證明及選殖p48 腫瘤相關抗原基因
Identification and cloning p48 as a tumor-associated antigen gene.
中文摘要
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。
英文摘要
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.
