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Triple inactivated vaccine for porcine epidemic diarrhea, porcine transmissible gastroenteritis and porcine deltacoronavirus and preparation method thereof

A technology for porcine epidemic diarrhea and inactivated vaccines, which is applied in the field of multiple inactivated vaccines for animals and its preparation, can solve the problems of no effective treatment measures and vaccines, and losses in pig farming, and achieve reduced epidemic prevention costs and simple immunization methods , The effect of simplifying the immunization procedure

Inactive Publication Date: 2018-12-28
YULIN UNIV +1
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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, TGEV, PDCoV, etc. are a major problem in the global pig industry, causing great losses to the pig industry
There are two vaccines for infectious gastroenteritis and epidemic diarrhea in China, but there is still no effective treatment and vaccine for PDCoV. Therefore, a vaccine that can effectively prevent and treat porcine epidemic diarrhea, porcine infectious gastroenteritis and Porcine deltacoronavirus vaccine has become a top priority

Method used

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  • Triple inactivated vaccine for porcine epidemic diarrhea, porcine transmissible gastroenteritis and porcine deltacoronavirus and preparation method thereof
  • Triple inactivated vaccine for porcine epidemic diarrhea, porcine transmissible gastroenteritis and porcine deltacoronavirus and preparation method thereof
  • Triple inactivated vaccine for porcine epidemic diarrhea, porcine transmissible gastroenteritis and porcine deltacoronavirus and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] The source of embodiment 1 strain

[0031] 1. Porcine epidemic diarrhea virus PEDV-KB2013-4 strain

[0032] The disease material collected from a pig farm 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 μm filter, and store the filtrate below -20°C for future use.

[0033] The Vero cells covered with a dense monolayer were inoculated with the filtrate at a dose of 10%, and after adsorption, a 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 appeared at the second generation ( figure 1 ), the lesions were basically stable after the fifth generation.

[0034] 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-R: 5'-...

Embodiment 2

[0050] The characteristic of embodiment 2 strains

[0051] 1. Specificity

[0052] Dilute porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus and porcine deltacoronavirus to 200TCID respectively with MEM medium 50 / 0.1ml, mixed with the corresponding amount of PEDV, TGEV, PDCoV-specific positive serum, neutralized at 37°C for 1 hour, and respectively inoculated into 96-well cells of Vero cells, ST cells, and ST cells that had grown into a good monolayer Culture plate, inoculate 4 wells, 200μl / well, set normal cell control and unneutralized virus control at the same time, set at 37°C, 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 neutralization group nor the normal cell control group had cytopathic changes; all the unneutralized virus control groups had cytopathic changes.

[0053] 2. Virulence

[0054] A total of 15 healthy susceptible p...

Embodiment 3

[0057] Preparation and inspection of the triple inactivated vaccine of porcine epidemic diarrhea, porcine transmissible gastroenteritis and porcine delta coronavirus

[0058] 1. Materials and methods

[0059] 1.1 cells

[0060] Vero cells and ST cells were purchased from China Veterinary Drug Administration.

[0061] 1.2 Preparation of virus liquid for seedling production

[0062] Preparation of Porcine Epidemic Diarrhea Virus Seeds: The virus was prepared by the spinner bottle cell culture method. Inoculate Vero cells covered with a 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.

[0063] Preparation of Porcine Tran...

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Abstract

The invention provides a triple inactivated vaccine for porcine epidemic diarrhea (PED), porcine transmissible gastroenteritis (TGE) and porcine delta coronavirus (PDCoV) and a preparation method of the triple inactivated vaccine, before inactivation, the contents of the three viruses are greater than or equal to 10<7.0>TCID50 / mL, and after inactivation, the volume ratio of the antigens is 1 to 1to 1. With the triple inactivated vaccine disclosed by the invention, the problem that an effective multivalent vaccine for preventing and treating three diseases including porcine epidemic diarrhea (PED), porcine transmissible gastroenteritis (TGE) and porcine delta coronavirus (PDCoV) is not available on the market is solved, especially, the problem that the vaccine for the porcine delta coronavirus (PDCoV) epidemic in recent years is not available is solved. The triple inactivated vaccine provided by the invention is economical and practical, the immunizing procedure is simplified, the epidemic prevention cost can be effectively reduced, and a novel method for simultaneously preventing the occurrence of the three diseases is provided for domestic breeding enterprises.

Description

technical field [0001] The invention relates to an animal multiple inactivated vaccine and a preparation method thereof, in particular to a triple inactivated vaccine of porcine epidemic diarrhea, porcine transmissible gastroenteritis 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 transmissible gastroenteritis (Transmissible gastroenteritis, TGE) is also an important contact viral intestinal infectious disease that causes pig diarrhea. Although the olde...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/215A61K39/225A61P31/14
CPCA61K39/12A61K2039/5252A61K2039/70C12N2770/20034A61K2300/00
Inventor 董剑辉陈瑞张磊
Owner YULIN UNIV
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