La Valle R, Bromuro C, Ranucci L, Muller H M, Crisanti A, Cassone A
Department of Bacteriology and Medical Mycology, Istituto Superiore di Sanità, Rome, Italy.
Infect Immun. 1995 Oct;63(10):4039-45. doi: 10.1128/iai.63.10.4039-4045.1995.
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.
通过用抗完整真菌细胞的血清筛选白色念珠菌酵母形式的表达文库，克隆并测序了一个编码白色念珠菌应激蛋白的cDNA（2325 bp）。克隆的序列（CaRLV130）鉴定出一个单一的开放阅读框，长度为1968 bp，编码一个含有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基因的序列相似性为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]）定位于白色念珠菌的最高分子量（R1；约3.8 Mb）染色体上。如通过Northern（RNA）印迹以及使用特定的寡核苷酸序列对和基因探针进行逆转录酶-PCR所证明的，CaHSP70在热休克后表达。将完整编码序列克隆到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血清有效识别。此外，针对纯化重组蛋白的免疫小鼠血清在白色念珠菌全细胞蛋白提取物中识别出大小约为70 kDa的天然热诱导成分。总体而言，我们的数据表明CaHSP70编码一个蛋白质家族（Hsp70）的一个成员，这些蛋白质通常代表感染因子的高度保守的免疫显性抗原。
