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Effective renaturation liquid of human interferon inclusion body comprising D-sorbiol and renaturation method

A sorbitol and solution technology, applied in the field of renaturation solution, can solve the problems of low renaturation rate of inclusion bodies, complicated preparation process, etc., achieve huge social and economic benefits, simple equipment requirements, and improve the effect of renaturation rate

Inactive Publication Date: 2003-06-11
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] The present invention aims at the problems of low renaturation rate of inclusion bodies and complex preparation process in the production of recombinant human interferon, and obtains a renaturation liquid formula and renaturation method that can significantly improve the renaturation rate and require very simple equipment

Method used

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  • Effective renaturation liquid of human interferon inclusion body comprising D-sorbiol and renaturation method
  • Effective renaturation liquid of human interferon inclusion body comprising D-sorbiol and renaturation method
  • Effective renaturation liquid of human interferon inclusion body comprising D-sorbiol and renaturation method

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Embodiment 1

[0029] The present invention is further illustrated by the following examples, but the examples do not limit the scope of the claims of the present invention in any way. Example 1: Preparation of Denaturing Washing Refolding Solution

[0030] A. Dosing of denaturing washing and dissolving system:

[0031] 1. Washing solution 1 preparation:

[0032] 1M Tris-HCl (pH8.0) solution 10ml

[0033] 0.5M EDTA (pH8.0) solution 20ml

[0034] 2M NaCl 50ml

[0035] Add double distilled water (or deionized water) to 1000ml

[0036] 2.6M urea washing solution

[0037] 1M Tris-HCl (pH8.0) solution 10ml

[0038]0.5M EDTA (pH8.0) solution 20ml

[0039] 2M NaCl 50ml

[0040] Urea 360g

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

[0042] 3. Preparation of washing solution 2

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

[0044] 0.5M EDTA (pH8.0) solution 2ml

[0045] NaCl...

Embodiment 3

[0056] 5) Harvest the refolded recombinant human interferon α2a protein. After determination, the total amount was 613 mg / L culture, and the specific activity was 1.15×10 8 U / mg, confirmed by TLC scanning, the purity is over 90%, and the renaturation rate is 43%. Example 3 Isolation, purification and renaturation of human gene recombinant interferon α2b

[0057] 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 gr...

Embodiment 4

[0061] 5) Harvest the recombinant human interferon α2b protein. After determination, the total amount of IFN protein after renaturation is 861 mg / liter culture, and the specific activity is 1.31×10 8 U / mg, confirmed by TLC scanning, the purity is above 90%, and the renaturation rate is 41%. Embodiment 4 Purification and renaturation of human interferon alpha 2a gene expression product

[0062] Basically proceed according to the steps described in Example 1, except that refolding solution 2 is used.

[0063] Cultivate 8.1 grams of wet thalli, obtain human interferon alpha 2a protein 575 mg / liter culture after renaturation, specific activity reaches 1.16×10 8 IU / mg, confirmed by TLC scanning, the purity is above 90%, and the refolding rate is 42%. Example 5 Isolation, purification and renaturation of human gene recombinant interferon α2b

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Abstract

The invention refers to the plural solution used to reform human-interferon inclusion-body protein and the plural method. The solution comprises Tris-HCL solution, EDTA, reductive-type and oxidizing-type glutathione, D-sorbierite and double-vaporing or deionized water. The method: sub-step adding sample and once plural.

Description

technical field [0001] 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 (produced by Roche) and IFNα2b (produced by Schering) in 198...

Claims

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Application Information

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Patent Type & Authority Applications(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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