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Separation of porcine deltacoronavirus strain, preparation and application of inactivated vaccine

A coronavirus and inactivated vaccine technology, which is applied in the field of animal virology and zoonosis, can solve the problem of pathogenicity that has not been studied in depth, and achieve the effect of wide application range, good immunogenicity and good safety performance

Inactive Publication Date: 2018-03-20
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2011, Hong Kong scholars detected PDCoV in pig feces for the first time when conducting molecular epidemiological investigations on poultry and mammals, but did not conduct in-depth studies on its pathogenicity (Woo, P.C., et al., Discovery of seven novel Mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus. J Virol,2012,86(7):3995-4008)
At present, there is no particularly effective prevention and treatment method for the newly-occurring porcine delta coronavirus, so the development of PDCoV vaccine is imminent

Method used

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  • Separation of porcine deltacoronavirus strain, preparation and application of inactivated vaccine
  • Separation of porcine deltacoronavirus strain, preparation and application of inactivated vaccine
  • Separation of porcine deltacoronavirus strain, preparation and application of inactivated vaccine

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Embodiment 1

[0018] Embodiment 1: the acquisition of porcine delta coronavirus PDCoV CHN-HN-2014 strain

[0019] 1. Collection and processing of disease materials:

[0020]The contents of the small intestine were collected from piglets with severe diarrhea that were submitted to a pig farm in Henan Province, China, diluted with 10 times the volume of DMEM medium, fully homogenized and shaken, and centrifuged at 4200×g for 10 minutes at 4°C. The supernatant was sterilized by filtration with a 0.45-micron microporous membrane, and then used as an inoculum and frozen at -70°C for later use.

[0021] 2. RT-PCR detection of disease materials:

[0022] Get 200 microliters of the inoculum obtained in step 1, use the Trizol method (the kit is purchased from Treasure Bioengineering Dalian Co., Ltd., and operate according to the kit instructions) to extract total RNA, and use the RNA PCR Kit to reverse transcribe the extracted total RNA into After the cDNA was amplified by PCR, the upstream and do...

Embodiment 2

[0033] Embodiment 2: the application of porcine delta coronavirus CHN-HN-2014 strain in the preparation of porcine delta coronavirus inactivated vaccine:

[0034] 1. Take PDCoV CHN-HN-2014 strain (preservation number: CCTCC NO: V201650) and inoculate it into LLC-PK1 cells (purchased from ATCC cell bank, USA). TCID 50 Assay method Determine virus titer and adjust virus content to 10 8.0 TCID 50 / ml, inactivated with formaldehyde at a final concentration of 0.1%, mixed with the same quality of Montanide ISA 201 (purchased from SEPPIC, France) adjuvant, fully stirred and emulsified under sterile conditions, the emulsification temperature is 31 ° C, emulsified The time is 15 minutes. After the emulsification is complete, it is aseptically subpackaged and sealed for future use.

[0035] (1) Safety inspection of porcine delta coronavirus inactivated vaccine

[0036] Test method: choose 6 healthy piglets of age of 3-5 days, each piglet intramuscularly injects 2ml of porcine delta...

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Abstract

The invention belongs to the technical field of animal virology and epizootiology, particularly relates to separation of a porcine deltacoronavirus strain, preparation and application of an inactivated vaccine, and discloses a separating cultivation method of the porcine deltacoronavirus strain, preparation and application of the inactivated vaccine. According to the invention, the separated porcine deltacoronavirus CHN-HN-2014 strain is preserved in China Center for Type Culture Collection with the preservation number of CCTCC NO: V201650. The preserved inactivated vaccine prepared by porcinedeltacoronavirus has good immunogenicity, can induce piglets to generate neutralizing antibody with higher level, and can effectively protect immunized piglets from attack of the porcine deltacoronavirus.

Description

technical field [0001] The invention belongs to the technical field of animal virology and animal infectious disease. It specifically relates to the isolation of porcine delta coronavirus strains, the preparation and application of inactivated vaccines. Background technique [0002] Porcine deltacoronavirus (PDCoV) is a newly discovered porcine enteric coronavirus that can cause diarrheal disease in piglets. In 2011, Hong Kong scholars detected PDCoV in pig feces for the first time when conducting molecular epidemiological investigations on poultry and mammals, but did not conduct in-depth studies on its pathogenicity (Woo, P.C., et al., Discovery of seven novel Mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus. J Virol,2012,86(7):3995-4008). In February 2014, a large-scale diarrheal disease ...

Claims

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Application Information

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IPC IPC(8): C12N7/00A61K39/215A61P31/14C12R1/93
CPCA61K39/12A61K2039/5252A61K2039/552C12N7/00C12N2770/20021C12N2770/20034
Inventor 方六荣肖少波董楠王荡
Owner HUAZHONG AGRI UNIV
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