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An activating factor, human-derived technology, applied in the field of biomedicine, can solve the problems of high patient mortality and lack of treatment methods
Inactive Publication Date: 2010-02-17
RENJI HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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The clinical manifestations are severe jaundice, ascites, coagulation disorders, and hepatic encephalopathy. The disease is dangerous, but internal medicine c
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Embodiment 1
[0034] Example 1: Preparation of recombinant human HGFA-Fc fusion protein
[0035] Use RT-PCR to catch the HGFA active region DNA fragment (GeneID: 3083, 1104bp ~ 1962bp) from the liver tissue cDNA library with a size of 858bp, and use Overlap overlapping PCR (see Cao Yang et al., "Hebei Medicine", Volume 27, Issue 11, 2005) Fusion with human immunoglobulin Fc (GeneID: 2209) 693bp fragment to obtain 1575bp HGFA-Fc fragment, Xho I / Hind III double digestion and cloning into pcDNA3.1(+) vector. After the enzyme digestion and identification are correct, a large amount of extraction, purification, and concentration determination are used for later use. Specific steps are as follows:
[0036] 1. Construction of recombinant plasmid pcDNA3.1-HGFA-Fc:
[0037] 1. Primer synthesis for amplifying the HGFA-Fc chimeric gene
[0038] The human HGFA gene (ID: 3083) was determined according to the gene library, and primers were designed to amplify the range of 1102bp to 1965bp of the human...
Embodiment 2
[0071] Example 2: Enzyme cleavage activity and anti-apoptosis effect of rhHGFA-Fc recombinant fusion protein
[0072] 1. Detection of pro-HGF enzyme activity activated by HGFA-Fc:
[0073] In the pro-HGF reaction buffer with a final concentration of 50ug / ml, add HGFA-Fc concentration gradients of 0.15ug / ml, 0.3ug / ml, 0.6ug / ml, 0.12ug / ml, 0.24ug / ml, 4.8 ug / ml, Western blot was used to detect the expression of proHGF (92kD) and HGF (62kD) in each reaction system after 1 hour of reaction. The results show that rhHGFA-Fc recombinant protein can digest pro-HGF into α chain and β chain, and the enzymatic activity increases with the increase of rhHGFA-Fc concentration. The results are shown in Figure 4 . After calculation, the IC50 enzyme activity value of the recombinant protein is 20nM.
[0074] In the pro-HGF solution with a concentration of 50ug / ml, HGFA-Fc fusion protein with a final concentration of 2.4ug / ml was added, and HAI-1 with a final concentration of 0.5uM, 2uM and ...
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Abstract
The invention relates to the field of biological medicines. A hepatocyte growth factor (HGF) activating factor is plasma protein secreted by hepatocyte, and an activated HGFA is 33Kd polypeptide, is just distributed in the damaged tissues and organs, can activate the monomer HGF secreted by a liver nonparenchyma cell into the HGF at the damaged local part and plays roles of cell protection and tissue repair at the local parts of the damaged tissues and organs. The acute and chronic liver function failure has the main pathological characteristics of large-area hepatocyte apoptosis and necrosisand hepatocyte reproducing function inhibition. The clinical manifestation is dangerous, and the mortality of a patient is as high as 70-90 percent because of lacking an effective curing approach. Theinvention provides a recombinant anthropogenic HGF activating factor which has a nucleotide sequence and a coded amino acid sequence shown as SEQ ID NO:3 and can be used for an application of preparing a medicine for treating acute or subacute severe hepatitis hepatocyte necrosis.
Description
technical field [0001] The invention relates to the field of biomedicine, in particular to a recombinant human HGF activator (rhHGFA) and the application of rhHGFA in the preparation of medicines for treating acute or subacute severe hepatitis liver cell necrosis. Background technique [0002] Hepatocyte growth factor activator (HGFA) is a plasma protein secreted by liver cells, GeneID: 3083, activated HGFA is a 33Kd polypeptide, which is only distributed in damaged tissues and organs, and can hepatic nonparenchymal cells in damaged parts The secreted monomeric hepatocyte growth factor (pro-HGF) is activated into HGF, which plays a role in cell protection and tissue repair in damaged tissues and organs (see literature Shia S, Stamos J, Kirchhofer D, et al.Conformational ability in serineprotease active sites: structures of hepatocyte growth factor activator (HGFA) alone and with the inhibitory domain from HGFA inhibitor-1B. J Mol Biol. 2005 Mar 11; 346(5): 1335-49.; Miyazawa...
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IPC IPC(8): C12N15/12C07K14/435A61K38/17A61P1/16
Inventor 薛峰夏强
Owner RENJI HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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