pCDNA3-AADC真核表达载体的构建及其在原代培养骨骼肌细胞中的表达

Construction of eukaryotic expression vector pCDNA3-AADC and it's expression in cultured primary muscle cells.

目的 将人类神经元性芳香族氨基酸脱羧酶（AADC）基因构建于含CMV启动子的真核表达载体pCD-NA3上,并检测其在原代培养骨骼肌细胞中的表达。方法 采用分子克隆技术将经测序证实的人神经元性AADC基因片段同真核表达载体pCDNA3连接,酶切鉴定并筛选出正向连接重组体,并采用脂质体转染法将其导入原代培养的骨骼肌细胞,免疫印记检测该基因的表达。结果 酶切证实基因片断正向连接于pCDNA3上,并在原代骨骼肌细胞中获得表达。结论 含人类神经元AADC基因的真核表达重组体可在原代培养的骨骼肌细胞中有效的表达。

Objective To construe eukaryotic expression recombinant human neuronal aromatic-l-amino-acid decarboxylase (AADC) gene and to detect it's expression in cultured primary muscle cells. Methods Molecular cloning techniques were used to combine eukaryotic expression vector pCDNA3 and AADC gene which was confirmed by gene sequencing. Idenified by enzyme cutting reaction, the recombinants were transfected into primary muscle cells using CLONfectin reagent. Western blot was used to detect the gene expression. Results Enzymes cutting confirmed the positive directed ligation between AADC gene and pCDNA3. The recombinants were expressed successfully in primary muscle cells. Conclusions Eukaryotic expression vector pCDNA3-AADC can be efficiently expressed in primary muscle cells.