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Efficient production method and application of antimicrobial peptide PR-39 of pig small intestine in pichia pastoris

The technology of PR-39 and Pichia pastoris, which is applied in the field of genetic engineering, can solve the problems of complex production process, difficult purification, and low expression of PR-39 gene, and achieve the effects of simple separation and purification, low production cost and low price.

Inactive Publication Date: 2011-11-02
SOUTH CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] The purpose of the present invention is to provide a method for efficiently expressing porcine small intestine antimicrobial peptide PR-39 using the Pichia pastoris expression system in view of the defects of the existing PR-39 gene, such as low expression level, difficult purification, and complicated production process

Method used

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  • Efficient production method and application of antimicrobial peptide PR-39 of pig small intestine in pichia pastoris
  • Efficient production method and application of antimicrobial peptide PR-39 of pig small intestine in pichia pastoris
  • Efficient production method and application of antimicrobial peptide PR-39 of pig small intestine in pichia pastoris

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Embodiment 1

[0027] (1) Construct the expression vector: the amino acid sequence of antimicrobial peptide PR39 published on GenBank [GI:242304], select the partial tropism codon of Pichia pastoris, design 4 primers that serve as templates for each other, through two rounds of PCR, use P2, P3 Primers were subjected to the first round of PCR amplification. PCR reaction conditions: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 45 s, and after 30 cycles, extension at 72°C for 10 min; The PCR product of the first round was used as a template, and the second round of PCR amplification was carried out with P1 and P4 as primers. The PCR reaction conditions were the same as those of the first round, and the product was the target gene. The gene of the PR-39 that obtains is connected on the expression vector, obtains the nucleotide sequence of the described primer of recombinant expression vector as follows:

[0028] P1:

[0029...

Embodiment 2

[0046] (1) Antibacterial identification by agarose diffusion (refer to Agarose diffusion antimicrobial assay, US Patent 6,337,314, January 8, 2002).

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Abstract

The invention discloses an efficient manufacturing method and application of antimicrobial peptide PR-39 of pig small intestine in pichia pastoris. The efficient manufacturing method provided by the invention comprises the following steps of: synthesizing a DNA (Deoxyribonucleic Acid) for coding PR-39 according to the feeding preference of the pichia pastoris; connecting an expression vector pGAPZalphaA, constructing a pGAPZalphaA-PR-39 eukaryotic expression vector; transforming a recombinant expression vector into a host cell and constructing an expression engineering bacterium; fermenting and culturing recombinant saccharomycete and expressing. The method provided by the invention can be used for efficiently expressing the antimicrobial peptide PR-39 in the host cell of the pichia pastoris by utilizing a gene engineering technology, has the advantages of high expression efficiency, simple separation and purification, low production cost, easiness in amplification and good stability, is suitable for large-scale industrial production and has a wide application prospect in the fields of preparing antibacterial medicines, fodder additives or food preservatives.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to the expression of antibacterial peptides, in particular to a high-efficiency production method of porcine small intestine antibacterial peptide PR-39. Background technique [0002] At present, almost all conventional antibiotics have corresponding drug-resistant pathogenic strains, pathogenic bacteria [0003] The problem of antimicrobial resistance has become an increasingly serious threat to the health of animals and humans. At the same time, biosafety and food safety have attracted human attention. Finding new types of antibacterial agents is an effective way to solve the problem of drug resistance. [0004] Antimicrobial peptides are a class of biologically active small peptides that widely exist in the biological world. They are a kind of antibacterial active peptides produced by cells and tissues of microorganisms, plants, invertebrates and various an...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/81C07K14/47A61K38/17A61P31/04
Inventor 黄毓茂何俊刘德辉
Owner SOUTH CHINA AGRI UNIV
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