| 摘要: | nanobody;phage display;aflatoxin b1現今市面上偵測有毒物質小分子的方式主要是仰賴酵素結合免疫吸附法或是質譜法分析，除了耗時費工外也經常需要透過繁瑣的動物免疫來得到所需的多株抗體，因此，在診斷用的抗體開發上，仍缺乏可以快速製備出特異性單株抗體的有效方法。本研究建立了兩個新穎性合成噬菌體呈現奈米抗體庫，經設計可特別針對小分子進行篩選而分離出高專一性的單株奈米抗體，作為後續快速篩檢的應用。奈米抗體庫建構的基礎抗體模板是來自於蛋白質資料庫中的特別奈米抗體結構，分別是具有特殊手套般凹洞構型（編號3QXV）以及具有像雙峽谷般的雙凹洞構型（編號1QD0）。藉由分析出模板奈米抗體上主導與小分子結合的胺基酸，透過點突變法使特定區域胺基酸產生突變，進而建構出獨特性並具有多樣化的奈米抗體基因庫（複雜度~1010）。之後，藉由噬菌體呈現技術（phage display）將奈米抗體庫表達於噬菌體上成為噬菌體呈現奈米抗體庫，即可進行後續生物親和性篩選。為了驗證並測試抗體庫對於小分子篩選上的獨特性，我們選用生活中常見的小分子毒素，具有強烈的肝毒性的黃麴毒素B1（Aflatoxin B1, AFB1）作為目標進行所建構的獨特奈米抗體庫的篩選測試。 研究結果顯示經由生物親和性篩選後，我們分離出了三株高親和性奈米抗體，分別是來自3QXV抗體庫的奈米抗體A1和F2，以及來自1QD0抗體庫的奈米抗體2G，在實驗中我們確認了這些奈米抗體對於AFB1的特異性結合力。並且，經由電腦輔助分析抗體結構後指出，在奈米抗體獨特的CDR-H4 loop位置上帶正電的胺基酸主導了奈米抗體與AFB1結合的交互作用。在這個研究中，我們確認了藉由電腦輔助設計合成的噬菌體呈現奈米抗體庫，確實可以有效的篩選出能特異性結合小分子的奈米抗體。在未來，可以利用這兩個抗體庫對不同的小分子進行快速的生物親和性篩選來獲得特異性的單株奈米抗體，這將會利於快篩系統檢測材料的開發。
Nowadays, strategize for detecting toxic small molecules are mainly based on enzyme-linked immunosorbent assay (ELISA) or mass spectrometry, which are time-consuming, labor and resources demanding techniques. Also, primary detection polyclonal antibodies for ELISA are usually obtained by animal immunization, which are expensive and impractical due to the high background of non-specific binding. Researchers still lack an effective method for developing specific monoclonal antibodies. In this study, two novel synthetic phage-displayed nanobody libraries were constructed, which were designed to screen toxic small molecules and isolate high-specific monoclonal nanobody for rapid screening of toxins. The antibody template for constructing libraries is a special nanobody structure from a protein data bank (PDB), which has a special glove-like recess configuration (PDB ID: 3QXV) and a double-cave configuration similar to double canyon (PDB ID: 1QD0). By analyzing amino acids in Complementarity determining regions (CDRs) that dominate the binding to small molecules, we plan to mutate the selected amino acids by Kunkel mutagenesis method, thereby constructing a unique and diverse synthetic nanobody library (complexity ~1010). Thereafter, utilizing phage display technique, a phage-displayed nanobody library is applied to four round of bio-panning. To verify the ability of synthetic nanobody libraries for screening small molecules, we select a common small molecule with hepatotoxicity, Aflatoxin B1 (AFB1), as a screening target for bio-panning.
We isolated three high-affinity nanobodies after four rounds of bio-panning, two nanobodies named A1 and F2 from 3QXV derived nanobody library, and one nanobody named 2G from 1QD0 derived nanobody library. We confirmed the specific binding of these nanobodies to AFB1 by ELISA. Furthermore, assist by homology modeling and docking, we point out that the positively charged amino acid locates on the CDR-H4 loop of nanobodies dominate the binding with AFB1. As a result, we confirme that synthetic phage-displayed nanobody libraries aided by computer design can effectively screen small molecules and isolate high-affinity nanobodies. In the future, these two nanobody libraries can be used to perform screening of different small molecules to obtain monoclonal nanobodies with high specificity, which will facilitate the development of rapid screening systems. |
