Preparation method and application of fused bovine antibacterial peptide and interleukin 2 co-expression recombinant yeast preparation

A technology of interleukins and antimicrobial peptides, applied in the biological field, can solve problems such as the emergence of drug resistance of bacteria, hindering the development of aquaculture, and the failure of drugs to treat antibiotics.

Active Publication Date: 2018-06-08
四川三优康生物技术有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The abuse of feed antibiotic additives not only increases the cost of feeding, but also leads to a significant increase in the drug resistance and pathogenicity of pathogenic microorganisms, and the health of livestock and poultry bodies is weakened due to the side effects of drugs, and the immunity and disease resistance are significantly reduced
In such a vicious cycle, various severe infectious diseases of livestock and poultry frequently occur, making it difficult to control and treat
On the other hand, drug residues and the increasingly serious food safety problems of livestock and poultry products not only directly threaten the health of human beings themselves, but also hinder the development of the breeding industry.
[0006] The addition of antibiotics to animal feed, long-term or inappropriate use will not only induce drug resistance in bacteria, but also cause drug residues in animal products, causing serious food safety hazards, damaging human health and inducing the failure of drugs used to treat antibiotics

Method used

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  • Preparation method and application of fused bovine antibacterial peptide and interleukin 2 co-expression recombinant yeast preparation
  • Preparation method and application of fused bovine antibacterial peptide and interleukin 2 co-expression recombinant yeast preparation
  • Preparation method and application of fused bovine antibacterial peptide and interleukin 2 co-expression recombinant yeast preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0120] Embodiment 1, bovine antimicrobial peptide interleukin 2 fusion protein (FBAPIL2) and its coding gene

[0121] 1. Acquisition of the fusion protein FBAPIL2 and its coding gene

[0122] The bovine antimicrobial peptide and interleukin 2 were fused to obtain a fusion protein named FBAPIL2.

[0123] The amino acid sequence of the fusion protein is shown as sequence 1 in the sequence listing, the fusion gene encoding the fusion protein FBAPIL2 is named FBAPIL2, and the nucleotide sequence of the fusion gene is sequence 2.

[0124] Among them, the 265th-353rd position of the sequence 1 is a bovine antibacterial peptide, the 155th-264th position is a connecting peptide, and the 1st-154th position is an interleukin 2;

[0125] The 793-1059th position of sequence 2 is the bovine antimicrobial peptide coding nucleic acid, the 463-792nd position is the connecting peptide coding nucleic acid, and the 1-462nd position is the interleukin-2 coding nucleic acid.

[0126] The above-m...

Embodiment 2

[0146] Embodiment 2, the influence of bovine antimicrobial peptide interleukin 2 fusion protein (FBAPIL2) on lymphocyte proliferation

[0147] 1. Preparation of recombinant bacteria SMDpG-2B fermentation supernatant

[0148] (1) the recombinant bacterium SMDpG-2B (hereinafter referred to as SG2B) that embodiment 1 obtains is inoculated in 3mL substratum 1 (substratum 1 is the liquid medium that adds bleomycin (Zeocin) to obtain in YPD substratum, and The concentration of bleomycin was 100mg / mL), and the activated strain was cultivated overnight at 28°C and 200rpm.

[0149] (2) Take 300 μL of the bacterial solution obtained in step (1) and inoculate it into a 100 mL Erlenmeyer flask containing 30 mL of YPD medium, ferment at 28 °C and 200 rpm on a shaker for 48 hours (OD 600 for 25).

[0150] (3) Take 5 mL of the bacterial liquid obtained in step (2), centrifuge at 12 000×g for 2 min, and name the obtained supernatant as SG2B fermentation supernatant.

[0151] 2. Protease tr...

Embodiment 3

[0168] Example 3, Bacteriostatic Activity Detection of Bovine Antibacterial Peptide Interleukin 2 Fusion Protein (FBAPIL2)

[0169] Determination of bovine antimicrobial peptide interleukin 2 fusion protein FBAPIL2 on Escherichia coli standard bacteria (G - ) (hereinafter referred to as S-G - ), Escherichia coli resistant bacteria (G - ) (hereinafter referred to as R-G - ), Staphylococcus aureus standard bacteria (G + ) (hereinafter referred to as S-G + ), drug-resistant Staphylococcus aureus (G + ) (hereinafter referred to as R-G + ) antibacterial situation, the specific method is as follows:

[0170] First, the four bacterial strains were inoculated and activated and cultured in the exponential growth phase (OD 600 about 0.5), then diluted to OD with LB medium 600 About 0.005, the diluted bacterial solution was inoculated on a 96-well cell culture plate, 100 μL / well, one kind of bacteria per 96-well cell culture plate.

[0171] For each 96-well cell culture plate co...

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Abstract

The invention discloses a preparation method and an application of an antibacterial peptide fused cytokine FBAPIL2 co-expression biological preparation. The antibacterial peptide fused cytokine FBAPIL2 is a protein A1) with the amino acid sequence represented by sequence 1, or a protein A2) obtained by substituting and / or deleting and / or adding one or more amino acid residues to an amino acid sequence represented by a sequence 1 in a sequence table and having same functions, or a fusion protein A3) obtained through connecting a label to the N end or / and the C end of the protein A1) or A2). Experiments prove that the FBAPL2 can promote the proliferation of lymphocytes, erythrocyte and leukocytes, inhibit the growth of pathogenic microorganisms, promote the secretion of non-specific antibodies (IgG, IgG1 and IgG2a) and disease-specific antibodies and improve the immunity and survival rate of animals.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the preparation and application of a recombinant yeast preparation fused with bovine antimicrobial peptide and interleukin-2 co-expression. Background technique [0002] The aquaculture industry (animal husbandry and aquatic products) is an important part of large-scale agriculture. It is one of the important foundations for my country's agricultural modernization, rural economic development, and social stability. The aquaculture industry in advanced countries in Europe and the United States is very developed, accounting for 50% of large-scale agriculture. % above the specific gravity. It provides human beings with meat, milk, eggs, fur and other high-grade foods and important textile raw materials. Animal products are the main source of high-quality protein, minerals, vitamins and other nutrients for people. Their safety is directly related to human health. In recent yea...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62A61K38/20A61K38/17A61P37/04
CPCA61K38/00C07K14/47C07K14/55C07K2319/00
Inventor 高荣万小平魏泓肖永乐马常俊黎淩朱玉华胡立博刘建华田玉虎王泽洲吕学斌李江淩
Owner 四川三优康生物技术有限公司
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