A nanobody or antigen-binding fragment thereof specifically binding to porcine IgM Fc receptor FcμR and uses thereof

Abstract

The application belongs to the technical field of biology and relates to a nanobody or antigen-binding fragment thereof specifically binding to pig IgM Fc receptor FcμR and application. The application clones full-length cDNA of pig FcμR, expresses the extracellular region in prokaryotes, and constructs a nanobody library by camel immunization; a strain of specific anti-pig FcμR nanobody is obtained through three rounds of panning. The nanobody (FcμR-VHH) is expressed by E. coli, and it is proved that the nanobody can target pig macrophages (PAMs) by taking zsGreen as a model. In vitro and in vivo tests show that the targeting and immune enhancement effect of the FcμR-VHH fusion protein at the C-terminal of an antigen is better than that at the N-terminal. After immunizing animals, ELISA proves that the fusion protein significantly improves humoral and cellular immune responses.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to a Nanobody or antigen-binding fragment thereof specifically binding to porcine IgM Fc receptor FcμR and applications thereof. BACKGROUND

[0002] Vaccination is an effective strategy to prevent the spread of infectious diseases. Current vaccines are mostly composed of antigens derived from pathogens, such as purified proteins or inactivated pathogens, which stimulate the body to produce antigen-specific memory B and T cells and provide long-term protection for the vaccinated individuals. Vaccination has greatly reduced or eliminated the spread of serious diseases around the world. However, there are still many diseases that cannot be effectively prevented by vaccines, so more advanced strategies for vaccine preparation are needed. The technology of in vivo targeted delivery of antigens to antigen-presenting cells (APCs) as a method to improve the immune effect of genetically engineered vaccines has attracted much attention. The effect of antigen targeting to APCs depends largely on the choice of the targeting receptor, in addition to the nature of the immune itself. The choice of receptor directly affects the response triggered after antigen uptake. Targeting methods for different receptors of APCs have been explored in various animals, among which pigs, cattle, sheep and poultry are the most common.

[0003] In adaptive immune response, IgM is the first antibody type to react to pathogens, and is the earliest antibody produced in viral infection. Unlike IgG, which binds antigen mainly through a single variable region with a single affinity to the recognized epitope, IgM has a higher total avidity for the target antigen than IgG. Therefore, IgM can better tolerate mutations in viral targets, which is of great significance for the recognition of pathogens with high mutation rates. IgM regulates the function of the immune system through interaction with Fc receptors. Three IgM Fc receptors have been found, including Fcα / μ receptor (Fcα / μR), polymeric immunoglobulin receptor (pIgR), and Fcμ receptor (FcμR / FCMR). Fcα / μR is expressed by B cells and macrophages and plays a role in local and systemic mucosal immunity. pIgR is expressed in mucosal epithelium and mediates epithelial transport of J chain dimer IgA and multimer IgM molecules in the lamina propria. FcμR is an IgM-specific Fc receptor, also known as IgM Fc fragment receptor (FCMR) or TOSO / Fas apoptosis inhibitor molecule 3 (FAIM3), and the amino-terminal extracellular region of FcμR contains a V-type immunoglobulin-like domain similar to pIgR and Fcα / μR, which is likely to be the binding site for IgM Fc fragment. The cytoplasmic region of FcμR contains multiple conserved serine and tyrosine residues, and cross-linking of FcμR by IgM immune complexes can lead to phosphorylation of these serine and tyrosine residues, which are phosphorylation sites that initiate or mediate signal cascades, suggesting that FcμR can transmit extracellular information to the intracellular. In humans and mice, FcμR is mainly expressed by B cells, T cells and NK cells, and is considered to be a truly specific receptor for IgM. FcμR has high affinity for the Fc portion of multimeric IgM (~10 nM), and is widely expressed on macrophages, dendritic cells and lymphocytes, mediating the phagocytosis of immune complexes formed by IgM and antigen by target cells and the biological effects of IgM.

[0004] Previous studies by the present inventors have shown that immunizing piglets with inactivated PRRSV and specific murine IgM antibodies to form immune complexes can significantly improve the protective efficacy of PRRSV inactivated vaccine, reduce the damage to the lungs of piglets caused by PRRSV challenge, and significantly increase the level of IFN-γ in peripheral blood serum, suggesting that IgM may act as a special adjuvant that, when combined with antigen to form immune complexes, binds to the IgM Fc receptor FcμR to enhance the body's immune response to the antigen. However, due to the special nature of the IgM multimer structure, it is difficult to use it for vaccine production through recombinant expression. Therefore, how to enhance the targeting of vaccines is an important issue currently faced in vaccine research and production. SUMMARY

[0005] The application provides a nanobody or antigen-binding fragment thereof specifically binding to a porcine IgM Fc receptor FcμR and application, aiming to use the anti-porcine FcμR molecule nanobody as a ligand targeting the FcμR molecule, target the antigen to the APCs to improve the efficiency of antigen presentation and enhance the immune response of the body.

[0006] To solve the above technical problems, the application adopts the following technical means:

[0007] A nanobody or antigen-binding fragment thereof specifically binding to a porcine IgM Fc receptor FcμR, comprising the following CDRs: CDR1 with an amino acid sequence as shown in SEQ ID NO: 3, CDR2 with an amino acid sequence as shown in SEQ ID NO: 4, and CDR3 with an amino acid sequence as shown in SEQ ID NO: 5.

[0008] Preferably, it further comprises four framework regions FR1-FR4, which are staggered in order with the CDR1, CDR2 and CDR3; preferably, the FR1-FR4 are respectively as shown in SEQ ID NO: 6, 7, 8, 9.

[0009] A nanobody specifically binding to a porcine IgM Fc receptor FcμR, with an amino acid sequence as shown in SEQ ID NO: 1.

[0010] A polynucleotide encoding the above-mentioned nanobody or antigen-binding fragment thereof specifically binding to a porcine IgM Fc receptor FcμR.

[0011] Preferably, a polynucleotide encoding the above-mentioned nanobody specifically binding to a porcine IgM Fc receptor FcμR, with a nucleotide sequence as shown in SEQ ID NO: 2.

[0012] A nucleic acid construct comprising the polynucleotide encoding the nanobody or antigen-binding fragment thereof specifically binding to a porcine IgM Fc receptor FcμR; preferably, comprising the polynucleotide as shown in SEQ ID NO: 2; preferably, further comprising at least one expression regulatory element operably linked to the polynucleotide.

[0013] An expression vector comprising the above-mentioned nucleic acid construct.

[0014] A transformed cell comprising the above-mentioned polynucleotide, the above-mentioned nucleic acid construct or the above-mentioned expression vector.

[0015] The application also claims the use of the porcine IgM Fc receptor FcμR nanobody in the preparation of a porcine antigen-presenting cell targeting drug.

[0016] Based on the above technical solutions, the application has the following advantages and beneficial effects:

[0017] Based on the cloning and sequencing of the full-length cDNA of pig FcμR, the extracellular region thereof is expressed in prokaryotes for camel immunization; by extracting the total RNA of peripheral blood lymphocytes of the immunized camel and reversely converting the cDNA, a phage nanobody library is successfully constructed. By using the bio-panning technology, 1 strain of nanobody against the extracellular region of pig FcμR with highly consistent sequence is obtained from 96 ELISA positive bacterial colonies after 3 rounds of panning enrichment, and the sequence thereof is obtained, so that the specific nanobody of the pig FcμR molecule is screened, and the antigen targets the pig FcμR molecule through the ligand-receptor interaction.

[0018] The nanobody against pig FcμR (FcμR-VHH) is expressed by using the E. coli prokaryotic expression system, and the targeting property thereof on PAMs is verified; by using the green fluorescent protein zsGreen as a model antigen, the FcμR-VHH is fused to the C terminal and N terminal of the zsGreen respectively, the adsorption test is performed on different cells, different detection methods are used, and it is determined that the FcμR-VHH fused at different positions of the antigen can target the antigen, and it is verified that the targeting effect is better when the FcμR-VHH is fused to the C terminal of the antigen in vitro and in vivo. The results also show that the antigen can be targeted to APCs by fusing the FcμR-VHH, and the specific humoral immune response and cellular immune response of the antigen can be improved at the same time. The zsGreen fusion protein is used for immunizing animals, the level of specific antibody against the zsGreen in the serum of the animals after immunization is detected by using the indirect ELISA method, it is verified in vivo that the FcμR-VHH can target the APCs, enhance the humoral and cellular immune responses of the antigen, and it is verified that the FcμR-VHH fused to the C terminal of the antigen has better effect on enhancing the humoral and cellular immune responses. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0020] Figure 1SDS-PAGE and Western blot identification of the results of recombinant protein FcμR expression and purification in pigs: (a) SDS-PAGE identification of the purification conditions of recombinant FcμR protein, M: protein Marker; 1: before purification; 2: flow-through; 3: 5 mM imidazole elution; 4: 10 mM imidazole elution; 5: 100 mM imidazole elution; (b) SDS-PAGE identification of the recombinant FcμR protein after purification and dialysis.

[0021] Figure 2 Indirect ELISA detection of the titer of anti-porcine FcμR protein antibodies in camel serum.

[0022] Figure 3 Amplification of VHH genes and identification of the library: (a) and (b) nested PCR amplification of VHH genes; (c) bacterial liquid PCR identification of the positive rate of the library.

[0023] Figure 4 Indirect ELISA method for detecting the reactivity of recombinant nanobodies with porcine FcμR protein.

[0024] Figure 5 Amino acid sequence of anti-porcine FcμR nanobodies.

[0025] Figure 6 SDS-PAGE and Western blot identification of the results of prokaryotic expression and purification of recombinant FcμR-VHH: (a) SDS-PAGE identification of the purification conditions of recombinant FcμR-VHH protein, M: protein Marker; 1: before purification; 2: flow-through; 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 the recombinant FcμR-VHH protein after purification and dialysis; (c) Western blot identification of the recombinant FcμR-VHH protein after purification and dialysis, M: protein Marker; 1: recombinant FcμR-VHH protein.

[0026] Figure 7 Western blot detection of FcμR-VHH targeting: M: Marker; 1: MOCK; 2: F165R; 3: I215L; 4: HERC5; 5: FcμR-VHH.

[0027] Figure 8Amplification 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.

[0028] Figure 9 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.

[0029] Figure 10 SDS-PAGE analysis of purified protein: M: Marker; 1: zsGreen-VHH; 2: VHH-zsGreen.

[0030] Figure 11 Results of zsGreen and VHH fusion protein adsorption on APCs: (a) Fluorescence results (PAMs) (10×10); (b) Flow cytometry results (BM-DCs).

[0031] Figure 12 Detection of zsGreen after lysosome extraction following adsorption of PAMs by fusion protein.

[0032] Figure 13 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).

[0033] Figure 14 ELISpot assay was used to detect IFN-γ secretion levels in piglets' PBMCs 14 days after immunization. * indicates a difference. P<0.05). Detailed Implementation

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] 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.

[0036] Example 1: Expression and purification of porcine FcμR protein

[0037] 1.1 Transformation of porcine pET-28a-FcμR recombinant expression plasmid

[0038] 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

[0040] (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 culture at 37℃ and 220 r / min for 16 h to restore bacterial activity;

[0041] (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.

[0042] (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.

[0043] (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.

[0044] (5) Centrifuge at 12000 g for 25 min at 4℃, retain the supernatant, and filter the supernatant through a 0.45μm filter membrane;

[0045] (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 ℃.

[0046] (7) Use 5 mM, 10 mM, 50 mM, 100 mM, and 250 mM imidazole 8M urea solutions to elute proteins in a gradient manner;

[0047] (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;

[0048] (9) SDS-PAGE analysis of the purification status of porcine FcμR recombinant protein, such as... Figure 1 As shown.

[0049] (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℃.

[0050] 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.

[0051] Example 2: Construction of a porcine FcμR protein nanobody library

[0052] 2.1 Immunity of Bactrian camels

[0053] 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.

[0054] 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.

[0055] 2.2 Amplification of the VHH gene fragment

[0056] (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.

[0057] (2) Two rounds of nested PCR:

[0058] 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.

[0059] Table 1. Primer sequences

[0060]

[0061] Note: The underlined and italicized sequences are restriction enzyme sites, R = A / G.

[0062] 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.

[0063] 2.3 Double digestion of VHH fragment and pMECS vector

[0064] Two rounds of enzyme digestion were performed. The second round nested PCR product and the pMECS empty vector were double-digested with XbaI and Pst I-HF and Pst I-HF and Not I-HF, respectively. The digestion conditions were 37℃ for 16 h, and the products were recovered by gel extraction.

[0065] 2.4 Construction of VHH phage display vector

[0066] 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.

[0067] 2.5 Preparation of Electrocompetent States

[0068] (1) TG1 glycerol bacteria were streaked onto M9 plates and incubated at 37 ℃ for 36 h;

[0069] (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;

[0070] (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.

[0071] (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.

[0072] (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.

[0073] 2.6 Ligation Product Transformation and Library Capacity Identification

[0074] (1) Take TG1 electroporation competent cells, add 50 µL (5 µg) of ligation product, and perform electroporation transformation using an Eppendorf electroporator;

[0075] (2) After the electroporation is complete, place the conversion product on a shaker and incubate at 37°C and 120 rpm for 1 h.

[0076] (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 -5100 μL of each sample was spread on LB / AMP-GLU plates and incubated at 37 °C for 8 h for library identification.

[0077] (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;

[0078] (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 ℃.

[0079] 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).

[0080] Example 3: Preparation of M13KO7 helper phage

[0081] 3.1 Preparation of M13KO7 auxiliary plaques

[0082] (1) The method for preparing TG1 monoclonal colony culture medium is as described in steps (1) and (2) of 2.5 in Example 2.

[0083] (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 incubate at 37 °C for 15 min.

[0084] (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.

[0085] 3.2 M13KO7 helper phage amplification

[0086] (1) Prepare TG1 bacterial culture in the logarithmic growth phase for later use;

[0087] (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;

[0088] (3) Inoculate the phage suspension into 4 mL of TG1 in the logarithmic growth phase, incubate at 37°C for 15-20 min, and then place it in a shaker at 37°C and shake at 200 rpm for 2 h.

[0089] (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.

[0090] (5) Add Kan antibiotics to the conical flask at a ratio of 1:1000 and continue culturing for 16 h.

[0091] 3.3 Phage sedimentation

[0092] (1) After phage amplification culture, take the supernatant and add 1 / 5 volume of PEG / NaCl to settle on ice overnight;

[0093] (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 ℃;

[0094] (3) Centrifuge at 12000 g for 15 min at 4℃ and collect the supernatant. The supernatant is white and oily.

[0095] 3.4 Titration

[0096] 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.

[0097] Example 4: Screening of anti-pig FcμR protein nanobodies

[0098] 4.1 Rescue and titer determination of VHH phage library

[0099] (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;

[0100] (2) Inoculate with the helper phage amplified in Example 3 (greater than 20 MOI), mix well, and let stand at 25 ℃ for 30 min;

[0101] (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.

[0102] (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.

[0103] (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.

[0104] (6) Take 10 μL of product, dilute it 10 times in 9 gradients, soak it in EP tubes at 25℃ for 20 min, take 100 μL of each product and spread it on LB / AMP-GLU plates, and incubate at 37℃ for 10 h.

[0105] (7) Count the colonies and determine the library titer.

[0106] 4.2 Screening of anti-pig FcμR protein-specific nanobodies

[0107] (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;

[0108] (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.

[0109] (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.

[0110] (4) First wash the tube with PBS'T 15 times, 2 min each time, then wash with PBS (K + Rinse 15 times;

[0111] (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;

[0112] (6) Determine the titer of the elution product and repeat the rescue, enrichment and screening process twice.

[0113] 4.3 Enrichment of specific recombinant phages by phage ELISA detection

[0114] Detection of recombinant phages using ELISA:

[0115] (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;

[0116] (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 solution, 100 μL / well, and incubated at room temperature for 2h.

[0117] (3) The secondary antibody was M13 mouse anti-HRP antibody, diluted to 100 μL / well, and incubated at room temperature for 2 h;

[0118] (4) After incubation, TMB color development and termination are performed, and the OD450nm value is read.

[0119] 4.4 Preparation and Identification of Crude Nanobody Substances from Screening

[0120] (1) Randomly pick 96 single colonies from the plates of the third round of titer determination and label them as 1#~96#. Inoculate them into 96-well plates and incubate at 37℃ and 200rpm for 8h.

[0121] (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;

[0122] (3) Add TB medium containing 10mM IPTG, 100 μL / well, and incubate overnight at 37 ℃ and 200 rpm;

[0123] (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;

[0124] (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.

[0125] (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.

[0126] 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.

[0127] Table 2. Enrichment of porcine FcμR protein-specific nanobodies

[0128]

[0129] 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.

[0130] 4.5 Sequencing analysis of anti-pig FcμR protein-specific nanobodies

[0131] (1) Streak the plates of ELISA-positive clones from section 4.3 and incubate overnight at 37 °C;

[0132] (2) Single colonies were picked and amplified in 1.5 mL EP tubes with LB aspirin for 2 h. The bacterial culture was then sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results showed that the sequences of the 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. Its structure is shown in the diagram below. Figure 5 As shown.

[0133] Example 5: Recombinant expression of anti-pig FcμR protein-specific nanobodies

[0134] 5.1 Construction of FcμR-VHH recombinant expression vector and preparation of recombinant bacteria

[0135] (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:

[0136] Table 3 Primer sequences for amplifying the FcμR-VHH gene

[0137]

[0138] Note: The sequences marked with underlined italics are restriction enzyme sites.

[0139] (2) The FcμR-VHH gene was amplified by PCR using Takara GXL enzyme, and its nucleotide sequence is shown in SEQ ID NO: 2.

[0140] (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.

[0141] (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).

[0142] 5.2 Prokaryotic expression and purification of the anti-porcine FcμR protein-specific nanobody FcμR-VHH

[0143] (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;

[0144] (2) Take the activated bacterial solution 1:100 and inoculate it into LB medium containing Amp resistance. Use IPTG concentration (0.5mM / L) to induce the recombinant Escherichia coli that has reached the logarithmic growth phase and incubate at 37℃ for 8h.

[0145] (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.

[0146] (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 12 h.

[0147] (5) After dissolving, centrifuge at 4℃, 12000 g for 25 min, retain the supernatant, and filter the supernatant through a 0.45μm filter membrane;

[0148] (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.

[0149] (7) Use 5mM, 10mM, 50mM, 100mM, and 250mM imidazole 8M urea solutions to elute proteins in a gradient manner;

[0150] (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.

[0151] (9) SDS-PAGE analysis of the purification status of porcine FcμR-VHH recombinant protein, such as... Figure 5 As shown.

[0152] (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℃.

[0153] 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 6 (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 6 (b) and 6(c), the concentration was detected as 0.8 mg / mL using the BCA kit.

[0154] Example 6. Targeting test of porcine FcμR nanobody FcμR-VHH

[0155] 6.1 Detection of the targeting effect of FcμR-VHH on PAMs cells

[0156] (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;

[0157] (2) Replace 10% 1640 with PBS (K + ) buffer, add 5 μg FcμR-VHH or control protein to each well;

[0158] (3) Place the cell culture plate in a 37 ℃ incubator and incubate for 30 min;

[0159] (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.

[0160] (5) Repeat step (4) twice;

[0161] (6) Collect samples with 2×sample buffer and perform Western blot detection.

[0162] 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 7 The results show that the FcμR-VHH nanobody can target and adsorb PAMs.

[0163] Example 7. Targeting test of FcμR-VHH nanobody and zsGreen fusion protein

[0164] 7.1 Prokaryotic expression and purification of FcμR-VHH nanobody and zsGreen fusion protein

[0165] 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.

[0166] 7.1.1 Construction of pET-21b-VHH-zsGreen and pET-21b-zsGreen-VHH prokaryotic expression plasmids

[0167] 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 8 As 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.

[0168] 7.1.2 Induction and purification of VHH-zsGreen and zsGreen-VHH recombinant proteins

[0169] (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.

[0170] 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 9 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.

[0171] (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 3 to 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.

[0172] 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 10 The final protein buffer was PBS solution, and the concentration was detected using a BCA kit to be 0.5 mg / mL.

[0173] 7.2 Targeting effect test at the fusion site of FcμR-VHH and zsGreen

[0174] 7.2.1 Detecting the targeting effect of FcμR-VHH at different fusion sites on PAMs

[0175] (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;

[0176] (2) Replace 10% 1640 with PBS (K + ) buffer, add 5 μg zsGreen-VHH, VHH-zsGreen and control protein (zsGreen) to each well respectively;

[0177] (3) Place the cell culture plate in a 37 ℃ incubator and incubate for 30 min;

[0178] (4) Discard the supernatant in the well, treat the cells with proteinase K for 30 seconds, and wash away unbound proteins;

[0179] (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.

[0180] 7.2.2 Detecting the targeting effect of FcμR-VHH at different fusion sites on BM-DCs

[0181] (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;

[0182] (2) Centrifuge at 1600 rpm for 6 min at 4℃ and discard the culture medium;

[0183] (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;

[0184] (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.

[0185] (5) Centrifuge at 4℃ and 1600 rpm for 6 min, and discard the supernatant;

[0186] (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.

[0187] (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.

[0188] result Figure 11 The 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 an adsorption 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 also exhibited better targeting and adsorption effects.

[0189] Example 8. Internalization ability of PAMs on FcμR-VHH fused zsGreen

[0190] (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;

[0191] (2) Replace 10% 1640 with PBS (K +) buffer, add 5 μg zsGreen-VHH, VHH-zsGreen and control protein (zsGreen) to each well respectively;

[0192] (3) Place the cell culture plate in a 37 ℃ incubator and incubate for 30 min;

[0193] (4) Discard the supernatant in the well, treat the cells with proteinase K for 30 seconds, and wash away the internalized proteins;

[0194] (5) Gently wash the cells 3 times with PBS and centrifuge at 1500 rpm for 5 min;

[0195] (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.

[0196] 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 12 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.

[0197] Example 9. In vivo immunization of zsGreen and VHH fusion protein in animals

[0198] 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.

[0199] result Figure 13 (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 13 (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.

[0200] 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 14 (a) Results of the Spot Number Difference Analysis Figure 14(b) After stimulation with zsGreen, the number of IFN-γ cells produced by pig 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 cellular immune response during the immunization process, resulting in a higher level of cellular immune response after immunization.

Claims

1. A Nanobody or antigen binding fragment thereof that specifically binds to porcine IgM Fc receptor FcμR, characterized in that, CDRs comprising a CDR1 having an amino acid sequence of SEQ ID NO: 3, a CDR2 having an amino acid sequence of SEQ ID NO: 4, and a CDR3 having an amino acid sequence of SEQ ID NO:

5.

2. The Nanobody or antigen binding fragment thereof specifically binding to the porcine IgM Fc receptor FcμR according to claim 1, characterized in that, Further comprising four framework regions FR1-FR4, which are interleaved with the CDR1, CDR2 and CDR3 in order; the FR1-FR4 are respectively shown as SEQ ID NO: 6, 7, 8, 9.

3. A polynucleotide encoding the Nanobody or antigen-binding fragment thereof of specific binding to porcine IgM Fc receptor FcμR according to claim 1 or 2.

4. A Nanobody specifically binding to porcine IgM Fc receptor FcμR, characterized in that, The amino acid sequence of the Nanobody is shown as SEQ ID NO:

1.

5. A polynucleotide encoding the Nanobody of specific binding to porcine IgM Fc receptor FcμR according to claim 4, the nucleotide sequence of which is shown as SEQ ID NO:

2.

6. A nucleic acid construct comprising the polynucleotide of claim 3 or 5, further comprising at least one expression regulatory element operably linked to the polynucleotide.

7. An expression vector comprising the nucleic acid construct of claim 6.

8. A transformed cell comprising the polynucleotide of claim 3 or 5, or the nucleic acid construct of claim 6, or the expression vector of claim 7.

9. Use of the Nanobody of specific binding to porcine IgM Fc receptor FcμR according to claim 4 in the preparation of a porcine antigen-presenting cell targeting drug.

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