Equine herpesvirus-1 and application thereof
A equine herpes virus, XJYM2019 technology, applied in the direction of virus, virus/phage, double-stranded DNA virus, etc., to achieve the best proliferation effect, high yield and high safety effect
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Embodiment 1
[0022] Embodiment 1 Isolation and cultivation of Przewalski's equine herpesvirus type 1
[0023] (1) Collection and processing of disease materials
[0024] Cut the lung tissue of the collected dead horse into 1-2cm 3 Place the fragments in a sterile glass plate; grind to powder and add serum-free α-MEM medium to dilute at a ratio of 1:3 to make a virus suspension; freeze and thaw three times in a low-temperature refrigerator at -20°C ;Centrifuge at 4000r / min for 15min, take the supernatant and sterilize it with a 0.22μm filter; add 1000IU (μg / mL) double antibodies (penicillin and streptomycin), and store at -80℃ for later use;
[0025] (2) Virus isolation and subculture
[0026]Take well-grown Vero, BHK-21, MDBK, and RK13 monolayer cells, discard the culture medium, and wash twice with sterile PBS; take 700uL of the supernatant of the disease sample suspension, filter it with a 0.22μm filter, and inoculate the cells. Add 7ml of 10% cell culture solution, and place it in a ...
Embodiment 2
[0035] Example 2 Purification of Przewalski's Equine Herpesvirus Type 1 Virus and Optimization of Culture Conditions
[0036] (1) Plaque purification of virus
[0037] The virus liquid was diluted with culture medium (10 -1 ~10 -9 ), 900 μL of each dilution was inoculated in a 6-well cell plate BHK-21 monolayer cell, incubated in a cell culture incubator for 2 hours, washed the plate twice with sterile PBS (37°C), added nutrient agar gel (55°C low melting point Mix agarose with 1% α-MEM, 2% fetal bovine serum, and 1% antibiotics at a ratio of 3:1) 2.5ml / well, place it in a cell incubator after solidification and culture it upside down for 3-4 days, and 10 -1 ~10 -6 When obvious cell lesions appear in the wells, lay 1.5 ml / well of color-developing agar gel (nutrient agar gel with 2 / 1000 neutral red dye added) under weak light, and place it in a cell culture incubator for upside-down culture for 1 day after solidification. When milky white plaques are observed under fluoresc...
Embodiment 3
[0040] Example 3 Establishment of a Whole-Gene Sequencing Platform and Etiological Rapid Diagnosis Method for Przewalski's Equine Herpesvirus Type 1
[0041] (1) Design 70 pairs of specific primers according to the conserved region of the whole genome sequence of EHV-1 registered in GenBank, the EHV-1 genome can be amplified by the polymerase chain reaction (PCR) method, and the primer sequences are as shown in Table 1, The reaction system is shown in Table 2. And high-fidelity polymerase was used, and the reaction conditions were: denaturation at 98°C for 10s; annealing at 49-64°C for 7-15s, extension at 72°C for 30s, a total of 35 cycles; final extension at 72°C for 2min. The PCR product was purified and cloned into the pEASY-Blunt 3 vector using conventional methods, transformed into Trans1-T1 competent cells, extracted the plasmid, and sequenced at Shanghai Sangon Biotechnology Co., Ltd. The sequencing results were spliced using DNAMAN software. ORF finder was used to p...
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