全合成人源抗体库的构建及抗BHL-1单链抗体的初步筛选

发布时间：2018-01-13 20:38

本文关键词：全合成人源抗体库的构建及抗BHL-1单链抗体的初步筛选　出处：《吉林大学》2006年硕士论文　论文类型：学位论文

【摘要】：通过抗体库筛选针对特异抗原的噬菌体展示技术是目前获得人源性抗体最有效的方法,抗体库依据基因序列来源的不同,又可分为免疫库和天然库。利用全合成的方法可以获得序列随机分布的DNA文库,利用Kabat数据库公布的人抗体序列信息并结合已发表体骨架区序列,我们设计了全合成抗体库的骨架序列和CDR3随机区序列。根据统计和归纳,现有的人抗体的可变区基因可按重链和轻链分组,分别代表人抗体序列的七个胚系基因。其中重链分为VH1A、VH1B、VH2、VH3、VH4、VH5和VH6;轻链则按Kappa链和Lamda链的不同,将Kappa链和Lamda链各分为四个组和三个组,通过轻、重链的组合构成的单链抗体库可以涵盖超过95%的抗体胚系序列。在随机化过程中,利用DNA组合固相合成法来实现各位点氨基酸种类和频率的控制,以PCR的原理,无引物或低引物浓度来控制库冗余度,降低差错,经电击转化大肠杆菌最终建成了1.5×109库容的噬菌体展示文库,该抗体库的阳性率超过85%,正确序列超过50%,符合预期的目标。全合成抗体库的设计特征、构建方法和质控标准都可以保证其实用性。为进一步鉴定此抗体库的效率,我们以BHL-1(抗人卵巢癌×抗人CD3双特异抗体)蛋白为抗原,经生物淘选获得了数个具有较高亲和力的克隆,对其中C8进行亚克隆,转入表达载体进行表达,表达产物以包含体为主,经复性后进行活性检测,对靶抗原的ELISA测定具有特异的结合能力(OD490=0.727),推测其结合位点在抗卵巢癌单链抗体部分,表明获得了有意义的“人源化”抗体,此抗体经进一步优化可用于测定BHL-1蛋白的样品浓度和残留。理论上,此抗体成库可针对任意抗原筛选单链抗体,有望成为科研用抗体制备、抗体人源化的工具,经序列优化还可以用来制备诊断试剂或治疗性中和抗体。
[Abstract]:The library was screened against specific antigen by antibody phage display technology is the method of obtaining human antibody most effective, antibody library based on gene sequences from different, can be divided into natural immune library and DNA library library. You can use the full sequence of random distribution of synthetic methods, the use of human antibody sequences Kabat database published information combined with the published sequence frame area, we designed the framework of CDR3 sequence and random sequence fully synthetic antibody library. According to the statistics and induction variable region gene of human antibody can be divided into groups according to the heavy chain and light chain, seven germline genes representing human antibody sequences. The heavy chain divided into VH1A, VH1B, VH2, VH3, VH4, VH5 and VH6; light chain according to Kappa chain and Lamda chain, Kappa chain and Lamda chain were divided into four groups and three groups by light, single chain antibody library combined heavy chain which can Covering more than 95% of the antibody germline sequence. In the randomization process, to achieve control of all amino acid types and frequency of the use of DNA combined with solid phase synthesis method, the principle of PCR, no primer or low primer concentration to control database redundancy, reduce errors, Escherichia coli by electroporation into the final of the 1.5 x 109 capacity the phage display library, the positive rate of the antibody library of more than 85%, the correct sequence of more than 50%, in line with the expected target. The design features a fully synthetic antibody library, construction method and quality control standards can ensure the practicability. For further identification of the efficiency of antibody library, we use BHL-1 (double specific antibody against human ovarian cancer * anti human CD3) protein as antigen by biopanning and obtained a number of high affinity, in which C8 was subcloned into the expression vector, and expressed in inclusion body. After renaturation of Activity detection, determination of binding ability with specific target antigens of ELISA (OD490=0.727), the binding sites in the single chain antibody against ovarian cancer, that have the meaning of "humanized" antibody, the antibody after further optimization can be used for determination of BHL-1 protein sample concentration and residue. Theoretically, this as for any screening of antigen antibody library of scFv antibody preparation, is expected to be used in scientific research, humanized antibody sequence optimization by tools, can also be used for the preparation of diagnostic reagents or treatment of neutralizing antibody.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

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