CHIP基因及其突变体的克隆及在人卵巢癌细胞SKOV3中的表达

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栽体。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栽体，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-Tagl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。

Construction and expression of CHIP and its mutants in human ovarian carcinoma SKOV3 cells

Objective:CHIP,carboxy terminus of Hsc70 inter ac ting 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 wit h its possible substrates proteins, we constructed the eukaryotic expression vec tor 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 mutant s or deletion. Methods: Total RNA isolated from human non-sma ll cell lung cancer H322 cells which had high level expression of endogenous CHI P was used as template and RT-nested-PCR technique was used to amplify CHIP fu ll-length coding sequence. The CHIP-K30A, CHIP-H260Q and CHIP-U-box(-) mut ants were generated by using mutant primers in site-specific mutagenesis PCR. Results: Various pCMV-Tag1 expression constructs encoding the Myc epitope-tagged 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 succ essfully cloned. The overexpression of CHIP and its mutants in eukaryotic cell w ill offer a useful platform for further studying the function and regulation of CHIP.