t-PA突变体乳腺表达载体的构建及其在乳腺中的瞬时表达

Construction of mutant t-PA mammary gland expression vector and transient expression in mammary gland tissues

目的 :探索建立乳腺瞬时性表达系统 ,用于快速检测所构建的乳腺表达载体的有效性 ,为进行转基因动物实验奠定基础。方法 :DNA常规操作 ;孕小鼠乳腺注射 ;纤维蛋白琼脂糖平板法检测Mut PA。结果 :构建了含有Mut PA基因的乳腺表达载体pB3mt PA ,将此载体直接注射到孕小鼠乳腺中进行暂时性表达 ,产仔后第 4天采乳汁 ,用纤维蛋白琼脂糖平板法检测t PA的活性 ,表达活性可达 2 0～ 4 0U/ml,其活性能被天然的t PA多克隆抗体所中和 ,表达持续期 8d。结论 :所克隆的牛BLG基因 70 5bp的 5′端调控序列包含了必需的调控元件 ,并具有调控外源基因的表达的功能 ,克隆的 3′端序列是适合的。构建的表达载体可用于制备转基因动物。

Objective:To study on establishing instant expression system of mammary gland for examining quickly the quality of the expression vector of mammary gland in order to lay a foundation for the transgenic animal experiment. Methods:DNA Technique,injection into mammary glands of pregnant mice and detecting the biological activity of mutant tissue-type plasminogen activator(Mut-PA) by fibrin agarose plate assay(FAPA)were used.Results:The mammary gland expression vector pB3mt-PA containing Mut-PA sequence was constructed, and directly injected into mammary glands of pregnant mice. On the fourth day after delivery, the milk was obtained and the biological activity Mut-PA was detected by fibrin agarose plate assay and the fibrin-dissolved activity can be neutralized by the polyclonal antibody against natural t-PA. 20-40 U/ml of Mut-PA was expressed in milk, and the expression lasted longer than 8 days.Conclusions: This experiment shows that the -705 bp 5′end flanking region contains the necessary control sequence and 3′end flanking sequence is right, and the expression vector pB3mt-PA can be used to produce transgenic animal. [