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Production and application of fused antibacterial protein

A technology of fusion protein and recombinant bacteria, which can be used in antibacterial drugs, fusion peptides, peptide/protein components, etc., and can solve problems such as unstable lysis

Active Publication Date: 2020-06-05
中科纳迪(苏州)科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chinese patent CN201510844661.7 discloses a broad-spectrum phage chimeric lyase of Staphylococcus, which is used to prevent and treat a variety of Staphylococcus, and has a significant preventive effect on dairy cow mastitis infection caused by Staphylococcus, but the lyase Unstable lysis against different host bacteria, especially Staphylococcus aureus

Method used

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  • Production and application of fused antibacterial protein
  • Production and application of fused antibacterial protein
  • Production and application of fused antibacterial protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Synthesis of anti-staphylococcus fusion protein gene and construction of prokaryotic expression vector

[0046] a. Download the full-length Staphylococcus aureus phage K genome (AY176327) from GenBank, intercept the nucleotide sequences at 27072-27762 and 28638-29435 bp, and obtain the full-length LysK gene. The amino acid sequence of the antimicrobial peptide Mersacidin was obtained from the APD database (The Antimicrobial Peptide Database, http: / / aps.unmc.edu / AP / ). The LysK gene is connected in series with the nucleotide sequence encoding the amino acid sequence of the antimicrobial peptide Mersacidin, and the nucleotide sequence is optimized using the codon usage preference of Escherichia coli, and it is named LysK-Mer, and its nucleotide sequence is as shown in SEQ ID NO: 1.

[0047] b. Add BamH I and Xho I restriction site sequences to both ends of the optimized anti-staphylococcal fusion protein gene and send it to Suzhou Jinweizhi Biotechnology Co., Lt...

Embodiment 2

[0049] Example 2 Induced expression and purification of anti-staphylococcal fusion protein

[0050] a. The recombinant plasmid pET32a-LysK-Mer obtained in Example 1 was transformed into Escherichia coli BL21 (DE3) competent according to conventional molecular biology methods, spread on a plate containing 50 μg / mL ampicillin and cultured overnight at 37°C, positive clones After amplification and shaking, the recombinant bacteria were obtained and named as BL21(pET32a-LysK-Mer).

[0051] b. Transfer BL21 (pET32a-LysK-Mer) into 100mL LB medium, culture at 37°C, wait until OD 600 When it reached 0.5, IPTG was added at final concentrations of 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mmol / L, respectively, and protein expression was induced at 4°C, 16°C, 25°C, and 37°C, respectively.

[0052] c. After the induction, the bacteria were collected, the cells were disrupted by ultrasonic waves, the supernatant was collected after centrifugation (12000 rpm), and the protein expression in the supern...

Embodiment 3

[0055] The plate antibacterial test of embodiment 3 anti-staphylococcal fusion proteins

[0056] Staphylococcus aureus (CICC 10001, CICC 10145, CICC 10201), Staphylococcus epidermidis (CICC10294), Staphylococcus pseudointermediate (CICC 10499), Escherichia coli (CICC 10389), Bacillus subtilis (CICC10275), fecal Enterococcus (CICC 23658), Salmonella enteritidis (CICC 21482) and Clostridium perfringens (CICC22949) were recovered, and 100 μL of fresh bacterial solution (bacteria content 109 CFU / mL) was evenly spread on LB solid medium. Take 100 μL of the anti-staphylococcal fusion protein obtained in Example 2 (adjust the concentration to 1 mg / mL), drop it in the center of the LB solid medium and spread it evenly, and add dropwise an equal amount of PBS buffer to dissolve the anti-staphylococcal fusion protein After cultured at 37°C for 12 hours, the lysing effect of the anti-staphylococcal fusion protein on different host bacteria was observed, and the results are shown in Table...

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Abstract

The invention provides production and application of staphylococcus bacteriophage lysins LysK and antibacterial peptide Mersacidin fused protein. A nucleotide sequence which expresses the fused protein is shown in SEQ ID NO:1 as shown in the description, and an amino acid sequence of the fused protein is shown in SEQ ID NO:2 as shown in the description. According to production and application of the staphylococcus bacteriophage lysins LysK and antibacterial peptide Mersacidin fused protein, the fused protein is subjected to heterologous expression and purification, it is found through detection that the fused protein is stable, operative temperatures and pH values are wide in range, and the fused protein has specific sterilization effects on multiple kinds of staphylococcocci, including staphylococcus aureus. According to the provided antistaphylococcic fused protein, a safe protein preparation source is provided for control of staphylococcocci in food, especially dairy products, so that the provided antistaphylococcic fused protein has wide application prospects.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a preparation method and application of a staphylococcal phage lyase LysK and an antimicrobial peptide Mersacidin fusion protein. Background technique [0002] Staphylococcus is widely distributed in the air, ground, water and livestock and poultry feed. It is a Gram-positive bacterium with a round or oval shape and a diameter of about 0.8 μm. It has no flagella, no capsule, and cannot produce spores. A small number of staphylococci can cause zoonotic diseases, such as Staphylococcus aureus, which has always been the second most pathogenic bacteria in hospital-acquired infections after Escherichia coli, and can cause arthritis, osteomyelitis, wound infection, sepsis and Toxic shock syndrome and other diseases. Staphylococcus aureus can infect meat, eggs, and milk foods, causing food poisoning in humans, and can also infect livestock, poultry, and even aquatic animals. ...

Claims

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Application Information

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IPC IPC(8): C12N9/00C12N15/62C12N15/70C12N1/21C07K14/32A61K38/16A61K38/53A61P31/04C12R1/19
CPCA61K38/00A61P31/04C07K14/32C07K2319/00C12N9/93C12N15/70C12Y601/01006
Inventor 苏鑫崔新
Owner 中科纳迪(苏州)科技有限公司
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