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Protective Effect Of Recombinant Human Hepassocin On Experimental Hepatic Injury In Rat

Posted on:2007-07-29Degree:DoctorType:Dissertation
Country:ChinaCandidate:C Z CaoFull Text:PDF
GTID:1100360185479481Subject:Biochemistry and Molecular Biology
Abstract/Summary:PDF Full Text Request
Hepassocin (HPS) is a liver-specific gene which is upregulated during liver regeneration. The Hepassocin cDNA contained an open reading frame (ORF) encoding a protein of 312 amino acids. The Hepassocin is a 34kDa protein and has mitogenic activitiy in hepatocytes as a homodimer (66kDa). We cloned the Hepassocin cDNA from human fetal liver cDNA library and inserted it into the pBV220 vector.At present, the recominant plasmid was transformed into the E.coli strain BL21(DE3). After temperature induction, the expressed HPS protein which is about 40% of total bacterial protein existed mainly in the form of unsoluble inclusion body. In our study, we use an appropriate washing process and get more than 80% pure inclusion bodies containing HPS. Such purified inclusion bodies were used for subsequent solubilization and refolding for the recovery of bioactive protein. By using the AKTA FPLC protein purification system, we obtained more than 95% pure HPS protein.Recombinant human HPS produced in E.coli BL21-pBV220-HPS has mitogenic activities in hepatocytes including normal liver cell line L02 and primary rat hepatocytes, but it induces growth inhibition of hepatocellular carcinoma cell line HepG2 , SMMC7721 and other tissue cell lines. We further investigate the signaling pathway involved in the proliferation effect of HPS on the normal liver cell line L02. The result shows that HPS induced activation of ERK in L02 at 5min after induction of HPS, but the phosphorylation of Akt and p38 was not detected. Furthermore, the phosphorylation of ERK1/2 and proliferation activation of HPS was suppressed by treatment with a MEK inhibitor U0126 which indicate that the MAPK/ERK pathway...
Keywords/Search Tags:Hepassocin, prokaryotic expression, liver regeneration, prevent liver injury, molecular mechanism
PDF Full Text Request
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