Porcine epizootic diarrhea virus variant strain and application thereof

A porcine epidemic diarrhea and virus technology, applied in the field of microbiology, can solve the problems of detoxification, high fatality rate, and different effects, and achieve the effects of rapid antibody production, improved immune effect, and high safety

Active Publication Date: 2018-09-11
HENAN CENT FOR ANIMAL DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Since 2014, the epidemic of porcine epidemic diarrhea in my country is still serious. It mainly harms newborn piglets and has a high mortality rate, which has brought huge losses to the pig industry. Control the disease, but the effect is different, it is easy to cause detoxification and detoxification

Method used

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  • Porcine epizootic diarrhea virus variant strain and application thereof
  • Porcine epizootic diarrhea virus variant strain and application thereof
  • Porcine epizootic diarrhea virus variant strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] This example is used to illustrate the isolation and identification of PEDV-HNCADC-2017 strain.

[0044] 1. Main reagents

[0045] African green monkey kidney cells (vero) were purchased from China Center for Type Culture Collection of Wuhan University; cell culture medium DMEM was purchased from Boster Biological Company; fetal bovine serum was purchased from Sera Company; viral genome DNA / RNA rapid extraction kit was purchased from Axygen Biotechnology Co., Ltd.; Competent strains were purchased from Dalian Bao Biological Company; penicillin and streptomycin were purchased from Suleibao Company.

[0046] 2. Sample Processing

[0047] Take the feces samples or intestinal contents of piglets with diarrhea that are positive for PEDV fluorescent quantitative PCR, prepare a 1:5 suspension with DMEM, shake and mix, use a homogenizer for 5 minutes, and use a homogenizer at 10,000 r / min at 4°C Centrifuge for 10 minutes, take the supernatant and filter it with a 0.22 μm filt...

Embodiment 2

[0079] This example is used to illustrate the application of the PEDV-HNCADC-2017 strain, which is embodied in the preparation of a live attenuated porcine epidemic diarrhea vaccine by using the PEDV-HNCADC-2017 strain.

[0080] 1. Preparation of attenuated live vaccine

[0081] 1.1 Prepare Vero cells according to conventional methods (Vero cells are purchased from ATCC in the United States), the growth medium is MEM containing 8% newborn bovine serum, and SD10 strain virus is inoculated when the cultured cells grow into a monolayer;

[0082] 1.2 Take PEDV-HNCADC-2017 strain virus seeds (≥10 7.5 TCID 50 / 0.1mL) Inoculate a monolayer of Vero cells with good growth at 1%, place at 37°C for 1.5-2 hours, add DMEM maintenance solution containing 2% newborn bovine serum to continue culturing;

[0083] 1.3 After inoculation, observe twice a day, record the cell lesion, harvest when the cell lesion reaches more than 80% to 90%, and freeze and thaw three times repeatedly;

[0084] 1...

Embodiment 3

[0109] This example is used to illustrate the application of the PEDV-HNCADC-2017 strain, which is embodied in the preparation of porcine epidemic diarrhea inactivated vaccine by using the PEDV-HNCADC-2017 strain.

[0110] 1. Preparation of PEDV inactivated vaccine

[0111] 1.1 Virus culture and inactivation

[0112] Freeze and thaw the harvested virus cell culture solution twice, centrifuge at 6000rpm for 10 minutes, collect the supernatant, which is the virus solution, and add β-propiolactone inactivation for 36 hours according to a certain proportion to obtain the inactivated PEDV culture solution;

[0113] Verification of inactivation effect: The inactivated cell culture medium was blindly passed on VERO cells for 3 generations to observe the cell state, and there was no pathological change in the cell culture, indicating that the virus inactivation was sufficient.

[0114] 1.2 Emulsification of inactivated virus and white oil adjuvant

[0115] Adjuvant preparation: The ...

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Abstract

The invention belongs to the technical field of microbes, and particularly relates to a porcine epizootic diarrhea virus variant strain (PEDV-HNCADC-2017, preservation number: CCTCC NO:V201766) and application thereof. The variant strain provided by the invention realizes the generation passage to 156th generation in VERO cells of 10-percent blood serum medium; the TCID50 of the VERO cells can reach 107.7 / 0.1mL; the toxic valence is high; the reaction can be performed with porcine positive blood serum and Balb / c mouse positive blood serum; the immunofluorescence reaction is stronger. The prepared weak toxicity live vaccine can be used for preventing the porcine epizootic diarrhea diseases; the antibody generation is fast; the high antibody level can be generated after the immunization for2 weeks. The prepared inactivated vaccine has high safety; toxin expelling is avoided after the immunization; the strain can be used for preventing and controlling the porcine epizootic diarrhea diseases of sows and one-day-old piglets. When the combined immunization is used, the immunization effect can be obviously improved.

Description

technical field [0001] The invention belongs to the technical field of microorganisms, and in particular relates to a mutant strain of porcine epidemic diarrhea virus and an application thereof. Background technique [0002] Porcine epidemic diarrhea (Porcine epidemic diarrhea, PED) is caused by porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus, PEDV) and is characterized by diarrhea, vomiting and dehydration. The disease mostly occurs in the cold seasons of winter and spring, and suckling piglets, shelf pigs or fattening pigs and other age groups can suffer from the disease, but the suckling piglets are the most serious and the mortality rate is as high as 90% to 100%. serious damage and economic loss. In the 1970s, PED was first reported in the UK, and the occurrence and prevalence of the disease were reported successively in Belgium, Germany, Canada, Japan, Switzerland and other countries. After the 1980s, the disease began to spread in some pig-raising c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A61K39/215A61P31/14C12R1/93
CPCA61K39/12A61K2039/5252A61K2039/5254A61K2039/542A61K2039/552A61P31/14C12N7/00C12N2770/20021C12N2770/20034
Inventor 闫若潜班付国王华俊赵雪丽谢彩华马震原王淑娟刘梅芬陈慧娟赵月龙杨朋霞
Owner HENAN CENT FOR ANIMAL DISEASE CONTROL & PREVENTION
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