| Type IV collagenase with two subtypes of 72kDa/MMP-2 and 92kDa/MMP-9 plays an important role in tumor invasion and metastasis, which based on the mechanism of proteolytic degradation of collagen IV in basement membrane by which malignant tumor initiates its invasion and spread. It has been demonstrated that natural and synthetic inhibitors of type IV collagenase such as TIMPs, BB94 have reduced the metastasis of murine Lewis lung carcinoma and neovascularization in tumor tissues, so antibodies against type IV collagenase may have therapeutic use on antitumor invasion and metastasis. However, intact murine antibody has disadvantages of immunogenicity and low tissue penetration in clinical application. To overcome these shortings and based on recombinant phage display techniques, the construction, screening and expression of functional single-chain Fv fragment (scFv) from murine hybridoma M97 which secrets antibody specifically binding to 92kDa type IV collagenase/MMP-9 were performed. mRNA extracted from hybridoma M97 was used as a template to synthesize the first strand of cDNA. Light and heavy chain variable region genes (VL, VH) were separately amplified by RT-PCR. The scFv genes were assembled by using linker primers and re-amplified by using RS primers which hybridized to the conserved segment at the two ends of the scFv gene and contained Sfi I and Not I restriction sites for cloning into expression phagemid. The recombinant vector pCANTAB5E was introduced into competent E coli TG1 cells by CaCI2 transformation. In transformed TG1 cells, scFv was displayed on the tips of the phage as a fusion protein with g3p protein, because the function of amber coden between the scFv and g3p genes was suppressed. After M13KO7 helper rescue, a recombinant phage scFv library with titre of 5 X 109pfu/ml was established. Panning against MMP-9 coated on a tissue...
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