The Study Of Single-chain Fv Fragment Directed Against Type Ⅳ Collagenase And Its Fusion Protein

Type IV collagenase, also termed gelatinase, can be classified into 72 kDa and 92 kDa subtypes according to the molecular mass. Both are members of the matrix metalloproteinase gene family, and share structural homologies with other members of this family. Type IV collagenase degrades type IV collagen which is the major component of basement membrane and other collagens, destroys the integrity of basement membrane and extracellular matrix, which is in favor of the invasion and metastasis of tumor cells. The invasion potency of tumor cells is correlated with the activity of the secreted type IV collagenase. In addition, the activity of type IV collagenase is higher in endothelial cells stimulated by angiogenesis factors than normal cells. Inhibition of the activity of MMPs, especially type IV collagenase, can inhibit tumor invasion and metastasis, and angiogenesis.Murine monoclonal antibody 3D6 can specially bind to type IV collagenase, and some targeted drugs based on 3D6 have led to some striking antitumor effects. To reduce the molecular weight of immunoconjugate, single-chain Fv fragment (scFv) was constructed using phage display technique. We extracted and purified mRNA from the hybridoma cells secreted monoclonal antibody 3D6 and then cloned the heavy chain variable domain (V_H) and light chain variable domain (V_L) genes using RT-PCR. The V_H and V_L genes were assembled into scFv (V_H^-) Linker-V_L) genes by the connection with the linker DNA which encodes a flexible peptides (Gly₄Ser)₃ using overlap PCR. The PCR amplified scFv gene was digested with Sfi I and Not I, and then inserted into plasmid pCANTAB5E and transformed into E. coli TG1. With the presence of helper phage M13KO7, recombinant phage antibodies were produced as fusion proteins displayed on the tips of phages. Using collagenase IV as antigen, we screened the recombinant phage antibodies by immunoaffinity and obtained the strain 43, which has the highest affinity. The recombinant plasmid pCANscFv-43 was isolated and digested with Nco I and EcoR I. The scFv gene fragment was cloned into expression vector pET-30a (+) and inducible expressed in E. coli BL21 (DE3). The yield of expressed proteins mainly as inclusion bodies was about 15% of total cellular proteins. After denaturation and renaturation, the...