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

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, so as to improve solubility and expression level, reduce Expression and purification process costs, improve the effect of a wide range of applications

Active Publication Date: 2022-08-09
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 its application
  • Fusion protein of flagellin mutant and African swine fever antigen and its application
  • Fusion protein of flagellin mutant and African swine fever antigen and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Acquisition of Flagellin Mutants

[0041] 1. Design and construction of 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 amino acids ranging from 178-320 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 amino acids in the 178-320 range of the full-length Flagellin variable region with the flexible Linker sequence GSGPGG, retains the backbone sequence for activating the TLR5 pathway, and deletes the 6 amino acids VLSLLR at the carboxyl terminus. Its nucleotide sequence is as shown in SEQ ID As show...

Embodiment 2

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

[0051] 1. PCR amplification of Fm fusion antigen

[0052] Flagellin mutant gene Fm codon and synthesis, 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 was 828 bp.

[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 was 360 bp.

[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-R

[0067] (...

Embodiment 3

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

[0081] 1. Small test expression of Fm fusion antigen

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

[0083]The recombinant transformants were picked for activation and inoculated in 10 mL of LB liquid medium containing 50ug / ml the next day, 37°C, 200rpm shaking culture to about 0.6 when the OD600 was about 0.6, add 0.2-0.5mM IPTG, and culture at 20°C for 12- For 16 hours, the cells were harvested by centrifugation at 8000 rpm, and the cells were resuspended in 1 mL of PBS. At 4°C, ultrasonically disrupted for 5 minutes, working for 2 s, and intermittently for 3 s; the total protein solution was refrigerated and centrifuged at 12,000 rpm for 20 min, and the supernatant and precipitate were separated for detection by...

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Abstract

The invention belongs to the field of biotechnology and discloses a fusion protein of a flagellin mutant and an African swine fever antigen and its application. The invention fuses the African swine fever protective antigen and the flagellin mutant, which can be expressed and prepared in large quantities. Although the autoimmunity of p22 and p62 antigens are different, the fusion of flagellin can significantly improve the mucosal immunity of p22 and p62 antigens. Afterwards, it is helpful for the supernatant expression of p22 and p62.

Description

technical field [0001] The invention belongs to the field of biotechnology, and particularly relates to a fusion protein of a flagellin mutant and an African swine fever antigen and its application. Background technique [0002] The immune response of the body to antigens can be improved by mixing or fusing the flagellin protein with the target protein, and the immune response to the antigen can be improved. Vaccines are only researched in the laboratory stage. Although the protein purification process is not required, nucleic acid vaccines themselves 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 responses, mainly due to the high immunogenicity of the flagellin variable region. The amino and carboxyl termini of flagellin proteins of the same species of Salmonella are r...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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