蚓激酶基因乳腺表达载体构建及在牛乳腺中的瞬时表达

Construction of Recombinant Vectors Carrying Lumbrokinase Gene and Its Transient Expression in Mammary Gland of Cow

在稀有密码子的改造基础上,人工合成了849 bp的蚓激酶F-Ⅲ-1全长cDNA序列,并以其为目的基因,构建了以山羊β-酪蛋白启动子为上游调控序列的乳腺组织特异性表达载体pBLK和pLN-bCP-LK.2种质粒分别以200,500,1 000,2 000 μg的剂量梯度注入泌乳期黑白花奶牛乳腺,以纤维蛋白平板溶圈法（FAPA）检测该基因表达后奶样的纤溶活性.结果表明：注射后3～78 h,奶样有明显纤溶活性,其中6～16 h活性最高.500 μgpBLK质粒与其他3个剂量溶圈直径之间差异显著（P＜0.05）,而其他3个剂量之间差异不显著;pLN-bCP-LK质粒的各剂量溶圈直径之间差异均不显著,且2种质粒的同剂量溶圈直径之间差异也不显著.以上结果表明,改造后的蚓激酶基因可以在泌乳期黑白花奶牛乳腺中实现瞬时表达,以500 μg的注射剂量表达效果最佳.

The cDNA of lumbrokinase F-Ⅲ-1, 849 base pair in length, based on the modification of rare codons, was synthesized and subcloned into a mammary-gland-speeific expression vector, forming plasmid pBLK, in which the upstream promoter of caprine beta-casein was used as the regulatory sequence. Then the expression cassette was cloned into pLNCL to construct the recombinant vector pLN-BCP-LK. The resuits of FAPA showed that transient expression of lumbrokinase was gained by injecting the two plasmids into the lactaing mammary gland of cows. The fibrinolysis activity of the milk was the highest in 6 - 16 hours after injection. Statistic analysis showed that there was significant difference between 500 μg pBLK and others. But there was no significant difference between pBLK and pLN-BCP-LK. The study showed that the reconstructed lumbrokinase gene could be expressed transiently in the lactaing mammary gland of cows and the best expressed effect was gained in 500 μg dose iniection.