Lytic and high-titer vibrio parahaemolyticus bacteriophage RDP-VP-21010 and application thereof
A technology of RDP-VP-21010 and Vibrio hemolyticus, which is applied in the direction of phage, virus/phage, medical raw materials derived from virus/phage, etc. It can solve the problems of acute onset of aquatic animals, drug resistance of pathogenic bacteria, untimely treatment, etc. problems, to achieve good application prospects, strong cracking activity, wide range of acid-base tolerance effect
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Embodiment 1
[0038] Embodiment 1 Isolation and identification of pathogenic bacteria and bacteriophage
[0039] (1) Isolation and identification of pathogenic Vibrio parahaemolyticus BV-21002
[0040] Take samples from the gills of the diseased prawns, spread them on TCBS medium by aseptic operation, and cultivate them at 37°C for 18-24 hours. On the TCBS agar plate, there will be round green colonies with neat edges and a diameter of about 2-3mm; Then pick a typical colony and streak it on the Chromagar Vibrio chromogenic medium. After culturing at 37°C for 18-24 hours, purple colonies will appear on the Chromagar Vibrio chromogenic medium. Pick typical colonies and continue to streak and purify 3 times, then pick a single colony and inoculate it in 5 mL of 2216E liquid medium, shake and culture at 37°C for 6 hours at 200 rpm to obtain a uniform turbid bacterial suspension. Through 16sRNA molecular identification, it was determined to be Vibrio parahaemolyticus, and one of the strains wa...
Embodiment 2
[0050] Electron microscope observation of embodiment 2 phage
[0051] Take 20 μL of the liquid containing crude phage particles and drop them on the copper grid, let it settle naturally for 15 minutes, and absorb the excess liquid from the side with filter paper, add a drop of 2% phosphotungstic acid (PTA) on the copper grid to stain the phage for 10 minutes, and then use the filter paper to remove the The staining solution was sucked off from the side, and the morphology of the phage was observed with an electron microscope after the sample was dried.
[0052] Depend on figure 1 It can be seen that the phage RDP-VP-21010 is a long-tailed phage with a regular hexagonal head, a head diameter of about 56 nm, and a tail length of 103 nm.
Embodiment 3
[0053] Example 3 Phage Genome Sequencing
[0054] 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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