Cloning And Expression Of The Tumstatin Active Peptides-T₇ And Its Derivant-T₇-NGR

The growth and metastasis of tumors are dependent on neovascularization, tumor cells will apoptosis or necrosis if we can inhibit the formation of new blood vessels. In recent discoveries, it is shown that some macromolecular protein fragments with biological activity which are separated from tissue or body fluids are angiogenesis inhibitors. Tumstatin, which is composed of 244 amino acids, with the molecular weight of 28 kD, is one kind of protein from the C-terminal NC1 fragment of type IV collagenα3 chain. Tumstatin is able to inhibits pathological angiogenesis and suppresses the proliferation of endothelial cells and the growth of tumors. It was reported in recent studies that there are two main active sites of tumstatin: (1) inhibition of angiogenesis formation, thereby inhibiting tumor growth, invasion and metastasis; (2) direct inhibition of the tumor cell proliferation. These effects is achieved mainly through the combination of tumstatin and integrinαvβ3, which is tumor-specific expression. Tumstatin's anti-angiogenic activity was gradually localized in 25 residues of the amino acid, tumstatin74-98 residues, it was named T₇ peptide. Because of its small molecular weight, it is able to penetrate the tumor cells easily, with low immunogenicity and more stability, it is expected to become a marker of tumor-specific diagnosis and substances of the cell-targeted treatment.NGR is an oligopeptide with 13 amino acids (CNGRCVSGCAGRC). It was screened from the phage display library. NGR have the ablity of tumor angiogenesis specific binding through the surface expression of aminopeptidaseN (CD13) in tumor angiogenesis. With its highly binding affinity, NGR peptide can be used as radioactive tracer and targeting-transport vector.By using gene recombination technology, we successfully constructed the cloning vector and expression vector of T₇ and T₇-NGR peptide respectively. And obtained the expression products-T₇ peptide and its derivant T₇-NGR peptide. T₇-NGR is the fusion of T₇ and NGR, with NGR in the C-terminal of T₇. This study will lay the foundation for further activity experiments.