Pasteurella bacteriophage vB_PmuP_PS02 and bacteriophage composition and application thereof
A Pasteurella and phage technology, applied in the field of microbiology, can solve the problems such as the absence of Pasteurella phage, and achieve the effect of wide application and cost reduction
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Embodiment 1
[0036] The isolation culture and biological characteristic of embodiment 1 bacteriophage
[0037] (1) Recovery culture of strains and preparation of bacterial suspension
[0038] Pick the frozen bacteria solution of Pasteurella and draw a three-section line on the TSA plate (plus 5% newborn bovine serum), separate single colonies, and culture them in a 37°C incubator for 16-24 hours. A single colony was picked, inoculated into 5 ml of TSB broth (with 5% newborn bovine serum), and cultured in an air shaker at 37° C. at 180 rpm for 19 hours to obtain a single bacterial suspension.
[0039] (2) Isolation and purification of phage
[0040] Take pig manure samples, litter, sewage, fur and other samples, add appropriate amount of TSB broth, shake at 37°C, 170rpm for 30min, centrifuge at 10000rpm for 5min, take the supernatant and filter it with a 0.22μm bacterial filter for later use.
[0041] Add each strain of Pasteurella in an amount of 80% in each Erlenmeyer flask, add the cen...
Embodiment 2
[0058] The mensuration of embodiment 2 phage lysis spectrum and in vitro lysis test
[0059] (1) Determination of cleavage profile of phage vB_PmuP_PS02
[0060] (1) The lysis experiment of the phage on Pasteurella porcine
[0061] In this example, 50 strains of Pasteurella swine origin from Shandong, Henan, Guangxi, Guangdong and other places were selected as host bacteria to detect the lysis profile of phages.
[0062] 1. Test of phage lysis profile: The lysis profile of phage was determined by double-layer plate method, the steps are as follows: prepare the bacterial suspension of the host bacteria according to the method in Example 1. After incubating 300 μl of bacterial suspension and 300 μl of Pasteurella phage at 37°C for 5 minutes, add it to the upper layer of agar (with 5% newborn bovine serum) to prepare a double-layer plate, and place it in a 37°C incubator after the agar solidifies Inverted culture overnight to observe the lysis results, the statistical results a...
Embodiment 3
[0096] The safety test of embodiment 3 bacteriophage
[0097] Select 20 healthy SPF mice with a body weight of 20-22 g, half male and half female, and divide them into experimental group and control group. The number of male and female mice in each group is half and half, and the mice in the experimental group are injected intraperitoneally with purified phage proliferation solution (200 μl 10 9 PFU / ml), the same amount of saline (200 μl) was injected intraperitoneally to the mice in the experimental group for 3 days, the behavior of the mice was observed for 7 days, and the changes in the organs of the mice were observed by autopsy.
[0098] The results showed that the behavior of the mice in the experimental group and the control group was normal, and there was no death phenomenon; the necropsy found that the liver, lung, heart, spleen, kidney and other organs of the mice in the realization group were normal, and there was no significant difference from the control group.
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