Fusion protein of salmonella typhimurium flagellin derivative and Seneca virus antigen and application thereof

Abstract

The invention belongs to the technical field of biology, and discloses a fusion protein of a salmonella typhimurium flagellin derivative and a Seneca virus antigen and application of the fusion protein. A salmonella typhimurium flagellin derivative gene and a Seneca virus antigen gene are fused, codon optimization is carried out, the fusion protein is cloned to a pET-28a (+) vector, an escherichia coli system is utilized to express the fusion protein, and in-vivo experiments prove that the fusion protein shows good biological activity, can activate the activity of a TLR5 ligand and promote the activation of macrophages, and can be used for preparing a recombinant vector. And a foundation is laid for the development of Seneca virus subunit vaccines.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a fusion protein of a Salmonella typhimurium flagellin derivative and a Seneca virus antigen and an application thereof. Background Art

[0002] Senecavirus A (SVA) is a new type of small RNA virus that has caused vesicular diseases in many countries and regions in recent years, seriously endangering the development of the pig farming industry. SVA virus particles are icosahedral in structure, without an envelope, and have a diameter of about 25 to 30 nm. The four viral structural proteins (VP) VP1 to VP4 encoded form the capsid of the virus. Among them, VP2 protein is an important structural protein exposed on the surface of SVA virus particles, has strong antigenicity, and can induce a strong immune response in the body. [1] Currently, there is no commercial SVA vaccine available, so there is an urgent need to develop an effective vaccine to prevent and control SVA. [2] .

[0003] In recent years, SVA subunit vaccines have become a research hotspot, but their disadvantage is that they have poor immunogenicity, so adjuvants are needed to enhance their ability to stimulate the body's immune response. Although traditional adjuvants (aluminum salt adjuvants, oil emulsion adjuvants) can improve the immune response of subunit vaccines to a certain extent, they have high requirements for processing, storage and transportation, and a small range of use, which can no longer meet the needs of new vaccines. With the development of molecular biology technology, new adjuvants based on pathogen-associated molecular patterns (PAMPs) have gradually become a research hotspot.

[0004] Among them, flagellin, as a PAMPs that can activate the natural immune response, induces the production of proinflammatory factors in the body by stimulating the TLR5 pathway on the cell surface and / or the intracellular NLRC4 pathway, stimulates the body's innate immune response, and shows strong adjuvant activity. However, after flagellin acts on the TLR5 receptor, the strong natural immune response stimulated can lead to systemic adverse reactions, such as sepsis, etc. This side effect restricts its application prospects as an adjuvant. The typhoid Salmonella flagellin derivative of the present invention can not only retain the characteristics of the wild-type flagellin immune adjuvant, but also reduce the natural immune response stimulated by it, and reduce its side effects on the body, which is of practical significance for the development of new vaccines. By mixing or fusing flagellin with the target protein, the body's immune response to the antigen can be improved, and the immune effect of flagellin fusion or coupling antigen is more significant. However, multiple factors need to be considered in the design of the fusion protein, and improper design may lead to instability or incorrect folding of the protein structure, thereby affecting its biological function.

[0005] In the E. coli prokaryotic expression system, the SVAVP2 protein is mainly expressed in the form of inclusion bodies in existing research reports. [4-5] , resulting in the inability of the recombinant protein to fold correctly, thereby affecting its normal biological function. However, the Salmonella typhimurium flagellin derivative of the present invention can be efficiently expressed in the supernatant, and after being fused with the Seneca virus antigen, it is helpful for the supernatant expression of VP2. The fusion protein of the Salmonella typhimurium flagellin derivative and the Seneca virus antigen provided by the present invention shows good biological activity, and the production process is simple and low-cost, which lays a foundation for the development of Seneca virus subunit vaccines.

[0006] References

[0007] [1] Hales LM, Knowles NJ, Reddy PS, et al. 2008. Complete genome sequence analysis ofSeneca valley virus-001, a novel oncolytic picornavirus[J]. The Journal of General Virology, 89(Pt 5):1265-1275.

[0008] [2] Zhang T, Wang HB, Sun YY, et al. 2022. Preparation of monoclonal antibody based on VP1protein against Senecavirus A(SVA) and trial production of colloidal gold test strips for SVA detection[J]. Journal of Agricultural Biotechnology, 30(11):22552266.)

[0009] [3][1]MIZEL SB,BATES J T.Flagellin as an adjuvant:cellularmechanisms and potential[J].JImmunol,2010,185(10):5677-5682.

[0010] [4]Dvorak CM, Akkutay-Yoldar Z, Stone SR, et al. 2017. An indirectenzyme-linked immunosorbent assay for the identification of antibodies toSenecavirus A in swine[J]. BMC Veterinary Research, 13(1):50.

[0011] [5]Maggioli MF,Lawson S,de Lima M,et al.2018.Adaptive immune responses following SenecavirusAinfection inpigs[J].Journal Virology,92(3):e01717-17. Summary of the invention

[0012] The first object of the present invention is to provide a fusion protein of a Salmonella typhimurium flagellin derivative and a Seneca virus antigen.

[0013] The second object of the present invention is to provide a method for preparing a fusion protein of a Salmonella typhimurium flagellin derivative and a Seneca virus antigen.

[0014] The third object of the present invention is to provide an application of a fusion protein in the preparation of a Seneca virus vaccine.

[0015] Specifically include the following:

[0016] In a first aspect, the present invention provides a fusion protein of a Salmonella typhimurium flagellin derivative and a Senecavirus antigen, wherein the fusion protein is formed by fusing a Salmonella typhimurium flagellin derivative Δdublin with a Senecavirus VP2 antigen type A. Furthermore, the fusion protein is formed by linking the Senecavirus VP2 antigen to the N-terminus of the Salmonella typhimurium flagellin derivative Δdublin via a linker.

[0017] Preferably, the linker sequence is GGGGSGGGGSGGGGS.

[0018] Preferably, the amino acid sequence of the fusion protein is shown as SEQ ID No. 1. The nucleotide sequence of the fusion protein is shown as SEQ ID No. 2.

[0019] In a second aspect, the present invention provides a method for preparing a fusion protein of a Salmonella typhimurium flagellin derivative and a Seneca virus antigen, the method comprising the following steps:

[0020] S1: constructing a pET-28a(+) expression vector containing the nucleotide sequence encoding the nucleotide sequence as claimed in claim 4, transforming the recombinant expression vector into Rosetta (DE3), and obtaining a recombinant strain for producing a fusion protein;

[0021] S2: Induce the expression of the recombinant strain obtained in step S1;

[0022] S3: Separate and purify the bacterial solution obtained in step S2 to obtain the fusion protein.

[0023] Furthermore, the step S2 is:

[0024] (1) Seed culture: streak the strain on an LB plate and culture at 37°C for 12-15 h. Pick a single colony and inoculate it into 20 mL LB medium and culture it at 37°C, 220 rpm overnight to obtain an activated seed solution.

[0025] (2) The activated seed solution obtained in step (1) was inoculated into the culture medium at a rate of 3%, and cultured at 37°C and 220 rpm until the OD 600 To 0.4-0.6, add IPTG to a concentration of 0.1mM.

[0026] (3) Continue to induce expression at 16°C and 160 rpm for 20 h.

[0027] Furthermore, the step S3 is:

[0028] (1) centrifuging the bacterial solution obtained in step S3 to obtain bacterial cells, disrupting the bacterial cells with ultrasound, and centrifuging to obtain the supernatant after the bacterial cell disruption for later use;

[0029] (2) The supernatant after bacterial cell disruption is subjected to nickel column affinity chromatography to elute the fraction containing the fusion protein.

[0030] In a third aspect, the present invention provides an application of a fusion protein in the preparation of a Seneca virus vaccine.

[0031] Beneficial effects of the present invention:

[0032] 1. The present invention uses a prokaryotic expression system to express a fusion protein of a Salmonella typhimurium flagellin derivative and a Seneca virus antigen in the form of fusion expression, which is the first invention.

[0033] 2. The fusion protein of the Salmonella typhimurium flagellin derivative and the Seneca virus antigen of the present invention is expressed in a soluble form, which improves the solubility and expression level of the antigen, has a simple production process, low production conditions and production costs, and is easy to scale up and mass produce.

[0034] 3. The fusion protein of the Salmonella typhimurium flagellin derivative and the Seneca virus antigen of the present invention shows good biological activity, can activate TLR5 ligand activity, and promote macrophage activation, laying a foundation for the development of Seneca virus subunit vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The schematic diagram of the fusion protein plasmid of the flagellin derivative of Salmonella typhimurium and the Seneca virus antigen. Figure A is pET-28a(+)-VP2-Δdublin, Figure B is pET-28a(+)-Δdublin-VP2, and Figure C is pET-28a(+)-FliC Δ29-164 -VP2-FliC Δ412-483 .

[0036] Figure 2 This is the SDS-PAGE diagram of the expression and purification of the fusion protein of the flagellin derivative of Salmonella typhimurium and the Seneca virus antigen. Lane M is the protein marker; Lane 1 is the bacterial solution after VP2-Δdublin induction; Lane 2 is the bacterial solution after Δdublin-VP2 induction; Lane 3 is FliC Δ29-164 -VP2-FliC Δ412-483 Bacterial liquid after induction; Lane 4 is the purified VP2-Δdublin; Lane 5 is the purified Δdublin-VP2; Lane 6 is the purified FliC Δ29-164 -VP2-FliC Δ412-483 .

[0037] Figure 3 The in vitro TLR5 receptor activity test of the fusion protein of Salmonella typhimurium flagellin derivative and Seneca virus antigen was performed. 5 μg / mL of endotoxin-free VP2-Δdublin, Δdublin-VP2, FliCΔ 29-164 -VP2-FliCΔ 412-483 Human colon cancer cell Caco2 cells were stimulated for 6 hours, and unstimulated Caco2 cells were used as negative control group. Cells were collected and RNA was extracted and reverse transcribed into cDNA, and RT-PCR was used to detect cytokine mRNA expression.

[0038] Figure 4It is a fusion protein of Salmonella typhimurium flagellin derivative and Seneca virus antigen for in vitro activation of mouse macrophages. 5 μg / mL of endotoxin-free VP2-Δdublin, Δdublin-VP2, FliCΔ 29-164 -VP2-FliCΔ 412-483 Mouse macrophage RAW264.7 cells were stimulated for 6 hours, and unstimulated RAW264.7 cells were used as negative control group. Cells were collected and RNA was extracted and reverse transcribed into cDNA, and RT-PCR was used to detect cytokine mRNA expression. DETAILED DESCRIPTION

[0039] In order to make the present invention easier to understand, the embodiments of the present invention will be further described below. The present invention is further described and demonstrated in conjunction with the embodiments. However, this embodiment is not a limitation of the present invention. The technical solutions described in the present invention, unless otherwise specified, are conventional solutions in the art; the reagents or materials shown, unless otherwise specified, are from commercial channels.

[0040] Example 1 pET-28a(+)-VP2-Δdublin, pET-28a(+)-Δdublin-VP2, pET-28a(+)-FliCΔ 29-164 -VP2-FliCΔ 412-483 Recombinant plasmid construction

[0041] The genes encoding VP2-Δdublin, Δdublin-VP2, and FliCΔ 29-164 -VP2-FliCΔ 412-483 The gene sequence was sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for codon optimization and full gene synthesis, and then connected to the pET-28a(+) vector. The ligation product was transformed into DH 5α competent cells, added to LB liquid medium and shaken for 1 hour, and an appropriate amount of culture product was coated on LB solid medium with kanamycin (Kan+), and cultured overnight in a 37°C constant temperature incubator. The next day, several single colonies were picked and sent to the company for sequencing after PCR identification. Finally, DNA sequencing confirmed that the clone construction was correct, and the recombinant plasmids were named pET-28a(+)-VP2-Δdublin, pET-28a(+)-Δdublin-VP2, pET-28a(+)-FliCΔ 29-164 -VP2-FliC Δ412-483 Schematic diagram of plasmid construction is shown in Figure 1 shown.

[0042] Example 2VP2-Δdublin, Δdublin-VP2, FliCΔ 29-164 -VP2-FliCΔ 412-483 Prokaryotic expression and purification

[0043] 1. The pET-28a(+)-VP2-Δdublin, pET-28a(+)-Δdublin-VP2, and pET-28a(+)-FliCΔ 29-164 -VP2-FliCΔ 412-483 The recombinant expression plasmid was transformed into Rosetta (DE3) competent cells, then spread on LB solid medium containing kanamycin and chloramphenicol, and cultured overnight in a 37°C constant temperature incubator. A single positive clone was picked from the overnight cultured LB plate, placed in 20 mL liquid LB medium containing kanamycin and chloramphenicol, and cultured overnight in a 37°C shaker. The inoculation amount was 3.0% and inoculated into 500 mL LB liquid medium containing kanamycin and chloramphenicol, and cultured at 37°C 200rpm until OD 600nm When the concentration reaches 0.4-0.6, add IPTG (final concentration 0.1mmol / L) and continue to induce expression at 16℃ and 160rpm for 20h. After the expression is completed, collect the bacteria by centrifugation at 10000rpm for 20min, then suspend the bacterial precipitate, mix well, and ultrasonically disrupt the cells on ice (power 40%, 3s working and 7s rest) until the bacterial solution remains basically clear. Purify the protein with Ni NTA material: add an appropriate amount of deionized water to the filled Ni NTA column to rinse the ethanol, add 8 column volumes of Binding buffer to balance, and load the sample after the balance is completed; use 15 column volumes of Washbuffer to rinse the column; use 5 column volumes of Elution Buffer to elute and collect the eluate; wash with 3 column volumes of Binding Buffer and 5 column volumes of deionized water in turn, and then balance with 3 column volumes of 20% ethanol.

[0044] 2. Perform ultrafiltration concentration and desalination on the purified samples. Take an ultrafiltration tube with a molecular weight cutoff of 10kDa, add distilled water to the inner tube, centrifuge at 4000rpm / min for 3min, and repeat three times. Add the collected elution solution to the inner tube of the ultrafiltration tube, centrifuge at 4000rpm / min, and repeat the operation until all purified samples are concentrated to about 1mL. Add 10 times the volume of PBS buffer to the concentrated product, mix well, and centrifuge at 4000rpm / min until the target protein preservation system is replaced with phosphate buffer. After centrifugation, collect the sample in the ultrafiltration tube. Use an endotoxin removal kit to remove lipopolysaccharide (LPS) from the protein to ensure that the LPS content is less than 0.1EU / mL, and then use the BCA method to determine VP2-Δdublin, Δdublin-VP2, and FliCΔ 29-164 -VP2-FliCΔ 412-483The concentrations of the proteins were 1 mg / mL, 0.8 mg / mL, and 0.5 mg / mL, respectively.

[0045] like Figure 2 As shown, the electrophoresis results showed that VP2-Δdublin (61.5 kDa), Δdublin-VP2 (61.5 kDa), and FliCΔ 29-164 -VP2-FliCΔ 412-483 (61.9 kDa), consistent with the expected size.

[0046] Example 3 In vitro TLR5 receptor activity detection of the fusion protein of Salmonella typhimurium flagellin derivative and Seneca virus antigen

[0047] Human colorectal adenocarcinoma cells Caco2 were cultured in complete DMEM medium at 37°C, 5% CO2 overnight (6-well plate, 2×10 5 The next day, VP2-Δdublin, Δdublin-VP2, FliCΔ 29-164 -VP2-FliCΔ 412-483 Stimulate cells; collect cells after 6 hours for RNA extraction; detect mRNA level of cytokine IL-8 by qRT-PCR.

[0048] Refer to the steps in the RNAiso Plus (takara, catalog number: 9109) manual to extract total RNA. When collecting cells, add 1000μL RNAiso Plus to each well, transfer the lysate containing cells to a centrifuge tube, and repeatedly pipette until there is no obvious precipitation in the lysate. Let it stand at room temperature (15-30℃) for 5 minutes, and then separate RNA from nuclear protein. Add 200μL chloroform to the above homogenized lysate, cover the centrifuge tube tightly, and mix until the solution is emulsified and milky white. Let it stand at room temperature for 5 minutes. Centrifuge at 12000g at 4℃ for 15 minutes. Carefully remove the centrifuge tube from the centrifuge. At this time, the homogenate is divided into three layers. Aspirate the supernatant and transfer it to another new centrifuge tube. Add 1000μL isopropanol to the supernatant, turn the centrifuge tube upside down to mix thoroughly, and let it stand at room temperature for 10 minutes. Centrifuge at 12000g at 4℃ for 10 minutes. After centrifugation, RNA precipitate appears at the bottom of the test tube. Discard the supernatant, add 1000 μL of 75% ethanol, gently invert the tube to wash the wall, centrifuge at 7500g at 4℃ for 5 minutes, and carefully discard the supernatant. Open the centrifuge tube cover and dry the precipitate at room temperature for a few minutes. After the precipitate is dry, add an appropriate amount of RNase-free water to dissolve the precipitate.

[0049] Using PrimeScript TMFAST RT reagent Kit with gDNA Eraser (takara, catalog number: RR092A) was used to reverse transcribe the extracted RNA into cDNA. The reaction system (20 μL) was as follows: 2 μL 8X gDNA Eraser Premix, 1 μL RT Primer Mix, 4 μL 5X RT Premix, 1000 ng RNA template, and RNase-free H2O was added to 20 μL. The reaction conditions were: 37°C, 10 min; 85°C, 5 s.

[0050] The obtained cDNA was used as a template for qRT-PCR detection of cytokines. The specific primers for IL-8 are IL-8-F (SEQ ID No. 3) and IL-8-R (SEQ ID No. 4). The qRT-PCR reaction system (25 μL) is: 1 μL cDNA, 12.5 μL TB Green Premix Ex Taq II Fast qPCR (takara, catalog number: CN830A), 1 μI upstream primer (10 μM), 1 μL downstream primer (10 μM), 9.5 μL RNase-free H2O. The reaction mixture was placed in a fluorescent quantitative PCR instrument for gene amplification. The qRT-PCR reaction procedure was: 95°C pre-denaturation for 30s; 95°C for 5s, 60°C for 10s, and 40 cycles. -ΔΔCT Methods The fluorescence value was calculated and the relative expression level was calculated using the housekeeping gene as the internal reference.

[0051] The results are as follows Figure 3 As shown, the fusion proteins of Salmonella typhimurium flagellin derivatives and Seneca virus antigens VP2-Δdublin, Δdublin-VP2, FliCΔ 29-164 -VP2-FliCΔ 412-483 Significant stimulation of Caco2 secretion of IL-8 indicated good TLR5 receptor activity. The protein VP2-Δdublin, which fused the antigen to the N-terminus, had significant advantages in activating TLR5-mediated immune responses.

[0052] Example 4 Activation of mouse macrophages in vitro by fusion protein of Salmonella typhimurium flagellin derivative and Seneca virus antigen

[0053] Mouse macrophage cell line RAW264.7 was cultured with complete DMEM medium at 37°C, 5% CO2 overnight (6-well plate, 1×10 6 The next day, VP2-Δdublin, Δdublin-VP2, FliCΔ 29-164 -VP2-FliCΔ 412-483Stimulate cells; collect cells after 6 hours for RNA extraction; and evaluate the ability of recombinant proteins to activate macrophages in vitro by detecting the mRNA levels of cytokines IL-6 and IL-1β by qRT-PCR. The specific primers for IL-6 are IL-6-F (SEQ ID No.5) and IL-6-R (SEQ ID No.6). The specific primers for IL-1β are IL-1β-F (SEQ ID No.7) and IL-1β-R (SEQ ID No.8). The results are shown in Figure 4 As shown, the activation levels of mouse macrophages stimulated by VP2-Δdublin and Δdublin-VP2 were comparable and significantly higher than those of the control group. 29-164 -VP2-FliCΔ 412-483 The levels of IL-1β secretion by mouse macrophages were significant, but the levels of IL-6 secretion were not significant. The combined in vivo experimental results showed that the protein VP2-Δdublin, which fused the antigen to the N-terminus, had significant advantages in activating the TLR5 signaling pathway and stimulating macrophage activation, which may provide important guidance for vaccine design.

Claims

1. A fusion protein of a Salmonella typhimurium flagellin derivative and a Seneca virus antigen, characterized in that: The fusion protein is formed by fusing a Salmonella typhimurium flagellin derivative Δdublin with a type A Seneca virus VP2 antigen.

2. The fusion protein of the Salmonella typhimurium flagellin derivative and Seneca virus antigen according to claim 1, characterized in that: The fusion protein is composed of Seneca virus VP2 antigen connected to the N-terminus of the Salmonella typhimurium flagellin derivative Δdublin through a linker.

3. The fusion protein of the Salmonella typhimurium flagellin derivative and Seneca virus antigen according to claim 2, characterized in that: The amino acid sequence of the Linker is GGGGSGGGGSGGGGS.

4. The fusion protein of the Salmonella typhimurium flagellin derivative and Seneca virus antigen according to claims 1-3, characterized in that: The amino acid sequence of the fusion protein is shown in SEQ ID No.1, and the nucleotide sequence of the fusion protein is shown in SEQ ID No.

2.

5. A method for preparing a fusion protein of a Salmonella typhimurium flagellin derivative and a Seneca virus antigen, characterized in that: The following steps are involved: S1: constructing a pET-28a(+) expression vector containing the nucleotide sequence encoding the nucleotide sequence as claimed in claim 4, transforming the recombinant expression vector into Rosetta (DE3), and obtaining a recombinant strain for producing a fusion protein; S2: Induce the expression of the recombinant strain obtained in step S1; S3: Separate and purify the bacterial solution obtained in step S2 to obtain the fusion protein.

6. The method for preparing a fusion protein according to claim 6, wherein the step S2 comprises: (1) Seed culture: streak the strain on an LB plate and culture at 37°C for 12-15 h. Pick a single colony and inoculate it into 20 mL of LB medium and culture it at 37°C, 220 rpm overnight to obtain an activated seed solution. (2) The activated seed solution obtained in step (1) was inoculated into the culture medium at a rate of 3%, and cultured at 37°C and 220 rpm until the OD 600 To 0.4-0.6, add IPTG to a concentration of 0.1mM. (3) Continue to induce expression at 16°C and 160 rpm for 20 h.

7. The method for preparing a fusion protein according to claim 6, wherein the step S3 comprises: (1) centrifuging the bacterial solution obtained in step S3 to obtain bacterial cells, disrupting the bacterial cells with ultrasound, and centrifuging to obtain the supernatant after the bacterial cell disruption for later use; (2) The supernatant after bacterial cell disruption is subjected to nickel column affinity chromatography to elute the fraction containing the fusion protein.

8. Use of the fusion protein according to claims 5-7 in the preparation of Seneca virus vaccine.

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