- 學位論文
研究人類烯醇化酶單株抗體重鏈及輕鏈的重組
Investigation of promiscuity in heavy chain and light chain on anti–alpha enolase antibodies
摘要
中文摘要 西元1983年起癌症為我國十大死因之首，近年肺癌罹患率及死亡率皆有增加趨勢。西元2009年肺癌在男性癌症死因排行中僅次於大腸癌及直腸癌而位居第三。肺癌症狀不具特異性，咳嗽為其主要症狀，容易與其他肺部疾病混淆，往往讓患者不易察覺而錯失在早期診療之良機。烯醇化酶–Ι（alpha–enolase, ENO – 1）為重要之糖酵解酶，目前已被用作確定患者罹患肺癌之診斷指標。過去研究顯示早期非小細胞性肺癌病患在ENO – 1有較高表現量，其患者存活率較低，故可利用ENO – 1推測非小細胞性肺癌患者腫瘤的惡性程度。 本研究依據先前構築完成之L1–L6抗human alpha–enolase的scFv，由於蛋白的表現量和結合能力無法應用至臨床上，為改善此問題而使L1–L6抗human alpha–enolase的scFv在日後具有更佳應用性，使L1–L6抗human alpha–enolase之單株抗體具有高度的表現量和結合力，因此將原有L1–L6抗human alpha–enolase已構築好之單株抗體以聚合脢連鎖反應（Polymerase Chain Reaction, PCR）富化，其產生之重鏈及輕鏈基因片段分別為450–bp及400–bp並將其進行重組，以第二次PCR連接抗體重鏈和輕鏈DNA成為scFv的抗體片段，再經由DNA電泳可確認此抗體片段長度為750–bp。利用噬菌體展現技術製造帶有human alpha–enolase蛋白抗體片段的基因庫，將抗體的重鏈和輕鏈DNA送入pComb3x，以VCSM13感染帶有質體的大腸桿菌XL–1，建立scFv噬菌體基因庫，scFv short linker 大小為2.4×103，scFv long linker 大小為9×102。由建構之抗體基因庫利用四次週期篩選（panning）出具有與human alpha–enolase蛋白結合能力的單株抗體片段。篩選出的scFv片段以sfi1切出長度為750–bp的抗體片段，再將噬菌質體DNA以轉形方式送入TOP10F’之大腸桿菌進行表現，挑選分子量約28 kDa之單株抗體片段蛋白的菌株進行序列比對。由西方墨點法及酵素連結免疫分析結果篩選出對於human alpha–enolase的單株抗體具有高度表現量及結合力之抗體片段，未來在肺癌的臨床診療上，能夠有助於患病初期及早發現。
關鍵字
烯醇化酶並列摘要
Abstract Since A. D. 1982, cancer has been the first leading causes of death in Taiwan. The morbidity and mortality rates have been rising in the recent years. According to the recent report in A. D. 2009 by Department Of Health, R. O. C., lung cancer is the third most common cancer for male population in Taiwan. It is in urgent need to develop a rapid test with high specificity and sensitivity for diagnosis of lung cancer. Among men in Taiwan, lung cancer is the third most common cancer.It has non–specific symptoms of lung cancer. Cough is the main symptom, easy to be confused with other pulmonary diseases. Patients are not aware of this so that the lunge cancer cannot be found and treated early. Lung cancer is often so difficult to consciously and to enable find, So Patients cant early diagnosis and treatment . Alpha–enolase （ENO – 1） is a key glycolytic enzyme , that has been used as a diagnostic marker to identify human lung cancer. According to previous studies early stage of non–small cell lung cancer （NSCLC） patients with tumors expressing relatively higher ENO – 1 level were tightly correlated with poorer survival outcomes. Therefore, data strongly support a prognostic role of ENO – 1 in determining tumor malignancy of patients with NSCLC. This study is based on completion of L1–L6 anti–human ENO – 1 of monoclonal antibodies. We have previously isolated several anti–human ENO – 1 scFv antibodies from chicken. However, their binding affinity may not be satisfied for diagnostic/therapeutic application in clinic. Thus, this present study is to randomly combine the heavy and light chain variable region genes of L1 to L6 clones and to select any scFv antibodies with improved binding affinity to human ENO – 1 protein. To construct anti–Human alpha–enolase monoclonal antibodylibraries, overlap PCR was performed to generate a variable of DNA fragments containing heavy and light chain variable genes. These scFv DNA fragments were cloned into pComb3x VI vectors and then transfected into the XL–1 Blue E. coli following infected by VCSM13 phage to obtain recombinant phage. After four rounds panning by using recombinant phage, total DNA was transfected into TOP10F’ E. coli. We randomly select clones to express scFv fusion proteins and compare the sequence. And the scFv monoclonal antibody could bind to the Human alpha–enolase both in Western blot and ELISA analysis. Thus, we generated the anit–human alpha–enolase scFv monoclonal antibody with high protein expression and binding ability. The fragment of Human alpha enolase can be filtered out so that this can be helpful in the early detection or therapy of lung cancer in the clinical.