Antiangiogenic Activity of Peptide from NC1 Domain of Mouse Collagen TypeⅣ alpha 2 Chain

摘要

Mouse canstatin is a C-terminal globular non-collagenous domain of type Ⅳ collagen a 2 chain, which previously showed anti-angiogenic activity. To investigate in vivo angiostatic effects on chick chorioallantoic membrane (CAM) by recombinant mouse canstatin N-fragment(1-95aa), the cDNA for N-fragment of mouse canstatin, obtained from a cloning vector pMD18T-mCan by PCR, was introduced into an expression vector pET30a(+) to construct prokaryotic expression vector pET-mCanN. N-fragment of mouse canstatin efficiently expressed in E coli BL21 (DE3) after IPTG induction was monitored by SDS-PAGE and by Western blotting with an anti-hexahistidine tag antibody. The expressed N-fragment of mouse canstatin, mainly as inclusion bodies, accounted for approximately 18% of the total bacterial proteins as estimated by densitometry. The inclusion bodies were washed, lysed and purified by the nickel affinity chromatography to a purity of 92%. The refolded N-fragment of mouse canstatin was tested on the chicken embryo CAMs, and a large number of newly formed blood vessels were significantly regressed. In this report, we demonstrated for the first time that N-fragment derived from mouse canstatin, is an effective inhibitor of angiogenesis of the chicken embryo in a dose-dependent manner through CAM assay and a promising candidate for the treatment of cancer. These novel findings may add to our understanding of anti-angiogenesis effects of the N-fragment of mouse canstatin.