Preparation method for antimicrobial peptide-lysozyme fusion protein and application thereof

A technology of fusion protein and antibacterial peptide, applied in the fields of peptide/protein components, biochemical equipment and methods, antibacterial drugs, etc., can solve the problems of high construction cost of expression system, different sterilization mechanism, low protein expression, etc. Effects of respiratory disease incidence, survival rate improvement, and diarrhea rate reduction

Active Publication Date: 2015-11-18
杭州千岛湖生物谷高科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, antimicrobial peptides and lysozyme mainly exist alone to exert their antibacterial functions, but due to their different bactericidal mechanisms, they have certain limitations in the sterilization of different strains, resulting in their respective narrow antibacterial spectrums, which cannot be achieved. very good bactericidal effect
[0004] The Chinese invention patent announced as CN101649311B discloses a preparation method of human lysozyme-antimicrobial peptide fusion protein, which uses genetic engineering to construct human lysozyme-antimicrobial peptide recombinant plasmid, and then expresses human lysozyme-antimicrobial peptide in eukaryotic Fusion

Method used

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  • Preparation method for antimicrobial peptide-lysozyme fusion protein and application thereof
  • Preparation method for antimicrobial peptide-lysozyme fusion protein and application thereof
  • Preparation method for antimicrobial peptide-lysozyme fusion protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Tandem expression and protein purification of antimicrobial peptide Thanatin and T4 lysozyme

[0026] 1. Design and synthesis of fusion genes

[0027] Human thanatin (thanatian) and T4 lysozyme protein are connected in the form of thanatian-connecting peptide-T4 lysozyme, so the thanatin gene is in front and the T4 lysozyme gene is in the back.

[0028] The connecting peptide is a 3GSA fragment, which is inserted between the antimicrobial peptide and the lysozyme gene, as a flexible peptide, which reduces the structural impact of the two parts of the protein and increases the solubility of the fusion protein expression.

[0029] Adding sumo protein at the N-terminus of the fusion protein not only plays a melting role, but also helps to reduce the activity of the antibacterial protein on the toxicity of the host bacteria, so that it can be expressed in large quantities in the host bacteria.

[0030] The antimicrobial peptide thanatin gene sequence is shown in...

Embodiment 2

[0040] Embodiment 2: Antibacterial activity assay of antimicrobial peptide Thanatin-T4 lysozyme (tha-3GSA-T4) fusion protein

[0041] The antibacterial activity of the fusion protein was detected by the Oxford cup method:

[0042] Pour a layer of agar powder into the plate first, and after it solidifies, add 0.1% of the test bacteria in the logarithmic growth phase to 20m of LB solid medium at about 50°C, mix well, and spread the plate. Put an Oxford cup vertically on the plate, add 200 μL of sample into it, and put it in an oven at 37° C. for 12 hours. Thanatin and T4 lysozyme were used as controls alone.

[0043] The test bacteria include Gram-positive bacteria and Gram-negative bacteria, wherein Bacillus subtilis (ATCC6633) is selected as Gram-positive bacteria, and Escherichia coli (ATCC25922) is selected as Gram-negative bacteria.

[0044] The strength of the antibacterial activity was judged by the size of the inhibition zone.

[0045] The result is as image 3 , F...

Embodiment 3

[0046] Example 3: Study on the survival rate of piglets by tha-3GSA-T4 fusion protein

[0047] The breeds used in the experiment were triple hybrids of Duroc, Yorkshire and Landrace sows. During the experiment, all piglets were vaccinated according to the normal immunization program and managed in a conventional way.

[0048] Test group: tha-T4 fusion protein was added to the feed at a ratio of 3‰ (weight ratio, the same below). Piglets are added from the 7-day-old crevice until they are released from the nursery.

[0049] Control group: Piglets were added Thanatin, T4 lysozyme, Thanatin+lysozyme (thanatin and T4 lysozyme mixed in equimolar ratio), and conventional antibiotic amoxicillin in the feed at a ratio of 3‰.

[0050] The results are shown in Table 1.

[0051] Table 1 Statistics on the survival rate of piglets in lactation and nursery stages

[0052]

[0053] It can be seen from Table 1 that the tha-T4 fusion protein can significantly improve the survival rate of...

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Abstract

The present invention provides an antimicrobial peptide-lysozyme fusion protein. The protein comprises an antimicrobial peptide thanatin protein, 3GSA flexible peptide and T4 lysozyme protein from a terminal N to a terminal C. The preparation method comprises: obtaining a coding gene sequence comprising the antimicrobial peptide thanatin, 3GSA flexible peptide and T4 lysozyme by virtue of artificial synthesis; constructing a recombinant plasmid by taking pETDuet plasmid as a carrier; transforming the recombinant plasmid into BL21, and obtaining an expression strain by screening; putting the selected monoclone into a LB culture medium, and performing protein induction to obtain a fusion protein expressed in the supernatant; and purifying the fusion protein and cutting off an Sumo tag to obtain the objective antimicrobial peptide-lysozyme fusion protein. The protein provided by the present invention is proven to have remarkable effect in increasing the survival rate of piglets, reducing the diarrhea rate of nursery pigs, and decreasing the incidence of respiratory disease of nursery pigs, and is capable of being used for preparing related pharmaceutical preparations.

Description

technical field [0001] The invention relates to the technical field of biotechnology and pharmaceutical engineering, in particular to a preparation method of an antimicrobial peptide-lysozyme fusion protein, and the application of the fusion protein in improving the survival rate of piglets and reducing the incidence of diarrhea and respiratory diseases in nursery pigs. Background technique [0002] Since the advent of antibiotics, they have played a vital role in the treatment of human and animal diseases. Not only that, antibiotics have also been used more and more as human food additives and animal feed additives. It is precisely because antibiotics are widely used or even abused that the resistance of bacteria is getting stronger and stronger, and the bactericidal effect of antibiotics is getting worse and worse, and even some superbugs that cannot be killed by antibiotics appear. Therefore, the task of finding new antibacterial substances is imminent. [0003] Antimicr...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N9/36C12N15/62C12N15/70A61K38/47A61P31/04A61P1/12A61P11/00
Inventor 舒建洪王伟东吕正兵姜卫亮徐华强
Owner 杭州千岛湖生物谷高科技有限公司
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