Group I type 4 avian adenovirus subunit protein as well as preparation method and application thereof

A poultry adenovirus and subunit technology, applied in botany equipment and methods, biochemical equipment and methods, applications, etc., can solve the problems of low protein solubility, difficult amplification, and low yield, and achieve small immune doses and easy amplification , the effect of simple method

Active Publication Date: 2018-05-04
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES +1
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0006] In the Chinese invention patent application with the existing application number 201610777561.1, a subunit vaccine of group I avian adenovirus type 4 is proposed. The antigen in the subunit vaccine is the 248 amino acid sequence at the C-terminus of fibrin, and the antigen is expressed in Escherichia coli Sometimes it is expressed by inclusion bodies, and inclusion bodies need t

Method used

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  • Group I type 4 avian adenovirus subunit protein as well as preparation method and application thereof
  • Group I type 4 avian adenovirus subunit protein as well as preparation method and application thereof
  • Group I type 4 avian adenovirus subunit protein as well as preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0035] The preparation of embodiment 1 poultry adenovirus Fiber-2 (knob) protein

[0036] 1.1 Selection of avian adenovirus Fiber-2 (knob) protein

[0037] The spike protein of avian adenovirus can be divided into 3 parts: tail, shaft and knob. The tail region is connected to the base of the penton; the rod end is generally composed of 22 repeating subunits. These two parts Unable to generate neutralizing epitopes. The spherical region is a functional region that can bind to host cell receptors. Fibrin has type- and subgroup-specific antigenic determinants that can induce protective antibodies. However, in a natural state, the spherical region is in the form of a trimer (van Raaij ,M.J.,A.Mitraki,G.Lavigne andS.Cusack(1999)."A triple beta-spiral in the adenovirus fiber shaft reveals a new structural motif for a fibrous protein." Nature 401(6756):935-938.). Therefore, in theory, the expression of Fiber-2 (knob) protein is selected and can form a trimer, which can produce g...

Embodiment 2

[0164] Example 2 Preparation of avian adenovirus Fiber-2 (knob) protein subunit vaccine

[0165] 2.1 ISA 201 VG adjuvant (purchased from SEPPIC, France) vaccine preparation

[0166] Water phase preparation: according to the Fiber-2 (knob) protein content in the vaccine, use PBS (or physiological saline) to dilute the Fiber-2 (knob) protein prepared in Example 1 to an appropriate concentration, which is the water phase;

[0167] Preparation of oil phase: According to the total amount of prepared vaccine, according to the weight ratio of antigen phase and adjuvant is 1:1, and the volume ratio is 46:54, measure an appropriate amount of ISA 201 VG adjuvant;

[0168] Emulsification: Preheat both the water phase and the oil phase to 33°C, slowly add the water phase to the oil phase, stir at 200-500rpm for 20-30min, let stand at 20°C for 1 hour, then place at 4°C overnight;

[0169] Subpackage and storage: subpackage as needed, and store at 4°C for later use after passing the inspec...

Embodiment 3

[0180] Embodiment 3 Chicken immune challenge experiment

[0181] 3.1 Pre-challenge experiment

[0182] In order to verify the immunogenicity of the Fiber-2 (knob) protein, two batches of vaccines were prepared using ISA 201 VG adjuvant and Marcol52 white oil adjuvant (see Example 2 for specific preparation methods), and each batch of vaccines contained Fiber-2 ( knob) protein concentration was 100 μg / mL (0.5 ml for each immunization, so it was 50 μg / feather portion).

[0183] Fifteen 21-day-old SPF chickens were randomly divided into 3 groups with 5 chickens in each group. Group 1 was immunized with the vaccine prepared with ISA 201 VG adjuvant, group 2 was immunized with the vaccine prepared with Marcol 52 white oil adjuvant, and group 3 was not immunized as a control group, and 0.5 mL of vaccine was injected subcutaneously through the neck. After immunization, observe the state of the chicken, and challenge the virus on the 14th day after immunization, and inject 0.2mL gro...

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Abstract

The invention belongs to the technical field of biology, and discloses group I type 4 avian adenovirus subunit protein with an immunoprotection function. The subunit protein is group I type 4 avian adenovirus short fiber mutation protein spherical region truncated protein; the antigen from amino acid 283 site to amino acid 479 site determines the cluster amino acid sequence; better antigenicity and immunogenicity are realized. The group I type 4 avian adenovirus subunit protein and pharmaceutically acceptable adjuvant is prepared into vaccine; a preparation method of the vaccine mainly comprises the steps that firstly, the group I type 4 avian adenovirus short fiber mutation protein spherical region truncated protein nucleotide sequence subjected to codon optimization is cloned into the carrier; then, a recombinant vector is subjected to conversion, inducible expression and purification to obtain antigen protein; finally, the antigen protein and the adjuvant are emulsified to obtain the vaccine. The vaccine is safe and effective; the method for preparing the subunit vaccine is simple; the cost is low; the amplification is easy.

Description

technical field [0001] The invention belongs to the technical field of biological products, and in particular relates to a group I group 4 avian adenovirus subunit protein, a preparation method thereof and an application for preparing a vaccine. Background technique [0002] Fowl adenovirus (Fowl adenovirus, FAdV) is a common infectious pathogen in poultry, most of which replicate in poultry but do not cause disease or only cause mild symptoms. But when there are other incentives or mixed infection occurs, it will also affect the health of the flock. Certain avian adenoviruses are pathogenic in themselves, such as turkey hemorrhagic enteritis virus (HEV), quail bronchitis virus (Quil bronchitis virus, QBV), egg drop syndrome virus (Eggdrop syndrome virus, EDSV) Wait. Most avian adenoviruses can proliferate on the liver or kidney cells of birds. According to serotype differences, it is divided into three subgroups: most avian adenoviruses from chickens, turkeys, and geese ...

Claims

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Application Information

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IPC IPC(8): C07K14/075C12N15/34C12N15/70A61K39/235A61P31/20
CPCA61K39/12A61K2039/552C07K14/005C12N15/70C12N2710/10222C12N2710/10234
Inventor 林健钱泓吴有强张强杨志远刘立新徐玉兰邹高溪赵际成段会娟闻雪查银河王小蕾程慧敏潘洁刘月焕
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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