本文選題：CD40 + 抗原表位　； 參考：《蘇州大學》2011年碩士論文

【摘要】：本課題以本所自行成功研制的鼠抗人CD40激發(fā)型單克隆抗體5C11為研究對象,借助計算機輔助分子設計與蛋白質空間結構模擬技術,利用抗原-抗體相互作用原理,計算機模擬得到抗原CD40與抗體5C11的主要結合位點,從而推測出抗體識別的抗原表位;結合文獻報道的CD40-CD40L互相作用位點,通過定點突變技術、構建突變體基因轉染細胞株等生物學實驗方法,驗證計算機模建結果的可靠性,從而確定抗體識別的抗原表位,對5C11抗體進行人源化改造或研制抗人CD40抗體臨床用藥具有理論和潛在的臨床意義。 目的:通過計算機模擬與生物實驗,初步確定本所研制的抗人CD40激發(fā)型單抗5C11識別的抗原表位。 方法:利用InsightⅡ軟件分別模擬抗原、抗體結構,以及搭建抗原抗體復合物模型,通過計算并推測抗體所識別的抗原表位。通過RT-PCR的方法分別獲得人野生型CD40(wtCD40)和70位突變(70muCD40)及114位突變(114muCD40)的全長基因,構建重組真核表達載體pIRES2-EGFP/wtCD40、pIRES2-EGFP/70muCD40和pIRES2-EGFP/114muCD40并進行鑒定;脂質體轉染法將重組表達載體導入HEK293細胞;RT-PCR和FCM的方法鑒定重組表達載體;FCM、Western Blot法比較5C11分別與HEK293/wtCD40、HEK293/70muCD40和HEK293/114muCD40基因轉染細胞株的結合。 結果:(1)搭建合理的抗原CD40胞外段與抗體5C11 Fv結構的復合物模型。(2)酶切和測序結果均證實成功構建3個真核表達載體;RT-PCR和FCM檢測結果表明,表達載體成功轉染入HEK293細胞。(3) FCM結果表明5C11與HEK293/70muCD40和HEK293/114muCD40結合較之與HEK293/wtCD40結合的平均熒光強度有減弱的趨勢;Western blot檢測結果表明5C11僅識別HEK293/wtCD40,不識別HEK293/70muCD40和HEK293/114muCD40。由此表明5C11識別HEK293/70muCD40和HEK293/114muCD40的能力降低。 結論:預測5C11識別的抗原表位是15-17、23、43-56、68-80、85、87、100-115;成功構建HEK293/wtCD40、HEK293/70muCD40和HEK293/114muCD40基因轉染細胞株,證實人CD40序列的第70位蘇氨酸和第114位谷氨酸是其單抗5C11識別的抗原表位,同時驗證了計算機模建及預測結果的可靠性,為今后5C11抗體人源化改造的計算機輔助分子設計與實驗研究奠定了良好的基礎。
[Abstract]:In this study, the mouse anti-human CD40 stimulated monoclonal antibody (5C11), which was successfully developed by our institute, was used as the research object, and the principle of antigen-antibody interaction was used by computer aided molecular design and protein spatial structure simulation. The main binding sites of antigen CD40 and antibody 5C11 were obtained by computer simulation, and the antigen epitopes recognized by antibody were deduced. Combined with the interaction sites of CD40-CD40L reported in literature, the site-directed mutation technique was used. To construct mutant gene transfection cell line and other biological experimental methods to verify the reliability of the results of computer modeling, so as to determine the antigenic epitopes recognized by antibodies. It is of theoretical and potential clinical significance to humanize or develop anti-human CD40 antibody. Aim: to determine the antigenic epitopes recognized by human CD40 stimulated monoclonal antibody (5C11) by computer simulation and biological experiments. Methods: Insight 鈪，

本文編號：1854918