Porcine epidemic diarrhea virus inactivated vaccine and preparation method thereof

A porcine epidemic diarrhea and virus inactivation technology, applied in biochemical equipment and methods, vaccines, viruses, etc., can solve problems such as poor treatment effect, improve antibody production level, high antibody titer, and small immune dose. Effect

Active Publication Date: 2017-08-18
陕西诺威利华生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, there is no specific drug for the treatment of porcine epidemic diarrhea, and the effect of conventional treatment is not good. Therefore, vaccine prevention is still the main method, and the success of the virus strain and its cultivation is the key factor in the development of the vaccine.

Method used

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  • Porcine epidemic diarrhea virus inactivated vaccine and preparation method thereof
  • Porcine epidemic diarrhea virus inactivated vaccine and preparation method thereof
  • Porcine epidemic diarrhea virus inactivated vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Isolation and Identification of Porcine Epidemic Diarrhea Virus PEDV-KB2013-4 Strain

[0035] (1) Isolation of porcine epidemic diarrhea virus PEDV-KB2013-4 strain

[0036] During the epidemiological investigation, a pig farm in Shaanxi sent the small intestine of piglets with diarrhea for inspection, scraped the intestinal mucosa and contents, added PBS at a ratio of 1:5 (weight: volume), repeated freezing and thawing 3 times, and centrifuged to obtain the supernatant. Filter through a 0.22 μm filter membrane, add trypsin with a final concentration of 20 μg / ml to the filtrate, and treat at 37° C. for 1.5 hours.

[0037] Inoculate Vero cells with a monolayer according to the conventional method (wash three times with PBS of pH 7.4 before inoculation), inoculate the virus at a ratio of 10%, absorb at 37°C for 1 hour, and supplement the cell maintenance solution (containing 10 μg / ml trypsin) ), cultivated in a 37°C incubator. In this way, the cells were blind...

Embodiment 2

[0050] Embodiment 2: Prepare vaccine with PEDV-KB2013-4 strain

[0051] (1) Preparation of PEDV vaccine

[0052] a. Prepare Vero cells according to conventional methods (Vero cells are purchased from ATCC, USA), the growth medium is DMEM containing 10% newborn bovine serum, and inoculated with PEDV-KB2013-4 virus when the cells grow into a monolayer;

[0053] b. Take the PEDV-KB2013-4 strain (5×10 5 TCID 50 / ml) inoculated well-growing Vero monolayer cells at 1%, placed at 37°C for 1 hour, discarded the adsorption solution, and added DMEM containing 10 μg / mL trypsin as the maintenance solution to continue the culture;

[0054] c. After inoculation, observe twice a day and record the cell lesions. Harvest the virus when the cell lesions reach more than 80%. Freeze and thaw the harvested virus three times at -80°C, centrifuge at 6000 rpm for 20 min, and collect the supernatant The liquid is the virus liquid;

[0055] d. After the virus liquid was concentrated 20 times, it wa...

Embodiment 3

[0066] Example 3 Safety Test of Porcine Epidemic Diarrhea Virus PEDV-KB2013-4 Strain Inactivated Vaccine

[0067] 1. Materials and methods

[0068] 1.1 Single-dose test on pregnant sows

[0069] Ten pregnant sows that were negative for neutralizing antibodies and antigens in porcine epidemic diarrhea 5-6 weeks before delivery were randomly divided into 2 groups with 5 pigs in each group. The first group was intramuscularly injected with 20140101 batches of porcine epidemic diarrhea virus PEDV- The KB2013-4 strain inactivated vaccine was 1 dose per head, and the second group was not injected as the control group, and the sows were observed until the sows gave birth.

[0070] 1.2 Single-dose repeated test on pregnant sows

[0071] Ten pregnant sows that were negative for neutralizing antibodies and antigens in porcine epidemic diarrhea 5-6 weeks before delivery were randomly divided into 2 groups with 5 pigs in each group. The first group was intramuscularly injected with 201401...

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Abstract

The invention provides a porcine epidemic diarrhea virus inactivated vaccine. The porcine epidemic diarrhea virus inactivated vaccine contains the inactivated porcine epidemic diarrhea virus and an adjuvant, wherein the adjuvant is prepared from the following components in percentage by weight: 5% of squalane, 1% of oleic acid, 1% of Tween 80, 92% of 0.005M sodium citrate buffer solution, and 1% of beta-glucan, the porcine epidemic diarrhea virus is prepared by inactivating the porcine epidemic diarrhea virus strain PEDV-KB2013-4, is assigned with the microbial accession number of CGMCC No.12663, has the classification name of Porcine Epidemic Diarrhea Virus (PEDV), and is preserved in the China General Microbiological Culture Collection Center (CGMCC) on Aug 23, 2016, and the preservation address is the Institute of Microbiology of Chinese Academy of Sciences located in 3, Courtyard 1, West Beichen Road, Chaoyang District, Beijing.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to the field of veterinary biopharmaceuticals, and relates to an inactivated porcine epidemic diarrhea virus vaccine and a preparation method thereof. Background technique [0002] Porcine Epidemic Diarrhea (PED) is an acute, contagious viral intestinal infectious disease caused by Porcine Epidemic Diarrhea Virus (PEDV). Declining and dehydration are the common intestinal infectious diseases of pigs. Porcine epidemic diarrhea virus (PEDV) is a member of the Coronaviridae family Coronaviridae. Porcine epidemic diarrhea virus (PEDV) directly enters the small intestine after being infected by the mouth and nose, and proliferates in the epithelial cells of the small intestine, causing lesions, damage, Shedding and atrophy of small intestinal villi lead to decreased intestinal enzyme activity, impaired absorption, increased osmotic pressure in the intestinal tract, and osmotic diarrhea, whi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/215A61K39/39A61K38/20A61P31/14A61P37/04C12N7/06
CPCA61K38/2026A61K39/12A61K39/39A61K2039/5252A61K2039/545A61K2039/552A61K2039/55511A61K2039/55583C12N7/00C12N2770/20063C12N2770/22034A61K2300/00
Inventor 张磊薛青红陈瑞孙丰廷戚伟强
Owner 陕西诺威利华生物科技有限公司
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