Flagellin mutant for enhancing TLR5 ligand activity and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a flagellin mutant capable of enhancing TLR5 ligand activity and application of the flagellin mutant. An amino acid mutation site of salmonella typhimurium flagellin is selected, recombinant protein is expressed by using an escherichia coli system through a site-specific mutagenesis technology, in-vitro experiments prove that the recombinant protein shows good biological activity, and compared with a wild flagellin mutant, the recombinant protein can better activate the activity of a TLR5 ligand and can be used for preparing a TLR5 mutant. And a good foundation is provided for enhancing the activity of flagellin adjuvants and vaccines.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a Salmonella typhimurium flagellin mutant with enhanced TLR5 ligand activity and its applications. Background Technology

[0002] Toll-like receptors (TLRs) and NOD-like receptors (NLRs) are two important families of pattern recognition receptors for the innate immune system, playing crucial roles in pathogen recognition and innate immune activation. Flagellin proteins, major structural components of bacterial flagella, are important pathogen-associated molecular patterns (PAMPs). Extracellularly, they are specifically recognized by Toll-like receptor 5 (TLR5) on the cell surface, and intracellularly, they are specifically recognized by the NLR family CARD domain containing 4 (NLRC4), inducing the production of pro-inflammatory factors and thereby triggering the body's innate immune response. [1][2][3][4] .

[0003] The complete flagellin structure can be divided into four domains: D0, D1, D2, and D3. Among them, the D0 and D1 sequences are highly conserved, while D2 and D3 form the hypervariable region. [5] TLR5 recognizes only flagellin monomers. (Yoon) [6] The crystal structure of the TLR5 extracellular domain complex with flagellin was published, further elucidating the structural basis and mechanistic significance of TLR5-flagellate recognition. TLR5 primarily interacts with the three helices of the flagellin D1 domain on its outer side, then triggers an innate immune response through NF-κB activation signaling. However, due to variations in flagellin sequence and domains, flagellins from different bacterial species utilize different TLR5 recognition mechanisms.

[0004] Given the unique biological properties of flagellin, it shows significant potential for development in novel molecular immune adjuvants. Compared to traditional aluminum glue oil emulsion adjuvants, flagellin can simultaneously induce both humoral and cellular immune responses in the body. 7 ] [8] In some immunization regimens, co-administration of flagellin with antigen can induce an immune response against both the exogenous antigen and flagellin. [9]

[10] The unique structure of flagellin allows it to bind to exogenous antigens in multiple forms without affecting conformational epitopes, constructing fusion proteins that can significantly improve antigen immunization efficacy. 11 ]

[12] These findings indicate that flagellin and flagellin derivatives have many advantages as vaccine adjuvants, and flagellin, as the only agonist of TLR5, also has great potential for application in site-directed mutations of flagellin.

[0005] Current research on flagellin structural modification mostly involves truncation mutations and chimeric designs. However, the structural stability and solubility of modified flagellins may be significantly affected, and truncation mutations risk functional loss, while chimeric designs may introduce non-natural immunogenicity. Site-directed mutagenesis of flagellins can significantly improve the precision and controllability of flagellin modification, while preserving the native folded conformation to the greatest extent and reducing off-target risks. This strategy can not only accelerate the development of high-affinity flagellins but also be extended to the engineering research of other protein-receptor interactions.

[0006] References

[0007] [1]SongWS,JeonYJ,Namgung B,HongM,Yoon SI(2017)AconservedTLR5 binding and activation hot spot on flagellin.Sci Rep 7:40878.

[0008] [2]Miao EA, Andersen-Nissen E, Warren SE, et al. TLR5 and Ipaf: dualsensors ofbacterial flagellin in the innate immune system. SeminImmunopathol. 2007; 29(3):275–288.

[0009] [3] Mariathasan S, Newton K, Monack DM, et al. Differential activation of the inflammasome by caspase-1adaptorsASC and Ipaf. Nature. 2004; 430 (6996): 213–218.

[0010] [4]Miao EA,Alpuche-Aranda CM,Dors M,et al.Cytoplasmic flagellinactivates caspase-1and secretion of interleukin 1betavia Ipaf.NatImmunol.2006;7(6):569–575.

[0011] [5]Bode W.The bacterial flagella and the flagellar proteinflagellin.Angew Chem Int Ed Engl.1973;12(9):683–693.

[0012] [6]Yoon SI,Kurnasov O,Natarajan V,et al.Structural basis of TLR5-flagellin recognition and signaling.Science.2012;335:859-864.

[0013] [7]Vijay-Kumar M,Carvalho FA,Aitken JD,Fifadara NH,Gewirtz AT.TLR5 orNLRC4 is necessary and sufficientforpromotion ofhumoral immunitybyflagellin.EurJ Immunol.2010Dec;40(12):3528-34.

[0014] [8]Mizel SB,Bates JT.Flagellin as an adjuvant:cellular mechanisms andpotential.J Immunol.2010Nov15;185(10):5677-82.

[0015] [9]HajamIA,DarPA,Chandrasekar S,NandaRK,Kishore S,BhanuprakashV,GaneshK.Co-administration of flagellin augments immune responses toinactivated foot-and-mouth disease virus(FMDV)antigen.Res Vet Sci.2013Dec;95(3):936-41.

[0016]

[10] Flores-Langarica A,Bobat S,Marshall JL,Yam-Puc JC,Cook CN,SerreK,Kingsley RA,Flores-Romo L,Uematsu S,Akira S,Henderson IR,Toellner KM,Cunningham AF.Soluble flagellin coimmunization attenuates Th1 priming toSalmonella and clearance by modulating dendritic cell activation and cytokineproduction.EurJ Immunol.2015Aug;45(8):2299-311.

[0017]

[11] Yamayoshi S,Kawaoka Y.Current and future influenza vaccines.NatMed.2019Feb;25(2):212-220.doi:10.1038 / s41591-018-0340-z.Epub 2019Jan28.PMID:30692696.

[0018]

[12] Liu G,Tarbet B,Song L,Reiserova L,Weaver B,ChenY,Li H,Hou F,LiuX,Parent J,UmlaufS,ShawA,Tussey L.Immunogenicity and efficacy offlagellin-fusedvaccine candidates targeting 2009pandemic H1N1influenza inmice.PLoSOne.2011;6(6):e20928. Summary of the Invention

[0019] The primary objective of this invention is to provide a flagellin mutant of Salmonella typhimurium.

[0020] The second objective of this invention is to disclose the application of a Salmonella typhimurium flagellin mutant in the preparation of vaccine adjuvants and vaccines. This flagellin mutant, as an immune adjuvant, can enhance the efficacy of related vaccines. As a vaccine, this flagellin mutant provides an alternative to vaccines applicable in the relevant field. Specifically, it includes the following:

[0021] In a first aspect, the present invention discloses a Salmonella typhimurium flagellate protein mutant, the amino acid sequence of which is: SEQ ID NO:1.

[0022] Preferably, the method for preparing the Salmonella typhimurium flagellin mutant includes the following steps:

[0023] S1: Expression of the flagellin mutant of Salmonella Typhimurium: The Ser→Trp mutation was performed on amino acid 111 of the FliC gene and cloned into the prokaryotic expression vector pRSET-B for expression.

[0024] S2: Purify the Salmonella typhimurium flagellin mutant from S1.

[0025] Preferably, the application of the Salmonella typhimurium flagellin mutant in vaccine preparation.

[0026] Furthermore, the Salmonella typhimurium flagellin mutant is used as an immune adjuvant in vaccine preparation.

[0027] Secondly, the present invention discloses a vaccine adjuvant, wherein the active ingredient of the vaccine adjuvant contains the above-mentioned Salmonella typhimurium flagellin mutant.

[0028] Thirdly, the present invention discloses a vaccine comprising the above-mentioned Salmonella typhimurium flagellin mutant and one or more antigens.

[0029] The beneficial effects of this invention are:

[0030] 1. The Salmonella typhimurium flagellin mutant of the present invention exhibits good biological activity and can activate TLR5 ligand activity, laying the foundation for the development of vaccine adjuvants and vaccines.

[0031] 2. This invention utilizes a prokaryotic expression system to express the flagellated protein mutant of Salmonella Typhimurium in a soluble form. The production process is simple, with low production requirements and costs, and is easy to scale up and mass-produce. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the pRSET-B-Dublin recombinant plasmid.

[0033] 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.

[0034] Figure 3 Figure 1 shows the results of FliC Ser111Trp induction, expression, and purification (the red arrows indicate the FliC Ser111Trp protein). Figure A shows the induction results, with the marker indicating the protein molecular weight standard. Figures 5 and 7 show whole FliC Ser111Trp cells before induction, and figures 6 and 8 show whole FliC Ser111Trp cells after induction. Figure B shows the purification results, with the marker indicating the protein molecular weight standard. Figure 1 shows the supernatant after cell lysis, Figure 2 shows the precipitate after cell lysis, Figure 3 shows the flow-through buffer, Figure 4 shows the washing buffer, and Figures 5-8 show the elution buffer.

[0035] Figure 4 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

[0036] 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.

[0037] Example 1: Construction of pRSET-B-Dublin and pRSET-B-Dublin-Ser111Trp recombinant plasmids

[0038] The 111th amino acid of the Dublin flagellate protein FliC was selected as the mutation site. The Ser→Trp mutation was performed on the 111th amino acid of the FliC gene to obtain FliC Ser111Trp, and the pRSET-B-Dublin-Ser111Trp recombinant plasmid was constructed. The complete sequence of FliC Ser111Trp is SEQ ID NO:1: MAQVINTNSLSLLTQNNLNKSQSSLSSAIERLSSGLRI NSAKDDAAGQAIANRFTSNIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELSVQATNGTNSDSDLKWIQDEIQQRLEEIDRVSNQTQFNGVKVLSQDNQMKIQVGANDGET ITIDLQKIDVKSLGLDGFNVNGPKEATVGDLKSSFKNVTGYDTYAAGADKYRVDINSGAVVTDAVAPDKVYVNAANGQLTTDDAENNTAVDLFKTTKSTAGTAEAKAIAGAIKGGKE GDTFDYKGVTFTIDTKTGDDGNGKVSTTINGEKVTLTVADIAIGAADVNAATLQSSKNVYTSVVNGQFTFDDKTKNESAKLSDLEANNAVKGESKITVNGAEYTANATGDKITLAGK TMFIDKTASGVSTLINEDAAAAKKSTANPLasiDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNSARSRIEDADYATEVSNMSKAQILQQAGTSVLAQANQVPQNVLSLLR.

[0039] pRSET-B-Dublin plasmid ( Figure 1The 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 111 of FliC was mutated to Trp, yielding the pRSET-B-Dublin-Ser111Trp plasmid. Subsequently, the pRSET-B-Dublin-Ser111Trp plasmid was transformed into Top10 competent E. coli cells, plated on LB agar containing ampicillin, and cultured at 37°C for 16 hours. Single colonies were then picked and cultured in LB liquid agar containing ampicillin at 37°C and 220 rpm for 16 hours. The pRSET-B-Dublin-Ser111Trp plasmid was extracted using a plasmid extraction kit.

[0040] Example 2: Wild-type Dublin flagellin and Ser111Trp Dublin flagellin prokaryotic expression and purification

[0041] The recombinant plasmids pRSET-B-Dublin and pRSET-B-Dublin-Ser111Trp 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).

[0042] 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.

[0043] 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.

[0044] Example 3: Detection of the activation effect of wild-type Dublin flagellin and Ser111Trp Dublin flagellin on hTLR5

[0045] 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.

[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 experimental group and cultured for 5 h. RNA was then extracted from the cells.

[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 4 As shown, the mRNA level of IL-8 in the Ser111Trp Dublin group was significantly higher than that in the wild-type Dublin group.

[0050] The above experiments assessed the activation ability of flagellin on TLR5 by measuring the transcriptional level of IL-8 in Caco-2 cells. The results showed that, compared with wild-type typhoid flagellin, the Salmonella typhimurium flagellin mutant described in this invention significantly increased the transcriptional level of IL-8 in Caco-2 cells. Figure 4 This indicates that the Salmonella typhimurium flagellin mutant can effectively activate 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→Trp mutation was performed on amino acid 111 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. 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.

6. 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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