抗肿瘤侵袭与转移单链抗体scFV-M97基因克隆及分泌性表达

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5×109pfu/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732 bp.其中VH 351 bp,编码117个氨基酸;VL 336 bp,编码112个氨基酸,两者以连接肽(Gly4Ser)3相连.阳性噬菌体转染HB2151细胞,经1 mmol/L IPTG诱导培养20 h,培养液上清中有2 μg/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.

Construction and secretory expression of single-chain Fv fragment M97 with therapeutic potential against tumor invasion and metastasis

OBJECTIVE: To manufacture single chain Fv antibody fragment for cancer targeting therapy. METHODS: Based on recombinant phage display techniques, the construction, screening and expression of functional single-chain Fv fragment (scFv) from murine hybridoma C2H5 which secretes antibody specifically binding to (92 kD) type IV collagenase were performed. By RT-PCR for VH and VL genes, assembly of scFv genes and cloning into phagemid pCANTAB5E, transformation of TG1 cells and M13KO7 helper phage rescue, a recombinant phage scFv library with titre of 5 x 10(9) pfu/ml was established. Panning against immobilized type IV collagenase (92 kD) was performed by one round. Finally, 30 antigen-positive recombinant phage clones were isolated and identified by ELISA. RESULTS: DNA sequencing result showed that the entire gene of scFv against type IV collagenase (92 kD) designated as scFv-M97 is 732 bp, consisting of the 351 bp VH gene encoding 117 amino acids, the 336 bp VL gene encoding 112 amino acids and a 45 bp linker encoding (Gly4Ser)3. The antigen-positive phage with titre of 10(10) pfu/ml was used to infect HB2151 cells in which soluble scFv was expressed. After induction of Lac promotor by 1 mM IPTG for 20 h, the scFv-M97 yield of HB2151 in SBA medium is about 2 micrograms/ml. Immuno-blot indicated that the 27 kD scFv-M97 retained the affinity and specificity of the original intact antibody to type IV collagenase (92 kD). CONCLUSIONS: Single chain antibody scFv-M97 expressed by secretion was successfuly produced. As type IV collagenase plays an important role in tumor invasion and metastasis by the mechanism of proteolytic degradation of type IV collagen in the basement membrane, scFv-M97 may have therapeutic potential against tumor invasion and metastasis.