利用聯合基因庫技術來選殖和表達類風濕性因子

Molecular Cloning and Expression of Rheumatoid Factor Gene Using Combinatorial Library Technique

蔡演忠(Yen-Jung Tsai)

指導教授 ： 楊沂淵

臺北醫學大學/醫學院/醫學科學研究所/碩士(1996年)

摘要

類風濕性因子(RFs)是一種自體抗體, 能夠結合免疫球蛋白G(IgG)的 Fc部份, 在類風濕關節炎病人的發炎組織及其他自體免疫疾病病人身上都 可以被偵測到. 先前分析IgG RF的研究發現, 在抗體的變異區(variable region)出現許多變異(mutations), 顯示, IgG RF可能是經由抗原刺激所 造成. 為了解IgG RF在人體中所扮演的角色, 我們利用聚合脢連鎖反應( Polymerase Chain Reaction, PCR), 從周邊血液中的單核血球, 成功的 放大出抗體 (antibody)的重鏈(heavy chain)及輕鏈(light chain)基因, 並接入載體(vector) pCom3 而組成一個含有2*10^5個菌株的聯合基因庫( combinatorial library). 任意挑選10個菌株, 以內限制脢(restriction enzyme)來確認, 所有篩選的10個菌株的vector內都含有heavy chain及 light chain的基因片段, 利用對於IgG Fc的結合能力來篩選後, 再任選8 個菌株,利用IPTG來誘導抗體蛋白的表現, 再以酵素結合方式免疫吸附分 析法(ELISA)及西方式轉漬法(western blot)來確認, 結果顯示, 有超 過80%的菌株所表現之抗體已被誘導出來, 而且與IgG Fc具有結合能力. 就目前所得的結果而言: 以聯合基因庫技術來選殖和表達出RFs已得初步 的結果, 未來希望能把此單源抗體應用於臨床的診療上或者應用此聯合基 因庫技術去選殖其它的人類單源抗體.

關鍵字

聯合基因庫 ； 類風濕性因子 ； 基因 ； 免疫球蛋白G ； 聚合脢連鎖反應 ； 載體pComb3

並列摘要

Rheumatoid factors(RFs) are Ig which bind to the Fc regions of IgG molecules.They can be detected in the circulating blood and the inflammatory tissue ofpatients with rheumatoid arthritis(RA) and other autoimmune disorders. Previousanalysis of IgG rheumatoid factors(RFs) from patients wiht rheumatoid arthritis( RA) revealed that the majority contained signigicant numbers of skewed mutations per variable(V) region, suggestion that these RFs arouse from antigendriven responses. To further study the roles of IgG RFs in human body, we used pComb3 vector to construct a IgG;?amda combinatorial antibody library from theperipheral mononuclear cells(PMNC). Both heavy and light chains were successfully amplified using polymerase chain reacton (PCR). A combinatorial library was constructed and contained 2*10^5 clones in size. Restriction analysis of 10 randomly chosen clones revealed that the selected clones had H and L chain inserts. After panning against human IgG Fc molecule, Fab fragment in the total cellular lysates from 8 clones bound to Fc-coated wells on ELISA plates. The expressed Fab antibody of these 8 clones was further confirmed by Western blotting analysis. These results from this study demonstrated the combinatorial library technique is highly powerful in the cloning and selection of RF autoantibodies and should be considered as an alternative method other than conventional hybridoma technique in the generation of monoclonal antibodies.

並列關鍵字

Combinatorial library ； Rheumatoid factor ； Gene ； IgG ； PCR ； pComb3