摘要
目的:热休克蛋白70羧基端作用蛋白(CHIP)是近年来新发现的同时具有辅助伴侣分子和E3泛素连接酶活性的特殊蛋白分子,但其对相应底物蛋白代谢和活性的调控机制尚不十分清楚。本研究拟构建含CHIP全长编码基因的真核表达载体,并在此基础上构建含CHIP不同功能结构域突变体和缺失体基因的表达载体,以实现这些基因在真核细胞中的高效表达,为后续探讨CHIP相关功能的实验奠定基础。方法:从高表达CHIP的人非小细胞肺癌H322细胞中提取总RNA,以此为模板,采用逆转录巢式PCR(RT-nested-PCR)获得CHIP全长编码基因,经测序确定序列正确后将CHIP基因连入带有myc标签的克隆载体pCMV-Tag1中,并以此为模板采用点突变的方法构建CHIP基因N端TPR结构域突变体K30A和C端U-box结构域的突变体H260Q以及U-box结构域缺失体U-box(-)。结果:钓取的CHIP基因经测序鉴定序列与GenBank报道完全一致,瞬时转染证实所构建的CHIP及其突变体表达载体可以在人卵巢癌SKOV3细胞中实现高效表达。结论:成功地构建并获得能够在真核细胞内高表达的CHIP及其功能域突变体的表达载体,为进一步探讨真核细胞内CHIP对相关蛋白分子的调控机制奠定了前期基础。
Objective：CHIP, carboxy terminus of Hsc70 interacting protein, is a novel co-chaperone protein with E3-ubiquitin ligase activity,whereas the accurate mechanism for CHIP to mediate its substrates metabolism or activities is undefined. To investigate the function and correlation of CHIP with its possible substrates proteins, we constructed the eukaryotic expression vector containing CHIP full-length coding sequence and its mutants with either TPR or U-box domain mutation and its deletion with U-box domain and to set up the model with ectopically overexpression of CHIP and its various functional mutants or deletion. Methods： Total RNA isolated from human non-small cell lung cancer H322 cells which had high level expression of endogenous CHIP was used as template and RT-nested-PCR technique was used to amplify CHIP full-length coding sequence. The CHIP-K30A, CHIP-H260Q and CHIP-U-box（-） mutants were generated by using mutant primers in site-specific mutagenesis PCR. Results： Various pCMV-Tagl expression constructs encoding the Myc epitopetagged full-length CHIP, its TPR mutant CHIP-K30A, U-box domain mutant CHIP-H260Q or its U-box domain deletion CHIP-Ubox （-） were generated, and all constructs were sequence verified. Exogenous overexpression of CHIP and its mutants in SKVO3 cells were observed by transient transfection and Western blot detection. Conclusions：The CHIP and its mutants were successfully cloned. The overexpression of CHIP and its mutants in eukaryotic cell will offer a useful platform for further studying the function and regulation of CHIP.
出处
《军事医学科学院院刊》
CSCD
北大核心
2005年第4期316-320,共5页
Bulletin of the Academy of Military Medical Sciences
基金
军事医学科学院创新基金
国家自然科学基金重点项目(30330620)
国家自然科学基金面上项目(30470898)资助
