Characterization of the nucleotide sequence of the groE operon encoding heat shock proteins chaperone-60 and -10 of Francisella tularensis and determination of the T-cell response to the proteins in individuals vaccinated with F-tularensis
Characterization of the nucleotide sequence of the groE operon encoding heat shock proteins chaperone-60 and -10 of Francisella tularensis and determination of the T-cell response to the proteins in individuals vaccinated with F-tularensis
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DOI：
10.1128/iai.65.5.1824-1829.1997
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发表时间：
1997-05-01
影响因子：
3.1
通讯作者：
Sjostedt, A
中科院分区：
文献类型：
--
作者：
Ericsson, M;Golovliov, I;Sjostedt, A
对土拉菌LVS中编码热休克蛋白伴侣蛋白-10 （Cpn10）和Cpn60的groE操纵子进行了测序和表征，并对接种LVS的个体对这两个蛋白和第三个主要伴侣蛋白Ft-DnaK的t细胞应答进行了分析，利用这两个蛋白n端序列的退化寡核苷酸进行PCR扩增Cpn10和Cpn60基因，序列分析显示了预期的两个开放阅读框。编码M(r)s估计为10,300和57,400的蛋白质，推导出的氨基酸序列与其他原核生物的cpn10和Cpn60蛋白质非常相似。这些基因组成了一个双子操纵子，cpn10基因位于cpn60基因之前。在cpn10基因上游，倒置重复序列和基序与大肠杆菌sigma（70）依赖启动子的-35和-10序列相似，而与sigma（32）依赖启动子不同。该操纵子的倒置重复序列类似于在其他缺乏sigma（32）依赖启动子的原核groE操纵子中发现的所谓的发夹环。引物延伸分析显示，无论是否存在热或氧化应激，都有一个相同的转录起点。通过二维凝胶电泳分离土拉菌LVS有机体的裂解物后，提取DnaK， Cpn60和Cpn10作为t细胞测试的抗原，与未接种疫苗的个体相比，先前接种过活的土拉菌LVS的个体的T细胞对DnaK和Cpn60的增殖反应增加，而对Cpn10的增殖反应没有增加。目前的数据将有助于进一步研究热休克蛋白在土拉菌病保护性免疫中的作用。
The groE operon of Francisella tularensis LVS, encoding the heat shock proteins chaperone-10 (Cpn10) and Cpn60, was sequenced and characterized, and the T-cell response of LVS-vaccinated individuals to the two proteins and the third major chaperone, Ft-DnaK, was assayed, The cpn10 and cpn60 genes were amplified by PCR with degenerate oligonucleotides derived from the N-terminal sequence of the two proteins, The sequence analysis revealed the expected two open reading frames, encoding proteins with estimated M(r)s of 10,300 and 57,400, The deduced amino acid sequences closely resembled cpn10 and Cpn60 proteins of ether prokaryotes. The genes constituted a bicistronic operon, the cpn10 gene preceding the cpn60 gene. Upstream of the cpn10 gene, an inverted repeat and motifs similar to -35 and -10 sequences of sigma(70)-dependent but not of sigma(32)- dependent promoters of Escherichia coli were found, The inverted repeat of the operon resembled so-called hairpin loops identified in other characterized prokaryotic groE operons lacking sigma(32)-dependent promoters. Primer extension analysis disclosed one and the same transcription start, irrespective of the presence or absence of heat or oxidative stress, After separation of lysates of the F. tularensis LVS organism by two-dimensional gel electrophoresis, DnaK, Cpn60, and Cpn10 were extracted and used as antigens in T-cell tests, When compared to those from nonvaccinated individuals, T cells from individuals previously vaccinated with live F. tularensis LVS showed an increased proliferative response to DnaK and Cpn60 hut not to Cpn10, The present data will facilitate further studies of the involvement of the heat shock proteins in protective immunity to tularemia.