Sarina Nurgul, Abeldenov Sailau, Turgimbayeva Aigerim, Zhylkibayev Assylbek, Ramankulov Yerlan, Khassenov Bekbolat, Eskendirova Saule
Hum Antibodies. 2018 Feb 5;26(2):103-111. doi: 10.3233/HAB-170327.
Human epidermal growth factor receptor 2 (HER2) is an important biomarker for detection and treatment of different types of cancers such as breast, ovarian, stomach cancer. In this study, we developed a monoclonal antibody against the extracellular domain (ECD) of HER2 biomarker of breast cancer. For this purpose, the ECD-HER2 gene was amplified and cloned into an expression vector. Gene was generated in Escherichia coli BL21 (DE3) strain for expression of recombinant protein. The expressed protein was separated by SDS-PAGE and detected by anti-his monoclonal antibody in immunoblotting. Hybridoma cells were obtained by fusing myeloma cells with mouse spleen cells injected with recombinant ECD-HER2 and screened by ELISA for the production of monoclonal antibody. The results indicate that out of three candidate hybridoma cells one clone (1E7) was producing the highest titer and antibody specificity was envisioned in ELISA results. In vivo scaling up culture of hybridoma cells in BALB/C mice lead to significant increase in the monoclonal antibody concentration up to 16 mg/ml. Immunochemical methods demonstrated the specificity of developed antibody against ECD-HER2 protein.
人表皮生长因子受体2（HER2）是检测和治疗不同类型癌症（如乳腺癌、卵巢癌、胃癌）的重要生物标志物。在本研究中，我们开发了一种针对乳腺癌HER2生物标志物细胞外结构域（ECD）的单克隆抗体。为此，扩增了ECD-HER2基因并将其克隆到表达载体中。在大肠杆菌BL21（DE3）菌株中表达该基因以产生重组蛋白。通过SDS-PAGE分离表达的蛋白，并在免疫印迹中用抗组氨酸单克隆抗体进行检测。通过将骨髓瘤细胞与注射了重组ECD-HER2的小鼠脾细胞融合获得杂交瘤细胞，并通过ELISA筛选产生单克隆抗体的细胞。结果表明，在三个候选杂交瘤细胞中，一个克隆（1E7）产生的滴度最高，ELISA结果显示出抗体特异性。在BALB/C小鼠体内扩大杂交瘤细胞培养导致单克隆抗体浓度显著增加，高达16mg/ml。免疫化学方法证明了所开发抗体对ECD-HER2蛋白的特异性。
