Construction Of Fusion Expressive Plasmid PGEX-6p-1-hri And Oxidative Resistance Of GST-hRI On CCl₄ Damaged BALB/c

BACKGROUNDS: Human ribonuclease inhibitor (hRI) is ubiquitousin the cytoplasm of human cell with molecular weight of about 50 kD andits gene locates in the No.11 chromosome (11p15.5). hRI consists of 15leucine-rich repeat( LRP). Its tertiary structure is symmetricalhorseshoe-shape. Moreover, according to its primary structure, there are 32highly conserved cysteine residues in its molecule;this results in greatsensitivity to oxidants. And at least 30 cysteine residues should exist withreduced thiol groups which is necessary for hRI to maintain its biologicalactivities. It is well known that reduced glutathione protects organismsfrom peroxidation by its reduced thiol group. We predicted that RI mightalso have antioxidative functions. Our laboratory had carried out experiments in vitro about theantioxidative effect of hRI. We found that rat glial cell line C6 transfectedwith hRI cDNA can resist more injuries with H₂O₂ than the control C6 cells.The resistance to the oxidation stress of cells is monitored bymorphological observation and assays of LDH, MDA, GSH level. AIM: To find out the optimum expression condition of fusion proteinGST-RI in order to magnify the soluble yield of hRI. To evaluate theantioxidation protective effect of fusion protein GST-RI on CCl₄ damagedBALB/c mouse.METHODS: In this experiment, I constructed a fusion expressiveplasmid pGEX-6p-1-hRI and explored optimum inducing condition. ThecDNA sequence of human RI was inserted into the plasmid pGEX-6p-1immediately after the sequence of Glutathione S-Transferase (GST) tag.The small-scale product of fusion protein GST-hRI was induced at 26degree Celsius with 0.5mmol IPTG induced for 8 hrs. The fusion proteinwas purified through RNase A-Sepharose 4B affinity chromatography.Concentrated by dialysis against 20% PEG-6000, purified GST-hRI wasready for administration to peritoneum of BALB/c mice. Vitamin C is one of well-known antioxidative agent, which canprovide a standard of antioxidative effect. Vitamin C, GST-hRI, hRI, andsaline were pre-administered to BALB/c mice. Seven days later, carbontetrachloride (CCl4) was injected into all groups of mice to make an acutehepatic injure model. The blood and liver samples were collected after24hrs since the last injection of CCl4. Controlled by vitamin C and purifiedhRI protein, the protective antioxidation effect of GST-hRI fusion proteinon the injured liver was evaluated by the levels of serum GPT (GlutamicPyruvic Transaminase, ALT), GOT (Glutamic Oxaloacetic Transaminase,AST), liver SOD (Superoxide Dismutase), XOD (Xanthine Oxidase), MDA(malondialdehyde), and GSH-Px (Glutathione peroxidase). Pathologicalanalysis on H&E-stained slides was carried out to observe morphologicalchange of hepatocytes. RESULTS: 1. After treated by CCl4 for 24hrs, blood biochemical indices(including serum GPT and GOT in the groups of vitamin C, hRI andGST-hRI) are superior to the group of CCl4 damage, but inferior to thesaline group. Among three treatment groups, the protective effect ofGST-hRI is almost the same as that of Vitamin C by the student t-test. Theindices of these two groups are better than that of hRI treated group. 2. As for t0h11e indices SOD, MDA, GSH-Px of mice livers, thedifferences are significant between the CCl4 damage group and any of threetreatment groups. Moreover, among three treatment groups, the indices inthe vitamin C group and GST-hRI group is much better than that of hRIgroup. But there was no significant difference of XOD in the liver amongall groups. 3. According to pathological analysis on H&E-stained sections, bothfibrosis and cirrhosis were largely prevented in the treatment groups. Thelevel of hepatocyte damage in three treatment groups was much better thanthat of CCl4 damage group. Under light microscopic field, the hepatocytesin treatment groups looked swollen with ballooning degeneration, theyseemed distended and cytoplasm seems translucent and empty. Nucleilocated in the center of the cell with slightly dying. Hepatocytes corddisarrayed slightly. CONCLUSION: In this work, I found the protec...