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首页> 外文期刊>Infection and immunity >Cloning and sequencing of the genes coding for the 10- and 60-kDa heat shock proteins from Pseudomonas aeruginosa and mapping of a species-specific epitope.
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Cloning and sequencing of the genes coding for the 10- and 60-kDa heat shock proteins from Pseudomonas aeruginosa and mapping of a species-specific epitope.

机译：铜绿假单胞菌编码10-kDa和60-kDa热激蛋白的基因的克隆和测序以及物种特异性表位的作图。

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A genomic library of Pseudomonas aeruginosa DNA was screened with a monoclonal antibody (MAb 2528) specific for the P. aeruginosa 60-kDa heat shock protein. A positive clone, pAS-1, was isolated. The gene coding for P. aeruginosa chaperonin (hsp60) was localized to a 2-kb EcoRI fragment subcloned in pAS-2. A sequence analysis of pAS-2 and parts of pAS-1 identified two open reading frames that encoded proteins with calculated molecular masses of 10 and 57 kDa. In amino acid sequence comparison studies the sequences of these proteins, which were designated GroES and GroEL, exhibited up to 78% homology with known prokaryotic sequences of 10- and 60-kDa heat shock proteins (hsp10 and hsp60). In order to map the epitope recognized by MAb 2528, a series of GroEL nested carboxy-terminal deletion clones were tested with MAb 2528. We identified the clone with the shortest insertion that was still recognized by MAb 2528 and the clone with the largest insertion that was not recognized by MAb 2528. The 3' ends of the insertions were determined by sequencing and were found to delimit a region that encoded 25 amino acid residues. Synthetic oligonucleotides that coded for peptides possibly resembling the epitope within this region were ligated into expression vector pGEX-3X, and fusion proteins expressed by these clones were tested for reactivity with MAb 2528. By using this method we determined that the decapeptide QADIEARVLQ (positions 339 to 348 on GroEL) was responsible for the binding of P. aeruginosa-specific MAb 2528.
机译：用对铜绿假单胞菌60-kDa热激蛋白特异的单克隆抗体（MAb 2528）筛选铜绿假单胞菌DNA的基因组文库。分离出阳性克隆pAS-1。编码铜绿假单胞菌伴侣蛋白（hsp60）的基因位于pAS-2亚克隆的2kb EcoRI片段中。对pAS-2和pAS-1的部分进行序列分析，确定了两个开放阅读框，它们编码的蛋白质的分子质量分别为10和57 kDa。在氨基酸序列比较研究中，这些蛋白质的序列（称为GroES和GroEL）与已知的10 kDa和60 kDa热激蛋白（hsp10和hsp60）的原核生物序列具有高达78％的同源性。为了绘制MAb 2528识别的表位，用MAb 2528测试了一系列GroEL嵌套的羧基末端缺失克隆。我们鉴定了插入最短的克隆仍被MAb 2528识别，而克隆则克隆了最大的克隆。 MAb 2528不能识别插入序列。通过测序确定插入的3'端，发现其界定了一个编码25个氨基酸残基的区域。将编码可能与该区域内的表位相似的肽的合成寡核苷酸连接至表达载体pGEX-3X，并测试了由这些克隆表达的融合蛋白与MAb 2528的反应性。通过这种方法，我们确定了十肽QADIEARVLQ（位置339） （GroEL上的348）负责铜绿假单胞菌特异性MAb 2528的结合。

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