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Recombined protein specially combined with fiber protein and application thereof

A recombinant protein and protein technology, which is applied to the recombinant protein specifically binding to fibrin and its application field, can solve the problems of low accumulation of recombinant bFGF and difficult sustained action of drugs, etc.

Active Publication Date: 2013-03-13
INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Injection of recombinant bFGF can improve angiogenesis and blood circulation during injury and limb and myocardial ischemia. However, one of the reasons for the limited use of recombinant bFGF is that recombinant bFGF diffuses rapidly in the body, resulting in low accumulation of recombinant bFGF at the treatment site. Difficult to sustain (Rinsch, C., et al. (2001). Delivery of FGF 2 but not VEGF by encapsulated genetically engineered myoblasts improve survival and vascularization in a model of acute skin flap ischemia. Genetherapy 8: 523-533.)

Method used

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  • Recombined protein specially combined with fiber protein and application thereof
  • Recombined protein specially combined with fiber protein and application thereof
  • Recombined protein specially combined with fiber protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1, preparation recombinant K1bFGF and K4bFGF protein

[0048] 1) Acquisition of Kringle1 and Kringle4

[0049] The Kringle1 fragment was amplified by complementary sequence binding of primers K1-1, K1-2, K1-3, K1-4 and K1-5.

[0050] The order of adding primers is: K1-1 and K1-2 are used for the first amplification, K1-3 and K1-4 are used for the second time, and K1-1 and K1-5 are used for the third time.

[0051] The sequences of primers K1-1, K1-2, K1-3, K1-4 and K1-5 are as follows:

[0052] K1-1: 5’-AGA GGG ACG ATG TCC AAA ACA AAA AAT GGC ATC ACC TGT CAA AAATGG AGT TCC ACT TCT CCC CAC AGA CCT AGA TTC TCA CCT-3’;

[0053] K1-2: 5'-CCT GCG GAT CGT TGT CTG GAT TCC TGC AGT AGT TCT CCT CCA GTCCCT CTG AGG GGT GTG TAG CAG GTG AGA ATC TAG GTC TGT-3';

[0054] K1-3: 5'-TAT CTC TCA GAG TGC AAG ACT GGG AAT GGA AAG AAC TAC AGAGGGACG ATG TCC AAA AC-3';

[0055] K1-4: 5'-CAT ATC TCT TTT CTG GAT CAG TAG TAT AGC ACC AGG GCC CCT GCGGAT CGT TGT CTG GA-3';

[0056] K1-...

Embodiment 2

[0075] Example 2, in vitro verification of the biological activity of K1bFGF and K4bFGF

[0076] 1) Identification of mitogen activity of recombinant protein

[0077] Human fibroblasts were used to test the mitogenic activity of the recombinant proteins K1bFGF and K4bFGF.

[0078] Human fibroblasts were inoculated into a 48-well plate at 3000 cells / well, with 10% (volume ratio) fetal bovine serum (FBS), 1% (mass ratio) glutamine, 1% (mass ratio) non-essential amino acid , 100U penicillin / ml and 100μg streptomycin / ml high glucose DMEM medium (H-DMEM), at 37°C, 5% CO 2 and cultured at saturated humidity for 24 hours. After 24 hours, the FBS concentration of the culture medium was changed to 2% (volume ratio), and other conditions remained unchanged. Then add K1bFGF or K4bFGF with final concentrations of 0, 30, 120, 600, 1500 and 3000 pmol / L to each culture well in turn, set 4 parallel samples for each concentration, continue to cultivate for 3 days, and add The final concent...

Embodiment 3

[0102] Example 3, Functional Evaluation of Recombinant Proteins in Animals

[0103] 1) Effect evaluation of K1bFGF and K4bFG in rat skin random ischemia model

[0104] The model of random skin flap in rats can be used to detect angiogenesis. Due to the formation of plasma clots in this model, the recombinant protein can be directly applied to this model to observe the condition of angiogenesis.

[0105] The method of applying recombinant protein to rat skin random ischemia model is as follows:

[0106] 1) Animal model: Rat "random pattern skin flap", male Wistar rats, about 180g, were anesthetized by intraperitoneal injection (i.p.) of pentobarbital sodium at a dose of 40mg / kg body weight; Hair, disinfected with alcohol; cut 4 pieces of 15*15mm on the back 2 The side-based skin cover;

[0107] 2) Apply K1bFGF and bFGF in amounts of 580 pmol (approximately equivalent to 10 μg of natural bFGF) to the wound surface, respectively. The location of each sample was varied in diffe...

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Abstract

The invention discloses recombined protein specially combined with fiber protein and the application thereof. The recombined protein is fusion protein which is obtained by fusing polypeptide a, b, c or d to the amino terminal of target protein, wherein the polypeptide a is formed by an amino acid sequence which is shown by the 22nd-107th site from the amino terminal in the second amino acid sequence of an amino acid sequence table; the polypeptide b is formed by an amino acid sequence which is shown by the 22nd-109th site from amino terminal in the fourth amino acid sequence of the amino acid sequence table; the polypeptide c is deviated from the polypeptide a, is specially combined with the fiber protein and is formed by substituting and / or deleting one or a plurality of amino acid from the limited amino acid sequence of the polypeptide a and / or adding one or a plurality of amino acid to the limited amino acid sequence of the polypeptide a; and the polypeptide d is deviated from the polypeptide b, is specially combined with the fiber protein and is formed by substituting and / or deleting one or a plurality of amino acid from the limited amino acid sequence of the polypeptide b and / or adding one or a plurality of amino acid to the limited amino acid sequenceof the polypeptide b. The recombined protein specially combined with fiber protein can be used for wound healing and active tissue inducing regeneration.

Description

technical field [0001] The invention relates to a recombinant protein specifically combined with fibrin and its application. Background technique [0002] Basic fibroblast growth factor (bFGF) is a strong mitogen that can promote the proliferation of a variety of mesoderm-derived cells, including fibroblasts, vascular endothelial cells, and smooth muscle cells, so it is expected to be used for Therapeutic angiogenesis and injury repair. Endogenous bFGF is often insufficient to rapidly promote angiogenesis and injury repair during injury and ischemia. Injection of recombinant bFGF can improve angiogenesis and blood circulation during injury and limb and myocardial ischemia. However, one of the reasons for the limited use of recombinant bFGF is that recombinant bFGF diffuses rapidly in the body, resulting in low accumulation of recombinant bFGF at the treatment site. It is difficult to sustain the effect (Rinsch, C., et al. (2001). Delivery of FGF 2 but not VEGF by encapsula...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21A61K38/16A61P9/10A61P17/02A61L27/22
Inventor 戴建武赵文学
Owner INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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