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首页> 外文期刊>Infection and immunity >Molecular cloning and expression of a 70-kilodalton heat shock protein of Candida albicans.
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Molecular cloning and expression of a 70-kilodalton heat shock protein of Candida albicans.

机译：白色念珠菌的70千达热休克蛋白的分子克隆和表达。

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By screening an expression library of the yeast form of Candida albicans with a serum directed against whole fungal cells, a cDNA (2,325 bp) encoding a stress protein of C. albicans was cloned and sequenced. The cloned sequence (CaRLV130) identified a single open reading frame with a length of 1,968 bp coding for a protein containing 656 amino acid residues (70 kDa). The deduced amino acid sequence was 84% similar to the sequence of the Saccharomyces cerevisiae SSA1 gene, which encodes one member of the 70-kDa heat shock protein (Hsp70) family. The relevant gene (C. albicans HSP70 gene [CaHSP70]) was localized on the highest-M(r) (R1; approximately 3.8 Mb) chromosome of C. albicans as determined by pulse-field electrophoresis. CaHSP70 was expressed after heat shock, as demonstrated by Northern (RNA) blotting and reverse transcriptase-PCR with specific pairs of oligonucleotide sequences and gene probes. A recombinant protein was obtained in Escherichia coli after cloning of the full coding sequence into the BamHI site of the pDS56/RBSII6xhisE- plasmid and purification by nickel chelate affinity chromatography. The recombinant protein (6xhis-CaHsp70) was efficiently recognized in immunoblots by a monoclonal antibody directed against a common epitope of eukaryotic Hsp70 proteins, as well as by sera from normal human subjects. Moreover, immune mouse sera against the purified recombinant protein recognized native, heat-inducible constituents with sizes of around 70 kDa in whole-cell protein extracts of C. albicans. Overall, our data demonstrate that CaHSP70 encodes one member of a family of proteins (Hsp70) which usually represent highly conserved immunodominant antigens of infectious agents.
机译：通过用针对整个真菌细胞的血清筛选白色念珠菌酵母形式的表达文库，克隆了编码白色念珠菌应激蛋白的cDNA（2,325 bp）。克隆的序列（CaRLV130）鉴定出一个单一的开放阅读框，其长度为1,968 bp，编码包含656个氨基酸残基（70 kDa）的蛋白质。推导的氨基酸序列与酿酒酵母SSA1基因的序列相似，为84％，该基因编码70 kDa热休克蛋白（Hsp70）家族的一个成员。通过脉冲场电泳确定，相关基因（白色念珠菌HSP70基因[CaHSP70]）位于白色念珠菌的最高M（r）（R1;大约3.8 Mb）染色体上。 CaHSP70在热休克后表达，如Northern（RNA）印迹和逆转录酶-PCR用特定的寡核苷酸序列和基因探针对所证实。将完整的编码序列克隆到pDS56 / RBSII6xhisE-质粒的BamHI位点并通过镍螯合亲和层析纯化后，在大肠杆菌中获得了重组蛋白。重组蛋白（6xhis-CaHsp70）在免疫印迹中被针对真核Hsp70蛋白共同表位的单克隆抗体以及正常人的血清有效识别。此外，针对纯化的重组蛋白的免疫小鼠血清可识别白色念珠菌全细胞蛋白提取物中大小约为70 kDa的天然热诱导成分。总体而言，我们的数据表明，CaHSP70编码一个蛋白家族（Hsp70）的一个成员，该蛋白通常代表高度保守的传染原免疫优势抗原。

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