基于Mx启动子和荧光素酶报告基因定量检测鸡Ⅰ型干扰素生物学活性的研究  

Mx Promoter-Luciferase Reporter Gene Assay for the Quantification of Chicken Type Ⅰ Interferon

作　　者：陈伟业[1] 葛金英[2] 肇慧君[2] 胡森[2] 温志远[2] 曹文雁[2] 王喜军[2] 黄克和[1] 步志高[2] 

机构地区：[1]南京农业大学动物医学院,南京210095 [2]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室,哈尔滨150001

出　　处：《畜牧兽医学报》2008年第1期123-128,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家“973”项目(2005CB523200)

摘　　要：为了探索并建立快速、敏感、准确的Ⅰ型干扰素生物学活性定量分析方法,本研究克隆了鸡Mx启动子(Mxp),并在其下游连接荧光素酶(Luciferase,luc)报告基因,构建鸡Ⅰ型干扰素活性检测质粒pMxp-luc。以pMxp-luc瞬时转染鸡胚成纤维细胞系DF-1,24 h后用鸡IFN-α/β处理细胞,结果荧光素酶基因在Mx启动子的作用下获得转录表达;荧光素酶的表达量与干扰素的抗病毒活性有着良好的线性相关性,并在干扰素处理细胞6 h后就可以检测;对鸡IFN-α和IFN-β检测的敏感性分别约为抗病毒活性定量检测方法的10和100倍。该检测系统与传统抗病毒定量检测方法相比,具有快速简便、准确敏感、便于高通量检测等优势,在禽类乃至哺乳动物干扰素生物学基础和应用研究及产业化过程中具有重要应用价值。In this study,the chicken Mx promoter（Mxp）was cloned,and luciferase（luc） gene was inserted into downstream of Mxp.Then pMxpluc was constructed for the bioactivity assay of type Ⅰ IFN and the assay was developed and described as follows.Chicken embryo fibroblast cell line（DF-1） was transfected with pMxp-luc.24 h later,the cells were incubated with 10-fold diluted chicken IFN-α and IFN-β for another 6 h.The expression of luciferase was stimulated by Mxp and had a good linear correlation with the antiviral activity of IFN.This new method was 10-and 100-fold more sensitive than the antiviral assay to quantify chicken IFN-α and IFN-β,respectively.In conclusion,the reporter gene assay described here is more rapid,safe and accurate than antiviral assay,and has potential value for high-flux assay and great value in the fundamental and application study of avian and mammalian type Ⅰ IFN.

关 键 词：鸡Ⅰ型干扰素 生物活性检测 鸡Mx启动子 萤光素酶 

分 类 号：S852.4[农业科学—基础兽医学]