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Preparation and application of antibiotic peptide pichia yeast

A Pichia pastoris and antimicrobial peptide technology, applied in the biological field, can solve problems such as affecting human health, difficult treatment, and difficulty in large-scale cultivation, and achieve the effect of shortening production cycle and reducing cost

Inactive Publication Date: 2004-07-21
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the high cost of insect cell culture, large-scale cultivation is not easy for insect body expression
[0005] In recent years, the abuse of antibiotics in livestock and poultry feeds has resulted in residues in the milk, meat, and eggs of livestock and poultry, seriously affecting human health, and the emergence of drug-resistant strains due to the extensive use of antibiotics has brought difficulties to treatment

Method used

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  • Preparation and application of antibiotic peptide pichia yeast
  • Preparation and application of antibiotic peptide pichia yeast
  • Preparation and application of antibiotic peptide pichia yeast

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0061] 1. Preparation of antimicrobial peptides by Pichia pastoris

[0062] 1. Synthesis of antimicrobial peptide AD gene

[0063] According to the design of antibacterial peptide AD and its gene, synthesize F with a DNA synthesizer 1 (82 bases) and F 2 (78 bases), using primer 1 and primer 2 for PCR amplification. f 1 and F 2 Flake acid (25mmol / uL) 1uL, d NTP (pmol / L) 2.5uL, 10×PCR buffer 5uL, double distilled water 35.5uL, denature at 92°C for 1min, add Taq DNA polymerase 1uL (3u), press 52°C 60 Sec, 70°C 60Sec, 90°C 30Sec for a total of 35 cycles, and the PCR product was recovered after termination. Using the first PCR product as a template, primer 1 and primer 2 were used for the second PCR amplification, and the final step was extended at 72°C for 10 min. After the PCR product was recovered, it was electrophoresed on a low-melting point agarose gel to obtain the antibacterial peptide AD gene, which was stored at -20°C after recovery.

[0064] 2. Antimicrobial pepti...

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Abstract

The antheraea pernyi antibacterial peptide possesses broad-spectrum bacteriocidal action. According to amino acid sequences of 1-11 situs of natural antibacterial peptide A and amino acid sequences of 12-37 situs of antibacterial peptide D the antibacterial peptide AD gene is designed, and cloned in Pichia yeast and expressed. By optimizing formula of culture medium and culture condition the high-density culture and high-efficiency expression (500 unit / ml) can be obtained in a fermentation tank. The antibacterial peptide yeast product can be added in the feed for piglet in 5000 unit / kg or added in the drinking water for chicken in 60-100 unit / animal. day., can be used for preventing and curing animal diarrhea, and can be used as feed additive instead of partial vitamins.

Description

technical field [0001] The present invention relates to the field of biotechnology. In particular, the antimicrobial peptide gene is cloned in yeast to prepare feed additive technology. Background technique [0002] Tussah antimicrobial peptide (Cecropin) is a natural polypeptide induced by injecting Escherichia coli into tussah silkworm (Antheraea pernyi) pupae. It has been found that there are A, B and D, etc., and it has a broad-spectrum bactericidal effect. The yield of antimicrobial peptides extracted from tussah silkworm immune pupa is low (less than 0.02%), more raw materials are consumed, the process is complicated, the cost is high, and it is difficult to put into production. The pupal residue and cocoon shell after the antimicrobial peptides are extracted still need to be further processed. Such as 1000 tons of tussah cocoons (10-12 million yuan) can only extract 200-250Kg antimicrobial peptides, and the cost per Kg is more than 100,000 yuan....

Claims

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Application Information

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IPC IPC(8): A23K20/195C07K14/435C12N1/19C12N15/12C12N15/70C12N15/81
Inventor 黄自然黄亚东郑青姚汝华
Owner SOUTH CHINA AGRI UNIV
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