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Antibacterial peptide, preparation method and applications thereof

An antibacterial peptide and amino acid technology, applied in the fields of biotechnology and genetic engineering, can solve problems such as unsatisfactory antibacterial activity, and achieve the effect of enhancing bactericidal activity in vitro and in vivo and easy to prepare

Inactive Publication Date: 2013-10-02
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to develop a new type of safe antibacterial agent that can replace antibiotics, we have carried out a series of studies on the biologically active substance derived from mammalian milk - porcine lactoferrin; lactoferrin is derived from milk and has no effect on animals. Toxicity, relatively safe, but its antibacterial activity is not ideal

Method used

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  • Antibacterial peptide, preparation method and applications thereof
  • Antibacterial peptide, preparation method and applications thereof
  • Antibacterial peptide, preparation method and applications thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Example 1: Preparation and separation and purification of antimicrobial peptides

[0035] According to the above sequence, the antimicrobial peptide LFP-20 and the modified antimicrobial peptides LF-2 and LF-6 were synthesized by solid-phase chemical method using the Pioneer peptide synthesizer produced by Applied Biosystems. The solid-phase chemical method is automatically controlled by a peptide synthesizer, and the protected amino acids are coupled and deprotected one by one from the C-terminal to the N-terminal on the Fmoc-Arg (Pbf)-Wang Resin resin column in a pre-designed sequence. The synthesized peptide was cleaved by high-concentration TFA, then purified by reverse column, and the purified peptide was identified by mass spectrometry.

[0036] 1. Preparation of antimicrobial peptides (taking the preparation of 1mg LFP-20 as an example)

[0037]All the following reagents for the preparation of antimicrobial peptides were purchased from Applied Biosystems. The p...

Embodiment 2

[0050] Embodiment two: Bactericidal activity detection

[0051] Various bacterial strains used in the following examples were purchased from China Institute for the Control of Biological Products. The bactericidal activity of the antimicrobial peptides was detected by the improved micro broth dilution method to evaluate the bactericidal activities of the two modified antimicrobial peptides in the present invention.

[0052] Determination of antimicrobial peptide bactericidal activity was carried out according to the following steps: thaw the strain frozen at -80°C in ice, streak it on the MH agar plate with an inoculation loop, and culture it at 37°C to form a single colony; the single colony was inoculated on fresh MH In the broth medium, 37°C constant temperature shaking overnight culture activation; take the above overnight culture suspension and transfer it to fresh MH broth medium at a ratio of 1:100, and culture at 37°C 250rpm constant temperature shaking until OD 600nm...

Embodiment 3

[0055] Embodiment three: Sterilization speed detection

[0056] Set the concentration of the detected peptide according to the needs of the experiment, and generally use integer multiples of the MIC for detection. This experiment intends to measure the bactericidal curve of LFP-20 and its modified antimicrobial peptides against bacteria under the MIC concentration; the sampling time points are set at 0, 5, 10, 20, 30, 40, 50, 60, 90 , 120, 150, 180 and 240min. Add 1mL of the prepared bacterial suspension into the centrifuge tube, and at the same time, add the corresponding concentration of polypeptide to each tube, and set a blank control without polypeptide, and place it at 37°C for shaking culture; at the corresponding time point, take the bacterial suspension for appropriate dilution After overnight culture on MH agar plate medium, take 3 repeated total colony counts at a dilution concentration of 30-200 for colony counting, and calculate the bacterial concentration of the...

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Abstract

The present invention discloses an antibacterial peptide, a preparation method and applications thereof. The antibacterial peptide is a porcine lactoferrin peptide LFP-20 or a reconstruction antibacterial peptide LF-2 or a reconstruction antibacterial peptide LF-6. The antibacterial peptide preparation method comprises adopting a solid phase chemical synthesis method or a genetic engineering expression technology to clone encoding gene of the antibacterial peptide to a vector, and then expressing in host cells to obtain the antibacterial peptide, wherein engineering bacterial used in the genetic engineering expression technology is genetic engineering bacteria P.pastorisSMD1168, and an expression vector is an eukaryotic expression vector PICZalphaA. The invention further relates to an application of the antibacterial peptide in preparation of drugs for treatments of gram-positive bacteria or gram-negative bacteria infectious diseases. With the reconstruction antibacterial peptide, in vitro bacterial killing activity and bacterial killing speed of the LFP-20 can be increased, and permeability of the LFP-20 on inner membranes and outer membranes of bacterial can be enhanced so as to enhance bacterial killing capability of the LFP-20. The reconstruction antibacterial peptide LF-2 and the reconstruction antibacterial peptide LF-6 further have strong in vivo bacteria removing capability. The present invention further relates to a method for expressing the recombinant reconstruction antibacterial peptide LF-6 by using pichia pastoris yeast. According to the present invention, the process is simple, and the product has significant bacterial inhibition activity.

Description

technical field [0001] The invention relates to the technical fields of biotechnology and genetic engineering, in particular to a group of reconstructed antibacterial peptides and its preparation method and application. Specifically, it involves a molecular reconstruction of porcine lactoferrin peptide LFP-20 by means of amino acid replacement, and optimizes the acquisition of two modified antibacterial peptides LF-2 and LF-6 with better antibacterial activity. Yeast obtained recombinantly modified antimicrobial peptide LF-6. Background technique [0002] The discovery and application of traditional antibiotics has provided a very effective way to treat infectious diseases and also significantly increased the number of animal food products. Undoubtedly, antibiotics represent the most important scientific and medical discoveries of the 20th century (Gordon et al., 2005; McPhee et al., 2005). Although antibiotic treatment is still the first choice to deal with human and anim...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C12N15/81A61K38/10A61P31/04C12R1/84
Inventor 汪以真韩菲菲谢永刚张海文夏溪
Owner ZHEJIANG UNIV
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