抗人小细胞肺癌单抗2F7人-鼠嵌合Fab片段基因的构建和表达

Construction and expression of a recombinant human-mouse chimeric Fab fragment of mAb 2F7

目的 :通过基因工程抗体改造获得具有与人小细胞肺癌有特异性反应的人 鼠嵌合抗体 2F7Fab片段。方法 :采用RT PCR技术从抗人小细胞肺癌单克隆抗体 2F7杂交瘤中克隆该抗体的重、轻链可变区基因 ,将 2F7单抗的重、轻链可变区基因装入带有人恒定区基因的表达载体pSW1 Fab中 ,构建得到pSW1 2F7Fab ,转化受体菌E .coilXL1 Blue ,IPTG诱导表达 ,经Westernblot和ELISA鉴定表达蛋白的活性。结果 :从抗人小细胞肺癌单克隆抗体 2F7杂交瘤中克隆获得了抗体重、轻链可变区基因。测序证实 2F7单抗的重链 (VH)可变区基因全长 36 2bp ,编码 12 0个氨基酸 ;轻链 (VL)可变区基因全长 319bp ,编码10 5个氨基酸。构建的 2F7人 鼠嵌合Fab表达载体诱导后获得了目的蛋白的表达 ,主要分泌在菌液的上清中 ,表达量较高且稳定 ,具有与NCI H1 2 8细胞特异性结合的活性。结论 :构建和表达了抗人小细胞肺癌单抗 2F7人 鼠嵌合Fab片段 ,表达产物具有与抗原结合的特异性 ,为进一步研究和应用打下了基础。

Objective:To obtain human mouse chimeric Fab fragment of the anti human small cell lung cancer mAb 2F7 by means of engineering antibody.Methods:Using RT PCR, the genes of the heavy and light chain variable regions (V H and V L) of mAb 2F7 were obtained from anti human small cell lung cancer hybridoma 2F7. The genes of V H and V L of mAb 2F7 were inserted into expression vector pSW1 Fab which with human constant region C H1 and C κ gene, respectively. The expression plasmid pSW1 2F7 Fab was constructed. E. coil XL1 Blue was transformed with pSW1 2F7 Fab and induced by IPTG. The expression and activity of 2F7 Fab protein were detected by ELISA and Western blot.Results:The V H gene is 362 bp and encode 120 amino acids, the V L gene is 319 bp and encode 105 amino acids. The human mouse chimeric 2F7 Fab protein expressed by induction was secreted into culture media and confirmed by Western Blotting. ELISA demonstrated 2F7 Fab could interact with the antigen on NCI H128 cells.Conclusion:The recombinant human mouse chimeric 2F7 Fab expresseed in E. coil has the activity and specificity of interacting with 2F7 antigen.