Preparation method of fusion protein of salmonella typhimurium flagellin and Seneca virus antigen

Abstract

The invention belongs to the technical field of biology, and discloses a preparation method of fusion protein of salmonella typhimurium flagellin and Seneca virus antigen. Escherichia coli Rosetta (DE3) is selected as a host, a salmonella typhimurium flagellin gene and a Seneca virus antigen gene are fused and are cloned to a pET-28a (+) vector after codon optimization, the obtained fusion protein keeps the flagellin adjuvant activity, the solubility and the expression level of VP2 are improved, the yield can reach 40 mg / L, and the fusion protein can be used for preparing a recombinant vector. The expression and purification process cost is greatly reduced. The fusion protein provided by the invention can activate a TLR5 pathway and mouse macrophages, is simple in production process and low in cost, and has important significance on research and production of Seneca virus subunit vaccines.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing a fusion protein of Salmonella typhimurium flagellin and Seneca virus antigen. Background Art

[0002] Senecavirus A (SVA) causes vesicular lesions of the oral and nasal mucosa of adult pigs, anorexia, lethargy and lameness, and acute death in newborn piglets. Currently, the disease has become prevalent in many countries around the world, and pigs of all ages are susceptible to SVA, which seriously endangers the development of my country's animal husbandry and causes serious losses to my country's economy. So far, no commercial vaccine has been put into production and use, so the development of a safe and effective vaccine is of utmost importance.

[0003] The genome of SVA encodes four structural proteins, VP1, VP2, VP3 and VP4, and seven non-structural proteins. Among them, viral structural protein 2 (VP2) plays an important role in inducing the body's immune response. VP2 is one of the main antigens of SVA and has better immunogenicity than VP1 and VP3. [1-2] Studies have shown that VP2 has multiple antigenic determinants that can trigger humoral and cellular immune responses. [3-5] VP2 is one of the main antigens that induces the body to produce neutralizing antibodies, and therefore has become a hot candidate antigen in 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 the vaccine and enhance the immunogenicity of the antigen. Although traditional adjuvants, such as aluminum salts and oil-emulsion adjuvants, can enhance the immune response of subunit vaccines to a certain extent, they have problems such as insufficient safety and effectiveness, and it is difficult to meet the needs of modern vaccine development. With the continuous advancement of molecular biology technology, new adjuvants based on pathogen-associated molecular patterns (PAMPs) have gradually become a research hotspot, showing great application prospects. Among them, flagellin, as a new type of immune adjuvant, can induce the body's innate and acquired immune responses through the TLR5 and NLRC4 signaling pathways. Flagellin can be combined with exogenous antigens in various forms to enhance the immune effect of the antigen. Compared with the mixed inoculation of flagellin and the target antigen into the body, the fusion expression of the target antigen with flagellin is more conducive to the simultaneous delivery of the antigen and adjuvant to the host antigen-presenting cells (DCs) expressing TLR5, thereby improving the presentation efficiency of the antigen. [6] Therefore, this fusion form may become a better strategy for constructing vaccines. In addition, the fusion expression of flagellin and exogenous antigens can also greatly reduce vaccine production time and the amount of immune adjuvants used, thereby saving production costs.

[0005] Existing research reports show that the exogenous expression of SVA-VP2 protein mostly exists in the form of inclusion bodies, which results in the inability of the recombinant protein to fold correctly, thus affecting its normal biological function. [7] . However, after the present invention fuses the flagellin of Salmonella typhimurium with the Senecavirus antigen, the solubility and expression level of VP2 are improved while maintaining the adjuvant activity of the flagellin, greatly reducing the cost of the expression and purification process. The fusion protein of the flagellin of Salmonella typhimurium and the Senecavirus antigen provided by the present invention can activate the TLR5 pathway and activate mouse macrophages, and the production process is simple and low-cost, which is of great significance to the research and production of the Senecavirus subunit vaccine.

[0006] References

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

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

[0009] [3] Guo Liying, Feng Xia, Pan Songjia, et al. 2022. Screening and identification of T cell antigen epitopes of Seneca virus A structural protein[J]. Chinese Journal of Veterinary Science, 52(01):77-84.

[0010] [4] Yao Fei. 2020. Screening and identification of B cell epitopes of Seneca virus AVP1 and VP2 proteins[D]. Master's degree thesis, Chinese Academy of Agricultural Sciences, Supervisor: Pan Li. pp. 27-37.

[0011] [5] Wu Chunping. 2021. Identification of B cell antigen epitopes of the structural protein of Seneca virus type A[D]. Master's degree thesis, Chinese Academy of Agricultural Sciences, supervisor: Zheng Haixue, pp. 25-41.

[0012] [6]IZEL SB,BATES J T.Flagellin as an adjuvant: cellular mechanisms and potential[J].J Immunol,2010,185(10):56775682.

[0013] [7]Maggioli MF, Lawson S, Lima MD, et al. 2018. Adaptive immune responses following Senecavirus A infection in pigs[J]. Journal of Virology, 92(3):e01717-17. Summary of the invention

[0014] The first object of the present invention is to provide a fusion protein of Salmonella typhimurium flagellin and Seneca virus antigen.

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

[0016] The third object of the present invention is to provide an application of a fusion protein in the preparation of a Seneca virus vaccine.

[0017] Specifically include the following:

[0018] In a first aspect, the present invention provides a fusion protein of Salmonella typhimurium flagellin and Senecavirus antigen, wherein the fusion protein is formed by fusion of Salmonella typhimurium flagellin dublin and Senecavirus VP2 antigen type A. Furthermore, the fusion protein is formed by Senecavirus VP2 antigen connected to the N-terminus of Salmonella typhimurium flagellin dublin via 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] In a second aspect, 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: synthesizing a nucleotide sequence encoding the fusion protein according to claim 4, wherein the nucleotide sequence is 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(+) by infusion recombination technology to obtain a recombinant expression vector containing a nucleotide sequence encoding the VP2-dublin fusion protein;

[0024] S3: transforming the recombinant expression vector described in S2 into a 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 flagellin and Seneca virus antigen;

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

[0026] S5: centrifuging the bacterial solution described in S4 to obtain recombinant bacterial cells, breaking the cells, and taking the supernatant for later use;

[0027] S6: The supernatant of S5 was purified by affinity chromatography, and the fraction containing VP2-dublin protein was eluted.

[0028] Furthermore, the step S4 is:

[0029] (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.

[0030] (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 0.1 mM;

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

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

[0033] Beneficial effects of the present invention:

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

[0035] 2. The fusion protein of the Salmonella typhimurium flagellin and the Seneca virus antigen of the present invention improves the solubility and expression level of VP2 while maintaining the adjuvant activity of the flagellin protein, and the yield can reach 40 mg / L, which greatly reduces the cost of expression and purification process and is easy to scale up and mass produce.

[0036] 3. The fusion protein provided by the present invention shows good biological activity, and the levels of activation of TLR5 pathway and 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Schematic diagram of the fusion protein plasmid pET-28a(+)-VP2-dublin of Salmonella typhimurium flagellin and Seneca virus antigen.

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

[0039] Figure 3 This is a fusion protein of Salmonella typhimurium flagellin and Seneca virus antigen to detect TLR5 receptor activity in vitro. Human colon cancer cell Caco2 cells were stimulated with endotoxin-free VP2-dublin and VP2+dublin for 6 hours, respectively, 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.

[0040] Figure 4It is a fusion protein of Salmonella typhimurium flagellin and Seneca virus antigen for in vitro activation of mouse macrophages. VP2-dublin and VP2+dublin, which have been detoxified, were used to stimulate mouse macrophage RAW264.7 cells for 6 hours, respectively, with unstimulated RAW264.7 cells as a negative control group. The cells were collected and RNA was extracted and reverse transcribed into cDNA, and RT-PCR was used to detect cytokine mRNA expression. DETAILED DESCRIPTION

[0041] 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.

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

[0043] The gene sequence encoding VP2-dublin 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 culture medium and shaken for 1 hour, and an appropriate amount of culture product was coated on LB solid culture 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 correct PCR identification. Finally, DNA sequencing confirmed that the clone construction was correct, and the recombinant plasmid was named pET-28a(+)-VP2-dublin. The schematic diagram of plasmid construction is shown in the figure. Figure 1 shown.

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

[0045] 1. Transform the pET-28a(+)-VP2-dublin recombinant expression plasmid accurately identified by DNA sequencing into Rosetta (DE3) competent cells, then spread on LB solid culture medium containing kanamycin and chloramphenicol, and culture in a 37°C constant temperature incubator overnight. Pick a single positive clone from the LB plate cultured overnight, place it in 20mL liquid LB culture medium containing kanamycin and chloramphenicol, and culture it in a 37°C shaker overnight. Inoculate it into 500mL LB liquid culture medium containing kanamycin and chloramphenicol at a 3.0% inoculum, and culture at 37°C 200rpm until OD 600nmWhen it reaches 0.4-0.6, add IPTG (final concentration 0.1mmol / L) and continue to induce expression at 16℃160rpm for 20h. After the expression is completed, collect the bacteria by centrifugation at 10000rpm for 20min, then suspend the bacterial sediment, 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; rinse the column with 15 column volumes of Washbuffer; elute with 5 column volumes of Elution Buffer 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.

[0046] 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 solution eluted 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 the concentration of VP2-dublin protein to be 1mg / mL.

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

[0048] Example 3 In vitro TLR5 receptor activity detection of the fusion protein of Salmonella typhimurium flagellin and Seneca virus antigen

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

[0050] 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.

[0051] Using PrimeScript TM FAST 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.

[0052] 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 TBGreen 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. -ΔΔCTMethods The fluorescence value was calculated and the relative expression level was calculated using the housekeeping gene as the internal reference.

[0053] The results are as follows Figure 3 As shown, the fusion protein VP2-dublin of Salmonella typhimurium flagellin and Seneca virus antigen significantly stimulated Caco2 to secrete IL-8, indicating that it has good TLR5 receptor activity. Moreover, the fusion protein VP2-dublin has a significant advantage in activating TLR5-mediated immune response compared with the mixed group (VP2+dublin).

[0054] Example 4 Activation of mouse macrophages in vitro by fusion protein of Salmonella typhimurium flagellin and Seneca virus antigen

[0055] Mouse macrophage cell line RAW264.7 was cultured overnight in complete DMEM medium at 37°C, 5% CO2; the next day, cells were stimulated with VP2-dublin and VP2+dublin, respectively; cells were collected 6 hours later for RNA extraction; the mRNA levels of cytokines IL-6 and IL-1β were detected by qRT-PCR to evaluate the ability of recombinant proteins to activate macrophages in vitro. 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).

[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). The combined in vivo experimental results showed that the fusion protein VP2-dublin of Salmonella typhimurium flagellin and Seneca virus antigen had significant advantages over the mixed group (VP2+dublin) in activating the TLR5 signaling pathway and stimulating macrophage activation, which may provide important guidance for vaccine design.

Claims

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

2. The fusion protein of Salmonella typhimurium flagellin 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 Salmonella typhimurium flagellar protein dublin through a Linker.

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

4. The fusion protein of Salmonella typhimurium flagellin and Seneca virus antigen according to claim 2, 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 Salmonella typhimurium flagellin and Seneca virus antigen, comprising the following steps: S1: synthesizing a nucleotide sequence encoding the fusion protein according to claim 4, wherein the nucleotide sequence is shown as SEQ ID No. 2; S2: The VP2-dublin fusion protein gene sequence described in S1 is cloned into the prokaryotic expression vector pET-28a(+) by infusion recombination technology to obtain a recombinant expression vector containing a nucleotide sequence encoding the VP2-dublin fusion protein; S3: transforming the recombinant expression vector described in S2 into a 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 flagellin and Seneca virus antigen; S4: using the recombinant strain described in S3 as a seed strain to induce expression of the VP2-dublin fusion protein; S5: centrifuging the bacterial solution described in S4 to obtain recombinant bacterial cells, breaking the cells, and taking the supernatant for later use; S6: The supernatant of S5 was purified by affinity chromatography, and the fraction containing VP2-dublin protein was eluted.

6. The method for preparing the fusion protein of Salmonella typhimurium flagellin and Seneca virus antigen according to claim 5, wherein the step S4 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%, cultured at 37°C and 220 rpm until the OD600 reached 0.4-0.6, and IPTG was added to a concentration of 0.1 mM; (3) Continue to induce expression at 16°C and 160 rpm for 20 h.

7. Use of the fusion protein prepared by the preparation method according to claims 5-6 in preparing Seneca virus vaccine.

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