Actinobacillus pleuropneumoniae gene, and prokaryotic expression and application of protein

An actinomycetes, prokaryotic expression technology, applied in application, genetic engineering, plant genetic improvement and other directions, can solve the problems of APP infection damage to the respiratory system of pigs, aggravate the spread of African swine fever, and endanger the development of the pig industry.

Pending Publication Date: 2021-08-06
SICHUAN AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] To sum up, the problems in the existing technology are: most of the existing studies on AdhE focus on its function in ethanol metabolism, nitrification stress and ethanol tolerance, and there is no infectious pleuropneumonia radiation yet. Application of Bacillus (type 7) AdhE Protein in Subunit Vaccine
[0

Method used

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  • Actinobacillus pleuropneumoniae gene, and prokaryotic expression and application of protein
  • Actinobacillus pleuropneumoniae gene, and prokaryotic expression and application of protein
  • Actinobacillus pleuropneumoniae gene, and prokaryotic expression and application of protein

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Experimental program
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Embodiment

[0060] 1. Amplification and sequence analysis of AdhE gene

[0061] 1.1 Primer design

[0062] According to the complete App7 gene sequence published by NCBI with accession number CP001091.1, the upstream and downstream primers (SEQ ID NO: 3) of the AdhE protein gene were designed (Table 1).

[0063] Table 1 Specific primers for amplifying Actinobacillus pleuropneumoniae type 7

[0064]

[0065]

[0066] 1.2 AdhE gene amplification was connected with prokaryotic expression vector pET-32a(+).

[0067] The bacterial genomic DNA of App7 type was extracted according to the instructions of the bacterial genomic DNA extraction kit (Tiangen). The PCR amplification product of the AdhE gene (Appendix 1) was recovered and ligated with the expression plasmid pET-32a(+), and transformed into a DH5α strain for preservation. (Such as figure 2 shown)

[0068] 1.3 Prokaryotic expression, purification and WB identification of AdhE protein

[0069] Cultivate the expression strain B...

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Abstract

The invention belongs to the technical field of biological medicines, and discloses an Actinobacillus pleuropneumoniae gene, and prokaryotic expression and application of a protein. An App7 type bacterial strain genome is taken as a template to carry out amplification to obtain the whole-genome sequence of AdhE; and a pET-32a(+)-rAdhE prokaryotic expression carrier is successfully constructed, soluble inducible expression of the pET-32a(+)-rAdhE prokaryotic expression carrier in escherichia coli BL21 is realized, and the size of a fusion protein is 110kDa. ELISA detection proves that the prokaryotic expression rAdhE fusion protein can stimulate a mouse to generate a high-level specific antibody, and immunogenicity is good; and after the mouse is immunized by rAdhE, the mouse can resist App7 type lethality virus attacking and 70% of APP1 type virus attacking protection, and the in vivo acute inflammatory reaction of the mouse is lightened.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to prokaryotic expression and application of genes and proteins of Actinobacillus pleurodes. Background technique [0002] Currently, Actinobacillus pleuropneumoniae (App) is the causative agent of porcine pleuropneumonia, a highly contagious and severe swine disease that can be associated with respiratory pathogens such as pseudorabies virus and / or Mycoplasma hyopneumoniae. The mixed infection of the disease worsens. All cause great loss to the breeding industry all over the world. Porcine pleuropneumonia is characterized by hemorrhagic, fibrotic, and necrotizing lung cysts, with clinical manifestations ranging from acute to chronic. Exposure to this microbe can lead to chronic infection that prevents animals from thriving, and even if pigs do survive, they become asymptomatic carriers, spreading the disease to healthy herds. [0003] Aldehyde / alcohol dehydrogena...

Claims

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Application Information

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IPC IPC(8): C12N15/31C12N15/70C12N1/21A61K39/05A61P31/04C12R1/19
CPCC07K14/195C12N15/70A61K39/05A61P31/04
Inventor 徐志文朱玲蔡瑶赵军谷思睿张儒博
Owner SICHUAN AGRI UNIV
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