- 學位論文
抗白色念珠菌之α-異烯醇酶多株與單株抗體的製備與定性
Generation and Characterization of Polyclonal and Monoclonal Antibodies against Candida albicans Alpha-Enolase
摘要
中文摘要 白色念珠菌為一種伺機感染的人類病原菌，主要寄生在皮膚，口腔，腸胃道與陰道等部位中。白色念珠菌為造成臨床上念珠菌菌血症(50.4%) 的主要病原菌。念珠菌菌血症為真菌界中的念珠菌屬感染並進入病人的血液中，在健康的人是極為罕見。然而，近數十年來，患有免疫功能有缺陷病人族群如器官移植，血液腫瘤，接受免疫抑制劑或化學治療，愛滋病等病人的增加，念珠菌血症成為臨床上重要的課題，念珠菌菌血症會引起其他的併發症及造成高死亡率。在美國，念珠菌菌屬為造成院內血液感染的第四名，也造成了49%的死亡率。烯醇化酶–Ι（alpha–enolase, ENO-1）為重要之糖酵解酶。於患有念珠菌菌血症的病人血液中可以測到烯醇化酶–Ι，若將白色念珠菌的烯醇化酶(CaENO1)基因突變，也會抑制白色念珠菌的生長。CaENO1可以被用來發展針對念珠菌菌血症快速檢驗試劑以及治療藥物的重要指標蛋白。本實驗中，我們利用大腸桿菌表現和純化CaENO1重組蛋白，並將其表現與大量地純化出來，用這些重組的CaENO1免疫雞隻，產生高效價的IgY 抗體反應。這些多株抗體利用 Western blot、ELISA也都顯示它們對於CaENO1 蛋白有特異性的結合能力。為了製作針對CaENO1的單株抗體，我們也利用是菌體抗體基因庫的技術構建兩個分別具有2.4×106 與1.36×107 含有short或 long linker的抗體基因庫，並且進一步地，利用此兩個基因庫來篩選出對CaENO1有特異性結合的單株抗體。其中我們篩選到一株CaS1抗體，在Western blot的實驗中CaS1抗體可特異性的辨認到C. albicans 與Candida tropicalis 。以流式細胞儀分析(BD FACSCalibar)可得知，CaS1抗體和C. albicans可以特異性的結合。CaS1抗體和白色念珠菌作用後亦可抑制白色念株菌在YPD培養基上的生長，亦可抑制白色念珠菌對口腔上皮細胞(OECM-1)的貼附能力。在小鼠念珠菌血症模式中，實驗結果可得知CaS1抗體可延長念珠菌血症小鼠存活時間。於Fibrin matrix-gel degradation 分析，可知CaS1可抑制C. alicans 表面alpha-enolase 與plasminogen得結合。由以上實驗可知，CaS1抗體有發展成為臨床檢驗試劑的潛力。更待進一步實驗確認CaS1抗體用於臨床上白色念珠菌所引發的病症，其治療與應用的價值。
並列摘要
C. albicans is an opportunistic human pathogen, which colonizes at several sites including skin, oral, gastrointestinal track and vagina. C. albicans is also a major pathogen caused clinical candidemia of 50.4%. Candidemia is a fungal infection that can occur when Candida yeasts enter the bloodstream, is extremely rare in health people. In recent decades, due to a defective immune patient population such as organ transplantation, blood cancer, receiving immunosuppressive, or chemotherapy and AIDS patients increasing, Candidemia becomes important issue. Candidemia has a very significant morbidity and mortality. In the United States, Candida species are the fourth leading cause of nosocomial bloodstream infection, and the attributable mortality to be 49%. Alpha–enolase（ENO1） is a key glycolytic enzyme . C. albicans Alpha–enolase (CaENO1) can be detected in the sera of candidemia patient, and mutations on Candida alpha-enolase in C. albicans would inhibit cell growth. CaENO1 were used as an important marker protein in medical application. In this study, CaENO1 recombinant DNA transformed into E. coli, CaENO1 protein could be expressed and purified. After the chicken immunized with CaENO1 recombinant protein, it succeed to generate the polyclonal chicken IgY. These polyclonal antibodies could specifically bind to CaENO1 protein in Wesrern blot and ELISA. Following, we used phage display technology to construct two scFv（single chain variable fragment）antibody library (short linker and long linker, 2.4×106 and 1.36×107 , respectively ). After the fourth round of bio-panning, we selected a monoclonal antibody CaS1 with specific binding ability to CaENO1. In Western blot analysis, CaS1 antibody can specifitily recognize to alpha-enolase on C. albicans and Candida tropicalis. CaS1 antibody can bind to C. albicans surface alpha-enolase in flow cytometry analysis. CaS1 can also inhibit Candida albican growing on YPD agar plate and reduce Candida albican binding ability to oral epidermoid carcinoma cell line (OECM-1) cell. In vivo test showing that CaS1 can prolong survival of candidemia mice. CaS1 can inhibit C. albicans surface alpha-enolase binding to plasminogen by fibrin matrix-gel degradation analysis. According to the results, CaS1 antibodies could be used to detect and treat C. albicans infection in the future.