Preparation and application of recombinant yeast preparation fused with bovine antimicrobial peptide fbap

A technology of recombinant bacteria and nucleic acid molecules, used in fusion polypeptides, hybrid peptides, antibacterial drugs, etc.

Active Publication Date: 2020-11-10
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

[0005] Reports on the fusion cloning and expression of antimicrobial peptide genes and other related genes are gradually increasing, but there are few reports on the fusion expression of multiple short peptides with antibacterial activity, and self-cleavage after expression to form a single active molecule

Method used

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  • Preparation and application of recombinant yeast preparation fused with bovine antimicrobial peptide fbap
  • Preparation and application of recombinant yeast preparation fused with bovine antimicrobial peptide fbap
  • Preparation and application of recombinant yeast preparation fused with bovine antimicrobial peptide fbap

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0121] 1. Obtaining the fusion protein FBAP and its coding gene

[0122] Four kinds of bovine antimicrobial peptides were fused by connecting peptides to obtain a fusion protein named FBAP.

[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 FBAP is named FBAP, and the nucleotide sequence of the fusion gene is sequence 2.

[0124] Among them, the 1st-12th position of sequence 1 is bovine antimicrobial peptide F, the 13th-20th position is a linking peptide, the 21st-32nd position is bovine antimicrobial peptide B, the 33rd-40th position is a linking peptide, and the 41st-55th position is Bovine antimicrobial peptide A, the 56th-62nd is the connecting peptide, and the 63rd-75th is the bovine antimicrobial peptide P.

[0125] The 1st-36th position of sequence 2 is the nucleic acid encoding bo...

Embodiment 2

[0142] Embodiment 2, the influence of bovine antimicrobial peptide fusion protein (FBAP) on lymphocyte proliferation

[0143] 1. Preparation of recombinant bacteria SMDpG-B fermentation supernatant

[0144] (1) the recombinant bacterium SMDpG-B (hereinafter referred to as SGB) 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.

[0145] (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).

[0146] (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 SGB fermentation supernatant.

[0147] 2. Protease treatment of the fermen...

Embodiment 3

[0164] Embodiment 3, the antibacterial activity detection of bovine antimicrobial peptide fusion protein (FBAP)

[0165] Determination of bovine antimicrobial peptide fusion protein FBAP 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:

[0166] 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.

[0167] For each 96-well cell culture plate containing bacteria, it was pr...

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Abstract

The invention discloses preparation and application of a fusion bovine antibacterial peptide (FBAP) recombinant yeast preparation. The invention provides four kinds of bovine antibacterial peptides; amino acid sequences of the four kinds of bovine antibacterial peptides are 1-12 sites of a sequence 1, 21-32 sites of the sequence 1, 41-55 sites of the sequence 1 and 63-75 sites of the sequence 1 respectively. Proved by experiments, the FBAP provided by the invention can promote proliferation of lymphocytes, erythrocytes and leukocytes, inhibits growth of pathogenic microorganisms, promotes secretion of non-specific antibodies (IgG, IgG1 and IgG2a) and disease-specific antibodies, improves the immunity of animals and increases the survival rate of the animals.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to the preparation and application of a recombinant yeast preparation fused with bovine antimicrobial peptide FBAP. Background technique [0002] At present, China's livestock and poultry breeding industry is under the pressure of controlling the spread of more than 30 kinds of infectious pathogens, especially severe diseases such as avian influenza and respiratory reproductive syndrome, as the degree of intensification increases, and the scale and density of livestock and poultry breeding are increasing day by day. Diarrhea caused by drug-resistant bacteria in animal breeding, Salmonella, Escherichia coli, streptococcus, PRRSV (PRRSV), circovirus (PCV2), swine fever (CSFV) and other bacterial and viral diseases, For a long time, it has been a bottleneck problem in animal husbandry, which seriously hinders the development of animal husbandry; the abuse of drug additives such as antibiotic...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62A61K38/10A61K47/64A61P37/04A61P31/04A61P31/14
CPCA61K38/00C07K7/08C07K2319/00
Inventor 高荣万小平肖永乐马常俊黎淩朱玉华刘建华吕学斌王泽洲李江淩其他发明人请求不公开姓名
Owner 四川三优康生物技术有限公司
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