牛骨骼肌特异性启动子的筛选

摘要

分别构建含有牛骨骼肌α-肌动蛋白(α-actin)启动子、牛骨骼肌肌球蛋白轻链2(mylpf)启动子及牛肌酸激酶(ckm)启动子的荧光素酶报告基因表达质粒.将3种荧光素酶表达质粒分别与含有海肾荧光素酶的质粒共转染牛成肌细胞和牛成纤维细胞.经双荧光素酶检测得到转染成肌细胞,72 h后,α-actin启动子的表达量显著高于mylpf启动子和ckm启动子,转染成纤维细胞72 h后,这3种启动子的表达量和空载体相近.结果表明,α-actin启动子在成肌细胞中的表达效率高于mylpf启动子和ckm启动子;3种启动子在成纤维细胞中的表达量与空载体相近,证明了这3种启动子的骨骼肌特异性.通过对骨器肌特异性启动子的研究,为外源基因在骨骼肌组织的特异性表达提供了试验依据,为进一步研究转基因肉牛奠定了基础.%Three vectors were constructed, which were carrying firefly luciferase, driven by α-actin promoter, myosin light chain (mylpf) promoter and muscle creatine kinase (M-type,ckm) promoter, respectively. The three promoters were all from luxi cattle. The combined pRL-TK and three plasmids were transfected respectively into luxi cattle myoblasts and fibroblasts.After 72 h of transfection,dual luciferase reporter assay system was used to measure the activities of three promoters. We observed that the α-actin promoter provided luciferase reporter gene activity higher than mylpf promoter and ckm promoter in myoblasts and the luciferase reporter gene activities that were provided by the three promoters were similar to pGL3-Basic in fibroblasts,which certified that the three promoters were skeletal muscle-specific. This results provided an experimental basis for gene expression in skeletal muscle and establish the basis for gene efficient and specific expression in skeletal muscle.