大容量天然噬菌体抗体库的建立、筛选及抗DR5抗体的鉴定

发布时间：2018-06-19 19:28

本文选题：天然噬菌体抗体库 + 人源抗体　；参考：《中国人民解放军军事医学科学院》2008年博士论文

【摘要】： 上世纪90年代中期以来,抗体药物在新药中崭露头角。在治疗性应用中,全人抗体可以克服鼠源单克隆抗体在临床应用中的缺点:如诱发人体产生人抗小鼠抗体(human anti-mouse antibody, HAMA)反应、在人体内半衰期短、不能够有效地激活CDC及ADCC等。噬菌体展示抗体库技术是获取全人抗体的最主要手段之一。能否从库中筛选出针对任意抗原的特异性抗体,抗体库库容是主要的决定因素。此外,筛选出抗体亲和力大小和库容密切相关。因此,本研究尝试通过分子生物学技术,经Cre/LoxP定位重组系统构建超大容量天然噬菌体抗体库,并对筛选中得到的抗DR5抗体在原核、真核系统中表达,对表达抗体的抗原结合等特性进行了初步研究。 1人源天然噬菌体抗体库的建立、筛选从正常人外周血和新生儿脐血中分离淋巴细胞(180份),提取RNA,用RT-PCR分别扩增抗体可变区轻重链基因(VH和VL),通过重叠PCR技术将VH和VL连接为单链抗体ScFv形式,克隆插入到pDF噬菌粒载体,转化XL1-BLUE细菌得到ScFv初级抗体库。以高感染复数(MOI100:1)感染Cre +菌株BS1365,利用Cre/LoxP位点特异性重组原理,使VH和VL基因定向同源重组匹配,随后以低感染复数(MOI1:1)感染XL1-BLUE,获取次级工作库。结果表明:抗体V区基因得到有效扩增,初级库库容3.6×10~7,工作库库容1.8×10~(11),经Cre/LoxP定位重组系统成功构建了超大容量天然噬菌体抗体库;分别用人B淋巴细胞刺激因子(Blys)、人死亡受体胞外段(DR5)、肿瘤坏死因子α(TNFα)、白细胞介素6(IL-6)以及蓖麻毒素(Ricin)等5种不同抗原进行筛选均得到特异性结合的噬菌体抗体,测序结果进一步表明,所获取抗体基因涵盖了不同的基因亚群,证明该抗体库具有良好的多样性,可用于制备人源抗体。 2在原核、真核系统中表达的抗DR5抗体的抗原结合等特性的初步研究为了降低试验费用,缩短实验周期,借助生物信息学理论及计算机辅助分子模拟技术,通过多序列比对分析、抗体Fv构象稳定性分析,对抗体库中筛选出来的抗DR5单克隆抗体进行了合理性评估。结合筛选过程中的特异性结合实验结果,以抗体可变区构象稳定性、结合能力、出现频率为判据选取C11、B2、A2、A8共四株噬菌体抗体进行生物学实验。将四株噬菌体抗体以ScFv形式插入pET28a原核表达载体,转化BL21菌株后表达重组人ScFv抗体。试验结果表明ScFv抗体分子得到有效表达;此外,超声破碎后的表达上清,经ELISA、Western Blot试验证实:表达的ScFv抗体蛋白可以和预先纯化的DR5抗原特异性结合,证明了抗体库筛选得到的ScFv抗体具有特异性识别重组人DR5胞外区蛋白的活性。将四株噬菌体抗体的轻、重链可变区基因,插入到真核表达载体pCMV-Express中,等量瞬时转染293T细胞,表达完整的抗体分子。Western blot试验证实,表达上清中存在能被山羊抗人IgG酶标抗体特异性识别,且分子量与IgG标准品一致的人全分子抗体;并且表达抗体可特异性识别转印在NC膜上的DR5抗原;竞争ELISA结果显示,表达抗体与DR5的天然配体TRAIL存在竞争关系,其结合力随TRAIL剂量增加而下降。
[Abstract]:In the mid 90s of the last century, antibody drugs have emerged in new drugs. In therapeutic applications, human antibodies can overcome the shortcomings of mouse monoclonal antibodies in clinical application: such as inducing human human anti mouse antibody (human anti-mouse antibody, HAMA) reaction, short half life in human body, and not effectively activating CDC and ADC C and so on.
Phage display antibody library technology is one of the most important means of obtaining all human antibodies. Whether the specific antibody against any antigen is screened from the library, the capacity of the antibody library is the main determinant. In addition, the size of the antibody affinity is closely related to the size of the antibody. Therefore, this study tries to pass the molecular biology technology through the molecular biology technology, Cre/LoxP A large capacity natural phage antibody library was constructed by positioning and recombination system, and the characteristics of anti DR5 antibody in the prokaryotic, eukaryotic system and antigen binding of the expressed antibody were preliminarily studied.
Establishment of a natural phage antibody library of 1 human sources and screening
Lymphocytes (180 portions) were isolated from normal human peripheral blood and neonatal umbilical blood, and RNA was extracted and amplified by RT-PCR (VH and VL). VH and VL were linked to ScFv form of single chain antibody by overlapping PCR technology. The clones were inserted into the pDF phagocytic carrier and XL1-BLUE bacteria were converted to ScFv primary antibody library. The number (MOI100:1) infected with Cre + strain BS1365, using the principle of Cre/LoxP specific recombination, matching the VH and VL genes to homologous recombination, and then using the low infection complex (MOI1:1) to infect XL1-BLUE to obtain the secondary working library. The results showed that the antibody V region gene was effectively amplified, the storage capacity of the primary reservoir was 3.6 * 10~7, the storage capacity of the working library was 1.8 x 10~ (11), and Cre/Lo The xP location and recombination system successfully constructed a large capacity natural phage antibody library, and screened 5 different antigens, such as human B lymphocyte stimulator (Blys), human death receptor extracellular segment (DR5), tumor necrosis factor alpha (TNF alpha), interleukin 6 (IL-6) and ricin (Ricin). The sequence results further showed that the antibody genes covered different subsets of genes, which showed that the antibody library had good diversity and could be used for the preparation of human antibody.
2 preliminary study on antigen binding characteristics of anti DR5 antibodies expressed in prokaryotic and eukaryotic systems
In order to reduce the test cost and shorten the experimental period, by means of bioinformatics theory and computer aided molecular simulation technology, the Conformation Stability Analysis of antibody Fv was analyzed by multi sequence alignment, and the rationality evaluation of anti DR5 monoclonal antibodies screened from the antagonism body library was conducted. Four phage antibodies of C11, B2, A2 and A8 were selected for biological experiments. The four phage antibodies were inserted into the pET28a prokaryotic expression vector in the form of ScFv, and the BL21 strain was transformed to express the recombinant human ScFv antibody. The experimental results showed that the ScFv antibody molecules were expressed effectively. In addition, the expression supernatant after ultrasonic fragmentation was confirmed by ELISA, Western Blot test. The expression of ScFv antibody protein can be specifically combined with the pre purified DR5 antigen. It is proved that the ScFv antibody screened by the antibody library has the specific activity to identify the recombinant human DR5 extracellular domain protein.
The light, heavy chain variable region gene of four phage antibodies was inserted into the eukaryotic expression vector pCMV-Express, and the 293T cells were transiently transfected in the same amount. The expression of the complete antibody molecule.Western blot test proved that the expression of the expression supernatant could be identified by the Goat anti human IgG specific antibody specific recognition, and the molecular weight was consistent with the IgG standard. The antibody can be expressed specifically to identify the DR5 antigen transferred to the NC membrane; competitive ELISA results show that the expression antibody has a competitive relationship with the natural ligand TRAIL of DR5, and its binding force decreases with the increase of TRAIL dose.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392

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