含F/2A序列的抗 P185erbB2人鼠嵌合抗体慢病毒表达载体的构建

摘要

目的 构建含 F/2A 序列的抗 P185erbB2 人鼠嵌合抗体慢病毒表达载体,观察其在 293T 细胞中的表达.方法 用具有自我剪切能力的弗林蛋白酶(Furin)/口蹄疫病毒2A多肽(F/2A)连接人鼠嵌合抗体的重链和轻链,形成一个开放阅读框 (ORF),插入慢病毒表达载体pWPI,构建重组抗P185erbB2全长人鼠嵌合抗体表达载体pWPI/H-F2A-L.以已构建的慢病毒表达载体pWPI/H-IRES-L为对照质粒.应用磷酸钙沉淀法将慢病毒载体 3 质粒系统共转染入 293T 细胞进行包装,测定病毒滴度.再感染 293T 细胞,荧光显微镜下观察 GFP 的表达和转染效率,RT-PCR、ELISA 方法分别检测嵌合抗体 mRNA和蛋白的表达.结果 经测序鉴定,pWPI/H-F2A-L与预期设计一致;pWPI/H-F2A-L组的病毒滴度为4.3×105 TU/ml,而pW-PI/H-IRES-L 组的病毒滴度为3.5×105 TU/ml;两组重组慢病毒的转染效率分别为 87.68%和 79.08%:两组重组慢病毒感染 293T 细胞后,都有嵌合重链和嵌合轻链的表达,由F/2A介导的嵌合抗体的表达水平要高于由 IRES 介导的嵌合抗体.结论 成功构建了含F/2A序列的抗P185erbB2人鼠嵌合抗体慢病毒表达载体,为今后抗P185erbB2工程抗体的研究奠定了基础.%Objective To construct the lentiviral expression vector containing F/2A fragment for anti-P185erbB2 mouse/human chimeric antibody , and detect its expression in 293T cells. Methods The heavy and light chains of chimeric antibody were joined by Furin and 2A ( F/ 2A) self-cleavage peptide and cloned into a lentiviral vector of pWPI , to generate the lentiviral ex-pression vector, pWPI/H-F2A-L Another lentiviral expression vector , pWPI/H4RES-L that had been generated already , was used as control plasmid. 293 T cells were co-transfected with the 3 helper plasmid system by using calcium phosphate precipitation , and then the virus titer was exam-ined. The 293 T cells were infected by the obtained lentiviral particles. The expression of GFP and the transfection efficiency were examined under fluorescent microscope after transfection . The expression of chimeric antibody was examined by RT -PCR and ELISA. Results Sequence analysis showed that the lentiviral expression vector pWPI/H-F2A-L was constructed correctly. The viral titer was exam-ined as 4. 3×105 TU/mL for pWPI/H2A4, group, and 3. 5×105 TU/mL for pWPI/H4RES4, group. The transfection efficiency in these two groups reached to 87. 68% and 79. 08% , respective-ly. After 293T cells were transfected with lentiviral particles , both heavy and light chains of the chi-meric antibody genes could express in the two groups. The expression level of the chimeric anti body linked by F2A was higher than that of the chimeric anti body linked by IRES . Conclusion The lentiviral expression vector containing F/2A fragment for anti-P185erbB2 mouse/human chimeric antibody was successfully constructed for further study of anti -P185erbB2 engineered antibody.