补体成分C3及其缺失突变体蛋白的表达及与CLIC1蛋白共定位的研究  

The expression of human complement component C3 and its deletion mutants and the colocalization with CLIC1

作　　者：王二宁 陈丹丹[1] 刘晓颖[1] 范礼斌[1] 

机构地区：[1]安徽医科大学生命科学学院生物教研室,合肥230032

出　　处：《安徽医科大学学报》2015年第11期1533-1537,共5页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金青年基金(编号:81201368)

摘　　要：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pc DNA3.1-C3-FLAG、pc DNA3.1-C3(1-840)-FLAG、pc DNA3.1-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 construct three eukaryotic expression plasmids of pc DNA3. 1-C3-FLAG,pc DNA3. 1-C3（ 1-840）-FLAG and pc DNA3. 1-C3（ 824-1663）-FLAG（ according to C3 structure domain and splitting position）. The plasmids were transfected into HEK 293 T 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 pc DNA3. 1-C3-FLAG,pc DNA3. 1-C3（ 1-840）-FLAG and pc DNA3. 1-C3（ 824-1663）- FLAG were constructed successfully,which could be expressed in HEK 293 T and COS7 cells,and the cellular localization of C3 and C3（ 1-840）,C3（ 824-1663） appeared similar,mainly in the cytoplasm,and only C3（ 824-1663） co-localized in the cytoplasm with CLIC1. Conclusion Complement C3 and its deletion mutants C3（ 1-840）,C3（ 824-1663） can be effectively expressed in HEK 293 T and COS7 cells,and all of them are mainly distributed in the cytoplasm and C3（ 824-1663） co-localized in the cytoplasm with CLIC1.

关 键 词：C3 转染 基因表达 定位 

分 类 号：Q28[生物学—细胞生物学] R392.7[医药卫生—免疫学]