Method for preparing porcine circovirus inactivated vaccine

A porcine circovirus and inactivated vaccine technology, applied in biochemical equipment and methods, vaccines, viruses, etc., can solve the problem of no effective treatment for porcine circovirus disease, achieve good antigen recovery rate, and high antigen recovery rate , good effect of vaccine safety

Inactive Publication Date: 2016-07-27
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There is no effective treatment for porcine circovirus disease at present, and the application of porcine ci

Method used

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  • Method for preparing porcine circovirus inactivated vaccine
  • Method for preparing porcine circovirus inactivated vaccine
  • Method for preparing porcine circovirus inactivated vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] The cultivation and identification of embodiment 1 virus

[0030] Get the sample of PCV2WH strain and inoculate in the PK-15 cell suspension of fresh digestion simultaneously by 10%, 37 ℃ static culture 24h, after the cell forms monolayer, add appropriate amount 300mmolD-glucosamine to process according to Tischer method (in order to be able to completely Covering the cell monolayer shall prevail), cultured at 37°C for 30min, discarded D-glucosamine, added DMEM maintenance solution containing 2% newborn bovine serum to continue culturing for 48h, harvested the virus, and placed it in a -20°C refrigerator for temporary storage (long-term storage need to be placed at -80°C).

[0031] The identification of the virus is mainly determined by PCR method, indirect immunofluorescence method and electron microscope observation method.

[0032] Referring to the nucleotide sequence of the PCV2 strain (AY122275) published by GenBank, a pair of primers were designed to amplify the ...

Embodiment 2B

[0039] The preparation of embodiment 2BEI inactivator

[0040] BEA and 0.2 mol / L NaOH solution were placed in a water bath at 37°C for 1 hour to prepare a BEI inactivator, and stored at 4°C for future use.

[0041] The inactivation of the formaldehyde of embodiment 3BEI and 2‰ to virus liquid

[0042] Add BEI with a final concentration of 0.002mol / L, 0.001mol / L, 0.0005mol / Ll and 0.00025mol / L and formaldehyde with a concentration of 2‰ to the virus liquid, respectively, at 37°C, 32°C, 27°C and Place it at 22°C for 12 days, and add blocking agent to stop the inactivation. Samples were taken daily and virus levels were determined by indirect immunofluorescence.

[0043] Tables 1 to 4 show the inactivation effect of formaldehyde on PCV2 (WH strain) under different temperature conditions with a concentration of 0.002mol / L, 0.001mol / L, 0.0005mol / L, 0.00025mol / L and a concentration of 2‰.

[0044] Finished product inspection: According to the appendix of "The Veterinary Pharmacopo...

Embodiment 4

[0054] The sub-packaging of the seedlings of the inactivated virus liquid of embodiment 4

[0055] Inactivate BEI and 2‰ formaldehyde in four concentrations (0.002mol / L, 0.001mol / L, 0.0005mol / L, 0.00025mol / L) at different temperatures (37°C, 32°C, 27°C, 22°C) The virus liquid was mixed and subpackaged, and after the sterility test passed, the vaccines mixed with 4 concentrations of BEI and 2‰ formaldehyde inactivated vaccines were compiled into groups A, B, C, D, and E5 (4 kinds at 37°C) Vaccines formulated with concentrations of BEI and 2‰ formaldehyde inactivated are coded as A1, B1, C1, D1, E1, and vaccines formulated with 4 concentrations of BEI and 2‰ formaldehyde inactivated at 32°C are coded as A2, B2, C2, D2, E2, the vaccine obtained under each temperature condition is a group, and so on), and the vaccine is stored at 4°C for later use.

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Abstract

The invention discloses a method for preparing a porcine circovirus inactivated vaccine.The method comprises the steps that, PK15 and ST or BHK-21 are subjected to EDTA-trypsin digestion and then subjected to passage, and continue to be cultured with a cell growth solution, and the number of cells is maintained with a cell maintaining solution when the cells grow to reach 85-100%; a porcine circovirus 2b type strain is inoculated to a medium with the cells growing on 85-100% of the medium, and the strain is cultured and collected; toxins of the strain are collected and inoculated to the medium with the cells growing on 85-95% of the medium, and culture and collection are carried out to obtain a porcine circovirus 2b type strain antigen solution; an immunologic adjuvant is added after inactivation is carried out, and the inactivated vaccine is obtained through emulsification.The vaccine obtained through the method is high in antigen stability, immunogenicity, antigen recycling rate and vaccine safety.The inactivating method and a methanal inactivating method are compared, after inactivated vaccines prepared through the two inactivating methods are used for immunity of pigs, the serum antibody valence is high, and an inactivated vaccine obtained through the method of carrying out inactivating for 48 h with 0.002 mol/L BEI has the best antigen stability and immunogenicity and the highest antigen recycling rate and vaccine safety.

Description

technical field [0001] The invention relates to the field of veterinary biological products, in particular to a method for preparing porcine circovirus inactivated vaccine. Background technique [0002] Porcine circovirus (Porcine circovirus, PCV) belongs to Circoviridae (Circoviridae), Circovirus (Circovirus), a non-enveloped single-stranded circular DNA virus. It has two serotypes, PCV1 and PCV2 (Khampee K., 2005). PCV1 was first isolated from a porcine kidney cell line by Tischer (1974). Clark (1991) reported in Canada that PCV2 is the main pathogen of postweaning multisystemic wasting syndrome (postweaningmultisystemicwastingsyndrome, PMWS). Then it was first isolated from PMWS pigs by Ellis et al. (1998). It has been proved that when PCV2 infects gnotobiotic (GN) pigs, non-lactating (colostrum-deprived, CD) pigs and SPF pigs, it can lead to the occurrence of PMWS (Krakowka et al., 2000; 2001; Allan et al., 2000b), And the infectious clone of PCV2 genome can infect S...

Claims

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Application Information

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IPC IPC(8): A61K39/12A61P31/20C12N7/00
CPCA61K39/12A61K2039/5252A61K2039/552C12N7/00C12N2750/10034
Inventor 严伟东何启盖陈芳洲李梦莹叶十一李中华郭效珍陈焕春
Owner HUAZHONG AGRI UNIV
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