Porcine epidemic diarrhea virus attenuated strain, vaccine composition prepared therefrom, and application

A porcine epidemic diarrhea and vaccine composition technology, which is applied in antiviral agents, viruses/bacteriophages, biochemical equipment and methods, etc., can solve the problems of inability to resist the attack of epidemic strains, high cost, and limited immune protection effect, etc. question

Active Publication Date: 2017-04-05
PU LIKE BIO ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently live vaccines use classic strains such as domestic CV777 strain, Korean DR13 strain, Japanese 83P-5 strain, etc., which are quite different from popular strains. For the current field strains, their immune protection effect is limited and cannot be well tested. Resist attacks from prevalent strains
For inactivated vaccines, although there are vaccines prepared by adopting epidemic strains such as AJ1102 strains, their ability to stimulate the body to produce cellular immunity and mucosal immune responses is worse than that of live viruses, and the immune efficacy needs to be improved; A large number of strong viruses need to be cultivated, and the production environment needs to be strictly controlled to prevent the spread of strong viruses; the cost of preparing inactivated vaccines is much higher than that of attenuated live vaccines

Method used

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  • Porcine epidemic diarrhea virus attenuated strain, vaccine composition prepared therefrom, and application
  • Porcine epidemic diarrhea virus attenuated strain, vaccine composition prepared therefrom, and application
  • Porcine epidemic diarrhea virus attenuated strain, vaccine composition prepared therefrom, and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach

[0020] As an embodiment of the present invention, the nucleotide fragment encoding the last 9 amino acids EAFEKVHVQ in the S gene coding sequence of the attenuated porcine epidemic diarrhea virus strain of the present invention is deleted or terminated in translation.

[0021] As an embodiment of the present invention, the nucleotide fragment encoding the last 9 amino acids EVFEKVHVQ in the S gene coding sequence of the attenuated porcine epidemic diarrhea virus strain of the present invention is deleted or terminated in translation.

[0022] The attenuated strain of porcine epidemic diarrhea virus of the present invention can be subcultured and attenuated, and the nucleotide fragment encoding the last 9 amino acids of the S gene can be deleted or terminated by continuous passage on the passaged cells, or can be mutated or terminated by genetic engineering. Recombination deletes or terminates translation of the nucleotide fragment encoding the last 9 amino acids of the S gene. ...

Embodiment 1

[0097] Embodiment 1: Isolation, detection and propagation of porcine epidemic diarrhea virus strain HN1301 strain

[0098] 1. Isolation of porcine epidemic diarrhea virus HN1301 strain

[0099] The small intestine with the typical onset of porcine epidemic diarrhea collected clinically in Henan, together with the intestinal contents, was cut into pieces with sterile scissors, and sterile PBS (pH 7.4) was added at a mass-volume ratio of 1:3. Grind carefully on ice to complete. Collect the ground liquid and perform gradient centrifugation step by step according to the following steps: centrifuge at 3000rpm, 4°C for 15min, then centrifuge at 5000rpm, 4°C for 15min, then centrifuge at 7000rpm, 4°C for 15min, and finally centrifuge at 10000rpm, 4°C for 15min. Take the supernatant after centrifugation, inoculate Vero cells, and place the inoculated Vero cells at 37°C, 5% CO 2 Cultivate, harvest and freeze at -20°C after 3 days, and the harvested virus liquid is isolated porcine ep...

Embodiment 2

[0109] Embodiment 2: Virulence determination test of porcine epidemic diarrhea virus HN1301 strain

[0110] 1. Test animals

[0111] Blood samples from pregnant sows were used to detect PEDV antigen, neutralizing antibody and TGEV, and piglets born to sows negative for PEDV antigen, antibody and TGEV were selected for this experiment, and the piglets did not eat colostrum.

[0112] 2. Challenge test

[0113] 20 piglets above the age of 3 days without colostrum were selected and divided into 4 groups, 5 piglets in each group. The first group was orally inoculated with 4ml of HN1301 virus solution cultivated in Example 1; the second group was orally inoculated with 2ml of HN1301 virus solution; the third group was orally inoculated with 1ml of HN1301 virus solution;

[0114] 3. Clinical observation and pathological analysis

[0115] The clinical symptoms of piglets after challenge were observed every day. If the pigs infested with the poisonous virus were killed, an autopsy ...

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Abstract

The invention relates to a porcine epidemic diarrhea virus attenuated strain. The porcine epidemic diarrhea virus attenuated strain is a nucleotide fragment or termination translator in a porcine epidemic diarrhea virus S gene encoding sequence, for encoding last nine amino acids EAFEKVHVQ or a homologous fragment thereof. The porcine epidemic diarrhea virus attenuated strain is a porcine epidemic diarrhea virus epidemic strain attenuated strain, and has the advantages of substantially reduced pig pathogenicity, no return after pig immunization, good immunogenicity and realization of effective resistance of immunized pigs to virulent attack. The invention also relates to a vaccine composition obtained by adopting the porcine epidemic diarrhea virus attenuated strain as a live virus antigen, a porcine epidemic diarrhea virus attenuated strain mutation S protein, and a preparation method of the porcine epidemic diarrhea virus attenuated strain.

Description

technical field [0001] The invention belongs to the field of veterinary vaccines, and in particular relates to an attenuated strain of porcine epidemic diarrhea virus, a prepared vaccine composition and an application thereof. 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, especially suckling piglets are the most affected. The disease has become a serious problem in the development of the pig breeding industry. Since 2012, the disease has broken out rapidly worldwide, causing serious economic losses. [0003] After infection, the sick piglets often show vomiting, rapid diarrhea and dehydration, and the diarrhea feces are yellow or gray-yellow water samples. The body temperature is basically normal, and the body temperature of the piglets frequently drops before death. Nursery pig...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A61K39/215A61P31/14C12R1/93
Inventor 田克恭吕超超孙进忠张许科
Owner PU LIKE BIO ENG
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