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目的研制抗肿瘤侵袭和转移的单链抗体。方法应用重组噬菌体抗体技术,从小鼠抗Ⅳ型胶原酶杂交瘤C2H5细胞中提取mRNA,构建单链抗体基因并克隆到噬粒pCANTAB5E中,转化大肠杆菌TG1,经M13KO7援救后,得到滴度为5×10~9pfu/ml单链抗体库。对抗体库进行一轮抗原固相化亲和富集与ELISA筛选鉴定,得到30株阳性噬菌体。结果 DNA序列分析表明,抗Ⅳ型胶原酶单链抗体scFv-M97基因全长732bp。其中V_H351bp,编码117个氨基酸;V_L336bp,编码112个氨基酸,两者以连接肽(Gl_(y_4)Ser)_3相连。阳性噬菌体转染HB2151细胞,经1 mmol/L IPTG诱导培养20h,培养液上清中有2μg/ml可溶性单链抗体。免疫印迹证实所表达产物保留了原亲本抗体的特异性和亲合力。结论单链抗体scFv-M97可分泌性表达,为以Ⅳ型胶原酶为靶点的抗肿瘤侵袭与转移的治疗及新型导向药物的研制奠定了基础。
Objective To develop anti-tumor invasive and metastatic single-chain antibodies. Methods The recombinant phage antibody technique was used to extract mRNA from mouse anti-type IV collagenase hybridoma C2H5 cells. The single chain antibody gene was constructed and cloned into the phagemid pCANTAB5E. E. coli TG1 was transformed into E. coli TG1 and rescued by M13KO7. The titer was 5 × 10 to 9 pfu/ml scFv library. A round of antigen immobilization and affinity enrichment of antibody libraries was performed and screened by ELISA. 30 positive phage were obtained. Results DNA sequence analysis showed that the anti-type IV collagenase single-chain antibody scFv-M97 gene was 732 bp in length. Among them, V_H 351bp encodes 117 amino acids; V_L336bp encodes 112 amino acids, and the two are linked by a connecting peptide (Gl_(y_4)Ser)_3. Positive phage were transfected into HB2151 cells and induced by 1 mmol/L IPTG for 20 h. There were 2 μg/ml soluble single-chain antibodies in the culture supernatant. Western blotting confirmed that the expressed product retained the specificity and avidity of the parental antibody. Conclusion The single-chain antibody scFv-M97 can be expressed secretively, which lays the foundation for the treatment of anti-tumor invasion and metastasis with type IV collagenase as the target and the development of new-oriented drugs.