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Porcine epidemic diarrhea virus variant strain, and isolated culturing method and applications thereof

A porcine epidemic diarrhea and virus mutation technology, applied in the field of virus microorganisms, can solve problems such as the impact of PEDV isolation and the difficulty of in vitro isolation of PEDV

Inactive Publication Date: 2016-03-16
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The in vitro isolation of PEDV has always been relatively difficult. The source of Vero cells, the differences between strains and some details of the culture conditions will affect the isolation of PEDV.
Based on the experience of successfully isolating PEDV many times, the present invention summarizes a set of practical virus isolation and cultivation methods. At the same time, a large number of genome sequence determination and phylogenetic tree analysis found that the newly emerging epidemic strains are similar to the traditional ones such as CV777. Compared with other types of vaccine strains, large changes have taken place, making traditional vaccines unable to provide sufficient protection against new virus infections. Therefore, it is imminent to isolate new epidemic strains and prepare new vaccines on this basis , the present invention emerges at the historic moment under this background

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  • Porcine epidemic diarrhea virus variant strain, and isolated culturing method and applications thereof
  • Porcine epidemic diarrhea virus variant strain, and isolated culturing method and applications thereof
  • Porcine epidemic diarrhea virus variant strain, and isolated culturing method and applications thereof

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Embodiment 1

[0021] Embodiment 1: A variant strain of porcine epidemic diarrhea virus of the present invention:

[0022] A variant strain of porcine epidemic diarrhea virus of the present invention, the strain is named CH / ZMDZY / 11, was preserved in the General Microbiology Center of China Committee for the Collection of Microbial Cultures on June 18, 2015, and the preservation number is CGMCCNO.10893. Preservation address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0023] A method for isolating and cultivating a porcine epidemic diarrhea virus variant strain of the present invention, the detailed steps are:

[0024] a. Collection and processing of disease materials:

[0025] Collect the contents of the small intestine of piglets with watery diarrhea, dilute it with 1×PBS buffer at a volume ratio of 1:1, shake and mix well, and centrifuge at 10,000×g for 10 minutes, take the supernatant, and filter it out with...

Embodiment 2

[0044] Embodiment 2: Preparation and application of porcine epidemic diarrhea virus mutant strain CH / ZMDZY / 11 inactivated vaccine:

[0045] 1) Preparation of porcine epidemic diarrhea virus mutant strain CH / ZMDZY / 11 inactivated vaccine:

[0046] Virus culture: VERO cells are used for virus culture. When the cell growth coverage reaches 90%, the preferred multiplicity of infection is 0.1 to inoculate the virus. The preferred virus maintenance solution is 0.3% tryptone phosphate broth, 0.02% yeast extract and 5 μg / 1×DMEM medium with ml trypsin, culture at 37°C, harvest the virus liquid when the cytopathic effect reaches 80%, freeze and thaw three times at -70°C and room temperature, centrifuge at 10,000×g for 10 minutes, remove cell debris, and take the supernatant Store frozen at -70°C.

[0047] Inactivation of virus liquid: in virus liquid with qualified virus content (preferably ≥10 7.0 PFU / mL) formaldehyde was added to make the final concentration of formaldehyde reach 0....

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Abstract

The invention discloses a porcine epidemic diarrhea virus variant strain, and an isolated culturing method and applications thereof. The porcine epidemic diarrhea virus variant strain is named as CH / ZMDZY / 11, and is preserved in China General Microbiological Culture Collection Center; and preservation number is CGMCC NO.10893. The porcine epidemic diarrhea virus variant strain can be used for preparing an inactivated vaccine which can be used for preventing porcine epizootic diarrhea effectively, so that an effective control method is provided for controlling porcine epizootic diarrhea caused by porcine epidemic diarrhea virus variant strains.

Description

1. Technical field: [0001] The invention relates to the field of virus microorganisms and belongs to the field of veterinary biological products, in particular to a mutant strain of porcine epidemic diarrhea virus, its isolation and cultivation method and its application in the preparation of porcine epidemic diarrhea virus inactivated vaccine. 2. Background technology: [0002] Porcine epidemic diarrhea (porcineepidemicdiarrhea, PED) was first discovered in the UK in 1971 in newborn piglets and fattening pigs. In the 1880s, the occurrence of PED was also reported in Asia, and continued to be popular thereafter. With the widespread use of PED attenuated vaccines and inactivated vaccines, the prevalence of PED worldwide has been controlled to a certain extent within many years. However, at the end of 2010, PED broke out again in China. This time, the epidemic PED mainly affected piglets within one week of age, causing vomiting, diarrhea, dehydration and acute death of infecte...

Claims

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Application Information

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IPC IPC(8): C12N7/00A61K39/215A61P31/14C12R1/93
CPCC12N7/00A61K39/12A61K2039/54A61K2039/545C12N2770/20021C12N2770/20034C12N2770/20051
Inventor 赵军王川庆高冬生李永涛杨霞常洪涛陈陆王新卫王永生刘红英姚惠霞
Owner HENAN AGRICULTURAL UNIVERSITY
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