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Fusion protein of flagellin mutant and African swine fever antigen and application thereof

A technology of fusion protein and African swine fever virus, which is applied in the field of fusion protein of flagellin mutants and African swine fever antigen, can solve the problems of unpredictable safety risks and clinical supervision of nucleic acid vaccines, and achieve improved solubility and expression levels, strong Scalability, improving the effect of a wide range of applications

Active Publication Date: 2022-04-08
WUHAN KEQIAN BIOLOGY CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Immunization by mixing or fusing flagellin protein with the target protein can improve the body’s immune response to antigens, and the immune effect of flagellin fusion or coupled antigen is more significant [1] [2], nucleic acid with flagellin as adjuvant Vaccines are only researched in the laboratory stage. Although they do not need to go through the protein purification process, nucleic acid vaccines face unpredictable safety risks and clinical supervision; at present, the flagellin of many pathogenic bacteria may be potentially dangerous and toxic. Producing a large number of antibodies against self, leading to possible tolerance and inflammatory response, mainly due to the high immunogenicity of the variable region of flagellin

Method used

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  • Fusion protein of flagellin mutant and African swine fever antigen and application thereof
  • Fusion protein of flagellin mutant and African swine fever antigen and application thereof
  • Fusion protein of flagellin mutant and African swine fever antigen and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Obtaining of flagellin mutants

[0041] 1. Design and construction of the target gene

[0042] The full-length Flagellin sequence refers to the original sequence (WP_050188722), which was synthesized by Beijing Qingke Biotechnology, and its nucleotide sequence is shown in SEQ ID NO.1. Different truncations were performed on the full-length Flagellin. The truncated Fm-C6 replaced the 178-320 amino acids in the variable region of the full-length Flagellin with the flexible Linker sequence GSGPGG, and retained the backbone sequence for activating the TLR5 pathway. Its amino acid sequence is shown in SEQ ID NO.2 shown. The truncated Fm protein replaces the 178-320 amino acids in the variable region of the full-length Flagellin with the flexible Linker sequence GSGPGG, retains the backbone sequence for activating the TLR5 pathway, and deletes the 6 amino acids VLSLLR at the carboxy-terminal. Its nucleotide sequence is shown in SEQ ID As shown in NO.3, the amino ...

Embodiment 2

[0050] Example 2: Recombinant Construction of Flagellin Fusion Antigen

[0051] 1. PCR amplification of Fm fusion antigen

[0052] For the Fm codon and synthesis of the Flagellin mutant gene, see Sequence ID NO.3.

[0053] The primers used for Fm fusion PCR amplification were

[0054] Upstream primer: BamH-Fla-F (5'-ctgtatttccagggaggatc-3')

[0055] Downstream primer: Linker-Fla-R (5'-actgcctccagagccacc-3')

[0056] The length of the amplified fragment is 828bp.

[0057] The primers used for p62 fusion PCR amplification were

[0058] Upstream primer: Linker-p62-F

[0059] (5'-ggctctggaggcagtaaacatggtgtgacatttatctatc-3')

[0060] Downstream primer: HindIII-p62-R

[0061] (5'-ctcgagtgcggccgcaagcttaccttccagcggcatgcta-3')

[0062] The length of the amplified fragment is 360bp.

[0063] The primers used for p22 fusion PCR amplification were:

[0064] Upstream primer: Linker-p22-F

[0065] (5'-ggctctggaggcagtaagaagcagcagccgccg-3')

[0066] Downstream primer: HindIII-p22-...

Embodiment 3

[0080] Example 3: Expression and purification of flagellin fusion African swine fever antigen in Escherichia coli

[0081] 1. Small-scale expression of Fm fusion antigen

[0082] pET30t-Fm-p62 and pET30t-Fm-p22 were respectively transformed into Escherichia coli BL21 (DE3) competent, incubated for 20 minutes to activate, and coated with LB solid medium containing 50 μg / ml kanamycin.

[0083]Recombinant transformants were selected for activation, inoculated in 10mL LB liquid medium containing 50ug / ml the next day, 37°C, 200rpm shaking culture until OD600 was about 0.6, added 0.2-0.5mM IPTG, and cultured at 20°C for 12- After 16 hours, harvest the cells by centrifugation at 8000rpm, resuspend the cells in 1mL of PBS, 4°C, ultrasonicate for 5 minutes, work for 2s, and rest for 3s; the total protein solution was refrigerated and centrifuged at 12000rpm for 20min, the supernatant and precipitate were separated, and detected by SDS-PAGE For the expression of the target protein, see...

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Abstract

The invention belongs to the technical field of biology, and discloses fusion protein of flagellin mutant and African swine fever antigen and application thereof. The African swine fever protective antigen is fused with the flagellin mutant, and mass expression and preparation can be realized. Although the autoimmunogenicity of the p22 antigen and the autoimmunogenicity of the p62 antigen are different, the mucosal immune level of the p22 antigen and the mucosal immune level of the p62 antigen can be remarkably improved after the flagellin is fused, and meanwhile due to the fact that the constructed flagellin mutant can be efficiently expressed in supernate, supernate expression of the p22 antigen and the p62 antigen is facilitated after fusion.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a fusion protein of a flagellin mutant and an African swine fever antigen and an application thereof. Background technique [0002] Immunization by mixing or fusing flagellin protein with the target protein can improve the body’s immune response to antigens, and the immune effect of flagellin fusion or coupled antigen is more significant [1] [2], nucleic acid with flagellin as adjuvant Vaccines are only researched in the laboratory stage. Although they do not need to go through the protein purification process, nucleic acid vaccines face unpredictable safety risks and clinical supervision; at present, the flagellin of many pathogenic bacteria may be potentially dangerous and toxic. A large number of antibodies against self are produced, leading to possible tolerance and inflammatory response, which is mainly due to the high immunogenicity of the variable region of flagell...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/70A61K39/12A61P31/20
CPCY02A50/30
Inventor 邹忠左文峰金梅林康超杨于尚霄敏黎晶晶杨丽孙小美何兴林
Owner WUHAN KEQIAN BIOLOGY CO LTD
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