[1]施开创,李焕荣,杨汉春*,等.猪IL-4、IL-6和IL-10 SYBR Green I real-time PCR检测方法的建立及应用[J].中国预防兽医学报,2010,(02):102-107.
 SHI Kai-chuang,LI Huan-rong,YANG Han-chun*,et al.Development and application of a SYBR Green I-based real-time PCR assay for detection of porcine IL-4, IL-6 and IL-10[J].Chinese journal of preventive veterinary medicine,2010,(02):102-107.
点击复制

猪IL-4、IL-6和IL-10 SYBR Green I real-time PCR检测方法的建立及应用
分享到:

《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2010年02期
页码:
102-107
栏目:
诊断技术
出版日期:
2010-02-01

文章信息/Info

Title:
Development and application of a SYBR Green I-based real-time PCR assay for detection of porcine IL-4, IL-6 and IL-10
文章编号:
1008-0589(2010)02-0102-06
作者:
施开创12李焕荣13杨汉春1*郭 鑫1盖新娜1
1. 中国农业大学 动物医学院/农业部人畜共患病重点开放实验室,北京 100193;
2. 广西动物疫病预防控制中心,广西 南宁 530001;
3. 北京农学院 动物科学技术系,北京 102206
Author(s):
SHI Kai-chuang12 LI Huan-rong13 YANG Han-chun1* GUO Xin1 GE Xin-na1
1. Key Laboratory of Zoonosis of Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China;
2. Guangxi Center for Animal Disease Control and Prevention, Nanning 530001, China;
3. Department of Animal Science and Technology, Beijing University of Agriculture, Beijing 102206, China
关键词:
Th2型细胞因子荧光定量PCR检测方法
Keywords:
porcine Th2-type cytokine real-time PCR detection method
分类号:
S852.65
文献标志码:
A
摘要:
为探讨PRRSV感染后机体的体液免疫应答、从分子水平深入研究PRRSV的免疫机制,本研究针对Th2型细胞因子IL-4、IL-6、IL-10以及看家基因β-actin的基因序列分别设计一对特异性引物,构建含有各自引物扩增序列的重组质粒作为阳性标准品,建立了检测IL-4、IL-6、IL-10及β-actin的SYBR GreenⅠreal-time PCR方法。该方法线性关系好,各种细胞因子及β-actin标准曲线的相关系数均达到0.997以上;敏感性高,初始模板的检出下限均达到1×101 copies/μL;特异性强,扩增产物形成单一的特异性熔解峰;重复性好,组内与组间的变异系数均小于3 %。应用所建立的方法对猪繁殖与呼吸综合征病毒(PRRSV)感染仔猪外周血单个核细胞(PBMC)中IL-4、IL-6和IL-10 mRNA的表达水平进行了检测。结果表明,本研究建立的real-time PCR检测方法灵敏度高、特异性强、重复性好,可以用于猪Th2型细胞因子的检测及定量分析。
  
Abstract:
Real-time PCR assays based on SYBR GreenⅠfor detection of IL-4, IL-6, IL-10 and β-actin were established using primers derived from porcine Th2-type cytokines (IL-4, IL-6 and IL-10) gene. The assays were highly sensitive and had a detection limit of 1×101 copies/μL of initial templates. These assays were highly specific and there was single specific melting peak for every cytokine. It was highly reproducible and had a coefficient of variation less than 3 percent for both intra-and inter-assay. The established assays were successfully used to detect IL-4, IL-6 and IL-10 mRNA expression levels in peripheral blood mononuclear cells (PBMCs) in piglets experimentally infected with porcine reproductive and respiratory syndrome virus (PRRSV). The high sensitivity, specificity and reproducibility of the assays indicated that the SYBR GreenⅠreal-time PCR could be used as an effective tool for detection and quantification of Th2-type cytokines.
  

参考文献/References:

[1] Tian K, Yu X, Zhao T, et al. Emergence of fatal PRRSV variants: unparalleled outbreaks of atypical PRRS in China and molecular dissection of the unique hallmark [J]. PLoS ONE, 2007, 2 (6): 526.
[2] Suradhat S, Thanawongnuwech R. Upregulation of interleukin-10 gene expression in the leukocytes of pigs infected with porcine reproductive and respiratory syndrome virus [J]. J Gen Virol, 2003, 84 (10): 2755-2760.
[3] Feng W H, Tompkins M B, Xu J S, et al. Analysis of constitutive cytokine expression by pigs infected in-utero with porcine reproductive and respiratory syndrome virus [J]. Vet Immunol Immunopathol, 2003, 94(1-2): 35-45.
[4] Díaz I, Darwich L, Pappaterra G, et al. Immune responses of pigs after experimental infection with a European strain of porcine reproductive and respiratory syndrome virus [J]. J Gen Virol, 2005, 86(7): 1943-1951.
[5] Asai T, Mori M, Okada M, et al. Elevated serum haptoglobin in pigs infected with porcine reproductive and respiratory syndrome virus [J]. Vet Immunol Immunopathol, 1999, 70 (1-2): 143-148.
[6] Thanawongnuwech R, Thacker B, Halbur P, et al. Increased production of proinflammatory cytokines following infection with porcine reproductive and respiratory syndrome virus and Mycoplasma hyopneumoniae [J]. Clin Diagn Lab Immunol, 2004, 11 (5): 901-908.
[7] Liu C H, Chuang H C, Chang H L, et al. Expression of Toll-like receptor mRNA and cytokines in pigs infected with porcine reproductive and respiratory syndrome virus [J]. Vet Microbiol, 2009, 136(3-4): 266-276.
[8] 施开创,杨汉春,郭 鑫,等. 猪繁殖与呼吸综合征病毒和猪圆环病毒2型共感染对外周血单个核细胞促炎细胞因子mRNA表达的影响[J]. 畜牧兽医学报,2009,40(3): 363-370.
[9] Johnsen C K, Botner A, Kamstrup S, et al. Cytokine mRNA profiles in bronchoalveolar cells of piglets experimentally infected in utero with porcine reproductive and respiratory syndrome virus: association of sustained expression of IFN-γ and IL-10 after viral clearance [J]. Viral Immunol, 2002, 15(4): 549-556.
[10] Thanawongnuwech R, Thacker E L. Interleukin-10, interleukin-12, and interferon-γlevels in the respiratory tract following Mycoplasma hyopneumoniae and PRRSV infection in pigs [J]. Viral Immunol, 2003, 16(3): 357-367.
[11] 冯志新,姜平. 猪细胞因子SYBR green实时PCR检测方法的建立[J]. 现代生物医学进展,2007,7 (1): 108-111.
[12] 李焕荣,杨汉春,郭 鑫,等.PRRSV和PCV2共感染对猪肺泡巨噬细胞细胞因子IL-10、IL-12p40、IFN-γ mRNA转录的影响[J]. 畜牧兽医学报,2007,38 (4): 382-387.
[13] Kubista M, Andrade J M, Bengtsson M, et al. The real-time polymerase chain reaction [J]. Mol Aspects Med, 2006, 27(2-3): 95-125.
[14] Simpson D A, Feeney S, Boyle C, et al. Retinal VEGF mRNA measured by SYBR green I fluorescence: A versatile approach to quantitative PCR [J]. Mol Vis, 2000, 6: 178-183.
[15] Gao Z Q, Guo X, Yang H C. Genomic characterization of two Chinese isolates of porcine respiratory and reproductive syndrome virus [J]. Arch Virol, 2004, 149 (7): 1341-1351.
[16] Raymond C R, Wilkie B N. Th-1/Th-2 type cytokine profiles of pig T-cells cultured with antigen-treated monocyte-derived dendritic cells [J]. Vaccine, 2004, 22 (8): 1016-1023.
[17] Meier W, Galeota J, Osorio F A, et al. Gradual development of the interferon-gamma response of swine to porcine reproductive and respiratory syndrome virus infection or vaccination [J]. Virology, 2003, 309(1): 18-31.
[18] Diehl S, Rincon M. The two faces of IL-6 on Th1/Th2 differentiation [J]. Mol Immunol, 2002, 39(9): 531-536.
[19] Moore K W, de Waal Malefyt R, Coffman R L, et al. Interleukin-10 and the interleukin-10 receptor [J]. Annu Rev Immunol, 2001, 19: 683-765.
[20] Charerntantanakul W, Platt R, Roth J A. Effects of porcine reproductive and respiratory syndrome virus-infected antigen-presenting cells on T cell activation and antiviral cytokine production [J]. Viral Immunol, 2006, 19(4): 646-661.
[21] Wong G H W, Goeddel D V. Tumour necrosis factors αand β inhibit virus replication and synergize with interferons [J]. Nature, 1986, 323(6091): 819-822.

相似文献/References:

[1]蒋媛媛,郭军庆,颜焰,等.抗猪lgG单克隆抗体的制备与应用[J].中国预防兽医学报,2008,(09):727.
[2]高 明,魏 华*.重组猪IFN-α原核表达及其活性分析 [J].中国预防兽医学报,2009,(11):902.
 GAO Ming,WEI Hua*.Expression of recombinant porcine alpha-interferon in E.coli and the anti-virus activity analysis[J].Chinese journal of preventive veterinary medicine,2009,(02):902.
[3]张莉莉,白 娟,王先炜,等.猪Toll样受体3、7和8基因的克隆与序列分析[J].中国预防兽医学报,2010,(09):727.
 ZHANG Li-li,BAI Juan,WANG Xian-wei,et al.Cloning and sequencing analysis of porcine Toll-like receptor 3, 7 and 8 genes[J].Chinese journal of preventive veterinary medicine,2010,(02):727.
[4]王 新*,黄 山,周 婷,等.猪源耐甲氧西林金黄色葡萄球菌的耐药性及其SCCmec基因分型研究[J].中国预防兽医学报,2010,(12):975.[doi:10.3969/j.issn.1008-0589.2010.12.16]
 WANG Xin*,HUANG Shan,ZHOU Ting,et al.Antimicrobial susceptibility and SCCmec genetyping of methicillin-resistant Staphylococcus aureus isolates from swine[J].Chinese journal of preventive veterinary medicine,2010,(02):975.[doi:10.3969/j.issn.1008-0589.2010.12.16]
[5]汤 君,文心田,曹三杰*,等.猪传染性胸膜肺炎放线杆菌转铁结合蛋白B基因的克隆与表达[J].中国预防兽医学报,2007,(12):943.
 TANG Jun,WEN Xin-tian,CAO San-jie*,et al.Cloning and expression of the tbpB gene of Actinobacillus pleuropneumoniae[J].Chinese journal of preventive veterinary medicine,2007,(02):943.
[6]李永明,罗阿东,徐景峨,等.猪源多杀性巴氏杆菌的生物学鉴定与荚膜PCR分型[J].中国预防兽医学报,2007,(07):506.
 LI Yong-ming,LUO A-dong,XU Jing-e,et al.PCR identification and typing of Pasteurella multocida originated from pigs[J].Chinese journal of preventive veterinary medicine,2007,(02):506.
[7]田国宝,王红宁*,张安云,等.规模化猪场大肠杆菌β-内酰胺类药物耐药性检测及其超广谱β-内酰胺酶调查[J].中国预防兽医学报,2011,(10):776.[doi:10.3969/j.issn.1008-0589.2011.10.06]
 TIAN Guo-bao,WANG Hong-ning*,ZHANG An-yun,et al.Detection of resistance to β-lactams and characterization of extended-spectrum lactamases in Escherichia coli isolates from swine[J].Chinese journal of preventive veterinary medicine,2011,(02):776.[doi:10.3969/j.issn.1008-0589.2011.10.06]
[8]訾占超,夏应菊,韩 雪,等.2009年我国部分猪群输血传播病毒感染情况调查[J].中国预防兽医学报,2011,(10):759.[doi:10.3969/j.issn.1008-0589.2011.10.02]
 ZI Zhan-chao,XIA Ying-ju,HAN Xue,et al.Prevalence of Torque teno virus in China swine herds in 2009[J].Chinese journal of preventive veterinary medicine,2011,(02):759.[doi:10.3969/j.issn.1008-0589.2011.10.02]
[9]曾 博,王红宁*,邹立扣,等.西南地区猪肠外大肠杆菌毒力基因检测及耐药性研究[J].中国预防兽医学报,2013,(02):126.[doi:doi: 10.3969/j.issn.1008-0589.2013.02.11]
 ZENG bo,WANG Hong-ning*,ZOU Li-kou,et al.Virulence gene detection and antibiotic sensitivity of swine extraintestinal Eschericha coli from southwest China[J].Chinese journal of preventive veterinary medicine,2013,(02):126.[doi:doi: 10.3969/j.issn.1008-0589.2013.02.11]
[10]李玉明,王 刚,汪志艳,等.猪源凋亡相关基因SYBR Green I荧光定量RT-PCR方法的建立[J].中国预防兽医学报,2013,(05):369.[doi:10.3969/j.issn.1008-0589.2013.05.07]
 LI Yu-ming,WANG Gang,WANG Zhi-yan,et al.Development of real-time RT-PCR assay based on SYBR Green I for detection of porcine apoptosis-related cytokines[J].Chinese journal of preventive veterinary medicine,2013,(02):369.[doi:10.3969/j.issn.1008-0589.2013.05.07]

备注/Memo

备注/Memo:
收稿日期:2009-07-05
基金项目:国家973计划项目(2005CB523204)
作者简介:施开创(1968-),男,广西隆安人,副研究员,博士,主要从事重大动物疫病诊断与防控技术研究.
更新日期/Last Update: 2010-05-14