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Porcine epizootic diarrhea virus strain and vaccine composition, preparation method and application thereof

A technology for porcine epidemic diarrhea and virus strains, applied in the field of biomedicine, can solve the problems of not being able to resist epidemic strains well, and the immune effect of vaccines is not ideal, and achieve high immune efficacy and safety, good immunogenicity and The effect of stability

Active Publication Date: 2016-11-23
PU LIKE BIO ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the above-mentioned similarities and differences in neutralizing epitopes, although the vaccines of traditional strains have certain cross-protection against the current epidemic strains, they cannot resist the epidemic strains well.
[0004] The use of vaccines for immunization prevention is the fundamental means to control porcine epidemic diarrhea at home and abroad, and the commercial vaccines sold in the market are mainly porcine epidemic diarrhea-porcine transmissible gastroenteritis dual inactivated vaccines / live vaccines, and porcine epidemic diarrhea -Porcine transmissible gastroenteritis-porcine rotavirus triple inactivated vaccine / live vaccine, the application of these vaccines has played a certain preventive effect on porcine epidemic diarrhea, but due to the mutation of porcine epidemic diarrhea virus S gene, the existing The vaccine cannot completely prevent the occurrence of the disease, which leads to the unsatisfactory immune effect of the vaccine, and porcine epidemic diarrhea continues to break out in China and some countries and regions around the world

Method used

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  • Porcine epizootic diarrhea virus strain and vaccine composition, preparation method and application thereof
  • Porcine epizootic diarrhea virus strain and vaccine composition, preparation method and application thereof
  • Porcine epizootic diarrhea virus strain and vaccine composition, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1 Isolation and Proliferation of Porcine Epidemic Diarrhea Virus HN1303 Strain

[0073] 1 Virus isolation

[0074] Cut the clinically collected small intestine with typical porcine epidemic diarrhea together with the intestinal contents with sterile scissors, add sterile PBS (pH 7.4) at a mass volume ratio of 1:3, and use a sterile grinder on ice Be careful to grind completely. The ground liquid was collected and centrifuged at 10000rpm at 4°C for 15min. Take the supernatant after centrifugation, filter it with a 0.22 μm filter, take 1ml of the filtrate and inoculate Vero cells (25T cell flask), absorb at 37°C for 2h, then add maintenance solution to an appropriate volume, and place at 37°C in 5% CO 2 For culture, 80% of the maintenance solution was replaced every day. After 6 days of culture, the virus solution was harvested and frozen at -20°C. Obvious cytopathic changes were seen after 5 generations of continuous blind passage.

[0075] Specific primers we...

Embodiment 2

[0085] Example 2 Sequence Analysis of Porcine Epidemic Diarrhea Virus HN1303 Strain

[0086] According to the sequence design of PEDV CV777 strain (AF353511) published in GenBank, specific primers are used to amplify the isolate of the present invention. Since the S gene is longer, it is artificially divided into 3 parts (S1, S2, S3) for amplification , the primers are listed in Table 1 below:

[0087] Table 1 S gene amplification primer sequence

[0088]

[0089] Sequence comparison results show that compared with vaccine strains such as CV777, DR13, 83P-5, this isolate inserts "NQGV" at aa 58-61 at the N-terminal of the S protein, inserts "N" at aa140, and deletes at aa165-166 "GK". Among the 4 known neutralizing epitopes of the S protein, 2 neutralizing epitopes, SS2 and 2C10 epidemic strains, are completely consistent with the CV777 vaccine strain, namely "YSNIGVCK" and "GPRLQPY"; the other 2 neutralizing epitopes There are differences between the circulating strain ...

Embodiment 3

[0091] Example 3 Preparation of HN1303 Strain PEDV Inactivated Vaccines

[0092]1. The 15th generation of strain PEDV isolate HN1303 was isolated in the laboratory.

[0093] 2. Purity test of PEDV strain

[0094] Virus strains should be free from bacteria, mold (in accordance with the appendix of the Regulations of the People’s Republic of China on Veterinary Biological Products, hereinafter referred to as page 50 of the appendix), mycoplasma (page 53 of the appendix), and other foreign virus contamination (page 54 of the appendix).

[0095] 3. The preparation process of PEDV inactivated vaccine:

[0096] 3.1 The cells used for subculture of PEDV are African green monkey kidney cells (Vero cells, purchased from Shanghai Institute of Biochemistry). Take 1ml of the virus solution and dilute it 100 times, inoculate it into Vero cells that have grown into a good monolayer, absorb at 37°C for 2 hours, add maintenance solution to an appropriate volume, and place at 37°C in 5% CO ...

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Abstract

The invention discloses a porcine epizootic diarrhea virus strain with good immunogenicity and an inactivated vaccine prepared through the porcine epizootic diarrhea virus strain. The porcine epizootic diarrhea virus strain is a current epidemic strain, and good immunogenicity and stability are achieved; compared with a commercially available vaccine strain, the vaccine prepared through the strain has the advantages of being good in safety, high in immune protective capability, high in immune efficacy and the like, and porcine epizootic diarrhea can be comprehensively and effectively prevented and treated.

Description

technical field [0001] The invention relates to a porcine epidemic diarrhea virus strain, and also relates to a vaccine composition prepared by the virus strain, a preparation method and application of the vaccine composition, and belongs 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, especially suckling piglets suffer the most. 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] PEDV is a member of the genus Coronavirus of the Coronviridae family. The PEDV genome is a single-stranded positive-strand infectious RNA with a cap structure (cap) at the 5' end of the genome and a Poly(A) tail at the 3' end. The full leng...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/50C07K14/165A61K39/215A61P31/14A61P1/12
Inventor 田克恭张许科孙进忠高晓静
Owner PU LIKE BIO ENG
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