新疆多浪羊IL-1β基因地高辛探针的标记和敏感性检测  

IL-1β Probes Labeled with Digoxgenin of Duolang Sheep in Xinjiang and Detection

作　　者：曾国航 徐宏伟[1] 潘伊微[1] 贾山岭[1] 曹玉华[1] 李莲瑞[2,3] 

机构地区：[1]塔里木大学生命科学学院,新疆阿拉尔843300 [2]新疆兵团塔里木畜牧科技重点实验室,新疆阿拉尔843300 [3]塔里木大学动物科学学院,新疆阿拉尔843300

出　　处：《新疆农业科学》2015年第3期560-565,共6页Xinjiang Agricultural Sciences

基　　金：国家自然科学基金项目"棉酚对新疆多浪羊白细胞介素表达影响研究"(30960277)

摘　　要：【目的】IL-1β是一种多效的促炎因子,在细胞因子炎性反应中为重要介质,处于介导炎性反应的重要因子,在造血和免疫活性中都发挥重要的作用。该实验结果为进一步研究绵羊分子免疫学,从多浪羊外周血淋巴细胞c DNA文库的筛选基因,寻找与IL-1β相关的新基因奠定基础。【方法】将构建好的多浪羊的p MD-18T-IL-1β质粒[1]进行PCR扩增,经过切胶回收,目的基因的浓缩后,采用罗氏地高辛标记和检测试剂盒标记IL-1β基因,获得IL-1β地高辛探针,进行敏感性检测,在Hybond N+膜上显影,计算探针浓度。【结果】计算出标记后的探针浓度为17.1 g/m L。【结论】通过southern杂交可以得知,Hybond N+膜上有IL-1β基因的双链DNA,标准杂交液中为DIG标记的IL-1β基因单链DNA,通过避光显色,在Hybond N+膜有深紫色的杂交条带,证明探针的浓度达到标准浓度。【Objective】IL-1β is a pleiotropic pro-inflammatory cytokines,and is important factor leading to inflammatory cytokines,and also in hematopoietic and immune activity plays an important role. This study aims at laying a foundation for not only for further study of molecular immunology in sheep,but also providing a guarantee for c DNA library of peripheral blood lymphocyte cell in the hope of researching relative new genes of IL-1β.【Method】We carried out the experiment by constructing p MD-18T-IL-1β plasmid of Duolang sheep which was amplified by PCR. According to DNA gel extraction,after the target IL-1β DNA gene eviction,IL-1β probes was labeled with digoxgenin of Roche,and then detected by DIG DNA labeling and detection kit,all of results were displayed in Nylon N＋,so could calculate the concentration of the labeled probe.【Result】It was calculated that the concentration of the labeled probe was 17. 1ng / ml. 【Conclu-sion】By the Southern hybridization it could be learned that there was ds DNA IL-1β gene in Hybond N ＋membrane. In standard hybridization solution,IL-1 β gene ss DNA was labeled for DIG. Through the dark color,hybridization band deep purple was displayed in the Hybond N＋,which proved that the probe concentration reached the standard concentration

关 键 词：多浪羊IL-1β基因 地高辛 探针标记 检测 

分 类 号：S826[农业科学—畜牧学]