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Preparation and application of recombinant yeast preparation co-expressed with bovine antibacterial peptide and interleukin 4/6

A technology of recombinant bacteria and expression cassettes, applied in the direction of fusion peptides, hybrid peptides, antibacterial drugs, etc., can solve the problems of rare antibacterial peptide gene fusion cloning and expression reports

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

[0004] There are few reports on the fusion cloning and expression of antimicrobial peptide genes and other immune genes

Method used

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  • Preparation and application of recombinant yeast preparation co-expressed with bovine antibacterial peptide and interleukin 4/6
  • Preparation and application of recombinant yeast preparation co-expressed with bovine antibacterial peptide and interleukin 4/6
  • Preparation and application of recombinant yeast preparation co-expressed with bovine antibacterial peptide and interleukin 4/6

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0118] Embodiment 1, bovine antimicrobial peptide interleukin 4 / 6 fusion protein (FBAPIL46) and its coding gene

[0119] 1. Acquisition of the fusion protein FBAPIL46 and its coding gene

[0120] The bovine antimicrobial peptide and interleukin 4 / 6 were fused to obtain a fusion protein named FBAPIL46.

[0121] The amino acid sequence of the fusion protein is shown as sequence 1 in the sequence listing, the fusion gene encoding the fusion protein FBAPIL46 is named FBAPIL46, and the nucleotide sequence of the fusion gene is sequence 2.

[0122] Among them, the 387-475th position of sequence 1 is a bovine antimicrobial peptide, the 277-386th position is a connecting peptide, and the 1-276th position is an interleukin 4 / 6;

[0123] The 1159-1428th position of sequence 2 is the bovine antibacterial 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.

[0124] ...

Embodiment 2

[0140] Embodiment 2, the influence of bovine antimicrobial peptide interleukin 4 / 6 fusion protein (FBAPIL46) on lymphocyte proliferation

[0141] 1. Preparation of recombinant bacteria SMDpG-46B fermentation supernatant

[0142] (1) the recombinant bacterium SMDpG-46B (hereinafter referred to as SG46B) 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.

[0143] (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).

[0144] (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 SG46B fermentation supernatant.

[0145] 2. Prot...

Embodiment 3

[0162]Example 3, Bacteriostatic Activity Detection of Bovine Antimicrobial Peptide Interleukin 4 / 6 Fusion Protein (FBAPIL46)

[0163] Determination of bovine antimicrobial peptide interleukin 4 / 6 fusion protein FBAPIL46 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:

[0164] 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.

[0165] For each 96-well cell culture pla...

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Abstract

The invention discloses preparation and application of a biological preparation co-expressed by antimicrobial peptide fusion cytokine FBAPIL46. The antimicrobial peptide fusion cytokine FBAPIL46 is asthe following (A1), (A2) or (A3): (A1) protein of which an amino acid sequence is a sequence 1 in a sequence table; (A2) protein with the amino acid sequence as shown in the sequence 1 in the sequence table being subjected to substitution and / or deficiency and / or adding by one or more amino acid residues and with the same functions; (A3) fused protein formed by connecting tags at the end N or / andthe end C of the (A2) or the (A3). Experiments prove that the FBAPIL46 can promote the multiplication of lymphocyte, erythrocyte and hemameba, inhibit the growth of pathogenic microorganism, promotesecretion of nonspecific antibodies (IgG, IgG1 and IgG2a) and disease specific antibodies, and improve the immunocompetence 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 antibacterial peptide and interleukin 4 / 6 co-expression. 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 a...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/81A61K38/20A61K38/17A61P37/04A61P1/14A61P31/04A61P31/14
CPCA61K38/00C07K14/47C07K14/5406C07K14/5412C07K2319/00
Inventor 高荣万小平肖永乐马常俊黎淩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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