人類抗單純泡疹病毒第一型抗體片段的產生及其特性分析

Generation and Characterization of Human Anti-Herpes Simplex Virus Type-1 Fab Fragments

杜致明(Du Zhi-Ming)

指導教授 ： 楊沂淵

臺北醫學大學/醫學院/細胞及分子生物研究所/碩士(1998年)

摘要

單純泡疹病毒疾病(Herpes Simplex Viral Diseases) 是一種常見的疾病。單純泡疹病 毒(Herpes Simplex Virus; HSV)主要分成第一型(Type I)及第二型(Type II)，其中以第 一型的感染佔較大的比例及致死率。人類單株抗體(Human Monoclonal Antibodies)在病 毒疾病的預防及治療上有很重要的潛在性。聯合基因組庫技術(Combinatorial Library T echnique)是一種利用噬菌體表現系統(Phage Display System)來大量製造並篩選對特定 抗原特異性之抗體的技術，目前這種技術也被利用來製造一些單株抗體。我們由帶有抗HS V-1抗體的健康人的單核球，分離出RNAs並反轉錄成cDNAs，再以聚合酵素鏈鎖反應(Polym erase Chain Reaction)放大輕鏈(包含κ和λchain)及重鏈(Heavy chain)的基因片段， 並選殖入pComb3載體中，組成四個基因組庫。我們由λ輕鏈擊重鏈所建構的噬菌體基因組 庫中，篩選(Biopanning)出帶有抗HSV type I(HSV-1)抗體基因的噬菌體，經由富化(Enri chment)之後隨機挑選50個菌株，以膠體電泳分析來確認其質體DNA的大小，其中有28個菌 株是正確的。再由這28個菌株中挑選20個利用IPTG來誘導抗體蛋白片段(Fab)的表現，並 以西方點墨法(Western Blotting Assay)，酵素結合免疫吸附分析法(Enzyme-linked Imm unoabsorbent Assay; ELISA)，及免疫螢光法(Immunofluorescent Assay)來分析Fab的特 性。由結果得知，我們有將Fab片段由大腸桿菌中誘導出來且為人類的抗體，且這些Fab片 段和HSV-1 Ag有結合能力。以上述的結果，我們得知可由大腸桿菌中製造出具有和HSV-1 Ag結合的人類抗體蛋白片段。

關鍵字

單純泡疹病毒 ； 抗體片段

並列摘要

Herpes simplex viral disease is one of the most frequent diseases in human. H erpes simplex virus(HSV) includes two types : type 1 and type 2, and the infec tion of type 1 has occupied higher proportion and mortality. Human monoclonal antibodies have considerable potential in the prophylaxis and treatment of vir al disease. Combinatorial library technique uses phage display system to produ ce the most diversified immunological repertoire. In present days, this techni que has been used to produce some monoclonal antibodies. We isolated periphera l mononuclear cells from one healthy individual with anti-HSV-1 Ab and extract ed total RNAs, which were reversely transcribed to cDNAs. Polymerase chain rea ction was used to amplify the fragments of heavy chain and light chain(includi ngκandλ) which were then cloned into pComb3 vector. We constructed four comb inatorial antibody libraries from the individual. After biopanning, we used ag arose analysis to randomly check 50 clones and found that 28 contained correct size of recombinant DNA. Furthermore, we used IPTG to induce the expression o f Fab fragments. Twenty of these 28 clones were chosen to analysis the charact eristic of the Fabs using western blotting assay, enzyme-linked immunoabsorben t assay(ELISA), and immunofluorescent assay(IFA). According to the results, we found that the Fabs were expressed in E. coli and indicated that the Fab frag ments the binding capacity to HSV-1 Ag. In summary, we generate the human Fab fragments from E. coli and these fragments had the capacity to HSV-1 Ag.

並列關鍵字

Herpes Simplex Virus ； Fab Fragments