摘要
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.
出处
《中国生物工程杂志》
CAS
CSCD
2004年第9期49-52,共4页
China Biotechnology
基金
国家"8 63"计划资助项目 ( 2 0 0 2AA2 0 665 3 )