Chimeric protein of escherichia coli Nissle 1917 flagellin capable of effectively activating TLR5 signal channel and application of chimeric protein

Abstract

The invention provides a chimeric protein of escherichia coli Nissle 1917 flagellin capable of effectively activating a TLR5 (toll-like receptor 5) signal channel and application of the chimeric protein. The chimeric protein is formed by chimeric combination of an escherichia coli Nissle 1917 flagellin and a Seneca virus type A VP2 antigen through Linker (GGGGS) 3. Soluble expression of the chimeric protein is achieved through an escherichia coli expression system, and the yield can reach 100 mg / L. Further in-vitro cell model verification shows that the recombinant chimeric protein can effectively activate a TLR5 signal channel, which provides a data basis and a research reference for developing a safe and efficient Senecavirus type A subunit vaccine.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to the design, induction expression, purification method and in vitro biological activity verification of a chimeric protein of Escherichia coli Nissle 1917 flagellin that effectively activates a TLR5 signaling pathway. Background Art

[0002] Senecavirus type A (SenecavirusA, SVA) belongs to the genus Senecavirus of the family Picornaviridae and is contagious and pathogenic to pigs. In the early stages of infection, adult pigs show symptoms such as lethargy, anorexia and fever, followed by blisters on the nasal mirror, oral epithelium, tongue and hoof crown. Severe cases may lead to ulceration and lameness. The mortality rate of newborn piglets is high after the disease occurs. Since the disease was discovered in 2007, related infection cases have been reported in many different countries and regions. In 2015, a small-scale epidemic occurred in my country, bringing new challenges to the efficient, green and healthy development of the pig industry. [1] At present, the main vaccine for SVA prevention and control is inactivated vaccine, but this vaccine has some disadvantages, including the risk of anti-virus, the inability to effectively induce the body to produce cellular immune effects, and the slow activation of antibody levels, which requires regular repeated vaccination. Subunit vaccine has become the focus of vaccine research and development for this disease because of its simple preparation and high safety. [2] VP2 is the main antigenic epitope of SVA, which has strong immunogenicity and can effectively stimulate the body to produce specific antibodies. [1] However, adjuvants are usually required to improve the efficacy of VP2 subunit vaccines during their manufacture, and VP2 has poor solubility when expressed alone.

[0003] As a new type of immune adjuvant, flagellin is gradually replacing or supplementing the use of some traditional adjuvants due to its unique immune activation properties and safety. As a pathogen-associated molecular pattern (PAMP), flagellin can activate innate immunity and regulate adaptive immunity. Its unique structural characteristics give it effective and flexible adjuvant activity, allowing it to be designed as different types of vaccine strategies to prevent various diseases. Studies have found that the chimerism of flagellin and antigen is a preferred strategy for constructing vaccines. The recombinant protein produced by gene fusion tightly binds the antigen to flagellin while maintaining their relatively independent spatial structure and biological activity. The pre-existing anti-flagellin immune response does not inhibit the TLR5 activity of this recombinant chimeric protein, thereby promoting a strong response to weakly immunogenic antigens at extremely low immune doses. Since antigens and flagellin can be consistently delivered to the same antigen presenting cell (APC) and TLR5, the flagellin fusion strategy is superior to the co-mixing of flagellin and antigen. At present, the adjuvant activity of the chimeric antigen of Salmonella typhimurium flagellin has been widely studied, and its immune adjuvant effect has been applied in the research of vaccines for various pathogens. The candidate vaccines entering clinical research include plague vaccine with recombinant flagellin-F1-V protein, recombinant M2e-flagellin influenza vaccine and recombinant HA-flagellin influenza vaccine. [3] Recent studies have also shown that the flagellin of Escherichia coli Nissle 1917 has similar structure and function and may be used as a potential immune adjuvant. However, although some studies have supported the application of Nissle 1917 flagellin [4] However, it is still crucial to determine its structural domain that is suitable for the correct expression of heterologous antigens. Currently, there is limited research on the Nissle1917 flagellin gene, its mechanism of action has not yet been clarified, and there is still a lack of a large amount of research data to evaluate its effectiveness and safety.

[0004] [1]Ru Y,Hao R,Wu C,Li Y,Lu B,Liu H,Tian H,Li D,Shi Z,Luo J,Ma K,ZhangG,Liu X,Zheng H.Identification ofPotential Novel B-Cell Epitopes ofCapsidProtein VP2 in SenecavirusA.Microbiol Spectr.2023Aug 17;11(4):e0447222.doi:10.1128 / spectrum.04472-22.Epub 2023Jul 10.PMID:37428080;PMCID:PMC10433816.

[0005] [2] Mo Yaxia. Prokaryotic expression of Seneca virus type A capsid protein and in vitro assembly and effect evaluation of virus-like particles[D]. Sichuan Agricultural University, 2019. DOI: 10.27345 / d.cnki.gsnyu.2019.000781.

[0006] [3]Cui B, Liu 30.PMID:29580106.

[0007] [4] Yang Ying, Deng Yongjun, Zhang Jin, et al. Structural analysis and epitope prediction of flagellar protein of probiotic Escherichia coli Nissle1917[J]. Chinese Journal of Biologicals, 2022, 35(06): 672-677. DOI: 10.13200 / j.cnki.cjb.003627. Summary of the invention

[0008] The first object of the present invention is to provide a chimeric protein of Escherichia coli Nissle 1917 flagellin that effectively activates the TLR5 signaling pathway.

[0009] The second object of the present invention is to provide a method for preparing a chimeric protein of Escherichia coli Nissle 1917 flagellin that effectively activates the TLR5 signaling pathway.

[0010] The third purpose of the present invention is to disclose the use of a chimeric protein of Escherichia coli Nissle 1917 flagellin that effectively activates the TLR5 signaling pathway in protecting animals from type A Senecavirus infection, providing another alternative for applicable Senecavirus subunit vaccines. Specifically, it includes the following contents:

[0011] In a first aspect, the present invention provides a chimeric protein of Escherichia coli Nissle 1917 flagellin that effectively activates the TLR5 signaling pathway, wherein the chimeric protein is formed by combining Escherichia coli Nissle 1917 flagellin with type A Senecavirus VP2 antigen.

[0012] Furthermore, the type A Senecavirus VP2 antigen is at the C-terminus of the Escherichia coli Nissle 1917 flagellin.

[0013] Furthermore, the chimeric protein of Escherichia coli Nissle 1917 flagellin that effectively activates the TLR5 signaling pathway is formed by chimerizing the Escherichia coli Nissle 1917 flagellin with the type A Seneca virus VP2 antigen via a Linker.

[0014] Preferably, the amino acid sequence of the Linker is GGGGSGGGGSGGGGS.

[0015] Preferably, the amino acid sequence of the chimeric protein of Escherichia coli Nissle 1917 flagellin that effectively activates the TLR5 signaling pathway is shown in SEQ ID NO: 2. The nucleotide sequence of the chimeric protein of Escherichia coli Nissle 1917 flagellin that effectively activates the TLR5 signaling pathway is shown in SEQ ID NO: 1.

[0016] Furthermore, the chimeric protein of Escherichia coli Nissle 1917 flagellin that effectively activates the TLR5 signaling pathway is used in the preparation of a vaccine to prevent Seneca virus infection.

[0017] In a second aspect, the present invention provides a method for preparing a chimeric protein of Escherichia coli Nissle 1917 flagellin that effectively activates the TLR5 signaling pathway, comprising the following steps:

[0018] S1: The nucleotide sequence of the chimeric protein FliC-L-VP2 was cloned into the expression vector pET-28a(+) through the BamHI and SalI restriction sites;

[0019] S2: Transform the recombinant E. coli expression vector into competent BL21 (DE3) to obtain a recombinant strain for producing chimeric proteins;

[0020] S3: The recombinant strain obtained in step S2 was induced to express at 37°C;

[0021] S4: Separating and purifying the bacterial solution obtained in step S3, and obtaining the chimeric protein by nickel column affinity chromatography.

[0022] Furthermore, the chimeric protein of Escherichia coli Nissle1917 flagellin that effectively activates the TLR5 signaling pathway is prepared by the above preparation method.

[0023] In a third aspect, the present invention provides a chimeric protein of Escherichia coli Nissle 1917 flagellin that effectively activates the TLR5 signaling pathway and is used in the preparation of a vaccine for preventing Seneca virus infection.

[0024] The beneficial effects of the present invention are as follows: 1) The solubility of the VP2 antigen expressed alone is poor. It was unexpectedly discovered that the chimeric protein of the Escherichia coli Nissle 1917 flagellin that effectively activates the TLR5 signaling pathway can effectively solve the problem of VP2 antigen inclusion body expression, and the chimeric protein is basically expressed in a soluble form. 2) The soluble expression amount of the chimeric protein can reach about 100 mg / L. 3) The chimeric protein of the present invention can effectively activate the TLR5 signaling pathway in vitro. 4) The chimeric protein of the present invention can effectively activate T lymphocytes, improve the immunogenicity of VP2, and enhance the overall immune effect of the flagellin vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 : Plasmid map of the recombinant expression vector FliC-L-VP2 in pET28a(+).

[0026] Figure 2 : SDS-PAGE electrophoresis of the soluble expression levels of the three chimeric proteins. Figure a is FliC Δ195-320 -L-VP2, lane 1: protein molecular weight standard; lane 2: uninduced sample; lane 3: 10℃ induced sample; lane 4: bacterial lysate precipitate; lane 5: bacterial lysate supernatant. Δ274-406 -L-VP2, lane 1: sample induced at 10℃; lane 2: bacterial lysate precipitate; lane 3: bacterial lysate supernatant. c is FliC-L-VP2, lane 1: uninduced sample; lane 2: sample induced at 37℃; lane 3: bacterial lysate precipitate; lane 4: bacterial lysate supernatant. d is VP2 antigen, lane 1: bacterial lysate supernatant; lane 2: bacterial lysate precipitate.

[0027] Figure 3 : SDS-PAGE electrophoresis of the purified novel chimeric protein. Lane 1: uninduced sample; Lane 2: 37℃ induced sample; Lane 3: bacterial lysate precipitate; Lane 4: bacterial lysate supernatant; Lane 5: lysate supernatant flow-through sample; Lane 6: rinse sample 1; Lane 7: rinse sample 2; Lane 8: eluted sample 1; Lane 9: eluted sample 2; Lane 10: eluted sample 3; Lane 11: protein molecular weight standard.

[0028] Figure 4 : Western blot results of the purified chimeric protein.

[0029] Figure 5:Activity detection of the three chimeric proteins after purification to activate the TLR5 signaling pathway. Human colon cancer Caco2 cells and mouse macrophage Raw264.7 cells were stimulated with 20 μg / mL endotoxin-free chimeric proteins for 12 hours, respectively, and unstimulated cells were used as negative control groups. The cell culture supernatant was removed, and RNA was extracted after lysis using RNAiso Plus. After reverse transcription, qRTPCR was used to detect the gene expression levels of IL-8 and IL-6. DETAILED DESCRIPTION

[0030] The embodiments of the present invention will be clearly and completely described below in conjunction with the examples. Obviously, the described examples are only used to illustrate a part of the embodiments of the present invention and should not be regarded as limiting the scope of the present invention. If the specific conditions are not specified in the examples, they are carried out according to the normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are regarded as conventional products that can be purchased commercially.

[0031] Unless otherwise specified, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional molecular biology, biochemistry, protein recombinant expression technology, cell culture, and conventional techniques in related fields in the technical field.

[0032] In the specific implementation process of the present invention, three different forms of chimeric proteins were constructed to evaluate their effects on the flagellin adjuvant effect. The three chimeric flagellins are: VP2 is chimeric to the C-terminus of flagellin FliC to form FliC-L-VP2; VP2 is chimeric to the hypervariable region D3 of FliC to form FliC Δ274-406 -L-VP2; and inserting VP2 into the hypervariable region D2D3 of FliC to form FliC Δ195-320 -L-VP2.

[0033] Glossary

[0034] 1. Linker: In biochemistry and molecular biology, "Linker" or "linker peptide" refers to a short peptide sequence that connects two or more functional peptides or proteins. The design of linker peptides usually needs to consider their length, amino acid composition, and spatial conformation to ensure that they do not interfere with the function between the connected proteins while maintaining the desired biological activity.

[0035] 2. Chimerism: In biochemistry and molecular biology, "chimerism" usually refers to combining two or more biological molecules (such as proteins, DNA or RNA) from different sources to form a new molecule. For example, a chimeric protein is a protein composed of protein domains or functional domains from different sources.

[0036] Example 1 Construction of recombinant expression vector pET-28a(+) (containing FliC-L-VP2 sequence)

[0037] The gene sequence of the designed chimeric protein FliC-L-VP2 was provided to GENEWISE for codon optimization and full gene synthesis. The sequence was cloned into the vector pET-28a(+) through the BamHI and SalI restriction sites to construct the plasmid FliC-L-VP2inpET-28a(+). The plasmid map of the recombinant expression vector is shown in Figure 1 shown.

[0038] The construction methods of the other two chimeric protein recombinant expression vectors are the same as above.

[0039] Example 2 Prokaryotic Expression of FliC-L-VP2 Chimeric Protein

[0040] (1) The recombinant plasmid FliC-L-VP2 in pET-28a(+) synthesized by GENEWISE was transformed into competent Escherichia coli BL21(DE3) cells. The genetically engineered strain FliC-L-VP2 in pET-28a(+) / BL21(DE3) was streaked with a loop on a pre-prepared LB plate containing 80 μg / mL Kan. The plate was inverted at 37°C incubator for 12-16 hours, and a single colony was picked and inoculated into LB liquid culture medium containing 80 μg / mL Kan at 37°C, 200 rpm, and cultured for 12-16 hours to obtain the recombinant bacterial culture solution.

[0041] The empty plasmid pET-28a(+) was transformed into Escherichia coli BL21(DE3) competent cells to obtain the negative control strain pET-28a(+) / BL21(DE3).

[0042] (2) The recombinant bacterial culture fluid in (1) was inoculated at 5% inoculation into 500 mL of LB liquid medium containing 80 μg / mL Kan, and cultured at 37°C, 200 rpm, for 4 hours until its OD reached 0.6-0.8. IPTG was added to reach a final concentration of 0.8 mmol / L, and induced for expression at 37°C for 6 hours. Compared with the control strain, the genetically engineered recombinant strain FliC-L-VP2 in pET-28a(+) / BL21(DE3) showed a target protein band at about 90 kDa, which was consistent with the theoretical value of the chimeric protein, and the expression level could reach 100 mg / L. The induction conditions for the other two chimeric proteins were 10°C, a final IPTG concentration of 0.01 mmol / L, and an induction of 32 hours.

[0043] Example 3 Purification of FliC-L-VP2 chimeric protein

[0044] (1) After adding an appropriate amount of deionized water to the gravity column filled with Ni NTABeads 6FF to rinse the ethanol, add 10 column volumes of Binding buffer to balance, and then load the sample. After collecting the bacteria, add 30 mL of Binding Buffer for every 1 g of bacteria to resuspend and ultrasonically lyse the bacteria. Load the supernatant of the bacterial lysate onto the column, rinse the column with 10 column volumes of Wash buffer after 20 minutes, elute with 3 column volumes of Elution Buffer, and collect the eluted sample. Wash with 5 column volumes of Binding Buffer and 5 column volumes of deionized water in turn, and then balance with 2 column volumes of 20% ethanol (the ethanol should immerse the filler), and store at 4°C.

[0045] (2) SDS-PAGE electrophoresis was used to detect the purification effect of the chimeric protein FliC-L-VP2 and to determine the concentration of the purified protein. Figure 3 As shown, the soluble expression level of the chimeric protein FliC-L-VP2 is high, reaching 100 mg / L. A band of about 90 kDa appears in lanes 8-10, and the purity reaches more than 85%.

[0046] The purification methods of the other two chimeric proteins were the same as above.

[0047] Example 4 Western blot analysis of FliC-L-VP2 chimeric protein

[0048] The purified FliC-L-VP2 chimeric protein sample was subjected to 10% SDS-PAGE electrophoresis and then transferred to a PVDF membrane at a constant voltage of 100V for 90min. The transferred PVDF membrane was placed in PBST containing 10% skimmed milk powder, and blocked by slowly shaking on a shaker at room temperature for 2h, and then washed with PBST three times, 5min / time. Anti-His-tag mouse monoclonal antibody (Biyuntian, catalog number: AF2873) was used as the primary antibody, diluted with antibody diluent (1:2000), incubated slowly on a shaker at 4°C overnight, and then washed with PBST three times, 5min / time. The PVDF membrane was developed using the ECL luminescence method. Western blot results are shown in the figure below. Figure 4 As shown, the chimeric protein FliC-L-VP2 can react specifically with anti-His-tag mouse monoclonal antibody, and the protein size is about 90 kDa.

[0049] The Western blot analysis method for the other two chimeric proteins was the same as above.

[0050] Example 5 Detection of the activity of chimeric protein FliC-L-VP2 in activating TLR5 in vitro

[0051] (1) The chimeric protein purified in Example 3 was concentrated five times and dialyzed overnight. The buffer was replaced with PBS and the protein was purified by ToxinEraser TM Endotoxin Removal Kit (GenScript, Cat. No. L00338) removes lipopolysaccharide (LPS) from flagellar protein. ToxinSensor TM The Gel Clot Endotoxin Assay Kit (GenScript, Cat. No. L00351) was used to ensure that the LPS content was less than 0.1 EU / mL. The BCA method was then used to measure the protein concentration after endotoxin removal to be 400 mg / L.

[0052] (2) Human colorectal adenocarcinoma cells (Caco2) expressing TLR5 receptor were used as an in vitro cell model to detect the activity of recombinant protein FliC-L-VP2 in activating TLR5. Caco2 cells were grown at 10 cm 2 After the cells are grown in the culture dish, 3 × 10 5 Cells were seeded into 12-well cell culture plates and incubated at 37°C with 5% CO 2 After overnight culture, Caco2 cells were stimulated for 12 hours by adding purified chimeric proteins at a final concentration of 20 μg / mL. Untreated Caco2 cells were used as negative controls. The cells were removed from the supernatant, washed three times with PBS, and 400uL RNAiso Plus (Takla, Cat. No.: 9109) was added to each well. The lysate containing the cells was transferred to an enzyme-free centrifuge tube, and after standing at room temperature for 5 minutes, centrifuged at 4°C, 12000×g for 5 minutes. The supernatant was transferred to a new enzyme-free centrifuge tube, 80uL of chloroform was added, and after shaking evenly, the supernatant was allowed to stand at room temperature for 5 minutes, and centrifuged at 4°C, 12000×g for 15 minutes. The supernatant was transferred to a new enzyme-free centrifuge tube, 400uL of isopropanol was added, and the supernatant was allowed to stand at room temperature for 10 minutes, and centrifuged at 4°C, 12000×g for 10 minutes. The supernatant was discarded, and 400uL of 75% ethanol was added to wash the precipitate, and centrifuged at 4°C, 7500×g for 5 minutes. Discard the supernatant, dry the precipitate naturally, and add 30uL RNase-free water to dissolve the RNA. After measuring the RNA concentration, use 1ug of total RNA for reverse transcription. First add 8×gDNAEraser Premix (Takla, Catalog No.: RR092A), the system is 16uL, 42℃, 2min, to remove genomic DNA. Add 4uL 5×RTPremix (Takla, Catalog No.: RR092A), reverse transcribe for 10min at 37℃. Terminate the reaction at 85℃, 5sec. Finally, select TB Green PremixEx Taq II FAST qPCR (Takla, Catalog No.: CN830A) for real-time quantitative PCR reaction.

[0053] Primer sequences used for qRTPCR amplification.

[0054]

[0055] The qRTPCR reaction solution was prepared according to the following composition.

[0056]

[0057]

[0058] The qRT PCR reaction was performed according to the following two-step PCR procedure.

[0059]

[0060] The results are as follows Figure 5 As shown in a, it shows that the chimeric protein FliC-L-VP2 significantly stimulates the transcription of IL-8 in Caco2 cells, and the activation effect is significantly higher than that of the other two chimeric proteins, indicating that the recombinant protein FliC-L-VP2 has good TLR5 activation activity, and its activity is higher than that of FliC Δ274-406 -L-VP2 and FliC Δ195-320 -L-VP2.

[0061] (3) Furthermore, the activity of the chimeric protein FliC-L-VP2 in activating TLR5 was detected using mouse macrophages (Raw264.7) that can express TLR5 receptors as an in vitro cell model. The remaining methods were the same as described in (2). Figure 5 As shown in b, it shows that the recombinant protein FliC-L-VP2 significantly stimulates the transcription of IL-6 in Raw264.7 cells and is significantly higher than that of FliC Δ274-406 -L-VP2 activation effect, indicating that the recombinant protein FliC-L-VP2 has good TLR5 activation activity.

[0062] Primer sequences used for qRTPCR amplification.

[0063]

Claims

1. A chimeric protein of Escherichia coli Nissle 1917 flagellin that effectively activates the TLR5 signaling pathway, characterized in that: The chimeric protein is formed by combining the Escherichia coli Nissle 1917 flagellin with the type A Seneca virus VP2 antigen.

2. The chimeric protein according to claim 1, wherein The type A Senecavirus VP2 antigen is at the C-terminus of the Escherichia coli Nissle 1917 flagellin.

3. The chimeric protein according to claim 2, characterized in that The chimeric protein is obtained by chimerizing the Escherichia coli Nissle 1917 flagellin and the Seneca virus type A VP2 antigen through a linker.

4. The chimeric protein according to claim 3, characterized in that The amino acid sequence of the Linker is GGGGSGGGGSGGGGS.

5. The chimeric protein according to claim 1, wherein The amino acid sequence of the chimeric protein is shown in SEQ ID NO:

2.

6. The chimeric protein according to claim 1, wherein The nucleotide sequence of the chimeric protein is shown in SEQ ID NO:

1.

7. Use of the chimeric protein according to claims 1-6 in the preparation of a vaccine for preventing Seneca virus infection.

8. A method for preparing a chimeric protein, comprising the following steps: S1: The nucleotide sequence of the chimeric protein FliC-L-VP2 was cloned into the expression vector pET-28a(+) through the BamHI and SalI restriction sites; S2: Transform the recombinant E. coli expression vector into competent BL21 (DE3) to obtain a recombinant strain for producing chimeric proteins; S3: The recombinant strain obtained in step S2 was induced to express at 37°C; S4: Separating and purifying the bacterial solution obtained in step S3, and obtaining the chimeric protein by nickel column affinity chromatography.

9. A chimeric protein prepared by the preparation method according to claim 8.

10. Use of the chimeric protein according to claim 9 in preparing a vaccine for preventing Seneca virus infection.

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