Alloush H M, López-Ribot J L, Chaffin W L
Department of Microbiology and Immunology, Texas Tech University Health Sciences Center, Lubbock 79430, USA.
J Med Vet Mycol. 1996 Mar-Apr;34(2):91-7.
Five cDNA clones were selected from the positive clones detected by screening a germ tube expression library constructed in lambda gt11 with rabbit antisera raised against cell wall extracts of Candida albicans. The selected clones were amplified and used to obtain affinity purified antibodies by eluting from the expressed proteins that had been previously transferred onto nitrocellulose discs. The antibodies obtained were used as probes in immunoblots of the cell wall extracts separated by denaturing polyacrylamide electrophoresis. A single protein band was detected for each clone. Detection of products of the cloned sequences varied according to the extraction procedure and/or cell morphology. These products included bands exhibiting apparent molecular weights of 40, 58, 68 and 70 kDa present in beta-mercaptoethanol (beta ME) extracts from both yeast and germ tubes, and a 30 kDa beta ME extracted protein specific for germ tubes. The expression of these products at the cell surface was confirmed by indirect immunofluorescence. Expression of the mRNAs of the different cDNA clones varied according to growth- and morphology-related factors and showed no direct correlation between expression and presence in the cell wall. These observations suggest that complex mechanisms are involved in the regulation and expression of cell surface components of C. albicans.
从用抗白色念珠菌细胞壁提取物的兔抗血清筛选λgt11构建的芽管表达文库检测到的阳性克隆中挑选出5个cDNA克隆。对所选克隆进行扩增，并通过从先前转移到硝酸纤维素膜上的表达蛋白上洗脱来获得亲和纯化抗体。所获得的抗体用作变性聚丙烯酰胺电泳分离的细胞壁提取物免疫印迹中的探针。每个克隆检测到一条单一的蛋白带。根据提取程序和/或细胞形态，克隆序列产物的检测结果有所不同。这些产物包括在酵母和芽管的β-巯基乙醇（βME）提取物中出现的表观分子量为40、58、68和70 kDa的条带，以及芽管特有的30 kDaβME提取蛋白。通过间接免疫荧光证实了这些产物在细胞表面的表达。不同cDNA克隆的mRNA表达根据生长和形态相关因素而有所不同，并且在细胞壁中的表达与存在之间没有直接相关性。这些观察结果表明，白色念珠菌细胞表面成分的调节和表达涉及复杂机制。
