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Variable-conformation recombinant interferon crystal, and three-dimensional structure and use thereof

A technology of recombinant interferon and three-dimensional structure, applied in the field of recombinant interferon crystals, can solve the problems of spatial conformation and biological efficacy changes, and achieve the effects of low side effects, high antiviral activity, and reduced toxic and side effects

Inactive Publication Date: 2011-06-22
SICHUAN HUIYANG LIFE SCI & TECH CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The recombinant interferon has the same amino acid sequence as Ganfujin, but its spatial conformation and biological efficacy have changed

Method used

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  • Variable-conformation recombinant interferon crystal, and three-dimensional structure and use thereof
  • Variable-conformation recombinant interferon crystal, and three-dimensional structure and use thereof
  • Variable-conformation recombinant interferon crystal, and three-dimensional structure and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0116] Preparation of recombinant interferon rSIFN-co

[0117] This embodiment is a preparation method of recombinant interferon rSIFN-co (hereinafter also referred to as "rSIFN-co") (stock solution) (for details, refer to US Patent No. 7,364,724 Specifications Examples 1 and 2 and Chinese Patent Publication No. method described on pages 11-17).

[0118] 1. Gene cloning:

[0119]According to the published coding DNA sequence and deduced amino acid sequence data (Klein ML, Bartley TD, Lai PH, et al., Structural characterization of recombinant consensus interferon-alpha. Journal of Chromatography, 1988; 454: 205-215), the large intestine Bacillus expresses codons preferentially (The Wisconsin Package, by Genetics Computer Group, Inc. Copyright 1992, Medison, Wisconsin, USA). Under the condition that the amino acid sequence remains unchanged, molecular design is carried out on its DNA coding sequence, and then artificially synthesized. rSIFN-co full-length cDNA encoding gene....

Embodiment 2

[0188] Recombinant Interferon Preparations

[0189] Formula for freeze-dried injection (lyophilized powder)

[0190] rSIFN-co stock solution of the present invention 34.5 μg / ml

[0191] Phosphate buffer at pH 7.0 10mmol / L

[0192] Glycine 0.4mol / L

[0193] Preparation process: weigh according to the formula, dissolve in sterile pyrogen-free injection water, filter and sterilize through a 0.22 μm pore-diameter filter membrane, store at 6-10°C, take samples for sterility and pyrogen inspection, and then pack them into vials. A single dose of 0.3-0.5 vials is divided and placed in a lyophilizer for freeze-drying.

[0194] Formulation of aqueous solution injection

[0195] rSIFN-co stock solution of the present invention 34.5 μg / ml

[0196] Phosphate buffer at pH 7.0 25mmol / L

[0197] Sodium chloride 0.4mol / L

[0198] Preparation process: Weigh according to the formula, dissolve in sterile pyrogen-free injection water, filter and sterilize through a 0.22μm pore-diameter f...

Embodiment 3

[0200] Crystallization of recombinant interferon

[0201] Preparation of high-quality rSIFN-co protein single crystal is a prerequisite for determination of its crystal structure. The rSIFN-co samples used for crystal growth were obtained from the rSIFN-co of the present invention as described above. The single crystal preparation method, technical process and crystallization conditions of rSIFN-co, and its crystallographic parameters are as follows:

[0202] The above-mentioned rSIFN-co freeze-dried powder of the present invention was dissolved in pure water and stored at low temperature (-20° C.), with an initial protein concentration of 0.42 mg / ml. The rSIFN-co protein samples were concentrated to 3-3.5 mg / ml before crystallization and immediately used for crystal growth experiments. Crystal cultivation was carried out at room temperature (293K) by the hanging drop vapor phase diffusion method.

[0203] In the early stage of crystallization research, microcrystals of r...

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Abstract

The invention provides a variable-conformation recombinant interferon crystal, which has the same amino acid sequence as a human interferon and a three-dimensional structure which is different from that of interferon (IFN)-alpha2b. The interferon of the invention has improved bioactivity. The invention also provides a structural model for medicine screening and / or medicine design.

Description

technical field [0001] The present invention mainly relates to a recombinant interferon crystal with changed conformation, its crystallization method, its three-dimensional structure and the application of the crystal and the three-dimensional structure. Background technique [0002] Interferon (IFN) is a soluble protein produced by a variety of cells, which has a variety of important biological functions, including antiviral, antitumor and immunomodulatory functions. Interferon can be divided into type I, type II and type III interferon according to the difference of producing cell type, receptor, biological activity, etc. Type I interferons are mainly induced by viruses and synthetic double-stranded RNA, so they are also called antiviral interferons. There are three types of type I interferons: IFNα, INFβ, and IFNω. Type II interferon, also known as immune interferon or IFNγ, is produced by T cells and is an important immune regulator in the body. Type III interferons a...

Claims

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Application Information

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IPC IPC(8): C07K14/555C07K7/64G01N33/68G06F19/00A61K38/21A61P31/12A61P35/00A61P37/02G16B15/30
CPCG06F19/16C07K2299/00G01N2500/04G06F19/706C07K14/56A61K38/00G16C20/20G16B15/00G16C20/50A61P1/04A61P1/16A61P1/18A61P11/00A61P11/02A61P13/08A61P13/10A61P13/12A61P15/00A61P17/00A61P19/00A61P21/00A61P25/00A61P31/12A61P31/14A61P31/16A61P31/18A61P31/20A61P31/22A61P35/00A61P35/02A61P37/02A61P37/04A61P5/00A61P7/00A61P9/00Y02A50/30Y02A90/10G16B15/30C07K14/555
Inventor 魏光文王大成
Owner SICHUAN HUIYANG LIFE SCI & TECH CORP
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