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  • 學位論文

證明及選殖p48腫瘤相關抗原基因

Identification and cloning p48 as a tumor-associated antigen gene.

指導教授 : 施能耀 林鈺玲
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摘要


自從HOM-Mel-40抗原在皮膚黑色素瘤的病患中,以血清學方法被鑑定為腫瘤相關抗原之後,更加肯定在癌症病人體內具有對抗自體腫瘤的免疫力。因此,利用自體細胞毒素T淋巴細胞( autologous cytotoxic T lymphocytes )或其血清抗體去選殖腫瘤抗原,成為一種可行的手段。在本篇實驗中,所使用的抗體是從肺癌病患CA926的胸水所純化而來。此抗體辨識自體抗原p48具有專一性,而且是屬於免疫球蛋白G及M類別,而非免疫球蛋白A。西方點墨法分析以CA926抗體做為探針,顯示在非癌肺組織及代表正常的肺表皮細胞株WI38中,CA926抗體能辨識的p48抗原並未被偵測到,因此推測在癌化過程中p48蛋白可能是過量表現於腫瘤細胞中。同時,CA926抗體之標的物,p48抗原也出現於另一株肺腺癌腫瘤細胞株PE089中。經體外培養,其細胞裂解物可大量獲得,並利用DEAE陰離子交換樹脂及硫酸銨鹽沉澱等生化技術,將此p48抗原純化。隨後, p48蛋白再經由12.5 %含SDS之長蛋白膠中分離,硝酸銀染色使蛋白顯現,之後即切下p48蛋白並以質譜儀分析,來鑑定其蛋白。此結果認為p48蛋白為α-enolase。為了進一步確認α-enolase是CA926抗體的目標蛋白( target protein )。本研究中以分子選殖方式產生GST- enolase融合蛋白並表現於JM109大腸桿菌細胞中。再利用西方點墨法以CA926抗體為探針,其結果顯示CA926抗體能辨識GST- enolase融合蛋白與經thrombin蛋白酶切除GST後的enolase蛋白,但未能辨識GST tag蛋白。此證實了α-enolase確實為 CA926抗體之目標蛋白。而它的過量表現可能與癌化過程相關。在未來的實驗中,我們有興趣知道有多少癌症病患血清中含有對α-enolase有辨識能力之抗體,此外,並探討其存在是否與疾病的惡化有所關聯。因此,在本研究後,約100位癌症病患的胸水或腹水將會被篩檢以回答以上問題。

關鍵字

腫瘤抗原

並列摘要


After HOM-Mel-40 was serologically identified as a tumor associated antigen from melanoma patients, it confirmed that cancer patients contained immunity against their own tumors. Therefore, cloning of these tumor antigens by either their autologous cytotoxic T lymphocytes or serum antibodies become feasible. In my research, the antibodies were purified from pleural effusion of a lung cancer patient, CA926. The antibodies have recognition specificity for p48 autologous antigen and also demonstrated that CA926 antibodies recognizing this antigen belonged to IgG and IgM, but not IgA class. Western blotting analysis using CA926 antibodies as probe showed that the epitopes of p48 did not appear in non-cancer lung tissues and a “normal” lung epithelial cell line, WI38, suggesting that p48 might be overexpressed during tumorgenesis. The CA926 antibodies’ target is also found in a lung adenocarcinoma cell line, PE089. Using DEAE anionic column purification and ammonium sulfate “salting-out” precipitation, p48 antigen in PE089 cell lysate was successfully enriched in these processes. The enriched p48 fractionation subsequently separated by a long-ranged 12.5% SDS-containing acrylamide gel. The concentrated p48 band was visualized by silver staining and cut out for mass spectrometric analysis. The result indicated that p48 was α-enolase protein. Furthermore, we generated and expressed a GST-enolase fusion protein in JM109 E. coli. cells. The purified protein was immuno- blotted with CA926 antibodies and removed the GST tag by thrombin treatment, confirming that α-enolase was a target of CA926 antibodies. It is interesting to know the occurrence of cancer patients’ serum against α-enolase; therefore, in the near future, around 100 cancer patients’ effusion or ascites antibodies will be screened for estimating the occurrence.

並列關鍵字

無資料

參考文獻


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