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Basic fibroblast growth factor (bFGF) specifically bound with chitin as well as coding gene, preparation method and application thereof

A fibroblast, specific binding technology, applied in the field of genetic engineering, can solve the problems of unfavorable blood vessel formation, tissue regeneration, carcinogenic risk, etc., and achieve the effect of promoting proliferation, avoiding excessive release, and good slow release effect.

Active Publication Date: 2016-02-03
CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, high concentrations of bFGF are not conducive to angiogenesis and tissue regeneration, and there is a risk of cancer

Method used

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  • Basic fibroblast growth factor (bFGF) specifically bound with chitin as well as coding gene, preparation method and application thereof
  • Basic fibroblast growth factor (bFGF) specifically bound with chitin as well as coding gene, preparation method and application thereof
  • Basic fibroblast growth factor (bFGF) specifically bound with chitin as well as coding gene, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] The preparation of embodiment 1CBD-bFGF

[0049] The gene containing CBD-bFGF is cloned into PET21b expression vector, and the enzyme cutting sites are: NDEI and XholI.

[0050] This vector was transformed into E.coliBL21(DE3), and 0.2mMIPTG was induced to express CBD-bFGF protein at 37℃ for 12h. CBD-bFGF protein was obtained through His-tag packing affinity chromatography. As shown in the figure below: the electrophoresis figure shows that we have purified CBD-bFGF protein, the theoretical molecular weight is 23619.82 and the molecular weight of CBD-bFGF in the electrophoresis figure is basically the same, as figure 1 shown.

Embodiment 2

[0051] Example 2 Detection of CBD-bFGF and chitin sphere binding ability

[0052] CBD-bFGF and bFGF are used as detection objects, and PBS is used as control. The method is:

[0053] 1) Add chitin spheres to solutions containing CBD-bFGF (1 μg / mL) and bFGF (1 μg / mL) respectively, and incubate at 37°C for 2 hours;

[0054] 2) Wash the chitin spheres three times with PBS, 5 minutes each time;

[0055] 3) Add the chitin spheres bound with CBD-bFGF and bFGF to the solution containing 10% BSA respectively for sealing;

[0056] 4) Add the chitin spheres bound with CBD-bFGF and bFGF to the antibody containing mouse anti-bFGF respectively, and incubate overnight at 4°C;

[0057] 5), wash the chitin spheres with PBS three times, 5 minutes each time;

[0058] 6) Add the chitin spheres bound with CBD-bFGF and bFGF to the rabbit anti-mouse FITC-labeled antibody, and incubate at 37°C for 2 hours;

[0059] 7), wash the chitin spheres with PBS three times, 5 minutes each time;

[0060]...

Embodiment 3

[0065] The sustained release effect of embodiment 3CBD-bFGF

[0066] To detect the slow-release effect of CBD-bFGF, with bGFG as the control, the method is as follows:

[0067] The chitin membrane is prepared into a disc with a diameter of 8mm;

[0068] bFGF, CBD-bFGF cytokines are prepared into a 20ug / ml solution with PBS;

[0069] Soak the chitin membrane in bFGF and CBD-bFGF solution respectively, overnight at 4°C;

[0070] Take the membranes out of bFGF and CBD-bFGF solutions respectively, and place them in PBS solution;

[0071] Sampling at 1, 3, 5, 8, 11, 14, 17, 20, 24, 48 hour time points;

[0072] According to the method of bFGFElisa kit instructions, detect the content of bFGF in samples at different time points;

[0073] Use Origin to map, such as image 3 .

[0074] Depend on image 3 It can be seen that from 0 to 48 hours, the cytokine CBD-bFGF with chitin-binding domain has a better slow release ability than native bFGF.

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Abstract

The invention relates to the field of genetic engineering and in particular relates to a basic fibroblast growth factor (bFGF) specifically bound with chitin as well as a coding gene, a preparation method and application thereof. The bFGF specifically bound with chitin comprises an amino acid sequence shown in SEQ ID NO:1 and an amino acid sequence shown in SEQ ID NO:2. The bFGF can be specifically bound with chitin and the bonding capacity is far higher than that of the bFGF; therefore the slow-release effects are also stronger than the slow-release effects of the bFGF. In vivo experiments verify that the bFGF specifically bound with chitin has similar biological activity of promoting cell proliferation to the natural bFGF.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to basic fibroblast growth factor specifically combined with chitin and its coding gene, preparation method and application. Background technique [0002] Basic fibroblast growth factor (bFGF), secreted by endothelial cells, smooth muscle cells, and macrophages, is a polypeptide that transmits developmental signals and can promote cell division in mesoderm and neuroectoderm. bFGF is an important growth factor in the process of tissue damage repair in vivo. It can promote the formation of new blood vessels, promote the proliferation of fibroblasts and repair damaged endothelial cells. In vitro, bFGF can stimulate cell proliferation and migration, induce plasminogen activator and collagenase activity, and is a cell mitogen with high affinity to heparin. [0003] At present, bFGF is often mixed with scaffold materials for wound repair, and the most commonly used scaffold material is...

Claims

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Application Information

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IPC IPC(8): C07K14/50C12N15/12C12N15/70C12N5/073
CPCC07K14/503C12N5/0603C12N2501/115C12N2501/90
Inventor 章培标王宇王宗良史新翠黄晶
Owner CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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