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Porcine epidemic diarrhea, porcine deltacoronavirus dual inactivated vaccine and preparation method thereof

A technology of porcine epidemic diarrhea and dual inactivated vaccines, which is applied in biochemical equipment and methods, viruses, vaccines, etc., can solve the problems of no effective treatment measures and vaccines, and losses in pig farming, and reduce the cost of epidemic prevention, Simple Effects of Immunization Methods

Active Publication Date: 2019-05-07
陕西诺威利华生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, diseases caused by mixed infection of PEDV, PDCoV, etc. are a major problem in the global pig industry, causing great losses to the pig industry
There is a dual vaccine for infectious gastroenteritis and epidemic diarrhea in China, but there is still no effective treatment and vaccine for PDCoV. become a top priority

Method used

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  • Porcine epidemic diarrhea, porcine deltacoronavirus dual inactivated vaccine and preparation method thereof
  • Porcine epidemic diarrhea, porcine deltacoronavirus dual inactivated vaccine and preparation method thereof
  • Porcine epidemic diarrhea, porcine deltacoronavirus dual inactivated vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] The source of embodiment 1 strain

[0027] 1. Porcine epidemic diarrhea virus PEDV-KB2013-4 strain

[0028] The disease material collected from a pig farm in Shaanxi and tested positive for porcine epidemic diarrhea antigen by RT-PCR was added to PBS at a ratio of 1:10 (weight: volume). Filter twice with a 0.22 μm filter, and store the filtrate below -20°C for future use.

[0029] The Vero cells covered with a dense monolayer were inoculated with the filtrate at a dose of 10%, and after adsorption, the maintenance solution containing trypsin was added, and cultured at 37°C. At the same time, blank cells were set as a negative control. Obvious cytopathic changes in the second passage ( figure 1 ), the lesions were basically stable after the fifth generation.

[0030] Using Primer Premier 5.0 gene analysis software, refer to the PEDV M protein gene in Genbank to design detection primer pairs: upstream primer PEDV-F: 5'-AACGGTTCTATTCCCGTTGATG-3'; downstream primer PEDV-...

Embodiment 2

[0040] The characteristic of embodiment 2 virus strains

[0041] 1. Specificity

[0042] Dilute porcine epidemic diarrhea virus and porcine deltacoronavirus to 200TCID with MEM medium respectively50 / 0.1ml, mixed with the corresponding equivalent amount of PEDV and PDCoV specific positive serum, neutralized at 37°C for 1 hour, inoculated into a 96-well cell culture plate of Vero cells and ST cells that had grown into a good monolayer, and inoculated into 4 wells , 200μl / well, set normal cell control and unneutralized serum control at the same time, set at 37℃, containing 5% CO 2 The cells were cultured in an incubator for 7 days, and the cytopathic changes were observed and recorded daily. Neither the positive serum neutralizing group nor the normal cell control group had cytopathic changes; all the non-neutralizing serum control groups had cytopathic changes.

[0043] 2. Virulence

[0044] A total of 10 healthy susceptible piglets aged 17-19 days (both antigens and antibod...

Embodiment 3

[0047] Example 3 Preparation and inspection of porcine epidemic diarrhea and porcine deltacoronavirus dual inactivated vaccine

[0048] 1. Materials and methods

[0049] 1.1 cells

[0050] Vero cells and ST cells were purchased from China Veterinary Drug Administration.

[0051] 1.2 Preparation of virus liquid for seedling production

[0052] Preparation of Porcine Epidemic Diarrhea Virus Seeds: The virus was prepared by the spinner bottle cell culture method. Inoculate Vero cells covered with dense monolayer with 0.2% dose of qualified porcine epidemic diarrhea virus seed virus, and add MEM with a final concentration of 10 μg / mL of trypsin, 100 U / ml of penicillin, and 100 μg / ml of streptomycin The maintenance solution was cultured at 37°C for 48-72 hours. When the cytopathic effect reached more than 80%, the virus liquid was harvested, frozen and thawed twice, and kept below -20°C for later use.

[0053] Preparation of porcine deltacoronavirus seed: the virus was prepared...

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Abstract

The invention provides a dual inactivated vaccine of porcine epidemic diarrhea and porcine delta coronavirus and a preparation method thereof. Before inactivation, the contents of the two viruses are all ≥ 10 7.0 TCID50 / mL, the volume ratio of each antigen after inactivation is 1:1. The double inactivated vaccine of the present invention solves the problem of the lack of effective vaccines for the prevention and treatment of porcine epidemic diarrhea and porcine delta coronavirus disease in the current market, especially solves the lack of porcine delta coronavirus that has become popular in recent years disease vaccine. The dual inactivated vaccine of the invention is economical and practical, effectively reduces the cost of epidemic prevention, and provides a new method for domestic breeding enterprises to simultaneously prevent the occurrence of two diseases.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a dual inactivated vaccine for animals and a preparation method thereof, in particular to a dual inactivated vaccine for porcine epidemic diarrhea and porcine delta coronavirus and a preparation method thereof. Background technique [0002] Porcine epidemic diarrhea (PED) is an infectious intestinal disease of pigs characterized by vomiting, diarrhea and dehydration. It is mainly caused by PED virus infection, which is naturally transmitted mainly through the oral route of feces or pollutants excreted by infected pigs. Pigs of all ages are susceptible, especially suckling piglets, with a case fatality rate of up to 100%. is one of the most important viruses affecting the swine industry worldwide. [0003] Porcine Deltacoronavirus (PDCoV) is a new type of coronavirus, and its clinical characteristics mainly cause acute watery diarrhea in sows and piglets. After PDCoV was first detect...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/215A61P31/14
CPCA61K39/12A61K2039/70A61K2039/5252C12N2770/20034A61P31/14A61K2039/552
Inventor 董剑辉陈瑞张磊
Owner 陕西诺威利华生物科技有限公司
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