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Preparation method of Cathelicidins antimicrobial peptides of chicken

A technology of antimicrobial peptides and acquisition methods, applied in the biological field, can solve the problems of small molecular weight, high cost of antimicrobial peptides, and inability to meet needs

Inactive Publication Date: 2013-12-18
POULTRY INST SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] 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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  • Preparation method of Cathelicidins antimicrobial peptides of chicken
  • Preparation method of Cathelicidins antimicrobial peptides of chicken
  • Preparation method of Cathelicidins antimicrobial peptides of chicken

Examples

Experimental program
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Effect test

Embodiment 1

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

[0055] 1. Primer Design

[0056] According to the cathelicidins gene sequence (DQ092350, 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 to amplify chicken cathelicidin1 respectively. , 3 The cDNA sequence of the coding gene, the primers are respectively CathL1P1 / P2 and CathL3P1 / P2 in the table, wherein P1 is the forward primer, and P2 is the 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, CathL3S F / R primer pair, the underlined bases represent the EcoR I and Xho I restriction endonuclease sites introduced in the upstream and downstream primers, respectively.

[0058] T...

Embodiment 2

[0076] The genetic engineering preparation method of recombinant chicken Cathelicidins antimicrobial peptide fusion protein comprises the following steps:

[0077] 1. Preparation of chicken Cathelicidins 1, 3 mature peptide coding sequence and construction of its fusion expression vector

[0078] (1) Amplification of chicken Cathelicidins 1, 3 mature peptide coding sequence: According to the cloned chicken Cathelicidins 1, 3 antimicrobial peptide gene sequence and deduced amino acid sequence, design 3 pairs of specific primers CathL1S F / R, CathL3S F / R, the downstream primer has a stop codon TGA, and the primer sequence is shown in Table 1. Using the positive plasmids pMD-CathL1 and pMD-CathL3 containing the cDNA sequences of chicken Cathelicidins-1 and -3 genes as templates, the chicken Cathelicidins 1 and 3 mature peptide coding sequences (~100bp) were amplified by PCR.

[0079] The reaction system is: 10×PCR Buffer (containing Mg 2+ ) 5.0 μL, dNTP (2.5 mmol / L) 4.0 μL, pri...

Embodiment 3

[0133] Optimization of Cathelicidins Fusion Protein Expression Conditions and Affinity Chromatography Purification of Expression Products

[0134] 1. Optimization of fusion protein expression conditions

[0135] In order to increase the proportion of soluble components in the expressed total protein, reduce the induction temperature (down to 25°C or 15-20°C), prolong the induction time (3h-8h), and adjust the concentration of the inducer (the concentration of IPTG is 0.4mmol respectively) / 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.

[0136] Inhibit the formation of inclusion bodies and increase the soluble protein in the expression product by using hyperosmotic medium (i.e. LB / SB medium), low temperature (25°C or 15-20°C) long-term induction, and adjusting the concentration of the inducer IPTG. The purpose of the proportion. Experiments have shown that under the induction...

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Abstract

The invention discloses a group of Cathelicidins antimicrobial peptides of chicken, a preparation method and an application thereof. The nucleotide sequences and amino acid sequences of Cathelicidin 1 and Cathelicidin 3 antimicrobial peptides of the chicken are presented in a sequence table. The preparation method of the antimicrobial peptides is a solid phase chemical synthesis method or a gene engineering method, wherein the gene engineering method includes that coding genes of mature peptides of the Cathelicidin 1 and 3 antimicrobial peptides of the chicken are cloned on expression vectors respectively and then led to host cells for expression. According to the group of antimicrobial peptides, the preparation method and the application thereof, the Cathelicidins antimicrobial peptides of the chicken are efficiently expressed in pronucleus host cells by using the gene engineering technology, and experiments prove that the group of antimicrobial peptides has obvious inhibiting effects on a plurality of gram-positive bacteria and gram-negative bacteria.

Description

[0001] This application is a divisional application with the original application number 201010600240.7, the filing date is December 22, 2010, and the invention title is "a group of chicken Cathelicidins antimicrobial peptides and their preparation method and application". technical field [0002] 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 [0003] 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 ha...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C07K14/465C12N15/63C12N15/70A61K38/17A61P31/04A61P31/10
Inventor 于可响吴静宋敏训李玉峰马秀丽姜亦飞黄兵林树乾
Owner POULTRY INST SHANDONG ACADEMY OF AGRI SCI
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