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Pseudorabies/porcine parvovirus infection combined inactivate vaccine and suspension culture preparation method

A technology of porcine pseudorabies virus and dual inactivated vaccines, applied in biochemical equipment and methods, vaccines, viruses, etc., can solve the problem of increasing the number of artificial and animal stress, large area occupied by spinner bottle culture, and affecting the use effect of vaccines, etc. problems, to achieve the effect of reducing the number of animal stress, reducing the number of immunizations, and large batches of antigens

Inactive Publication Date: 2018-08-21
WUHAN KEQIAN BIOLOGY CO LTD
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The products currently on the market are all single vaccines, which need to be immunized separately to increase the number of artificial and animal stresses. They are all cultured by the spinner bottle process, which is backward in technology and labor-intensive. The spinner bottle culture takes up a large area and has low antigen content. , affecting the efficacy of the vaccine

Method used

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  • Pseudorabies/porcine parvovirus infection combined inactivate vaccine and suspension culture preparation method
  • Pseudorabies/porcine parvovirus infection combined inactivate vaccine and suspension culture preparation method
  • Pseudorabies/porcine parvovirus infection combined inactivate vaccine and suspension culture preparation method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] The preparation of embodiment 1 porcine pseudorabies virus suspension culture antigen

[0030] 1.1 Porcine pseudorabies virus gE gene deletion strain (HNX-12 strain)

[0031] 1.1.1 Preparation of virus seeds for production

[0032] 1.1.1.1 Breeding of poisonous seeds

[0033] The suspension type BHK-21 cells were inoculated into cell shaker flasks, cultured on a shaker at 37°C and 120r / min, the cell viability was over 95%, and the cell density was 4-6×10 6 Seed cells per ml were added to the inoculation bottle, mixed well, sampled and counted, and transferred to the bioreactor. Dissolved oxygen (DO) electrodes, pH electrodes, and temperature electrodes were calibrated before the bioreactor was inoculated with cells, and the tank was autoclaved. According to the culture volume, pump 70% of the culture medium in advance, and set the best culture conditions: temperature 37°C, inoculation density 5~7×10 5 cells / ml, rotation speed 50-60r / min, pH value 7.2-7.4, dissolved ...

Embodiment 2

[0078] Embodiment 2 Porcine pseudorabies / porcine parvovirus disease dual inactivated vaccine

[0079] 2 Vaccine preparation

[0080] 2.1 Preparation of aqueous phase Mix the two inactivated virus liquids (porcine pseudorabies virus: porcine parvovirus) uniformly at a ratio of 1:1 (volume ratio). Among them, the virus content of porcine pseudorabies virus before inactivation should be ≥10 8.0 TCID50 / ml, the porcine parvovirus content before inactivation should be ≥10 7.0 TCID 50 / ml or virus hemagglutination (HA) value ≥ 2 10 .

[0081] 2.2 Emulsification: Mix the water phase and the adjuvant according to the ratio (mass ratio) of 1:1 and then emulsify. Take a sample, draw 10.0ml of vaccine into a centrifuge tube, centrifuge at 3000r / min for 15 minutes, and the water precipitated at the bottom of the tube should not exceed 0.5ml.

[0082] 2.3 Subpackaging: Quantitatively subpackage the emulsified vaccines, crimp and label.

Embodiment 3

[0083] The toxicity determination of embodiment 3 antigen

[0084] 3 Porcine pseudorabies virus TCID 50 Determination of

[0085] 3.1.1 The virus was serially diluted 10 times with DMEM maintenance solution (containing 2% newborn bovine serum), and 10 -5 、10 -6 、10 -7 、10 -8 、10 -9 5 dilutions, each dilution was inoculated in a 96-well micro-cell culture plate in a vertical row with a total of 8 wells, and 8 wells of cell control were set at the same time, 100 μl per well.

[0086] 3.1.2 Add 100 μl of BHK-21 cell suspension to each well.

[0087] 3.1.3 5% CO at 37°C 2 Cultivate and observe in the incubator for 4 to 6 days. When 80% of the cells are lesioned (cells aggregate and bulge, the outline of the cells is blurred, round and shrink, and finally fall off and disintegrate), it is judged as infection, record the number of cells with lesion, and calculate the TCID according to the Reed-Muench method 50 . The results are shown in Table 1 below. The experimental data ...

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Abstract

The invention belongs to the technical field of veterinary biological products and particularly relates to a pseudorabies / porcine parvovirus infection combined inactivate vaccine and a suspension culture preparation method. The preparation method comprises the following steps: preparing a pseudorabies virus suspension culture antigen and a porcine parvovirus infection virus suspension culture antigen; proportionally mixing the inactivated pseudorabies virus and porcine parvovirus infection virus antigen solutions, and then adding an adjuvant to fully emulsify to obtain the pseudorabies / porcineparvovirus infection combined inactivate vaccine. The suspension culture processes of the pseudorabies virus antigen solution and the porcine parvovirus infection virus antigen solution are established, the virus antigen solutions prepared through the suspension culture process have the advantages of high antigen content, large batch of the antigen and stable batch, the preparation method greatlyreduces the use of manpower, the occupied area and space of a culture system is reduced, and the production cost of an enterprise is lowered; meanwhile, the pseudorabies / porcine parvovirus infectioncombined inactivate vaccine is used, two viruses are prevented through one syringe, the immunity times of an animal are reduced, the stress times of the animal are reduced, and the production cost ofthe vaccine and the culture cost of a farmer are greatly reduced.

Description

technical field [0001] The invention belongs to the technical field of veterinary biological products, and in particular relates to a porcine pseudorabies / porcine parvovirus dual inactivated vaccine and a suspension culture preparation method thereof. Background technique [0002] Porcine pseudorabies (Pseudorabies, PR) is an acute infection characterized by fever, itching (except pigs), respiratory and nervous system diseases caused by a variety of domestic and wild animals caused by pseudorabies virus (PRV) Among them, it is the most harmful to pigs, mainly causing abortion, stillbirth, mummification, return to oestrus and repeated infertility in pregnant sows; mass death of newborn piglets; slow growth of fattening pigs and loss of breeding ability of breeding boars. The World Health Organization (OIE) classifies it as a Class B animal disease, and my country classifies it as a Class II animal disease. The prevalence of this disease is on the rise worldwide. It has beco...

Claims

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Application Information

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IPC IPC(8): A61K39/295A61K39/125A61K39/205A61P31/20A61P31/22C12N7/00C12N7/04
CPCA61K39/12A61K2039/5252A61K2039/552A61K2039/55566A61K2039/70A61P31/20A61P31/22C12N7/00C12N2710/16734C12N2710/16751C12N2710/16761C12N2750/14034C12N2750/14051C12N2750/14061
Inventor 尹争艳徐高原周明光钟恩陈斌方玉林苏顺攀陈章表苏秀婵黄慧君洪灯陈关平曹毅张洁金建云
Owner WUHAN KEQIAN BIOLOGY CO LTD
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