A method for preparing a fusion protein of salmonella typhimurium flagellin and seneca virus antigen

Abstract

The application belongs to the technical field of biology, and discloses a preparation method of a Salmonella typhimurium flagellin and Senecavirus antigen fusion protein. The application selects Escherichia coli Rosetta (DE3) as a host, fuses a Salmonella typhimurium flagellin gene and a Senecavirus antigen gene, optimizes codons, and then clones the fusion protein into a pET-28a (+) vector, so that the obtained fusion protein can keep the adjuvant activity of the flagellin, improve the solubility and expression level of VP2, and the yield can reach 40 mg / L, thereby greatly reducing the expression and purification process cost. The fusion protein provided by the application can activate the TLR5 path and activate mouse macrophages, and the production process is simple and low in cost, so that the application has important significance for the research and production of Senecavirus subunit vaccines.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to a preparation method of a Salmonella typhimurium flagellin and Senecavirus A antigen fusion protein. BACKGROUND

[0002] Senecavirus A (SVA) causes vesicular lesions in the oral and nasal mucosa, anorexia, lethargy and lameness in adult pigs, and acute death in newborn piglets. Currently, the disease has been prevalent in many countries around the world, and pig populations of all ages are susceptible to SVA. So far, no commercial vaccine has been put into production and use, so developing a safe and effective vaccine is a top priority.

[0003] The genome of SVA encodes four structural proteins VP1, VP2, VP3 and VP4 and seven non-structural proteins, among which the viral structural protein 2 (VP2) plays an important role in inducing immune responses in the body. [1-2] Studies have shown that VP2 has multiple antigenic determinants and can induce humoral and cellular immune responses in the body [3-5] VP2 is one of the main dominant antigens of SVA that induces the body to produce neutralizing antibodies, and therefore has become a hot candidate antigen for SVA subunit vaccine research.

[0004] However, the challenge is that the immunogenicity of subunit vaccines is relatively low, so adjuvants need to be added to improve the quality of vaccines and enhance the immunogenicity of antigens. Traditional adjuvants, such as aluminum salts and oil emulsion adjuvants, can enhance the immune response of subunit vaccines to a certain extent, but have problems such as insufficient safety and effectiveness, which are difficult to meet the needs of modern vaccine development. With the continuous progress of molecular biology technology, new adjuvants based on pathogen-associated molecular patterns (PAMPs) have gradually become a research hotspot and shown great application prospects. Among them, flagellin as a new type of immune adjuvant can induce innate and acquired immune responses in the body through TLR5 and NLRC4 signaling pathways. Flagellin can be combined with foreign antigens in various forms to enhance the immune effect of antigens. Compared with mixing flagellin and target antigens and inoculating them into the body, fusion expression of target antigens and flagellin is more conducive to the simultaneous delivery of antigens and adjuvants to TLR5-expressing host antigen-presenting cells (DCs), thereby improving the presentation efficiency of antigens [6] Therefore, this fusion form may become a more optimal strategy for constructing vaccines. In addition, fusion expression of flagellin and foreign antigens can greatly reduce the production time of vaccines, the amount of immune adjuvants used, and thus save production costs.

[0005] Previous studies have reported that the exogenous expression of SVA-VP2 protein is mainly in the form of inclusion bodies, which leads to incorrect folding of the recombinant protein and affects its normal biological function [7] However, the present application fuses the Salmonella typhimurium flagellin protein with the Senecavirus antigen, which not only maintains the adjuvant activity of the flagellin protein, but also improves the solubility and expression level of VP2, greatly reducing the cost of expression and purification process. The fusion protein of Salmonella typhimurium flagellin protein and Senecavirus antigen provided by the present application can activate the TLR5 pathway and activate mouse macrophages, and the production process is simple and low in cost, which has important significance for the research and production of Senecavirus subunit vaccine.

[0006] REFERENCES

[0007] [1]Dvorak C M,Akkutay-Yoldar Z,Stone S R,et al.2017.An in directenzymelinked immunosorbent assay for the identification of antibodies to Senecavirus A in swine[J].BMC Veterinary Research,13(1):50.

[0008] [2]Gimenez-Lirola L G,Rademacher C,Linhares D,et al.2016.Serologicaland molecular detection of Senecavirus A associated with an outbreak of swineidiopathic vesicular disease and neonatal mortality[J].Journal of ClinicalMicrobiology,54(8):2082-2089.

[0009] [3]Guo L Y,Feng X,Pan S J,et al.2022.Senecavirus A structural protein T-cellantigen epitope screening and identification[J].China Animal Health Science,52(01):77-84.

[0010] [4]Yao F.2020.Senecavirus A VP1 and VP2 protein B cell epitope screening andidentification[D].Master's thesis,Chinese Academy of Agricultural Sciences,Supervisor:Pan Li.pp.27-37.

[0011] [5] Wu C P. 2021. Identification of B cell epitopes of structural proteins of Senecavirus A [D]. Master's thesis, Chinese Academy of Agricultural Sciences, Advisor: Zheng H X, pp. 25-41.

[0012] [6] IZEL S B, BATES J T. Flagellin as an adjuvant: cellular mechanisms and potential [J]. J Immunol, 2010, 185(10): 5677 5682.

[0013] [7] Maggioli M F, Lawson S, Lima M D, et al. 2018. Adaptive immune responses following Senecavirus A infection in pigs [J]. Journal of Virology, 92(3): e01717-17. SUMMARY

[0014] The first object of the present application is to provide a Salmonella typhimurium flagellin and Senecavirus A antigen fusion protein.

[0015] The second object of the present application is to provide a method for preparing a Salmonella typhimurium flagellin and Senecavirus A antigen fusion protein.

[0016] The third object of the present application is to provide a fusion protein for use in the preparation of a Senecavirus A vaccine.

[0017] Specifically comprising the following contents:

[0018] In a first aspect, the present application provides a Salmonella typhimurium flagellin and Senecavirus A antigen fusion protein, wherein the fusion protein is formed by fusing Salmonella typhimurium flagellin dublin and Senecavirus A VP2 antigen. Further, the fusion protein is connected to the N-terminus of Salmonella typhimurium flagellin dublin through a Linker.

[0019] Preferably, the Linker sequence is GGGGSGGGGSGGGGS.

[0020] 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.

[0021] Secondly, the present invention provides a method for preparing a fusion protein of Salmonella typhimurium flagellin and Seneca virus antigen, the method comprising the following steps:

[0022] S1: Synthesize a nucleotide sequence encoding the fusion protein as described in claim 4, wherein the nucleotide sequence is as shown in SEQ ID No. 2;

[0023] S2: The VP2-dublin fusion protein gene sequence described in S1 is cloned into the prokaryotic expression vector pET-28a(+) using Infusion recombination technology to obtain a recombinant expression vector containing a nucleotide sequence encoding the VP2-dublin fusion protein;

[0024] S3: Transform the recombinant expression vector described in S2 into competent Escherichia coli Rosetta (DE3) strain to obtain a recombinant Escherichia coli Rosetta (DE3) strain for producing the fusion protein VP2-dublin of Salmonella Typhimurium flagella protein and Seneca virus antigen.

[0025] S4: Using the recombinant strain described in S3 as the seed strain, induce expression of VP2-dublin fusion protein;

[0026] S5: Centrifuge the bacterial solution obtained in S4 to obtain recombinant bacterial cells, break the bacterial cells, and collect the supernatant for later use;

[0027] S6: Take the supernatant of S5 and perform affinity chromatography purification to elute and obtain the fraction containing VP2-dublin protein.

[0028] Further, step S4 is as follows:

[0029] (1) Seed culture: The bacterial strain was streaked onto LB plates and cultured at 37°C for 12-15 hours. A single colony was picked and inoculated into 20 mL of LB medium and cultured overnight at 37°C and 220 rpm to obtain activated seed culture.

[0030] (2) Inoculate the activated seed solution obtained in step (1) into the culture medium at an inoculation rate of 3%, and incubate at 37°C and 220 rpm until OD. 600 Add IPTG to a concentration of 0.1 mM, bringing the total concentration to 0.4–0.6.

[0031] (3) Continue to induce expression at 16℃ and 160rpm for 20h.

[0032] Thirdly, the present invention provides an application of a fusion protein in the preparation of a Seneca virus vaccine.

[0033] The beneficial effects of this invention are:

[0034] 1. This invention utilizes a prokaryotic expression system to express the fusion protein of Salmonella typhimurium flagella protein and Seneca virus antigen in a fusion expression form, which is a first invention.

[0035] 2. The fusion protein of Salmonella typhimurium flagellin and Seneca virus antigen of the present invention improves the solubility and expression level of VP2 while maintaining the adjuvant activity of flagellin, with a yield of up to 40 mg / L, greatly reducing the cost of expression and purification processes, and is easy to scale up and produce on a large scale.

[0036] 3. The fusion protein provided by this invention exhibits good biological activity, and its activation of the TLR5 pathway and the level of activation of mouse macrophages are significantly higher than those of the mixed group (VP2+dublin), which is of great significance for the research and production of Seneca virus subunit vaccines. Attached Figure Description

[0037] Figure 1 This is a schematic diagram of the fusion protein particle pET-28a(+)-VP2-dublin, which is a combination protein of Salmonella typhimurium flagella protein and Seneca virus antigen.

[0038] Figure 2 This is an SDS-PAGE image of the expression and purification of the fusion protein of Salmonella Typhimurium flagellar protein and Seneca virus antigen. Lane M is the protein marker; lane 1 is the bacterial culture before VP2-dublin induction; lane 2 is the bacterial culture after VP2-dublin induction; lane 3 is the total protein from VP2-dublin cell lysis; lane 4 is the VP2-dublin cell lysis supernatant; lane 5 is the VP2-dublin cell lysis precipitate; lane 6 is the NTA nickel column flow-through buffer; lane 7 is the washing buffer; lane 8 is the VP2-dublin sample washed with 50 mM imidazole; lane 9 is the VP2-dublin sample washed with 100 mM imidazole; lane 10 is the VP2-dublin sample washed with 250 mM imidazole; and lane 11 is the VP2-dublin sample washed with 500 mM imidazole.

[0039] Figure 3 This study aimed to detect the in vitro TLR5 receptor activity of a fusion protein of Salmonella typhimurium flagellin and Seneca virus antigen. Human colon cancer cells (Caco2 cells) were stimulated for 6 hours with endotoxin-removed VP2-dublin and VP2+dublin, respectively, with unstimulated Caco2 cells serving as a negative control. RNA was extracted from cells, reverse transcribed into cDNA, and cytokine mRNA expression was detected by RT-PCR.

[0040] Figure 4This study aimed to activate mouse macrophages in vitro using a fusion protein of Salmonella typhimurium flagellar protein and Seneca virus antigen. RAW264.7 mouse macrophage cells were stimulated for 6 hours with VP2-dublin and VP2+dublin (both endotoxin-free), with unstimulated RAW264.7 cells serving as a negative control. RNA was extracted from the cells and reverse transcribed into cDNA. Cytokine mRNA expression was detected by RT-PCR. Detailed Implementation

[0041] 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.

[0042] Example 1: Construction of pET-28a(+)-VP2-dublin recombinant plasmid

[0043] The VP2-dublin gene sequence was sent to Suzhou Genewise Biotechnology Co., Ltd. for codon optimization and full-gene synthesis, and then ligated into the pET-28a(+) vector. The ligation product was transformed into DH 5α competent cells, added to LB liquid medium, and cultured with shaking for 1 hour. An appropriate amount of culture product was spread onto LB solid medium containing kanamycin (Kan+) and cultured overnight at 37°C. The next day, several single colonies were picked, identified by PCR, and sent to the company for sequencing. Finally, DNA sequencing confirmed the correct cloning construction, and the recombinant plasmid was named pET-28a(+)-VP2-dublin. A schematic diagram of plasmid construction is shown below. Figure 1 As shown.

[0044] Example 2: Prokaryotic expression and purification of VP2-dublin

[0045] 1. The pET-28a(+)-VP2-dublin recombinant expression plasmid, accurately identified by DNA sequencing, was transformed into Rosetta(DE3) competent cells. The cells were then plated on LB agar plates containing kanamycin and chloramphenicol and incubated overnight at 37°C. Single positive clones were picked from the overnight LB plates and placed in 20 mL of LB liquid medium containing kanamycin and chloramphenicol, and incubated overnight on a shaker at 37°C. Inoculation was then performed at 3.0% of the target cell count into 500 mL of LB liquid medium containing kanamycin and chloramphenicol, and the cells were incubated at 37°C and 200 rpm until OD (outcome limit) was reached. 600nmWhen the concentration reached 0.4–0.6, IPTG (final concentration 0.1 mmol / L) was added, and expression was induced at 16℃ and 160 rpm for 20 h. After expression, the bacterial cells were collected by centrifugation at 10,000 rpm for 20 min. The bacterial cell pellet was then resuspended, mixed, and sonicated on ice (40% power, 3 s working, 7 s rest) until the bacterial culture was basically clear. Protein purification was performed using Ni NTA: An appropriate amount of deionized water was added to the packed Ni NTA column to rinse it with ethanol. Eight column volumes of binding buffer were added for equilibration. After equilibration, the sample was loaded. The column was washed with 15 column volumes of wash buffer. Elution was performed with 5 column volumes of Elution Buffer, and the eluent was collected. The column was washed sequentially with 3 column volumes of binding buffer and 5 column volumes of deionized water, and then equilibrated with 3 column volumes of 20% ethanol.

[0046] 2. The purified sample was concentrated and desalted by ultrafiltration. A 10 kDa ultrafiltration tube was used. Distilled water was added to the inner tube, and the sample was centrifuged at 4000 rpm for 3 min, repeated three times. The collected elution solution was added to the inner tube of the ultrafiltration tube, and the sample was centrifuged at 4000 rpm. This process was repeated until all purified samples were concentrated to approximately 1 mL. Ten volumes of PBS buffer were added to the concentrated product, and the mixture was centrifuged at 4000 rpm until the target protein storage system was replaced with phosphate buffer. Centrifugation was then completed, and the sample from the inner tube of the ultrafiltration tube was collected. Lipopolysaccharide (LPS) was removed from the protein using an endotoxin removal kit, ensuring the LPS content was less than 0.1 EU / mL. The concentration of VP2-dublin protein was then determined to be 1 mg / mL using the BCA method.

[0047] like Figure 2 As shown, the electrophoresis results indicate that VP2-dublin (90.7 kDa) with high purity was obtained, consistent with the expected size.

[0048] Example 3: Detection of in vitro TLR5 receptor activity of the fusion protein of Salmonella typhimurium flagellin and Seneca virus antigen.

[0049] Human colorectal adenocarcinoma cells Caco2 were cultured overnight in complete DMEM medium at 37°C and 5% CO2. The next day, the cells were stimulated with VP2-dublin and VP2+dublin, respectively. After 6 hours, the cells were collected for RNA extraction. The mRNA level of cytokine IL-8 was detected by qRT-PCR.

[0050] Total RNA was extracted following the instructions for RNAiso Plus (Takara, catalog number: 9109). When collecting cells, 1000 μL of RNAiso Plus was added to each well. The cell lysis buffer was transferred to a centrifuge tube, and the cells were repeatedly pipetted until no obvious precipitate was observed. The mixture was incubated at room temperature (15-30℃) for 5 min, and then RNA was separated from the nucleoproteins. 200 μL of chloroform was added to the homogenate lysis buffer, the centrifuge tube was tightly capped, and the mixture was stirred until the solution emulsified and turned milky white. The mixture was incubated at room temperature for 5 min. Centrifuged at 12000g at 4℃ for 15 min. The centrifuge tube was carefully removed from the centrifuge; the homogenate had separated into three layers. The supernatant was transferred to a new centrifuge tube. 1000 μL of isopropanol was added to the supernatant, and the tube was inverted to mix thoroughly. The mixture was incubated at room temperature for 10 min. Centrifuged at 12000g at 4℃ for 10 min. After centrifugation, RNA precipitate appeared at the bottom of the tube. Discard the supernatant, add 1000 μL of 75% ethanol, gently invert the centrifuge tube to wash the walls, centrifuge at 7500g at 4℃ 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. After the precipitate is dry, add an appropriate amount of RNase-free water to dissolve it.

[0051] Using PrimeScript TM The FAST RT reagent kit with gDNA Eraser (Takara, catalog number: RR092A) is used to reverse transcribe extracted RNA into cDNA. The reaction system (20 μL) is 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 to a final volume of 20 μL. The reaction conditions are: 37℃ for 10 min; 85℃ for 5 s.

[0052] Using the obtained cDNA as a template, qRT-PCR was performed to detect cytokines. The specific primers for IL-8 were IL-8-F (SEQ ID No. 3) and IL-8-R (SEQ ID No. 4). The qRT-PCR reaction system (25 μL) consisted of: 1 μL cDNA, 12.5 μL TB GreenPremix ExTaqII Fast qPCR (Takara, catalog number: CN830A), 1 μL upstream primer (10 μM), 1 μL downstream primer (10 μM), and 9.5 μL RNase-free H2O. The reaction mixture was placed in a real-time PCR instrument for gene amplification. The qRT-PCR reaction program was: 95℃ pre-denaturation for 30 s; 95℃ for 5 s, 60℃ for 10 s, for 40 cycles. -ΔΔCTThe fluorescence value was calculated using the method, and the relative expression level was calculated using the housekeeping gene as an internal reference.

[0053] The results are as follows Figure 3 As shown, the fusion protein VP2-dublin, composed of Salmonella typhimurium flagellin and Seneca virus antigen, significantly stimulated Caco2 secretion of IL-8, indicating good TLR5 receptor activity. Furthermore, the fusion protein VP2-dublin showed a significant advantage over the mixed group (VP2+dublin) in activating TLR5-mediated immune responses.

[0054] Example 4: Activation of the fusion protein of Salmonella Typhimurium flagellar protein and Seneca virus antigen in mouse macrophages in vitro.

[0055] Mouse macrophage line RAW264.7 was cultured overnight in complete DMEM medium at 37°C and 5% CO2. The next day, cells were stimulated with VP2-dublin and VP2+dublin, respectively. Cells were collected after 6 hours for RNA extraction. The ability of recombinant proteins to activate macrophages in vitro was evaluated by detecting the mRNA levels of cytokines IL-6 and IL-1β using qRT-PCR. Specific primers for IL-6 were IL-6-F (SEQ ID No. 5) and IL-6-R (SEQ ID No. 6). Specific primers for IL-1β were IL-1β-F (SEQ ID No. 7) and IL-1β-R (SEQ ID No. 8).

[0056] The results are as follows Figure 4 As shown, the activation level of mouse macrophages stimulated by VP2-dublin was significantly higher than that of the mixed group (VP2+dublin). Combined in vivo experimental results indicate that the fusion protein VP2-dublin, a fusion protein of Salmonella typhimurium flagellin and Seneca virus antigen, has a significant advantage over the mixed group (VP2+dublin) in activating the TLR5 signaling pathway and stimulating macrophage activation, potentially providing important guidance for vaccine design.

Claims

1. A fusion protein of Salmonella typhimurium flagella protein and Seneca virus antigen, 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.

2. A method for preparing a fusion protein of Salmonella typhimurium flagellin and Seneca virus antigen, comprising the following steps: S1: Synthesize a nucleotide sequence encoding the fusion protein as described in claim 1, wherein the nucleotide sequence is as shown in SEQ ID No. 2; S2: The fusion protein gene sequence described in S1 is cloned into the prokaryotic expression vector pET-28a(+) using Infusion recombination technology to obtain a recombinant expression vector containing the nucleotide sequence encoding the fusion protein; S3: Transform the recombinant expression vector described in S2 into competent Escherichia coli Rosetta (DE3) strain to obtain a recombinant Escherichia coli Rosetta (DE3) strain for producing a fusion protein of Salmonella typhimurium flagella protein and Seneca virus antigen. S4: Using the recombinant strain described in S3 as the seed strain, induce expression of the fusion protein to obtain the induced expression bacterial solution; S5: Centrifuge the bacterial solution obtained in S4 to obtain recombinant bacterial cells, break the bacterial cells, and collect the supernatant for later use; S6: Take the supernatant of S5 and perform affinity chromatography purification to elute and obtain the component containing the fusion protein.

3. The method for preparing the fusion protein of Salmonella typhimurium flagellin and Seneca virus antigen as described in claim 2, wherein step S4 is as follows: (1) Seed culture: The bacterial strain was streaked onto an LB plate and cultured at 37°C for 12-15 hours. A single colony was picked and inoculated into 20 mL of LB medium and cultured overnight at 37°C and 220 rpm to obtain activated seed liquid. (2) Inoculate the activated seed solution obtained in step (1) into the culture medium at an inoculation rate of 3%, and culture at 37℃ and 220 rpm until the OD600 reaches 0.4 to 0.

6. Add IPTG to a concentration of 0.1 mM. (3) Continue to induce expression at 16℃ and 160rpm for 20h.

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