鸡β-防御素-1 SYBR GreenⅠ实时荧光定量PCR检测方法的建立

中国畜牧兽医

鸡β-防御素-1 SYBR GreenⅠ实时荧光定量PCR检测方法的建立

李岩，王元心，隋欣，郑世民

（东北农业大学动物医学学院，黑龙江哈尔滨 150030）

收稿日期:2013-11-25 出版日期:2014-06-20 发布日期:2014-07-24
通讯作者: 郑世民（1959—），男，甘肃人，博士生导师，研究方向：畜禽免疫病理学。E-mail:zhengshiminbl@sohu.com；Tel: 0451-55190405
作者简介:李岩（1988—），男，吉林人，硕士，研究方向：畜禽免疫病理学。
基金资助:
国家自然基金（30972162）。

Establishment of SYBR Green Ⅰ Real-time Fluorescent Quantitation PCR for Detection of Gallinacin-1

LI Yan, WANG Yuan-xin, SUI Xin, ZHENG Shi-min

(College of Veterinary Medicine，Northeast Agricultural University，Harbin 150030，China)

Received:2013-11-25 Online:2014-06-20 Published:2014-07-24

摘要/Abstract

摘要： 以鸡β-防御素-1 (Gallinacin-1, Gal-1)为研究对象，据GenBank中已发表的鸡β-actin和Gal-1序列设计引物，构建重组质粒作为标准品，成功建立了检测鸡免疫器官组织中Gal-1 mRNA表达的实时荧光定量PCR方法，并进行灵敏度、重复性、特异性试验。结果显示，此方法具有快速、高通量、线性范围广、特异性强、灵敏度高等特点，为进一步定量研究Gal-1 mRNA表达及其与机体免疫机能的关系奠定了技术基础。

关键词:

鸡; Galmso-bidi-font-family: 宋体" lang="EN-US">-1基因; 实时荧光定量PCR

Abstract: The chicken beta-defensin-1 (Gallinacin-1, Gal-1) was regarded as the research object. According to the chicken β-actin and Gal-1 genes sequences published in GenBank, the recombinant plasmids were constructed for the standard, the study successfully established Real-time fluorescence quantitative PCR method for detecting the expression of Gal-1 mRNA in chicken, and tested the sensitivity, repetitiveness and specificity. The results showed that this method was characterized by rapidity, high-flux, wide linear range, good specificity, high sensitivity， and so on. So this method would provide the basis for making further efforts for quantitative research on the expression of Gal-1 mRNA and laying technical foundations for research on the related diseases.

Key words:

chicken；Galmso-bidi-font-family: 宋体" lang="EN-US">-1 gene；Realmso-bidi-font-family: 宋体" lang="EN-US">-time fluorescence quantitative PCR

李岩，王元心，隋欣，郑世民. 鸡β-防御素-1 SYBR GreenⅠ实时荧光定量PCR检测方法的建立[J]. 中国畜牧兽医.

LI Yan, WANG Yuan-xin, SUI Xin, ZHENG Shi-min. Establishment of SYBR Green Ⅰ Real-time Fluorescent Quantitation PCR for Detection of Gallinacin-1[J]. .

参考文献

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13 Xiao Y J, Hughes A L, Ando J, et al. A genome-wide screen identifies a single β-defensin gene cluster in the chicken: Implications for the origin and evolution of mammalian defensins［J］. BMC Genomics，2004a，5(1): 56～67.
14 Xiao L Q，Liu Z H，Zhang Y L. An effective method for raising antisera against β-defensins：Double-copy protein expression of mBinlb in E.coli［J］．Acta Biochim Biophys Sin(Shanghai)，2004b，36(8)： 571～576.
1 王璟，杨玉荣，梁宏德，等．禽类防御素作用机理及其基因工程研究进展［J］．动物医学进展，2007，28(10): 81～84.
2 刘贤华，白晓东，粟永萍．防御素的多向生物学活性特征:基因工程研究现状［J］．中国临床康复，2005，9(15)： 145～147.
3 钟江华，张光萍，柳小英. 实时荧光定量PCR技术的研究进展与应用［J］. 氨基酸和生物资源，2011，33(2)： 68～72.
4 Glulietti A，Overbergh L，Valckx D，et al．An overview of real-time quantitative PCR：Applications to quantify cytokine gene expression［J］. Methods，2011，25(4)： 386～401.
5 Harwig S S，Swiderekb K M，Kokryakova V N, et al.Gallinacins: Cysteine-rich antimicrobial peptides of chicken leukocytes［J］. FEBS Lett, 1994, 342(3): 281～285.
6 Haryadi S, Pak-Lam Y. Mechanisms of action of ostrich β-defensins against Escherichia coli［J］. FEMS Microbiology Letters，2007，270(2): 195～200.
7 Lehrer R I, Ganz T, Szklarek D, et al．Modulation of the in vitro candidacidal activity of human neutrophil defensins by target cell metabolism and divalent cations［J］. Clin Invest，1988，81：1829～1835.
8 Lynn D J, Higgs R, Gaines S, et al. Bioinformatic discovery and initial characterization of nine novel antimicrobial peptide genes in the chicken［J］.Immunogenetics，2004，56(3): 170～177.
9 Ma D Y, Lin L J, Zhang K X, et al. Three novel Anas platyrhynchos avian-defensins, upregulated by duck hepatitis virus, with antibacterial and antiviral activities［J］. Molecular Immunology，2011，3776: 1～13.
10 Ma D Y, Zhou C Y, Zhang M Y, et al. Functional analysis and induction of four novel goose (Anser cygnoides) avian β-defensins in response to Salmonella enteritidis infection［J］. Comparative Immunology, Microbiology and Infectious Diseases，2012，35(2): 197～207.
11 Sugiarto H，Yu P L. Avian antimicrobial peptides：The defense role of beta-defensins［J］. Biochem Biophys Res Commun，2004，323(3)： 721～727.
12 Van Dijk A，Veldhuizen E J，Haagsman H P. Avian defensins［J］. Veterinary Immunology and Immunopathology，2008，124 (1～2)： 1～18.
13 Xiao Y J, Hughes A L, Ando J, et al. A genome-wide screen identifies a single β-defensin gene cluster in the chicken: Implications for the origin and evolution of mammalian defensins［J］. BMC Genomics，2004a，5(1): 56～67.
14 Xiao L Q，Liu Z H，Zhang Y L. An effective method for raising antisera against β-defensins：Double-copy protein expression of mBinlb in E.coli［J］．Acta Biochim Biophys Sin(Shanghai)，2004b，36(8)： 571～576.