Thomas P W, Wyckoff E E, Pishko E J, Yu J J, Kirkland T N, Cole G T
Department of Microbiology and Immunology, Medical College of Ohio, Toledo 43614-5806, USA.
Gene. 1997 Oct 15;199(1-2):83-91. doi: 10.1016/s0378-1119(97)00351-x.
A heat shock protein-encoding gene (hsp60) from the human respiratory fungal pathogen, Coccidioides immitis (Ci), was cloned, sequenced, chromosome-mapped, expressed and immunolocalized in parasitic cells. Both the genomic and cDNA sequences are presented. The transcription start point and poly (A) addition site were confirmed. The hsp60 gene contains two introns and a 1782-bp ORF which translates a 594-amino acid (aa) protein of 62.4 kDa and pI of 5.6. The translated protein revealed two potential N-glycosylation sites. The deduced HSP60 showed 78-83% aa sequence similarity to reported fungal HSP60 proteins. The hsp60 gene was mapped to chromosome III of Ci and was shown to be a single copy gene by Southern and Northern hybridization. Expression of a 1737-bp cDNA fragment of the hsp60 gene in E. coli resulted in production of a recombinant protein. Amino acid sequence analysis of the recombinant protein confirmed that it was encoded by the Ci hsp60 gene. Antiserum raised in mice against the isolated recombinant protein immunolocalized HSP60 in the cytoplasm and wall of parasitic cells of Ci. The recombinant HSP60 was used to immunize BALB/c mice and was shown to induce proliferation of T cells isolated from lymph nodes of these animals. The hsp60 gene of Ci is the first reported heat-shock protein gene of this human pathogen.
克隆、测序、进行染色体定位、表达并在寄生细胞中进行免疫定位了来自人类呼吸道真菌病原体球孢子菌（Coccidioides immitis，Ci）的一个热休克蛋白编码基因（hsp60）。给出了基因组序列和cDNA序列。确认了转录起始点和聚腺苷酸（poly (A)）加尾位点。hsp60基因包含两个内含子和一个1782 bp的开放阅读框（ORF），该开放阅读框翻译出一个62.4 kDa、5.6的等电点（pI）的594个氨基酸（aa）的蛋白质。翻译后的蛋白质显示有两个潜在的N - 糖基化位点。推导的HSP60与已报道的真菌HSP60蛋白显示出78 - 83%的氨基酸序列相似性。hsp60基因被定位到Ci的第三条染色体上，并且通过Southern和Northern杂交显示为单拷贝基因。hsp60基因的一个1737 bp的cDNA片段在大肠杆菌中表达产生了一种重组蛋白。对该重组蛋白的氨基酸序列分析证实它是由Ci hsp60基因编码的。用针对分离出的重组蛋白在小鼠中产生的抗血清在Ci寄生细胞的细胞质和细胞壁中对HSP60进行了免疫定位。用重组HSP60免疫BALB/c小鼠，结果显示它能诱导从这些动物淋巴结中分离出的T细胞增殖。Ci的hsp60基因是该人类病原体中首个被报道的热休克蛋白基因。
