目的制备新型人源化抗CD20单克隆抗体并检测其与CD20抗原的亲和力和抗肿瘤活性。方法通过计算机模建技术,在蛋白质结构数据库中找到与利妥昔单抗(rituximab)空间结构最相近的人Ig G1为骨架,将利妥昔单抗的互补决定区(complementarity determining region,CDR)进行移植和改造,通过重叠PCR方法,扩增出目的基因片段。分别将轻链(L)、重链(H)基因片段克隆到载体pc DNA3.3、p Opti VEC质粒上。瞬时转染至293F细胞,收取细胞上清,用r Protein A亲和层析法纯化目的抗体,并用十二烷基硫酸钠(SDS)-聚丙烯酰胺凝胶电泳(PAGE)检验抗体的纯度和表达量,采用Fortebio技术检测抗体与抗原的结合能力,最后通过裸鼠移植瘤生长抑制实验检测抗体的抗肿瘤活性。结果构建了3种抗CD20人源化抗体。抗体在还原SDS-PAGE中表现为两条相对分子质量约为25×103和55×103的条带,与预计条带大小相符;Fortebio实验结果表明,3组抗体与抗原具有良好的亲和活性:利妥昔单抗、L4H7抗体、L5H5抗体和L5H7抗体的Kd值分别为6.48×10-9、1.91×10-9、7.35×10-10和1.91×10-9mol/L。裸鼠移植瘤生长抑制实验表明,L5H7抗体的抗肿瘤活性优于利妥昔单抗。结论成功构建并表达了3种抗CD20人源化抗体,其中L5H7抗体具有良好的抗原结合能力和抗肿瘤活性。 Objective To prepare a new type of humanized anti-CD20 monoclonal antibody and test its affinity with anti-CD20 antigen and anti-tumor activity. Methods Human Ig G1, which has the closest spatial structure to rituximab, was found in the protein structure database by computer modeling technique. The complementarity determining region (CDR) of rituximab, Transplantation and transformation, by overlapping PCR method, amplification of the target gene fragment. The light chain (L) and heavy chain (H) gene fragments were cloned into the vector pcDNA3.3, p Opti VEC plasmid respectively. The cells were transiently transfected into 293F cells and the cell supernatant was collected. The antibodies of interest were purified by r Protein A affinity chromatography and the purity of the antibodies was checked by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis The expression level of the antibody was detected by Fortebio technology. The antitumor activity of the antibody was detected by the growth inhibition assay in nude mice. Results Three anti-CD20 humanized antibodies were constructed. Antibodies showed two bands with relative molecular mass of about 25 × 103 and 55 × 103 in reducing SDS-PAGE, which was in accordance with the expected size of the bands. The results of Fortebio experiments showed that the three groups of antibodies had good affinity with the antigen : The Kd values ​​of rituximab, L4H7 antibody, L5H5 antibody and L5H7 antibody were 6.48 × 10-9, 1.91 × 10-9, 7.35 × 10-10, and 1.91 × 10-9 mol / L, respectively. The growth inhibition of transplanted tumor in nude mice shows that the anti-tumor activity of L5H7 antibody is better than rituximab. Conclusion Three anti-CD20 humanized antibodies were successfully constructed and expressed. L5H7 antibody has good antigen-binding ability and anti-tumor activity.