Cracking vibrio parahaemolyticus bacteriophage RDP-VP-21007 and application thereof
A technology of RDP-VP-21007 and Vibrio hemolyticus, which is applied in the direction of phage, virus/phage, and medical raw materials derived from virus/phage, and can solve problems such as ineffective medication, acute onset of aquatic animals, and drug resistance of pathogenic bacteria , to achieve strong cracking activity and good application prospects
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Embodiment 1
[0035] Embodiment 1 Isolation and identification of pathogenic bacteria and bacteriophage
[0036] (1) Isolation and identification of pathogenic Vibrio parahaemolyticus BV-21001
[0037] Sampling from the diseased sea cucumber lesions, using aseptic technique, streaking on TCBS medium, culturing at 37°C for 18-24 hours, showing round green colonies with neat edges on TCBS agar plates, with a diameter of about 2-4mm ; Then pick typical colonies and streak on the Chromagar Vibrio chromogenic medium. After culturing at 37°C for 18-24 hours, purple colonies appear on the Chromagar Vibrio chromogenic medium. Pick typical colonies and continue to streak After purification for 3 times, a single colony was picked and inoculated in 5 mL of 2216E liquid medium, and cultured with shaking at 200 rpm at 37°C for 8 hours to obtain a uniform turbid bacterial suspension. Through 16sRNA molecular identification, it was identified as Vibrio parahaemolyticus, and one of them was named BV-21001...
Embodiment 2
[0046] Electron microscope observation of embodiment 2 phage
[0047] Drop phage liquid (titer 10 10 pfu / m L) 100 μL, place the membrane side of the copper mesh on the phage droplet, take it off after 10 minutes, and let it dry naturally in the air for 2-3 minutes. Then drop a drop of 2% phosphotungstic acid (PTA) aqueous solution on the copper grid for staining, take it off after 10 minutes, dry it in the air for 10-15 minutes, observe with an electron microscope, and select a clear phage image to take pictures.
[0048] photo by electron microscope figure 1 It can be seen that the phage has a regular hexagonal head with a diameter of about 55 nm and a tail.
Embodiment 3
[0049] Example 3 Phage Genome Sequencing
[0050] After the enrichment culture of a single phage, centrifuge at 8000g for 15 minutes at 4°C, add 10% PEG8000 and 0.5M NaCl and let it stand overnight, then add an equal amount of chloroform to mix well, and centrifuge at 5000g for 10 minutes after standing for stratification , after removing the chloroform layer and PEG layer, add restriction endonuclease for digestion treatment, and use gradient density cesium chloride to suspend phage, and then use TM buffer to dialyze 3 times, each time for 30 minutes, and send it to Huada Gene for biological sequencing The company performs whole genome sequencing. The results are shown in the sequence listing.
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