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A method for improving the antibacterial performance of porcine lysozyme by fusing the hlh functional domain at the N-terminus

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: 2019-12-06
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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  • A method for improving the antibacterial performance of porcine lysozyme by fusing the hlh functional domain at the N-terminus
  • A method for improving the antibacterial performance of porcine lysozyme by fusing the hlh functional domain at the N-terminus
  • A method for improving the antibacterial performance of porcine lysozyme by fusing the hlh functional domain at the N-terminus

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Experimental program
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Effect test

Embodiment 1

[0031] Embodiment 1: the production of fusion porcine lysozyme

[0032] 1. The coding gene of the functional domain HLH (amino acid sequence shown in SEQ ID NO.2) simulated by Swiss-Model software is fused with the N-terminal of the porcine lysozyme coding gene, and no cleavage site is added between the two genes , the amino acid sequence and nucleotide sequence of the fused gene are respectively shown in SEQ ID NO.4 and SEQ ID NO.5.

[0033] 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.

[0034] 3. The inclusion bodies obtained above were refolded by the dilution method, and the solution u...

Embodiment 2

[0036] Example 2: Detection of antibacterial properties of fusion lysozyme

[0037] The antibacterial activity of the fusion porcine 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 600 0.6, take 0.2mL bacterial liquid and mix with 0.4mL TSB medium, add 0.2mL PBS (0.05mol / L, pH 7.0) buffer solution containing fusion lysozyme (porcine lysozyme SSL and antimicrobial peptide SP as control) and mix well , so that the final concentration of fusion lysozyme (or control) is 8.3 × 10 -8mol / 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. 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 o...

Embodiment 3

[0041] Embodiment 3: the application of fusion lysozyme

[0042] The fusion 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 instead of antibiotics.

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Abstract

The invention discloses a method for improving pig lysozyme by fusing an N-terminal with an HLH functional domain, and belongs to the field of bioengineering. The method comprises the steps of design of a fused pig lysozyme gene and expression and renaturation of an escherichia coli expression system. By means of detection of antibacterial activity, the fused pig lysozyme has obvious resistance to gram-positive bacteria and can kill various gram-positive bacteria simultaneously, and an excellent case is provided for improving the antibacterial activity of the lysozyme. The fused pig lysozyme preparation technology has the advantages of being simple and efficient, the product can reach electrophoretic purity, and the purity is 90% or above.

Description

technical field [0001] The invention relates to a method for improving the antibacterial performance of porcine lysozyme by fusing HLH functional domains at the N terminal, and 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-...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/36C12N1/21C12N15/70A61P31/04A23K20/189
CPCC07K2319/00C12N9/2462C12Y302/01017
Inventor 蔡国林陆健朱德伟
Owner JIANGNAN UNIV
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