【作者】 程巨龙；

【导师】 姚新生； 黄华梁； 王起振；

【作者基本信息】 沈阳药科大学 ， 药学， 2002， 博士

【摘要】 本研究的第一个目的是验证一种具有实际意义，简化易行构建改形抗体的新方法。与以往其它方法不同的是：该方法采用实验定向分子进化方法，不需要对抗体序列进行空间模拟，以确定人源抗体受体的FRs以及受体FRs上的哪个氨基酸残基需要进一步突变；该改形方法将抗体的改形及亲和力成熟于同一步骤完成，从而节省构建的时间及劳动强度。利用该方法构建了改形抗CD28重链单域抗体。根据鼠源抗CD28VH氨基酸序列，从GenBank中得到两个与该序列最同源的人源抗体，选用其中之一作为改形抗体的主框架FRs区。在保留鼠源CDRs氨基酸残基的同时，对主框架FRs一些氨基酸残基进行可选择替换突变，可选择替换原则根据对原始鼠源序列；鼠源抗体所在Kabat分类中的种属序列；两个人源抗体序列及人源抗体所在Kabat分类中的种属序列的比较而确定。选择大肠杆菌喜好密码子，推导出其基因序列；合成大小不同的核苷酸片段，采用重叠PCR扩增得到改形抗体全长基因。在合成片段时，将突变位点要被替换氨基酸残基和替换氨基酸残基的核苷酸采用简并合成，使二者都有机会在完整基因中出现。为了进一步提高改形抗体基因数目，在重叠PCR扩增改形抗体全长基因时，采用高浓度Mg²⁺和Taq DNA聚合酶，以便进一步随机引入突变。利用PCR产物构建了一个改形抗CD28VH抗体库，经过三轮淘选，获得了具有高结合活性的改形抗体基因。进一步将两个改形基因与c-myc及人IgG3’CL铰链区基因融合在大肠杆菌BL21（DE3）中表达，复性后的融合表达蛋白仍表现为具有较高与CD28分子的结合活性。这一结果表明，该构建改形抗体方法是有效和可行的，通过一步就可以获得满意的改形抗体。 本研究第二个目的是构建及表达一种新的三特异抗体：抗人卵巢癌scFv×改形抗CD3 scFv×改形抗CD28VH。构建的三特异抗体有两种类型：环形三特异抗体；非环形三特异抗体。在三特异抗体构建中使用了不同的连接肽序列（interlinker），这些序列的使用，引入了一些不同的生物学功能。两种类型的三博土论文 中文摘要特异抗体的基因在大肠杆菌BLZI pE3)中表达后，测定了表达蛋白与CD28分子的结合活性。更多还原

【Abstract】 The first aim of this study was to demonstrate a new and practical method for constructing reshaping antibodies. Different from other methods, it was not necessary to carry out modeling the sequences for determining the human acceptor and which amino acid residues in human acceptor FRs should be substituted, and reshaping and enhancing the antigen binding affinity shared one procedure at the same time. The reshaping anti-CD28 single domain antibodies were constructed by using this method. According to the amino acid sequence of a mouse anti-human CD28 monoclonal antibody VH, two most homologous sequences of human antibodies were searched from GenBank and one of them was used as a main framework residues for constructing the reshaping antibody FRs. The mouse anti-CD28 VH CDRs were still kept, but some amino acid residues in human FRs were substituted according to the comparison of searched human antibody sequences, original mouse antibody sequence, and their consensus sequences. In order to increase diversity of the gene, the amino acid residues planned to substitute were reserved in synthesizing the gene sequences by code degeneracy. Also, when the synthesized nucleotide fragments in different length were assembled and amplified to intact genes by overlap PCR, Taq DNA polymerase and high Mg2+ concentration were used for inducing more mutations randomly. A phage display library was constructed by using the PCR products. The genes of reshaping anti-CD28 VH that had high antigen binding activity were selected after three round panning selections. Two genes were fused with c-myc tag and the hinge region of human IgGS’CL gene, and expressed in E. coli BL21(DE3). The antigen binding affinity and specificity of renatured fusion protein were determined. The results suggested that the procedure we used was feasible and efficient, with which it could be gotten an ideal reshaping antibody in one step and so save the time and labor.The second aim of this study was to construct and express a novel trispecific antibody fragment: anti-human Ovarian Carcinoma scFvX reshaping anti-CD3 scFvX reshaping anti-CD28VH. Two types of the trispecific antibodies were constructed: one was constructed in a cycle formation; another was in a broken cycle formation. Different interlinkers were used in the construction, and some interlinkers could induce some biological properties beside the connection function. The molecular masses of the trispecific antibodies were about 84 kDa, which were suitable for deliver and tumor targeting. The genes of the two type trispecific antibodies were expressed in E.coli BL21(DE3), and the CD28 binding affinity of the expression proteins were determined.更多还原