山羊IL-1β IL-8和Mx1因子实时荧光定量检测方法的建立
Chinese Journal of Veterinary Medicine(2019)
Abstract
根据GenBank羊炎性细胞因子(IL-1β和IL-8)和Mx1保守序列,设计特异性引物,利用SYBR Green I染料法建立实时荧光定量检测方法.将羊IL-1β、IL-8和Mx1细胞因子基因保守区域克隆到pMD18-T载体,构建标准质粒.分别以标准质粒为模板建立荧光定量PCR反应的标准曲线、熔解曲线.结果 表明,羊IL-1β、IL-8和Mx1细胞因子实时荧光定量检测方法Ct值与标准品呈良好的线性关系,R2均大于0.990,所有稀释度标准品模板出现特异性熔解峰.应用所建立的方法对山羊痘病毒AV41感染山羊外周血淋巴细胞中IL-1β、IL-8和Mx1 mRNA表达水平进行检测,山羊痘病毒AV41可以刺激机体产生较高的炎性细胞因子.建立的羊IL-1β、IL-8和Mx1细胞因子荧光定量检测方法,可以为山羊痘病毒感染后分子免疫机制研究奠定基础.
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