Effective renaturation liquid of recombinant human interon inclusion body and renaturation method

A technology of recombinant human interferon and solution, which is applied in the direction of interferon, cytokine/lymphokine/interferon, drug combination, etc., can solve the problems of complex preparation process and low renaturation rate of inclusion body, and achieve simple equipment requirements, Huge social and economic benefits, the effect of reducing production costs

Inactive Publication Date: 2005-03-23
武汉柏傲生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Aiming at the problems of low renaturation rate of inclusion bodies and complex preparation process in the production of recombinant human interferon, the present invention proposes a renaturation liquid formula and a renaturation method that can significantly improve the renaturation rate and require very simple equipment

Method used

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  • Effective renaturation liquid of recombinant human interon inclusion body and renaturation method
  • Effective renaturation liquid of recombinant human interon inclusion body and renaturation method
  • Effective renaturation liquid of recombinant human interon inclusion body and renaturation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Preparation of Denaturing Washing Refolding Solution

[0036] A. Dosing of denaturing washing and dissolving system:

[0037] 1. Washing solution 1 preparation:

[0038] 1M Tris-HCl (pH8.0) solution 10ml

[0039] 0.5M EDTA (pH8.0) solution 20ml

[0040] 2M NaCl 50ml

[0041] Add double distilled water (or deionized water) to 1000ml

[0042] 2.6M urea washing solution

[0043] 1M Tris-HCl (pH8.0) solution 10ml

[0044] 0.5M EDTA (pH8.0) solution 20ml

[0045] 2M NaCl 50ml

[0046] Urea 360g

[0047] Add double distilled water (or deionized water) to 1000ml

[0048] 3. Preparation of washing solution 2

[0049] 1M Tris-HCl (pH8.0) solution 10ml

[0050] 0.5M EDTA (pH8.0) solution 2ml

[0051] NaCl 58.4g

[0052] Add double distilled water (or deionized water) to 1000ml

[0053] 4. Preparation of IFN inclusion body solutio...

Embodiment 2

[0059] Embodiment 2 Purification and renaturation of human interferon alpha 2a gene expression product

[0060] 1) The expression system used to express the novel human interferon α2a modified recombinant gene is the E.coli prokaryotic expression system, and the original plasmid is PBR32. Conventional culture conditions and conventional LB medium were used. Prepare 1 liter of medium, 10 grams of tryptone (Trypton), 10 grams of yeast extract (Yeast Extract), 5 grams of sodium chloride (NaCl), and 1000 ml of double distilled water. The culture adopts a constant temperature of 37°C, shaking culture, and induces expression with IPTG inducer. The whole process time from inoculating the strain to harvesting the culture was 7 hours, and 8.2 grams of wet bacteria were harvested by centrifugation.

[0061] 2) Preparation of renaturation solution: prepare 3 liters of renaturation solution 1 in total.

[0062] 3) Initially purify the insoluble interferon expression product according t...

Embodiment 3

[0065] Example 3 Isolation, purification and renaturation of human gene recombinant interferon α2b

[0066] 1) The expression system used to express the novel human interferon α2b modified recombinant gene is the E.coli prokaryotic expression system, and the original plasmid is PBR32. Conventional culture conditions and conventional LB medium were used. Prepare 1 liter of medium, 10 grams of peptone (Trypton), 10 grams of yeast extract (Yeast Extract), 5 grams of salt (NaCl), and 1000 ml of double distilled water. The culture adopts a constant temperature of 37°C, shaking culture, and induces expression with IPTG inducer. The whole process time from inoculating the strain to harvesting the culture was 7 hours, and 8.4 grams of wet bacteria were harvested by centrifugation.

[0067] 2) Preparation of renaturation solution: 3 liters of renaturation solution 1 in total.

[0068] 3) Initially purify the insoluble interferon expression product according to the method described i...

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Abstract

The invention refers to the plural solution used to reform human-interferon inclusion-body protein and the plural method, belonging to bio-pharmacy field. The solution consists of 100ml 0.05-0.20mol Tris-HCL solution, pH 7.0-8.5, 0.44-0.60mol EDTA, pH 7.0-8.51, 1.2-2.55mmol reductive-type glutathione, 0.1-0.31 mmol oxidizing-type glutathione, 0.4-0.60 oml L-arginine and the remaining double-vaporing or deionized water according to the final concentration of every litre. The method: sub-step adding sample and once plural.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals. The invention relates to a refolding solution for refolding recombinant human interferon inclusion bodies and a use method thereof. Background technique [0002] Interferon (English interferon, abbreviated as IFN) is a kind of highly active and multifunctional cytokine secreted by cells. Its main function is to inhibit virus proliferation and anti-tumor, and improve the human body's anti-virus ability. It has at least 20 different subtypes. Natural alpha interferon consists of 165-166 amino acid residues, and its molecular weight is between 16-27KD. Recombinant human alpha interferon is the earliest cytokine produced in large quantities by gene technology. The purified recombinant human alpha interferon has a molecular weight of 18.5-19.4KD and has two disulfide bonds (C1-C98, C29-C138 or C1-C99, C29-C139). [0003] The U.S. FDA first approved the marketing of genetically engineered IFNα2a (...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/21A61P31/12A61P35/00C07K14/555
Inventor 白宪鹤林雨霖
Owner 武汉柏傲生物工程有限公司
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