類風濕性因子的產生及其特性分析
Generation anf characteri{ation of rheumatoid factor
中文摘要
聯合基因組庫 (Combinatorial library) 技術是一種利用噬菌體表
現系統 (Phagedisplay system) 來篩選和富化對某抗原具特異性的抗體
的技術，近年來常以此技術開發一些單株抗體，其中包括多種對抗人類疾
病病毒的抗體及自體免疫疾病抗體，如：anti-HSV-1, anti-HBsAg 和
anti-HIV。類風濕性因子 (Rheumatoid factor) 是一種可和 IgG 免疫球蛋
白的
Fc 部分結合的自體抗體 (Autoantibodies)，常作為類風濕性關節炎(
Rheumatoid arthritis) 病人病程的一項重要指標，更可在一些自體免疫
疾病病人的體內偵測到。為了對類風濕性因子有更進一步的了解，我們由
特發性血小板減少性紫斑病人的單核球，分離其全部的
RNAs 反轉錄成
cDNAs，再進一步以聚合脢鏈鎖反應 (Polymerase chainreaction) 放大
重鏈 (Heavy chain) 和輕鏈 (包含λ和κchain) 片段基因，並選殖入
pComb3 載體中，組成一個約含 7.9 x 10^7 的菌株的抗體基因組庫。經過噬
菌體的篩選(Biopanning) 和富化 (Enrichment)，然後利用 In situ dot
blottng 的方法由 200 個菌株中，篩選出 14 個含有對 Fc 具特異性的 Fab 抗體
的菌株，並以西方轉漬法 (Western blotting)確認 Fab 抗 體的確是由一
段重鏈和一段輕鏈所組合而成的。而由
ELISA 的結果可知這些篩選所得到
的
Fab 抗體對 Fc 分子的結合能力比對 BSA 蛋白質來得好。為了確認它們對 Fc
的特異性，我們需要作
competitive assay 的實驗，目前所有的κ輕鏈和
重鏈的基因序列已經近乎完成，一旦完成這些抗體的基因序列，經過分析
和比較，我們將可進一步地了解各種不同抗體基因在
RF 的使用情形，這方
面的資料對於將來發展治療
RA 病人的藥物應該會有莫大的幫助。
英文摘要
Combinatorial Library technique using phage display system provides a meansof capturing the vast diversity of the immunological repertoire. The display oflibraries of small
peptides (Fab) on the filamentous phage has proven to bepowerful approach for selecting ligand of defined specificity.Rheumatoid factorsare autoantibidies that bind to the Fc portion of IgG, which are thought to bemore important indicator in the pathology of rheumatoid arthritis and often canbe found in some other autoimmune diseases. For further understanding of
cells were isolatedfrom a patient withidiopathic thromocytopenia purpura (ITP) with high titer ofserum RFs. Total RNA were reversely transcribed to cDNA. Using polymerasechain reaction, both heavy and light chain genes were amplified and
acombinatorial antibody library was constructed and found to contain7.9 x 10^7clones in size. In situ dot blotting analysis of 200 clones after bio-panningagainst Fc-coated plates showed that 14 clones could be identified by goatanti-human κ chain antibody, we select 14 specificFabs against Fc. Theexpressed antibody molecules are presumably consist of one heavy chain and lightchain as demonstrated by western blotting. Based on the result of ELISA, weconcluded that the panned Fab fragments had better binding capacity to Fc thanBSA. A competitive assay is requiredto further confirm their specificity to Fcmolecules. The nucleotide sequences of all the κ chain and heavy chain
genesare almost finished. After analyzing all the gene sequences, the gene usageof heavy and light chain in a RF antibody repertoire can be further delineatedand will provide a molecular basis of drug development for RA treatment.
