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A kind of recombinant lysozyme antibacterial peptide fusion protein and its preparation method and application

A technology of fusion protein and antimicrobial peptide, which is applied in the field of biogenetic engineering, can solve problems such as inability to directly express and apply, and achieve the effects of preventing significant decline in yield and activity, simple process, and broadening of biological resources

Active Publication Date: 2021-08-13
JINLING INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Antimicrobial peptides can make up for the shortcomings of lysozyme in terms of antibacterial range and stability in terms of performance, but they cannot be directly expressed and applied in microorganisms.

Method used

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  • A kind of recombinant lysozyme antibacterial peptide fusion protein and its preparation method and application
  • A kind of recombinant lysozyme antibacterial peptide fusion protein and its preparation method and application
  • A kind of recombinant lysozyme antibacterial peptide fusion protein and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Entrust a biotechnology company to artificially synthesize the nucleotide sequence SEQ ID NO.5 encoding the gene of SEQ ID NO.4, clone it into the Escherichia coli expression vector pET30a between the NdeI and HindIII restriction sites, and obtain the constructed recombinant large intestine Bacillus expression vector pET30-RA, the recombinant vector was used CaCl 2 The recombinant lysozyme antimicrobial peptide fusion protein gene engineering strain pET30-RA-BL21(DE3) was obtained by transforming into Escherichia coli host cell BL21(DE3). Pick the identified pET30-RA-BL21(DE3) glycerol-frozen strain and inoculate it into 5 mL LB containing 100 mg / L kanamycin culture solution, culture at 220 r / min, 37°C overnight. Take the bacterial solution and add it to 250mL shake flask fermentation medium containing 100mg / L kanamycin culture solution according to the volume ratio of 1 / 100, and cultivate it at 37°C until the logarithmic growth phase, when A 600 When it reached 0.7, I...

Embodiment 2

[0049] Pick the pET30-RA-BL21(DE3) glycerol frozen strain identified in Example 1 and inoculate it in 50mL LB containing 100mg / L kanamycin culture solution, culture at 220r / min, 37°C for 15 hours. The inoculum was transferred to a 5L automatic fermenter at a volume ratio of 1%. The culture temperature was 37°C, and the dissolved oxygen and pH were automatically controlled. The fermentation parameters were: 37°C, dissolved oxygen > 25%, and pH 7.5. Adjust the dissolved oxygen concentration by increasing the stirring speed and increasing the ventilation. Several hours after the start of fermentation, when the dissolved oxygen concentration increased rapidly, the glucose was fed at a rate of 0.05 g / (L·min) to a final concentration of 2 g / L. After the glucose was exhausted, lactose was fed to 10dL to induce expression for 6 hours. Wherein the fermentor culture medium contains glucose, a small amount of inorganic salt (glucose 5g / L, MgSO 4 ·7H 2 (2-YT substratum of 5g / L, pH7.0);...

Embodiment 3

[0053] Escherichia coli, Salmonella, Pseudomonas aeruginosa, Staphylococcus aureus and Pseudomonas aeruginosa were used as indicator bacteria, and the minimum inhibitory concentration (MIC) was determined by micro broth dilution method. Various indicator bacteria (CFU: 1X 10 6 ) were inoculated in LB medium, and then 10 μL of different concentrations of antibacterial proteins were added, (final concentrations were 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.125, 7.8125, 3.9 μg / mL) and incubated at 37°C for 12 hours, The absorbance of each tube was measured at 492 nm. The lowest concentration of antimicrobial peptide without significant change in absorbance is MIC. Apply each tube greater than the MIC on the LB solid medium plate, incubate at 37°C for 12 hours, and observe that the lowest concentration without bacterial growth is MBC. At the same time, two antibiotics, penicillin sodium and amoxicillin, were used as positive controls to compare the antibacterial peptide fusion...

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Abstract

The invention provides a recombinant lysozyme antibacterial peptide fusion protein and its preparation method and application, belonging to the technical field of biogenetic engineering. Compared with the prior art, the present invention has the following advantages: (1) the lysozyme antimicrobial peptide fusion protein prepared by the present invention has high antibacterial activity, and is effective against Gram-positive bacteria Staphylococcus aureus, Staphylococcus epidermidis and Gram-negative bacteria large intestine Escherichia, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella typhimurium all have obvious inhibitory effects, the antibacterial activity is significantly improved, the antibacterial spectrum is broadened, and there is no cell hemolysis and animal toxicity. (2) The purification and preparation process adopted in the present invention is simple because the chromatographic renaturation technology and the connecting peptide of the present invention can effectively prevent the yield and activity of refolded purified protein from significantly decreasing.

Description

technical field [0001] The invention belongs to the technical field of biological genetic engineering, and in particular relates to a recombinant lysozyme antibacterial peptide fusion protein and its preparation method and application. Background technique [0002] The abuse of antibiotics has led to the emergence of a large number of drug-resistant bacteria, which poses a direct threat to the human body and has become a worldwide problem. There is also a phenomenon of antibiotic abuse in livestock and aquaculture. The amount of antibiotics used in animal husbandry has far exceeded the sum of the amount used by humans. Antibiotic residues in food and animals and the resulting drug-resistant bacteria are transmitted to humans through food or contact between animals and humans, endangering human health. Long-term abuse of food preservatives will also lead to the emergence of drug-resistant strains, many of which have a strong risk of carcinogenicity, and cause harm to bone gr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C07K14/00C07K1/36C07K1/30C07K1/16C12N9/36C12N15/62C12N15/70A61P31/04
CPCA61P31/04C07K14/00C12N9/2462C12N15/70C12N2800/22C12Y302/01017
Inventor 蒋加进陈俊红陈涛方光远戴鼎震陆雨楠黄翔宇
Owner JINLING INST OF TECH
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