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Preparation and application of recombinant yeast preparation for co-expression of porcine interleukin 4/6 and fusion porcine antibacterial peptide

A technology for interleukin and antimicrobial peptides, applied in the direction of fusion peptides, cationic antimicrobial peptides, hybrid peptides, etc., can solve the problems of rare porcine interleukin fusion antimicrobial peptide gene recombination cloning and co-expression

Active Publication Date: 2018-09-21
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

[0007] There are few reports on recombinant cloning and co-expression of porcine interleukin fusion antimicrobial peptide gene

Method used

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  • Preparation and application of recombinant yeast preparation for co-expression of porcine interleukin 4/6 and fusion porcine antibacterial peptide
  • Preparation and application of recombinant yeast preparation for co-expression of porcine interleukin 4/6 and fusion porcine antibacterial peptide
  • Preparation and application of recombinant yeast preparation for co-expression of porcine interleukin 4/6 and fusion porcine antibacterial peptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0121] Embodiment 1, porcine antimicrobial peptide interleukin 4 / 6 fusion protein (FPAPIL46) and its coding gene

[0122] 1. Acquisition of the fusion protein FPAPIL46 and its coding gene

[0123] The porcine antimicrobial peptide and interleukin 4 / 6 are fused to obtain a fusion protein named FPAPIL46.

[0124] The amino acid sequence of the fusion protein is shown as sequence 1 in the sequence listing, the fusion gene encoding the fusion protein FPAPIL46 is named FPAPIL46, and the nucleotide sequence of the fusion gene is sequence 2.

[0125] Among them, the 387-475th position of the sequence 1 is a porcine antimicrobial peptide, the 277-386th position is a connecting peptide, and the 1-276th position is an interleukin 4 / 6;

[0126] The 1159-1428th position of sequence 2 is the porcine antimicrobial peptide encoding nucleic acid, the 829-1158th position is the connecting peptide encoding nucleic acid, and the 1-828th position is the interleukin 4 / 6 encoding nucleic acid.

...

Embodiment 2

[0143] Embodiment 2, the effect of porcine antimicrobial peptide interleukin 4 / 6 fusion protein (FPAPIL46) on lymphocyte proliferation

[0144] 1. Preparation of recombinant bacteria SMDpG-46P fermentation supernatant

[0145] (1) the recombinant bacterium SMDpG-46P (hereinafter referred to as SG46P) 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.

[0146] (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).

[0147] (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 SG46P fermentation supernatant.

[0148] 2. Protea...

Embodiment 3

[0165] Example 3, Antibacterial Activity Detection of Porcine Antibacterial Peptide Interleukin 4 / 6 Fusion Protein (FPAPIL46)

[0166] Determination of porcine antimicrobial peptide interleukin 4 / 6 fusion protein FPAPIL46 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:

[0167] 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.

[0168] For each 96-well cell culture p...

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Abstract

The invention discloses preparation and application of a recombinant yeast preparation for co-expression of porcine interleukin 4 / 6 and fusion porcine antibacterial peptide. The antibacterial peptidefusion cytokine FFPIL46 is as follows A1), A2) or A3); A1) is the protein of which an amino acid sequence is the sequence 1 in a sequence listing; A2) is the protein with the same functions as the amino acid sequence shown by the sequence 1 in the sequence listing subjected to substitution and / or deletion and / or addition of one or more amino acid residues; A3) is the fusion protein obtained by connecting the N-terminal or / and C-terminal of A1) or A2) with labels. Experiments show that the FPAPL46 can promote the proliferation of lymphocytes, red blood cells and white blood cells, inhibit the growth of pathogenic microorganisms, promote the secretion of non-specific antibodies (IgG, IgG1, 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 co-expressed with porcine interleukin 4 / 6 and fusion porcine antimicrobial peptide. 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 husband...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62A61K48/00A61K38/20A61K38/17A61P37/04A61P31/04A61P31/14A61P31/20
CPCA61K38/00A61K48/005C07K14/4723C07K14/5406C07K14/5412C07K2319/00
Inventor 高荣万小平魏泓肖永乐吴雪颖胡立博朱玉华刘建华田玉虎吕学斌王泽洲李江淩
Owner 四川三优康生物技术有限公司
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