本文選題：CD40 + 抗原表位　； 參考：《蘇州大學(xué)》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(duì)象,借助計(jì)算機(jī)輔助分子設(shè)計(jì)與蛋白質(zhì)空間結(jié)構(gòu)模擬技術(shù),利用抗原-抗體相互作用原理,計(jì)算機(jī)模擬得到抗原CD40與抗體5C11的主要結(jié)合位點(diǎn),從而推測(cè)出抗體識(shí)別的抗原表位;結(jié)合文獻(xiàn)報(bào)道的CD40-CD40L互相作用位點(diǎn),通過(guò)定點(diǎn)突變技術(shù)、構(gòu)建突變體基因轉(zhuǎn)染細(xì)胞株等生物學(xué)實(shí)驗(yàn)方法,驗(yàn)證計(jì)算機(jī)模建結(jié)果的可靠性,從而確定抗體識(shí)別的抗原表位,對(duì)5C11抗體進(jìn)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(guò)計(jì)算機(jī)模擬與生物實(shí)驗(yàn),初步確定本所研制的抗人CD40激發(fā)型單抗5C11識(shí)別的抗原表位。 方法:利用InsightⅡ軟件分別模擬抗原、抗體結(jié)構(gòu),以及搭建抗原抗體復(fù)合物模型,通過(guò)計(jì)算并推測(cè)抗體所識(shí)別的抗原表位。通過(guò)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(zhǎng)基因,構(gòu)建重組真核表達(dá)載體pIRES2-EGFP/wtCD40、pIRES2-EGFP/70muCD40和pIRES2-EGFP/114muCD40并進(jìn)行鑒定;脂質(zhì)體轉(zhuǎn)染法將重組表達(dá)載體導(dǎo)入HEK293細(xì)胞;RT-PCR和FCM的方法鑒定重組表達(dá)載體;FCM、Western Blot法比較5C11分別與HEK293/wtCD40、HEK293/70muCD40和HEK293/114muCD40基因轉(zhuǎn)染細(xì)胞株的結(jié)合。 結(jié)果:(1)搭建合理的抗原CD40胞外段與抗體5C11 Fv結(jié)構(gòu)的復(fù)合物模型。(2)酶切和測(cè)序結(jié)果均證實(shí)成功構(gòu)建3個(gè)真核表達(dá)載體;RT-PCR和FCM檢測(cè)結(jié)果表明,表達(dá)載體成功轉(zhuǎn)染入HEK293細(xì)胞。(3) FCM結(jié)果表明5C11與HEK293/70muCD40和HEK293/114muCD40結(jié)合較之與HEK293/wtCD40結(jié)合的平均熒光強(qiáng)度有減弱的趨勢(shì);Western blot檢測(cè)結(jié)果表明5C11僅識(shí)別HEK293/wtCD40,不識(shí)別HEK293/70muCD40和HEK293/114muCD40。由此表明5C11識(shí)別HEK293/70muCD40和HEK293/114muCD40的能力降低。 結(jié)論:預(yù)測(cè)5C11識(shí)別的抗原表位是15-17、23、43-56、68-80、85、87、100-115;成功構(gòu)建HEK293/wtCD40、HEK293/70muCD40和HEK293/114muCD40基因轉(zhuǎn)染細(xì)胞株,證實(shí)人CD40序列的第70位蘇氨酸和第114位谷氨酸是其單抗5C11識(shí)別的抗原表位,同時(shí)驗(yàn)證了計(jì)算機(jī)模建及預(yù)測(cè)結(jié)果的可靠性,為今后5C11抗體人源化改造的計(jì)算機(jī)輔助分子設(shè)計(jì)與實(shí)驗(yàn)研究奠定了良好的基礎(chǔ)。
[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(hào)：1854918