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Recombined human gama interferon gland virus and preparation of products therefrom

An adenovirus and interferon technology, applied in the field of genetic engineering, can solve the problems of unstable nature and easy inactivation, and achieve the effects of high titer, stable and lasting expression, and wide host range

Inactive Publication Date: 2004-12-08
黄文林
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, the γ-interferon currently used in clinical practice is a protein product produced by recombinant technology, which is unstable and easy to inactivate

Method used

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  • Recombined human gama interferon gland virus and preparation of products therefrom
  • Recombined human gama interferon gland virus and preparation of products therefrom
  • Recombined human gama interferon gland virus and preparation of products therefrom

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Recombinant human γ-interferon adenovirus (Ad-rhIFNγ) contains secreted human γ-interferon target gene and defective adenovirus. The defective adenovirus is adenovirus type 5 of subgroup C (Ad5).

[0037] Ad-hIFN-γ structure: the recombinant human γ-interferon adenoviral vector has deleted the E1 region (E1 Δ ), and part of the E3 region (partiteE3 Δ ), the human γ-interferon protein gene is the target gene, and the expression cassette expressing secreted human γ-interferon is inserted into the E1 region. The target gene expression cassette is composed of cell virus (CMV) promoter, human γ-interferon gene and Downstream polyA signaling components (see figure 1 ).

Embodiment 2

[0039] Preparation of recombinant human gamma-interferon adenovirus:

[0040] The production process of recombinant human γ-interferon adenovirus includes: construction of plasmid, transfection of bacteria, infection of 293 cells, separation and purification, etc. Specifically see its production process flow chart (see figure 2 ).

Embodiment 3

[0042] Expression detection of recombinant human gamma-interferon adenovirus target gene:

[0043] 1. Inoculate C6 cells into a 96-well plate, 2×10 4 / hole.

[0044] 2. After 24 hours, inoculate Ad-hIFN-γ. Method: Dilute the virus stock solution by 4 times with 1640 culture solution. When inoculating, first suck out the culture solution in the 96-well plate and discard it. After washing the wells with PBS once, add viruses of different dilutions. solution, add 6 wells for each dilution, 50 μL / well. At the same time, 1640 culture medium (without virus) was used as negative control. 37°C, 5% CO 2 , after incubating for 1 hour under saturated humidity conditions, add 1640 culture solution containing 5% newborn calf serum, 200 μL / well, 37 ° C, 5% CO 2 , cultivated under saturated humidity conditions.

[0045] 3. After that, collect the cell supernatant in 2 wells of each dilution every 24 hours, store at -80°C, and collect 3 times in a row.

[0046] 4. According to the instr...

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Abstract

A recombinant adenovirus with gene of human gamma-interferon by gene recombination, its preparation and application for antiviral medicine and tumor treatment medicine are disclosed. Adenovirus is taken as carrier to recombine gamma - interferon protein fragment with adenovirus by gene recombination, forming stable recombinant human gamma-interferon adenovirus, and by inducing gamma-interferon into organic tissue cells to make gamma-interferon protein steadily express in body in the way of copying defect adenovirus carrier. It achieves remarkable antiviral effect.

Description

1. Technical field [0001] The invention belongs to the field of genetic engineering, and specifically relates to the preparation and application of recombinant human gamma-interferon adenovirus obtained through gene recombination. Specifically, the present invention relates to an adenovirus vector, an interferon protein, a genetic engineering product combining the two through recombination, a preparation method of the product and an application in preparing antiviral drugs. 2. Background technology [0002] Interferon is a kind of protein produced in the body with anti-virus, anti-tumor and immunomodulatory effects. Among them, γ-interferon has the following biological effects: [0003] ① γ-interferon is a cytokine with pro-inflammatory effect, secreted by Th1 cells and NK cells, which can induce the body's non-specific immunity and protect the body from virus infection; [0004] ②Interferon-γ interacts significantly with the cytokine network in the body, which can induce c...

Claims

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Application Information

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IPC IPC(8): A61K38/00C07K14/57C12N7/01C12N15/23C12N15/861
CPCC07K14/57C12N15/86C12N2710/10343A61K38/00A61P31/12
Inventor 黄文林
Owner 黄文林
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