 藏绵羊基因OLA-DQA2第2外显子多态性分析[J]. 中国农业科学, 2009, 42(8): 2930-2936 .
[14]	. 孕酮受体基因多态性及其与济宁青山羊产羔数关系[J]. 中国农业科学, 2009, 42(5): 1768-1775 .
[15]	张小白,昝林森,王洪宝,郝瑞杰,杨彦杰 . THRSP基因C184T突变与秦川牛肉质性状相关性的研究[J]. 中国农业科学, 2009, 42(11): 4058-4063 .