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Porcine epizootic diarrhea virus strain, attenuated vaccine strain thereof and application thereof

A technology for porcine epidemic diarrhea and virus strains, which is applied in the field of biomedicine and can solve the problems of unsatisfactory vaccine immune effect and virus strain immune protection.

Active Publication Date: 2015-04-15
PU LIKE BIO ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, due to the mutation of the S gene of the currently circulating porcine epidemic diarrhea virus strains, the existing vaccines are not enough to produce comprehensive immune protection against the current circulating strains, resulting in a less than ideal immune effect of the vaccines. Porcine epidemic diarrhea virus in China Even some countries and regions around the world continue to erupt

Method used

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  • Porcine epizootic diarrhea virus strain, attenuated vaccine strain thereof and application thereof
  • Porcine epizootic diarrhea virus strain, attenuated vaccine strain thereof and application thereof
  • Porcine epizootic diarrhea virus strain, attenuated vaccine strain thereof and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Isolation, Detection and Propagation of Porcine Epidemic Diarrhea Virus HN1301 Strain

[0052] 1. Isolation of porcine epidemic diarrhea virus HN1301 strain

[0053] The small intestine of the typical porcine epidemic diarrhea collected from the clinic in Henan, together with the intestinal contents, was cut into pieces with sterile scissors, and sterile PBS (pH7.4) was added at a mass-volume ratio of 1:3. Grind carefully on ice to complete. The ground liquid was collected and subjected to gradient centrifugation according to the following steps: 3000rpm at 4°C for 15min, 5000rpm at 4°C for 15min, 7000rpm at 4°C for 15min, and 10000rpm at 4°C for 15min. The supernatant after centrifugation was taken, inoculated with Vero cells, and placed at 37°C, 5% CO 2 Cultivate, harvest and freeze at -20°C after 3 days, and the harvested virus liquid is the isolated porcine epidemic diarrhea virus HN1301 strain.

[0054] The porcine epidemic diarrhea virus strain is na...

Embodiment 2

[0064] Example 2: Sequence Analysis of Porcine Epidemic Diarrhea Virus HN1301 Strain

[0065]According to the sequence of the PEDV CV777 strain (AF353511) published in GenBank, a pair of primers were designed for amplifying the porcine epidemic diarrhea virus HN1301 strain of the present invention. The primers are as follows:

[0066] Upstream: 5'-GGTAAGTTGCTAGTGCGTAA-3'

[0067] Downstream: 5'-CACAGAAAGAACTAAACCC-3'

[0068] The analysis of the sequencing results shows that the total length of the genome of the isolate HN1301 described in the present invention is 28047 nt, and the full length of the S1 gene is 762 bp.

[0069] The genetic relationship between the isolated strains of the present invention and the Korean strains from 2007 to 2009, the Thai strains from 2007 to 2008, the Vietnamese strains from 2009 to 2010, the Japanese strains from 2011 and the popular strains from 2010 to 2012 in other regions of China close, but with European strains (CV777, Brl / 87), curre...

Embodiment 3

[0070] Embodiment three: the virulence assay test of porcine epidemic diarrhea virus HN1301 strain

[0071] 1. The PEDV strain is the porcine epidemic diarrhea virus HN1301 strain isolated in Example 1.

[0072] 2. Cell line Vero cells, from Shanghai Institute of Biochemistry.

[0073] 3. Virus Culture

[0074] Select a well-grown Vero cell monolayer, discard the culture medium, and inoculate the porcine epidemic diarrhea virus HN1301 strain virus solution isolated in Example 1 by 1% of the maintenance solution, and place it at 37°C and 5% CO 2 Culture in the incubator and observe the cytopathic changes (CPE) every day until 48-72 hours. When the CPE reaches more than 80%, it can be harvested. After 2 times of freezing and thawing, store at -20°C.

[0075] Passage the above porcine epidemic diarrhea virus HN1301 strain on Vero cells, take the 11th passage virus liquid, and carry out the challenge test on animals.

[0076] 4. The method of challenge test

[0077] 4.1 Strain...

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Abstract

The invention discloses a porcine epizootic diarrhea virus strain (HN1301) and an attenuated vaccine strain (HN1302) attenuated by passage of the porcine epizootic diarrhea virus. The attenuated vaccine strain of the porcine epizootic diarrhea virus is assigned the accession number CCTCC-V201342. The attenuated vaccine strain (HN1302) is good in safety, is strong in immune protective capability and is high in immune efficacy.

Description

technical field [0001] The invention relates to a virulent strain of porcine epidemic diarrhea virus HN1301, an attenuated vaccine strain HN1302 which is attenuated by the passage of the virulent strain HN1301, and also relates to a vaccine composition prepared from the attenuated strain of porcine epidemic diarrhea virus HN1302 and the The preparation method and application of the vaccine composition belong to the field of biomedicine. Background technique [0002] Porcine epidemic diarrhea (PED), caused by porcine epidemic diarrhea virus (PEDV), is an intestinal infectious disease of pigs characterized by diarrhea, vomiting and dehydration. Pigs of all ages are susceptible, and the incidence rate of suckling piglets, shelf pigs or fattening pigs can reach 100%, especially suckling piglets suffer the most. Infection with this virus has become a serious problem in the development of pig farming industry. The disease has broken out in many countries around the world and caus...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/50C12N7/00C12N7/08C07K14/165A61K39/215A61P31/14C12R1/93
Inventor 张许科孙进忠田克恭
Owner PU LIKE BIO ENG
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