蚓激酶基因的人工合成及山羊乳腺表达

Synthesis of Lumbrokinase cDNA and Its Expression in Goat Milk

为了实现蚓激酶在真核细胞的高效稳定表达 ,人工合成了全长 849bp ,不含罕用密码子的蚓激酶F Ⅲ 1cDNA序列 ,并以其为目的基因 ,构建以山羊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和重组逆转录病毒表达载体pLN bCP LK。质粒pBLK 40 0 μg直接注入稳定泌乳期奶山羊乳腺 ,以纤维蛋白平板溶圈法 (FAPA)检测奶样纤溶活性。结果显示 ,注射后 3～ 60h ,奶样有明显纤溶活性 ,其中 6～ 9h活性最高。脂质体介导质粒pLN bCP LK转染PA31 7细胞系 ,5 0 0mg LG41 8筛选后获得 4株高产毒细胞系 ,产毒滴度达 1× 1 0 4～ 1× 1 0 5CFU ml水平。产毒细胞上清 1 0ml直接注入临产前两周的山羊乳腺 ,隔日以等量再注 1次 ,产羔后其奶样即有明显纤溶活性 ,注后第

To achieve efficient and stable expression of lumbrokinase in eukaryotic cells,the cDNA of lumbrokianse F-III-1,849 base pair in length,with no rare codons,was synthesized and subcloned into a mammary-gland-specific expression vector,forming plasmid pBLK,in which the upstream promoter of caprine beta-casein was used as the regulatory sequence.Recombinant vector,pLN-bCP-LK,containing the expression cassette was also constructed and then packaged into retrovirus (1×10 4～1×10 5CFU/ml) in PA317 cells under G418(500μg /L) selection.Transient expression of the lumbrokinase was gained by injecting 400μg plasmid into lactating mammary gland.The fibrinolytic activity of the milk presented in 3 hours after injection and lasted for more than 60 hours.Stable expression was achieved by injecting 1×10ml recombinant retrovirus (two times, after an interval of one day) into mammary glands two weeks before parturition.The fibrinolytic activity of the milk has persisted for 45 days.