Chimeric flagellin and uses thereof

Abstract

The application provides a kind of chimeric flagellin and its application.The chimeric flagellin is formed by the chimeric of A type Seneca virus VP2 fragment and Escherichia coli Nissle 1917 flagellin FliC through Linker (GGGGS) 3, which realizes the soluble expression of chimeric flagellin in Escherichia coli system, and the yield can reach 200 mg / L at most. And through the detection of in vitro Caco2 and Raw264.7 cell models, the recombinant chimeric flagellin shows good TLR5 receptor activity, which can effectively activate TLR5 signal pathway.

Description

Technical fields:

[0001] This invention belongs to the field of biotechnology, specifically relating to the design, induced expression, purification, and in vitro bioactivity verification of a chimeric protein of type A Seneca virus VP2 fragment and Escherichia coli Nissle1917 flagellate protein. Background technology:

[0002] Seneca disease is a newly emerging infectious disease in pigs caused by Senecavirus A (SVA). Infected pigs typically exhibit symptoms such as depression, fever, anorexia, lameness, and vesicles on the hooves and snout. In recent years, SVA outbreaks have been increasing in many regions of China, leading to high mortality rates in infected piglets and causing significant economic losses to the pig farming industry. Currently, there is no commercially available vaccine for SVA infection control, and the inactivated and live attenuated vaccines that have been extensively studied also have some shortcomings, such as the risk of reversion in live attenuated vaccines and the problem of virus escape and incomplete inactivation during the production of inactivated vaccines. Subunit vaccines, due to their high safety, long-lasting immunity, and the ability to be mass-produced using engineered bacteria, have become a key focus of vaccine development. Research has found that the SVA precursor protein P1 can be ultimately broken down into four structural proteins, VP1-VP4, by proteases such as 3C. Among them, the VP2 structural protein has better immunogenicity, making the design of SVA subunit vaccines using VP2 as the antigen the preferred method for SVA prevention. [1] However, the manufacturing process of VP2 subunit vaccines typically requires the addition of adjuvants to improve vaccine efficacy, and VP2 expressed alone has poor solubility. Flagellin, as a novel antibody adjuvant, has the advantage of exerting its adjuvant effect at lower doses compared to other traditional aluminum glue oil emulsion adjuvants. It can effectively induce humoral and cellular immune responses in the host. [2] .

[0003] Bacterial flagellin, as an important pathogen-associated molecular pattern (PAMP), can induce pro-inflammatory responses and exert immune adjuvant activity by activating the TLR5 or NLRC4 signaling pathways in the body. Currently, research on the adjuvant activity of Salmonella typhimurium flagellin is extensive, and its immune adjuvant efficacy has been applied in the research of vaccines against various pathogens, including Yersinia pestis, Streptococcus mutans, Listeria monocytogenes, influenza A virus, West Nile virus, and Plasmodium vivax. [3] Recent studies have also found that the flagellin of *E. coli* Nissle 1917 has a similar crystal structure and can induce host cells to secrete a variety of cytokines, regulating immune responses. This demonstrates good adjuvant potential and could represent another novel vaccine design strategy. [4-5] .

[0004] Chimerism between flagellin and antigen is a preferred strategy for vaccine construction. Inserting exogenous antigens at different sites on the flagellin not only maintains immunomodulatory function and ensures effective delivery of antigens and adjuvants, thus enhancing the immune response, but also shortens vaccine production time and reduces costs. Currently, this chimerism strategy has been extensively studied in the application of Salmonella typhimurium flagellin, such as its chimerism with Plasmodium merozoon surface protein antigen, Yersinia pestis F1 antigen, and human papillomavirus antigen, all of which have shown good protective effects. [6] However, research on chimeric antigens of flagellin from *E. coli* Nissle 1917 is relatively lacking, possibly due to insufficient structural elucidation and incomplete mechanistic studies. Therefore, using chimeric strategies based on *Salmonella typhimurium* flagellin may not be effective or economical, leading to problems such as incorrect protein folding and low expression efficiency. Thus, Deng Yongjun's research involved immunization with a mixture of *E. coli* Nissle 1917 flagellin and antigen. The results showed that immunization with a mixture of *E. coli* Nissle 1917 flagellin and its mutants with the ALVP27 model antigen could exert an adjuvant effect. [5] This invention aims to address the aforementioned deficiencies and explore the effects of different chimeric forms on the adjuvant effect of flagellin. Flagellin from *E. coli* Nissle1917 was chimeric with the VP2 fragment of SVA. Unexpectedly, this chimeric form was found to solve the problem of poor VP2 solubility, significantly increasing the soluble expression yield. In vitro detection using Caco2 and Raw264.7 cell models showed that the recombinant chimeric flagellin exhibited good TLR5 receptor activity, effectively activating the TLR5 signaling pathway, while also improving the immunogenicity of VP2 and enhancing the overall efficacy of flagellin vaccines, showing promise for the prevention of Seneca disease.

[0005] [1]Ru Y,Hao R,Wu C,Li Y,Lu B,Liu H,Tian H,Li D,Shi Z,Luo J,Ma K,ZhangG,Liu X,Zheng H.Identification ofPotential Novel B-Cell Epitopes ofCapsidProtein VP2 in SenecavirusA.Microbiol Spectr.2023Aug 17;11(4):e0447222.doi:10.1128 / spectrum.04472-22.Epub 2023Jul 10.PMID:37428080;PMCID:PMC10433816.

[0006] [2]Hajam IA,Dar PA,Shahnawaz I,Jaume JC,Lee JH.Bacterial flagellin-apotent immunomodulatory agent.Exp Mol Med.2017Sep 1;49(9):e373.doi:10.1038 / emm.2017.172.PMID:28860663;PMCID:PMC5628280.

[0007] [3] Zhao Jing. Study on the enhancement of antibody response by flagellin of Salmonella typhimurium [D]. Second Military Medical University, 2011.

[0008] [4] Yang Ying, Deng Yongjun, Zhang Jin, et al. Structural analysis and epitope prediction of flagellate protein of probiotic Escherichia coli Nissle1917[J]. Chinese Journal of Biological Products, 2022, 35(06):672-677. DOI:10.13200 / j.cnki.cjb.003627.

[0009] [5] Deng Yongjun. Research on probiotic Nissle1917 flagellin as a novel immune adjuvant [D]. Guizhou University, 2021. DOI:10.27047 / d.cnki.ggudu.2021.003111.

[0010] [6] Wu Wenwen. Study on the immune adjuvant activity of different domains of Escherichia coli flagellin FliC [D]. Yangzhou University, 2020. DOI:10.27441 / d.cnki.gyzdu.2020.002019. Summary of the Invention:

[0011] The first objective of this invention is to provide a chimeric flagellin.

[0012] The second objective of this invention is to provide a method for preparing chimeric flagellin.

[0013] A third objective of this invention is to disclose the application of a chimeric flagellin in protecting animals from Seneca virus type A infection, providing an alternative to improve the effectiveness of Seneca virus vaccines and offering a viable vaccine alternative. Specifically, this includes the following:

[0014] In a first aspect, the present invention provides a chimeric flagellin, which is formed by chimerism of a type A Seneca virus VP2 fragment and an Escherichia coli Nissle 1917 flagellin.

[0015] Furthermore, the Seneca virus type A VP2 fragment is located at the N-terminus of the Nissle 1917 flagellin in Escherichia coli.

[0016] Furthermore, the chimeric flagellin is formed by linker-linked chimeric Seneca virus VP2 fragment and Escherichia coli Nissle 1917 flagellin.

[0017] Preferably, the amino acid sequence of the Linker is GGGGSGGGGSGGGGS.

[0018] Preferably, the amino acid sequence of the chimeric flagellin is shown in SEQ ID NO: 2. The nucleotide sequence of the chimeric flagellin is shown in SEQ ID NO: 1.

[0019] Furthermore, the chimeric flagellin is used in the preparation of a vaccine to prevent Seneca virus infection.

[0020] Secondly, the present invention provides a method for preparing chimeric flagellin, comprising the following steps:

[0021] S1: Complete genetic synthesis of chimeric flagellin VP2-L-FliC, the nucleotide sequence of which is shown in SEQ ID NO: 1;

[0022] S2: The nucleotide sequence of the chimeric flagellin VP2-L-FliC was cloned into the expression vector pET-28a(+) through BamHI and SalI restriction sites to construct the plasmid VP2-L-FliC in pET-28a(+). The recombinant Escherichia coli expression vector was transformed into competent cells BL21(DE3) to obtain a recombinant strain for producing chimeric flagellin.

[0023] S3: Induce expression of the recombinant strain obtained in step S2;

[0024] S4: The bacterial culture obtained in step S3 is separated and purified to obtain chimeric flagellin.

[0025] Furthermore, chimeric flagellin was prepared using the above preparation method.

[0026] Thirdly, the present invention provides the application of chimeric flagellin in the preparation of a vaccine to prevent Seneca virus infection.

[0027] The beneficial effects of this invention are as follows: Compared with the prior art, the chimeric flagellin of this invention exhibits the following significant advantages: 1) The VP2 fragment expressed alone has poor solubility. Unexpectedly, the chimeric flagellin of this invention can solve the problem of VP2 fragment inclusion body expression, and the chimeric flagellin is essentially soluble. 2) The soluble expression level of the chimeric flagellin can reach approximately 200 mg / L. 3) Compared with other chimeric proteins, the chimeric flagellin of this invention shows better in vitro TLR5 receptor activity. 4) The chimeric flagellin of this invention can activate T lymphocytes, improve the immunogenicity of VP2, and enhance the overall efficacy of flagellin vaccines. Attached Figure Description

[0028] Figure 1 Plasmid map of recombinant expression vector VP2-L-FliC in pET28a(+).

[0029] Figure 2 SDS-PAGE electrophoresis images of the soluble expression levels of three chimeric proteins. Figure a shows FliC. Δ195-320 -L-VP2, Lane 1: Protein molecular weight standard; Lane 2: Uninduced sample; Lane 3: Sample induced at 10℃; Lane 4: Cell lysate precipitate; Lane 5: Cell lysate supernatant. Figure b shows FliC. Δ274-406 -L-VP2, Lane 1: Sample induced at 10℃; Lane 2: Cell lysate precipitate; Lane 3: Cell lysate supernatant. Figure c shows VP2-L-FliC, Lane 1: Protein molecular weight standard; Lane 2: Uninduced sample; Lane 3: Sample induced at 37℃; Lane 4: Cell lysate precipitate; Lane 5: Cell lysate supernatant. Figure d shows the VP2 fragment, Lane 1: Cell lysate supernatant; Lane 2: Cell lysate precipitate.

[0030] Figure 3 SDS-PAGE electrophoresis image of purified chimeric protein. Lane 1: Protein molecular weight standard; Lane 2: Uninduced sample; Lane 3: Sample induced at 37℃; Lane 4: Cell lysate precipitate; Lane 5: Cell lysate supernatant; Lane 6: Lysate supernatant flow-through; Lane 7: Washed sample; Lane 8: Eluted sample 1; Lane 9: Eluted sample 2; Lane 10: Eluted sample 3; Lane 11: Eluted sample 4.

[0031] Figure 4 : Western blot results of the purified chimeric protein.

[0032] Figure 5 Dynamic light scattering (DLS) results of VP2-L-FliC protein

[0033] Figure 6Transmission electron microscopy (TEM) results of VP2-L-FliC protein

[0034] Figure 7 The in vitro TLR5 receptor activity of three purified chimeric proteins was detected. Human colon cancer cells (Caco2) and mouse macrophages (Raw264.7) were stimulated for 12 h with 20 μg / mL of the endotoxin-free chimeric proteins, with unstimulated cells serving as a negative control. Cell culture supernatants were collected, and the concentrations of IL-8 and TNF-α in the supernatants were detected using human IL-8 and mouse TNF-α ELISA kits, respectively. Detailed Implementation

[0035] The embodiments of the present invention will be clearly and completely described below with reference to the examples. Obviously, the described embodiments are only used to illustrate a part of the embodiments of the present invention and should not be regarded as limiting the scope of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Where the manufacturers of reagents or instruments used are not specified, they shall be regarded as conventional products that can be purchased commercially.

[0036] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, protein recombinant expression processes, cell culture, and related fields.

[0037] In the specific implementation of this invention, three different forms of chimeric flagellin were constructed to evaluate their effects on the adjuvant effect of flagellin. These three chimeric flagellins are: VP2 is intercalated into the N-terminus of flagellin FliC to form VP2-L-FliC; VP2 is intercalated into the hypervariable region D3 of FliC to form FliC. Δ274-406 -L-VP2; and embedding VP2 into the high-variance region D2D3 of FliC to form FliC Δ195-320 -L-VP2.

[0038] Glossary

[0039] 1. Linker: In biochemistry and molecular biology, a "linker" or "linking peptide" refers to a short peptide sequence that links two or more functional peptides or proteins. The design of linking peptides typically requires consideration of their length, amino acid composition, and spatial conformation to ensure that they do not interfere with the function of the linked proteins while maintaining the desired biological activity.

[0040] 2. Chimerism: In biochemistry and molecular biology, "chimerism" generally refers to the combination of two or more biomolecules (such as proteins, DNA, or RNA) from different sources to form a new molecule. For example, chimeric proteins are proteins composed of structural or functional domains from proteins of different origins.

[0041] Example 1 describes the construction and expression of the VP2-L-FliC chimeric protein, and the optimization of the recombinant expression process.

[0042] (1) Construction of recombinant plasmids

[0043] The complete amino acid sequence of the Seneca virus type A VP2 fragment chimeric with E. coli Nissle 1917 flagellin, as shown in SEQ ID NO.2, was provided to Genewiz for codon optimization. The codon-optimized VP2-L-FliC encoding gene was then synthesized by Genewiz. The sequence was cloned into the vector pET-28a(+) using BamHI and SalI restriction sites to construct the plasmid VP2-L-FliC inpET-28a(+).

[0044] The construction methods for the other two chimeric protein recombinant plasmids are the same as above.

[0045] (2) Construction and identification of recombinant bacteria

[0046] The obtained recombinant plasmid VP2-L-FliC inpET-28a(+) was transformed into *E. coli* BL21(DE3) competent cells, plated on LB agar plates containing kanamycin sulfate, and incubated at 37°C. Single positive clones were picked, cultured in LB liquid medium containing kanamycin sulfate, and the plasmid was extracted. The cells were identified by double digestion with BamHI and SalI, followed by agarose gel electrophoresis to identify the digestion products. After plasmid digestion, a band of approximately 2690 bp was observed. *E. coli* cells successfully infused with the recombinant plasmid VP2-L-FliCin pET-28a(+) were named VP2-L-FliC in pET-28a(+) / BL21(DE3). The construction and identification of the other two chimeric protein recombinant bacteria followed the same method.

[0047] The empty plasmid pET-28a(+) was transformed into Escherichia coli BL21(DE3) competent cells to obtain the negative control strain pET-28a(+) / BL21(DE3).

[0048] (3) Induced expression of chimeric proteins

[0049] The chimeric protein VP2-L-FliC was induced in the recombinant strain VP2-L-FliC inpET-28a(+) / BL21(DE3), with the control strain pET-28a(+) / BL21(DE3) serving as the negative control. The specific methods are as follows:

[0050] ① Select a single positive colony of the recombinant bacteria and inoculate it into LB liquid medium containing 80 μg / mL kanamycin sulfate (containing 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl), and incubate overnight at 37°C.

[0051] ② Take 300 μl of the recombinant bacterial culture obtained in step ① and inoculate it into 300 mL of fresh LB liquid medium containing 80 μg / mL kanamycin sulfate. Incubate at 37℃ for 3-4 h until the OD600 reaches 0.6-0.8.

[0052] ③ Add IPTG to a final concentration of 0.8 mmol / L to the bacterial culture with an OD600 of 0.6-0.8, and continue culturing at 37℃ for 6 h. Compared with the control strain, the genetically engineered recombinant strain VP2-L-FliC in pET-28a(+) / BL21(DE3) showed a target protein band at approximately 90 kDa, consistent with the theoretical value, and the expression level reached 200 mg / L.

[0053] The induction conditions for the other two chimeric proteins were 10℃, a final IPTG concentration of 0.01 mmol / L, and induction for 32 h.

[0054] (4) Purification of chimeric proteins

[0055] ① The recombinant strain VP2-L-FliC in pET-28a(+) / BL21(DE3) and the control strain pET-28a(+) / BL21(DE3) after induction expression were centrifuged at 4℃ and 12000rpm for 5min, and the bacterial cells were collected. The bacterial cells were washed twice with PBS buffer.

[0056] ② The bacterial cells were resuspended in binding buffer (20mM PB pH 7.4 + 500mM NaCl + 75mM imidazole + 0.1mM PMSF + 0.05mM MDT). The bacterial suspension was placed in an ice bath, and the cells were lysed using ultrasound at 60W for 3 seconds, followed by a 3-second pause, until the bacterial solution became clear, yielding the lysate of the recombinant bacteria. The chimeric protein was expressed in a predominantly soluble manner.

[0057] ③ Centrifuge the lysate of the recombinant bacteria at 4℃ and 10000rpm for 20min, and collect the supernatant of the lysate. Purify the chimeric protein VP2-L-FliC using a Ni NTABeads 6FF column (Tiandi Renhe, catalog number: SA005025) with gradient elution using Elution buffer (20mM PB pH7.4 + 500mM NaCl + 100-500mM imidazole).

[0058] ④ SDS-PAGE electrophoresis was used to detect the purification effect of the chimeric protein VP2-L-FliC and to determine the concentration of the purified protein. SDS-PAGE electrophoresis is as follows: Figure 3 As shown, a band of approximately 90 kDa appeared in lanes 8-11 of the purified chimeric protein, with a purity of over 85%.

[0059] The purification methods for the other two chimeric proteins are the same as above.

[0060] (5) Western blot validation analysis of chimeric proteins

[0061] The purified chimeric protein sample was subjected to SDS-PAGE electrophoresis and then transferred to a PVDF membrane under constant current of 300 mA for 90 min. The transferred PVDF membrane was then placed in 10% skim milk powder prepared with TBST (TBS + 0.5% Tween 20) and incubated on a shaker at room temperature for 2 h with gentle shaking. Anti-His-tag mouse monoclonal antibody (Beyotime, catalog number: AF2873) was used as the primary antibody, diluted with antibody dilution buffer (1:2000), and incubated on a shaker at room temperature for 2 h with gentle shaking. The membrane was washed three times with TBST for 5 min each time. The PVDF membrane was then developed using ECL luminescence. Western blot results are shown below. Figure 4 As shown, the chimeric protein VP2-L-FliC can specifically react with anti-His-tag mouse monoclonal antibody, and the protein size is approximately 90 kDa.

[0062] The Western blot validation analysis method for the other two chimeric proteins is the same as above.

[0063] Example 2: Structural confirmatory analysis of the chimeric protein VP2-L-FliC

[0064] (1) The chimeric protein VP2-L-FliC purified in Example 1 was concentrated five times, and the buffer was replaced with 20 mM PB pH 7.4. After assembly at 4°C overnight for 24 h, the particle size of the VP2-L-FliC protein of this invention was detected by dynamic light scattering (DLS) (Malvin particle size analyzer). The results are as follows: Figure 5As shown, dynamic light scattering (DLS) results indicate that the assembled VP2-L-FliC protein has a particle size of 10-30 nm, which is close to the theoretical values ​​in existing literature.

[0065] (2) The morphology and size of the VP2-L-FliC protein were simultaneously detected by TEM (Talos F200C). 5 μL of the VP2-L-FliC protein of this invention was added and adsorbed onto a copper grid for 1 min. The adsorption was continued with filter paper until a thin water film was formed. Then, 10 μL of the protein was added...

[0066] Stain with 2% phosphotungstic acid negative dye solution for 1 minute, absorb excess dye with filter paper, air dry, and observe under a transmission electron microscope (TEM). The results are as follows: Figure 6 As shown, the morphology and size of the particles formed by the self-assembly of VP2-L-FliC protein are consistent with the results of dynamic light scattering (DLS).

[0067] Example 3: Detection of in vitro TLR5 receptor activity of chimeric protein VP2-L-FliC

[0068] (1) The chimeric protein purified in Example 1 was concentrated five times and the buffer was replaced with PBS. The lipopolysaccharide (LPS) in the flagellar protein was removed using the ToxinEraser™ Endotoxin Removal Kit (GenScript, catalog number: L00338). The LPS content was ensured to be less than 0.1 EU / mL using the ToxinSensor™ Gel Clot Endotoxin Assay Kit (GenScript, catalog number: L00351). The protein concentration after endotoxin removal was then determined to be 800 mg / L using the BCA method.

[0069] (2) Using human colorectal adenocarcinoma cells (Caco2) that express the TLR5 receptor as an in vitro cell model, the TLR5 receptor activity of the chimeric protein was detected. Caco2 cells were cultured in 10 cm⁻¹ cells. 2 After the cells have grown to a confluence in the culture dish, divide them into groups of 3 × 10⁶ cells per well. 5 Caco2 cells were seeded into 12-well cell culture plates and cultured overnight at 37°C with 5% CO2. The cells were then stimulated for 12 h with purified chimeric protein at a final concentration of 20 μg / mL. Untreated Caco2 cells were used as a negative control. The cell culture supernatant was collected, centrifuged at 3000 rpm for 20 min at 4°C, and the concentration of IL-8 in the cell supernatant was detected using a human IL-8 ELISA kit (ELISA kit, catalog number: MM-1558H1). Results are shown below. Figure 7 As shown in figure a, the VP2-L-FliC chimeric protein significantly induced Caco2 cells to secrete IL-8, and its level was higher than that of FliC. Δ274-406 -L-VP2, and with FliCΔ195-320 There was no significant difference compared to -L-VP2. This indicates that VP2-L-FliC possesses good TLR5 receptor activity, and its activity is higher than that of FliC. Δ274-406 -L-VP2.

[0070] (3) Next, using mouse macrophages (Raw264.7) expressing the TLR5 receptor as an in vitro cell model, the TLR5 receptor activity of the chimeric protein was further detected. The experimental method followed the same procedure as Caco2 cells, but a mouse TNF-α ELISA kit (enzyme immunoassay, catalog number: MM-0132M2) was used. The ELISA results are as follows: Figure 7 As shown in b, VP2-L-FliC significantly stimulated Raw264.7 cells to secrete TNF-α, and FliC Δ195-320 -L-VP2 induction levels were significantly higher than FliC. Δ274-406 -L-VP2 further confirmed the good TLR5 receptor activity of VP2-L-FliC.

[0071] Despite VP2-L-FliC and FliC Δ195-320 -L-VP2 shows similar activity to TLR5 receptors, but to achieve the same adjuvant effect, FliC Δ195-320 The preparation of -L-VP2 is significantly more difficult than that of VP2-L-FliC. Specifically, FliC Δ195-320 The soluble expression level of -L-VP2 is only one-tenth that of VP2-L-FliC. Its preparation requires low-temperature induction, and the entire process is time-consuming and complex. Therefore, this invention preferentially selects VP2-L-FliC as the chimeric protein to improve the feasibility of research and application.

Claims

1. A chimeric flagellin, characterized in that, The chimeric flagellin is formed by embedding a type A Seneca virus VP2 fragment into the N-terminus of the Escherichia coli Nissle 1917 flagellin. The amino acid sequence of the chimeric flagellin is shown in SEQ ID NO: 2, and the nucleotide sequence of the chimeric flagellin is shown in SEQ ID NO:

1.

2. The use of the chimeric flagellin as described in claim 1 in the preparation of a vaccine to prevent Seneca virus infection.

3. A method for preparing a chimeric flagellin, comprising the following steps: S1: Complete genetic synthesis of chimeric flagellin VP2-L-FliC, the nucleotide sequence of which is shown in SEQ ID NO: 1; S2: The nucleotide sequence of the chimeric flagellin VP2-L-FliC was cloned into the expression vector pET-28a(+) through BamHI and SalI restriction sites to construct a recombinant Escherichia coli expression vector. The recombinant Escherichia coli expression vector was transformed into competent cells BL21(DE3) to obtain a recombinant strain for producing chimeric flagellin. S3: Induce expression of the recombinant strain obtained in step S2; S4: The bacterial culture obtained in step S3 is separated and purified to obtain chimeric flagellin.

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