非洲布魯氏錐蟲，俗稱非洲錐蟲，為一種單細胞真核類寄生性原生動物，可引起非洲昏睡病。其中新發現的TWD-1基因含有2154個核苷酸、可轉譯出589個胺基酸、分子量約65.3 kD之蛋白質，TWD。經比對資料庫顯示，TWD具有WD重覆區段、Leucine Zipper，以及ERM相似區，顯然是個多domain且多功能的蛋白質。ERM蛋白質其N端會與細胞膜上蛋白作用，如 : CD43、CD44、ICAM-1、-2、-3等，而C端會與actin binding，參與細胞骨架的作用，包含運動、附著、收縮及分裂。因此ERM蛋白質被認為是細胞膜及細胞骨架的連接蛋白質，在訊息傳遞與細胞運動上扮演極重要的角色。進一步比對蛋白質序列，顯示NCOⅠ限制酶片段內的胺基酸和ERM的actin binding區有極高的相似度，提供了TWD-1基因可能參與的生理作用。至目前為止，發現WD repeats參與許多種重要的生理現象，如細胞分裂、基因轉錄、成熟RNA的修飾與跨越細胞膜之訊息傳遞等，且至今已發現有許多含WD repeats之蛋白質會與細胞骨架之微管系統作用。更可說明TWD-1基因在生物體內可能扮演一重要角色。
本論文以NCOⅠ限制酶切出之DNA片段所隱含的36kD蛋白質為研究重心。利用pET質體特性，先確定表現36kD蛋白質在細菌內的條件，再大量收取蛋白質，並以鎳親和管柱配以兩次梯度法脫去尿素及脫離親和柱，得到純化的蛋白質。此蛋白質經Edman degradation及MALDI-TOF法均確認此蛋白質和DNA隱含的密碼相符。這個純化後的蛋白質接著被做為抗原，注入兔子體內得到多株抗體後，利用此抗體做了以下實驗:1.確認TWD蛋白質在錐蟲體內為一65kD之蛋白質，與電腦預估相當，沒有明顯改變分子量的轉譯後修飾作用(post-translational modification)在此蛋白質上。2.經過不同程度的處理，可以看出TWD蛋白質主要是在detergent-resisitant fraction內，符合屬於骨架蛋白質之相關蛋白質定義。��|以免疫沉澱法與不同的fraction作用，可以看出TWD和微管成分蛋白質tubulin連結，和actin蛋白質也有連結關係。由2與3看出，TWD是骨架蛋白質之一份子，和微管及微絲兩大細胞骨架蛋白質都有連結。至於TWD在連結兩大系統這中間的角色，甚至和細胞骨架主導的細胞型態、生長與增殖的生理機制有何影響就留得後續的研究了。

Trypanosoma brucei is a unicellular eukaryote that parasitizes in mammals and insects to cause African sleeping sickness of human being. A novel gene, TWD, is found in the organism that consists 2154 base of its full length cDNA and encodes a 65.3 kD protein. Similarity search showed that it contains 7 WD repeats protein at its entire C-terminal half and 7 leucine zipper and ERM protein motifs in WD repeat N-terminal extension. TWD obviously is a multi-domain protein which likely has a multi functions in the cell. WD repeat proteins have been found to participate in many important processes of a cell such as cell division, transcription, mRNA modification, proteasome-mediated protein degradation, signal transduction across cell membrane, and cell apoptosis. In database, TWD’s ortholog is PF20, a bridging protein of central tubules of axoneme in a green algae. In this thesis, the focus was set at an NCO I restriction fragment of TWD gene which contains motif of ERM protein, a protein family linking actin and cell membrane. The NCO fragment was expressed as inclusion body inside E. coli with pET system to have His-tag at its C terminus by 0.8mM IPTG induction at 37℃ for 5 hours. The expressed 36kD protein was collected and purified through Ni+2 charged affinity chromatography with two gradient steps to remove urea and later depart the adsorbed protein at 0.52M imidazole. Both Edman degradation and MALDI-TOF methods proved protein sequence as predicted from DNA sequence. This 36kD protein was then used to elicit rabbit polyclonal antiserum, NCO B, which was able to recognize a 65 kD protein in T. brucei cell lysate by Western method. Immuno-coprecipitation experiments showed that NCOB could pull down tubulin but not actin in the cytoplasm. In the cytoskeleton portion, TWD could be detected with tubulin and also with actin. Fractionation experiments showed that TWD resisted Triton X-100 wash and maintained with cytoskeleton. In summary, an antigen corresponding to part of TWD gene was prepared and used to elicit antiserum. Through this antiserum, TWD could be detected in the cell lysate without obvious modification and existed in the cytoskeleton. Most likely, TWD should be a tubulin and actin linker protein.

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