抗人CD40单抗5C11抗原识别表位的初步研究

发布时间：2018-05-07 02:08

  本文选题：CD40 + 抗原表位　； 参考：《苏州大学》2011年硕士论文

【摘要】：本课题以本所自行成功研制的鼠抗人CD40激发型单克隆抗体5C11为研究对象,借助计算机辅助分子设计与蛋白质空间结构模拟技术,利用抗原-抗体相互作用原理,计算机模拟得到抗原CD40与抗体5C11的主要结合位点,从而推测出抗体识别的抗原表位;结合文献报道的CD40-CD40L互相作用位点,通过定点突变技术、构建突变体基因转染细胞株等生物学实验方法,验证计算机模建结果的可靠性,从而确定抗体识别的抗原表位,对5C11抗体进行人源化改造或研制抗人CD40抗体临床用药具有理论和潜在的临床意义。 目的:通过计算机模拟与生物实验,初步确定本所研制的抗人CD40激发型单抗5C11识别的抗原表位。 方法:利用InsightⅡ软件分别模拟抗原、抗体结构,以及搭建抗原抗体复合物模型,通过计算并推测抗体所识别的抗原表位。通过RT-PCR的方法分别获得人野生型CD40(wtCD40)和70位突变(70muCD40)及114位突变(114muCD40)的全长基因,构建重组真核表达载体pIRES2-EGFP/wtCD40、pIRES2-EGFP/70muCD40和pIRES2-EGFP/114muCD40并进行鉴定;脂质体转染法将重组表达载体导入HEK293细胞;RT-PCR和FCM的方法鉴定重组表达载体;FCM、Western Blot法比较5C11分别与HEK293/wtCD40、HEK293/70muCD40和HEK293/114muCD40基因转染细胞株的结合。 结果:(1)搭建合理的抗原CD40胞外段与抗体5C11 Fv结构的复合物模型。(2)酶切和测序结果均证实成功构建3个真核表达载体;RT-PCR和FCM检测结果表明,表达载体成功转染入HEK293细胞。(3) FCM结果表明5C11与HEK293/70muCD40和HEK293/114muCD40结合较之与HEK293/wtCD40结合的平均荧光强度有减弱的趋势;Western blot检测结果表明5C11仅识别HEK293/wtCD40,不识别HEK293/70muCD40和HEK293/114muCD40。由此表明5C11识别HEK293/70muCD40和HEK293/114muCD40的能力降低。 结论:预测5C11识别的抗原表位是15-17、23、43-56、68-80、85、87、100-115;成功构建HEK293/wtCD40、HEK293/70muCD40和HEK293/114muCD40基因转染细胞株,证实人CD40序列的第70位苏氨酸和第114位谷氨酸是其单抗5C11识别的抗原表位,同时验证了计算机模建及预测结果的可靠性,为今后5C11抗体人源化改造的计算机辅助分子设计与实验研究奠定了良好的基础。
[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，

本文编号：1854918