Construction Of Fusion Expressive Plasmid PGEX-6p-1-hri As Well As Effect Of GST-hRI On BALB/C Carrying H22 Tumor And On The Chicken Embryo Angiogenesis

Human ribonuclease inhibitor (hRI) is ubiquitous in the cytoplasm of human cell with molecular weight of about 50 kD and its gene locates in the No.11 chromosome (11p15.5). hRI consists of 15 leucine-rich repeat(LRP). Its tertiary structure is symmetrical horseshoe-shape. The activity of ribonuclease A (RNase A) can be inhibited by hRI through tight combination with it. Angiogenin is a member of RNase A superfamily. Moreover, according to its primary structure, there are 32 highly conserved cysteine residues in its molecule ; This results in great sensitivity to oxidants. And at least 30 cysteine residues should exist with reduced thiol groups which is necessary for hRI to maintain its biological activities. RI is the inhibitor of Ribonuclease A in the hydrolysis to RNA. The amino acid sequence of angiogenin (Ang) is highly conservative compared with RNase A. Based on the analysis of cDNA sequence, Ang is 35% identical with human pancreatic RNase A. Ang forms more restricted complex with hRI than that formed by RI and RNase A. Ang is produced and secreted by tumor cells and normal cells and its molecular mass is 14.4 kDa. It has a robust capacity to induce blood vessel formation in vivo and in vitro. It is expressed widely in many kinds of cell and tissue, such as cultured human colon cancer cells and hepatic cells, gastric cancer, colonic cancer, mammary cancer, carcinoma of corpus uteri and so on. Ang forms more restricted complex with RI than that formed by RI and RNase A. X-ray analysis showed that Ang had a very similar space structure with RNase A.They can both form tight complex with RI. Because RI can form the tight complex with Ang, so it can inhibit the activity of Ang, Our investigation showed that exogenous ribonuclease inhibitor can inhibit the tumor growth in mice, including Ca761, S180 solid tumor. The fusion protein was purified through Sepharose 4B affinity chromatography. Both the two have the effect of inhibiting tumor, but GST-hRI is superior.we can see it from the chicken embryo experiment and GST-hRI is superior to the same dose of hRI at the aspect of antioxidation. Coupled GST may contribute to maintain the stabilization of fragile hRI against the oxygen and keep its activity.