Flagellin mutant and application thereof in preparation of African swine fever antigen fusion protein

A technology of fusion protein and African swine fever, which is applied in the direction of virus antigen components, vector-borne diseases, hybrid peptides, etc., can solve the unpredictable safety risks and clinical supervision of nucleic acid vaccines, so as to improve the solubility and expression level, Reduce the cost of expression and purification process and improve the effect of wide application

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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  • Flagellin mutant and application thereof in preparation of African swine fever antigen fusion protein
  • Flagellin mutant and application thereof in preparation of African swine fever antigen fusion protein
  • Flagellin mutant and application thereof in preparation of African swine fever antigen fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Obtaining of flagellin mutants

[0046] 1. Design and construction of the target gene

[0047] 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

[0055] Example 2: Recombinant Construction of Flagellin Fusion Antigen

[0056] 1. PCR amplification of Fm fusion antigen

[0057] Flagellin mutant gene Fm codon and synthesis, see sequence Sequence ID NO.3,

[0058] The primers used for Fm fusion PCR amplification were

[0059] Upstream primer: BamH-Fm-F (5'-ctgtatttccagggaggatc-3')

[0060] Downstream primer: Linker-Fm-R (5'-actgcctccagagccacc-3')

[0061] The length of the amplified fragment is 828bp.

[0062] The primers used for p30 fusion PCR amplification were

[0063] Upstream primer: Linker-p30-F:

[0064] (5'-ggtggctctggaggcagtatggattttatcctgaacatcag-3')

[0065] Downstream primer: HindIII-p30-R:

[0066] (5'-ctcgagtgcggccgcaagcttaataaccatcagacgaacaac-3')

[0067] The length of the amplified fragment is 621bp.

[0068] The primers used for p54 fusion PCR amplification were:

[0069] Upstream primer: Linker-p54-F:

[0070] (5'-ggtggctctggaggcagtgctatcgaagaagaagacatc-3')

[0071] Downstream primer: HindIII-...

Embodiment 4

[0085] Example 4: Expression and purification of flagellin fusion African swine fever antigen in Escherichia coli

[0086] 1. Small-scale expression of Fm fusion antigen

[0087] pET30t-Fm-p54 and pET30t-Fm-p30 were respectively transformed into competent Escherichia coli BL21(DE3), incubated and activated for 20 minutes, and coated with LB solid medium containing 50 μg / ml kanamycin.

[0088] 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 a flagellin mutant and application thereof in preparation of African swine fever antigen fusion protein. The amino acid sequence of the flagellin mutant is as shown in SEQ ID NO. 4. The flagellin truncated mutant Fm provided by the invention can realize efficient supernatant expression, the protein yield after shake-flask culture and purification per liter can reach 40mg, the flagellin truncated mutant Fm is easy to amplify and produce on a large scale, and the wide application of the flagellin protein can be improved. After the flagellin adjuvant is fused with African swine fever antigen protein, while the activity of the flagellin adjuvant is maintained, the solubility and expression level of a recombinant antigen are improved, and the cost of expression and purification processes is greatly reduced.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a flagellin mutant and its application in preparing African swine fever antigen fusion protein. 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 flagellin. T...

Claims

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Application Information

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IPC IPC(8): C07K14/255C07K19/00C12N15/70A61K39/12A61P31/20
CPCY02A50/30
Inventor 金梅林邹忠左文峰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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