Application of nano antibody of anti-porcine IgM Fc receptor Fc [mu] R in preparation of PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) vaccine

Abstract

The invention belongs to the technical field of biology, and relates to application of an anti-porcine IgM Fc receptor Fc [mu] R nano antibody in preparation of a PRRSV vaccine. According to the invention, four structural proteins (M, N, GP3 and GP4) of an HP-PRRSV-JXA1 strain are used as protective antigens of the PRRSV and are subjected to fusion expression with a nano antibody FcmuR-VHH, and a PAMs adsorption test is utilized to verify that the fusion antigen has a targeting effect on PAMs. The expressed fusion protein is further used for immunizing animals twice, and then a challenge test is carried out. Results show that the antigen fused with the nano antibody FcmuR-VHH can target APCs, the presentation efficiency of the antigen is improved, and compared with a monomer structure protein, the antigen can better stimulate an animal body to generate a specific antibody. In the middle and later periods of infection, the blood toxicity removal level of a VHH fusion protein immune group is even superior to that of an attenuated vaccine group, which prompts that the removal of viruses in peripheral blood is accelerated by a high-level antibody.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to the application of nanobodies against porcine IgM Fc receptor FcμR in the preparation of PRRSV vaccines. Background Technology

[0002] Vaccination is an effective strategy for preventing the spread of infectious diseases. Current vaccines mostly consist of pathogen-derived antigens, such as purified proteins or inactivated pathogens. These antigens stimulate the body to produce antigen-specific memory B and T cells, providing long-term protection for the vaccinated individual. Vaccination has significantly reduced or eliminated the spread of serious diseases worldwide. However, many diseases remain unpreventable by vaccines, necessitating more advanced vaccine preparation strategies. In vivo targeted delivery of antigens to antigen-presenting cells (APCs) is a method to enhance the immunogenicity of genetically engineered vaccines, and its research progress has attracted considerable attention. The effectiveness of antigen-targeted APCs depends largely on the chosen target receptor, in addition to the properties of the immunogen itself. The choice of receptor directly affects the response triggered after antigen uptake. Targeting methods against different receptors of APCs have been explored in various animals, most commonly pigs, cattle, sheep, and poultry.

[0003] IgM antibodies are the first type of antibody produced by the body during infection. Unlike other antibody types, IgM antibodies have a pentamer structure, thus possessing unique biological activities. IgM antibodies can bind to complement C1 to activate the complement response via the classical pathway, stimulating a local innate immune response. Simultaneously, IgM antibodies can bind to Fc receptors on the surface of antigen-presenting cells (APCs) through their constant region (Fc), promoting the antigen-presenting capacity of APCs and initiating adaptive immune responses (humoral and cellular immune responses). FcμR is an IgM-specific Fc receptor, also known as the IgM Fc fragment receptor (FCMR) or TOSO / Fas apoptosis inhibitor molecule 3 (FAIM3). The extracellular N-terminus of FcμR contains a type V immunoglobulin-like domain similar to pIgR and Fcα / μR, which may be the binding site for the IgM Fc fragment. The cytoplasmic region of FcμR contains multiple conserved serine and tyrosine residues. Crosslinking of FcμR with IgM immune complexes leads to phosphorylation of these serine and tyrosine residues, serving as phosphorylation sites that initiate or mediate signaling cascades, suggesting that FcμR can transmit extracellular information into the cell. In humans and mice, FcμR is mainly expressed by B cells, T cells, and NK cells, and is considered a truly IgM-specific receptor. FcμR has a high affinity (~10 nM) for the Fc region of multimeric IgM and is widely expressed on macrophages, dendritic cells, and lymphocytes, mediating the phagocytosis of IgM-antigen immune complexes by target cells and the biological effects of IgM.

[0004] Previous research by the inventors has shown that immunizing piglets with an inactivated PRRSV vaccine after incubating it with its specific murine IgM antibody to form an immune complex significantly improves the protective efficacy of the inactivated PRRSV vaccine, reduces lung damage caused by PRRSV challenge, and significantly increases the level of IFN-γ in peripheral blood serum. This suggests that IgM may act as a special adjuvant, forming an immune complex with the antigen and then binding to the IgM Fc receptor FcμR, thereby enhancing the body's immune response to the antigen. However, due to the unique structure of the IgM polymer, it is difficult to use it for vaccine preparation through recombinant expression. Therefore, how to enhance the targeting of vaccines is an important issue currently facing vaccine research and production. Summary of the Invention

[0005] This invention provides the application of anti-pig IgM Fc receptor FcμR nanobodies in the preparation of PRRSV vaccines. The aim is to use anti-pig FcμR molecule nanobodies as ligands targeting FcμR molecules to target antigens to APCs, thereby improving the efficiency of antigen presentation and enhancing the body's immune response.

[0006] To solve the above technical problems, the present invention adopts the following technical means: This invention seeks protection for the use of a nanobody that specifically binds to the porcine IgM Fc receptor FcμR in the preparation of a porcine vaccine. The amino acid sequence of the nanobody is shown in SEQ ID NO:1. The nanobody is fused with an antigen protein to prepare a fusion protein, which is then used in the preparation of the vaccine.

[0007] A fusion protein, the fusion protein comprising: (1) Nanobody, wherein the nanobody can specifically bind to porcine IgM Fc receptor FcμR, and its amino acid sequence is shown in SEQ ID NO:1; (2) Antigen protein, said antigen protein being derived from bacteria or viruses capable of infecting pigs; And / or (3) Linker, i.e., linker peptide.

[0008] Preferably, the nanobody is linked to the C-terminus or N-terminus of the antigen protein via a linker.

[0009] Preferably, the antigen protein is the PRRSV M protein, N protein, GP3 protein and / or GP4 protein.

[0010] Preferably, the amino acid sequence of the fusion protein is as shown in SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18 or SEQ ID NO:20.

[0011] The polynucleotide encoding the fusion protein has the sequence shown in SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19 or SEQ ID NO:21.

[0012] An expression vector comprising the above-described polynucleotide sequence encoding the fusion protein.

[0013] A vaccine for porcine reproductive and respiratory syndrome, wherein the vaccine comprises a fusion protein with the amino acid sequences shown in SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18 and / or SEQ ID NO:20.

[0014] Preferably, the antigen protein of the vaccine is composed of the fusion proteins shown in SEQ ID NO:14, SEQ ID NO:16 and SEQ ID NO:20.

[0015] Based on the above technical solutions, the present invention has the following advantages and beneficial effects: This invention, based on the cloning and sequencing of the full-length cDNA of porcine FcμR, expresses its extracellular region in prokaryotes for camel immunization. A phage nanobody library was successfully constructed by extracting total RNA from peripheral blood lymphocytes of immunized camels and reversing it into cDNA. Using a biopanning technique, after three rounds of enrichment, a highly sequence-identical nanobody targeting the extracellular region of porcine FcμR was obtained from 96 ELISA-positive colonies, and its sequence was obtained. Specific nanobodies targeting the porcine FcμR molecule were then screened, achieving antigen targeting of the porcine FcμR molecule through ligand-receptor interactions.

[0016] Anti-porcine FcμR nanobody (FcμR-VHH) was expressed using an E. coli prokaryotic expression system, and its targeting ability for PAMs was verified. Using green fluorescent protein zsGreen as a model antigen, FcμR-VHH was fused to the C-terminus and N-terminus of zsGreen, respectively. Adsorption assays were performed on different cells, and different detection methods were used to determine that FcμR-VHH fused to different positions of the antigen could target the antigen. In vitro and in vivo tests verified that the targeting effect was better when FcμR-VHH was fused to the C-terminus of the antigen. The results also showed that targeting APCs with antigen by fusing FcμR-VHH can simultaneously improve antigen-specific humoral immune response and cellular immune response.

[0017] This invention uses four structural proteins (M, N, GP3, GP4) of the HP-PRRSV-JXA1 strain as protective antigens of PRRSV, fused with the nanobody FcμR-VHH for expression. The PAMs adsorption assay was used to verify that the fusion antigen targets PAMs. Animals were then immunized twice with the expressed fusion protein, followed by a challenge experiment. Results showed that, compared to the other monomeric structural protein immunization groups, the VHH fusion protein immunization group rapidly reached higher levels of specific antibodies in the early stage of immunization (first immunization), and these levels remained stable at a high level after challenge. This demonstrates that the antigen fused with the FcμR-VHH nanobody can target APCs, improve antigen presentation efficiency, and better stimulate the production of specific antibodies in animals compared to monomeric structural proteins. In the mid-to-late stages of infection, the blood toxin clearance level in the VHH fusion protein immunization group was even better than that in the attenuated vaccine group, suggesting that high antibody levels accelerated the clearance of the virus from peripheral blood. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 SDS-PAGE and Western blot identification of porcine FcμR recombinant protein induced expression and purification results: (a) SDS-PAGE identification of purification conditions of recombinant FcμR protein, M: protein marker; 1: before purification; 2: effluent; 3: 5 mM imidazole elution; 4: 10 mM imidazole elution; 5: 100 mM imidazole elution; (b) SDS-PAGE identification of recombinant FcμR protein after purification and dialysis.

[0020] Figure 2 Indirect ELISA was used to detect the antibody titer against porcine FcμR protein in camel serum.

[0021] Figure 3 Amplification of the VHH gene and identification of the library: (a) (b) Nested PCR amplification of the VHH gene; (c) PCR identification of the positive rate of the library.

[0022] Figure 4 : Indirect ELISA method to detect the reactivity of recombinant nanobodies with porcine FcμR protein.

[0023] Figure 5SDS-PAGE and Western blot identification of recombinant FcμR-VHH prokaryotic expression and purification results: (a) SDS-PAGE identification of purification conditions of recombinant FcμR-VHH protein, M: protein marker; 1: before purification; 2: effluent; 3: 5 mM imidazole elution; 4: 10 mM imidazole elution; 5: 100 mM imidazole elution; 6: 250 mM imidazole elution; (b) SDS-PAGE identification of recombinant FcμR-VHH protein after purification and dialysis; (c) Western blot identification of recombinant FcμR-VHH protein after purification and dialysis, M: protein marker; 1: recombinant FcμR-VHH protein.

[0024] Figure 6 Western blot detection of FcμR-VHH targeting: M: Marker; 1: MOCK; 2: F165R; 3: I215L; 4: HERC5; 5: FcμR-VHH.

[0025] Figure 7 Amplification and PCR identification of the zsGreen and VHH fusion fragments and schematic diagram of fusion positions: (a) Amplification of the zsGreen and VHH fusion fragments; (b) PCR identification of zsGreen and VHH, where M: nucleic acid marker; 1: zsGreen-VHH; 2: VHH-zsGreen; (c) Schematic diagram of the fusion of FcμR-VHH with zsGreen at different positions.

[0026] Figure 8 Results of prokaryotic expression and purification of recombinant FcμR-VHH zsGreen fusion protein identified by SDS-PAGE and Western blot: (a) and (c) SDS-PAGE and Western blot identification of recombinant zsGreen-VHH fusion protein induced expression; (b) and (d) SDS-PAGE and Western blot identification of recombinant VHH-zsGreen protein induced expression. Wherein M: protein marker; 1, 3, 5, 7: supernatant of recombinant bacterial product induced by IPTG concentrations of 0.1 mM / L, 0.2 mM / L, 0.5 mM / L, and 1.0 mM / L; 2, 4, 6, 8: precipitate of recombinant bacterial product induced by IPTG concentrations of 0.1 mM / L, 0.2 mM / L, 0.5 mM / L, and 1.0 mM / L.

[0027] Figure 9 SDS-PAGE analysis of purified protein: M: Marker; 1: zsGreen-VHH; 2: VHH-zsGreen.

[0028] Figure 10 Results of zsGreen and VHH fusion protein adsorption on APCs: (a) Fluorescence results (PAMs) (10×10); (b) Flow cytometry results (BM-DCs).

[0029] Figure 11 Detection of zsGreen after lysosome extraction following adsorption of PAMs by fusion protein.

[0030] Figure 12 Indirect ELISA detection of zsGreen antibody levels in serum after immunization: (a) Detection of zsGreen antibody levels in serum of piglets in each immunization group 7 days after immunization; (b) Detection of zsGreen antibody titers in serum of piglets in each immunization group 7 days after immunization. "*" indicates differences. P <0.05).

[0031] Figure 13 ELISpot assay was used to detect IFN-γ secretion levels in piglets' PBMCs 14 days after immunization. * indicates a difference. P <0.05).

[0032] Figure 14 Optimization of IPTG induction concentration for VHH and PRRSV antigen recombinant fusion proteins: (a) M-VHH protein; (b) N-VHH protein; (c) VHH-GP3; (d) VHH-GP4. Wherein M: protein marker; 1, 3: supernatant of recombinant bacterial products induced at 16℃ with IPTG concentrations of 0.5 mM / L and 1.0 mM / L; 2, 4: precipitate of recombinant bacterial products induced at 16℃ with IPTG concentrations of 0.5 mM / L and 1.0 mM / L; 5, 7: supernatant of recombinant bacterial products induced at 37℃ with IPTG concentrations of 0.5 mM / L and 1.0 mM / L; 6, 8: precipitate of recombinant bacterial products induced at 37℃ with IPTG concentrations of 0.5 mM / L and 1.0 mM / L.

[0033] Figure 15 SDS-PAGE identification of VHH recombinant fusion protein purification results: Lane 1: M-VHH protein purification; Lane 2: N-VHH protein purification; Lane 3: VHH-GP3 protein purification; Lane 4: VHH-GP4 protein purification.

[0034] Figure 16 Western blot was used to detect the targeting of the FcμR-VHH fusion protein with the PRRSV antigen.

[0035] Figure 17 The level of structural protein-specific antibodies in the serum of each experimental group after immunization.

[0036] Figure 18 Serum viral load: "*" indicates a difference ( P <0.05); "**" indicates a significant difference ( P <0.01); "***" indicates a highly significant difference ( P <0.001). Detailed Implementation

[0037] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, the embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that equivalent substitutions, combinations, improvements or modifications can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all of these fall within the protection scope of the present invention.

[0038] Example 1: Expression and purification of porcine FcμR protein 1.1 Transformation of porcine pET-28a-FcμR recombinant expression plasmid Based on previous research, the porcine FcμR cDNA sequence was cloned for the first time (Genbank Accession: OM721658.1). A recombinant expression plasmid pET-28a-FcμR for the FcμR protein was constructed and transformed into... E. coli Recombinant bacteria pET-28a-FcμR were obtained from BL21(D3) competent cells. E. coli BL21 (D3), after identification, should be frozen at -80℃.

[0039] 1.2 Induction, expression and purification of porcine FcμR recombinant protein (1) Take 100 μL of pET-28a-FcμR stored at -80℃ E. coli Add BL21(D3) glycerol bacterial suspension to 10 mL of LB medium containing Kan resistance, and activate the bacteria by incubation at 37℃ and 220 r / min for 12-16 h to restore bacterial activity. (2) Take the activated bacterial solution 1:100 and inoculate it into a 500 mL Erlenmeyer flask for culture. Use IPTG concentration (0.5 mM / L) to induce the recombinant Escherichia coli that has reached the logarithmic growth phase. Culture at 37℃ for 8 h to induce large-scale expression. (3) Collect bacterial cells by centrifuging at 6000 rpm for 6 min. Add 10 mL of PBS buffer to every 200 mL of bacterial solution to resuspend the cells and then sonicate them. (4) After sonication, centrifuge at 4℃, 10000g for 10 min, collect the precipitate, resuspend it in 8M urea solution, and dissolve it at room temperature for 8~12 h. (5) Centrifuge at 12000 g for 25 min at 4℃, retain the supernatant, and filter the supernatant through a 0.45μm filter membrane; (6) Take Ni-NTA, equilibrate the column with 5 times the volume of ultrapure water, and then equilibrate the column with 5 times the volume of 8M urea solution; incubate the filtered supernatant with Ni-NTA overnight in a shaker at 4 ℃. (7) Use 5 mM, 10 mM, 50 mM, 100 mM, and 250 mM imidazole 8M urea solutions to elute proteins in a gradient manner; (8) Add the eluted recombinant FcμR protein to an 8-4 kDa dialysis bag and dialyze sequentially with 8M, 6M, 4M and 2M urea buffer at 4 °C for 2 h for each gradient; (9) SDS-PAGE analysis of the purification status of porcine FcμR recombinant protein, such as... Figure 1 As shown.

[0040] (10) After dialysis, the recombinant FcμR protein was filtered through a 0.45 μm filter membrane, and the protein concentration was determined using the Thermo Scientific BCA Protein Quantitative Kit. The protein was stored at -80℃.

[0041] The results are as follows Figure 1 As shown, a large amount of crude protein product, after induction and expression, was collected, sonicated, and dissolved in 8M urea. This product was then incubated with a Ni column, and the Ni column was eluted with 100mM imidazole urea solution. The CBB indicator turned blue, indicating that protein had eluted at this imidazole concentration. The pre-loading, eluent, washing buffer, and elution buffer were collected and subjected to SDS-PAGE for identification. The Coomassie brilliant blue staining results were observed. Figure 1 (a) The molecular weight is 35 kDa, and the target band in the eluent is single, which is as expected. After dialysis to 2 M urea, the protein purity after dialysis was determined by SDS-PAGE. Figure 1 (b) The concentration was detected by the BCA kit as 1 mg / mL.

[0042] Example 2: Construction of a porcine FcμR protein nanobody library 2.1 Immunity of Bactrian camels The purified porcine FcμR protein was mixed with adjuvant and immunized Bactrian camels 5 times. The first immunization used complete Freund's adjuvant, and the second to fifth immunizations used incomplete Freund's adjuvant. After the last immunization, wait 4 days and collect anticoagulated blood to separate camel peripheral blood lymphocytes.

[0043] Serum was collected from Bactrian camels after five immunizations, and the antibody titer against porcine FcμR protein in the camel serum was detected using an indirect ELISA method. Figure 2 The results showed that the serum titer reached 1:1048576, which can be used for the construction of phage libraries.

[0044] 2.2 Amplification of the VHH gene fragment (1) Lymphocyte RNA was extracted using the QIAGEN Neasy® Plus Mini RNA Extraction Kit and first-strand cDNA was synthesized by reverse transcription using the PrimeScript™ RT Reagent Kit.

[0045] (2) Two rounds of nested PCR: Round 1: Using the cDNA obtained from reverse mixing as a template, amplification was performed using primers CALL001 and CALL002, resulting in a band of approximately 700 bp. Round 2: Using the gel recovery product from Round 1 as a template, amplification was performed using primers VHH-FOR and VHH-REV, resulting in a band of approximately 400 bp. The primer sequences are shown in Table 1 below.

[0046] Table 1. Primer sequences

[0047] Note: The underlined and italicized sequences are restriction enzyme sites, R = A / G.

[0048] The reaction system for both rounds of PCR was as follows: 5 μL template DNA (cDNA or PCR amplification product), 2 μL each of upstream and downstream primers, 4 μL dNTPs, 5 μL 10 × HiFi Buffer II, 1 μL HiFi DNA polymerase, and dd H2O to a final volume of 50 μL.

[0049] 2.3 Double digestion of VHH fragment and pMECS vector Two rounds of enzyme digestion were performed. The second round nested PCR product and the pMECS empty vector were digested with XbaI and Pst I-HF and Pst I-HF and Not I-HF, respectively. The digestion conditions were 37 °C for 16 h. The products were then recovered by gel extraction.

[0050] 2.4 Construction of VHH phage display vector The VHH fragment was ligated to the pMECS phage display vector using T4 DNA ligase at 16°C. After 16 hours, the ligation product was purified and eluted using a PCR product recovery kit, and the concentration was determined for subsequent experiments.

[0051] 2.5 Preparation of Electrocompetent States (1) TG1 glycerol bacteria were streaked onto M9 plates and incubated at 37 ℃ for 36 h; (2) Pick a single TG1 colony and inoculate it into 10 mL of 2×YT antibiotic-free medium. Separately pick colonies and inoculate them into Kan / 2×YT medium and Amp / 2×YT medium (ensure that bacteria can proliferate only in 2×YT medium). Incubate at 37 ℃ with shaking at 200 rpm until the bacterial culture reaches OD. 600nm Up to 0.6~0.8; (3) TG1 was amplified and cultured in four 1L Erlenmeyer flasks using 2×YT medium, 250 mL / flask, at 37 ℃ with shaking at 200 rpm. The OD of the bacterial culture was measured. 600nm Once the pH reaches 0.4–0.6, immediately place it on ice for 30 minutes. (4) Centrifuge 2500g of culture medium at 4 ℃ for 15 min and discard the supernatant. Gently resuspend the bacterial cells in an equal volume of 10% pre-cooled glycerol. Continue centrifuging and discard the supernatant. Reduce the volume of 10% glycerol by half for the second centrifugation. Repeat 3 times. (5) After discarding the supernatant for the last time, gently resuspend the bacterial cells with 1.5 mL of 10% pre-cooled glycerol to obtain competent cells that can be used directly for electroconversion experiments or stored at -80 °C.

[0052] 2.6 Ligation Product Transformation and Library Capacity Identification (1) Take TG1 electroporation competent cells, add 50 µL (5 µg) of ligation product, and perform electroporation transformation using an Eppendorf electroporator; (2) After the electroporation is complete, place the conversion product on a shaker and incubate at 37°C and 120 rpm for 1 h. (3) Take 100 µl of culture medium and dilute it 10-fold with LB / AMP-GLU medium. The dilution is 10. -2 10 -3 10 -4 10 -5 100 μL of each sample was spread on LB / AMP-GLU plates and incubated at 37 °C for 8 h for library identification. (4) Spread 1.5 mL of the remaining culture evenly on 10 LB / AMP-GLU plates (135×135mm square petri dishes) and incubate at 37 ℃ for 6 h; (5) After the bacterial growth is complete, add 2 ml of LB / AMP-GLU medium to each petri dish, scrape off the bacterial growth, add 1 / 3 volume of 50% glycerol, mix well, dispense, and store at -80 ℃.

[0053] Peripheral blood lymphocytes were isolated from camel anticoagulated blood, and total RNA was extracted and reverse transcribed into cDNA. This cDNA was then amplified by two rounds of nested PCR. The first round of PCR yielded a band of approximately 700 bp. Figure 3 (a)), the second round of PCR yielded a band of approximately 400 bp. Figure 3 (b) The target band was recovered from the gel, cloned into the phage display vector pMECS, and electroporated to obtain a library with a final volume of 8.15 × 10⁻⁶. 7 The VHH phage display library was used; single clones from the library were selected and subjected to PCR to identify library diversity, with a positive rate of 88.2%. The results are as follows: Figure 3 (c).

[0054] Example 3: Preparation of M13KO7 helper phage 3.1 Preparation of M13KO7 auxiliary plaques (1) The method for preparing TG1 monoclonal colony culture medium is as described in steps (1) and (2) of 2.5 in Example 2.

[0055] (2) Serially dilute the phage stock solution purchased from NEB: 10 -1 10 -2 10 -3 , ... 10 -9 Take 10 respectively -3 10 -5 10 -7 10 -8 10 -9 Add 100 μL of M13 dilution at five different dilutions to 5 mL test tubes, add 100 μL of TG1 from the logarithmic phase in step (1) to each tube, and infect at 37 °C for 15 min. (3) After the top agar is prepared, it is temporarily stored in a 55℃ water bath to prevent solidification. In step (2), 3 mL of top agar is added to each test tube along the wall. The mixture is quickly and gently mixed and spread on an antibiotic-free LB plate. After standing for 5 min, the top agar is placed in a 37℃ incubator and incubated for 8-14 h.

[0056] 3.2 M13KO7 helper phage amplification (1) Prepare TG1 bacterial culture in the logarithmic growth phase for later use; (2) Using the large end of a sterilized yellow nozzle, pick up a single intact plaque from the 3.1 (3) plate and place it in 1 mL of 2×YT medium, and incubate at 4 ℃ for 1 h; (3) Inoculate the phage suspension into 4 mL of TG1 in the logarithmic growth phase, infect at 37°C for 15-20 min, and then place in a shaker at 37°C and shake at 200 rpm for 2 h. (4) Transfer the culture to four 1 L Erlenmeyer flasks, add 500 mL of 2×YT medium to each Erlenmeyer flask, and incubate at 37℃ and 200 rpm for 1 h with shaking. (5) Add Kan antibiotics to the conical flask at a ratio of 1:1000 and continue culturing for 16 h.

[0057] 3.3 Phage sedimentation (1) After phage amplification culture, take the supernatant and add 1 / 5 volume of PEG / NaCl to settle on ice overnight; (2) Centrifuge at 5000 g for 30 min at 4℃, collect the precipitate, and rinse with 3 mL PBS (K) + Resuspend and incubate overnight at 4 ℃; (3) Centrifuge at 12000 g for 15 min at 4℃ and collect the supernatant. The supernatant is white and oily.

[0058] 3.4 Titration Dilute the phage according to the steps in 3.1, incubate on top agar plates, count the phage plaques, calculate the titer of the prepared M13KO7 helper phage, and store at 4 ℃ for later use.

[0059] Example 4: Screening of anti-pig FcμR protein nanobodies 4.1 Rescue and titer determination of VHH phage library (1) Take 1 mL of the phage display library constructed in step 2.6 (5) of Example 2, and amplify and culture it at 37℃ and 200 rpm for 2-3 h until OD. 600 nm is 0.6~0.8; (2) Inoculate with the helper phage amplified in Example 3 (greater than 20 MOI), mix well, and let stand at 25 ℃ for 30 min; (3) Collect the bacterial cells and resuspend them. Amplify and culture them in 500 mL of 2×YT / AMP-KAN medium at 37℃ and 200 rpm for 13~16 h. (4) Collect the culture supernatant, add 1 / 5 volume of pre-cooled PEG / NaCl, mix well, and let it stand on ice for more than 12 h to settle. (5) Collect the sedimentation products, PBS (K) + The phage was resuspended in an EP tube, incubated overnight at 4°C, and the supernatant was collected by centrifugation to obtain a concentrated phage library product. (6) Take 10 μL of product, dilute it 10 times in 9 gradients, infect TG1 in EP tubes at 25℃ for 20 min, take 100 μL of each and spread it on LB / AMP-GLU plates, and incubate at 37℃ for 10 h. (7) Count the colonies and determine the library titer.

[0060] 4.2 Screening of anti-pig FcμR protein-specific nanobodies (1) Coat 50 μg / tube of the screening agent (the porcine FcμR recombinant protein prepared in Example 1) in an immunoassay tube, incubate overnight at 4 °C, and then incubate with PBS (K) + () serves as a negative control; (2) Add 1 mL of 5% skim milk powder to the tube, and block it in a shaker at 37 ℃ for 2 h. After blocking, wash with PBS'T 3 times, 5 min each time. (3) Add 100 μL to the tube to achieve a final concentration of 5 × 10⁻⁶. 11 A rescued phage library at pfu / mL, PBS (K + Add to a final volume of 800 μL and incubate at 25°C for 2 hours. (4) First wash the tube with PBS'T 15 times, 2 min each time, then wash with PBS (K + Rinse 15 times; (5) Add 0.1M triethylamine to 1 mL of the tube, incubate at room temperature for 15 min to elute the bound recombinant phage, aspirate the triethylamine mixture, and add it to a new EP tube containing an equal volume of 1M Tris-HCl (pH=7.4) to neutralize; (6) Determine the titer of the elution product and repeat the rescue, enrichment and screening process twice.

[0061] 4.3 Enrichment of specific recombinant phages by phage ELISA detection Detection of recombinant phages using ELISA: (1) The purified porcine FcμR recombinant protein was coated onto ELISA plates (400 ng / well), coated at 4 ℃ for 12-14 h and then blocked; (2) The primary antibody is the original phage library and the phage solution amplified and concentrated after each round of enrichment. It is diluted 1:1 with blocking buffer, 100 μL / well, and incubated at room temperature for 2 h. (3) The secondary antibody was M13 mouse anti-HRP antibody, diluted to 100 μL / well, and incubated at room temperature for 2 h; (4) After incubation, TMB color development and termination are performed, and the OD450nm value is read.

[0062] 4.4 Preparation and Identification of Crude Nanobody Substances from Screening (1) 96 single colonies were randomly picked from the plates of the third round of titer determination and labeled as 1#~96#. They were inoculated into 96-well plates and cultured at 37°C and 200 rpm for 8 hours. (2) The culture was amplified in TB medium in 24-well plates and cultured at 37 °C and 200 r / min until the bacteria were in the logarithmic growth phase; (3) Add TB medium containing 10mM IPTG, 100 μL / well, and incubate overnight at 37 ℃ and 200 rpm; (4) Collect the culture medium from each well into a 1.5 mL EP tube, label them in order, centrifuge at 3200 g / min for 10 min at 4 ℃, discard the supernatant, and freeze and thaw in a -20 ℃ freezer; (5) Add 500 μL of PBS (K) to each EP tube. + The bacterial cells were resuspended by shaking at room temperature for 1 hour, and the supernatant was collected by centrifugation to obtain a crude extract of soluble recombinant nanobodies. (6) Specificity of crude extract was detected by ELISA: the coated protein was purified porcine FcμR recombinant protein, and PBS (K) was coated without antigen control. + Add 100 μL of crude extract containing recombinant nanobodies to each well and incubate at 37°C for 1 hour. Dilute the primary antibody (HA antibody) 1:5000 with 5% skim milk powder, 100 μL / well. Dilute the secondary antibody (Goat Anti-Mouse HRP antibody) 1:5000 with 5% skim milk powder, 100 μL / well. Perform TMB color development and read the OD values ​​after termination. 450 nm value.

[0063] Purified FcμR extracellular protein was used as the coating antigen, and specific nanobodies were screened through three rounds of panning. The results are shown in Table 2. The recovery rate of specific phages increased with each round of screening, reaching 476 after the third round. Simultaneously, the phage ELISA OD... 450 The nm value also increased in turn, indicating that the specific recombinant phage was significantly enriched.

[0064] Table 2. Enrichment of porcine FcμR protein-specific nanobodies

[0065] Ninety-six soluble recombinant nanobody crude extracts were prepared by amplification and IPTG induction. The reactivity with porcine FcμR recombinant protein was identified by ELISA, and the results are as follows: Figure 4 As shown, all 96 selected clones were positive clones (OD). 450nm A value more than 3 times greater than the PBS control is considered positive.

[0066] 4.5 Sequencing analysis of anti-pig FcμR protein-specific nanobodies (1) Streak the plates of ELISA-positive clones from section 4.3 and incubate overnight at 37 °C; (2) Single colonies were picked and amplified in 1.5 mL EP tubes with LB aspirin for 2 h. The bacterial culture was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results showed that the sequences of 96 positive colonies were highly consistent, with the amino acid sequences as shown in SEQ ID NO:1 and the nucleotide sequences as shown in SEQ ID NO:2. Thus, the anti-porcine FcμR protein specific nanobody sequence was obtained.

[0067] Example 5: Recombinant expression of anti-pig FcμR protein-specific nanobodies 5.1 Construction of FcμR-VHH recombinant expression vector and preparation of recombinant bacteria (1) Primers were designed using SnapGene gene editing software based on the FcμR-VHH sequence obtained from Beijing Qingke Biotechnology Co., Ltd., as shown in Table 3 below: Table 3 Primer sequences for amplifying the FcμR-VHH gene

[0068] Note: The sequences marked with underlined italics are restriction enzyme sites.

[0069] (2) The FcμR-VHH gene was amplified by PCR using Takara GXL enzyme, and its nucleotide sequence is shown in SEQ ID NO:2.

[0070] (3) Selection BamHI and XhoI Enzyme cleavage site, NEB BamHI and XhoI The pET21b vector and the FcμR-VHH fragment were digested with restriction endonucleases. The pET-21b vector was ligated to the target fragment using GenStar T4 ligase to obtain the porcine pET-21b-FcμR-VHH recombinant expression plasmid.

[0071] (4) Transform the pET-21b-FcμR-VHH recombinant expression plasmid E. coli Recombinant bacteria pET-21b-FcμR-VHH were prepared from BL21(D3) competent cells. E. coli BL21 (D3).

[0072] 5.2 Prokaryotic expression and purification of the anti-porcine FcμR protein-specific nanobody FcμR-VHH (1) Take 100 μL of pET-21b-FcμR-VHH stored at -80℃ E. coli Add BL21(D3) glycerol bacterial suspension to 10 mL of LB medium containing Amp resistance, and activate the culture at 37°C and 220 rpm for 12-16 h to restore bacterial activity; (2) Take the activated bacterial solution and inoculate it at a ratio of 1:100 into LB medium containing Amp resistance. Use IPTG concentration (0.5 mM / L) to induce the recombinant Escherichia coli that has reached the logarithmic growth phase and incubate at 37℃ for 8 h. (3) Collect bacterial cells by centrifuging at 6000 rpm for 6 min. Add 10 mL of PBS buffer to each 200 mL of bacterial solution to resuspend the bacterial cells and then sonicate them. The sonication power is 30W, the working time is 3s, the interval is 3s, and the sonication time is 45 min. (4) After sonication, centrifuge at 10000 g for 10 min at 4℃, collect the precipitate, resuspend it in 8M urea solution, and dissolve it at room temperature for 8~12 h. (5) After dissolving, centrifuge at 4℃, 12000 g for 25 min, retain the supernatant, and filter the supernatant through a 0.45μm filter membrane; (6) Take Ni-NTA, equilibrate the column with 5 times the volume of ultrapure water, and then equilibrate the column with 5 times the volume of 8M urea solution; incubate the filtered supernatant with Ni-NTA overnight in a shaker at 4°C. (7) Use 5mM, 10mM, 50mM, 100mM, and 250mM imidazole 8M urea solutions to elute proteins in a gradient manner; (8) Add the eluted recombinant FcμR protein to an 8~14 kDa dialysis bag and dialyze sequentially with 8M, 6M, 4M, 2M, 1M urea buffer and 1M arginine buffer at 4°C for 2 hours for each gradient. (9) SDS-PAGE analysis of the purification status of porcine FcμR-VHH recombinant protein, such as... Figure 5 As shown.

[0073] (10) After dialysis, the FcμR-VHH protein was filtered through a 0.45μm filter membrane, and the protein concentration was determined using the Thermo Scientific BCA Protein Quantitative Kit. The protein was stored at -80℃.

[0074] A large quantity of crude protein product, obtained after induced expression, was collected, sonicated, and dissolved in 8M urea. This product was then incubated with a Ni column. The Ni column was eluted with 50 mM, 100 mM, and 250 mM imidazole urea solutions. The CBB indicator turned blue in all three solutions, indicating that protein was eluted at these three imidazole concentrations. The pre-loading solution, eluent, washing buffer, and elution buffer were collected and subjected to SDS-PAGE for identification. The Coomassie brilliant blue staining results were observed. Figure 5 (a) The molecular weight is 15 kDa, and the target band in the eluent is single, as expected. After dialyzing to 1M arginine PBS buffer, the protein purity was identified by SDS-PAGE and Western blot. Figure 5 (b) and 5(c), the concentration was detected as 0.8 mg / mL using the BCA kit.

[0075] Example 6. Targeting test of porcine FcμR nanobody FcμR-VHH 6.1 Detection of the targeting effect of FcμR-VHH on PAMs cells (1) Resuscitate PAMs in 6-well plates and incubate them in 10% 1640 medium at 37°C for 3 hours to allow the cells to recover and settle to the bottom of the plate; (2) Replace 10% 1640 with PBS (K+ ) buffer, add 5 μg FcμR-VHH or control protein to each well; (3) Place the cell culture plate in a 37 ℃ incubator and incubate for 30 min; (4) Discard the supernatant in the well and rinse with ice-cold PBS (K). + Resuspend the cells and collect them in EP tubes. Centrifuge at 1600 rpm for 6 min. (5) Repeat step (4) twice; (6) Collect samples with 2×sample buffer and perform Western blot detection.

[0076] If the selected FcμR nanobodies exhibit APC targeting, they can target FcμR molecules on the surface of PAMs and BM-DCs, and nanobodies adsorption can be detected on PAMs and BM-DCs. Several unrelated proteins (F165R, I215L, and HERC5) carrying His tags and also using pET 21b as a carrier, stored in our laboratory, were selected as controls. FcμR-VHH targeting was tested on PAMs (PAMs are more readily available in our laboratory than BM-DCs). Western blot results were obtained. Figure 6 The results show that the FcμR-VHH nanobody can target and adsorb PAMs.

[0077] Example 7. Targeting test of FcμR-VHH nanobody and zsGreen fusion protein 7.1 Prokaryotic expression and purification of FcμR-VHH nanobody and zsGreen fusion protein Using green fluorescent protein zsGreen as a model antigen, FcμR-VHH was fused to the C-terminus and N-terminus of the protein, respectively, and the recombinant protein was used to adsorb PAMs. Since zsGreen has green fluorescence, the targeting of the fusion protein was preliminarily determined by observing whether the cells had green fluorescence under a fluorescence microscope and by qualitative analysis of fluorescence intensity under the same conditions and quantitative analysis of fluorescence intensity by flow cytometry.

[0078] 7.1.1 Construction of pET-21b-VHH-zsGreen and pET-21b-zsGreen-VHH prokaryotic expression plasmids FcμR-VHH was fused to the C-terminus and N-terminus of zsGreen using overlap extension PCR to construct pET-21b-VHH-zsGreen and pET-21b-zsGreen-VHH recombinant fusion plasmids. Specific identification results are as follows: Figure 7As shown, the amino acid sequence of ZSGREEN-VHH is SEQ ID NO:10, and the nucleotide sequence is SEQ ID NO:11; the amino acid sequence of VHH-ZSGREEN is SEQ ID NO:12, and the nucleotide sequence is SEQ ID NO:13.

[0079] 7.1.2 Induction and purification of VHH-zsGreen and zsGreen-VHH recombinant proteins (1) Plasmid transformation and induced expression: The constructed plasmids are transformed. E. coli Recombinant bacteria were prepared from BL21(D3) competent cells and then induced to express their contents. Recombinant *E. coli* bacteria that had reached the logarithmic growth phase were induced with different IPTG concentrations (0.1 mM / L, 0.2 mM / L, 0.5 mM / L, and 1 mM / L), cultured at 37°C for 8 h, and then detected by SDS-PAGE and Western blot. Results ( Figure 8 The results showed that the target band appeared at around 50 kDa, which was in line with expectations. The expression level of zsGreen-VHH was the highest when the IPTG concentration was 0.2 mM / L, and the expression level of VHH-zsGreen was the highest when the IPTG concentration was 0.5 mM / L. Both protein expression bands appeared in the sonication precipitate, expressed in the form of inclusion bodies, and the bands were relatively simple. The inclusion body washing method can be used for purification.

[0080] (2) Inclusion body purification: When exploring the induction expression conditions, it was found that inclusion body bands could be purified by washing inclusion bodies. Therefore, 5 mL of inclusion body washing solution was used to resuspend 200 mL of bacteria, vortexed for 5 min, centrifuged at 8000 g for 15 min, and the supernatant was discarded. This step was repeated 5 times. Then, the precipitate was resuspended with 8M urea, dissolved overnight, and 40 μL was taken for SDS-PAGE gel chromatography to identify the purification results.

[0081] Both proteins were expressed as inclusion bodies with relatively simple bands; therefore, the inclusion body washing method was used for purification. The results of gradient renaturation after purification are shown below. Figure 9 The final protein buffer was PBS solution, and the concentration was detected using a BCA kit to be 0.5 mg / mL.

[0082] 7.2 Targeting effect test at the fusion site of FcμR-VHH and zsGreen 7.2.1 Detecting the targeting effect of FcμR-VHH at different fusion sites on PAMs (1) Resuscitate PAMs in 6-well plates, culture in 10% 1640 medium at 37 ℃ for 3 h to allow the cells to recover and settle to the bottom of the plate; (2) Replace 10% 1640 with PBS (K + ) buffer, add 5 μg zsGreen-VHH, VHH-zsGreen and control protein (zsGreen) to each well respectively; (3) Place the cell culture plate in a 37 ℃ incubator and incubate for 30 min; (4) Discard the supernatant in the well, treat the cells with proteinase K for 30 seconds, and wash away unbound proteins; (5) Use PBS (K) + Gently rinse the cells three times, place the cell plate under a fluorescence microscope, and observe the fluorescence intensity under the same exposure conditions.

[0083] 7.2.2 Detecting the targeting effect of FcμR-VHH at different fusion sites on BM-DCs (1) The induced BM-DCs were dispensed into 1.5 mL EP tubes and cultured in 10% 1640 medium at 37 °C for 1 h to allow the cells to recover their state; (2) Centrifuge at 1600 rpm for 6 min at 4℃ and discard the culture medium; (3) Replace 10% 1640 with PBS (K + ) buffer, add 5 μg zsGreen-VHH, VHH-zsGreen, control protein zsGreen and an equal volume of PBS (K) to each well. + ), ultimately resulting in a total volume of 150 μL / tube; (4) Place the cell culture plate at 4 ℃ to allow the protein to adsorb for 1 hour. During this period, gently tap the tube wall every 15 minutes to prevent cell sedimentation. (5) Centrifuge at 4℃ and 1600 rpm for 6 min, and discard the supernatant; (6) Add 1 mL of PBS (K) + Wash cells with buffer, centrifuge at 1600 rpm for 6 min at 4°C, and discard the supernatant; repeat 3 times to wash away unadsorbed proteins. (7) After the last centrifugation, the cells were resuspended in Facs buffer and the targeting effect of different fusion sites of FcμR-VHH was detected by flow cytometry.

[0084] result Figure 10The results showed that the fusion proteins obtained by fusing the screened nanobodies FcμR-VHH with zsGreen at the N-terminus or C-terminus could specifically target PAM and BM-DCs cells, producing fluorescence on the cell surface. However, the fluorescence intensity of the zsGreen-VHH fusion protein on PAMs was observed to be greater than that of the VHH-zsGreen fusion protein, indicating that the zsGreen-VHH fusion protein (i.e., FcμR-VHH nanobodies fused to the C-terminus of zsGreen) had a stronger ability to target FcμR molecules on PAMs than the VHH-zsGreen fusion protein (i.e., FcμR-VHH nanobodies fused to the N-terminus of zsGreen). Quantitative detection by flow cytometry on BM-DCs also demonstrated that the FcμR-VHH nanobodies fused to the C-terminus of zsGreen exhibited better targeting and adsorption effects.

[0085] Example 8. Internalization ability of PAMs on FcμR-VHH fused zsGreen (1) Resuscitate PAMs in 6-well plates and incubate them in 10% 1640 medium at 37 ℃ for 3 h to allow the cells to recover and settle to the bottom of the plate; (2) Replace 10% 1640 with PBS (K + ) buffer, add 5 μg zsGreen-VHH, VHH-zsGreen and control protein (zsGreen) to each well respectively; (3) Place the cell culture plate in a 37 ℃ incubator and incubate for 30 min; (4) Discard the supernatant in the well, treat the cells with proteinase K for 30 seconds, and wash away the internalized proteins; (5) Gently wash the cells 3 times with PBS and centrifuge at 1500 rpm for 5 min; (6) Extract lysosomes and endosomes, and operate on ice throughout the process; resuspend the cell pellet from (5) in 1 mL of pre-cooled reactionation buffer (pre-added with 1000×cocktail), and mix by pipetting; add the cell suspension to a cell grinder and grind on ice for 25 min to ensure that more than 90% of the cells are ground and broken; centrifuge at 8000 rpm for 12 min at 4 ℃ to remove unbroken cells and broken cell fragments; aspirate the supernatant, centrifuge again in a new EP tube, collect the supernatant again, balance it, and centrifuge at 100,000 g for 1 h. The resulting pellet is the endosome and lysosome; lyse the sample with 2×Sample buffer and perform Western blot identification.

[0086] PAMs were selected for endosome / lysosomal extraction. After the fusion protein was adsorbed and internalized, lysosomes were extracted from the PAMs. The difference in zsGreen protein content in the extracted lysosomes was detected by Western blot using zsGreen monoclonal antibody, with Rab7 as a lysosomal marker. The results showed ( Figure 11 After adsorbing PAMs, both zsGreen-VHH and VHH-zsGreen fusion proteins showed detectable zsGreen protein in their lysosomes, with the zsGreen-VHH fusion protein containing more zsGreen protein in the lysosomes of PAMs. This indicates that zsGreen-VHH and VHH-zsGreen fusion proteins can specifically target FcμR molecules on the surface of PAMs.

[0087] Example 9. In vivo immunization of zsGreen and VHH fusion protein in animals Nine 5-6 week old Landrace piglets were selected as immunization animals and divided into three groups of three piglets each. These three groups were named: zsGreen immunization group, zsGreen-VHH fusion protein immunization group, and VHH-zsGreen fusion protein immunization group. zsGreen monomeric protein, zsGreen-VHH fusion protein, and VHH-zsGreen fusion protein were mixed with 206 adjuvant at a 1:1 volume ratio and thoroughly emulsified in an emulsifier. Each piglet was immunized according to its assigned group, with 30 μg of protein administered. Serum was collected at 0, 3, 7, 10, and 14 days after immunization, and the level and titer of zsGreen antibodies in the serum were detected by ELISA. Anticoagulated blood was collected 14 days after immunization, and peripheral blood mononuclear cells (PBMCs) were isolated from the piglets. The activation level of T lymphocytes in the immunized piglets was detected using the Mabtech IFN-γ ELISpot assay kit.

[0088] result Figure 12 (a) shows that on day 7, the ELISA OD of the two zsGreen-VHH and VHH-zsGreen fusion protein immunization groups was... 450 The nm values ​​were all higher than those of the zsGreen monomer protein immunization group, and the ELISAOD values ​​of the zsGreen-VHH fusion protein immunization group were also higher. 450 The nm value was significantly higher than that of the zsGreen monomer protein immunization group (P<0.05). Serum was serially diluted, and the serum zsGreen antibody titer of each immunization group was measured. The results are as follows: Figure 12(b) Seven days after immunization, the serum titer of zsGreen antibody in the zsGreen-VHH fusion protein immunization group was significantly higher than that in the zsGreen monomer protein immunization group (P<0.05). This demonstrates that the FcμR-VHH nanobody can target antigens to APCs to enhance humoral immune responses, and that the immune enhancement effect is better when the FcμR-VHH nanobody is fused to the C-terminus of the antigen.

[0089] Fourteen days after immunization, anticoagulated blood was collected from piglets, and PBMCs were isolated for porcine IFN-γ ELISpot detection. ConA-stimulated cells were used as a positive control. Representative ELISpot results are shown below. Figure 13 (a) Results of the Spot Number Difference Analysis Figure 13 (b) After stimulation with zsGreen, the number of IFN-γ cells produced by piglet PBMCs in the zsGreen-VHH and VHH-zsGreen fusion protein immunization groups tended to be higher than that in the zsGreen monomer protein immunization group, and the zsGreen-VHH fusion protein immunization group produced significantly more IFN-γ cells than the zsGreen monomer group (P<0.05). This further demonstrates that the FcμR-VHH nanobody can target antigens to APCs, and that fusing the FcμR-VHH nanobody to the C-terminus of the antigen can enhance the Th1-type cellular immune response during the immunization process, resulting in a higher level of cellular immune response after immunization.

[0090] Example 10. Expression and purification of FcμR-VHH and PRRSV fusion antigen Using overlap extension PCR, FcμR-VHH was fused with PRRSV M, N, GP3, and GP4, and cloned into the pET21b prokaryotic expression vector to construct four fusion expression plasmids, named M-VHH (amino acid sequence SEQ ID NO:20, nucleotide sequence encoding the amino acid SEQ ID NO:21), N-VHH (amino acid sequence SEQ ID NO:22, nucleotide sequence encoding the amino acid SEQ ID NO:23), VHH-GP3 (amino acid sequence SEQ ID NO:24, nucleotide sequence encoding the amino acid SEQ ID NO:25), and VHH-GP4 (amino acid sequence SEQ ID NO:26, nucleotide sequence encoding the amino acid SEQ ID NO:27). These plasmids were then transformed into... E. coliRecombinant bacteria were prepared from BL21(D3) competent cells, and then induced for expression and purified (referring to the method in 7.1.2 of Example 7). The purified refolded inclusion bodies were then added. The eluted recombinant fusion protein was added to an 8-14 kDa dialysis bag and dialyzed sequentially at 4°C with 8M, 6M, 4M, 2M, 1M urea buffer, and 1M arginine buffer for 2 hours at each gradient. SDS-PAGE was used to analyze the purification status of each recombinant fusion protein. The purified protein was filtered through a 0.45 μm filter membrane, and the protein concentration was determined using a Thermo Scientific BCA protein quantification kit. The protein was stored at -80°C.

[0091] Preliminary induction experiments revealed that when FcμR-VHH was fused to the C-terminus of GP3 and GP4, the fusion protein could not be induced in the prokaryotic expression system. Therefore, FcμR-VHH was fused to the N-terminus of GP3 and GP4 for induction. Protein expression was induced at 37℃ and 16℃, respectively. Different concentrations of IPTG (0.5 mM / L and 1.0 mM / L) were added at different temperatures to induce protein expression. The expression conditions were tested by SDS-PAGE and Western blot. The results ( Figure 14 The results showed that M-VHH and N-VHH were induced to the highest levels at 16℃ and 1 mM / L IPTG concentration, so these two proteins were selected for amplified expression. VHH-GP3 showed no visible bands on SDS-PAGE at 37℃, but its expression level was slightly higher at 16℃ and 1 mM / L IPTG concentration, so this expression condition was selected for this protein. According to the identification results, the induction expression conditions for VHH-GP4 were the same as those for VHH-GP3.

[0092] After gradient dialysis, VHH-M and VHH-N were finally buffered in 1M arginine PBS solution, GP4-VHH in 0.5M arginine PBS solution, and GP3-VHH, due to its strong hydrophobicity, could not be renatured and was finally placed in 6M urea buffer. The purity of each protein was determined by SDS-PAGE after dialysis. The results were as follows (…). Figure 15 The results showed that the VHH-M, VHH-N, GP3-VHH, and GP4-VHH fusion proteins all reached the purity required for subsequent experiments.

[0093] Example 11. Targeting test of FcμR-VHH and PRRSV fusion antigen Following the method described in Example 6, Western blot analysis was used to examine the PAMs targeting of the expressed M-VHH, N-VHH, and VHH-GP4 (VHH-GP3 inclusion body refolding buffer contained urea, making cell-based assays impossible). Results ( Figure 16The results showed that all three proteins could target PAMs, verifying the in vitro targeting of the FcμR-VHH and PRRSV fusion antigen.

[0094] Example 12. In vivo immune enhancement assay of FcμR-VHH and PRRSV fusion antigen 12.1 Preparation of laboratory animals Five-week-old Landrace piglets were selected as candidate experimental animals. Twenty piglets that were negative for PRRSV antigen and antibody, ASFV antigen and antibody, CSFV and PCV antigen were randomly divided into four groups of five piglets each. The four groups were named VHH immunization group, inactivated virus immunization group, TJM-F92 attenuated live vaccine immunization group, and PRRSV challenge group, respectively.

[0095] 12.2 Immunization regimen of FcμR-VHH and PRRSV fusion antigen (1) Preparation of immunization proteins: The immunization dose of each protein per piglet was 100 μg. The required protein volume of the four fusion proteins (M-VHH, N-VHH, VHH-GP3 and VHH-GP4) was calculated according to the protein concentration, and mixed with ISA 206 adjuvant at a volume ratio of 1:1. After thorough emulsification, immunization was carried out according to the division of each experimental group. The prokaryotic PRRSV monomeric structural proteins (N, M, GP3, GP4) were purified and preserved in the laboratory. The PRRSV-JXA1 strain and TJM-F92 attenuated vaccine were preserved in the laboratory.

[0096] (2) PRRSV challenge: 21 days after the second immunization, the experimental animals in each group were intramuscularly injected with PRRSV-JXA1 strain, 1×10 3 TCID 50 / head.

[0097] 12.3 Assess the level of humoral immune response 12.3.1 Serum sample collection time points: Day 0, 7, and 14 of the first immunization; Day 7 and 14 of the second immunization; Day 0, 3, 7, 14, 21, and 28 after challenge. 5 mL per pig each time, placed at 4 ℃ for agglutination, centrifuged at 3500 rpm for 5 min, and serum was collected.

[0098] 12.3.2 Serum antibody levels in each immunization group were detected using an indirect ELISA method: (1) Antigen coating: Take 4 PRRSV monomer structural proteins (N, M, GP3, GP4) stored in the laboratory, and coat each protein in a 96-well microplate with 400 ng / well of the prepared buffer at 4 ℃ for 12-14 h; (2) Block with 5% skim milk powder at 37 ℃ for more than 4 h (200 μL / well). After blocking, wash the plate 3 times with PBS'T and pat dry any residual liquid in the wells. (3) Dilution of primary antibody: The primary antibody is the serum sample collected in 4.2.3.3 (1), diluted with 5% skim milk powder at 1:40, 100 μL / well, and incubated at 37 ℃ for 1 h; (4) Wash the plate three times with PBS'T and pat dry any remaining liquid in the wells; (5) Diluting the secondary antibody: The secondary antibody was Jackson's sheep anti-pig HRP-labeled antibody, diluted 1:5000 with 5% skim milk powder, 100 μL / well, and incubated at 37 ℃ for 1 h; (6) Wash the plate three times with PBS'T and pat dry any remaining liquid in the wells; (7) TMB color development, reaction at room temperature in the dark for 15 min; (8) Termination: The reaction was terminated with 50 μL / well of 3M H2SO4. The result was obtained by measuring the OD value using a UV spectrophotometer. 450 The value read at nm.

[0099] Before PRRSV challenge, each experimental group underwent two immunizations according to their respective categories. On day 21 of the second immunization, animals in each group were challenged. Serum samples were collected on days 0, 7, 10, 14, 7, 14, and 21 of the second immunization, and on days 3, 7, 14, 21, and 28 of challenge. Antibody levels in the serum were detected using an indirect ELISA method to assess the protective effect of the humoral immune response established by each immunization group against PRRSV challenge. Simultaneously, to compare the differences in humoral immune levels between the PRRSV monomeric structural protein and the FcμR-VHH nanobody fusion structural protein, serum from previous laboratory experiments using the PRRSV monomeric structural protein and post-challenge animal serum (the immunization methods and dosages for each monomeric protein in the animal experiments were the same as in this experiment) were added for comparison.

[0100] ELISA results ( Figure 17The results showed that for structural protein M, from day 0 to day 7 of the second immunization, the level of specific antibodies against M protein rapidly increased after VHH fusion, and was significantly higher than that of the monomeric structural protein immunization group and the TJM-F92 vaccine immunization group. In contrast, the inactivated virus immunization group produced almost no specific antibodies against M protein from day 0 to day 14 of the first immunization, and only began to produce low levels of specific antibodies on day 7 of the second immunization. After challenge, the level of M protein antibodies in the monomeric immunization group decreased, while the levels of specific antibodies against structural protein M in the other groups further increased. For structural protein N, from the start of immunization to day 7 of the second immunization, the specific antibody level in the VHH immunization group rapidly increased and reached a high level, and then remained stable at a high level. The specific antibody levels in the monomeric immunization group and the inactivated virus immunization group only began to increase after the second immunization. After challenge, the specific antibody levels in the VHH immunization group and the TJM-F92 vaccine immunization group remained stable, while the level in the inactivated virus immunization group rapidly increased, and the level of specific antibodies in the monomeric group showed a decreasing trend. For the GP3 protein, the TJM-F92 vaccine immunization group showed higher levels of specific antibodies against the second immunization compared to other groups. After challenge, the antibody levels in the TJM-F92 vaccine immunization group decreased slightly on day 7 and then rebounded. The VHH fusion GP3 group failed to effectively increase GP3-specific antibody levels. The inactivated virus immunization group showed a slight increase in antibody levels, while the monomeric immunization group showed a significant decrease. For the GP4 protein, except for the inactivated virus immunization group, all other groups produced specific antibodies during immunization, and the VHH fusion GP4 immunization group showed significantly higher levels of specific antibodies than other groups. The VHH immunization group rapidly produced high levels of specific antibodies during the first immunization, while the monomeric immunization group only began to rise in antibody levels in the later stages of the second immunization. After challenge, the specific antibody levels in the VHH immunization group and the TJM-F92 vaccine immunization group remained relatively stable. The inactivated virus immunization group, which did not produce specific antibodies during immunization, began to show an increase in antibody levels against GP4 after challenge, while the antibody levels in the monomeric immunization group continued to decrease after challenge.

[0101] In this invention, when evaluating humoral immune response levels using an indirect ELISA method, serum from piglets immunized only with GP3 / GP4 / M / N monomeric proteins was added as a control in previous animal experiments. It was found that when immunizing with the same mass of antigen (100 μg), except for GP3, the VHH fusion protein immunization group showed a rapid increase in specific antibody levels in the serum compared to other monomeric structural protein immunization groups in the early stage of immunization (primary immunization). Furthermore, the level of specific antibodies remained stable at a high level after challenge, demonstrating that the antigen fused with the nanobody FcμR-VHH can target APCs, improving antigen presentation efficiency and better stimulating the animal body to produce specific antibodies compared to monomeric structural proteins. On the other hand, since the VHH nanobody itself has a molecular weight of 15 kDa, after fusion with the PRRSV structural protein, the net content of PRRSV structural protein antigen in 100 μg of VHH fusion protein is less than that of the monomeric protein. This further illustrates that using FcμR-VHH to target the antigen with the nanobody can reduce the amount of antigen used.

[0102] 12.4 Blood Toxin Detection The serum collected in 12.3.1 (200 μL) was used to extract viral RNA from the serum using the GeneJET Viral DNA and RNA Purification Kit. The PRRSV load in the serum after challenge was detected by qPCR to assess the rate of virus clearance in the animal.

[0103] Figure 18 The results showed that the viral load in the VHH immunization group decreased significantly on day 14 after challenge, showing a difference compared to the challenge group (P<0.05) and was slightly lower than that in the TJM-F92 vaccine immunization group. On day 28 after challenge, the viral load in the VHH immunization group was low and significantly lower than that in the challenge group (P<0.01), and was also significantly lower than that in the TJM-F92 vaccine immunization group (P<0.001). The TJM-F92 vaccine immunization group cleared the virus the fastest, and the viral load remained at a low level from day 3 after challenge, which was consistent with the clinical manifestations in the animals. The serum viral load in the inactivated virus immunization group also gradually decreased with the extension of the challenge time, but there were significant differences in viral load among the experimental animals within the group. The results show that although the blood toxin levels in the VHH fusion protein immunization group were not significantly different from those in the control group in the early stages of infection (within 7 days), the rate of toxin clearance was faster in the subsequent time points. Furthermore, in the mid-to-late stages of infection, the toxin clearance level in the VHH fusion protein immunization group was even better than that in the live attenuated vaccine group, suggesting that high antibody levels accelerated the clearance of the virus from peripheral blood.

[0104] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Use of a Nanobody specifically binding to the porcine IgM Fc receptor FcμR for the manufacture of a porcine vaccine, characterized in that, The amino acid sequence of the nanobody is shown as SEQ ID NO: 1, and the nanobody is used for preparing a vaccine after being fused with an antigen protein to prepare a fusion protein.

2. Use according to claim 1, characterized in that, The pig vaccine is a vaccine for porcine reproductive and respiratory syndrome, and the antigen protein is M, N, GP3, GP4 protein or a fragment thereof of PRRSV.

3. A fusion protein, characterized in that, The fusion protein comprises: (1) a nanobody capable of specifically binding to pig IgM Fc receptor FcμR, and the amino acid sequence of the nanobody is shown as SEQ ID NO: 1; (2) an antigen protein derived from a bacterium or virus capable of infecting pigs; and / or (3) a linker, i.e. a connecting peptide segment.

4. The fusion protein of claim 3, wherein, The nanobody is connected to the C-terminal or N-terminal of the antigen protein through the linker.

5. The fusion protein according to claim 3 or 4, characterized in that, The antigen protein is M protein, N protein, GP3 protein and / or GP4 protein of PRRSV.

6. The fusion protein of claim 3, wherein The amino acid sequence of the fusion protein is shown as SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18 or SEQ ID NO:

20.

7. A polynucleotide encoding the fusion protein of claim 6, characterized in that, The sequence of the polynucleotide is shown as SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19 or SEQ ID NO:

21.

8. An expression vector, characterized by, The expression vector comprises the polynucleotide sequence encoding the fusion protein according to claim 7.

9. A vaccine for porcine reproductive and respiratory syndrome, wherein the vaccine comprises a fusion protein with an amino acid sequence shown as SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18 and / or SEQ ID NO:

20.

10. The vaccine of claim 9, characterized in that, The antigen protein of the vaccine is composed of the fusion protein shown as SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 20.

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