Cloning, Expression And Purification Of Recombinant Human Augmenter Of Liver Regeneration

IntroductionAs is known to us, Acute or chronic hepatic failure due to all kinds of pathogenies attack people's health badly. A great deal of hepatic cells become necrotic in a very short time and result in a high mortality of hepatic failure. The pathogenesis is unknown and we have no effective therapy means yet. Liver has powerful regeneration ability. Researches in the cytokines contribute to the liver regeneration become a hot point these years. Many cytokines regulate the regeneration process of liver. Till now we have found more than 20 kinds of cytokines which promote or restrain the liver regeneration process. Such as Hepatocyte growth factor (HGF), Transforming growth factor a (TGF-a), Epidermal growth factor (EGF), insulin, But they all can not explain the apparatus specific mechanism of liver regeneration. In 1994 Hygiya cloned a new cytokine from the cytosol of weanling rat livers and named it as augmenter of liver regeneration (ALR). ALR is the only cytokine which can specially stimulate hepatic origin cells to grow regardless of genus. It has proved by experiment that ALR can promote the regeneration of liver and protect liver from all kinds of injury. We found higher expression of ALR after liver injury or partial excision of liver.In this research we cloned a full length cDNA sequence of human augmenter of liver regeneration (hALR) from human liver tissue and then inserted the hALR cDNA into the prokaryotic expression vector of pET28a(+) to construct the recombinant expression plasmid of pET28a(+)-ALR. Then weobtained and purified the recombinant hALR protein from E.coli BL21 (DE3) and it could be developed into a new anti-hepatic damage product later.Materials and methodsTotal RNA was obtained from normal human liver tissue. Primer was desighed according to the hALR coding sequence. Full length hALR cDNA was got by one-step RT-PCR and then the hALR cDNA was inserten into the prokaryotic expression vector pET28a(+). The correct insertion site was confirmed by restriction endonuclease digesting and the correct DNA sequence of the insertion fragment was determined by DNA sequencing. The recombinant hALR protein was expressed in Escherichia coli BL21 (DE3) under IPTG induction. Change the expression conduction of temperature and time to increase the expression of solube protein. Purify the objective protein by His Tag bind kits. Detect the purified protein by SDS-Page and Western-blot.ResultsThe full length cDNA we obtained by one-step RT-PCR encoding hALR and histidine label was got and detected by agarose gel electrophoresis of about 420 bp. The correct insertion site was confirmed by restriction endonuclease digesting and the correct DNA sequence of the insertion fragment was determined by DNA sequencing of 418 bp, which contained the full length coding sequence of hALR cDNA and its initiate codon and the determination codon. The hALR cDNA sequence was in accordance with the sequence displayed in Genbank . The insertion fragment also contained the histidine label sequence and the protease Factor Xa sequence. The recombinant hALR protein was expressed successfully in E.coli BL21 (DE3) . The molecular weight of the recombinant hALR protein including histidine label was about 23 kD detected by SDS-Page and Western-blot. We saw the objective protein laybetween 20 kD and 30 kD in SDS-Page and had a apparent exposure line in Western-blot, which was accordance to our expect. Lower temperature and longer time can increase the expression of solube protein. 23℃ was proved to be the best conduction temperature to obtain more solube protein. Purify the solube part of objective protein by His bind kits. The protein strape in SDS-Page was a single line of 23 about kD after His Tag bind kits purification which was correspond to our expect.conclusion1. Full length hALR cDNA was obtained from human liver tissue by one-step RT-PCR and it was inserted correctly into the prokaryotic expression plasmid pET28a(+).2. Recombinant hALR protein was successfully expressed in E.coli BL21 (DE3). Change condition of conduct...