Cloning Of Human Insulin-like Growth Factor Binding Protein-3 Gene And Its Expression In E.coli

Insulin like growth factor binding protein 3 is an important member in multifunctional endocrine factor family, and it has the largest number and the strongest effect in the blood.95%-99% IGF-1 in the blood bind can combine with IGFBP-3. Recombinat human IGF-1 has been used into the treatment of dwarfishness for a long time. And an investigation found that it can cure diabetes mellitus, and osteoporosis. But It will take some gentle and moderate side effects when use IGF-1 is used solo for treatment, such as puffiness of the limbs, headache, artache, jaw tenderness, postural hypotension, optic fasciculus oedema. IGFBP-3 can reduce the untoward reaction by prolonging the half life and controling the biological availability of the IGF-1.So in order to raise the drug action and degrade the adverse effect,the combination of rhIGF-1/rhIGFBP-3 could be the best proposal.In addition IGFBP-3 has the hope to be exploited as a kind of new drug, and has extensive prospect for application. After researching for many years IGFBP-3 has been identified as a apoptotic factor that is independent from IGFs. IGFBP-3 takes effects in includs inducing apoptotic, interfering growth of cells and reinforcing the radio sensitivity of cancer cell. The mechanism of IGFBP-3 influencing the cells which growth out of control has became a new target of antineoplaston. IGFBP-3 has been used to the treatment of tumor alone or jointly used with chemotherapeutics,and it has entered the first stage of clinical trail. But the report of IGFBP-3 at home is fewer.The efficient expression system of recombinant human IGF-1 has been constructed successfully in our laboratory. The work in this paper is to construct efficient expression system of recombinant human IGFBP-3, and to make the basis for further study in investigating the biological activity of IGFBP-3. Tatal RNA was extracted from human liver tissue with Trizol Reagent. The cDNA encoding the IGFBP-3 was amplified in using RT-PCR,and then was inserted into expression vector pQE-30Xa, named pQE-30Xa/IGFBP-3. As expressing cell, E. coli M15[PREP4] was transformed with constructed recombinant plasmid pQE-30Xa/IGFBP-3 and its expression was induced by IPTG. The expressed product was verified by Western blot. The recombinant protein was purified by using Affinity Chromatography on nickel-nitrilotriacetic acid column. The combination between IGF-1 and rhIGFBP-3 was detected by ELISA. And the inhibition of proliferation of Hela cell was detected by using method MTT. After purification,the purity of protein exceed 95% and has bioactivity.