A kind of industrial preparation method and application of recombinant interferon gamma

A technology of porcine interferon and purification method, which is applied in the direction of peptide preparation method, biochemical equipment and method, interferon, etc., can solve the problems of lowering the specific activity rate of recombinant protein, unqualified product quality, precipitation and other problems, and achieve easy scale-up and repeatable, reduced content, easy-to-amplify effects

Active Publication Date: 2021-02-09
GENSUN INST OF BIOMEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If the renaturation conditions are not suitable, there will be covalent bonding or hydrophobic bonding between molecules to form aggregates, which will reduce the specific activity rate of the recombinant protein, resulting in unqualified product quality, and at the same time, it is easy to produce precipitation and affect the yield.

Method used

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  • A kind of industrial preparation method and application of recombinant interferon gamma
  • A kind of industrial preparation method and application of recombinant interferon gamma
  • A kind of industrial preparation method and application of recombinant interferon gamma

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Construction and expression identification of rpIFNγ Escherichia coli strain

[0053] The inventor has previously completed codon optimization of the rpIFNγ gene for E. coli and mRNA free energy optimization, and inserted the optimized rpIFNγ gene into the pET21b vector to construct a recombinant expression plasmid, denoted as pET21b-rpIFNγ; the expression plasmid pET21b-rpIFNγ It was introduced into the expression host of E. coli BL21(DE3) (denoted as BL21(DE3)-pET21b-rpIFNγ) by means of molecular cloning to achieve high protein expression, as described in the patent (application number: 201210593705.X). To clone the rpIFNγ gene from the pET21b vector into pET28a, see the following steps:

[0054] 1. Take glycerol tubes of BL21(DE3)-pET21b-rpIFNγ and DH5α-pET28a strains frozen in a -80°C refrigerator, inoculate them in LB liquid medium after thawing, and use the plasmid mini-extraction reagent after culturing overnight on a shaker at 37°C at 220 rpm The plas...

Embodiment 2

[0062] Example 2 High-density fermentation of rpIFNγ

[0063] This example mainly describes the high-density fermentation process of the BL21(DE3)-pET28a-rpIFNγ strain. In the fermentation process, glucose and glycerol with clear components are used as the carbon source for bacterial metabolism, and ammonia water (used for pH adjustment during the fermentation process) It is also used as a nitrogen source for cell metabolism) and diammonium hydrogen phosphate as a nitrogen source for cell metabolism; since no compound medium similar to yeast powder and peptone is used, the compound nitrogen source / carbon source is avoided. Therefore, the medium composition of different fermentation batches can be easily controlled, and because the cost of glucose and glycerol is low, the overall cost of the fermentation process is well controlled. Fermentation cell OD after fermentation 600 It can reach between 90-100, and the expression level of the target protein can reach more than 1.5g / L....

preparation example 1

[0064] Preparation example 1 concrete steps are as follows:

[0065] 1. Preparation of fermented seed liquid: inoculate the cryopreservation tube of the rpIFNγ E. coli strain constructed in Example 1 into LB solid medium containing 50 μg / mL kanamycin using the method of three-section line, and overnight at 37°C culture for activation;

[0066] 2. Pick a single colony with a plump shape and a moderate size from the solid medium and inoculate it into LB liquid medium containing 50 μg / mL kanamycin, and culture it on a shaker at 220 rpm at 37°C for 8 hours. This is the primary seed solution.

[0067] 3. Transfer the primary seed solution in step 2 to fresh LB liquid medium containing 50 μg / mL kanamycin according to the inoculum amount of 1%, and culture it on a shaker at 37°C and 220 rpm until OD600≈3-5 Between, this is the preparation of secondary seed solution.

[0068] 4. Composition of batch fermentation medium: citric acid monohydrate 1.5g / L, potassium dihydrogen phosphate ...

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Abstract

The invention provides a recombinant interferon gamma (rpIFN gamma) Escherichia coli expression, inclusion body denaturation, renaturation and purification industrial production method. The industrial production method particularly includes the steps: 1 building rpIFN gamma Escherichia coli expression strains; 2 developing an rpIFN gamma high-density fermentation process; 3 developing an rpIFN gamma inclusion body denaturation and renaturation process; 4 purifying an rpIFN gamma sample after renaturation. By technical improvement of four levels, rpIFN gamma produced by the method has high biological activity (specific activity reaching 5-7*107U / mg), and the method is high in yield (reaching 1.5g / L or more), low in cost and easy to amplify.

Description

technical field [0001] The invention belongs to the field of genetic engineering and protein purification, and specifically relates to an industrialized production method of recombinant porcine interferon gamma (rpIFN gamma) expressed in Escherichia coli, inclusion body renaturation and purification. Background technique [0002] Interferon (Interferon, IFN) is a cytokine produced by monocytes and lymphocytes. They have broad-spectrum anti-virus, affect cell growth, differentiation, and regulate immune function on the same type of cells. . According to the different antigenicity of IFN, it can be divided into three types: IFNγ, IFNβ, and IFNγ. Among them, only one type II IFN has been found so far, that is, IFN-γ, which is mainly produced by activated T cells and NK cells, and its biological activity is involved in immune regulation. IFNγ is a group of low-molecular-weight glycoproteins with similar structures and close functions produced by immune cells through antiviral ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P21/00C07K1/14C07K1/18C12R1/19
CPCC07K14/57
Inventor 马永赵百学王安良
Owner GENSUN INST OF BIOMEDICINE
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