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Method for constructing fusion protein TD-bFGF efficiently expressed in CHO cells and application of fusion protein TD-BFGF

A technology of fusion protein and expression cassette, which is applied in the direction of medical preparations containing active ingredients, fusion polypeptides, peptide/protein components, etc., and can solve problems such as low activity, skin allergies, and poor expression of bFGF protein

Inactive Publication Date: 2017-05-24
北京佳华戴维生物技术有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is how to improve the existing problems such as poor expression effect of bFGF protein in Escherichia coli, low activity during use and skin allergy caused by prokaryotic endotoxin residue of transdermal peptide

Method used

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  • Method for constructing fusion protein TD-bFGF efficiently expressed in CHO cells and application of fusion protein TD-BFGF
  • Method for constructing fusion protein TD-bFGF efficiently expressed in CHO cells and application of fusion protein TD-BFGF
  • Method for constructing fusion protein TD-bFGF efficiently expressed in CHO cells and application of fusion protein TD-BFGF

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Experimental program
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Effect test

Embodiment 1

[0073] Example 1. Obtaining of Transdermal Peptide Fragment TD and Human bFGF Fusion Protein Mutant and Its Encoding Gene

[0074] In order to improve the transdermal effect of wild-type bFGF, the protein sequence transdermal peptide transdermal peptide fragment TD fragment of bFGF is fused. The specific protein sites of specific fusion proteins are shown in Table 2.

[0075] Table 2 Fusion protein design

[0076]

[0077]The synthesis of the transdermal peptide fragment and bFGF fusion protein gene shown in SEQ ID No.1 is to fuse the N-terminal fusion fragment of bFGF protein, its coding sequence is shown in SEQ ID No.1, and the coding sequence shown in SEQ ID No.2 Protein transdermal peptide fragment and bFGF fusion protein. Other transdermal peptides can also be fused to the C-terminus of the bFGF protein, and the bFGF amino acid residues remain unchanged to obtain a bFGF mutant.

Embodiment 2

[0078] Embodiment 2, the preparation of bFGF expression

[0079] 1. Construction of recombinant expression vector of recombinant transdermal peptide fragment TD and bFGF fusion protein

[0080] Use NheI and XhoI to double digest the PET32TD-bFGF vector to obtain the DNA synthesis DNA nucleic acid molecule shown in SEQ ID No.1. The fragment of about 600bp is the target fragment, which contains the DNA fragment of the coding gene of TD-bFGF or the DNA fragment of the mutation coding gene Carry out recovery and purification. The result is as figure 1 .

[0081] The purified DNA fragment containing the coding gene of TD-bFGF was respectively digested with NheI and XhoI double restriction enzyme expression vector pcCND3.1(-), and the effect was as follows: figure 2 , the synthetic gene was ligated to the vector segment (the ligation system was 0.5 μL pcCND3.1(-); 4.5 μL TD-bFGF enzyme-digested fragment, 2xT4 quick ligase, room temperature for 3 hours). The ligation product wa...

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Abstract

The invention relates to a fusion cytokine production technology in the technical field of biology, and particularly relates to a method for constructing fusion protein TD-bFGF efficiently expressed in CHO (Chinese Hamster Ovary) cells by means of genetic engineering and application of the fusion protein TD-bFGF. The fusion protein TD-bFGF provided by the invention has an amino acid sequence as shown in SEQ ID No. 2, or has at least 75% of identity compared with the sequence as shown in SEQ ID No. 2. The invention further provides a biological material associated with the fusion protein TD-bFGF and a method for constructing a recombinant bFGF protein in the CHO cells. The TD-bFGF fusion protein obtained by the invention can penetrate the skin, has activity higher than that of the market product, can be used for cosmetic skin medicines, and has effects of reducing the production cost and improving the effect of bFGF; the method constructs CHO cell strains for stably and efficiently expressing a transdermal peptide fragment and cytokine bFGF fusion protein and provides a new application idea for the application of bFGF.

Description

technical field [0001] The invention relates to fusion cytokine production technology in the field of biotechnology, in particular to a method and application for constructing CHO cells to efficiently express bFGF fusion protein by means of genetic engineering. Background technique [0002] Transdermal drug delivery system (Transdermal therapeutic system, TTS or Transdermaldelivery, TDS) refers to the administration on the surface of the skin, the drug passes through the layers of the skin at a constant speed (or close to a constant speed), enters the systemic circulation, and produces systemic or local therapeutic effects. Novel drug delivery methods. Compared with traditional oral and subcutaneous intravenous injection, transdermal drug delivery has the advantages of convenience, sustainability and controllability, and can facilitate the entry of bFGF and other macromolecular protein drugs through simple mixing, smearing or spraying. Animal blood circulation exerts its th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/85A61K8/64A61K9/12A61K8/04A61Q19/00A61P17/00
CPCC07K14/503A61K8/046A61K8/64A61K9/0014A61K9/12A61K38/00A61K2800/10A61K2800/86A61Q19/00C07K2319/00C12N15/85C12N2800/107
Inventor 不公告发明人
Owner 北京佳华戴维生物技术有限公司
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