Recombinant fusion protein based on PCV2 nano antibody and bacterial flagellin as well as preparation method and application of recombinant fusion protein

Abstract

The invention provides a recombinant fusion protein based on a porcine circovirus type 2 (PCV2) nano antibody and salmonella flagellin as well as a preparation method and application of the recombinant fusion protein. According to the invention, a PCV2 capsid protein (Cap) specific nano antibody gene and a salmonella flagellin gene are fused, and an escherichia coli prokaryotic expression system is used for expression so as to prepare the Nbcapp-flagellin recombinant fusion protein. The recombinant fusion protein can realize dynamic coupling of a flagellin adjuvant and an antigen protein by utilizing the targeted binding capacity of a Cap protein specific nano antibody, so as to form an antigen-adjuvant compound of which the adjuvant dosage can be regulated and controlled; while the function of flagellin as an immunologic adjuvant is fully played, the fixed adjuvant-antigen proportion limitation caused by fusion expression of traditional flagellin and antigen protein is broken through, the flexibility of vaccine design is improved, and the inflammatory reaction possibly caused by excessive bacterial flagellin is avoided; and a new technical path is provided for developing an efficient, safe and broad-spectrum PCV2 subunit vaccine.

Description

Technical Field

[0001] The present application belongs to the field of biotechnology, and particularly relates to a recombinant fusion protein based on PCV2 nanoantibodies and bacterial flagellin, and a preparation method and application thereof. Background Art

[0002] Porcine circovirus type 2 (PCV2) is a non-enveloped, single-stranded circular DNA virus belonging to the Circoviridae family. It primarily infects the respiratory, urinary, nervous, and reproductive systems of domestic and wild pigs, causing post-weaning multisystemic wasting syndrome (PMWS) and reproductive failure, among other circovirus-associated diseases (PCVAD), resulting in billions of dollars in annual economic losses for the global swine industry. The virus exhibits significant genetic variability, resulting in eight genotypes, PCV2a to PCV2h. Since 2010, PCV2d has become the predominant strain globally due to its ecological advantages. Some strains harbor key epitopes in the capsid protein (Cap), such as the R→K mutation at position 59, which significantly reduces the protective efficacy of traditional vaccines. Furthermore, PCV2 spreads rapidly through fecal-oral, respiratory, and vertical routes of transmission, and infection often leads to secondary bacterial infections, further complicating clinical symptoms and making prevention and control difficult, making it a core public health challenge for the global swine industry.

[0003] Currently, PCV2 prevention and control mainly rely on inactivated vaccines and virus-like particles (VLPs) subunit vaccines based on Cap protein. Cap protein can spontaneously assemble into VLPs with a natural conformation, making it the preferred antigen for subunit vaccine development, which can efficiently induce neutralizing antibodies and T cell immune responses. However, the immunogenicity of subunit vaccines is limited by the efficiency of antigen delivery, and it is necessary to rely on adjuvants to enhance the immune response and achieve effective protection through multiple immunizations or increased antigen doses. Although traditional aluminum salt adjuvants can enhance humoral immune responses, they have problems such as local inflammatory reactions and insufficient activation of cellular immunity, and it is difficult to meet the cross-protection requirements brought about by the diversity of PCV2 genotypes. Clinical data show that the neutralizing potency of existing vaccines against emerging genotypes such as PCV2d is significantly reduced by about 1.5-2 times. Therefore, there is an urgent need to develop new adjuvant systems with strong adaptability and dynamic optimization to improve the broad spectrum of vaccines.

[0004] Flagellin, the primary structural protein of bacterial flagella, has been extensively studied as a vaccine adjuvant due to its ability to activate the Toll-like receptor 5 (TLR5) signaling pathway. By inducing both mucosal and systemic immune responses, it can significantly enhance the immunogenicity of co-delivered antigens. Previous studies have demonstrated the ability to directly fusion the PCV2 Cap protein with Salmonella flagellin, enhancing neutralizing antibody levels and lymphocyte proliferation. However, the antigen-adjuvant ratio in such fusion proteins is fixed and cannot be flexibly adjusted to meet immune needs, leading to an imbalance between immune stimulation intensity and antigen loading. For example, excessive flagellin may trigger an excessive inflammatory response, while a low-ratio fusion may fail to activate sufficient TLR5 signaling. Furthermore, when used alone as an adjuvant, flagellin requires physical mixing with the antigen or multiple inoculations, resulting in low delivery efficiency and insufficient immune memory persistence, limiting its potential for clinical application. Summary of the Invention

[0005] To address these issues, the present invention leverages the advantages of Nanobodies (Nbs)—high antigen affinity, excellent stability, and a small molecular weight of 15 kDa—to construct a novel immune adjuvant based on the PCV2-Cap protein-specific Nanobodies fused to flagellin via a flexible linker peptide. This novel adjuvant couples flagellin to the antigen protein via the Cap protein-specific Nanobodies, forming an antigen-adjuvant complex. This design overcomes the fixed ratio limitations of traditional fusion proteins, enabling dynamic adjustment of the antigen-adjuvant ratio while also avoiding inflammatory responses induced by excessive flagellin. This provides an innovative solution for the development of highly effective, broad-spectrum, and safe PCV2 subunit vaccines.

[0006] The first object of the present invention is to provide a recombinant fusion protein based on PCV2 nanobody and bacterial flagellin.

[0007] The second object of the present invention is to provide a method for preparing a recombinant fusion protein based on PCV2 nanobody and bacterial flagellin.

[0008] The third object of the present invention is to provide an application of a recombinant fusion protein based on PCV2 nanobody and bacterial flagellin.

[0009] To achieve the above object, the present invention adopts the following technical solutions:

[0010] A recombinant fusion protein based on a PCV2 nanobody and bacterial flagellin, wherein the recombinant fusion protein is formed by fusing a PCV2 Cap protein-specific nanobody with Salmonella flagellin via a linker peptide. The amino acid sequence of the recombinant fusion protein is shown in SEQ ID NO. 1 or SEQ ID NO. 2.

[0011] Furthermore, the PCV2 Cap protein-specific nanobody in the recombinant fusion protein is connected to the N-terminus or C-terminus of the Salmonella flagellin through a linker.

[0012] Furthermore, the amino acid sequence of the Linker is shown in SEQ ID NO.3.

[0013] The present invention also provides a method for preparing a recombinant fusion protein based on PCV2 nanobody and bacterial flagellin, the method comprising the following steps:

[0014] S1: The flagellin gene sequence of Salmonella and the Linker gene sequence were subjected to Escherichia coli rare codon expression analysis and optimal codon optimization for the flagellin gene nucleotide sequence and the Linker gene nucleotide sequence to obtain a gene sequence for the fusion expression of flagellin and Linker as shown in SEQ ID NO.4; the sequence shown in SEQ ID NO.4 was obtained through artificial codon optimization and screening, which can ensure to the greatest extent that the two protein fragments upstream and downstream of the Linker fragment do not affect each other, thereby achieving high-level expression of the recombinant fusion protein of Salmonella flagellin and PCV2-Cap protein specific nanoantibody.

[0015] S2: Synthesize the nucleotide sequence of the recombinant fusion protein shown in SEQ ID NO. 4 and clone it into the pCold-SUMO vector to obtain the recombinant expression vector pCold-SUMO-flagellin-linker containing the nucleotide sequence encoding the flagellin-linker recombinant protein;

[0016] S3: Using NcoI / HindIII restriction endonucleases, the PCV2-Cap protein-specific nanobody gene nucleotide sequence Nbcap was excised from the constructed prokaryotic recombinant expression vector pET32a-Nbcap. The PCV2-Cap protein-specific nanobody gene nucleotide sequence Nbcap is shown in SEQ ID NO. 5;

[0017] S4: Linearize the recombinant expression vector pCold-SUMO-flagellin-linker using NcoI / HindIII restriction endonucleases, clone the Nbcap gene sequence into the linearized recombinant expression vector pCold-SUMO-flagellin-linker, and obtain a recombinant expression vector containing the nucleotide sequence encoding the Nbcap-flagellin recombinant fusion protein;

[0018] S5: Transform the recombinant expression vector pCold-SUMO-flagellin-linker described in S4 into the competent Escherichia coli BL21 (DE3) strain to obtain a recombinant Escherichia coli BL21 (DE3) strain for expressing the Nbcap-flagellin recombinant fusion protein;

[0019] S6: Using the recombinant E. coli BL21 (DE3) strain described in S5 as the seed strain, induce expression of the Nbcap-flagellin recombinant fusion protein, obtain the recombinant bacteria by centrifugation, and then ultrasonically disrupt them, and retain the supernatant for later use;

[0020] S7: The supernatant of the bacterial cell disruption described in S6 is subjected to affinity chromatography purification, and the purified product of the Nbcap-flagellin recombinant fusion protein is eluted.

[0021] Furthermore, the step S6 is:

[0022] A. Streak the recombinant engineered bacteria onto ampicillin-resistant LB solid medium and incubate at 37°C for 14-16 hours. Pick a single colony and inoculate it into 20 mL of LB liquid medium. Incubate the culture overnight at 37°C with shaking at 180 rpm to obtain an activated seed solution.

[0023] B. Transfer the activated seed solution obtained in step A to fresh LB liquid medium at a 1%-5% inoculum volume and culture at 37°C and 180 rpm with shaking until the OD600 value reaches 0.5-0.8;

[0024] C. Add IPTG to the culture medium to a final concentration of 0.01mM-1mM;

[0025] D. Adjust the culture conditions to 18°C ​​and 160 rpm, and continue inducing expression for 8 hours.

[0026] Furthermore, the step S8 is:

[0027] A. Filter the supernatant of bacterial cell disruption using a 0.45 μm filter membrane and add it to a Ni-NTA purification column;

[0028] B. Wash the column with 10 volumes of loading buffer to remove non-specifically bound proteins, and then further remove non-target proteins with 10-15 column volumes of wash buffer, wherein the loading buffer comprises 20 mM PB, 500 mM NaCl, 10 mM imidazole, pH 8.0; and the wash buffer comprises 20 mM PB, 500 mM NaCl, 30 mM imidazole, pH 8.0;

[0029] C. collecting the target protein using an elution buffer comprising 20 mM PB, 500 mM NaCl, 250 mM imidazole, pH 8.0;

[0030] D. The target protein obtained in step C was dialyzed against PBS buffer at pH 8.0 to remove imidazole.

[0031] The present invention also provides a use of the recombinant fusion protein in preparing PCV2 subunit vaccines.

[0032] The beneficial effects of the present invention are:

[0033] 1. The present invention utilizes a prokaryotic expression system to express and prepare a recombinant fusion protein of PCV2-Cap protein-specific nanoantibodies and Salmonella flagellin in the form of fusion expression, which is a first invention.

[0034] 2. The Nbcap-flagellin recombinant fusion protein of the present invention can form an antigen-adjuvant complex with the Cap protein, effectively solving the problem of the inability to optimize the antigen-adjuvant ratio caused by the fusion expression of the antigen and adjuvant.

[0035] 3. The Nbcap-flagellin recombinant fusion protein of the present invention exhibits good biological activity. The generated complex can effectively stimulate the body to produce higher levels of specific antibodies and is an ideal candidate adjuvant for enhancing the efficacy of PCV2 subunit vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the agarose gel electrophoresis diagram of the recombinant PCR product after the Nbcap gene fragment and the linearized vector pCold-SUMO-flagellin-linker were transformed into Escherichia coli DH5α.

[0037] In the figure: M is DNA marker DL2000, N is PCR negative control, and 1-6 are PCR identification products of 6 randomly picked single colonies.

[0038] Figure 2 This is the SDS-PAGE analysis result after purification of Nbcap-flagellin recombinant fusion protein.

[0039] In the figure: M is a protein marker, 1 is the flow-through of protein purification column, 2-5 are the eluates of protein purification.

[0040] Figure 3 This is the Western blot analysis result of Nbcap-flagellin recombinant fusion protein.

[0041] In the figure: M is a protein marker, and 1 is the result of identifying the Nbcap-flagellin recombinant fusion protein using a His tag antibody.

[0042] Figure 4 The results of SDS-PAGE analysis of the purified flagellin recombinant protein are shown in Figure 3.

[0043] In the figure: M is a protein marker, 1 is the supernatant after sonication of the expressing bacteria, and 2-9 are the eluates of purified flagellin recombinant protein.

[0044] Figure 5 These are the changes in Cap protein-specific antibody levels in the serum of each group of mice after vaccination.

[0045] Figure 6 These are the results of the PCV2 neutralizing antibody titer test in the serum of each group of mice after vaccination.

[0046] Figure 7 These are the analysis results of cytokine levels in the serum of each group of mice after vaccination.

[0047] In the figure: A is the analysis result of IFN-γ level in mouse serum; B is the analysis result of TNF-α level in mouse serum.

[0048] Figure 8 These are the test results of viral load levels in the serum of mice in each immunized group after being challenged with PCV2. DETAILED DESCRIPTION

[0049] To make the present invention easier to understand, the following examples are further described. The present invention is further described and demonstrated in conjunction with the examples, but these examples are not intended to limit the present invention. The technical solutions described in the present invention are conventional solutions in the field unless otherwise specified; the reagents or materials shown are all from commercial sources unless otherwise specified.

[0050] Example 1

[0051] S1. The flagellin gene sequence fused to the linker gene sequence (flagellin-linker) of Salmonella was codon-optimized using the online codon optimization tool (https: / / www.jcat.de / ) and synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0052] S2. Use SacI / XbaI restriction endonucleases to cut the synthetic flagellin-linker gene fragment from the pUC57-flagellin-linker recombinant expression vector and then clone it into the pCold-SUMO vector. Transform the ligation product into DH5α competent cells, add LB medium and culture for 30 minutes. Take an appropriate amount of culture medium and apply ampicillin (Amp + )-resistant LB agar plates were cultured in a 37°C constant temperature incubator overnight. The next day, 6 single colonies were randomly picked and identified as correct by PCR and sent to a sequencing company for sequencing verification. The correct intermediate recombinant expression vector identified by DNA sequencing was named pCold-SUMO-flagellin-linker.

[0053] S3. Use HindIII / NotI restriction endonuclease to cut and recover the PCV2-Cap protein-specific nanobody gene (Nbcap) fragment from the recombinant vector pET-32a-Nbcap constructed earlier in the laboratory.

[0054] S4. The Nbcap gene fragment was ligated to the linearized pCold-SUMO-flagellin-linker recombinant vector treated with HindIII / NotI restriction enzymes. The ligation product was transformed into DH5α competent cells. After adding LB medium, the cells were revived and cultured for 30 minutes. An appropriate amount of the culture was coated with ampicillin (Amp). + ) resistant LB agar plates, cultured overnight in a 37°C constant temperature incubator, and randomly picked 6 single colonies the next day for PCR identification. The size of the PCR amplification band of the positive recombinant bacteria was 750bp. The gel electrophoresis results of the PCR products were as follows Figure 1 The plasmid extracted from the recombinant bacteria identified as correct by PCR was sent to a sequencing company for sequencing verification. The correct intermediate recombinant expression vector identified by DNA sequencing was named pCold-SUMO-Nbcap-flagellin.

[0055] S5. The correctly sequenced pCold-SUMO-Nbcap-flagellin recombinant expression vector was transformed into BL21 (DE3) competent cells to obtain recombinant engineered bacteria.

[0056] S6. Spread the recombinant engineered strain pCold-SUMO-Nbcap-flagellin / BL21(DE3) obtained in S5 onto LB solid medium containing 100 μg / mL ampicillin and incubate inverted at 37°C for 12-16 hours. Pick a single colony and inoculate it into 20 mL of LB liquid medium containing the same concentration of ampicillin. Incubate the culture at 37°C with shaking at 180 rpm overnight. The next day, transfer a 1% inoculum to fresh LB medium and incubate at 37°C until the OD600 reaches 0.6. Add IPTG to a final concentration of 0.1 mmol / L, lower the culture temperature to 18°C, and continue induction at 160 rpm for 8 hours.

[0057] S7. Collect the cells by centrifugation at 10,000 rpm for 10 min at 4°C. Resuspend in pre-chilled protein loading buffer and disrupt by sonication in an ice bath. Centrifuge the cell suspension at 12,000 rpm for 20 min at 4°C. Collect the supernatant and filter through a 0.45 μm filter. The loading buffer consists of 20 mM PB, 500 mM NaCl, and 10 mM imidazole, pH 8.0.

[0058] S8. After the Ni-NTA purification column was equilibrated with 10 column volumes of deionized water and 10 column volumes of loading buffer, the filtered supernatant after cell disruption was added to the Ni-NTA purification column. Non-specific binding proteins were then washed with 10 column volumes of loading buffer, and non-target proteins were further removed with 10 to 15 column volumes of wash buffer. Finally, the target protein was collected with elution buffer. After purification, the chromatography column was rinsed with 10 column volumes of loading buffer and 10 column volumes of deionized water, and the filler was immersed in 20% ethanol and stored at 4°C until use. The wash buffer included 20mM PB, 500mM NaCl, 30mM imidazole, pH 8.0, and the elution buffer included 20mM PB, 500mM NaCl, 250mM imidazole, pH 8.0.

[0059] After protein expression and purification, samples were identified by SDS-PAGE and Western blot. Figure 2 As shown in the figure, the Nbcap-flagellin recombinant fusion protein can be efficiently purified by Ni-NTA affinity chromatography column, and SDS-PAGE analysis shows that the target protein has an apparent molecular weight of approximately 38 kDa, which is consistent with the expected size. Figure 3 As shown, the target protein was verified by Western blot using a His tag antibody, and a specific band was observed at 38 kDa, confirming that the recombinant fusion protein was successfully expressed.

[0060] Example 2

[0061] Application of recombinant fusion protein based on PCV2 nanoantibody and bacterial flagellin in the preparation of PCV2 subunit vaccine.

[0062] In this example, a recombinant fusion protein of an anti-PCV2 nanobody and Salmonella flagellin was used as an immune adjuvant in combination with the PCV2 Cap protein to construct a subunit vaccine for the prevention and control of porcine circovirus type 2 (PCV2). The specific preparation process of the subunit vaccine includes mixing the purified PCV2 Cap protein and the recombinant fusion protein (comprising the anti-PCV2 nanobody and the Salmonella flagellin protein connected by a flexible linker) in a mass ratio of 2.5:1, stirring at 4°C for 30 minutes to form a homogenous solution, filtering through a 0.22μm sterile filter for sterilization, and storing at 2-8°C in the dark until use. The final vaccine formulation contains 10μg of Cap protein per dose, 4μg of recombinant fusion protein per dose, and 28.6% of the adjuvant by weight.

[0063] To validate the vaccine's efficacy, an immunization trial was conducted in a BALB / c mouse model. This recombinant fusion protein adjuvant-based vaccine offers an innovative solution for PCV2 subunit vaccine design by integrating the antigen-targeting properties of nanobodies with the inherent adjuvant effect of flagellin.

[0064] Comparative Example 1

[0065] The synthesized flagellin gene fragment was cloned into the pET-32a expression vector using the restriction endonucleases BamHI / HindIII to construct the recombinant expression vector pET-32a-flagellin. The identified recombinant expression vector was transformed into BL21(DE3) competent cells, plated onto LB solid medium containing 100 μg / mL ampicillin, and incubated upside down at 37°C for 12-16 hours. A single colony was picked and inoculated into 20 mL of LB liquid medium containing 100 μg / mL ampicillin and incubated at 37°C with shaking at 180 rpm overnight. A 1% inoculum was transferred to 200 mL of fresh LB medium and incubated at 37°C until the OD600 reached 0.6-0.8. IPTG was then added to a final concentration of 0.1 mmol / L and expression was induced at 37°C at 180 rpm for 8 hours. After expression was complete, the cells were harvested by centrifugation at 10,000 rpm at 4°C for 10 minutes, resuspended in pre-chilled lysis buffer, and disrupted by sonication on ice. The disrupted liquid was centrifuged at 4°C and 12000 rpm for 30 min, and the supernatant was collected and filtered through a 0.45 μm filter membrane for subsequent purification. The pre-cooled lysis buffer included 20 mM PB, 500 mM NaCl, 10 mM imidazole, and pH 8.0.

[0066] Recombinant flagellin protein was purified using Ni-NTA affinity chromatography. The supernatant after filtration and sonication was loaded onto a pre-equilibrated Ni-NTA purification column. Nonspecifically bound proteins were removed using 10 column volumes of equilibration buffer, followed by 10 column volumes of wash buffer. The target protein was eluted with elution buffer, collected, and dialyzed into PBS (pH 8.0) to remove imidazole. Protein purity was verified by SDS-PAGE. The equilibration buffer consisted of 20 mM Tris-HCl, 300 mM NaCl, and 10 mM imidazole, pH 8.0. The wash buffer consisted of 20 mM PB, 500 mM NaCl, and 30 mM imidazole, pH 8.0. The elution buffer consisted of 20 mM PB, 500 mM NaCl, and 250 mM imidazole, pH 8.0.

[0067] like Figure 4 As shown, recombinant flagellin protein was expressed in a soluble form in E. coli BL21(DE3) with an apparent molecular weight of approximately 25 kDa, consistent with the expected size. High-purity recombinant flagellin protein was obtained by purification using a Ni-NTA affinity chromatography column.

[0068] Experiments were conducted based on the products of Example 1 and Comparative Example 1:

[0069] 1. Immunity and challenge protection test of mice:

[0070] The purified Nbcap-flagellin recombinant fusion protein obtained in Example 1 was dialyzed into PBS buffer at pH 8.0. The protein and flagellin recombinant protein obtained in Comparative Example 1 were treated with Triton-X114 to remove endotoxins. The protein concentrations were then determined using a BCA protein quantification kit and used for further analysis. The Cap protein, purified by prokaryotic expression using conventional methods and stored in this laboratory, was used as an antigen, endotoxin-free, and quantified for further analysis.

[0071] Sixty 8-week-old SPF female BALB / c mice were randomly divided into 5 groups, with 12 mice in each group. Groups 1 to 3 were experimental groups: Group 1 was injected intramuscularly with 10 μg of unadjuvanted Cap recombinant protein; Group 2 was injected with 10 μg of Cap recombinant protein combined with 4 μg of flagellin recombinant protein adjuvant obtained in Comparative Example 1; Group 3 was injected with 10 μg of Cap recombinant protein and 4 μg of Nbcap-flagellin fusion adjuvant obtained in Example 1; Group 4 was injected with 100 μL PBS as the attack control group; Group 5 was the negative control group with the same dose of PBS. All groups were immunized twice, with an interval of 2 weeks. Blood was collected through the retroorbital venous plexus on days 7, 14, 21, and 28 after the first immunization, and the Cap-specific IgG antibodies (ELISA method), neutralizing antibody titers (microneutralization test), and IFN-γ and TNF-α cytokine levels in the serum were systematically detected. Two weeks after the booster immunization, the first 4 groups were injected intraperitoneally with 10 5 TCID 50 Group 5 was challenged with 100 μL of virus suspension, and group 5 was injected with an equal amount of PBS as an uninfected control.

[0072] 2. Detection of Cap protein-specific antibody levels in immunized mice:

[0073] The prokaryotic expressed Cap protein was diluted to 5 μg / mL and 100 μL was added to each well of the ELISA plate for overnight coating at 4°C. The next day, the wells were discarded and washed six times with PBST wash buffer. Nonspecific sites were blocked with 200 μL of 3% MPBS blocking buffer added to each well and incubated at 37°C for 1 hour. The plates were then washed again six times with PBST. Serially diluted serum samples were then added at 100 μL per well and incubated at 37°C for 1 hour. A 1:8000 dilution of horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (Goat anti-mouse IgG-HRP) was then added at 100 μL per well and incubated at 37°C for 1 hour. Unbound secondary antibody was then removed by thorough washing. A color reaction was developed with 100 μL of TMB substrate solution added to each well for 10 minutes at 37°C in the dark. Upon completion of color development, the reaction was terminated with 50 μL of 2 mol / L H₂SO₄ stop solution. Finally, the absorbance of each well was measured using an enzyme-labeled instrument at a wavelength of 450 nm.

[0074] The levels of Cap protein-specific antibodies in the serum of mice in each group were as follows: Figure 5As shown. On the 7th day after the first immunization, no specific antibodies were detected in the first group of Cap protein immunization without adjuvant, while the second and third groups containing adjuvants both showed positive antibody signals. In the dynamic monitoring from the 7th day to the 28th day, the Cap-specific antibody levels of the first three groups of mice showed a continuous upward trend with the increase in the number of immunizations. By the 28th day, that is, 14 days after the second immunization, the PCV2-specific antibody titer of the third group using the Nbcap-flagellin fusion adjuvant obtained in Example 1 was significantly higher than that of the flagellin single adjuvant group obtained in Control Example 1 in the second group and the adjuvant-free group in the first group. The results show that the Nbcap-flagellin fusion adjuvant can significantly enhance the immunogenicity of the Cap protein and induce a higher titer antibody response.

[0075] 3. Detection of PCV2 neutralizing antibody levels in immunized mice:

[0076] After inactivation at 56°C for 30 minutes, serum samples were serially diluted in DMEM (1:2 to 1:256), with 50 μL per well in triplicate. PCV2 virus was diluted to 200 TCID50 / mL and added to the serum wells in equal volumes. Neutralization occurred at 37°C for 1 hour. After discarding the PK15 cell monolayer culture medium, 100 μL of the mixture was added, with duplicate wells for each dilution. Negative / positive serum controls, virus controls, and blank controls were also included. After adsorption at 37°C for 1 hour, the cells were replaced with a maintenance medium containing 2%-3% serum and incubated for 48-72 hours. After fixation of the cells, a mouse anti-PCV2 monoclonal antibody (1:400) and a FITC-conjugated secondary antibody (1:200) were added sequentially. Incubation was performed at 37°C for 1 hour each, and observation was performed under a fluorescence microscope. The reciprocal of the highest serum dilution that completely inhibited PCV2 infection was designated as the neutralization titer.

[0077] The results are as follows Figure 6 As shown, no neutralizing antibodies were detected in the PBS control group throughout the whole process; neutralizing antibodies appeared in the Cap unadjuvanted group on the 21st day after the first immunization, with a titer of 1:6.72, while the flagellin adjuvanted group obtained in Comparative Example 1 responded as early as the 14th day; the neutralizing antibodies in the Nbcap-flagellin adjuvanted group obtained in Example 1 were positive on the 7th day, and the titer was significantly higher than that in the unadjuvanted group on the 21st day, and rose to 1:38 on the 28th day, which was statistically different from the flagellin adjuvanted group and the unadjuvanted group.

[0078] 4. Detection of cytokine levels in the serum of immune mice:

[0079] Mouse serum samples were collected on days 7 and 14 after immunization. Interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α) in mouse serum were quantitatively detected using the mouse IFN-γ ELISA detection kit and mouse TNF-α ELISA detection kit from Thermo Fisher according to the kit instructions. All samples were tested in triplicate, and cytokine concentrations were expressed in pg / mL. The results are shown in Figure 2. Figure 7 As shown, the TNF-α and IFN-γ levels in the PBS control group did not change significantly throughout the entire immunization process. The Nbcap-flagellin recombinant fusion protein adjuvant group obtained in Example 1 showed significantly higher TNF-α levels than the no-adjuvant group on day 7 of immunization. Over time, its levels of both cytokines continued to be higher than those in the flagellin recombinant protein adjuvant group obtained in Comparative Example 1 and the no-adjuvant group, reaching a peak on day 28. TNF-α and IFN-γ levels were significantly higher than those in the flagellin recombinant protein adjuvant group obtained in Comparative Example 1, and were the only group significantly higher than the negative control group. This indicates that the Nbcap-flagellin recombinant fusion protein adjuvant can more effectively induce cytokine secretion and enhance cellular immune responses, with better effects than flagellin adjuvant.

[0080] 5. Determination of virus content in mouse serum after challenge:

[0081] Blood samples from infected mice were collected three weeks after infection, and total nucleic acid was extracted using the Tiangen Virus DNA / RNA Extraction Kit. PCV2 DNA was detected by SYBR Green I fluorescent quantitative real-time PCR, with primer sequences of 5'-ATAACCCAGCCCTTCTCCTACC-3' (upstream) and 5'-GGCCTACGTGGTCTACATTTCC-3' (downstream). The reaction procedure was pre-denaturation at 95°C for 30 seconds, followed by 40 cycles of denaturation at 95°C for 5 seconds and annealing at 60°C for 30 seconds. The results are shown in Figure 2. Figure 8 As shown, PCV2 viremia was detected in all mice in the challenge control group, and the number of copies of serum viral genomes reached 10^(5.0±1.2)copies / mL. Compared with the data of 10^(4.2±1.5)copies / mL in the challenge control group, the viral load of the Cap recombinant protein-free adjuvant immunization group did not show a significant decrease. The load of the flagellin recombinant protein adjuvant immunization group obtained in Comparative Example 1 was 10^(3.8±1.2)copies / mL, and the Nbcap-flagellin recombinant fusion protein adjuvant group obtained in Example 1 showed a significant antiviral effect, and the viral load was reduced to 10^(3.1±1.5)copies / mL, which was the lowest level among all experimental groups. This shows that the Nbcap-flagellin recombinant fusion protein can be used as a potential candidate immune adjuvant for PCV2 subunit vaccine.

Claims

1. A recombinant fusion protein based on PCV2 nanobody and bacterial flagellin, characterized by: The recombinant fusion protein is formed by fusing PCV2Cap protein-specific nanobody with Salmonella truncated flagellin flagellin via a linker peptide.

2. The recombinant fusion protein based on PCV2 nanobody and bacterial flagellin according to claim 1, characterized in that: The amino acid sequence of the recombinant fusion protein is shown in SEQ ID NO.1 or SEQ ID NO.

2.

3. The recombinant fusion protein based on PCV2 nanobody and bacterial flagellin according to claim 1, characterized in that: The PCV2 Cap protein-specific nanobody is connected to the N-terminus or C-terminus of the truncated flagellin protein of Salmonella through a linker peptide.

4. The recombinant fusion protein based on PCV2 nanobody and bacterial flagellin according to claim 3, characterized in that: The amino acid sequence of the connecting peptide Linker is shown in SEQ ID NO.

3.

5. The method for preparing a recombinant fusion protein based on PCV2 nanobody and bacterial flagellin according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1: The truncated flagellin gene flagellin from Salmonella was fused with the linker peptide gene, and the flagellin-linker gene fragment was synthesized after codon optimization. The nucleotide sequence of the flagellin-linker gene fragment is shown in SEQ ID NO. 4; S2: Clone the flagellin-Linker gene fragment described in S1 into the prokaryotic expression vector pCold-SUMO to obtain the recombinant expression vector pCold-SUMO-flagellin-Linker; S3: Using NcoI / HindIII restriction endonucleases, the PCV2 Cap protein-specific nanobody gene fragment Nbcap was excised from the prokaryotic recombinant expression vector pET32a-Nbcap. The nucleotide sequence of Nbcap is shown in SEQ ID NO.

5. S4: The recombinant expression vector pCold-SUMO-flagellin-Linker described in S2 was linearized using NcoI / HindIII restriction endonucleases, and the Nbcap gene fragment described in S3 was cloned into the linearized vector to obtain the recombinant expression vector pCold-SUMO-Nbcap-flagellin; S5: The recombinant expression vector pCold-SUMO-Nbcap-flagellin described in S4 was transformed into Escherichia coli BL21 (DE3) competent cells to construct the recombinant engineered bacteria pCold-SUMO-Nbcap-flagellin / BL21 (DE3); S6: Induce the expression of the Nbcap-flagellin recombinant fusion protein in the recombinant engineering bacteria pCold-SUMO-Nbcap-flagellin / BL21 (DE3) described in S5; S7: Collect the bacteria induced by S6 by centrifugation, disrupt the bacteria and take the supernatant; S8: The supernatant described in S7 is purified by affinity chromatography, and the Nbcap-flagellin recombinant fusion protein is eluted.

6. The method for preparing a recombinant fusion protein based on PCV2 nanobody and bacterial flagellin according to claim 5, characterized in that: The S6 step is: A. Streak the recombinant engineered bacteria pCold-SUMO-Nbcap-flagellin / BL21(DE3) onto ampicillin-resistant LB solid medium. After cultivation, pick a single colony and inoculate it into LB liquid medium. Incubate with shaking overnight to obtain an activated seed solution. B. Transfer the activated seed solution obtained in step A to fresh LB liquid medium at a 1%-5% inoculum volume and culture with shaking until the OD600 value reaches 0.5-0.8; C. Add inducer IPTG to the culture medium to a final concentration of 0.01mM-1mM; D. Adjust culture conditions to continuously induce expression.

7. The method for preparing a recombinant fusion protein based on PCV2 nanobody and bacterial flagellin according to claim 5, characterized in that: The step S8 is as follows: A. Filter the supernatant obtained in S7 using a membrane filter and add it to a Ni-NTA purification column; B. Wash the column with 10 volumes of loading buffer to remove non-specific binding proteins, and then use 10-15 column volumes of wash buffer to further remove non-target proteins; C. Collect the target protein using elution buffer; D. The target protein obtained in step C was dialyzed against PBS buffer.

8. Use of the recombinant fusion protein based on PCV2 nanobody and bacterial flagellin according to claims 1-4 in the preparation of PCV2 subunit vaccine.

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