Porcine pseudorabies virus, vaccine composition and preparation method and use thereof
a pseudorabies virus and vaccine technology, applied in the field of vaccine composition, can solve the problems of high death rate of piglets, neurological signs, stillborn or mummified fetuses, etc., and achieve the effect of good immune function of porcine pseudorabies
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example 1
[0059]Collection and Isolation of Viruses
[0060]Porcine brain tissue was collected under aseptic conditions from samples isolated from samples from Henan province suspected of having pseudorabies infection, added in MEM liquid medium in a ratio of 1:10(V:V), and ground to prepare a tissue suspension. After 3 times of repeated freezing-thawing, the tissue suspension was centrifuged at 2000r / min for 15 minutes. The supernatant was then collected, filtered through a 0.2 μm pore filter, subcultured on PK-15 cells and incubated at 37° C. for lh, and then the medium was changed by adding MEM liquid medium supplemented with 2% fetal bovine serum, and incubated at 37° C. for 5 days. PRV was detected by PRV PCR detection kit (Beijing Anheal Laboratories Co., Ltd), and the result was positive; PCR kit was employed to detect the exotic virus contamination (porcine reproductive and respiratory syndrome virus RT-PCR detection kit kit, porcine parvovirus PCR detection kit and classical swine fever...
example 2
[0062]Genetic Characteristics of the Isolated Virus
[0063]Genetic characteristics of the isolated virus in Example 1 were determined by means of gene analysis. Genomic DNA prepared from the pseudorabies virus isolated from PK-15 cells was used as template with primers shown in Table 1 for PCR amplification reactions. The Primer Primer 5.0 was used for designing the primer sequence for amplifying gB, gC and gD gene, respectively.
[0064]The genomic DNA extracted was used as template to prepare the following PCR amplification system: 100 μg template DNA, 0.5 μL PrimerSTAR HS DNA Polymerase (2.5U / μL), 25 μL 2 ×PrimerSTAR GC Buffer, 1 μL of each upstream and downstream primer (10 pmol / μL), 4 μL dNTP Mix (2.5 mM each), adjusted to a final volume of 50 μL with distilled water. Two-step PCR reaction was carried out by an initial denaturation for 10 sec at 98° C. followed by annealing and extension at 68° C. (all the time is calculated by 1 kb / min) and there were 30 cycles in total. The PCR re...
example 3
[0065]Pathogenicity Test of the Virus
[0066]3.1 Pathogenicity of the Virus in Piglets at Different Days of Age
[0067]6 piglets at 34˜35 days of age which were negative for pseudorabies antibodies were randomly divided into 2 groups, one with 4 piglets (experimental group) and the other with 2 piglets (control group), wherein the experimental group was inoculated with PRV HN1201 strain by intranasal instillation (challenge dosage is 2×108.0TCID50 / piglet) and the control group was inoculated with DMEM medium. Meanwhile 4 piglets at 49 days of age were inoculated with third passage of the virulent HN1201 strain after preservation (challenge dose was 2×108.0TCID50 / piglet), and the control is still piglets at 35 days of age. After inoculation of virus, the temperature of piglets was determined daily, and clinical signs and death status were observed. The results are shown in Table 2.
TABLE 2Pathogenicity of PRV HN1201 strain in piglets at different days of ageNum-Inoculation GroupberDaysDos...
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