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Preparation and application of enhanced serum avian adenovirus type 4 subunit vaccine

A technology of spike protein and fiber-2, applied in the field of immunology and vaccinology, can solve problems such as inappropriate adjuvant mix, uneven quality of inactivated virus, and inappropriate attenuation methods

Active Publication Date: 2022-03-15
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, traditional inactivated and attenuated vaccines have problems such as uneven quality of inactivated viruses, inappropriate adjuvant mix, inappropriate attenuation methods, and expensive labor costs.

Method used

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  • Preparation and application of enhanced serum avian adenovirus type 4 subunit vaccine
  • Preparation and application of enhanced serum avian adenovirus type 4 subunit vaccine
  • Preparation and application of enhanced serum avian adenovirus type 4 subunit vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1, the construction of recombinant baculovirus

[0051] In the previous research of the present inventors, in order to reduce the cost of recombinant expression, they tried to express Fiber-2 in prokaryotic, using Escherichia coli. However, the expressed protein cannot be folded accurately. After selection of the expression system, it was determined to adopt the recombinant baculovirus expression system. In this embodiment, this expression method is provided.

[0052] 1. Obtaining the target gene

[0053] The construction and expression of Fiber-2 is based on the insect baculovirus expression system, and the target sequence encoding Fiber-2 (the gene sequence is optimized according to the insect cell expression system) is cloned into pFastBac TM 1 shuttle plasmid with 6 His-tags at its C-terminus, named pFastBac1-Fiber-2.

[0054] The amino acid sequence of the Fiber-2 protein is as follows (SEQ ID NO: 1):

[0055]

[0056]

[0057] 2. Construction...

Embodiment 2

[0066] Embodiment 2, the preparation of recombinant Fiber-2 protein

[0067] 1. Expression test of recombinant protein Fiber-2

[0068] The above-mentioned orifice plate prepared for the P3 generation virus was processed as follows: absorb 1ml of the culture supernatant from the well plate of the control group and the experimental group, centrifuge at 10000g for 15min at 4°C, and then take the centrifuged supernatant for sample preparation; Add 2ml of 1% TritonX-100 to the medium to lyse the cells. After the lysate is fully lysed, centrifuge the lysate at 10,000g for 15min at 4°C, take a sample from the centrifuged supernatant, suspend the precipitate with 2ml of PBS and take a sample, and detect the above sample by Western blot.

[0069] The result is as figure 2 , indicating that the target protein will not be secreted into the medium supernatant, and most of the target protein is located in the cell lysate supernatant in a soluble form after cell disruption.

[0070] 2. ...

Embodiment 3

[0077]Embodiment 3, expression and preparation of interleukin 2 and interferon gamma

[0078] 1. Obtaining the target gene

[0079] The sequences of chicken interleukin-2 (chIL-2) and chicken interferon gamma (chIFN-γ) were synthesized by Nanjing Jinsurui, and the target sequences encoding chIL-2 and chIFN-γ (gene sequences optimized by E. coli expression vector) were respectively cloned into pET28a (+), the recombinant plasmids pET28a-chIL-2 and pET28a-chIFN-γ were obtained respectively, and transformed into Escherichia coli BL21(DE3) for expression.

[0080] The amino acid sequence of chIL-2 is as follows (SEQ ID NO:2):

[0081]

[0082] The amino acid sequence of chIFN-γ is as follows (SEQ ID NO: 3):

[0083]

[0084] 2. Expression of recombinant chIL-2 and chIFN-γ

[0085] Inoculate the positive strain with 1% inoculum into the culture medium containing Kanna resistance, culture at 37°C until OD=0.6-0.8, add IPTG at a final concentration of 1mmol / L for induction, ...

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Abstract

The invention relates to the preparation and application of enhanced serum avian adenovirus type 4 subunit vaccine. In the present invention, a baculovirus expression system is used to express avian adenovirus type 4 spike protein (Fiber-2), and an Escherichia coli expression system is used to construct and express two cytokines: interleukin 2 (IL-2) and interferon gamma (IFN ‑γ), and apply a mixture of the three. The vaccine prepared by the method of the invention has low cost, and the obtained vaccine has good immune effect, and can effectively prevent the infection of avian adenovirus type 4.

Description

technical field [0001] The invention belongs to the field of immunology and vaccinology, and more specifically, the invention relates to the preparation and application of the enhanced serum avian adenovirus type 4 subunit vaccine. Background technique [0002] Fowl adenovirus (fowl adenovirus, FAdVs) can be divided into three groups: I group, II group and III group. Group I has 12 serotypes, which are divided into 5 branches of A, B, C, D, and E according to genotype. Inclusion body hepatitis (Inclusion Body Hepatitis, IBH) and pericardial effusion syndrome (Hepatitis–hydropericardium Syndrome, HHS) are two main diseases in chicken flocks in China, and they are all caused by group I adenovirus, wherein IBH is caused by FAdV- 8b and FAdV-11 are caused, and HHS is caused by FAdV-4. [0003] The avian adenovirus has a non-encapsulated spherical structure, and the virus particles are arranged in a lattice-like arrangement in the infected cell nucleus. The particles contain a ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/235A61P31/20
CPCA61K39/12A61P31/20A61K2039/552C12N2710/10234
Inventor 刘琴季国荣王蓬勃张元兴
Owner EAST CHINA UNIV OF SCI & TECH
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