Binary inactivated vaccine against Japanese encephalitis virus and porcine parvovirus and preparation method thereof

A dual inactivated vaccine, swine Japanese encephalitis technology, applied in antiviral agents, viral antigen components, pharmaceutical formulations, etc., can solve the problems of cumbersome operating procedures, affecting the quality of vaccines, and high cell loss rate, reducing clinical Effects of stress response, overcoming high rate of cell attrition, simplifying immunization program

Active Publication Date: 2013-01-23
PU LIKE BIO ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are some disadvantages common to the traditional spinner bottle culture in this technical scheme of spinner bottle culture: (1) spinner bottle cell culture cannot adjust the culture conditions such as nutrient, pH and dissolved oxygen of the culture medium in real time, so it cannot Ensure that the cells are in the best culture state; (2) spinner bottle cell culture, the operation procedures are cumbersome, there are many exposure points with pollution risks, and the production process is difficult to scale up; (3) spinner bottle cell culture, the batch-to-batch variation is large, and the production quality is difficult to control , the product quality is unstable
[0005] Chinese patents "A method for the industrial production of porcine Japanese encephalitis vaccine using a bioreactor" (CN102038943A) and "A method for industrially producing porcine parvovirus vaccine using a bioreactor "(CN102038945A) used the microcarrier bioreactor to cultivate porcine encephalitis virus and porcine parvovirus, which further improved the scale of production. ,

Method used

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  • Binary inactivated vaccine against Japanese encephalitis virus and porcine parvovirus and preparation method thereof
  • Binary inactivated vaccine against Japanese encephalitis virus and porcine parvovirus and preparation method thereof
  • Binary inactivated vaccine against Japanese encephalitis virus and porcine parvovirus and preparation method thereof

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preparation example Construction

[0073] (1) Cell seeding, adsorption and culture

[0074] Dilute the animal cell suspension at 0.5×10 6 ~5×10 6 The final density of cells / g carrier is inoculated in the carrier tank, and the parameters of the bioreactor are set to carry out the cell adsorption and culture process. Among them, animal cells for cultivating porcine Japanese encephalitis virus include BHK-21, IBRS-2, ST, Vero, C6 / 36 and chicken embryo fibroblasts, preferably BHK-21 cells. The cultured cells of porcine parvovirus include ST, PK-15, IBRS-2, CPK and MVPK cells, preferably ST cells.

[0075] (2) Virus inoculation, adsorption and proliferation

[0076] After the cells grow to 0.5×10 7 ~1.0×10 8 When the density of cells / g carrier is reached, replace the cell culture medium, then inoculate the porcine Japanese encephalitis virus and porcine parvovirus liquid into the carrier tank respectively, and set the parameters of the bioreactor to carry out the process of virus adsorption and multiplication. ...

Embodiment 1

[0097] The microcarrier used in this embodiment is polyester fiber, and the cell lines used for making seedlings are BHK-21 cells and ST cells, wherein, porcine Japanese encephalitis virus uses BHK-21 cell proliferation, and porcine parvovirus uses ST cell proliferation .

[0098] The bioreactor is a 10L carrier bottle Tidecell tidal bioreactor.

[0099] The formula of cell growth liquid: the growth liquid of BHK-21 cell is the DMEM (Invitrogen company) containing 5% (V / V) fetal bovine serum; The growth liquid of ST cell is the DMEM containing 5% (V / V) fetal bovine serum α-MEM (Invitrogen Corporation).

[0100] The formula of cell maintenance solution: the growth solution of BHK-21 cells is DMEM (Invitrogen Company) containing 1% (V / V) fetal bovine serum; the growth solution of ST cells is DMEM containing 1% (V / V) fetal bovine serum α-MEM (Invitrogen Corporation).

[0101] (1) Cell seeding, adsorption and culture

[0102] In a 10L bioreactor, add 650g of sterile microcarri...

Embodiment 2

[0118] 1. Finished product inspection of dual inactivated vaccine:

[0119] Physical property test:

[0120] a) Appearance: milky white or light red homogeneous emulsion.

[0121] b) Dosage form: bi-directional, that is, water-in-oil-in-water (W / O / W). Take a clean straw, suck a small amount of vaccine and drop it in clean cold water, except for the first drop, it is in the shape of oil droplets and does not spread.

[0122] c) Stability: Take 10ml of the vaccine and put it into a centrifuge tube, centrifuge at 3,000rmp / min for 15min, no stratification occurs, and the water phase ≤0.5ml precipitates at the bottom of the tube; take the vaccine and place it at about 37°C for 21 days, the result shows no stratification, no Demulsification phenomenon.

[0123] d) Viscosity: use a 1ml clean straw (1.2mm lower diameter, 2.7mm upper diameter) to draw 1ml of vaccine at about 25°C to let it flow out vertically and naturally, and record the time required to flow out 0.4ml. The results...

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Abstract

The invention provides a preparation method of a binary inactivated vaccine against a Japanese encephalitis virus and a porcine parvovirus, which comprises the following steps of: (1) inoculating the animal cells to a microcarrier for performing the absorption culture and multiplication culture processes of the cells in a tidal biological reactor; (2) inoculating the Japanese encephalitis virus and the porcine parvovirus on the cells being subjected to multiplication culture; (3) when the inoculated animal cell CPE (Cytopathic Effect) reaches above 75%, harvesting a viral solution; and (4) freezing and thawing the harvested viral solution twice at minus 20 DEG C, inactivating after the concentration, and adding an emulsifier for emulsification to obtain the binary inactivated vaccine against Japanese encephalitis virus and porcine parvovirus. The binary inactivated vaccine provided by the invention has the advantages of good immunogenicity, stable growth and high titer of the preserved seed virus; and the tidal biological reactor is adopted to respectively culture and harvest two viruses with high titers, and the two viruses are directly emulsified and prepared after the concentration and the inactivation, so that the binary inactivated vaccine can be continuously produced in a large scale.

Description

technical field [0001] The invention relates to a porcine dual inactivated vaccine, in particular to a dual inactivated vaccine for treating and preventing porcine Japanese encephalitis and porcine parvovirus infection, belonging to the field of veterinary vaccines. Background technique [0002] Porcine encephalitis virus (Swine Japanese Encephalitis Virus, JEV) and porcine parvovirus (Porcine Parvovirus Virus, PPV) are one of the important pathogens that cause reproductive disorders in pigs. The acute inflammatory reaction or infertility of the boar testes has caused huge losses to the pig industry. Except for single infection, JEV and PPV are often co-infected. The survey of mixed infection in large-scale pig farms in Sichuan, Chongqing, Hubei, and Henan found that the positive rate of mixed infection was ≥ 60%. [0003] In "Porcine Parvovirus Infection-Japanese Encephalitis Dual Oil Emulsion Inactivated Vaccine Research", it is disclosed that porcine parvovirus inactivat...

Claims

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Application Information

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IPC IPC(8): A61K39/295A61P31/14A61P31/20A61K39/12A61K39/23
CPCY02A50/30
Inventor 张许科孙进忠白朝勇
Owner PU LIKE BIO ENG
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