Purification, Identification And Activity Of Recombinant Human Augmentor Of Liver Regeneration

Liver diseases such as hepatitis and hepatocirrhosis seriously damage human's health. Recent results showed augmentor of liver regeneration (ALR), a cytokine existed in cytosol of liver cell, had the ability of promoting hepatic cell proliferation. ALR was believed to be a hopeful candidate drug in treating serious liver diseases.In this thesis, process of purifying S-rhALR (soluble form in cytoplasm) from recombinant strain pJL3-pET22b-rhALR/BL21-GOLD (DE3) was established. Proper process was sonification, centrifugation, precipitation with 40% (NH4)2SO4, Butyl SFF chromatography, dialysis, CM SFF chromatography, protein concentration and Superdex-75 chromatography.The purification process of IB-rhALR (IB) from pET22b- rhALR/BL21- GOLD (DE3) was also established. The craft included sonification, IB acquirement, lysis of IB, Q SFF chromatography, renaturation, concentration, CM SFF and Superdex-75 chromatography.The results of analysis showed that the purity of S-rhALR and IB-rhALR had reached 95% and both existed as the form of homodimer. Their sequences of the N-terminus of 15 amino acids were the same and consistent with the deduced codes from cDNA of hALR. Both proteins showed immunity activity to the anti-serium extracted from fetal liver. Coenzyme FAD was proved to bind with S-rhALR but not to IB-rhALR.In vitro experiment showed S-rhALR had thioloxidase activity, but IB-rhALR showed no thioloxidase activity. In in vivo experiment, S-rhALR significantly promoted the DNA synthesis of hepatocytes and effectively protected hepatocytes from acute hepatic injury by CCl4. On the contrary, IB-rhALR didn't show the activities mentioned above.This research provides basis on developing ALR to drug in treatment of serious liver diseases.