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Method for high-density fermentation of recombinant human hepatocyte growth factor naked plasmids

A hepatocyte growth factor, high-density fermentation technology, applied in biochemical equipment and methods, fermentation, recombinant DNA technology, etc., can solve the problem of large dosage, low bacterial concentration and plasmid content, and the total yield cannot reach the scale requirements and other issues, to achieve the effect of high efficiency and strong practicability

Active Publication Date: 2017-01-04
BEIJING NORTHLAND BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

All in all, it can be seen from the reports on the plasmid DNA fermentation process that due to the influence of factors such as the strains and the characteristics of the plasmid itself, although most of the existing fermentation processes can obtain higher cell concentrations during small-scale fermentation and plasmid content, but only lower cell concentration and plasmid content can be obtained during large-scale fermentation. Therefore, the problems in many plasmid fermentation processes reported now are the three factors of fermentation scale, cell concentration and plasmid content. Can not be presented at a higher level at the same time, resulting in a total yield that cannot meet the scale requirements
However, plasmid DNA has disadvantages such as low cell transfection efficiency, short expression duration, low expression level, and large drug dosage. Therefore, whether large-scale plasmid DNA preparation can be realized has become one of the key steps for plasmids to be used in gene therapy.

Method used

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  • Method for high-density fermentation of recombinant human hepatocyte growth factor naked plasmids
  • Method for high-density fermentation of recombinant human hepatocyte growth factor naked plasmids
  • Method for high-density fermentation of recombinant human hepatocyte growth factor naked plasmids

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] 1. 30L fermenter pilot high-density fermentation:

[0031] 1) Working seed culture

[0032] Take a strain from the working seed bank stored at -80°C, and inoculate 100 µL of the strain into the first-grade seed medium (50 mL in a 250 mL Erlenmeyer flask, containing 50 µg / mL kanamycin) after it has completely melted. Cultivate in a shaker, control the temperature at 37°C, rotate at 230rpm, and cultivate for 8 hours to obtain the primary seed solution; the primary seed solution is inoculated into the secondary seed medium (400mL in 2L Erlenmeyer flasks containing 50 μg / mL kanamycin), placed on a shaker for cultivation, controlled temperature at 37°C, rotation speed at 230 rpm, and cultivated for 15-17 h to obtain the working seed liquid for fermentation.

[0033] 2) Inoculate in a 30L fermenter for fermentation

[0034]Inoculate the prepared fermented working seed solution with 5% inoculum in a 30L fermenter containing 14L fermentation medium for fermentation; the mecha...

Embodiment 2

[0066] 1. 100L fermenter pilot high-density fermentation:

[0067] 1) Working seed culture

[0068] Take a strain from the working seed bank at -80°C. After it is completely melted, take 200 µL of the strain and inoculate it into the primary seed medium (100 mL in a 500 mL Erlenmeyer flask, containing 50 µg / mL kanamycin), and place in a shaker. Cultivate on a bed, control the temperature at 37°C, rotate at 230rpm, and cultivate for 8 hours to obtain the primary seed liquid; inoculate the primary seed liquid with a 4% inoculation amount into the secondary seed medium (4 2L Erlenmeyer flasks are filled with 650mL, containing 50 μg / mL kanamycin), placed on a shaker for cultivation, controlled temperature at 37°C, rotation speed at 230 rpm, and cultivated for 15-17 h to obtain the working seed liquid for fermentation.

[0069] 2) Inoculate in a 100L fermenter for fermentation

[0070] Inoculate the prepared fermentation working seed solution with 5% inoculum in a 100L fermenter ...

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Abstract

The invention relates to the technical field of biological pharmacy, in particular to a method for high-density fermentation of recombinant human hepatocyte growth factor naked plasmids. The method for high-density fermentation of the recombinant human hepatocyte growth factor naked plasmids comprises a step 1) of cultivting working seeds and a step 2) of performing inoculation to a fermentation tank for fermentation. According to the fermentation method, high expression of the recombinant human hepatocyte growth factor naked plasmids in 50 L scale fermentation can be achieved, the final cell concentration OD600 is 55 or above, the naked plasmid content can be 420 mg / L or above, so that the technical problem that the recombinant human hepatocyte growth factor naked plasmids are low in content and the yield cannot meet the large scale requirements is solved, and a solid basis is laid for industrialization of the recombinant human hepatocyte growth factor naked plasmids.

Description

technical field [0001] The invention relates to the technical field of biopharmaceuticals, in particular to a high-density fermentation method of recombinant human hepatocyte growth factor naked plasmid. Background technique [0002] Recombinant human hepatocyte growth factor naked plasmid is a gene therapy drug for the treatment of vascular ischemic diseases. It is injected into the muscle of the ischemic site, transfected into striated muscle cells, and expresses and secretes hepatocyte growth factor (HGF), which has the effect of angiogenesis. ), form collateral circulation locally, establish a "molecular bypass" mechanism, and increase blood supply to the ischemic site, so as to achieve the purpose of treating arterial ischemic diseases. [0003] The preparation method of recombinant human hepatocyte growth factor naked plasmid adopts genetic engineering method, that is, the use of bioengineering technology to construct a gene transfer system, which is mainly replicated ...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/12
Inventor 聂李亚许松山马素永
Owner BEIJING NORTHLAND BIOTECH