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Human acid fibroblast growth factor fusion protein, and coding gene and applications thereof

A technology for fibroblasts and fusion proteins, applied in human acidic fibroblast growth factor fusion proteins and their coding genes and application fields

Active Publication Date: 2013-10-02
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0018] However, so far, no one has used the P19 protein encoded by tomato bushy dwarf virus as a silencing suppressor for exogenous gene expression, and a fusion protein composed of 2A polypeptide and human acidic fibroblast factor has been co-expressed in transgenic Salvia miltiorrhiza

Method used

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  • Human acid fibroblast growth factor fusion protein, and coding gene and applications thereof
  • Human acid fibroblast growth factor fusion protein, and coding gene and applications thereof
  • Human acid fibroblast growth factor fusion protein, and coding gene and applications thereof

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Experimental program
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Effect test

Embodiment 1

[0078] 1. Artificial synthesis of tomato bushy dwarf virus P19 protein-2A polypeptide-haFGF fusion protein gene (P19-2A-haFGF fusion protein gene)

[0079] The nucleotide sequence encoding tomato bushy dwarf virus P19 protein is derived from the 3888-4403 sites in GenBank NC_001554 (accession number), and its DNA length is 516bp, encoding 172 amino acid residues. Because the gene originally came from a plant virus genome, it is suitable for efficient transcription and translation in plant cells, so its codons were not further optimized.

[0080] The nucleotide sequence encoding foot-and-mouth disease virus 2A polypeptide is derived from GenBank AJ251476 (accession number) 3472-3537 sites, its DNA length is 66bp, encoding 22 amino acid residues.

[0081] Although both humans and plants belong to eukaryotes, they still have certain differences in gene codon preference, which may affect the stability and high efficiency of human acidic fibroblast factor gene in transgenic Danshen...

Embodiment 2

[0085] 1. Construction of plant expression vector pBI-P19-2A-haFGF

[0086] The pT-P19-2A-haFGF plasmid vector was extracted from the above bacterial clones, and the vector pT-P19-2A-haFGF and pBIl21 were digested with restriction enzymes XbaI and SacI to recover the P19 protein-2A polypeptide-haFGF The gene fragment encoding the fusion protein and the pBI121 carrier fragment are connected, and the gene fragment encoding the P19 protein-2A polypeptide-haFGF fusion protein is inserted into the original GUS gene site of the plasmid pBI121 (by double digestion with XbaI and SacI), and the The binary plant expression vector pBI-P19-2A-haFGF (see Figure 4 ).

[0087] Plasmid pBIl21 was purchased from Clonetech, USA. Its XbaI restriction site is located between the cauliflower mosaic virus 35S promoter and the start codon of the GUS gene, and SacI is located between the stop codon of the GUS gene and the NOS terminator. Plasmid pBI121 was selected because the original GUS gene ca...

Embodiment 3

[0116] 1. Modeling of burns and scalds in mice

[0117] Five male SD rats (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were purchased one week before each experiment, free to drink and eat (the experiment was repeated 3 times, a total of 15 rats were required). The day before the experiment, the rats were anesthetized with ether, the hair on the back of the animal was cut short with scissors, and depilated with 8% sodium sulfide, with an area of ​​9 cm × 8 cm, disinfected with active iodine, and washed with normal saline. On the day of the experiment, they were fasted and anesthetized with ether. A piece of gauze was placed under the abdomen of the rat to make the back flat, and the rat was placed on a fixed frame. Connect the YLS-5Q desktop super temperature-controlled scald instrument to the 220V power supply, adjust the temperature to 80°C, and adjust the pressure to 0. The weight of the head itself (0.5kg) is used as the injury pressure, ...

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Abstract

The invention provides a fusion protein. The fusion protein comprises suppressors of gene silencing, 2A polypeptide and a human acid fibroblast growth factor protein. All or part of the human acid fibroblast growth factor protein is a natural human acid fibroblast growth factor protein; or the human acid fibroblast growth factor protein is a fusion protein formed by co-expression or splicing of the natural human acid fibroblast growth factor protein with another fibroblast factor protein, or with another protein which possesses completely different biological functions. The invention also provides the coding gene of the fusion protein, and a preparation method of genetically modified radix salviae miltiorrhizae which is used for high-efficiency expression of the human acid fibroblast factor. The genetically modified radix salviae miltiorrhizae can be used for effective treatment of operative incisions, nerve injuries, body mechanical injuries, diabetic foot, anabrosis, burned and scalded injuries, and the like, and is also applied in developing a new generation of recombinant human acidic fibroblast factor drugs.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a human acidic fibroblast growth factor fusion protein and its coding gene and application. Background technique [0002] Human fibroblast growth factor belongs to a large protein family, at least 23 members have been discovered so far. Among them, acidic fibroblast growth factor (aFGF) was first isolated from bovine brain by Thomas et al. in 1984. Because its isoelectric point is 5-7, it is acidic, so it is named . [0003] Human acidic fibroblast growth factor (haFGF) is mainly distributed in the kidney and brain tissue. It is a cytoplasmic protein that lacks an N-terminal signal peptide structure. It acts on surrounding cells mainly through autocrine and paracrine. . The haFGF polypeptide consists of 154 amino acid residues and has a molecular weight of about 16kDa. It is a complete β-sheet protein without disulfide bonds, and its secondary structure is a clover structure...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N5/10C12N15/82A61K38/19A61P17/02
Inventor 刘德虎谭亚清李刚强王楠
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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