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目的构建和表达抗CD20Fab-LDP(力达霉素辅基蛋白)融合蛋白,制备强化融合蛋白抗CD20Fab-LDM(力达霉素),并测定该融合蛋白的生物学活性。方法采用PCR和overlapPCR方法构建抗CD20Fab-LDP融合蛋白,并用双脱氧终止法测定DNA序列;采用亲和层析法纯化该产物,并用Western blot鉴定纯化产物;采用FACS方法鉴定纯化产物与靶细胞的结合活性;采用冷乙二醇法制备强化融合蛋白抗CD20Fab-LDM;采用MTT法鉴定抗CD20Fab-LDM对CD20+Raji细胞特异性的细胞毒作用。结果DNA序列测定结果表明抗CD20Fab-LDP融合蛋白已构建成功,可溶性表达产物的产量可达4mg.L-1以上,具有与Raji细胞(CD20+)结合的活性,与抗CD20Fab的亲合常数相当,抗CD20Fab-LDM体外能特异性杀伤Raji细胞,IC50值为0.9×10-10mol.L-1。结论成功地构建了抗CD20Fab-LDP融合蛋白,并获得可溶性高效表达,表达产物具有与相应靶抗原结合的活性,并成功制备了抗CD20Fab-LDM强化融合蛋白,体外能特异性杀伤CD20+的Raji细胞。
Objective To construct and express anti-CD20Fab-LDP (lidamycin-dependent protein) fusion protein and prepare the enhanced fusion protein anti-CD20Fab-LDM (lidamycin). The biological activity of the fusion protein was determined. Methods The anti-CD20Fab-LDP fusion protein was constructed by PCR and overlapPCR. The DNA sequence was determined by dideoxy terminator method. The product was purified by affinity chromatography and identified by Western blot. The purified product was identified by FACS The anti-CD20 Fab-LDM was prepared by cold glycol method. The cytotoxicity of anti-CD20 Fab-LDM against CD20 + Raji cells was identified by MTT assay. Results The results of DNA sequencing showed that the anti-CD20 Fab-LDP fusion protein was successfully constructed and the yield of soluble expression product was up to 4 mg.L-1. The activity of the fusion protein with Raji cells (CD20 +) was comparable to that of anti-CD20 Fab. Anti-CD20 Fab-LDM can specifically kill Raji cells in vitro with an IC50 value of 0.9 × 10 -10 mol·L -1. Conclusion The anti-CD20Fab-LDP fusion protein was successfully constructed and expressed efficiently and efficiently. The expressed product had the activity of binding to the corresponding target antigen and the anti-CD20Fab-LDM enhanced fusion protein was successfully prepared. The Raji cells that could specifically kill CD20 + .