稳定表达Caveolin-3基因突变型P104L和P47L的C2C12骨骼肌细胞株的构建

Establishment of C2C12 skeletal muscle cell lines stably overexpressing Caveolin-3 gene P104L and P47L mutations

目的构建稳定表达Caveolin-3(CAV3)基因突变型P104L和P47L的C2C12骨骼肌细胞株。方法将野生型CAV3(CAV3-WT)、CAV3-P104L、CAV3-P47L装在带有GFP标签的质粒中,构建目的基因与GFP基因融合的表达质粒,以只含GFP的载体为对照。将C2C12细胞随机分为A、B、C、D组,分别转染对照空载体(NC)、CAV3-WT、CAV3-P47L及CAV3-P104L。以遗传霉素(G418)进行阳性克隆筛选,荧光显微镜下挑选稳定转染的细胞系,Western blotting法检测各组细胞中的CAV3-GFP融合蛋白,免疫荧光法检测各组细胞中的CAV3蛋白,以荧光强度代表目的蛋白相对表达量。结果经酶切和测序验证,证实4种表达质粒构建成功。B、C、D组细胞中测得CAV3-GFP融合蛋白,A组未测得融合蛋白。A、B、C、D组CAV3蛋白相对表达量分别为23.0±2.25、32.3±0.35、25.6±0.92、24.3±1.42,B组CAV3蛋白表达增强,与A组相比,P<0.01;A、C、D组CAV3蛋白表达下调,与B组相比,P均<0.01。结论成功构建了稳定表达CAV3-P47L和CAV3-P104L的C2C12细胞,两种细胞中CAV3蛋白表达下调。

Objective To construct C2C12 skeletal muscle cell lines stably expressing Caveolin-3（ CAV3） gene P104 L and P47 L mutations. Methods The wild type CAV3（ CAV3-WT）,CAV3-P104 L,CAV3-P47 L were installed in the plasmid with GFP tag,and then we constructed the target gene and GFP gene fusion expression plasmid,with only GFP carrier as the control. C2C12 cells were randomly divided into four groups： groups A,B,C and D which were transfected with empty vector（ NC）,CAV3-WT,CAV3-P47 L and CAV3-P104 L. After treated with geneticin（ G418）,we selected the stable transfected cell lines under the fluorescence microscope. The expression of CAV3 and GFP fusion protein was detected by Western blotting,and the expression of CAV3 protein was detected by immunofluorescence. The relative expression of the target protein was expressed by the fluorescence intensity. Results After enzyme digestion and sequencing,the four expression vectors proved to be successfully constructed. Cell lines of groups B,C and D contained the fusion protein expression of CAV3 and GFP,but fusion protein was not detected in group A. The relative expression of CAV3 in groups A,B,C and D was 23. 0 ± 2. 25,32. 3 ± 0. 35,25. 6 ± 0. 92 and 24. 3 ± 1. 42. The expression of CAV3 in group B was increased compared with that of group A,P 0. 01,and the expression of CAV3 in groups A,C and D was significantly decreased compared with that of B group,all P 0. 01. Conclusion We successfully constructed the C2C12 cells stably expressing CAV3-P104 L and CAV3-P47 L,and the expression of CAV3 protein was down-regulated in these two cells.