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Porcine epizootic diarrhea, transmissible gastroenteritis and porcine deltacoronavirus triple subunit vaccine

A porcine epidemic diarrhea and triple vaccine technology, applied in the field of veterinary biological products, can solve the problems of the epidemic situation of recombinant and mutant field viruses, limited clinical application effect, and increased purification difficulty, and achieves good immunogenicity, high safety, and high efficiency. The effect of a small dose of immunity

Active Publication Date: 2018-04-13
陕西诺威利华生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, foreign countries mainly use attenuated vaccines from South Korea, domestically mainly TGE, PED and rotavirus triple attenuated live vaccines developed by Harbin Animal Research, TGE-PED dual attenuated live vaccines from Dabeinong Company, and TGE from Wuhan Keqian Company. - Vaccines such as PED dual inactivated vaccines, but the clinical application effect is limited; in addition, there is a risk of dispersing the virus in the production of attenuated vaccines and inactivated vaccines. On the one hand, it may cause recombination and mutation of epidemic strains and vaccine strains, complicating the field virus epidemic situation, and further increasing the difficulty of purifying the above-mentioned diseases. The continued healthy development of the

Method used

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  • Porcine epizootic diarrhea, transmissible gastroenteritis and porcine deltacoronavirus triple subunit vaccine
  • Porcine epizootic diarrhea, transmissible gastroenteritis and porcine deltacoronavirus triple subunit vaccine
  • Porcine epizootic diarrhea, transmissible gastroenteritis and porcine deltacoronavirus triple subunit vaccine

Examples

Experimental program
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Embodiment 1

[0023] Example 1: Screening of Porcine Epidemic Diarrhea Virus (PEDV) S1 Gene, Porcine Transmissible Gastroenteritis Virus (TGEV) S1 Gene and Porcine Dicoronavirus (PDCoV) S1 Gene

[0024] Since 2010, a large number of piglets with diarrhea have appeared in many provinces and cities in mainland China, and some affected pigs have been injected with the diarrhea vaccine that was available in the market at that time. Therefore, it is speculated that the field virus is a mutated strain. After the detection of clinical samples in 2016, different porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus and porcine D-coronavirus were isolated from the diseased pigs for whole genome sequencing and genetic evolution analysis ( image 3 , Figure 5 and Figure 7 ), the PEDV KB2013-4 strain, the TGEV BD strain and the PDCoV SD strain were selected as reference strains of the current field strains; among them, the PEDVKB2013-4 strain (Porcine epidemic diarrhea virus) ...

Embodiment 2

[0028] Embodiment 2: Preparation of recombinant porcine epidemic diarrhea virus S1 protein, porcine transmissible gastroenteritis virus S1 protein and porcine D-coronavirus S1 protein,

[0029] The method comprises the following steps: a. constructing an expression vector; b. constructing an expression strain; c. inducing, extracting and purifying recombinant porcine epidemic diarrhea virus S1 protein, porcine transmissible gastroenteritis virus S1 protein and porcine D-coronavirus S1 protein. The details are as follows: the positive clone plasmid pEASY-T1-PEDV-S1 or pEASY-T1-TGEV-S1 or pEASY-T1-PDCoV-S1 and the expression vector pPICZα vector were digested with Bam HI and Hind III respectively, and passed through 1.0% agar After sugar gel electrophoresis, purify and recover with a DNA gel recovery kit to obtain 1.9 kb or 1.9 kb or 1.6 kb and 3.3 kb fragments, respectively, and construct pPICZα-PEDV-S1 or pPICZα-TGEV-S1 or pPICZα by directional ligation at 16°C - PDCoV-S1 expr...

Embodiment 3

[0040] Embodiment 3: the preparation of porcine epidemic diarrhea, porcine transmissible gastroenteritis, porcine D-type coronavirus disease triple subunit vaccine

[0041] The semi-finished PEDV-S1, TGEV-S1 and PDCoV-S1 protein antigens after passing the inspection are used for vaccine preparation (the liquid components in the following preparations are calculated by volume ratio).

[0042] 1 Adjuvant Preparation Disperse 0.5% volume Span-85 in 5% volume squalene phase, mix with 0.5% volume Tween-80 into 94% volume 10mM sodium citrate buffer, then mix at high speed A crude emulsion is obtained. This coarse emulsion is then repeatedly passed through a high-pressure homogenizer (1200bar, 6-8 cycles) to produce an emulsion with uniform oil droplet size; the emulsion is filtered through a 0.22μm filter membrane to remove large oil droplets to obtain an average Droplet size remained unchanged for at least 3 years at 4°C.

[0043] 2 Antigen preparation Porcine epidemic diarrhea v...

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Abstract

The invention provides a porcine epizootic diarrhea, transmissible gastroenteritis and porcine deltacoronavirus triple subunit vaccine, which consists of an antigen and a vaccine adjuvant, wherein theantigen includes porcine epizootic diarrhea virus S1 protein which is expressed by an X33-PEDV-S1 strain that preservation number of CGMCC No.14372, transmissible gastroenteritis virus S1 protein which is expressed by an X33-TGEV-S1 strain that preservation number is CGMCC No.14371 and porcine deltacoronavirus S1 protein which is expressed by an X33-PDCoV-S1 strain that preservation number is CGMCC No.14370. The porcine epizootic diarrhea, transmissible gastroenteritis and porcine deltacoronavirus triple subunit vaccine provided by the invention is high in safety and good in immunogenicity, and the triple vaccine, after being applied to immunization, can rapidly generate an antibody and can keep antibody titer at a relatively high level for a long time; the vaccine is long in preservationperiod and low in immunizing dose; the adopted adjuvant is easy to inject; and three major diarrhea-related viral diseases can be prevented and treated by conducting injection once, so that the survival rate of piglets is improved.

Description

technical field [0001] The invention belongs to the technical field of veterinary biological products, in particular to a triple subunit vaccine for porcine epidemic diarrhea, porcine transmissible gastroenteritis and porcine D-coronavirus disease Background technique [0002] Porcine epidemic diarrhea (Porcineepidemicdiarrhea, PED) is a highly contagious intestinal infectious disease of pigs caused by porcine epidemic diarrhea virus. It is characterized by diarrhea, vomiting and loss of appetite. The disease is susceptible to pigs of different ages. Infection, especially in suckling piglets, is the most serious and is a highly contagious infectious disease. The disease first appeared in the United Kingdom in 1971, and appeared in my country for the first time in 1986. Since 2010, porcine epidemic diarrhea has broken out in the whole country, and the popular field strains have mutated compared with the strains before 2010, which has caused huge economic losses to the breedi...

Claims

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Application Information

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IPC IPC(8): A61K39/215A61K39/225A61P31/14
CPCA61K39/12A61K2039/5252A61K2039/55566A61K2039/70C12N2770/20034A61K2300/00
Inventor 刘灿张磊陈瑞
Owner 陕西诺威利华生物科技有限公司
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