CAJ | 학술논문

目的研究人类胞内氯离子通道蛋白3(CLIC3)在真核细胞中的定位和表达,及其GST融合蛋白在原核细胞中的表达。方法以含人CLIC3的全长cDNA序列的质粒为模板,PCR扩增CLIC3片段,构建真核表达载体pcDNA3.1-CLIC3-FLAG,检测其定位及表达；构建原核表达载体pGEX-5X-3-CLIC3,转化到大肠杆菌 BL21菌株,IPTG诱导融合蛋白GST-CLIC3表达。结果细胞免疫荧光结果表明 CLIC3在COS7细胞质和细胞核中均有分布；Western blot结果显示CLIC3在HEK-293T细胞中能有效表达；考马斯亮蓝染色结果表明融合蛋白GST-CLIC3在BL21菌株中能有效表达。结论人类的CLIC3蛋白COS7、HEK-293T及大肠杆菌BL21菌株均能有效表达,为进一步了解CLIC3的功能奠定了一定的基础。
목적연구인류포내록리자통도단백3(CLIC3)재진핵세포중적정위화표체,급기GST융합단백재원핵세포중적표체。방법이함인CLIC3적전장cDNA서렬적질립위모판,PCR확증CLIC3편단,구건진핵표체재체pcDNA3.1-CLIC3-FLAG,검측기정위급표체；구건원핵표체재체pGEX-5X-3-CLIC3,전화도대장간균 BL21균주,IPTG유도융합단백GST-CLIC3표체。결과세포면역형광결과표명 CLIC3재COS7세포질화세포핵중균유분포；Western blot결과현시CLIC3재HEK-293T세포중능유효표체；고마사량람염색결과표명융합단백GST-CLIC3재BL21균주중능유효표체。결론인류적CLIC3단백COS7、HEK-293T급대장간균BL21균주균능유효표체,위진일보료해CLIC3적공능전정료일정적기출。
Objective To investigate the expression and localization of the human chloride channel protein 3 (CLIC3) in eukaryotic cells, and the expression of GST fusion protein in prokaryotic cells. Methods Plasmids containing full length of human CLIC3 cDNA was used as PCR template to construct the prokaryotic and eukaryotic expression vectors. The pcDNA3. 1-CLIC3-FLAG was transfected into COS7 and HEK-293T cells respectively to detect the localization and expression of CLIC3. The pGEX-5X-3-CLIC3 was transformed into E. coli BL21 to inves-tigate the expression of fusion protein GST-CLIC3 . Results The immunofluorescence results indicated that CLIC3 was distributed in both cytoplasm and nucleus of COS7 cells;Western blot showed CLIC3 could be effectively ex-pressed in HEK-293T cells;Coomassie blue staining proved GST-CLIC3 could be expressed in E. coli BL21. Con-clusion Human CLIC3 protein can be expressed effectively both in eukaryotic and prokaryotic cells, which is im-portant for further research on the function of human CLIC3 .