三种鸡细胞因子mRNA实时荧光定量RT-PCR检测方法的建立与应用  

Establishment and application of real time fluorescent quantitative RT-PCR method for the detection of three chicken cytokines

作　　者：高铭扬[1] 马春丽[2] 马德星[1] 李广兴[1] 张瑞莉[1] 

机构地区：[1]东北农业大学动物医学学院,黑龙江哈尔滨150030 [2]东北农业大学食品学院,黑龙江哈尔滨150030

出　　处：《中国兽医科学》2013年第2期184-189,共6页Chinese Veterinary Science

基　　金：国家自然科学基金资助项目(30901061);中国博士后科学基金项目(2011M501007);东北农业大学博士启动基金项目(2010RCB38);哈尔滨市青年科技创新基金项目(2012RFQXN003)

摘　　要：为建立鸡细胞因子SYBR GreenⅠ实时荧光定量RT-PCR检测方法,根据GenBank中三种重要的鸡细胞因子即ChIL-12、ChIL-15、ChIL-18的基因序列,以鸡3-磷酸甘油脱氢酶(ChGAPDH)基因为内参,设计特异引物扩增目的基因。将四种基因克隆至pMD18-T载体上,得到各自阳性克隆质粒,以四种阳性质粒为标准品建立标准曲线并进行熔解曲线分析以及灵敏性、特异性和重复性试验。结果显示,当标准品稀释度为1×102～1×1010 copies/μL时,四种基因的Ct值与浓度间具有良好的线性关系,相关系数均大于0.985。熔解曲线分析表明,产物为特异性单峰且重复性较好。应用建立的方法对堆型艾美耳球虫核酸疫苗pcDNA-Ea3-1E免疫的SPF雏鸡脾中ChIL-12mRNA和ChIL-18 mRNA的相对表达量进行检测,免疫组ChIL-12mRNA和ChIL-18mRNA的相对表达量均极显著高于空白对照组(P<0.01)。研究结果为ChIL-12、ChIL-15、ChIL-18的定量分析提供了技术平台。In order to establish the assay of SYBR GreenⅠreal-time fluorescent quantitative RT-PCR for detecting three important chicken cytokines ChIL-12,ChIL-15 and ChIL-18,four pairs of specific primers were designed according to the sequences of ChIL-12,ChIL-15,ChIL-18 and reference gene ChGAPDH from GenBank to amplify the objective gene.The four genes were respectively cloned to the pMD18-T vector.The corresponding plasmids were identified by sequencing,and they were then used as quantitative template to construct the standard curve and to analyze the melting curve,detection sensitivity,specificity and repeatability.The results showed that Ct value of the four gene had good linear relationship（R20.985） with the dilution ranging from 1×102 to 1×1010 copies/μL.The melting curve displayed a single peak and good repeatability.The established assay was used to detect the transcriptional level of ChIL-12 and ChIL-18 mRNA in the spleen of chickens immunized by nucleic acid vaccine pcDNA-Ea3-1E.The results showed that mRNA level of ChIL-12 and ChIL-18 in immunized group were both significantly higher than that in control group（P0.01）.The research provides the platform for quantitative analysis of chicken cytokine ChIL-12,ChIL-15 and ChIL-18.

关 键 词：鸡 细胞因子 SYBR GreenⅠ 实时荧光定量RT-PCR 

分 类 号：S852.42[农业科学—基础兽医学]