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Fusion protein with alpha-interferon activity and its coded gene and use

A technology of fusion protein and alpha interferon, which can be used in medical preparations containing active ingredients, peptide/protein components, hybrid peptides, etc. Mass production, etc.

Inactive Publication Date: 2007-04-11
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although human alpha interferon has been obtained through expression and purification in Escherichia coli, this expression method has overcome the relevant problems and potential dangers of producing this interferon from natural sources, but this method also has its own defects: one On the one hand, in some cases, the expressed protein cannot be processed correctly; on the other hand, in order to remove the bacterial endotoxin, a purification step is required, which is difficult to scale up for large-scale production because the purification method includes multiple chromatographic steps
However, the yield of human interferon-α in Saccharomyces cerevisiae is very low, and it is difficult to carry out large-scale production

Method used

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  • Fusion protein with alpha-interferon activity and its coded gene and use
  • Fusion protein with alpha-interferon activity and its coded gene and use
  • Fusion protein with alpha-interferon activity and its coded gene and use

Examples

Experimental program
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Effect test

Embodiment 1

[0059] Example 1. Acquisition of a gene encoding a fusion protein with interferon-α activity

[0060] Amplify the coding gene of the fusion protein with alpha interferon activity of the present invention by PCR method, and the specific process comprises the following steps:

[0061] 1. Amplification of IFNα-2b gene

[0062] 1) Amplification of human IFNα-2b gene connected with α-factor and IgGγ1 fragment with Kex2 single protease cleavage site

[0063]The plasmid pGEM-7Z-Fa2-T7 [pGEM-7Z-Fa2-T7] containing the human IFNα-2b gene was constructed by cloning the human IFNα-2b gene (GenBank number: 184581) into the vector pGEM-7Zf(+) (Promega ) between the EcoR I and Hind III restriction endonuclease sites of the multiple cloning site to obtain a recombinant vector] as a template, in the upstream primer IFNα-2b-K-Fw (5'-CTCGAGAAAAGATGTGATCTGCCTCAAAACCCAC-3') and downstream Under the guidance of primer IFNα2b / γ1-Rv (5'-GATTTGGGCTCTTCCTTACTTCTTAAACTTTCTTG-3'), PCR amplified human I...

Embodiment 2

[0131] Example 2, Expression of fusion protein gene with alpha interferon activity in Pichia pastoris and purification of expression product

[0132] 1. Construction of a Pichia pastoris expression vector having a fusion protein gene with alpha interferon activity

[0133] Referring to Fig. 1 constructing the Pichia pastoris expression vector of the fusion protein gene with alpha interferon activity, specific method is: use restriction endonuclease BamH I and EcoR I to the 8 gene fragments that embodiment 1 step seven obtains (collectively referred to as alpha -Factor-IFNα-Fc) were double-enzymatically digested, and then the 8 digested fragments were respectively connected with the plasmid pPic9k that had been double-digested with the same enzyme with T4 DNA ligase, and the ligated products were transformed into E. coli DH5α competent cells, and screened Positive recombinants, plasmids were extracted, sequenced, and the sequencing results were consistent with the expected resu...

Embodiment 3

[0165] Example 3, Detection of IFN-α activity of the fusion protein of the present invention

[0166] The fusion protein obtained through expression in Example 2 was tested for IFN-α activity using the human amniotic cell Wish strain (Wish cell) / vesicular stomatitis virus (VSV virus) system. 10% calf serum DMEM medium (purchased from Invitrogen Company) was made into a suspension with a cell concentration of 3.5 × 105 / mL, added to a 96-well cell culture plate, 100ul / well, in 5% CO 2 After culturing for 4-6h in a 37° C. incubator, add 100 ul of the fusion protein sample obtained through expression in Example 2 through 4-fold gradient dilution into each well (the sample diluent is DMEM culture fluid containing 7% calf serum), and at the same time Set up IFNα-2b standard substance control and blank control wells, and incubate at 37°C for 18-24h. After the cultivation, the supernatant was discarded, and the VSV virus (100TCID50) diluted in DMEM medium containing 3% calf serum was...

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Abstract

The present invention discloses one kind of fusion protein with alpha-interferon activity and its coding gene, expression mode and application in preparing medicine for raising body's immunity. The fusion protein is human IFN-alpha-2b fusion protein with human IgG Fc-gamma-1 or human IgG Fc-gamma-2 connected to the carboxyl group end, the human IFN-alpha-2b has the amino acid residue sequence as shown in SEQ ID No. 13 of the sequence list, the human IgG Fc-gamma-1 is wild human IgG Fc-gamma-1 or its mutant, and the human IgG Fc-gamma-2 is wild human IgG Fc-gamma-2 or its mutant. The fusion protein has the following advantages: high alpha-interferon activity, long half life and small ADCC damaging effect. The present invention is significant and possesses wide application foreground in medicine and biomedicine preparing fields, especially in preparing medicine for raising immunity.

Description

technical field [0001] The present invention relates to a fusion protein and its encoding gene and application, in particular to the fusion protein with alpha interferon activity and its encoding gene, as well as the expression method of the fusion protein and its application in the preparation of medicines for improving the body's immunity. Background technique [0002] Interferon-alpha (IFN-α) is a group of cytokines that can induce the expression of a series of intracellular proteins, and then play an anti-virus, anti-cell proliferation and immune response role. Natural IFN-α is mainly produced by leukocytes, fibroblasts, etc. under the induction of stimuli such as bacteria, DNA, RNA viruses, polyinosinic acid polycytidylic acid (Poly I-C) or polynucleotides. According to statistics, there are at least 20 subtypes of IFN-α, and these family members have similar structural characteristics: the molecular weight is between 19-26kD, the isoelectric point (pI) is about 5-7, an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N1/19A61K38/16A61P37/00
Inventor 肖卫华王磊
Owner UNIV OF SCI & TECH OF CHINA
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