構築及表現白色念珠菌ORF19.2730的重組DNA及其功能性研究

Abstract

白色念珠菌（Candida albicans）是人體重要的伺機性致病真菌之一，具有多形態的特性。當人體免疫力下降或受藥物副作用影響而改變體內菌落生態時，便會伺機感染黏膜組織；局部性臨床症狀如鵝口瘡、陰道炎等，嚴重者會造成全身性系統感染而死亡。近年來因為免疫不全患者增加及使用抗生素不當而產生抗藥性等因素，導致院內感染的病例不斷攀升。對白色念珠菌的基因研究將有助於感染的預防、治療、及解決抗藥性等問題。 Braun等人（2005）綜合Standford、CandidaDB、CGD等主要的白色念珠菌基因資料庫，比對總數11615個推定的ORF片段，排除重複及錯誤的部份，確認出6354個基因片段，其中包括功能不明或未確定的部分基因。實驗室先前經抑制相減雜交之比對發現orf19.2730的序列可能涉及形態變化。orf19.2730是位於第四對染色體上的ORF片段，屬於Contig19-10158，和orf19.10244為對偶基因，計有1821個鹼基對。本研究以目前電腦資料庫推定而未經實驗確認的orf19.2730為標的，因其編碼蛋白質與啤酒酵母菌的RPH1有同源性，與DNA修補有關。以啤酒酵母（Saccharomyces cerevisiae）為表現平台，藉著偵測其蛋白質表現來推測此ORF的功能性。首先以PCR的方式自白色念珠菌的Genomic DNA 擷取此ORF片段及其上游的promoter共2192個鹼基對插入YEP363質體，構築成YEP363-TA1質體。在E.coli增殖後轉殖到啤酒酵母，以Coomassie blue staining檢測，未觀察到明顯的表現。另由實驗室已構築的pETΔ5T系列質體擷取HAHis Tag序列做為標籤，連接在去除3’端終止碼的orf19.2730下游，再以西方轉漬分析偵測，發現其確實是一個能產生蛋白質的ORF。又將orf19.2730上游371個鹼基的序列分成三個片段進行β-galactosidase filter assay觀察，發現在中段-257個鹼基到-315個鹼基的區域可能有未知的負向調控之調控子存在。

Candida albicans is one of the important opportunistic fungal pathogens in human body. It has various morphologies. When body immunity deteriorates or microbial ecology in the body changes due to medical reasons, the fungus would have opportunity to infect mucous tissue; typical clinical symptoms include thrush and vaginitis. In the worst case, it would lead to general system infection and death. Recently, because of the increase of immune deficiency patients and drug resistance due to improper use of antibiotics, the cases of nosocomial infection keeps rising. Genetic and functional studies on Candida albicans may help in issues of infection prevention, treatment, and drug resistance. Previously, the laboratory has conducted suppression subtractive hybridization and found the sequence of orf19.2730 might have involved in the morphogenesis of Candida albicans. Orf19.2730 is a hypothetical gene consisting 1821 nucleotides and is in Contig19-10158 on the fourth chromosome of C. albicans. The homologous allele is orf19.10244. The sequence of orf19.2730 and the upstream promoter, 2192 nucleotides in total, was amplified by PCR. The ORF was then cloned into 2μ vector for expression in Saccharomyces cerevisiae. The promoter sequence was fused to lacZ gene and then was transformed into Saccharomyces cerevisiae for the purpose of identifying cis-acting sequences regulating the promoter activity. Western blot analysis showed that the ORF indeed could manifest, able to produce protein. β-galactosidase filter assay also showed that there may have inhibitory sequence from -257 to -315 nucleotides.