Lysozyme derived from phases and gene and application of lysozyme

A technology of lysozyme and gene, which is applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of narrow action temperature range, narrow action pH range, and few species, and achieve wide action temperature range, excellent genetic resources and enzymes resources, the effect of good thermal stability

Active Publication Date: 2019-11-15
THIRD INST OF OCEANOGRAPHY MINIST OF NATURAL RESOURCES +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, there are few types of phage lysozymes in existence, and most of them have shortcomings such as narrow action temperature range and narrow action pH range. Personnel in the field have been committed to finding new phage lysozymes with excellent biological activity.

Method used

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  • Lysozyme derived from phases and gene and application of lysozyme
  • Lysozyme derived from phases and gene and application of lysozyme
  • Lysozyme derived from phases and gene and application of lysozyme

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] The acquisition of embodiment 1 lysK1 gene

[0027] The phage used in this example was isolated from seawater at a depth of 2800 m in the western Pacific Ocean, and the phage was long-tailed phage WP3 (phage WP3). Primer F (5'-ATCGGATCCATGAGCCCGACTGTAAGCGCCA-3') and primer R (5'-GGACTCGAGTCAGATCTTCGGTCCCTCCAGCCT-3') were used to PCR amplify the lysK1 gene fragment of the deep-sea phage, and BamH1 and Xho1 restriction sites were introduced into its upstream and downstream, respectively. The full-length CDS (Coding sequence, coding sequence) of the lysK1 gene is 1221 bp, and its nucleotide sequence is shown in SEQ ID NO: 2, encoding 406 amino acids.

Embodiment 2

[0028] Cloning expression of embodiment 2lysK1 gene

[0029] The lysK1 gene fragment obtained in Example 1 was connected between the BamH1 and Xho1 restriction sites of the pET28a vector to obtain the pET28a-lysK1 recombinant vector; the obtained pET28a-lysK1 recombinant vector was transformed into Escherichia coli E.coliBL21 (DE3) competent State cells, and smeared on LB solid culture plate containing 10mg / ml kanamycin and 10% agar, cultured at 37°C for 24 hours; In LB liquid medium, culture overnight at 37°C with shaking at 200rpm; inoculate the positive clones cultured overnight at a dilution ratio of 1:100 into 500ml of LB liquid medium (containing 10mg / ml kanamycin), at 37°C , shake culture at 200rpm to OD 600 When it reaches 0.6, add isopropylthio-β-D-galactoside (IPTG) at a final concentration of 1 mM, and induce the expression of LysK1 lysozyme at 18° C. for 12 hours.

Embodiment 3

[0030] The separation and purification of embodiment 3LysK1 protein

[0031] 15000rpm centrifugation collects the thalli that embodiment 2 induces to express, after washing three times with PBS, with lysis buffer (0.3mol / L NaCl, 10mmol / L imidazole, 50mmol / L NaH 2 PO 4 , pH 8.0) resuspended cells. Use the sonication method (power 80%, 5s / 5s pulse cycle) to break and lyse the bacteria on ice, centrifuge at 15000rpm at a high speed, and collect the supernatant; rotate and mix the collected supernatant with Ni-NTA Agarose for 30min According to the instructions of the Ni-NTA Agarose kit (QIAGEN), the lysK1 lysozyme was purified to obtain the LysK1 protein, whose amino acid sequence is shown in SEQ ID NO: 1, and analyzed by SDS-PAGE electrophoresis. figure 1 , Lanes 1 and 2 are the purified recombinant LysK1 protein, and its size is consistent with the predicted molecular weight (48.5kD).

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Abstract

The invention relates to a lysozyme derived from phases and a gene and application of the lysozyme. The amino acid sequence of the lysozyme is shown in SEQ ID NO:1. The gene encoding the lysozyme is derived from the phages WP3 collected from deep sea. The lysozyme is subjected to heterologous expression and purification. As is detected, the optimum temperature of the lysozyme is 40 DEG C, more than 50% of vitality can be maintained within the temperature range of 20-60 DEG C, and the lysozyme has a wide acting temperature range; the optimum pH value is 6.0, more than 50% of vitality can be maintained within the pH range of 4.0-10.0, and the lysozyme has a wide acting pH range. The lysozyme has a broad-spectrum bactericidal activity, and can efficiently kill various Gram-positive bacteria and Gram-negative bacteria, excellent gene resources and enzyme resources are provided for developing lysozyme-type antibacterial products, and the lysozyme has a wide application prospect.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a lysozyme derived from bacteriophage and its gene and application. Background technique [0002] In recent years, due to the abuse of antibiotics, there are more and more antibiotic-resistant bacteria in the environment and in the human body. Traditional antibiotics cannot kill antibiotic-resistant bacteria, so it is urgent to find new antibacterial drugs that can replace antibiotics. At present, due to the specific and efficient characteristics of killing host bacteria, bacteriophages have become one of the hotspots in the development of new biocidal agents. [0003] A phage is a virus that can infect microorganisms such as bacteria. Like other viruses, a phage is an obligate parasite that must rely on host cells for its own survival and reproduction. When a phage infects a host bacterium, the phage will inject genetic material such as DNA into the host bacterium, and use the hos...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/36C12N15/56C12N15/70C12N1/21A61K38/47A61P31/04
CPCA61K38/47A61P31/04C12N9/2462C12N15/70C12Y302/01017
Inventor 金敏郭讯易志伟产竹华陈兴麟曾润颖
Owner THIRD INST OF OCEANOGRAPHY MINIST OF NATURAL RESOURCES
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