Productive method of recombinant human liver regenerated enhanced factor

A technology for enhancing factors and production methods, which can be used in drug combinations, pharmaceutical formulations, medical preparations containing active ingredients, etc., can solve problems such as lack of liver specificity, and achieve the effect of improving survival rate

Inactive Publication Date: 2005-12-28
INFECTIOUS INST OF PLA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Unlikely to be key regulators of liver regeneration due to the lack of liver-specific actions of most factors

Method used

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  • Productive method of recombinant human liver regenerated enhanced factor
  • Productive method of recombinant human liver regenerated enhanced factor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Production method of recombinant full-length human enhancer of liver regeneration factor

[0032] One, the technical points of this embodiment:

[0033] 1. Bacteria are fermented at low density;

[0034] 2. The inclusion body denaturant is urea;

[0035] 3. Purify the inclusion body by cation exchange chromatography after renaturation;

[0036] 2. Operation steps;

[0037] 1. Activation of engineering strains: Recombinant pBV220-hALRL / JM109 was constructed in our laboratory. Pick a well-separated single colony from the bacterial plate preserved at 4°C, inoculate it in 5ml LB / Amp (100ug / ml) medium, shake and cultivate at 200rpm at 30°C for 12h; Shake culture at 200 rpm at 30°C for 12 hours as seed solution.

[0038] 2. Fermentation: Inoculate the seed liquid in 500ml LB / Amp (100ug / ml) medium according to 2.5%, shake and culture at 200rpm at 30°C for 3-4h, wait for the OD 600 When =0.3-0.4, raise the culture temperature to 42°C, and continue shaking cultu...

Embodiment 2

[0045] Example 2: Recombinant full-length human enhancer of liver regeneration promotes DNA synthesis of the hepatic cell line HepG2 in vitro

[0046] One, the technical points of this embodiment:

[0047] Recombinant full-length human enhancer of liver regeneration promotes DNA synthesis in the hepatic cell line HepG2 in vitro.

[0048] 2. Operation steps

[0049] 1. HepG2 cell culture: HepG2 cells were grown in high-glucose DMEM+10% fetal bovine serum, 37°C, 5% CO 2 to cultivate. After the cells grow to the logarithmic phase, trypsinize and adjust the cell density to 4×10 5 / ml, inoculate 100ul per well in a 96-well culture plate, and continue culturing for 6h.

[0050] 2. Adding samples: Add the samples to be tested in the 96-well culture plate, set 3 replicate wells for each concentration, and continue to culture for 24 hours. If 2.0×10 4 Q 3 H-TdR, continue to culture for 3h.

[0051] 3. Detection: Use the cell harvester to collect the cells on the filter paper, a...

Embodiment 3

[0053] Example 3: Recombinant full-length human enhancer of liver regeneration promotes DNA synthesis in rat liver cells after partial hepatectomy in vivo

[0054] One, the technical points of this embodiment:

[0055] Recombinant full-length human enhancer of liver regeneration promotes DNA synthesis in rat liver cells after partial hepatectomy in vivo.

[0056] 2. Operation steps:

[0057] 1. Establishment of rat partial hepatectomy model: Rats were reared in a single cage for 1wk, fasted overnight before the experiment, and pentobarbital sodium (40mg kg -1 ) anesthetized with 750mL·L after epigastric hair removal -1 Disinfect the skin with alcohol, make a midline incision in the upper abdomen, and expose the liver. After the ligaments of the left and middle lobes of the liver are fully freed according to Hoggin’s method, the roots of each liver lobe are firmly ligated, and the left and middle lobes of the liver are removed quickly at the distal end of the ligature. That ...

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Abstract

The invention relates to a production method of recombinant full-length human liver regeneration enhancing factor and its use for treating severe liver diseases, and belongs to the field of production methods and uses of biological products. On the basis of obtaining the full-length human enhancer regeneration factor gene sequence and successfully constructing high-efficiency expression strains, a process of prokaryotic expression of recombinant full-length enhancer of human liver regeneration factor, separation and lysis of inclusion bodies, protein purification and renaturation was established, and finally the recombinant whole-body gene was obtained. A pure product of long-term human liver regeneration enhancing factor. The latter can promote DNA synthesis of the hepatic cell line HepG2 in vitro, promote liver regeneration in rats after partial hepatectomy in vivo, and promote CCl 4 DNA synthesis and repair in rat hepatocytes after poisoning, increase in CCl 4 The survival rate of rats after poisoning indicates that the recombinant full-length human liver regeneration enhancing factor has the utility of treating severe liver diseases.

Description

field of invention [0001] The invention belongs to the field of production method and application of bioengineering products, in particular the production method of recombinant full-length human liver regeneration enhancing factor and its application for treating severe liver diseases. Background technique [0002] The liver is one of the organs with the most powerful regeneration ability in the human body. The regeneration of the liver is of great significance in the pathogenesis and treatment of various liver diseases such as acute / chronic viral hepatitis and severe hepatitis. The regulatory mechanism has been continuously studied, and dozens of regulatory factors have been found, such as hepatocyte growth factor (HGF), epidermal growth factor (EGF), transforming growth factor-α (TGF-α), etc. . Since the actions of most factors lack liver specificity, they are unlikely to be key regulators of liver regeneration. In 1975, Labrecque et al. reported for the first time that ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/18A61P1/16C12P21/02
Inventor 成军王刚夏小兵洪源刘妍钟彦伟王琳董菁
Owner INFECTIOUS INST OF PLA
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