本文選題：白念珠菌　切入點：基因表達序列分析　出處：《第三軍醫(yī)大學》2005年碩士論文

【摘要】： 白念珠菌是一種重要的條件性致病真菌,在各種真菌感染中占了首位。感染人的粘膜表面可引起鵝口瘡、念珠菌性陰道炎疾病;在系統(tǒng)性疾病的病人和免疫缺陷的人群中,白念珠菌可以引起全身的播散性感染并導致死亡。白念珠菌的毒力因子和發(fā)病機理有關,主要包括促進白念珠菌粘附于宿主細胞的生物分子(黏附素),與侵入有關的酶SAP(分泌型天冬氨酸蛋白酶)和PL(磷脂酶),及其菌相轉(zhuǎn)換(生長狀態(tài),可逆的單個酵母細胞和菌絲的轉(zhuǎn)換)。并且,菌相轉(zhuǎn)換和抗原表達的改變有關。 為更全面的尋找白念珠菌酵母相和菌絲相致病相關基因及其毒力因子,本實驗室前期應用LongSAGE技術,通過構建白念珠菌酵母相和菌絲相細胞LongSAGE標簽庫,來定性定量檢測白念珠菌酵母相和菌絲相細胞表達基因的改變,并聚類分析表達基因功能,探討酵母相和菌絲相細胞表達基因與菌相轉(zhuǎn)換、菌株毒力的相關性。 為了驗證LongSAGE標簽,我們利用SAGE標簽產(chǎn)生長片段cDNA應用于基因識別技術(generation of longer fragments from serial analysis of gene espression(SAGE)tags for gene identification GLGI),將17bp的SAGE標簽向3’端擴增到數(shù)百bp。17bp的LongSAGE標簽作為正向引物,3’端使用的是單一堿基dA、dC或dG與oligo-dT結合的引物進行第1次PCR擴增,只有與LongSAGE標簽相匹配的序列才能退火并得以擴增;在第2次循環(huán)中,只有5’端oligo-dA的前一堿基與錨定的oligo-dT相匹配的序列才能擴增,而單純的oligo-dA與oligo-dT結合的片段因處于錨定狀態(tài)則受抑制,因而最終得到的是含有LongSAGE標簽的長片段DNA擴增產(chǎn)物,產(chǎn)物長度在200～1000bp之間。 GLGI技術提供了幾種潛在的結果。首先,它為LongSAGE技術提供了發(fā)展策略;其次,結合應用LongSAGE-GLGI技術可以作為人或其它物種的基因的選擇和表達;再次,它可以鑒定任何基因的3’端序列的外顯子;最后,結合應用LongSAGE-GLGI技術可以確定人或其它物種的3’端在基因家族中所表達基因。 20個多重匹配標簽中有15個獲得穩(wěn)定有效的擴增,長度在200～1000bp之間,獲得的長片段進行序列分析后,有超過95%的堿基與已知基因相同,并且包括原來17bp的SAGE標簽,表明獲得的序列為此已知基因。其中酵母相多重匹配標簽17是基因
[Abstract]:Candida albicans is an important opportunistic fungus, leading the way in fungal infections. Infected mucosal surfaces can cause thrush, candida vaginitis; in patients with systemic diseases and in people with immune deficiency, Candida albicans can cause systemic disseminated infections and lead to death. The virulence factor of Candida albicans is related to the pathogenesis of Candida albicans. These include biological molecules that promote Candida albicans adhesion to host cells (adhesin), invasion-related enzymes SAP (secretory aspartate protease) and PLL (phospholipase), and their phase transition (growth state). Reversible transformation of individual yeast cells and hyphae. In order to search for the pathogenicity genes and virulence factors of Candida albicans in yeast and hyphal phase more comprehensively, LongSAGE technique was used to construct the LongSAGE tagging library of Candida albicans yeast and hyphae cells. The expression genes in yeast and hyphal phase of Candida albicans were detected qualitatively and quantitatively, and the function of expressed genes was analyzed by cluster analysis, and the relationship between the expression genes of yeast phase and hyphae phase and the virulence of the strain was discussed. To verify the LongSAGE tag, We use SAGE tag to produce long fragment cDNA and apply it to gene recognition technique: generation of longer fragments from serial analysis of gene espression(SAGE)tags for gene identification GLGIN. 17bp SAGE tag is amplified to 3'end of 17bp and hundreds of bp.17bp LongSAGE tags are used as forward primer. The primers combined with oligo-dT were amplified by PCR for the first time. Only the sequence matching the LongSAGE tag can be annealed and amplified; in the second cycle, only the first base of the 5'terminal oligo-dA can be amplified by matching the anchored oligo-dT. However, the simple oligo-dA and oligo-dT binding fragments were inhibited because they were in the anchoring state, so the final product was the DNA amplification product containing the LongSAGE tag, and the length of the product was between 200~1000bp. GLGI technology offers several potential results. First, it provides a development strategy for LongSAGE technology; secondly, the combination of LongSAGE-GLGI technology can be used as gene selection and expression of human or other species. It can identify the exons of the 3'terminal sequence of any gene. Finally, the 3'terminal gene expressed in the gene family can be identified by using LongSAGE-GLGI technique. Fifteen of the 20 multiple matching tags were stably and effectively amplified, and the length of the fragments was between 200~1000bp. After sequence analysis, more than 95% of the bases were identical to the known genes and included the original 17bp SAGE tag. The obtained sequence is a known gene, and yeast phase multiple matching label 17 is a gene.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2005
【分類號】：R379

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本文編號：1662620