分别抗HER2和FGFR2的人源单域抗体的筛选及鉴定研究

发布时间：2018-06-13 04:22

本文选题：单域抗体 + HER2　；参考：《暨南大学》2016年博士论文

【摘要】：目的:HER2和FGFR2蛋白在多种癌细胞内过表达,与多种癌症的发生、发展和转移有关,是癌症治疗的理想靶标。虽然已有抗HER2的抗体药上市用于癌症治疗,但这些都是传统的单克隆抗体。由于单克隆抗体是大分子蛋白,其应用受到很大限制。本论文筛选抗HER2的新型的小分子人源单域抗体,希望能代替上市的单克隆抗体药物。另外,也筛选抗FGFR2的新型的小分子人源单域抗体。方法:第一部分,筛选抗HER2的人源单域抗体。采用辅助噬菌体侵染制备人源单域抗体噬菌体文库,采用原核细胞包涵体表达方法制备和纯化HER2-domain II蛋白片段。采用噬菌体展示技术和HER2-domain II蛋白片段作为抗原筛选人源单域抗体文库。经过5轮文库的筛选富集后,采用多克隆噬菌体ELISA和单克隆噬菌体ELISA方法进一步筛选抗HER2-domain II的单域抗体,并获得单域抗体的DNA序列。采用原核细胞可溶性表达方法制备和纯化抗HER2-domain II的单域抗体蛋白,采用ELISA鉴定纯化的单域抗体与HER2-domain II的结合能力,采用非竞争性ELISA检测单域抗体与HER2-domain II的亲和力。第二部分,筛选抗FGFR2的人源单域抗体。采用辅助噬菌体侵染制备人源单域抗体噬菌体文库,通过人工合成的方法制备FGFR2蛋白片段。采用噬菌体展示技术筛选抗FGFR2蛋白片段的单域抗体,经过5轮文库的筛选富集后,采用多克隆噬菌体ELISA和单克隆噬菌体ELISA方法进一步筛选抗FGFR2蛋白片段的单域抗体,并获得单域抗体的DNA序列。结果:第一部分,筛选抗HER2的人源单域抗体。利用辅助噬菌体侵染制备了滴度为3.41×1013/ml的人源单域抗体噬菌体文库。通过原核细胞包涵体表达纯化得到了HER2-domain II蛋白,SDS-PAGE电泳图显示较高的蛋白纯度;非还原性SDS-PAGE电泳图显示HER2-domain II蛋白约为12 kDa。经过噬菌体展示技术5轮筛选后,抗HER2-domain II的单域抗体的滴度逐渐上升。采用多克隆噬菌体ELISA检测发现第3轮富集的单域抗体具有最强的的结合能力。从筛选后的文库里随机挑选196个单域抗体克隆,采用单克隆噬菌体ELISA检测得到了8个阳性单域抗体;采用单克隆噬菌体ELISA和多个无关抗原检测发现这8个阳性单域抗体能与HER2-domain II特异性结合。对于这8个阳性单域抗体的DNA测序发现只有2个不同的单域抗体(分别命名为aHER2-1C1和aHER2-2B4)。将这2个单域抗体克隆到pET22b表达载体内进行可溶性表达纯化,SDS-PAGE电泳图显示得到了较高纯度的蛋白。ELISA检测发现纯化的a HER2-1C1能与HER2-domain II结合,而纯化的aHER2-2B4与HER2-domain II结合的能力弱。采用非竞争性ELISA检测发现aHER2-1C1与HER2-domain II结合的亲和力为3.76 uM。第二部分,筛选抗FGFR2的人源单域抗体。通过人工合成的方法得到了FGFR2蛋白片段,高效液相色谱显示较高的蛋白纯度,质谱分析显示FGFR2蛋白片段约为4 kDa。经过噬菌体展示技术对人源单域抗体噬菌体文库进行了5轮筛选,采用多克隆噬菌体ELISA检测发现第5轮富集的单域抗体具有最强的的结合能力。从筛选后的文库里随机挑选168个单域抗体克隆,采用单克隆噬菌体ELISA检测得到了20个阳性单域抗体;采用单克隆噬菌体ELISA和多个无关抗原检测发现这20个阳性单域抗体能与FGFR2蛋白片段特异性结合。对于这20个阳性单域抗体的DNA测序发现只有2个不同的单域抗体(分别命名为aFG-7E5和aFG-9F8)。结论:本研究采用噬菌体展示技术分别获得了与HER2-domain II和FGFR2蛋白片段特异性结合的人源单域抗体,为癌症的早期诊断与靶向治疗奠定了较好的基础。
[Abstract]:Objective: HER2 and FGFR2 protein are overexpressed in a variety of cancer cells, which are related to the occurrence, development and metastasis of various cancers. It is an ideal target for cancer treatment. Although anti HER2 antibody drugs have been used for cancer treatment, these are traditional monoclonal antibodies. The application of monoclonal antibodies is large, and its application is greatly limited. In this paper, a new type of small molecular human single domain antibody against HER2 is screened in the hope of replacing the listed monoclonal antibody drugs. In addition, a new type of small molecular human single domain antibody against FGFR2 is screened. Method: the first part is to screen the human single domain antibody against HER2. HER2-domain II protein fragment was prepared and purified by the expression of prokaryotic cell inclusion body. The phage display technique and HER2-domain II protein fragment were used as antigen to screen the single domain antibody library of human source. After screening and enrichment of 5 rounds of library, polyclonal phage ELISA and monoclonal phage ELISA were used to further screen the anti HER2-dom. The single domain antibody of AIN II and the DNA sequence of the single domain antibody were obtained. The single domain antibody protein of anti HER2-domain II was prepared and purified by the prokaryotic expression method, and the binding ability of the purified single domain antibody to HER2-domain II was identified by ELISA, and the affinity of the non competitive ELISA detection single domain antibody to HER2-domain II was used. Second Part, screening anti FGFR2 human single domain antibody. Using phage infection to prepare human single domain antibody phage library, FGFR2 protein fragment was prepared by artificial synthesis method. Single domain antibody of anti FGFR2 protein fragment was screened by phage display technique. After screening and enrichment of 5 rounds of library, polyclonal phage ELISA was used. The monoclonal phage ELISA method further screened the single domain antibody against the FGFR2 protein fragment and obtained the DNA sequence of the single domain antibody. Results: the first part was to screen the human single domain antibody against HER2. The phage library of human single domain antibody of 3.41 x 1013/ml was prepared by the auxiliary phage infection. The expression of the inclusion body of the prokaryotic cell was expressed. The purified HER2-domain II protein was obtained, and the SDS-PAGE electrophoresis map showed high protein purity, and the non reductive SDS-PAGE electrophoresis map showed that the HER2-domain II protein was about 12 kDa. after 5 rounds of phage display technology, the titer of the single domain antibody against HER2-domain II increased gradually. The polyclonal phage ELISA detection found third rounds of enrichment. The single domain antibody had the strongest binding ability. 196 single domain antibody clones were randomly selected from the screened library and 8 positive single domain antibodies were detected by the monoclonal phage ELISA detection. The 8 positive single domain antibodies were found to be specific to HER2-domain II by monoclonal phage ELISA and multiple independent antigens. DNA sequencing of these 8 positive single domain antibodies found only 2 different single domain antibodies (named aHER2-1C1 and aHER2-2B4 respectively). The 2 single domain antibodies were cloned into the pET22b expression vector for soluble expression and purification. The SDS-PAGE electrophoresis map showed that the purified egg white.ELISA detection was found to be the purified a HER2-1C1 and HER2. -domain II was combined, and the ability of the purified aHER2-2B4 to bind to HER2-domain II was weak. The affinity of aHER2-1C1 to HER2-domain II was found to be 3.76 uM. second by non competitive ELISA detection, and the anti FGFR2 human single domain antibody was screened. The FGFR2 protein fragment was obtained by artificial synthesis, and high performance liquid chromatography showed higher performance. The protein purity and mass spectrometry analysis showed that the FGFR2 protein fragment was about 4 kDa., and the phage display technique was used to screen the human single domain antibody phage library by 5 rounds. The polyclonal phage ELISA detection showed that the fifth round of single domain antibodies had the strongest binding ability. 168 single domains were randomly selected from the selected library. 20 positive single domain antibodies were obtained by monoclonal phage ELISA detection. The 20 positive single domain antibodies were identified by monoclonal phage ELISA and multiple independent antigens. The DNA sequencing of the 20 positive single domain antibodies found only 2 different single domain antibodies (named respectively. AFG-7E5 and aFG-9F8) conclusion: This study uses phage display technique to obtain a human single domain antibody with specific binding of HER2-domain II and FGFR2 protein fragments, which lays a good foundation for early diagnosis and targeting therapy of cancer.
【学位授予单位】：暨南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R730.51

【相似文献】