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Yeast expressed chicken Cathelicidin antibacterial peptide as well as preparation method and application thereof

An antimicrobial peptide and expression vector technology, applied in the field of recombinant chicken Cathelicidin1 antimicrobial peptide, can solve the problems of lack of processing and modification ability of gene expression regulation mechanism, difficulty in expressing antimicrobial peptides, affecting the expression of antimicrobial peptides, etc., achieving convenient artificial synthesis and easy amplification , the effect of simple structure

Pending Publication Date: 2016-07-06
POULTRY INST SHANDONG ACADEMY OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The initial research on the gene expression of antimicrobial peptides was carried out in the prokaryotic expression system, but it is difficult to express antimicrobial peptides using this system, mainly in three aspects: (1) Escherichia coli is a prokaryotic organism and does not have the characteristics of eukaryotic organisms. Gene expression regulation mechanism and protein post-translational processing and modification ability, the products often form inactive inclusion bodies, which must be denatured, refolded, etc. before they can be used; (2) The toxicity of antimicrobial peptides to host cells, the antimicrobial peptides expressed by host cells It will feed back the proliferation of Escherichia coli and affect the further expression of antimicrobial peptides; (3) it is easy to be degraded. The antimicrobial peptide itself has a large number of positive charges and is very sensitive to proteases. It is easy to degrade in Escherichia coli and difficult to degrade. achieve mass expression
Therefore, antimicrobial peptide genes can only be expressed in Escherichia coli in the form of fusion proteins

Method used

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  • Yeast expressed chicken Cathelicidin antibacterial peptide as well as preparation method and application thereof
  • Yeast expressed chicken Cathelicidin antibacterial peptide as well as preparation method and application thereof
  • Yeast expressed chicken Cathelicidin antibacterial peptide as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] The cloning of the mature peptide coding sequence of the black-bone Cathelicidin1 antimicrobial peptide and the construction and identification of the secretory yeast expression vector include the following steps:

[0039] 1. Design of primers

[0040] According to the Cathelicidin1 gene sequence of silky chicken (GenBank sequence number: FJ938357) published in GenBank and the design principles of PCR primers, and with the help of computer software DNASTAR for auxiliary analysis, a pair of PCR gene-specific primers were designed for amplifying chicken The mature peptide coding sequence of Cathelicidin1 gene, the primers are pCathL1F / R in the table, where pCathL1F is the forward primer, and pCathL1R is the reverse primer. At the same time, according to the known gene sequence structure, a pair of universal primers 5'AOX1 / 3'AOX1 for the eukaryotic expression vector pPICZαA was synthesized, and the primers were all synthesized by Invitrogen.

[0041] The primer sequences ...

Embodiment 2

[0050] The genetic engineering preparation method of recombinant chicken Cathelicidin1 antimicrobial peptide fusion protein comprises the following steps:

[0051] 1. Construction of Pichia pastoris genetically engineered bacteria and screening of transformants

[0052] Recombinant expression plasmids pPICZαA-CathL1 and pPICZαA plasmids were digested with endonuclease SacI respectively, and the results were as follows: figure 2 As shown, the linearized plasmids can be obtained, and the linearized plasmids pPICZαA-CathL1 and pPICZαA are mixed with the prepared yeast X-33 competent cells in a quartz cuvette in turn, and then placed in the electroporator tank, and the parameters are adjusted to 1.5kV respectively , 25μF, 200Ω, and 0°C for electric shock, and the above plasmids were sequentially integrated into the Pichiapastoris X-33 genome.

[0053] Take 80 μL of the transformation solution and evenly spread it on a YPDS (containing 100 μg / mL Zeocin) plate, and culture at 28°C...

Embodiment 3

[0063] Example 3 Bacteriostatic activity detection of recombinant chicken antimicrobial peptide Cathelicidin1

[0064] The indicator strains used in the antibacterial test: Micrococcus luteus (CMCC28001), Salmonella pullorum (Salmonella pullorum CVCC79201, C79-1), Pasteurella multocida (CVCC474, C48-7) standard strains were purchased from the China Veterinary Drug Administration ; Pseudomonaspyocyanea and Escherichia coli (Escherichia coli1503) strains are clinically isolated and identified strains preserved in our laboratory. Specific steps are as follows:

[0065] 1. In vitro drug susceptibility test of standard strains such as Escherichia coli and Salmonella

[0066] According to the instructions of the drug susceptibility kit, 9 kinds of drug susceptibility test strips including amoxicillin (10 μg / tablet), amikacin (30 μg / tablet) and azithromycin (15 μg / tablet) were selected to detect Escherichia coli, Salmonella, multocida Pasteurella and other 5 strains were tested for...

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Abstract

The invention belongs to the field of biotechnology and particularly relates to pichia pastoris expressed recombinant chicken Cathelicidin antibacterial peptide and further discloses a preparation method and application thereof. In the method for preparing recombinant chicken Cathelicidin antibacterial peptide disclosed by the invention, a pPICZalphaA secreted expression vector is selected as a eukaryotic expression system of the exogenous gene so that the recombinant chicken Cathelicidin antibacterial peptide is efficiently expressed and direct killing of host cells caused by the expression product is avoided. Meanwhile, the method has the advantages of high expression efficiency, simple separation and purification of expression products, low production cost, easiness in magnification and good stability and is suitable for large-scale industrial production. According to experimental verification, the combination of Cathelicidin antibacterial peptide and antibiotic realizes an obvious synergistic antibacterial effect on multiple kinds of gram-positive bacteria and gram-negative bacteria, and the application and promotion prospects are broad.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a recombinant chicken Cathelicidin1 antibacterial peptide expressed by Pichia pastoris, and further discloses its preparation method and application. Background technique [0002] For a long time, my country's poultry breeding industry has been plagued by a variety of infectious diseases, which have seriously affected the development of the poultry industry. Since the discovery of antibiotics, they have played an important role in the prevention of bacterial infection and the prevention and treatment of livestock and poultry diseases, and have played a huge role in promoting the healthy and rapid development of animal husbandry and poultry industry. However, the long-term and extensive use of antibiotics has also brought a series of negative hidden dangers. On the one hand, the extensive use of antibiotics makes pathogenic bacteria gradually develop drug resistance, which increases ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/465C12N15/12C12N15/81C12N1/19C12P21/02A61K38/17A61P31/04C12R1/84
CPCC07K14/465A61K38/00C12N15/815C12N2800/102C12P21/02
Inventor 黄兵马秀丽于可响刘存霞吴静史玉颖胡峰李玉峰宋敏训
Owner POULTRY INST SHANDONG ACADEMY OF AGRI SCI
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