Production and Characterization of Recombinant Aspergillus fumigatus Cu,Zn Superoxide Dismutase and Its Recognition by Immune Human Sera

The Cu,Zn superoxide dismutase (SOD) of Aspergillus fumigatus has previously been purified and shown to be immunoreactive to the sera of patients with aspergillosis; however, the purification of large quantities of the enzyme for expanded immunological analysis is both difficult and time-consuming. Accordingly, a λEMBL3 A. fumigatus genomic library was screened with degenerate oligonucleotides based on N-terminal amino acid sequence data; from this initial screen a 1,400-bp fragment was identified, labelled, and used to screen an A. fumigatus λgt11 cDNA library. A full-length cDNA encoding Cu,Zn SOD was subsequently identified and cloned. The cDNA encodes a protein of 154 amino acids, which does not have a signal peptide. The A. fumigatus Cu,Zn SOD possesses the typical metal binding ligands of fungal Cu,Zn SODs (six histidines and one aspartic acid) and has significant overall homology with Cu,Zn SODs in general. A recombinant A. fumigatus Cu,Zn SOD has been expressed in Pichia pastoris, is enzymatically active, and has biochemical and biophysical properties that are similar to those of the native enzyme. A sheep polyclonal antibody raised against purified native A. fumigatus Cu,Zn SOD was reactive to the recombinant enzyme by immunoenzyme development of Western blots. Sixty percent of serum samples from patients with A. fumigatus infections were reactive against the recombinant Cu,Zn SOD via immunoenzyme development of Western blots, indicating that the recombinant protein may be useful in the serodiagnostic identification of A. fumigatus infections.

此前已有研究对烟曲霉（Aspergillus fumigatus）的Cu,Zn超氧化物歧化酶（Cu,Zn superoxide dismutase, SOD）进行了纯化，并证实其可与曲霉病患者血清发生免疫反应；然而，为开展扩展型免疫学分析而大量纯化该酶的过程既困难又耗时。据此，研究人员基于N端氨基酸序列数据，利用简并寡核苷酸对λEMBL3载体构建的烟曲霉基因组文库进行了筛选；通过初次筛选获得了一段1400 bp的片段，将其标记后用于筛选烟曲霉λgt11 cDNA文库。后续研究成功鉴定并克隆了一段编码Cu,Zn SOD的全长cDNA。该cDNA编码一条含154个氨基酸的蛋白质，且不含有信号肽。烟曲霉Cu,Zn SOD具备真菌Cu,Zn SOD典型的金属结合配体（6个组氨酸残基与1个天冬氨酸残基），且整体上与各类Cu,Zn SOD具有显著的序列同源性。研究人员在巴斯德毕赤酵母（Pichia pastoris）中成功表达了重组烟曲霉Cu,Zn SOD，该重组酶具备酶活性，其生化与生物物理特性与天然酶高度相似。以纯化的天然烟曲霉Cu,Zn SOD为抗原制备的绵羊多克隆抗体，可通过免疫酶法在蛋白质印迹（Western blots）实验中与重组酶发生反应。经蛋白质印迹的免疫酶法检测显示，60%的烟曲霉感染患者血清样本可与重组Cu,Zn SOD发生反应，这表明该重组蛋白可用于烟曲霉感染的血清学诊断。