抗凝血酶Ⅲ转基因山羊外源基因拷贝数及其蛋白表达量的测定

Determination of Exogenous Gene Copy Number and Protein Expression in Antithrombin Ⅲ Transgenic Goat

目的:检测抗凝血酶Ⅲ(ATⅢ)转基因山羊外源基因的拷贝数及ATⅢ蛋白的表达量,分析拷贝数与蛋白表达量的相关性。方法:用Primer 5.0软件设计外源基因ATⅢ及山羊管家基因GAPDH的引物,以倍比稀释的标准品进行实时荧光定量PCR,制作标准曲线,通过标准曲线计算外源基因拷贝数;通过酶显色底物法检测ATⅢ蛋白的含量。结果:依据ATⅢ和GAPDH基因标准曲线方程计算得到的山羊个体间外源基因的拷贝数相差很大,最少的为5,最多为25,且不同转基因山羊间ATⅢ表达水平的差别达10倍以上。结论:外源基因表达水平在受到拷贝数的影响外,也受到整合位点等其他因素的重要影响。

Objective：To determinate the copy number and protein expression in antithrombin Ⅲ（ATⅢ） trans-genic goat.Methods：The primers of ATⅢ and GAPDH gene were prepared with Primer 5.0.The standard curve was prepared based on the liner relationship between the amount of input sampals diluationed,then the copy number was calculated.The ATⅢ protein content was determinated through enzymatic chromogenic substrate.Re-sults：Transgenic goats which prepared in the same carrier,whose copy number varied widely,from 5 to 25.Be-tween different transgenic goats,the difference among the expression levels of transgenic ATⅢ goats was reached more than 10 times.However,there is no obvious correlation between the amount of copy number and the expres-sion levels of ATⅢ.Conclusion：What had been demonstrated was the expression of exogenous gene was impacted by the copy number,but also by other factors such as integration sites of the exogenous gene.