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Recombinant Bacillus subtilis expressing highly pathogenic avian influenza h5n1 hemagglutinin ha protein

A Bacillus subtilis, highly pathogenic technology, applied in the field of biotechnology genetic engineering, can solve the problems of pathogenicity, attenuated bacteria virulence, and somatic cell carcinogenesis.

Active Publication Date: 2020-08-11
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Firstly, the attenuated bacteria may have the risk of strong virulence; secondly, the plasmid DNA may be integrated into the host cell genome, which will cause the disorder of genes regulating cell growth, the uncontrolled expression of proto-oncogenes and tumor suppressor genes, somatic cell carcinogenesis, A series of issues such as cell transformation
In addition, attenuated bacteria may be potentially pathogenic in some elderly, infirm, and disabled individuals

Method used

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  • Recombinant Bacillus subtilis expressing highly pathogenic avian influenza h5n1 hemagglutinin ha protein
  • Recombinant Bacillus subtilis expressing highly pathogenic avian influenza h5n1 hemagglutinin ha protein
  • Recombinant Bacillus subtilis expressing highly pathogenic avian influenza h5n1 hemagglutinin ha protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0051] 1 Construction of expression plasmid pHT43-HA

[0052] The methods used in the following examples are conventional methods unless otherwise specified. The specific materials and reagents involved are as follows:

[0053] Bacillus subtilis WB800N strain was donated by Gao Xuewen, School of Plant Protection, Nanjing Agricultural University;

[0054] The pMD19-HA plasmid was donated by Wang Zhisheng, Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences;

[0055] The highly pathogenic avian influenza H5N1 strain was donated by Fan Gencheng of Qingdao Tianbang Biological Co., Ltd.;

[0056] E.coli JM109 strain and plasmid pMD19-T were purchased from Takara; plasmid pHT43 was purchased from LifeTechnologies;

[0057] Reagents: Q5 high-fidelity enzyme was purchased from NEB Company; agarose gel recovery kit, restriction endonuclease, T4 ligase, Trizol, RNA reverse transcription kit, fluorescence quantitative kit, etc. were purchased from Takara Company...

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Abstract

The invention relates to recombinational bacillus subtilis(B.S.-HA) of expressing highly pathogenic avian influenza(AIV) HSN1 hemagglutinin HA protein, and belongs to the field of biotechnology genetic engineering. The recombinational bacillus subtilis(B.S.-HA) of expressing the highly pathogenic avian influenza(AIV) HSN1 hemagglutinin HA protein successfully establishes recombinational bacillus subtilis expression plasmid pHT43 of the highly pathogenic avian influenza hemagglutinin HA protein, and successfully conducts elec-transformation of the recombinational bacillus subtilis expression plasmid pHT43 of the highly pathogenic avian influenza hemagglutinin HA protein into bacillus subtilis WB800N bacterial strain (B.S.). As verified by Western-blot, the highly pathogenic avian influenza hemagglutinin HA protein can be successfully expressed in the recombinational bacillus subtilis. Oral immunization can stimulate chicken body of 10-day old chicken to generate an effective AIV specific immune response level. The recombinational bacillus subtilis(B.S.-HA) is hopeful to be developed to genetic engineering oral vaccine in preventing the highly pathogenic avian influenza.

Description

[0001] 1. Technical field [0002] The invention relates to a recombinant bacillus subtilis expressing the hemagglutinin HA protein of highly pathogenic avian influenza, which belongs to the field of biotechnology genetic engineering. Specifically include: the construction of the highly pathogenic avian influenza hemagglutinin HA protein expression plasmid pHT43, the expression and identification of the highly pathogenic avian influenza hemagglutinin HA protein, and the recombinant Bacillus subtilis that can promote the growth of the body of 10-day-old chicks. Mucosal immunity and the level of AIV-specific immune response. [0003] 2. Background technology [0004] 1. Highly pathogenic avian influenza (HPAIV) protective antigen - hemagglutinin (HA) [0005] Hemagglutinin (HA) is one of the main components constituting the envelope fibrils of influenza virus. HA plays a key role in the process of virus adsorption and virus membrane penetration, and can stimulate the body to pr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/75C12N15/66C12N1/21C12Q1/6851G01N33/68A61K39/145A61P31/16C12R1/125
CPCA61K39/12A61K2039/523A61K2039/542A61K2039/552C07K14/005C12N15/66C12N15/75C12N2760/16122C12N2800/101C12Q1/6888C12Q2600/158G01N33/6866G01N33/6869G01N2333/5428G01N2333/5434
Inventor 杨倩牟春晓朱立麒杨晶晶
Owner NANJING AGRICULTURAL UNIVERSITY
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