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Children antimicrobial peptides Cathelicidins and preparation method and applications thereof

A pet30-cathl2s and pet30-cathl3s technology is applied in the field of chicken cathelicidins antibacterial peptide and its preparation, and can solve the problems of small molecular weight, inability to meet needs, difficulty in separation and purification, and the like

Inactive Publication Date: 2011-07-20
POULTRY INST SHANDONG ACADEMY OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, the content of natural antimicrobial peptides in poultry is extremely low, the molecular weight is small, and separation and purification are difficult, which cannot meet the needs
Chemical synthesis and genetic engineering methods have become the main means of obtaining antimicrobial peptides, but the cost of chemical synthesis of antimicrobial peptides is too high, and it is difficult to apply them clinically; way

Method used

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  • Children antimicrobial peptides Cathelicidins and preparation method and applications thereof
  • Children antimicrobial peptides Cathelicidins and preparation method and applications thereof
  • Children antimicrobial peptides Cathelicidins and preparation method and applications thereof

Examples

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Embodiment 1

[0054] Cloning and sequencing analysis of the complete coding gene of chicken cathelicidin1, 2, 3 antimicrobial peptides, including:

[0055] 1. Primer Design

[0056] According to the cathelicidins antimicrobial peptide gene sequence (DQ092350, DQ092351, DQ092352, DQ092353) published in GenBank and the design principles of PCR primers, and with the help of computer software DNASTAR for auxiliary analysis, three pairs of PCR gene-specific primers were designed for Amplify the cDNA sequences of chicken Cathelicidin1, 2, and 3 coding genes, and the primers are respectively CathL1 P1 / P2, CathL2 P1 / P2, and CathL3 P1 / P2 in the table, wherein P1 is a forward primer and P2 is a reverse primer. Primers were synthesized by Shanghai Boshang Company.

[0057]The primer sequences used in this example and Example 2 are shown in Table 1. In the CathL1S F / R, CathL2S F / R, and CathL3S F / R primer pairs, the underlined bases represent the EcoR I and Xho I restriction endonuclease sites introdu...

Embodiment 2

[0077] The genetic engineering preparation method of recombinant chicken Cathelicidins antimicrobial peptide fusion protein comprises the following steps:

[0078] 1. Preparation of chicken Cathelicidins 1, 2, 3 mature peptide coding sequence and construction of its fusion expression vector (1) Amplification of chicken Cathelicidins 1, 2, 3 mature peptide coding sequence: According to the cloned chicken Cathelicidins 1, 2, 3 antimicrobial peptide gene sequence and deduced amino acid sequence, design 3 pairs of specific primers CathL1S F / R, CathL2S F / R, CathL3S F / R with restriction sites, downstream primers with stop codon TGA, primer sequence See Table 1. Using the positive plasmids pMD-CathL1, pMD-CathL2, and pMD-CathL3 containing chicken Cathelicidins-1, -2, and -3 gene cDNA sequences as templates, the chicken Cathelicidins 1, 2, and 3 mature peptide coding sequences were amplified by PCR ( ~100bp).

[0079] The reaction system is: 10×PCR Buffer (containing Mg 2+ ) 5.0 μL...

Embodiment 3

[0135] Optimization of Cathelicidins Fusion Protein Expression Conditions and Affinity Chromatography Purification of Expression Products

[0136] 1. Optimization of fusion protein expression conditions

[0137] In order to increase the proportion of soluble components in the total protein expression, by reducing the induction temperature (down to 25°C or 15-20°C), prolonging the induction time (3h-8h), adjusting the concentration of the inducer (the concentration of IPTG is 0.4mmol / L, 0.8mmol / L, 1.0mmol / L, 1.2mmol / L) and other methods to inhibit the formation of inclusion bodies, thereby simplifying the purification operation.

[0138] By using hyperosmotic medium (i.e. LB / SB medium), long-term induction at low temperature (25°C or 15-20°C), and adjusting the concentration of the inducer IPTG, etc., the formation of inclusion bodies can be inhibited and the soluble protein in the expression product can be increased. The purpose of the proportion. Experiments have shown tha...

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Abstract

The invention discloses children antimicrobial peptides cathelicidins, a preparation method and the applications thereof. The nucleotide sequence and the amino acid sequence of children antimicrobial peptides Cathelicidin1, Cathelicidin2 and Cathelicidin3 are shown as a sequence table. The preparation method of the antimicrobial peptides adopts a solid-phase chemical synthesis method or a geneticengineering method. The genetic engineering method comprises the following steps of: respectively cloning the encoding genes of mature children antimicrobial peptides Cathelicidin1-3 to expression vectors; and then, guiding into host cells to express. By applying the genetic engineering technology in the invention, the children antimicrobial peptides Cathelicidins are expressed in the prokaryotichost cells with high efficiency. Proved by experiments, the antimicrobial peptides have obvious inhibition on various gram-positive bacteria and negative bacteria.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a group of chicken Cathelicidins antimicrobial peptides and its preparation method and application. Background technique [0002] In recent years, a variety of infectious diseases have plagued my country's breeding industry. Since the discovery of antibiotics, they have played an important role in preventing bacterial infections and preventing and controlling livestock and poultry diseases, and have played a huge role in promoting the rapid development of animal husbandry production. However, The long-term and extensive use of antibiotics has caused pathogenic bacteria to gradually develop drug resistance, which has increased the difficulty of disease prevention and control. At the same time, the drug residues caused by antibiotics have made the safety of animal foods increasingly prominent, and drug-resistant plasmids are easily cross-spread to humans, posing a threat to human health...

Claims

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Application Information

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IPC IPC(8): C12N15/12C07K14/465C12N15/09A61K38/17A61P31/04A61P31/10
Inventor 吴静宋敏训李玉峰马秀丽姜亦飞黄兵林树乾于可响
Owner POULTRY INST SHANDONG ACADEMY OF AGRI SCI
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