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Vaccine composition containing Avian egg drop syndrome virus gene engineering subunit vaccine, preparation method for vaccine composition and application of vaccine composition

A technology for egg drop syndrome and vaccine composition, applied in biochemical equipment and methods, medical preparations containing active ingredients, viruses, etc. Live incomplete biosafety and other issues, to achieve good prevention and control effect, good immunogenicity, good biosafety effect

Active Publication Date: 2021-04-20
PU LIKE BIO ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because it is difficult to obtain high-titer virus in duck embryos, the prepared vaccine is often difficult to provide the ideal immune effect; in addition, the production method of the virus antigen is completely dependent on duck eggs. When infectious diseases such as influenza occur, the supply of duck breeding eggs is difficult, which seriously affects the prevention and control of poultry egg drop syndrome
In addition, there are biosafety risks caused by incomplete virus inactivation during the production of whole virus vaccines.
[0005] Subunit vaccine is a relatively feasible new type of genetic engineering vaccine developed in recent years. The main research object of subunit vaccine research on avian egg drop syndrome is Hexon (240 / 252). However, the immune efficiency has been low for a long time. Not yet developed into a product
In the prior art, there is no avian egg drop syndrome subunit vaccine listed so far

Method used

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  • Vaccine composition containing Avian egg drop syndrome virus gene engineering subunit vaccine, preparation method for vaccine composition and application of vaccine composition
  • Vaccine composition containing Avian egg drop syndrome virus gene engineering subunit vaccine, preparation method for vaccine composition and application of vaccine composition
  • Vaccine composition containing Avian egg drop syndrome virus gene engineering subunit vaccine, preparation method for vaccine composition and application of vaccine composition

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Experimental program
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Effect test

Embodiment approach

[0038] As an embodiment of the present invention, the pharmaceutically acceptable carrier includes an adjuvant, and the adjuvant includes: (1) aluminum gel adjuvant, saponin, avridine, DDA; (2) water-in-oil emulsion , oil-in-water emulsions, water-in-oil-in-water emulsions; or (3) polymers of acrylic acid or methacrylic acid, copolymers of maleic anhydride and alkenyl derivatives; and RIBI adjuvant systems, Block co- One or more of polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, Escherichia coli heat-labile enterotoxin, cholera toxin, IMS 1314, muramyl dipeptide, Gel adjuvant kind;

[0039] Preferably, the saponin is Quil A, QS-21, GPI-0100;

[0040] Preferably, the emulsion is an SPT emulsion, an MF59 emulsion, or an emulsion formed by combining an oil with an emulsifier, the emulsion may be based on light liquid paraffin oil, isoprenoid oils such as squalane or squalene Oils, olefins, especially oils resulting from the oligomerization of isobutene or...

Embodiment 1

[0069] Construction of embodiment 1pET28a-EDSV-tFiber expression vector

[0070] 1. Extraction of EDSV virus DNA

[0071] According to the instructions of the virus DNA extraction kit, take 0.2ml of infected poultry egg drop syndrome virus HX strain (Egg Drop Syndrome Virus, Strain HX), the preservation number is CCTCC NO: V201942, and the preservation date is 2019 On June 19, 2009, the SPF duck embryo allantoic fluid stored at Wuhan University, China) was placed in a sterile 1.5ml centrifuge tube, and 0.4ml of VB was added to the sample solution, vortexed, and allowed to stand at room temperature for 10 minutes. Add 0.45ml AD buffer to the sample solution and mix vigorously. Put the VB column into a 2ml collection tube, add 0.6ml of the mixed solution to the VB column, centrifuge at 14000g for 1 minute, add the remaining mixed solution to the VB column, and centrifuge at 14000g for 1 minute, discard the 2ml collection tube, put the VB column in Put into a new 2ml collection...

Embodiment 2

[0084] The preparation of embodiment 2tFiber protein fragment

[0085]The expression strain containing pET28a-EDS-tFiber prepared in Example 1 was inoculated into LB medium containing 50-100 μg / ml kanamycin, the inoculum size was 1% (V / V), and cultured with shaking at 37°C. When OD 600 = 0.4 to 0.6, stand at 28°C for 30 minutes. Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to make the final concentration 0.1-1.0 mM, and cultured with shaking at 28°C for 24 hours. After the cultivation, the cells were collected and resuspended with PBS (sodium chloride, 8g, potassium chloride, 0.2g, disodium hydrogen phosphate, 1.44g, potassium dihydrogen phosphate, 0.24g, adjusted to pH 7.4, constant volume 1L) Bacteria were sonicated and centrifuged to obtain the supernatant. The content of soluble target protein in the expression product is high, the content of soluble tFiber protein fragment is high, the AGP titer of tFiber protein fragment reaches 1:512, and the content of endot...

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Abstract

The invention provides a nucleotide sequence represented by SEQ ID NO. 2 or an Avian egg drop syndrome virus tFiber protein fragment encoded by a degenerate sequence of the nucleotide sequence and a vaccine composition prepared from the protein fragment. The fragment has good immunogenicity, can generate a good immunization effect in case of a low immunization amount and has the protective rate of 100%, the immunogenicity is better than that of a Fiber full-length protein, and a combined vaccine can be prepared from the fragment and a variety of antigens so as to generate complete protection on chickens and ducks.

Description

technical field [0001] The invention relates to a vaccine composition comprising a genetically engineered subunit vaccine of avian egg drop syndrome virus, which belongs to the field of biomedicine. Background technique [0002] Egg Drop Syndrome Virus (EDSV) belongs to avian adenovirus group III. After the virus infects poultry, there are no obvious clinical symptoms, or mild diarrhea can be seen. Soft-shell, thin-shell, and shellless eggs have a serious decline in egg production, causing serious economic losses. [0003] EDSV has a typical shape of adenovirus, without capsule, has hemagglutinating activity, and proliferates in the oviduct of avian. The virus genome is a linear double-stranded DNA of about 33kb. The virus particles are composed of structural proteins. The diameter of the nucleocapsid is 70-80nm, which is icosahedral and symmetrical, and the DNA is enclosed in the capsid. The nucleocapsid consists of 252 capsomers, 240 of which are hexons (Hexon proteins),...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/075A61K39/00A61K39/39A61K39/17A61K39/12A61K39/145A61K39/215A61K39/235A61P31/20A61P31/14
CPCA61K39/12A61P31/20C07K14/075Y02A50/30C12N2710/10134A61K2039/53A61K2039/552C12N2760/18134C12N2760/16134C12N2770/20034C12N2720/10034C12N2710/10234A61K2039/70A61P31/14A61P31/16A61K39/145A61K39/17A61K39/215A61K39/235A61K39/39
Inventor 田克恭王孟月刘武杰张许科
Owner PU LIKE BIO ENG
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