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Preparation method and product of chicken infectious bursal disease virus antigen

A technology for chicken infectivity and bursal disease, applied in the direction of virus antigen components, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problems of low expression level and cumbersome experimental operation process, and achieve effective immunity The effect of protection

Active Publication Date: 2015-07-08
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using transgenic plants to produce IBDV vaccine, the expression level is not high, and the experimental operation process is cumbersome

Method used

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  • Preparation method and product of chicken infectious bursal disease virus antigen
  • Preparation method and product of chicken infectious bursal disease virus antigen
  • Preparation method and product of chicken infectious bursal disease virus antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Expression and detection of chicken infectious bursal disease virus VP2 / 4 / 3 polyprotein original sequence in silkworm bioreactor

[0057] 1 About the configuration of solution and medium

[0058] For the configuration methods of the solution and medium, refer to relevant reference books (Joseph et al., Molecular Cloning Experiment Guide, Third Edition, 2002; Osper, et al., Molecular Biology Guide, 1998).

[0059] 2 The original sequence of chicken infectious bursal disease virus VP2 / 4 / 3 polyprotein gene was obtained

[0060] 2.1 Extraction of total RNA

[0061] Take a certain amount of chicken lesion tissue in 1mL Trizol reagent, grind it thoroughly, and let it stand at room temperature for 10 minutes; add 200 μL of chloroform, vortex and shake for 15 seconds, and let it stand at room temperature for 5 minutes; Add 500μL of isopropanol, ice bath for 10min; centrifuge at 12000rpm at 4°C for 10min; wash the pellet once with 70% ethanol, centrifuge at 12000rpm ...

Embodiment 2

[0131] Example 2 Expression and detection of chicken infectious bursal virus VP2 protein original sequence in silkworm bioreactor

[0132] 1 About the configuration of solution and medium

[0133] The configuration method of relevant solution and culture medium is the same as embodiment 1.

[0134] 2 Amplification of the original sequence of chicken infectious bursal virus VP2 protein and connection with T vector

[0135] Using the pT-IBDV-VP2 / 4 / 3 plasmid obtained in Example 1 as a template, design specific primers to amplify the gene sequence of the IBDV-VP2 protein, the upstream primer is the same as IBDV-VP2 / 4 / 3-F, and the downstream The primer is IBDV-VP2-R: 5'-GATCTAGATTACCTCCGTAGGGCCCGGATTATG-3', which introduces Xba Ⅰ restriction enzyme cutting site. Amplify the VP2 target sequence according to the following PCR amplification system and parameters.

[0136] The PCR reaction system is:

[0137]

[0138] PCR parameter settings:

[0139]

[0140] Configure 1% (w...

Embodiment 3

[0177] Example 3 Expression and detection of chicken infectious bursal virus polyprotein optimized sequence VP2 / 4 / 3-O in silkworm bioreactor.

[0178] 1 About the configuration of solution and medium

[0179] The configuration method of relevant solution and culture medium is the same as embodiment 1.

[0180] 2 Screening, optimization and synthesis of IBDV-VP2 / 4 / 3 sequences

[0181]The polyprotein sequences of 24 IBDV strains popular in recent years were selected, including 8 super-virulent strains (GenBank accession numbers are JF907702.1, X92760, AY444873.3, AF322444.1, ABI52866.1, JQ403646. 1, JF907703.1, AF092943.1), 8 mutant strains (GenBank accession numbers are D00867.1, EF418033.1, AY368653.1, JQ411012.1, AF321055.1, AF051837.1, EU595671.1, EU595672 .1), 5 classic strains (GenBank accession numbers are D00869, X16107, X84034, D00499, AY319768.2) and 3 vaccine strains (GenBank accession numbers are DQ906921.1, DQ403248.1, AF499929.1). Because IBDV is easy to mutate ...

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Abstract

The invention discloses a preparation method and a product of a chicken infectious bursal disease virus antigen. A chicken infectious bursal disease virus VP2/4/3 polyprotein gene and a VP2 gene are optimized. The optimized chicken infectious bursal disease virus VP2/4/3 polyprotein gene or the VP2 gene is expressed in a silkworm bioreactor. The invention further discloses a preparation method of a chicken infectious bursal disease nucleic acid vaccine. The preparation method comprises the following steps: cloning the optimized chicken infectious bursal disease virus VP2/4/3 polyprotein gene or the VP2 gene into a baculovirus transfer vector controlled by a mammal promoter to be recombined with baculovirus to infect an insect host, presenting an exogenous gene in the insect host to obtain the nucleic acid vaccine. The expressed antigen is used for preparing a vaccine for immunizing animals or the nucleic acid vaccine is used for immunizing animals through injection or oral taking, so that effective immune protection is provided for the animals.

Description

technical field [0001] The present invention relates to a chicken infectious bursal disease virus antigen, in particular to a method for expressing an infectious bursal disease virus antigen in insects using a silkworm baculovirus expression system and its products, and the present invention also relates to a method for using a baculovirus expression system The invention discloses a method for preparing chicken infectious bursal disease nucleic acid vaccine in a silkworm bioreactor, belonging to the field of preparation of chicken infectious bursal disease virus antigen and nucleic acid vaccine. Background technique [0002] Chicken infectious bursal disease (Infectious bursal disease, IBD) is an acute contagious disease caused by infectious bursal disease virus (Infectious bursal disease virus, IBDV). [0003] At present, the main vaccines for preventing IBD are attenuated vaccines and inactivated vaccines. However, with the emergence of IBDV mutant strains and super-virul...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/08C12N15/40C12N15/866C12N5/10A61K39/12A61P31/14
Inventor 胡小元李轶女张志芳李田田杨鑫易咏竹吕言娜王智权
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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