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c DNA为模板,通过PCR法扩增得到PVALB,经酶切后与MTS、pc DNA3.1(+)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MHCC97H,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 pc DNA3.1(+)-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pc DNA3.1(+)-MTS-PVALB线粒体靶向真核表达载体,MTSPVALB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 MHCC97 H and its effect on mitochondrial calcium homeostasis. Methods Using skeletal muscle cells c DNA as the template,the PVALB was amplified by PCR,and then the digested PVALB was connected with MTS and pc DNA3. 1( +) vector. After identifying by enzyme digestion analysis and sequencing,the recombinant plasmid was transfected into MHCC97 H by liposome. The expression and localization of PVALB,calcium concentration in MHCC97 H were detected by laser scanning confocal microscopy after transfection. Results pc DNA3. 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 pc DNA3. 1( +)-MTS-PVALB was successfully constructed.The expressed MTS-PVALB was located in MHCC97 H mitochondria and regulated mitochondrial calcium homeostasis.
出处
《山东医药》
CAS
北大核心
2016年第9期4-6,共3页
Shandong Medical Journal
基金
国家自然科学基金资助项目(81572727
81572304)
关键词
肝肿瘤
小清蛋白
线粒体
钙稳态
载体构建
liver neoplasms
parvalbumin
mitochondria
calcium homeostasis
vector construction