Method for improving antibacterial performance of porcine lysozyme through N-terminal fusion with poly-hexapeptide

A lysozyme, N-terminal technology, applied in the field of bioengineering, can solve the problems of limited application and no bacteriostatic effect, and achieve the effect of broadening the antibacterial spectrum and high product purity

Active Publication Date: 2016-10-26
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, similar to other C-type lysozymes, the antibacterial spectrum of porcine lysozyme is mainly concentrated on Gram-positive bacteria, and has basically no antibacterial effect on Gram-negative bacteria, which also limits its application.

Method used

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  • Method for improving antibacterial performance of porcine lysozyme through N-terminal fusion with poly-hexapeptide
  • Method for improving antibacterial performance of porcine lysozyme through N-terminal fusion with poly-hexapeptide
  • Method for improving antibacterial performance of porcine lysozyme through N-terminal fusion with poly-hexapeptide

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Experimental program
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Effect test

Embodiment 1

[0033] Embodiment 1: the production of fusion lysozyme

[0034] 1. The six-molecule hexapeptide (A-W-V-A-W-K, SEQ ID NO.1) coding gene isolated from porcine lysozyme is sequentially connected and then fused with the N-terminal of the porcine lysozyme coding gene, without adding The cleavage site, the fused gene sequence is shown in SEQ ID NO.4, named 6SP-SSL.

[0035] 2. Ligate the fused gene sequence with the expression vector pET-28a(+), use restriction enzymes BamH Ⅰ and Hind Ⅲ for double digestion, transfer it into the host E.coli BL21(DE3) for expression, and store at 30°C, Under the condition of 200r / min, 0.1mmol / L IPTG was used to induce for 8h, and the fermentation broth was centrifuged to obtain bacterial cells. It was ultrasonically crushed to obtain recombinant protein inclusion bodies.

[0036] 3. The inclusion bodies obtained above were refolded by the dilution method, and the solution used was 50mM Tris-HCl, 0.15M NaCl, 5mM EDTA, 1M Urea, 0.5M L-Arg, 2mM GSH, 0...

Embodiment 2

[0039] Example 2: Detection of antibacterial properties of fusion lysozyme

[0040] The antibacterial activity of the fusion lysozyme was determined according to the method in the literature. After secondary activation, the test bacteria were inoculated into a Erlenmeyer flask containing 30mL TSB medium with 1% inoculum and cultivated to OD 6000.6, take 0.2mL of bacterial liquid and mix with 0.4mL of TSB medium, add 0.2mL of PBS (0.05mol / L, pH 7.0) buffer solution containing fusion lysozyme (porcine lysozyme SSL and hexapeptide SP as a control) and mix well, Make the final concentration of fusion porcine lysozyme (or control) be 8.3×10 -8 mol / L. After the mixed system was cultured at 37°C and 200r / min for 2 hours, it was diluted and spread on a TSB plate, and counted after the colonies grew out. Calculation of the inhibition coefficient log N 0 / N 1 , where N 0 Refers to the number of colonies in the blank group, that is, only PBS solution is added; N 1 is the number of...

Embodiment 3

[0044] Embodiment 3: the application of fusion lysozyme

[0045] The fusion porcine lysozyme of the invention can not only resist various Gram-positive bacteria, but also kill various Gram-negative bacteria, including various pathogenic bacteria, and can be used as a feed additive to replace antibiotics.

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Abstract

The invention discloses a method for improving antibacterial performance of porcine lysozyme through N-terminal fusion with poly-hexapeptide and belongs to the field of bioengineering. The method comprises steps as follows: design of a fusion porcine lysozyme gene, expression by an escherichia coli expression system as well as renaturation. Antimicrobial activity detection proves that the fusion porcine lysozyme not only has obvious resistance to Gram-positive bacteria, but also can kill various Gram-negative bacteria, and a good case is provided for improving the antibacterial activity of lysozyme. The preparation technology for the porcine lysozyme has the characteristics of being simple and efficient, a product can be electrophoretically pure, and the purity is 90% or higher.

Description

technical field [0001] The invention relates to a method for improving the antibacterial performance of porcine lysozyme by fusing N-terminal polyhexapeptide, which belongs to the field of bioengineering. Background technique [0002] For decades, antibiotics have played a positive role in preventing and treating animal diseases, promoting animal growth, and increasing the output of livestock and poultry products by inhibiting the proliferation of pathogenic microorganisms and reducing the toxic effects of their metabolic toxins on animals. However, the long-term use of antibiotics, especially the use of anti-disease drugs and growth-promoting additives in the breeding process, will cause excessive drug residues, environmental pollution in the production area, and lead to a decline in the quality of muscle and processed products. In addition, the long-term use of antibiotics as feed additives in poultry and livestock can cause some bacterial flora to become drug-resistant st...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N9/36C07K7/06A61K38/47A61P31/04
CPCA61K38/47C07K7/06C12N9/2462C12Y302/01017
Inventor 陆健蔡国林朱德伟
Owner JIANGNAN UNIV
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