Improved flagellin and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to salmonella typhimurium flagellin capable of enhancing human TLR5 binding affinity and application of the salmonella typhimurium flagellin. A key amino acid mutation site of flagellin is screened and determined, and recombinant expression is realized in an escherichia coli system by adopting a site-directed mutagenesis technology. In-vitro experiments prove that the derivative can significantly activate the function of a TLR5 ligand. The invention solves the problem that the existing flagellin has a limited effect on activating the activity of the human TLR5, and provides a new technical scheme and theoretical basis for the application of the flagellin in the fields of vaccines, immunotherapeutic agents and radioprotectants.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a modified flagellin that can enhance the binding affinity of human TLR5 and its applications. Background Technology

[0002] Flagellins are the main components of bacterial flagella, consisting of four domains: D0, D1, D2, and D3. The D0 and D1 domains are highly conserved among different bacteria and are primarily responsible for the aggregation and stabilization of flagellar filaments, containing key sites for host receptor recognition. [1] The D2 and D3 domains exhibit high diversity due to species differences. Flagellins can activate innate and adaptive immunity through the TLR5 and NLRC4 signaling pathways. Extracellular flagellins can bind to and activate Toll-like receptor 5 (TLR5) on the cell surface, inducing the secretion of inflammatory factors such as IL-8 and TNF-α. Intracellular flagellins bind to and activate the NAIP5-NLRC4 complex, inducing the production of IL-1β and IL-18 and apoptosis. [2] .

[0003] In recent years, the flagellin-TLR5 pathway has attracted widespread attention due to its unique role in immune regulation. Existing studies have shown that flagellin, as a novel immune adjuvant, possesses strong adjuvant activity, not only activating the body's innate immune response system but also simultaneously inducing cellular and humoral immunity. [3] In tumor immunotherapy, Zheng JH et al. [4] Salmonella bacteria that secrete flagellin targeting the tumor microenvironment have been found to exert anti-tumor immune responses through the flagellin-TLR5 signaling pathway. Furthermore, flagellin has shown potential value in radiation protection research. [5][6] .

[0004] However, naturally derived flagellin proteins still have limitations in their efficacy in activating human TLR5. Firstly, although TLRs are relatively conserved across species, it has become clear in recent years that there are potentially functionally important species differences in both ligand binding and the TIR domain. 7 This affects the immunostimulatory efficiency of flagellin in different species. Therefore, the use of TLR ligands as potent adjuvants in vaccines may need to be modified based on species-specific sequence differences in key residues within the TLR molecule. 8 Secondly, some pathogens' natural flagellin proteins have evolved relatively mild activation capabilities to avoid excessive inflammatory responses in the body. For example, Helicobacter pylori mutates to evade TLR5 detection. [9]These sites responsible for TLR5 escape are located at amino acid residues 89-96 of the flagellin D1 domain. Therefore, how to rationally modify flagellins has become a current research hotspot.

[0005] This invention improves the binding affinity and immune activation capacity of flagellin to human TLR5 by screening and modifying key amino acid residues that bind to TLR5, combined with site-directed mutagenesis and recombinant expression. This overcomes the limitations of natural proteins and provides a new technical approach for developing novel and highly effective vaccines, immunotherapeutic agents and radioprotective agents.

[0006] References

[0007] [1] Song WS, Yoon SI. Crystal structure of FliC flagellin from Pseudomonas aeruginosa and its implication in TLR5 binding and formation of the flagellar filament. Biochem Biophys Res Commun. 2014; 444(2): 109-115.

[0008] [2]Hajam IA, Dar PA, Shahnawaz I, Jaume JC, Lee JH. Bacterial flagellin-apotent immunomodulatory agent. Exp Mol Med. 2017; 49(9): e373.

[0009] [3]Cui B, Liu

[0010] [4] Zheng JH, Nguyen VH, Jiang S, Park S, Tan W, Hong SH et al. Two-stepenhanced cancer immunotherapy with engineered Salmonella typhimurium secreting heterologous flagellin. Science Transl Med 2017;9537:1–11.

[0011] [5]BurdelyaLG,Krivokrysenko VI,TallantTC,StromE,GleibermanAS,GuptaD,etal.An agonistoftoll-like receptor 5has radioprotective activity inmouseandprimate models.Science.2008;320:226–30.

[0012] [6] Brackett CM, Greene KF, Aldrich AR, et al. Signaling through TLR5mitigates lethal radiation damage by neutrophil-dependentrelease ofMMP-9. CellDeathDiscov.2021;7(1):266.Published2021 Sep 28.

[0013] [7]Tahoun A, Jensen K, Corripio-Miyar Y, et al. Host species adaptation of TLR5 signaling and flagellin recognition. Sci Rep. 2017;7(1):17677.Published2017 Dec 15.

[0014] [8] Smith SA, Jann OC, Haig D, et al. Adaptive evolution of Toll-likereceptor 5in domesticated mammals. BMC EvolBiol. 2012; 12:122. Published2012Jul24.

[0015] [9]TegtmeyerN,Neddermann M,Lind J,et al.Toll-like Receptor5Activation by the CagYRepeatDomains of Helicobacterpylori.CellRep.2020;32(11):108159. Summary of the Invention

[0016] The primary objective of this invention is to provide a modified flagellin.

[0017] The second objective of this invention is to provide a modified method for preparing flagellin.

[0018] The third objective of this invention is to disclose the use of a Salmonella Typhimurium flagellin mutant in the preparation of vaccines, immunotherapeutic agents, and radioprotective agents. Specifically, it includes the following:

[0019] In a first aspect, the present invention provides a Salmonella typhimurium flagellate protein mutant, which contains the Ser445Phe mutation compared with the wild-type Salmonella typhimurium flagellate protein. The Salmonella typhimurium flagellate protein mutant is formed by mutating the amino acid at position 445 of the FliC gene from Ser to Phe. The amino acid sequence of the Salmonella typhimurium flagellate protein mutant is: SEQ ID NO:1.

[0020] Preferably, the method for preparing the Salmonella typhimurium flagellin mutant includes the following steps:

[0021] S1: Expression of the flagellin mutant of Salmonella typhimurium: The Ser→Phe mutation was performed on amino acid 445 of the FliC gene and cloned into the prokaryotic expression vector pRSET-B for expression;

[0022] S2: Purify the Salmonella typhimurium flagellin mutant from S1.

[0023] Preferably, the application of the Salmonella typhimurium flagellin mutant in vaccine preparation.

[0024] Furthermore, the Salmonella typhimurium flagellin mutant is used as an immune adjuvant in vaccine preparation.

[0025] Preferably, the application of the Salmonella typhimurium flagellin mutant in the preparation of immunotherapeutic agents and radioprotective agents.

[0026] In a second aspect, the present invention provides a vaccine adjuvant, characterized in that the active ingredient of the vaccine adjuvant contains the Salmonella typhimurium flagellin mutant as described in any one of claims 1-2.

[0027] Thirdly, the present invention provides a vaccine, characterized in that the vaccine comprises the Salmonella typhimurium flagellin mutant as described in any one of claims 1-2 and one or more antigens.

[0028] The beneficial effects of this invention are:

[0029] 1. The flagellin mutant of the present invention exhibits good biological activity and can activate hTLR5 ligand activity, laying the foundation for the development of vaccines and vaccine adjuvants in related fields. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the pRSET-B-Dublin recombinant plasmid.

[0031] Figure 2 Figure A shows the results of wild-type FliC induction, expression, and purification (the red arrows indicate wild-type FliC protein). Figure A shows the induction results, with the marker representing the protein molecular weight standard; 1 is whole cells before induction, and 2 is whole cells after induction. Figure B shows the purification results, with the marker representing the protein molecular weight standard; 1 is the supernatant after cell lysis, 2 is the flow-through solution, 3 is the washing solution, and 4-7 are the elution solutions.

[0032] Figure 3 Figure 1 shows the results of FliC Ser445Phe induction, expression, and purification (the red arrows indicate the FliC Ser445Phe protein). Figure A shows the induction results, with the marker indicating the protein molecular weight standard. 9 and 11 are whole FliC Ser445Phe cells before induction, and 10 and 12 are whole FliC Ser445Phe cells after induction. Figure B shows the purification results, with the marker indicating the protein molecular weight standard. 1 is the supernatant after cell lysis, 2 is the precipitate after cell lysis, 3 is the flow-through buffer, 4 is the washing buffer, and 5-8 are the elution buffers.

[0033] Figure 4 To validate biological activity using flow cytometry.

[0034] Figure 5 The mRNA expression level of the cytokine IL-8 was monitored by qRT-PCR. Compared with the control group, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001. Detailed Implementation

[0035] To make the present invention easier to understand, embodiments of the present invention will be further described below. The present invention will be further described and demonstrated in conjunction with the embodiments. However, these embodiments are not intended to limit the present invention. Unless otherwise specified, the technical solutions described in this invention are conventional solutions in the art; the reagents or materials shown, unless otherwise specified, are all from commercial sources.

[0036] Example 1: Construction of pRSET-B-Dublin and pRSET-B-Dublin-Ser445Phe recombinant plasmids

[0037] The 445th amino acid of Dublin flagellate protein FliC was selected as the mutation site to mutate Ser to Phe, constructing the pRSET-B-Dublin-Ser445Phe recombinant plasmid. The complete sequence of FliC Ser445Phe is SEQ ID NO:1: MAQ VINTNSLSLLTQNNLNKSQSSLSSAIERLSSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELSVQATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQ TQFNGVKVLSQDNQMKIQVGANDGETITIDLQKIDVKSLGLDGFNVNGPKEATVGDLKSSFKNVTGYDTYAAGADKYRVDINSGAVVTDAVAPDKVYVNAANGQLTTDDAENNTAVDLFKTTKSTAGTAEAKAIAGAIKGGKEGDTFDYKGVTFTIDTKTGDDGNGKVSTTINGEKVTLTVADIAIGA ADVNAATLQSSKNVYTSVVNGQFTFDDKTKNESAKLSDLEANNAVKGESKITVNGAEYTANATGDKITLAGKTMFIDKTASGVSTLINEDAAAAKKSTANPLasiDSALSKVDAVRSSLGAIQNRFDFAITNLGNTVTNLNSARSRIEDADYATEVSNMSKAQILQQAGTSVLAQANQVPQNVLSLLR.

[0038] pRSET-B-Dublin plasmid ( Figure 1 The plasmid, provided by our laboratory, carries the FliC gene of Salmonella Typhimurium Dublin flagellate protein, the ampicillin resistance gene, and a 6×His tag. The FliC flagellate protein can be purified using a nickel column. pRSET-B-Dublin was sent to Sangon Biotech Co., Ltd., where the Ser gene at position 445 of FliC was mutated to Phe, yielding the pRSET-B-Dublin-Ser445Phe plasmid. Subsequently, the pRSET-B-Dublin-Ser445Phe plasmid was transformed into Top10 competent E. coli cells, plated on LB solid medium containing ampicillin, and cultured at 37°C for 16 hours. Single colonies were then picked and cultured on LB liquid medium containing ampicillin at 37°C and 220 rpm for 16 hours. The pRSET-B-Dublin-Ser445Phe plasmid was extracted using a plasmid extraction kit.

[0039] Example 2: Prokaryotic expression and purification of wild-type Dublin flagellin and Ser445Phe Dublin flagellin

[0040] The recombinant plasmids pRSET-B-Dublin and pRSET-B-Dublin-Ser445Phe were transformed into *E. coli* competent cells BL21(DE3)Plyss strain, respectively. The cells were plated on LB agar containing ampicillin and cultured overnight at 37°C. The next day, single colonies were picked and cultured in 20 mL of LB liquid agar containing ampicillin at 37°C and 220 rpm for 16 h. Then, 2% of the colonies were inoculated into LB liquid agar and cultured until the OD value was between 0.4 and 0.6. 1 M IPTG was added to a final concentration of 0.5 mM, and expression was induced for another 6 h. SDS-PAGE was used to determine whether flagellin was expressed. Figure 2 A, Figure 3 A).

[0041] The harvested bacterial culture was centrifuged at 8000 rpm and 4°C for 5 min to collect the bacterial cells. The cells were then washed with PBS, centrifuged again, and the supernatant was discarded. This process was repeated twice. The bacterial cells were then resuspended in Lysis buffer and sonicated until the solution was clear and transparent with no visible bacterial cells. The solution was then centrifuged at 12000 rpm for 10 min at 4°C, and the supernatant was collected. The nickel column was washed with distilled water, and then equilibrated twice with 5 column volumes of binding buffer. The supernatant containing soluble target protein was filtered through a 0.45 μm filter before loading the sample. Washing with wash buffer was performed until the G250 solution no longer turned noticeably blue. Elution was then performed using a linear gradient of Elusion buffer containing 20 mM-500 mM imidazole, and the eluent was collected. Finally, the nickel column was washed with 5 column volumes of binding buffer and distilled water, and the packing material was preserved with 20% ethanol and stored at 4°C.

[0042] SDS-PAGE electrophoresis was used to detect protein purification results. Figure 2 B Figure 3 B). The purified target protein was concentrated using an ultrafiltration tube with a molecular weight cutoff of 10 kDa (3500 x g, 10 min, 4 °C) to a sample volume of 1-2 mL. Then, 10 times the volume of PBS buffer was added to the concentrate, and the mixture was centrifuged at 3500 x g for 10 min at 4 °C until a sample volume of 1-2 mL was obtained. The protein preservation system was then replaced with PBS buffer.

[0043] Example 3: Detection of hTLR5 activation effects of wild-type Dublin flagellin and Ser445Phe Dublin flagellin

[0044] Lipopolysaccharide (LPS) in flagellar protein was removed using an endotoxin removal kit, and residual endotoxin levels were measured to ensure that the LPS content was less than 0.1 EU / mL. Flagellar protein concentration was then determined using the BCA method.

[0045] HEK293T-hTLR5-EGFP cells stably expressing human TLR5 (hTLR5) without treatment were used as the control group. HEK293T-hTLR5-EGFP cells treated with 1 μg / mL of different flagellin proteins were used as the experimental group, with three replicates per group. HEK293T-hTLR5-EGFP cells were seeded in 6-well plates, with 1 × 10⁶ cells per well. 6 After overnight culture at 37℃ and 5% CO2, the drug-treated group was stimulated with 1 μg / mL flagellar protein, followed by 6 h of further culture. Cell suspensions were prepared by trypsin digestion, PBS washing, and filtration through a 300-mesh nylon screen, and analyzed by flow cytometry. The relative fluorescence intensity of FITC was used as a quantitative indicator of hTLR5 expression. Experimental parameters were adjusted according to the negative control, and these were used as the experimental parameters for the experimental groups to detect differences in hTLR5 expression levels. Results are as follows: Figure 4 As shown, the hTLR5 expression level in the Ser445Phe Dublin group was higher than that in the wild-type Dublin flagellin group.

[0046] Untreated Caco-2 cells served as the control group, while cells treated with different flagellin proteins at 5 μg / mL were designated as the experimental group, with three replicates per group. Caco-2 cells were cultured at 5 × 10⁶ cells / year. 5 Cells were seeded into 6-well plates and cultured overnight. After cell adhesion, 5 μg / mL flagellar protein was added to each of the drug-treated groups and cultured for 5 h. Cell RNA was then extracted.

[0047] Remove the culture medium, wash once with PBS buffer, add 1 mL of RNAiso Plus to each well of a 6-well plate to lyse the cells, and transfer the lysis buffer to a 1.5 mL centrifuge tube. Repeatedly pipetting and refluxing until no obvious cell pellet appears in the lysis buffer, and incubate at room temperature for 5 min. Add 200 μL of chloroform to the homogenate lysis buffer, mix by pipetting and refluxing until the solution emulsifies and turns milky white, incubate at room temperature for 5 min, and centrifuge at 12,000 x g at 4 °C for 15 min. The homogenate will separate into three layers: a colorless supernatant (containing RNA), a middle white protein layer (mostly DNA), and a colored lower organic phase. Transfer the supernatant to another new centrifuge tube, add 1 mL of isopropanol, invert the centrifuge tube to mix thoroughly, and incubate at room temperature for 10 min. Centrifuge at 12,000 x g at 4 °C for 10 min; RNA pellet will appear at the bottom of the tube. Remove the supernatant, add 500 μL of 75% ethanol, gently invert the centrifuge tube to wash the walls, centrifuge at 7500 x g at 4 °C for 5 min, and carefully discard the supernatant. Open the centrifuge tube cap and allow the precipitate to dry at room temperature for a few minutes to remove the ethanol. After the precipitate has dried, add 40 μL of RNase-free water to dissolve it. Measure the RNA concentration using Nanodrop.

[0048] Add 1 μg of RNA sample and 2 μL of 8×gDNAEraser Premix to a 1.5 mL enzyme-free centrifuge tube, then add RNase-free H2O to a final volume of 16 μL. Incubate at 42 °C for 2 min to remove genomic DNA. Then, on ice, add 4 μL of 5×RTPremix to the reaction mixture, gently mix, and incubate at 37 °C for 10 min, followed by 85 °C for 5 s to reverse transcribe RNA into cDNA. Store the cDNA at -20 °C for later use.

[0049] Prepare Real-Time PCR reaction solution on ice using eight-tube strips according to the table. The internal control gene is human GAPDH, and the target gene is IL-8. After preparation, start the Real-Time PCR reaction. First, pre-denature the template at 95℃ for 30 seconds; then denature at 95℃ for 5 seconds, anneal / extend at 60℃ for 10 seconds, repeating for 40 cycles; finally, obtain the melting curve at 95℃ for 15 seconds, 60℃ for 30 seconds, and 95℃ for 15 seconds. Confirm the amplification and melting curves of the Real-Time PCR reaction using a 23... -ΔΔCT Fluorescence values ​​were calculated using the method, and relative expression levels were calculated using housekeeping genes as internal references. Results are as follows: Figure 5 As shown, the mRNA level of IL-8 in the Ser445Phe Dublin group was significantly higher than that in the wild-type Dublin group.

[0050] The above experiments first used the relative fluorescence intensity of FITC as a quantitative indicator of hTLR5 expression to evaluate the activation ability of flagellin on hTLR5. The results showed that the hTLR5 expression level in the Ser445Phe Dublin group was higher than that in the wild type.

[0051] The Dublin flagellin genome was then analyzed. The activation capacity of flagellin for hTLR5 was assessed by measuring the transcriptional level of IL-8 in Caco-2 cells. The results showed that, compared to the wild-type Dublin flagellin genome, the Salmonella typhimurium flagellin mutant described in this invention (mutating amino acid 445 of the FliC gene from Ser to Phe) increased the transcriptional level of IL-8 in Caco-2 cells, indicating that this flagellin mutant effectively activated human TLR5 activity.

Claims

1. A Salmonella typhimurium flagellin mutant, characterized in that, The amino acid sequence of the Salmonella typhimurium flagellin mutant is: SEQ ID NO:

1.

2. The method for preparing the Salmonella typhimurium flagellin mutant as described in claim 1, comprising the following steps: S1: Expression of the flagellin mutant of Salmonella typhimurium: The Ser→Phe mutation was performed on amino acid 445 of the FliC gene and cloned into the prokaryotic expression vector pRSET-B for expression; S2: Purify the Salmonella typhimurium flagellin mutant from S1.

3. The use of the Salmonella typhimurium flagellin mutant as described in any one of claims 1-2 in the preparation of vaccines.

4. The application as described in claim 3, characterized in that, The Salmonella typhimurium flagellin mutant is used as an immune adjuvant in vaccine preparation.

5. The use of the Salmonella typhimurium flagellin mutant as described in any one of claims 1-2 in the preparation of immunotherapeutic agents and radioprotective agents.

6. A vaccine adjuvant, characterized in that, The active ingredient of the vaccine adjuvant contains the Salmonella flagellin mutant as described in any one of claims 1-2.

7. A vaccine, characterized in that, The vaccine comprises the Salmonella typhimurium flagellin mutant as described in any one of claims 1-2 and one or more antigens.

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