线粒体靶向小清蛋白表达载体构建及其对肝癌细胞线粒体钙稳态的影响

摘要

目的 构建线粒体靶向小清蛋白(PVALB)表达载体,探讨线粒体靶向PVALB在肝癌细胞(MHCC97H)中的表达及对线粒体内钙稳态的影响.方法 以骨骼肌细胞的cDNA为模板,通过PCR法扩增得到PVALB,经酶切后与MTS、pcDNA3.1(+)载体连接;重组质粒经酶切分析及测序鉴定后,用脂质体转染法转染肝癌细胞MH-CC97H,用激光共聚焦显微镜检测PVALB定位及转染后MHCC97H线粒体中钙离子浓度.结果 pcDNA3.1(+)-MTS-PVALB经酶切、测序鉴定完全正确,真核表达载体构建成功;表达的MTS-PVALB定位于线粒体;MTS-PVALB可下调线粒体内钙离子浓度.结论 成功构建pcDNA3.1(+)-MTS-PVALB线粒体靶向真核表达载体,MTS-PVALB定位于MHCC97H线粒体并调节线粒体钙稳态.%Objective To construct mitochondria targeted parvalbumin ( PVALB) expression vector and to investigate the mitochondrial targeting PVALB expression in human hepatocellular carcinoma cells MHCC 97H and its effect on mito-chondrial calcium homeostasis .Methods Using skeletal muscle cells cDNA as the template , the PVALB was amplified by PCR, and then the digested PVALB was connected with MTS and pcDNA 3.1 (+) vector.After identifying by enzyme di-gestion analysis and sequencing , the recombinant plasmid was transfected into MHCC 97H by liposome.The expression and localization of PVALB, calcium concentration in MHCC97H were detected by laser scanning confocal microscopy after transfection.Results pcDNA3.1 (+)-MTS-PVALB was successfully constructed and was verified by restriction enzyme digestion and DNA sequencing .The expression of MTS-PVALB was located in the mitochondria , and MTS-PVALB could down-regulate the concentration of calcium .Conclusion pcDNA3.1 ( +)-MTS-PVALB was successfully constructed . The expressed MTS-PVALB was located in MHCC97H mitochondria and regulated mitochondrial calcium homeostasis .