尼古丁型乙醯膽鹼受器（nAchR）是一種具有神經傳導物質受器和離子通道功能的膜蛋白。過去的研究指出，在尼古丁的長期暴露下，nAchR會大量表現並造成癌症如肺癌、大腸癌、膀胱癌等。其中的α9 nAchR的大量表現則跟乳癌形成相關。故此蛋白未來在乳癌篩檢或治療上是一個相當有潛力的標的。而為了偵測α9 nAchR的表現，能夠專一性辨識此蛋白的抗體就變得十分重要。
　　本計畫以重組之人類α9 nAchR膜外片段作為抗原，對雞隻進行免疫注射後將雞隻犧牲取脾臟以噬菌體展示（phage display）技術建立重組單鏈抗體片段（single chain variable fragment，scFv）基因庫。之後將此基因庫進行四次篩選（panning）後把篩選好的基因群轉殖到top10F’大腸桿菌表現抗α9 nAchR scFv。之後再用重組之人類α9 nAchR以酵素結合免疫分析法 (ELISA) 和西方墨點法 (Western blot) 篩選出具專一性辨識能力之scFv。最後再以α9 nAchR高表現量的MDA-MB-231人類乳腺癌細胞株以西方墨點法、流式細胞儀分析 (Flow cytometry) 以及MTT測試scFv是否能辨識細胞所表現的α9 nAchR以及是否能抑制癌細胞生長。最後我們得到一株具有明顯專一性結合細胞原生α9 nAchR能力之scFv。希望這株抗體在未來可以應用在乳癌臨床篩檢甚至是標靶治療上。

Nicotine acetylcholine receptor (nAchR) is a kind of membrane proteins with neurotransmitter receptor and ion channel function. Previous studies have pointed out that long-term exposure to nicotine, nAchR will be up-regulated expression and cause cancers such as lung cancer, colon cancer, and bladder cancer. Overexpression of α9 nAchR is associated with the formation of breast cancer. Therefore, α9 nAchR is a considerable potential molecule target in screening or treatment breast cancer in the future. In order to detect the overexpression of α9 nAchR, specific α9 nAchR antibody is very important.
We produce truncated recombinant human α9 nAchR outer membrane fragments as antigen for chicken immunization. Chicken will be sacrificed after immunization, and be established recombinant antibody fragments (single chain variable fragment, scFv) library from spleen cell by phage display technology. After four panning, enriched anti-α9 nAchR scFv phage library will be transformed to Top10F' E. coli to express anti-α9 nAchR scFv. α9 nAchR specific scFv will be selected with recombinant α9 nAchR by ELISA and Western blot. Then, we test native α9 nAchR binding ability of scFv with high α9 nAchR expression MDA-MB-231 breast cancer cell by Western blot, Flow cytometry, and immunofluorescence staining. Further, scFv will be test if can inhibit MDA-MB-231 cell proliferation in MTT test. We get a clone scFv with obvious specific binding ability to native α9 nAchR of MDA-MB-231 cell. Hope this scFv can be applied in clinical screening and treatment for breast cancer in the future.

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