A salmonella typhimurium flagellin derivative and a senecavirus antigen fusion protein and application thereof

Abstract

The application belongs to the technical field of biology, and discloses a Salmonella typhimurium flagellin derivative and Senecavirus antigen fusion protein and application thereof. The Salmonella typhimurium flagellin derivative gene and the Senecavirus antigen gene are fused, and after codon optimization, the fusion protein is cloned into a pET-28a(+) vector, and the fusion protein is expressed by using an Escherichia coli system. In-vivo experiment verification shows that the fusion protein has good biological activity, can activate TLR5 ligand activity, and promote macrophage activation, and lays a foundation for development of a Senecavirus subunit vaccine.

Description

TECHNICAL FIELD

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

[0002] Senecavirus A (SVA) is a new type of small RNA virus, which has caused vesicular disease in many countries and regions in recent years, and has seriously harmed the development of the pig industry. SVA virus particles are icosahedral structures without envelope, with a diameter of about 25-30 nm, and four viral structural proteins (VP) VP1-VP4 encoded by SVA are formed into the capsid of the virus. The VP2 protein is an important structural protein exposed on the surface of the SVA virus particle and has strong antigenicity, and can induce a strong immune response in the body [1] . At present, there is no commercial SVA vaccine available, so it is urgent to develop an effective vaccine for prevention and control [2] .

[0003] In recent years, SVA subunit vaccines have become a research hotspot, but the problem is that they have poor immunogenicity, so adjuvants are needed to enhance their ability to stimulate the body's immune response. Traditional adjuvants (aluminum salt adjuvants, oil emulsion adjuvants) can improve the immune response of subunit vaccines to a certain extent, but they have high requirements for processing, storage and transportation, and have a small range of use, which cannot 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 is a PAMP that can activate natural immune responses, which can induce the production of pro-inflammatory factors in the body by stimulating the TLR5 pathway on the cell surface and / or the NLRC4 pathway in the cytoplasm, and stimulate the innate immune response of the body, showing strong adjuvant activity. However, after flagellin acts on the TLR5 receptor, the strong natural immune response produced will cause systemic adverse reactions such as sepsis, which restricts its application prospects as an adjuvant. The Salmonella typhimurium flagellin derivative of the application can not only retain the characteristics of wild-type flagellin immunoadjuvants, but also reduce the natural immune response it stimulates and reduce its side effects on the body, which has practical significance for the development of new vaccines. Immunization with mixed or fused flagellin and target proteins can improve the immune response of the body to antigens, and the immune effect of flagellin fusion or coupling antigens is more significant. However, multiple factors need to be considered in the design of fusion proteins, and improper design may lead to unstable or incorrect folding of the protein structure, thereby affecting its biological function.

[0005] In the E. coli prokaryotic expression system, the existing research reports show that the SVAVP2 protein is mainly expressed in the form of inclusion bodies [4-5] , which leads to the incorrect folding of the recombinant protein and further affects its normal biological function. However, the Salmonella typhimurium flagellin derivative of the present application can be efficiently expressed in the supernatant, and after being fused with the Seneca virus antigen, it helps to express VP2 in the supernatant. The fusion protein of the Salmonella typhimurium flagellin derivative and the Seneca virus antigen provided by the present application shows good biological activity, and the production process is simple and the cost is low, which lays a foundation for the development of Seneca virus subunit vaccine.

[0006] REFERENCES

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

[0008] [2]Zhang T,Wang H B,Sun Y Y,et al.2022.Preparation of monoclonal antibody based on VP1 protein 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 S B,BATES J T.Flagellin as an adjuvant:cellular mechanisms and potential[J].J Immunol,2010,185(10):5677-5682.

[0010] [4] Dvorak C M, Akkutay-Yoldar Z, Stone S R, et al. 2017. An indirect enzyme-linked immunosorbent assay for the identification of antibodies to Senecavirus A in swine [J]. BMC Veterinary Research, 13(1): 50.

[0011] [5] Maggioli M F, Lawson S, de Lima M, et al. 2018. Adaptive immune responses following Senecavirus A infection in pigs [J]. Journal Virology, 92(3): e01717-17. SUMMARY

[0012] A first object of the present application is to provide a fusion protein of Salmonella typhimurium flagellin derivative and Senecavirus antigen.

[0013] A second object of the present application is to provide a method for preparing a fusion protein of Salmonella typhimurium flagellin derivative and Senecavirus antigen.

[0014] A third object of the present application is to provide an application of the fusion protein in preparing Senecavirus vaccine.

[0015] Specifically comprising the following contents:

[0016] In a first aspect, the present application provides a fusion protein of Salmonella typhimurium flagellin derivative and Senecavirus antigen, wherein the fusion protein is formed by fusing Salmonella typhimurium flagellin derivative Δdublin and Senecavirus type A VP2 antigen. Further, the fusion protein is connected to the N-terminus of Salmonella typhimurium flagellin derivative Δdublin through Linker by Senecavirus VP2 antigen.

[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 application provides a method for preparing a fusion protein of Salmonella typhimurium flagellin derivative and Senecavirus antigen, the method comprising the following steps:

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

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

[0022] S3: separating and purifying the bacterial liquid obtained in step S2 to obtain the fusion protein.

[0023] Further, the step S2 is:

[0024] (1) seed culture, inoculating the bacterial strain on an LB plate and culturing at 37℃ for 12-15h, picking a single colony and inoculating in 20mL LB medium, culturing at 37℃, 220rpm overnight to obtain an activated seed liquid;

[0025] (2) inoculating the activated seed liquid obtained in step (1) into the medium at an inoculation amount of 3%, culturing at 37℃, 220rpm until OD 600 0.4-0.6, and adding IPTG to a concentration of 0.1mM.

[0026] (3) continuing to induce expression at 16℃, 160rpm for 20h.

[0027] Further, the step S3 is:

[0028] (1) centrifuging the bacterial liquid obtained in step S3 to obtain bacterial bodies, ultrasonically crushing the bacterial bodies, centrifuging to obtain the supernatant after crushing the bacterial bodies, and reserving;

[0029] (2) subjecting the supernatant after crushing the bacterial bodies to nickel column affinity chromatography to elute a component containing the fusion protein.

[0030] In a third aspect, the present application provides a use of the fusion protein in preparing a Senecavirus vaccine.

[0031] The present application has the following beneficial effects:

[0032] 1. The present application expresses the fusion protein of Salmonella typhimurium flagellin derivative and Senecavirus antigen in the form of fusion expression by means of a prokaryotic expression system, which is the first invention.

[0033] 2. The fusion protein of Salmonella typhimurium flagellar protein derivative and Seneca virus antigen of the present invention is expressed in a soluble form, which improves the solubility and expression level of the antigen. The production process is simple, the production conditions are not required and the production cost is low, and it is easy to scale up and produce on a large scale.

[0034] 3. The fusion protein of the Salmonella typhimurium flagellin derivative and Seneca virus antigen of the present invention exhibits good biological activity, can activate TLR5 ligand activity, and promote macrophage activation, laying the foundation for the development of Seneca virus subunit vaccines. Attached Figure Description

[0035] Figure 1 This diagram illustrates the fusion protein particle of Salmonella typhimurium flagellar protein derivative and Seneca virus antigen. Figure A shows pET-28a(+)-VP2-Δdublin, Figure B shows pET-28a(+)-Δdublin-VP2, and Figure C shows pET-28a(+)-FliC. Δ29-164 -VP2-FliC Δ412-483 .

[0036] Figure 2 This is an SDS-PAGE image showing the expression and purification of the fusion protein of Salmonella Typhimurium flagellar protein derivative and Seneca virus antigen. Lane M is the Protein marker; lane 1 is the bacterial culture after VP2-Δdublin induction; lane 2 is the bacterial culture after Δdublin-VP2 induction; lane 3 is the FliC... Δ29-164 -VP2-FliC Δ412-483 Post-induction bacterial culture; Lane 4: purified VP2-Δdublin; Lane 5: purified Δdublin-VP2; Lane 6: purified FliC Δ29-164 -VP2-FliC Δ412-483 .

[0037] Figure 3 The in vitro TLR5 receptor activity of a fusion protein of Salmonella typhimurium flagellin derivative and Seneca virus antigen was detected. The activity was assessed using 5 μg / mL of endotoxin-removed VP2-Δdublin, Δdublin-VP2, and FliCΔ... 29-164 -VP2-FliCΔ 412-483 Human colon cancer cells (Caco2 cells) were stimulated for 6 hours, with unstimulated Caco2 cells serving as a negative control. RNA was extracted from the cells and reverse transcribed into cDNA, and cytokine mRNA expression was detected by RT-PCR.

[0038] Figure 4Activation of mouse macrophage cells by Salmonella typhimurium flagellin derivative and Senecavirus antigen fusion protein in vitro. Mouse macrophage RAW264.7 cells were stimulated for 6h with 5μg / mL endotoxin-removed VP2-Δdublin, Δdublin-VP2, FliCΔ 29-164 -VP2-FliCΔ 412-483 respectively, with unstimulated RAW264.7 cells as negative control group. Cells were collected to extract RNA and reverse transcribed into cDNA, and the expression of cytokine mRNA was detected by RT-PCR. DETAILED DESCRIPTION

[0039] In order to make the present application easier to understand, the embodiments of the present application will be further described below. The present application will be further described and demonstrated in combination with the embodiments. However, the present embodiments are not a limitation on the present application. The technical solutions described in the present application are conventional solutions in the art, unless specifically stated; and the reagents or materials shown are from commercial channels, unless specifically stated.

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

[0041] The coding sequences of VP2-Δdublin, Δdublin-VP2, FliCΔ 29-164 -VP2-FliCΔ 412-483 were sent to Suzhou Jinyuizhi Biotechnology Co., Ltd. for codon optimization and whole gene synthesis, and then ligated into pET-28a(+) vector. The ligation product was transformed into DH 5α competent cells, added to LB liquid medium and shaken for 1h, and then an appropriate amount of culture product was spread on kanamycin (Kan+) LB solid medium and cultured overnight in a 37℃ incubator. The next day, several single colonies were picked and identified by PCR, and then sent for sequencing. Finally, DNA sequencing confirmed that the cloning was correctly constructed, and the recombinant plasmids were named pET-28a(+)-VP2-Δdublin, pET-28a(+)-Δdublin-VP2, pET-28a(+)-FliCΔ 29-164 -VP2-FliC Δ412-483 . The schematic diagram of plasmid construction is shown in Figure 1 .

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

[0043] 1. The pET-28a(+)-VP2-Adublin, pET-28a(+)-Adublin-VP2, pET-28a(+)-FliCAdublin-VP2, pET-28a(+)-VP2-FliCAdublin recombinant expression plasmids identified accurately by DNA sequencing were transformed into Rosetta (DE3) competent cells, then coated on LB solid medium containing kanamycin and chloramphenicol, and incubated in a 37°C constant temperature incubator overnight. Single positive clones were picked from the LB plate of the overnight culture, and placed in 20 mL liquid LB medium containing kanamycin and chloramphenicol, and incubated in a 37°C shaker overnight. Inoculated into 500 mL LB liquid medium containing kanamycin and chloramphenicol at a 3.0% inoculation amount, and incubated at 37°C and 200 rpm until the OD600 reached 0.4-0.6. 29-164 -VP2-FliCΔ 412-483 The recombinant expression plasmids were transformed into Rosetta (DE3) competent cells, then coated on LB solid medium containing kanamycin and chloramphenicol, and incubated in a 37°C constant temperature incubator overnight. Single positive clones were picked from the LB plate of the overnight culture, and placed in 20 mL liquid LB medium containing kanamycin and chloramphenicol, and incubated in a 37°C shaker overnight. Inoculated into 500 mL LB liquid medium containing kanamycin and chloramphenicol at a 3.0% inoculation amount, and incubated at 37°C and 200 rpm until the OD600 reached 0.4-0.6. 600nm When the OD600 reached 0.4-0.6, IPTG was added (final concentration 0.1 mmol / L), and the expression was induced at 16°C and 160 rpm for 20 h. After the expression was completed, the bacterial cells were collected by centrifugation at 10,000 rpm for 20 min, then the bacterial cell pellet was resuspended, mixed well, and the cells were broken by ultrasonic on ice (power 40%, 3 s working and 7 s intermittent). The protein was purified by Ni NTA: After the loaded Ni NTA column was washed with deionized water to remove ethanol, 8 times the column volume of Binding buffer was added for equilibration. After equilibration, the sample was loaded. 15 times the column volume of Wash buffer was used to wash the column. 5 times the column volume of Elution Buffer was used for elution, and the eluate was collected. After washing with 3 times the column volume of Binding Buffer and 5 times the column volume of deionized water, the column was equilibrated with 3 times the column volume of 20% ethanol.

[0044] 2. The purified sample was concentrated and desalted by ultrafiltration. A 10 kDa molecular weight cut-off ultrafiltration tube was used, and distilled water was added to the inner tube. Centrifugation was performed at 4000 rpm / min for 3 min, and the operation was repeated three times. The collected solution was added to the inner tube of the ultrafiltration tube, and centrifugation was performed at 4000 rpm / min. The operation was repeated until all the purified sample was concentrated to about 1 mL. 10 times the volume of PBS buffer was added to the concentrated product, mixed well, and centrifuged at 4000 rpm / min until the desired protein storage system was replaced with phosphate buffer. After centrifugation, the sample in the inner tube of the ultrafiltration tube was collected. Endotoxin removal kit was used to remove lipopolysaccharide (LPS) in the protein, and the LPS content was less than 0.1 EU / mL. The VP2-Adublin, Adublin-VP2, FliCAdublin-VP2, VP2-FliCAdublin proteins were determined by BCA method. 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] As shown in Figure 2 Figure 2, the electrophoresis results showed that VP2-Adublin (61.5 kDa), Adublin-VP2 (61.5 kDa), FliCΔ 29-164 -VP2-FliCΔ 412-483 (61.9 kDa) with high purity could be obtained, which were consistent with the expected sizes.

[0046] Example 3 In vitro TLR5 receptor activity detection of a Salmonella typhimurium flagellin derivative fused with Senecavirus antigen

[0047] Human colorectal adenocarcinoma cells Caco2 were cultured overnight (6-well plate, 2 x 10 5 cells / well) in complete DMEM medium at 37°C, 5% CO2; the next day, the cells were stimulated with VP2-Adublin, Adublin-VP2, FliCΔ 29-164 -VP2-FliCΔ 412-483 -VP2-FliCΔ, respectively; 6 h later, the cells were collected for RNA extraction; the mRNA level of cytokine IL-8 was detected by qRT-PCR.

[0048] Total RNA was extracted according to the steps in the instructions of RNAiso Plus (takara, item number: 9109). When the cells were collected, 1000 μL of RNAiso Plus was added to each well, and the cell-containing lysate was transferred to a centrifuge tube and repeatedly blown and sucked with a pipette gun until there was no obvious precipitate in the lysate. After standing at room temperature (15-30°C) for 5 min, the RNA was separated from the nuclear protein. 200 μL of chloroform was added to the above homogenate lysate, the centrifuge tube cap was tightly covered, and the solution was mixed until it was emulsified to a milky white color. It was allowed to stand at room temperature for 5 min. 12000 g centrifugation at 4°C for 15 min. The centrifuge tube was carefully removed from the centrifuge, and at this time the homogenate was divided into three layers. The supernatant was transferred to another new centrifuge tube. 1000 μL of isopropanol was added to the supernatant, and after mixing well, it was allowed to stand at room temperature for 10 min. 12000 g centrifugation at 4°C for 10 min. After centrifugation, RNA precipitate appeared at the bottom of the test tube. The supernatant was discarded, 1000 μL of 75% ethanol was added, the centrifuge tube wall was gently inverted up and down, and after 7500 g centrifugation at 4°C for 5 min, the supernatant was carefully discarded. The centrifuge tube cap was opened, and the precipitate was dried at room temperature for a few minutes. After the precipitate was dried, an appropriate amount of RNase-free water was added to dissolve the precipitate.

[0049] PrimeScript TMThe extracted RNA was reversely transcribed into cDNA by FAST RT reagent Kit with gDNA Eraser (takara, item number: RR092A), and 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, RNase-free H2O, and the total volume was made up to 20 μL. The reaction condition was as follows: 37 ℃, 10 min; 85 ℃, 5 s.

[0050] qRT-PCR was used to detect the cytokines with the obtained cDNA as the template. The specific primers of 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) was as follows: 1 μL cDNA, 12.5 μL TB Green Premix Ex Taq II Fast qPCR (takara, item number: CN830A), 1 μL upstream primer (10 μM), 1 μL downstream primer (10 μM), 9.5 μL RNase-free H2O, and the reaction mixture was placed in a fluorescence quantitative PCR instrument for gene amplification. The qRT-PCR reaction program was as follows: 95 ℃ pre-denaturation for 30 s; 95 ℃ for 5 s, 60 ℃ for 10 s, 40 cycles. 2 -ΔΔCT The fluorescence value was calculated by the method, and the relative expression amount was calculated by taking the housekeeping gene as the internal reference.

[0051] The results are shown in Table 1. Figure 3 The Salmonella typhimurium flagellin derivative and the Seneca Valley virus antigen fusion protein VP2-Δdublin, Δdublin-VP2, FliCΔ 29-164 -VP2-FliCΔ 412-483 significantly stimulated the secretion of IL-8 of Caco2, indicating that it had good TLR5 receptor activity. The antigen fusion protein VP2-Δdublin at the N-terminal had a significant advantage in activating the TLR5-mediated immune response.

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

[0053] The mouse macrophage cell line RAW264.7 was cultured overnight (6-well plate, 1x10 6 The next day, VP2-Δdublin, Δdublin-VP2, FliCΔ 29-164 -VP2-FliCΔ 412-483Stimulate cells; collect cells after 6h for RNA extraction; evaluate the ability of recombinant proteins to activate macrophages in vitro by detecting 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 in Figure 6, VP2-Adublin and Adublin-VP2 stimulated the activation of mouse macrophages at a similar level and significantly higher than the control group. FliCΔ 29-164 -VP2-FliCΔ 412-483 Stimulate mouse macrophages to secrete IL-1β at a significant level and not significantly secrete IL-6. The results of in vivo experiments show that the protein VP2-Adublin with antigen fused at the N-terminus has a significant advantage in activating the TLR5 signaling pathway and stimulating the activation of macrophages, 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 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 derivative and Seneca virus antigen, characterized in that, Includes the following steps: S1: Construct a pET-28a(+) expression vector encoding the nucleotide sequence as described in claim 1, transform the recombinant expression vector into Rosetta(DE3), and obtain a recombinant strain for producing fusion proteins; S2: Induce expression of the recombinant strain obtained in step S1; S3: The bacterial culture obtained in step S2 is separated and purified to obtain the fusion protein.

3. The method for preparing the fusion protein according to claim 2, wherein step S2 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) The activated seed liquid obtained in step (1) is inoculated into the culture medium at an inoculation amount of 3%, and cultured at 37°C and 220 rpm until OD 600 0.4~0.6, and IPTG is added to a concentration of 0.1 mM; (3) Continue to induce expression at 16℃ and 160rpm for 20h.

4. The method for preparing the fusion protein according to claim 2, wherein step S3 is as follows: (1) Centrifuge the bacterial solution obtained in step S3 to obtain bacterial cells, sonicate the bacterial cells to break them up, and centrifuge to obtain the supernatant after the bacterial cells are broken up for later use. (2) The supernatant after bacterial cell disruption was subjected to nickel column affinity chromatography to elute the fraction containing the fusion protein.

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