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目的:从人骨骼肌cDNA文库中,分离、鉴定视网膜感光受体外周蛋白的结合蛋白(PBP)-DnaJ(HSP40)类分子伴侣,并初步分析其在转染的哺乳动物细胞中的表达及分布。方法:以33p-dCTP标记探针杂交筛选人骨骼肌cDNA文库,对阳性克隆进行序列分析,并以脂质体介导转染哺乳动物细胞,用免疫印迹及间接免疫荧光染色法检测目的蛋白的表达。结果:筛选出全长pbp基因,经与GenBank中的序列比较分析证实,与DnaJ家族的1个成员-mrj(1.5 kb)的序列相同,且含140 bp左右富含GC的非编码区上游序列。在COS-7细胞 中瞬时表达的PBP,大多呈典型的胞浆分布,而J-domain缺失的pbp片段多为胞核分布。而在CHO细胞中稳定表达的PBP,在处于不同细胞周期时相的细胞中的分布不同。结论:DnaJ除与HSP70(DnaK)协同发挥重要功能外,其本身作为分子伴侣也可能发挥着重要作用,且与细胞周期可能存在某种密切联系。
OBJECTIVE: To isolate and characterize the peripheral retinal binding protein (PBP) -DnaJ (HSP40) chaperones from human skeletal muscle cDNA library and to analyze the expression and distribution of PBP-DnaJ (HSP40) in mammalian cells . Methods: Human skeletal muscle cDNA library was screened by hybridization with 33p-dCTP-labeled probe. The positive clones were sequenced and transfected into mammalian cells by liposome. Western blot and indirect immunofluorescence staining were used to detect the expression of target protein expression. Results: The full-length pbp gene was screened out and compared with the GenBank. The sequence of mrj (1.5 kb) was the same as that of one member of DnaJ family and contained about 140 bp GC-rich non-coding region upstream sequences . PBP transiently expressed in COS-7 cells mostly showed typical cytoplasmic distribution, while the majority of p-bp fragments lacking J-domain were mostly nuclear. Whereas PBP stably expressed in CHO cells differed in the distribution of cells in different phase of the cell cycle. Conclusion: DnaJ may play an important role as a molecular chaperone in addition to its synergistic effect with HSP70 (DnaK), and may be closely related to cell cycle.