Chicken infectious bursal disease virus VP2 gene, expression product and subunit vaccine thereof, and application of vaccine

A subunit vaccine, chicken infectious technology, applied in applications, vaccines, viruses, etc., can solve the problems that vaccines cannot completely control the epidemic and change, and achieve the effects of low cost, good safety, and increased yield

Inactive Publication Date: 2018-11-16
HENAN ACAD OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Changes in the molecular structure lead to changes in the pathogenicity of the virus and the host's response to the vaccine, making traditional vaccines unable to completely control its prevalence

Method used

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  • Chicken infectious bursal disease virus VP2 gene, expression product and subunit vaccine thereof, and application of vaccine
  • Chicken infectious bursal disease virus VP2 gene, expression product and subunit vaccine thereof, and application of vaccine
  • Chicken infectious bursal disease virus VP2 gene, expression product and subunit vaccine thereof, and application of vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Construction of recombinant vector pET28a-VP2

[0041] 1. Synthesis of VP2 Gene

[0042] Referring to the VP2 gene sequence of IBDV B87 strain (GenBank no. DQ906921), the sequence was optimized by comprehensive factors, such as the codon bias of Escherichia coli. The VP2 gene was artificially synthesized, its nucleotide sequence is shown in SEQ ID NO.1, its amino acid sequence is shown in SEQ ID NO.2, and it was ligated into the vector to obtain pUC57-VP2.

[0043] 2. PCR amplification of VP2 gene

[0044] (1) Based on the sequence of the synthetic gene VP2 as a template, design a pair of primers to amplify the VP2 fragment. The primers were synthesized by Shanghai Sangong Company. The primers are as follows:

[0045] EF: cgGAATTCATGACCAATCTGCAAG

[0046] ER: cgCTCGAGCAATGCGACGAATA

[0047] (2) Introduce a restriction enzyme site at the 5' end of the upstream primer EF Bam HI, introduce a restriction enzyme site at the 3' end of the downstream primer ER...

Embodiment 2

[0061] Example 2: Construction of IBDV recombinant Escherichia coli and expression of VP2 protein

[0062] 1. Construction and expression of recombinant strains

[0063] (1) Transform the correctly identified recombinant plasmid pET28a-VP2 into Escherichia coli BL21 (DE3) pLysS to construct a recombinant expression strain BL21 (DE3) pLysS (pET28a-VP2).

[0064] (2) After BL21 (DE3) pLysS (pET28a-VP2) was activated, it was inoculated into liquid LB (Kan, 100 μg / ml) at a ratio of 1:100, cultured on a shaker at 37°C for about 2 hours, and added IPTG to The final concentration was 1 mM. After overnight induction, the bacterial solution was centrifuged, the LB supernatant was removed, and the remaining bacterial cells were resuspended in PBS and ultrasonically disrupted.

[0065] (3) Centrifuge the sonicated cells to separate the supernatant and precipitate, and conduct solubility analysis by SDS-PAGE and Western Blot.

[0066] 2. Optimization of Expression Conditions of Recombin...

Embodiment 3

[0075] Example 3: Purification of IBDV VP2 protein and preparation of virus particles thereof

[0076] 1. Purification of IBDV VP2 Protein

[0077] Purify the VP2 protein expressed by the recombinant strain through a Ni-NTA chromatographic column as follows:

[0078] (1) Filling with nickel material: Take 10 ml of nickel packing to fill the column, and use ddH 2 O slowly flows through the nickel column, and the 20% ethanol solution is flowed out, and the column is washed repeatedly;

[0079] (2) Equilibrium nickel column: 20 ml Binding Buffer equilibrium column;

[0080] (3) Sample loading: add 10 ml VP2 protein to the nickel column, and repeat loading 3 times;

[0081] (4) Washing protein: 50 ml Washing Buffer continuously washes miscellaneous proteins;

[0082] (5) Elution protein: 10 ml Elution Buffer to elute target protein;

[0083] The purified samples were identified by SDS-PAGE and Western Blot, and the purity of VP2 protein could reach more than 90% after purific...

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Abstract

The invention discloses a chicken infectious bursal disease virus VP2 gene, an expression product and a subunit vaccine thereof, and an application of the vaccine. The invention further provides a nucleic acid sequence of the chicken infectious bursal disease virus VP2 gene, a primer for cloning the nucleic acid sequence, an expression vector containing the nucleic acid sequence, an engineering bacterium obtained through conversion of the expression vector, a chicken infectious bursal disease virus VP2 protein encoded by the nucleic acid sequence, expressed by the expression vector or separated from the engineering bacterium, and a chicken infectious bursal disease virus-like particle formed through self-assembling the chicken infectious bursal disease virus VP2 protein. The invention further provides the subunit vaccine prepared from the chicken infectious bursal disease virus VP2 protein. The subunit vaccine is prepared from the chicken infectious bursal disease virus-like particle.The subunit vaccine is applied to prevent chicken related diseases caused by the chicken infectious bursal disease virus. The subunit vaccine has the advantages of low production cost, simplicity in operation, good biological security, and effectiveness in prevention of infection of the chicken infectious bursal disease virus to chickens.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a chicken infectious bursal disease virus VP2 gene, its expression product, its subunit vaccine and application. Background technique [0002] Chicken infectious bursal disease (Infectious Bursal Disease, IBD) is caused by infectious bursal disease virus (Infectious Bursal Disease virus, IBDV). acute and high-contact viral infectious diseases. The characteristics of its onset are: after the chickens are infected with IBDV, the incidence rate is high (80% to 100%), the course of the disease is short (recovery lasts for one week), and the recovered chickens are prone to immunosuppression after infection. The main clinical symptoms include loss of appetite, high depression, intermittent diarrhea, bloody stools, and weight loss. Necropsy may reveal features such as muscle hemorrhage, renal enlargement, urate deposits, hemorrhage in the bursa, atrophy or enlargement. Early i...

Claims

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Application Information

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IPC IPC(8): C12N15/40C12N15/11C12N15/70C12N1/21C07K14/08A61K39/12A61P31/14C12R1/19
CPCA61K39/12A61K2039/5258A61K2039/552A61P31/14C07K14/005C12N2720/10022C12N2720/10023C12N2720/10034
Inventor 张改平刘运超蒋大伟陈玉梅刘东民赵建国魏蔷冯华周景明刘燕凯邓瑞广
Owner HENAN ACAD OF AGRI SCI
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