一种构建改形单域抗体的方法

为验证一种构建改形单域抗体的实用新方法，与以往方法不同的是，该方法不需要对抗体进行空间结构模拟，以确定人源抗体的FRs接受序列及在人源FRs接受序列中哪些氨基酸残基需要突变，并且该方法将抗体的改形与亲和力成熟于同一过程完成，利用该方法构建了改形抗CD28重链单域抗体，根据一种鼠源抗CD28重链单域抗体的氨基酸序列，于GenBank中查得两条与之最同源的人源抗体序列，利用其中一条的FRs作为改形抗体的主框架进行改形构建，将鼠源抗体的CDR区插入到人源FR区后，对人源FR区的一些氨基酸残基进行替换突变，替换的氨基酸残基数及替换原则主要是根据对查到的人源抗体序列，鼠源抗体序列，以及这些序列与Kabat分类中的种属序列进行的比较，为了增加改形抗体基因的多样性，对要被替换的氨基酸残基在基因合成中采用简并的方式，使要被替换的氨基酸残基和替换的氨基酸残基都有机会出现，二者出现的几率各为50%，同时，在将大小不同的合成核苷酸片段采用重叠PCR扩增以获得完整改形抗体基因时，采用高Mg^2 浓度下和使用TaqDNA聚合酶，以进一步随机引入突变，利用重叠PCR产物构建了一个噬菌体抗体库，经过3轮淘选后，获得了几个具有较高免疫学活性的改形抗体，对其中的两个抗体进行了进一步研究，将两个抗体的基因在大肠杆菌BL21（DE3）中表达，复性后的表达蛋白仍具有较高的免疫学活性，结果表明该方法是有效可行的。

A Method for Constructing Reshaping Single-Domain Antibody

The aim of this research was to demonstrate a novel and practical method for constructing reshaping Single domain antibodies. Different from other methods, our method does not need to model the configuration of antibodies with specific sequences to determine the sequences of human acceptor FRs and then determine which amino acid residues in human acceptor FRs should be substituted. Most importantly, reshaping and enhancing the antigen binding affinity shared one procedure at the same time. Using this method, the reshaping anti CD28 single domain antibodies were constructed. According to the amino acid sequence of a mouse anti human CD28 monoclonal antibody VH, two most homologous sequences of human antibodies were selected from GenBank and one of them was used as a main framework region for constructing the reshaping antibody. Before the original mouse antibody CDRs were inserted into the human acceptor FRs, some amino acid residues which were different from those of the original mouse antibody in the corresponding positions ofthe human acceptor FRs were determined or alternatively mutated by their conservative properties in Kabat classification. When the synthesized nucleotide fragments in different length were spliced by overlap PCR into the entire reshaping genes, Taq DNA polymerase and high Mg 2+ concentration were used to introduce more mutation in FRs and CDRs randomly. A phage library was constructed using these PCR products and several reshaping Single domain antibodies with high antigen binding affinity were selected after three rounds of panning. Two of them were expressed in E. coli BL21(DE3). The antigen binding affinity of refolded proteins was still in a high level measured by ELISA. These results suggested that this method was feasible and efficient for constructing reshaping Single domain antibodies.