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Recombinant soluble human-derived bone targeted interferon gamma-1b and preparation method thereof

An interferon and bone-targeted technology, applied in the field of molecular biology, can solve the problems of low biological activity and complicated preparation process, and achieve the effect of reducing production cost and simplifying the preparation process

Active Publication Date: 2018-09-04
SHANDONG MEDICAL BIO TECH RES CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2000, the U.S. FDA approved recombinant human interferon γ-1b (rhIFNγ-1b, trade name Actimmune) to be marketed for the treatment of osteosclerosis (there is currently no drug for the treatment of osteosclerosis in my country)
Moreover, IFNγ-1b drugs form inclusion bodies during the production process, which has the disadvantages of complex preparation process and low biological activity.

Method used

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  • Recombinant soluble human-derived bone targeted interferon gamma-1b and preparation method thereof
  • Recombinant soluble human-derived bone targeted interferon gamma-1b and preparation method thereof
  • Recombinant soluble human-derived bone targeted interferon gamma-1b and preparation method thereof

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preparation example Construction

[0051] The invention provides a recombinant soluble human bone-targeted interferon gamma-1b and a preparation method thereof. The bone-targeting molecule is fused with the interferon gamma-1b, and the bone-targeting property of the bone-targeting molecule is used to make the interferon The interferon gamma-1b is enriched in bone tissue, thereby improving the specificity of interferon gamma-1b in treating bone tissue diseases, reducing the dosage of drugs, and reducing toxic and side effects. At the same time, the present invention uses the principle of site-directed mutagenesis to replace key hydrophobic amino acids in the amino acid sequence of interferon gamma-1b, and optimizes codons, induced expression conditions, etc., thereby realizing the soluble expression of interferon gamma-1b in Escherichia coli , greatly improving the biological activity of interferon gamma-1b, the preparation method is simple, and has good application and development prospects.

[0052] In order t...

Embodiment 1

[0078] Example 1 Design of bone-targeted recombinant human interferon gamma-1b (rhIFN gamma-1b-D10) and mutants thereof

[0079] Download the amino acid sequence and gene sequence of human interferon γ-1b from the Gene Bank and Drμg Bank databases; DNA Star software analyzes the amino acid sequence characteristics and affinity and hydrophobicity of human interferon γ-1b; replaces the key hydrophobic amino acids in the hydrophobic region It is a hydrophilic amino acid, and check the SWISS-MODEL website (https: / / swissmodel.expasy.org / ) at the same time to ensure that the three-dimensional conformation of interferon does not change after amino acid substitution. Add 10 aspartic acid tags to the carboxy-terminal of the amino acid sequence of human interferon γ-1b and its mutant proteins as bone targeting molecules. Using Escherichia coli preferred codons, introducing His tags and corresponding restriction sites, DNAStar software was used to analyze the gene sequences of bone-targe...

Embodiment 2

[0080] The construction of embodiment 2 recombinant expression bacteria

[0081] The four recombinant plasmids constructed (including pET3c-IFNγ-1b-D10, pET3c-IFNγ-1b-D10 gene mutant 1, pET3c-IFNγ-1b-D10 gene mutant 2, pET3c-IFNγ-1b-D10 gene mutant Mutant 3) and empty plasmid were transformed into Escherichia coli BL21(DE3) and Rosetta(DE3) respectively.

[0082] Induced expression of recombinant proteins

[0083] (a) Single colonies of recombinant bacteria were picked respectively, inoculated in LB liquid medium containing ampicillin (50 μg / mL), and cultured overnight at 37° C. with shaking at 200 rpm;

[0084] (b) Inoculate 30 μl of the above cultured bacterial solution into 3 ml LB liquid medium (containing 50 μg / mL ampicillin), culture at 37°C, 200 rpm until OD600 reaches 0.5-0.6;

[0085] (c) adding IPTG with a final concentration of 1.0 mmol / L to the bacterial solution obtained in step (b) to induce expression, the induction temperature is 37°C, and the induction time ...

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Abstract

The invention provides recombinant soluble human-derived bone targeted interferon gamma-1b and a preparation method thereof. The amino acid sequence of the recombinant soluble human-derived targeted interferon gamma-1b comprises recombinant interferon gamma-1 or an active fragment thereof and a bone targeted molecule; and the bone targeted molecule is a polypeptide aptamer screened out of osteoidstem cells. The bone targeted molecule and the interferon gamma-1b are fused and the interferon gamma-1b is enriched in a bone tissue by utilizing the bone targeted characteristic of the bone targetedmolecule, so that the specificity of treating bone tissue disease by the interferon gamma-1b is improved, the dosage is reduced and the toxic or side effect is reduced. Meanwhile, hydrophobic amino acid positioned at the key point in the amino acid sequence of the interferon gamma-1b is replaced by adopting site-specific mutagenesis technical principle, escherichia coli preferred codons are further adopted, soluble expression of the interferon is finally realized by optimizing the induction condition, the biological activity of the interferon gamma-1b is greatly improved, the preparation method is simple, and good application and development prospect is achieved.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, in particular to a recombinant soluble human bone-targeted interferon gamma-1b and its preparation method and application. Background technique [0002] Interferon (Interferon, IFN) is a cytokine that was first discovered by the British scientist Isaacs in 1957 when he used chicken embryo chorioallantoic membrane to study the interference phenomenon of influenza virus. It has the functions of inhibiting cell proliferation, regulating immunity, anti-virus and anti-tumor. and many other functions. The IFN family includes three main members: IFNα, IFNβ and IFNγ, among which Interferon γ (Interferon gamma, IFNγ) is a cytokine with antiviral, antitumor and immunomodulatory effects, mainly produced by activated T cells and NK cells , Play an important role in the body's immune system. IFNγ-1b is currently used to treat rheumatoid arthritis, liver fibrosis, tumors and other diseases. In 200...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/57C12N15/70C12N1/21A61P19/08
CPCA61P19/08C07K14/57C12N15/70
Inventor 王志宇曹广祥付加芳宗工理王世立
Owner SHANDONG MEDICAL BIO TECH RES CENT
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