目的：研究补体成分 C3及其缺失突变体 C3（1-840）、C3（824-1663）在真核细胞内的表达及与氯离子通道蛋白（CLIC1）的共定位。方法构建 pcDNA3．1-C3-FLAG、pcDNA3．1-C3（1-840）-FLAG、pcDNA3．1-C3（824-1663）-FLAG 三个真核表达质粒（缺失突变体根据 C3的结构域及其裂解断裂位置设计），并分别转染至 HEK 293T 细胞中， Western blot 检测表达情况；上述质粒分别瞬时单转至 COS7细胞和分别与 GFP-CLIC1共转至 COS7细胞内，观察共定位情况。结果成功构建带 FLAG 标签的 C3基因及其两个缺失突变体[C3（1-840）、C3（824-1663）]的真核表达载体， Western blot 结果显示它们在 HEK 293T 细胞中均能成功表达；免疫荧光显示它们在 COS7细胞中均主要分布于细胞质，且三个真核表达载体中只有 C3（824-1663）与 CLIC1有共定位。结论补体 C3及其缺失突变体 C3（1-840）和 C3（824-1663）在 HEK 293T、COS7细胞中均能高效表达，且主要分布在细胞质内，C3（824-1663）与 CLIC1蛋白有共定位。%Objective To study the expression and cell localization of complement component C3 and its deletion mutants C3(1-840)and C3(824-1663)in eukaryotic cells and the colocalization with CLIC1.Methods To con-struct three eukaryotic expression plasmids of pcDNA3.1-C3-FLAG,pcDNA3.1-C3(1-840)-FLAG and pcDNA3.1-C3(824-1663)-FLAG(according to C3 structure domain and splitting position).The plasmids were transfected into HEK 293T cells.Then the expression was detected by Western blot,and their cellular localization was detected in COS7 cells by fluorescence microscopy.Results The eukaryotic expression plasmids of pcDNA3.1-C3-FLAG, pcDNA3.1-C3(1-840)-FLAG and pcDNA3.1-C3(824-1663)-FLAG were constructed successfully,which could be expressed in HEK 293T and COS7 cells,and the cellular localization of C3 and C3(1-840),C3(824-1663)ap-peared similar,mainly in the cytoplasm,and only C3(824-1663)co-localized in the cytoplasm with CLIC1.Con-clusion Complement C3 and its deletion mutants C3 (1-840),C3 (824 -1663)can be effectively expressed in HEK 293T and COS7 cells,and all of them are mainly distributed in the cytoplasm and C3(824-1663)co-localized in the cytoplasm with CLIC1.
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