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Polyethylene glycol modified recombinant human basic fibroblast growth factor

A fibroblast and growth factor technology, applied in the direction of fibroblast growth factor, growth factor/inducer, drug combination, etc., can solve problems such as unexplained modification sites, resistance to protease degradation, activity, apparent molecular weight, etc. , to achieve the effect of easy separation and purification and high uniformity

Active Publication Date: 2020-08-04
北京翼方生物科技有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the PEG-bFGF prepared according to this technique has not clarified the modification site and other information, and the modified product is not a pure product of a single site-specific modification chemically, but may be a mixture of single modifications at different sites. Point modification has a significant impact on the product's resistance to protease degradation, activity, apparent molecular weight, and other pharmaceutical and pharmacological indicators

Method used

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  • Polyethylene glycol modified recombinant human basic fibroblast growth factor
  • Polyethylene glycol modified recombinant human basic fibroblast growth factor
  • Polyethylene glycol modified recombinant human basic fibroblast growth factor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Preparation of recombinant human basic fibroblast growth factor

[0050] 1) Construction of high-efficiency expression strains:

[0051] Refer to the method provided in the article "Cloning and Expression of Human Basic Fibroblast Growth Factor Gene in Escherichia coli" in the Chinese Journal of Biochemical Pharmaceutics, 1996, issue, clone the DNA sequence shown in SEQ ID NO.1 into the pBV220 vector Construct a high-efficiency expression vector pBV-hbFGF, the structure is as follows figure 1 Shown. The expression vector was transformed into Escherichia coli DH5α, and a high-efficiency expression host strain was established. See the amino acid sequence and coding DNA sequence of recombinant human bFGF image 3 .

[0052] The seed medium used ordinary LB medium. The fermentation medium adopts M9 medium plus casein hydrolysate, the formula is as follows:

[0053] Reagent name Content (g / L) Na 2 HPO 4 ·12H 2 O

12.0 KH 2 PO 4

3.0 NH 4 Cl

1.0 NaCl0.5 Peptone5....

Embodiment 2

[0059] Example 2. PEGylation reaction

[0060] Reaction conditions: pH 5.0 (usually 4.0-6.0 is optional), 4 degrees Celsius (usually 2-8°C is optional), rhbFGF concentration is 5 mg / ml (the optional range is usually 2-10 mg / ml).

[0061] Steps: Take the bFGF concentrate obtained in Example 1, add 3 times the moles of bFGF (usually 2-10 times) of excess mPEG20kDa-ALD, shake until the PEG is completely dissolved; add NaCNBH 3 To the concentration of 20mM / L (usually 10-100mM) and quickly shake to dissolve; 2-8 ℃ reaction for 15 hours (usually 5-20 hours is optional); add glycine 2 times the amount of PEG to terminate the reaction . The reaction product can be represented by the following formula:

[0062]

Embodiment 3

[0063] Example 3. Purification of reaction products

[0064] 1) Molecular sieve chromatography

[0065] A 5.6cm×100cm Sephacryl S-200 chromatographic column is used, and the balance buffer is a weak acid solution, namely 20mM NaNc-HAc, pH4.0. Add 0.1M NaCl to increase ionic strength. The loading volume is 2-5% of the column volume, and the flow rate is 30ml / min. Peristaltic pump, model 100, product of Baoding Lange Constant Flow Pump Co., Ltd.; UV detector, model 8823B, Beijing Binda Yingchuang Technology Co., Ltd.;

[0066] Equilibrate the molecular sieve column with 20 mM NaAc-HAc pH 4.0 buffer until the effluent pH is 4.0. The PEG reactant was sampled on the chromatography column, and the molecular sieve chromatography column was equilibrated with buffer 20mM NaAc-HAc pH4.0. The absorption peak of the target protein was collected according to the ultraviolet absorption value for protein quantification and electrophoresis detection.

[0067] 2) Heparin column chromatography

[006...

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Abstract

The invention relates to the field of genetic engineering medicines, in particular to a novel polyethylene glycol modified recombinant human basic fibroblast growth factor and a preparation method thereof. According to the invention, straight-chain monomethoxy polyethylene glycol aldehyde is adopted to modify the human basic fibroblast growth factor. The modification reaction is carried out in anacidic reaction system under the conditions that the mole number of PEG is excessive, the protein concentration is 2-10 mg / ml and the like, and the reaction mixture is purified through molecular sievechromatography and other steps to obtain a pure product. The polyethylene glycol modified recombinant human basic fibroblast growth factor disclosed by the invention has a wide application prospect in preparation of medicines for promoting wound repair.

Description

Technical field [0001] The invention relates to the field of genetic engineering medicines, in particular to a novel polyethylene glycol modified recombinant human basic fibroblast growth factor and a preparation method thereof. Background technique [0002] In 1986, scientists cloned and characterized the human gene encoding FGF-2. In the following decades, some laboratories obtained recombinant FGF-2, also known as basic fibroblast growth factor (bFGF) and FGF-β, from E. coli cells, which is a kind of through FGF-2 gene The encoded growth factor and signal protein are members of the fibroblast growth factor family. It is known that there are 24 FGF members, and four receptors (FGFR) for these cytokines have been identified. [0003] Various cells synthesize FGF-2 to regulate the proliferation and differentiation of specific types of cells. FGF-2 shows effective angiogenesis in vivo and in vitro, stimulates the growth of smooth muscle cells, promotes wound healing and tissue re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/50C07K1/107A61K38/18A61K47/60A61P17/02A61P27/02A61P19/00A61P25/00A61P9/00
CPCC07K14/503A61K47/60A61P17/02A61P27/02A61P19/00A61P25/00A61P9/00A61K38/00
Inventor 姜海业
Owner 北京翼方生物科技有限责任公司
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