Porcine reproductive and respiratory syndrome, porcine pseudorabies dual live vaccine and its preparation method and application
A technology for respiratory syndrome and porcine pseudorabies, which is applied in antiviral agents, pharmaceutical formulas, virus antigen components, etc., can solve the problems of reducing pig immunity and reducing the immune effect of other vaccines, and achieve good immune promotion and good immunity original effect
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[0041] The present invention relates to a kind of preparation method of the vaccine composition of preventing or treating porcine reproductive and respiratory syndrome and / or porcine pseudorabies, wherein, said method comprises:
[0042] (1) cultivating and proliferating porcine reproductive and respiratory syndrome virus and porcine pseudorabies virus respectively;
[0043] (2) Mix in proportion, add lyoprotectant, and lyophilize.
[0044] Preferably, in the step (1), cultivating and proliferating porcine reproductive and respiratory syndrome virus and porcine pseudorabies virus selects susceptible cells, and the susceptible cells may be passaged cell lines or primary cells. Susceptible cells suitable for porcine reproductive and respiratory syndrome virus include, but are not limited to, African green monkey kidney cells Marc-145 cell line, MA-104 cell line, Vero cell line, CL-2621 cell line and other passage cell lines, Or primary cells such as PAM cells. Susceptible cell...
Embodiment 1
[0056] Proliferation and Culture of Porcine Reproductive and Respiratory Syndrome Virus JXA1-R Strain
[0057]The virus species used for production is the highly pathogenic porcine reproductive and respiratory syndrome JXA1-R strain, select the required amount of Marc-145 cells (cell spinner bottles that have grown into a monolayer), discard the growth solution, and press 1% ( V / V) Inoculate the JXA1-R strain of the virus seed virus used for production, add DMEM cell culture fluid containing 2% fetal bovine serum, cultivate at 37°C with rotation (9 to 12 revolutions / hour), and observe the cells caused by the virus under a microscope Lesions (CPE), when the CPE reaches more than 70%, the cell culture is harvested, frozen and thawed once, and the cell debris is removed by centrifugation or filtration, and frozen at -40°C; according to the appendix of "Chinese Veterinary Pharmacopoeia", the vaccine antigens are tested without Bacteria testing and virus content determination.
Embodiment 2
[0059] Proliferation and Culture of Porcine Pseudorabies Virus HN1201-R Strain
[0060] The virus species for production is porcine pseudorabies virus HN1201-R strain, select the required number of PK15 cells (cell spinner bottles that have grown into a single layer), discard the growth solution, and inoculate the virus for production at 1% (V / V). A virus, add DMEM cell culture medium containing 2% fetal bovine serum, cultivate at 37°C with rotation (9-12 revolutions / hour), observe the cytopathic effect (CPE) caused by the virus under a microscope, when the CPE reaches more than 70% Harvest the cell culture, freeze and thaw once, remove cell debris by centrifugation or filtration, and store frozen at below -40°C; perform sterility test and virus content determination on the vaccine antigen according to the appendix of "Chinese Veterinary Pharmacopoeia".
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