The Study Of Cloning, Expression And Bioassay Of Human Tumstatin

Tumor growth and metastasis are dependent on angiogenesis generation. If theformation of new blood vessels was inhibited, tumor cells will apoptosis or necrosis.At present, there is a variety of inhibiting angiogenesis drugs for anti-tumor research.Recently discoveries show that some macromolecular biological activity proteinfragments which are separated from the organization or body fluids are angiogenesisinhibitors. Tumstatin is 28 kDa protein from the C-terminal NC1 fragment of typeâ…£collagenα3 chain that inhibits pathological angiogenesis and suppresses proliferationof endothelial cells and growth of tumors. Recent research shows that there are twodistinct activity regions; one is the N-terminal amino acid 54～132 ofα3 chain possessanti-angiogenic activity. Another is the C-terminal amino acid 185～203 havedirectly anti-tumor activity. Thestudy indicates tumstatin is anαvβ3 integrin-dependent angiogenic inhibitor.Integrinαvβ3 is a desirable target which expression a lot on tumor angiogenesis. Astumstatin has a specific binding capacity to integrinαvβ3, we can use of ¹³¹I,⁹⁹Tc^mand other radioisotope labeled tumstatin for the radiological receptor imaging, whichhas an important and potential clinical value.Total RNA was extracted from HEK293 cell. The tumstatin gene was amplifiedby RT-PCR, then cloned into pMD19-T and sequenced. Subsequently, the tumstatingene was cloned into the pET28a (+) and transformed into E.coli BL21 (DE3) whereit was induced to express by IPTG. The fusion protein was expressed in E.coliefficiently and amounted to 30% of totalprotein content. The SDS-PAGE demonstratesthat the expression of tumstatin mainly in the form of inclusion bodies. A certainconcentration of protein was obtained from Ni-affinity chromatography and dialysisfor further study. The biological activities of tumatatin were determined by MTTassay, it can inhibit tumor cell proliferation.