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Cloning and characterization of the Brucella ovis heat shock protein DnaK functionally expressed in Escherichia coli.

机译:在大肠杆菌中功能表达的布鲁氏菌热休克蛋白DnaK的克隆和鉴定。

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摘要

The Brucella ovis dnaK gene, homolog to the eukaryotic hsp70 genes, was cloned by using a Drosophila melanogaster probe. Comparison of B. ovis and Escherichia coli sequences revealed a similar organization for the dnaK and dnaJ genes and putative regulatory signals. In E. coli transfected with the cloned fragment, B. ovis hsp70 was expressed at 30 and 50 degrees C apparently under the control of its own promoter. The recombinant protein and a B. ovis native protein displaying the same molecular weight were both recognized by anti-E. coli DnaK serum. Native B. ovis protein was also recognized by sera of sheep either infected or vaccinated with an attenuated Brucella strain, suggesting that Brucella hsp70 could be up-regulated during host colonization. A thermosensitive E. coli dnaK mutant transfected with the cloned fragment recovered colony-forming ability at 42 degrees C, showing that the B. ovis DnaK protein could behave as a functional heat shock protein in E. coli.
机译:通过使用果蝇黑色素瘤探针克隆了与真核hsp70基因同源的布鲁氏菌dnaK基因。牛双歧杆菌和大肠杆菌序列的比较显示了dnaK和dnaJ基因以及推定的调节信号的相似组织。在用克隆的片段转染的大肠杆菌中,B。ovis hsp70显然在其自身启动子的控制下于30和50摄氏度表达。重组蛋白和显示相同分子量的牛双歧杆菌天然蛋白都被抗E蛋白识别。大肠杆菌DnaK血清。绵羊血清也可以识别天然的牛肝杆菌蛋白,用减毒的布鲁氏菌菌株进行接种或接种,这表明布鲁氏菌hsp70可能在宿主定殖过程中被上调。转染了克隆片段的热敏大肠杆菌dnaK突变体在42摄氏度下恢复了菌落形成能力,这表明B. ovis DnaK蛋白可以在大肠杆菌中充当功能性热激蛋白。

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