论文部分内容阅读
目的：减少鼠源性单抗的免疫原性，获得最接近于天然构型的高表达基因工程抗体。方法：将抗人纤维蛋白单抗SZ－63的轻链可变区基因VK和重链可变区基因VH分别与人IgG恒区基因CK、CH进行拼接、扩增，构建了真核表达载体α－lys17－63VK／Hu和α－lys30－63VH／Hu，并应用linofectin方法将其相继导入小鼠骨髓瘤细胞SP2／0中，通过加压筛选获得一抗性细胞克隆，经体外长期传代培养和三次克隆化得到一稳定表达的阳性细胞株。结果：培养上清液中的抗体蛋白表达量为1mg／L，腹水中为40mg／L经Westernblot和竞争实验证实表达的嵌合抗体具有与亲本鼠源单抗相一致的纤维蛋白结合特性。结论：SZ－63嵌合抗体可用于血栓性疾病的导向显像和治疗。
OBJECTIVE: To reduce the immunogenicity of murine monoclonal antibodies and obtain highly engineered antibodies that are closest to the native configuration. METHODS: The anti-human fibrin monoclonal antibody SZ-63 VK and VH heavy chain variable region genes were spliced and amplified with human IgG constant region genes CK and CH, respectively. The eukaryotic expression vector was constructed. α-lys17-63VK/Hu and α-lys30-63VH/Hu were sequentially introduced into mouse myeloma cells SP2/0 using the linofectin method. A resistant cell clone was obtained by pressure screening and was in vitro long-term subculture. After three times of cloning, a stable positive cell line was obtained. RESULTS: The expression level of antibody protein in culture supernatant was 1 mg/L and 40 mg/L in ascites. The chimeric antibodies confirmed by Western blot and competition experiments had fibrin-binding properties consistent with the parental mouse monoclonal antibody. CONCLUSIONS: SZ-63 chimeric antibodies can be used for guided imaging and treatment of thrombotic diseases.