Dissertation > Medicine, health > Basic Medical > Human biochemistry, molecular biology

Studies on the Cloning, Expression, Bioactivities and Application of Human Augmenter of Liver Regeneration Gene

Author HaoYanQiu
Tutor LiJingPeng
School Northeast Agricultural University
Course Microbiology and Immunology
Keywords Augmenter of liver regeneration Clone Expression Biological Activity
CLC R346
Type PhD thesis
Year 2003
Downloads 152
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Augmenter of liver regeneration (Augmenter of liver regeneration, ALR) is a special kind of promoting hepatocyte mitogen, play an important role in the repair process of liver injury. Liver disease is one of the major diseases that endanger human health. China's hepatitis, liver cirrhosis and liver cancer patients, the number of about 100 million. And the incidence increased year by year, according to 1999 statistics, the incidence rate is about 12%. Therefore, in order to relieve the suffering of patients with liver disease to study a new drug to treat liver disease is imminent. Since 1987, Chinese scholars application of the suspension of the fetal liver, the treatment of various liver diseases and achieved remarkable results. Thus inspired and explains the proliferation of liver cells containing fetal liver stimulating factor. In 1994, Hagiya et al. Isolated from weanling rat liver cytosol factor of a material which promotes a thermal stability of the cell proliferation of hepatocytes, named hepatocyte regeneration enhancement factor. In this thesis, the application of genetic engineering means Cloning, expression and purification of human augmenter of liver regeneration factor gene (hALR was). Laid the foundation for the further study of its biological function. Established liver fibrosis in rats and mice the fat liver injury model, a more systematic study of the biological activity of the cloning method to obtain the regeneration enhancement factor gene, laid to hALR used in the clinical treatment of liver disease as early as possible the foundation. The results are as follows: 1. The the extract hALR gene for the first time from 6 months fetal liver total RNA by RT-PCR, cloned cDNA fragment of the complete reading frame of the hALR. By the sequencing results indicated that the recombinant fragment is 378 nucleotides consisting of 2 bases mutated, did not result in an amino acid change. 2. The first time constructed the recombinant ALR prokaryotic expression vector, to get the pET28a-hALR the recombinant plasmid, and sequencing confirmed. 3. IPTG inducible expression of the gradient, the first time the pET28a-hALR gene different concentrations of the inducer IPTG induced expression that, when the IPTG concentration of 1M, the highest expression of inducible expression of the time gradient; Initial the pET28a-hALR gene. After the induction of expression of the different time showed that the highest expression level of the induction time in 3 hours. 4. For the first time on the pET28a-hALR gene expression sites were analyzed. Prokaryotic expression of the protein in either the precipitate or in the supernatant were expressed. More expression than in the supernatant the precipitate. Lots i.e. protein expression in E. coli cells, and mainly exist in the form that contains the body. 5. Through the expression of the protein detected by Western blotting (Western-blot detection), that express 18.7KD fusion target protein. Westen-blot hybridization color reaction, results in the near 20.1KD there is a distinct black spots, it is hALR its antibody binding expression of the target protein. 6. NI ion exchange resin purification Expression the pET28a-hALR protein. SDS-PAGE gel electrophoresis of the purified protein to prove that the target protein is purified. 7. Through the establishment of human serum albumin immune injury in rat liver fibrosis model for the first time, research the rhALR biological activity of liver fibrosis. The results show that the rhALR significantly inhibited immune injury in rat liver fibrosis tissue, in autoimmune liver fibrosis, given rhALR can serum in ALT, AST, LDH level was significantly lower. Protect the human serum albumin-induced rat liver cells in chronic autoimmune damage. 8. Through the establishment of Ethionine for the first time in mice caused by fat F-injury model was rhALR biological activity of fatty liver injury. The results show that the rhALR could significantly inhibit fatty liver ALT, AST, LDH activity increased, at the same time reduce the TG tCH content, hALR have a therapeutic effect on the the ethionine cause of fatty liver in mice. Science, Northeast Agricultural University doctoral thesis 9. Production by genetic engineering methods can 11Hut, not only greatly Dow costs, and overcome the raw u agent Disadvantages: low purity, containing impurities, high cost, and prone to allergic reactions. But also solve the problem of limited sources of fetal liver. The drug development and clinical application of recombinant human augmenter of liver regeneration sub-gene laid out. 10 ALR level in the ELISA method detected in the serum of patients with different types of liver disease. Try a faster check now 9 serum ALR level. The experimental results show that the serum of patients with liver disease Film's ALR level of concentration can be detected a 0.2 u speaker, the maximum is 9.2 ug / L Found the reaction of ALR antibody ALR antigen of good linear positive correlation. Prompt the ELISA j3i:. ; Y the measured peripheral serum ALR level with good sensitivity and specificity.

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