Pseudorabies virus gE/gI double gene deleted vaccine strain, as well as construction method and application thereof
A porcine pseudorabies and double-gene technology, applied in the field of medicine, can solve problems such as stillbirth, sows giving birth to weak piglets, miscarriage, etc., and achieve the effects of rapid antibody production, long-lasting immunity period, and wide application prospects
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Embodiment 1
[0067] The isolation and identification of embodiment 1 porcine pseudorabies virus BJ strain
[0068] 1 Materials and methods
[0069] 1.1 Disease data
[0070] 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
[0120] The construction of embodiment 2 porcine pseudorabies virus gE / gI double gene deletion strain vaccine strain (BJC strain)
[0121] 1 Materials and methods
[0122] 1.1 Viruses and Cells
[0123] 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.
[0124] 1.2 Plasmid
[0125] pEGFP-C1 is preserved by our laboratory. The pMD18-T vector was purchased from Takara.
[0126] 1.3 Primer design
[0127] The primers are listed in Table 6 below.
[0128] Primers used in table 6PRV gE / gI knockout
[0129]
[0130] 1.4 Construction strategy
[0131] 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 with Bsp EI and BamH I to remove unused restriction sites, and the comp...
Embodiment 3
[0181] Example 3 Study on the pathogenicity of porcine pseudorabies virus gE / gI double gene deletion vaccine strain (BJC strain) to piglets
[0182] 1 Materials and methods
[0183] 1.1 Viruses and Cells
[0184] PRVBJC strain (prepared in Example 2); ST cells are preserved by our laboratory.
[0185] 1.2 Experimental animals
[0186] Healthy susceptible piglets aged 2 to 3 weeks should be negative for PRV antigen by PCR, and their neutralizing antibody titers should be ≤1:4, and they should be acclimatized in animal houses for at least 3 days after purchase.
[0187] 1.3gE antibody detection kit IDEXX company products.
[0188] 1.4 Experimental method
[0189] 1.4.1 Inoculation and observation
[0190] Take 10 healthy susceptible piglets aged 2 to 3 weeks (the PCR antigen test is negative; the neutralizing antibody titer of porcine pseudorabies serum is not higher than 1:4), and divide them into 2 groups randomly, 5 pigs in each group. Each head of the group received na...
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