肌肉中特异表达真核载体的构建及表达特异性验证

Construction and verification of muscle-specific expression eukaryotic vector

为构建肌肉中特异表达真核载体,本试验以PGL4.10质粒为骨架,利用基因合成、酶切连接方法获得重组载体,同时利用红色荧光蛋白基因DsRed2作为标记基因来检测重组载体在非肌源细胞和肌细胞中的表达情况。结果表明,成功构建了含有肌球蛋白轻链基因MLC启动子及其增强子的肌肉中特异表达载体,通过mRNA和蛋白水平检测,该表达载体在非肌源性细胞和肌细胞中均不表达DsRed2,只在分化的肌细胞中表达DsRed2。该表达载体的构建为制备肌肉中特异表达的转基因动物奠定了基础。

To construct a muscle-specific eukaryotic expression vector and to verify its specific expression,a recom-binant vector was constructed using gene synthesis and enzyme digestion ligation method based on PGL4. 10 plasmid as a backbone. Meanwhile,a red fluorescent protein （ DsRed2） was used as a marker to detect the expression of the recombi-nant vector in non-muscle-derived cells and muscle cells. The results showed that the muscle-specific eukaryotic expression vector containing the promoter and enhancer of myosin light chain （ MLC） gene was successfully constructed. This expres-sion vector did not express DsRed2 in non-myogenic cells and muscle cells, and could only express in differentiated muscle cells at mRNA and protein levels. The results provide a solid foundation for the preparation of muscle-specific expression for transgenic animal.