Yeast expressed chicken antibacterial peptide Cathelicidin 2 and preparation method and application thereof

An antibacterial peptide and expression vector technology, applied in the field of recombinant chicken Cathelicidin2 antibacterial peptides, can solve the problems of lack of processing and modification ability of gene expression regulation mechanism, difficulty in expressing antibacterial peptides, affecting the expression of antibacterial peptides, etc., achieving convenient artificial synthesis and easy amplification , the effect of simple structure

Inactive Publication Date: 2016-07-13
POULTRY INST SHANDONG ACADEMY OF AGRI SCI
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  • Abstract
  • Description
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  • 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 antibacterial peptide Cathelicidin 2 and preparation method and application thereof
  • Yeast expressed chicken antibacterial peptide Cathelicidin 2 and preparation method and application thereof
  • Yeast expressed chicken antibacterial peptide Cathelicidin 2 and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] The cloning of the mature peptide coding sequence of the silky chicken Cathelicidin2 antimicrobial peptide and the construction and identification of the secreted yeast expression vector specifically include the following steps:

[0040] 1. Design of primers

[0041] According to the Cathelicidin2 gene sequence of silky fowl published in GenBank (GenBank serial number: FJ938358) and the design principles of PCR primers, and with the aid of computer software DNASTAR for auxiliary analysis, a pair of PCR gene-specific primers were designed to amplify chickens. The mature peptide coding sequence of Cathelicidin2 gene, the primers are respectively pCathL2F / R in the table, where pCathL2F is the forward primer and pCathL2R is the reverse primer. At the same time, according to the known gene sequence structure, a pair of universal primers 5'AOX1 / 3'AOX1 of the eukaryotic expression vector pPICZαA were synthesized, and the primers were all synthesized by Invitrogen.

[0042] The prime...

Embodiment 2

[0051] The genetic engineering preparation method of recombinant chicken Cathelicidin2 antimicrobial peptide fusion protein includes the following steps:

[0052] 1. Construction of Pichia pastoris genetically engineered bacteria and selection of transformants

[0053] The recombinant expression plasmid pPICZαA-CathL2 and pPICZαA plasmid were subjected to single enzyme digestion treatment with endonuclease SacI, and the results are as follows figure 2 As shown, the linearized plasmid can be obtained. The linearized plasmids pPICZαA-CathL2 and pPICZαA are mixed with the prepared yeast X-33 competent cells in a quartz cup, and then placed in the electrotransmitter tank, and the parameters are adjusted to 1.5kV. , 25μF, 200Ω, 0°C electric shock, the above plasmids were integrated into the PichiapastorisX-33 genome.

[0054] Take 80μL of the transformation solution and evenly spread it on a YPDS (containing 100μg / mL Zeocin) plate, culture at 28°C for 3-5 days until white colonies grow. ...

Embodiment 3

[0064] Example 3 Detection of antibacterial activity of recombinant chicken antibacterial peptide Cathelicidin2

[0065] The indicator strains used in the bacteriostasis test: Micrococcus luteus CMCC28001, Salmonella pullorum CVCC79201 (C79-1), Pasteurella multocida (CVCC474, C48-7) standard strains were purchased from the China Veterinary Drug Administration ; Pseudomonaspyocyanea and Escherichiacoli 1503 are clinically isolated and identified strains preserved in this laboratory. Specific steps are as follows:

[0066] 1. In vitro drug susceptibility test of standard strains such as Escherichia coli and Salmonella

[0067] According to the instructions of the drug susceptibility kit, 9 kinds of drug susceptibility test strips, including amoxicillin (10μg / tablet), amikacin (30μg / tablet), azithromycin (15μg / tablet), etc., are selected to protect against Escherichia coli, Salmonella, and more. 5 strains of Pasteurella sexta were tested for drug susceptibility as a control for the an...

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Abstract

The invention belongs to the biotechnology field, particularly relates to a pichia pastoris-expressed recombinant chicken antibacterial peptide Cathelicidin 2, and further discloses a preparation method and application thereof.According to the preparation method of the recombinant chicken antibacterial peptide Cathelicidin 2, pPICZ alpha A secreting type expression vector is selected as an eukaryotic expression system of exogenous genes, the recombinant chicken antibacterial peptide Cathelicidin 2 is effectively expressed, and direct destruction caused by an expression product on host cells is avoided.Meanwhile, the method is high expression efficiency, the expression product is easy to separate and purify, the production cost is low, enlarging is easy, stability is good, and the method is suitable for large-scale industrial production.Experiments prove that the antibacterial peptide Cathelicidin 2 has the obvious synergistic antibacterial effect when used in cooperation with antibiotics, and has the wide application and popularization prospect.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a recombinant chicken Cathelicidin2 antibacterial peptide expressed by Pichia pastoris, and further discloses its preparation method and application. Background technique [0002] Since the discovery of antibiotics, they have played a very important role in preventing bacterial infections and preventing and curing livestock and poultry diseases, and have played a huge role in promoting the health and rapid development of the animal husbandry and poultry industry. However, the long-term massive use of antibiotics has also brought a series of negative risks. The extensive use of antibiotics makes pathogenic bacteria gradually develop resistance, posing threats to human health and disease prevention and treatment. The resulting drug residues also make animal food safety issues increasingly prominent. The international community has clearly proposed reducing the use of antibiotic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/465C12N15/12C12N15/81C12N1/19A61K38/17A61P31/04C12R1/84
CPCC07K14/465A61K38/00C12N15/815C12N2800/102
Inventor 黄兵于可响马秀丽刘存霞吴静刘玉山胡峰李玉峰宋敏训
Owner POULTRY INST SHANDONG ACADEMY OF AGRI SCI
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