新疆多浪羊IFN-γ基因地高辛探针的标记与敏感性检测  

Digoxin Probe Labeling and Sensitivity Detection of IFN γ Gene in Xinjiang Duolang Sheep

作　　者：艾东旭 徐宏伟[1] 李玮[1] 张晶[1] 宋显明[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：《西北农业学报》2016年第10期1436-1441,共6页Acta Agriculturae Boreali-occidentalis Sinica

基　　金：国家自然科学基金(30960277);新疆生产建设兵团应用基础研究(2007JC07);塔里木大学校长基金(TDZKCX201401)~~

摘　　要：为获得多浪羊IFN-γ地高辛探针,并对其标记效率进行检测。将多浪羊pMD-18T-IFN-γ克隆菌进行PCR扩增,经切胶回收及目的基因浓缩后获得目的基因IFN-γ,然后用地高辛标记和检测试剂盒对IFN-γ基因进行标记,获得IFN-γ地高辛探针。IFN-γ的PCR扩增产物经8g/L琼脂糖电泳分离后转移至Hybond N+膜,膜上显影后进行敏感性检测并计算探针质量浓度,结果显示,IFN-γ地高辛探针在杂交液中的质量浓度为30μg/L。Southern杂交显示,Hybond N+膜上有IFN-γ基因的双链DNA,且浓缩后的DNA(2.5μg)标记的特异性探针质量浓度较高,可以在Hybond N+膜上呈现出清晰的杂交条带,证明获得的探针质量浓度符合要求。In order to obtain Duolang sheep IFN-γ digoxin probe,and detecting its labeling efficiency, the pMD-18T-IFN-γ colonies of Duolang sheep was amplified by PCR. After cutting and recovering the gel and concentrating of target gene, the final desired IFN-γ was obtained, and then the IFN-γ gene was labeled by digoxin and detection kit and the IFN-γ digoxin probe was obtained. The IFN-γ, PCR product was isolated by 8 g/L agarose gel electrophoresis,and then transferred to Hybond N＋ membrane, after the membrane developing perform, sensitivity was detected and the probe mass concentration was calculated. The results showed that the mass concentration probe was 30 μg/L in the hybridization solution. The southern hybridization indicated that there is the double-stranded DNA IFN-γ, gene of Hybond N＋ membrane,and higher concentration of labeled specific probe was detected from mass concentrated DNA （2.5 μg） ,there was a clear hybridizing band in Hybond N＋ membrane, which proved that the probe mass concentration was up to standard mass concentrations.

关 键 词：多浪羊 IFN-Γ基因 地高辛 探针标记 检测 

分 类 号：S826[农业科学—畜牧学]