Chicken Cathelicidins antibacterial peptide and preparation method and application for same

A technology of pet30-cathl2s and antimicrobial peptides, which is applied in the field of chicken Cathelicidins antimicrobial peptides and its preparation, can solve the problems of high cost of antimicrobial peptides, inability to meet needs, and small molecular weight

Inactive Publication Date: 2013-02-06
POULTRY INST SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 an

Method used

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  • Chicken Cathelicidins antibacterial peptide and preparation method and application for same
  • Chicken Cathelicidins antibacterial peptide and preparation method and application for same
  • Chicken Cathelicidins antibacterial peptide and preparation method and application for same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Cloning and sequencing analysis of the complete coding gene of chicken Cathelicidin1, 2 antimicrobial peptides, including:

[0055] 1. Primer Design

[0056] According to the cathelicidins gene sequence (DQ092350, DQ092351) 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 , 2 The cDNA sequence of the coding gene, the primers are respectively CathL1 P1 / P2 and CathL2 P1 / 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, CathL2S F / R, primer pairs, the underlined bases represent the EcoR I and Xho I restriction endonuclease sites introduced in the upstream and downstream primers, respectively.

[0058] Table 1 Prim...

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, 2 Mature Peptide Coding Sequence and Construction of Fusion Expression Vector

[0078] 1) Amplification of chicken Cathelicidins 1, 2 mature peptide coding sequence: According to the cloned chicken Cathelicidins 1, 2 antimicrobial peptide gene sequence and deduced amino acid sequence, design 2 pairs of specific primers CathL1S F / R, CathL2S 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-CathL2 containing the cDNA sequences of chicken Cathelicidins-1 and -2 genes as templates, the mature peptide coding sequences (~100bp) of chicken Cathelicidins 1 and 2 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, prim...

Embodiment 3

[0134] Optimization of Cathelicidins Fusion Protein Expression Conditions and Affinity Chromatography Purification of Expression Products

[0135] 1. Optimization of fusion protein expression conditions

[0136] In order to increase the proportion of soluble components in the total protein expression, reduce the induction temperature (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.

[0137] By using hypertonic medium (ie LB / SB medium), low temperature (25°C or 15~20°C) long-term induction, 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 that und...

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Abstract

The invention discloses a chicken Cathelicidins antibacterial peptide and a preparation method and application for the same. A nucleotide sequence of Cathelicidins 1 and a nucleotide sequence of Cathelicidins 2 of the chicken antibacterial peptide are shown in a sequence table. The preparation method for the antibacterial peptide is solid-phase chemical synthesis or a genetic engineering method. The genetic engineering method includes respectively cloning coding genes of mature peptides of the Cathelicidins 1 and coding genes of mature peptides of the Cathelicidins 2 of the chicken antibacterial peptide on expression vectors; and leading the expression vectors into host cells for expression. A genetic engineering technology is applied to efficiently express the chicken Cathelicidins antibacterial peptide in the host cells of a pronucleus, and as proved by experiments, the antibacterial peptide has an obvious suppression effect on various 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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IPC IPC(8): C12N15/12C07K14/465C12N15/63C12N15/70A61K38/17A61P31/04A61P31/10
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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