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A fusion interferon and its application in the preparation of mucosal immune enhancer

An immune enhancer and protein technology, applied in the direction of interferon, cytokines/lymphokines/interferons, fusion polypeptides, etc., can solve the problems of economic losses in the pig industry and the inability to effectively prevent disease outbreaks and epidemics, and achieve major applications The effect of promoting value

Active Publication Date: 2020-08-18
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there are many kinds of pig disease vaccines and frequent immunizations, they still cannot effectively prevent disease outbreaks and epidemics. The above-mentioned viral infectious diseases have caused relatively large economic losses to the pig industry every year.

Method used

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  • A fusion interferon and its application in the preparation of mucosal immune enhancer
  • A fusion interferon and its application in the preparation of mucosal immune enhancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1, the construction of recombinant bacteria and the preparation of target protein

[0031] 1. Construction of recombinant plasmids

[0032] The double-stranded DNA molecule shown in Sequence 2 of the sequence listing was inserted between the NdeI and BamHI restriction sites of the pET28a(+) vector to obtain the recombinant plasmid pET28a-PoIFNλ1-IL2. According to the sequencing results, the structure of the recombinant plasmid pET28a-PoIFNλ1-IL2 is described as follows: a DNA molecule shown in Sequence 2 of the sequence table is inserted between the NdeI and BamHI restriction sites of the pET28a(+) vector. The DNA molecule shown in Sequence 2 of the Sequence Listing encodes the protein shown in Sequence 1 of the Sequence Listing. The protein shown in Sequence 1 of the sequence listing is named as the optimized PoIFNλ1-IL2 fusion protein. In sequence 1 of the sequence listing, amino acid residues 1-172 have an optimized porcine PoIFNλ1 protein, and amino aci...

Embodiment 2

[0078] Embodiment 2, the function of target protein (animal test verification)

[0079] The experimental animals were Landrace pigs (40 days old, each with a body weight of 10 kg).

[0080] The optimized PoIFNλ1-IL2 fusion protein used in this example was prepared by Escherichia coli BL21 / pET28a-PolFNλ1-IL2 in Step 2 of Example 1. The pre-optimized PoIFNλ1-IL2 fusion protein used in this example was prepared by recombinant Bacteria in Step 3 of Example 1. The pre-optimized porcine PoIFNλ1 protein used in this example was prepared by recombinant bacteria B in Step 4 of Example 1.

[0081] Composition I is composed of optimized PoIFNλ1-IL2 fusion protein, white oil and diluent, which are fully mixed and emulsified. Composition II is composed of pre-optimized PoIFNλ1-IL2 fusion protein, white oil and diluent, fully mixed and emulsified. Composition III is composed of porcine PoIFNλ1 protein before optimization, white oil and diluent, fully mixed and emulsified. White oil func...

Embodiment 3

[0093] Embodiment 3, interferon activity detection

[0094] Cell culture medium: DMEM medium containing 10% FBS.

[0095] 1. Interferon activity detection method

[0096] 1. Cell preparation

[0097] Take well-grown PK-15 cells, suspend them with cell culture medium after digestion, and obtain a cell concentration of 5×10 5 cells / mL of cell suspension; add the cell suspension to a 96-well cell culture plate (100 μl / well), at 37°C, 5% CO 2 Under the condition of culture 8-10h to make it into monolayer cells.

[0098] 2. Using cell culture medium as a solvent, first dissolve or dilute the substance to be tested so that the protein concentration is 0.001mg / ml, as the mother solution, and then dilute the mother solution by 4 times and dilute 6 gradients to obtain various dilutions.

[0099] 3. Take the cell culture plate that completed step 1, aspirate and discard the supernatant; add cell culture medium (100 μl / well) to 3 positive control wells, add cell culture medium (100 μl / ...

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Abstract

The invention discloses fusion interferon and application thereof in preparation of a mucosa immune enhancer. Firstly, a kind of protein is provided, and the protein comprises two segments, namely pigprotein PoIFNgamma1 and pig protein IL2; the protein also comprises a connecting peptide, and the connecting peptide is positioned between the pig protein PoIFNgamma1 and the pig protein IL2. The invention also protects application of the protein in the preparation of pig virus infectious disease vaccines. The invention also protects a pig virus infectious disease vaccine, and the pig virus infectious disease vaccine comprises the protein. The fusion interferon has important application and popularization value in the prevention and controlling of the pig virus infectious diseases.

Description

technical field [0001] The invention relates to a fusion interferon and its application in preparing a mucosal immune enhancer. Background technique [0002] In the pig industry, many viral infectious diseases such as porcine reproductive and respiratory syndrome, porcine pseudorabies, and swine flu occur frequently. Although swine disease vaccines are various and frequently immunized, they still cannot effectively prevent disease outbreaks and epidemics. The above-mentioned viral infectious diseases have caused relatively large economic losses to the pig industry every year. It is an urgent need in the pig industry to improve the immune efficacy of vaccines and to develop efficient and safe drugs for the prevention and treatment of swine diseases. [0003] Due to the actual production needs of intensive farming, more and more researchers have realized the importance of mucosal immunity in anti-virus. Vaccination through mucosa not only generates immune response in mucosal ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62A61K38/21A61K38/20A61P31/16
CPCA61K38/00A61P31/16C07K14/55C07K14/555C07K2319/00
Inventor 李晶刘文军范文辉刘丽蓉
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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