Porcine pseudorabies virus virulent strain and its application in preparation of inactivated vaccine
A porcine pseudorabies and inactivated vaccine technology, applied in the direction of viruses, antiviral agents, virus antigen components, etc., can solve the problems of stillbirth, loss, abortion, etc., and achieve the effect of long-lasting immunity and rapid antibody production
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Embodiment 1
[0043] The isolation and identification of embodiment 1 porcine pseudorabies virus BJ strain
[0044] 1 Materials and methods
[0045] 1.1 Disease data
[0046] The disease materials used in the experiment came from piglets or mummified fetuses from 6 affected pig farms in Hebei, Northeast China, and Beijing. The affected piglets showed the following clinical symptoms: elevated body temperature, mental fatigue, severe emaciation, uncoordinated movement, trembling, convulsions, etc. . For necropsy, the brain tissue, heart, liver, spleen, lung, kidney, and tonsil of each pig were taken as disease materials; the brain tissue was used as a sample, and the internal organs were mixed as disease materials for those without brain tissue; the disease materials were cut Grind thoroughly with a mortar, add a certain amount of PBS, freeze and thaw three times, centrifuge at 4000r / min for 15min, take the supernatant, filter it through a 0.22μm filter membrane and store it below -20℃ or i...
Embodiment 2
[0096] Example 2 Preparation and efficacy test of porcine pseudorabies virus virulent strain BJ strain inactivated vaccine
[0097] 1 poison seed
[0098] The virus seeds used for making the vaccine and the virus seeds used for testing are the virulent strain BJ of porcine pseudorabies virus. No bacteria, mold, mycoplasma and exogenous virus pollution.
[0099] 2 Vaccine manufacturing and inspection of semi-finished products
[0100] 2.1 Preparation of poisonous seeds for production
[0101] 2.1.1 Breeding of poisonous seeds
[0102] Discard the growth medium of the ST cells that have grown into a single layer, inoculate the virus strain BJ strain according to the amount of 2%, add DMEM cell culture medium containing 2% newborn bovine serum, and culture at 37°C for 2 to 3 days, when 75% or more Harvest the cells when lesions appear, freeze and thaw once at -15°C, aliquot quantitatively, indicate the date of harvest, virus generation (should not exceed 5 generations), etc.,...
Embodiment 3
[0132] Example 3 The application of porcine pseudorabies virus virulent strain BJ strain in the preparation of porcine pseudorabies virus gE / gI double gene deletion strain vaccine strain (BJC strain)
[0133] 1 Materials and methods
[0134] 1.1 Viruses and Cells
[0135] The PRV BJ strain virus was isolated from Example 1; ST cells and Vero cells were preserved by our laboratory. DH5α competent cells were purchased from Beijing Quanshijin Biotechnology Co., Ltd.
[0136] 1.2 Plasmid
[0137] pEGFP-C1 is preserved by our laboratory. The pMD18-T vector was purchased from Takara.
[0138] 1.3 Primer design
[0139] The primers are listed in Table 6 below.
[0140] Primers used in table 6 PRV gE / gI knockout
[0141]
[0142] 1.4 Construction strategy
[0143] The primers for amplifying the left and right homology arms of PRV were used for PCR amplification to obtain the left and right homology arms of the transfer vector. At the same time, pEGFP-C1 was double-digested...
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