Pig antigen-presenting cell targeting peptides and uses thereof

Abstract

The application discloses a pig antigen-presenting cell targeting peptide and application thereof, and the amino acid sequence of the pig antigen-presenting cell targeting peptide is shown as SEQ ID NO. 1 or SEQ ID NO. 6. The FcMR specific 12-peptide sequence of the pig is screened by using a phage random display peptide library, the obtained FcMR specific 12-peptide sequence is fused with a model antigen for expression, and the FcMR specific 12-peptide sequence with the APC targeting property is further screened. In subsequent animal experiments, it is determined that the specific targeting peptide has an obvious enhancement effect on an immune response, and has important significance for the research and development of a future pig APC targeting drug.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a porcine antigen-presenting cell targeting peptide and application thereof. BACKGROUND

[0002] Subunit vaccines are widely used in the prevention and control of pig diseases. They stimulate the immune system using specific antigens (such as proteins, polysaccharides) of pathogens, rather than using whole pathogens, reducing the risk of introducing diseases and being more secure. However, a number of experiments have shown that porcine subunit vaccines based on recombinant proteins generally have low immunogenicity, weak immune response, and cannot stimulate the body to produce a cellular immune response, resulting in poor protection after immunization with porcine subunit vaccines. Therefore, when using porcine subunit vaccines, a large amount of vaccine adjuvant is needed to assist in stimulating the immune system of the body, and multiple immunizations are needed to obtain sustained protection.

[0003] Antigen-presenting cells (APCs) can extract, process and handle antigens, present them to antigen-specific immune cells (T / B cells) and initiate adaptive immune responses, and play a key role in connecting innate immunity and initiating adaptive immune responses. When the body is invaded by pathogenic microorganisms or cells in the body become malignant, APCs can quickly recognize these antigens and internalize them, process and process them into antigen peptides, which are then presented to MHC molecules and expressed on the surface of APCs. Initial CD4 + T lymphocytes or CD8 + T cells recognize antigen peptides presented by MHC molecules on the surface of APCs through T cell receptors. MHC-I class molecules usually present endogenous antigen peptides presented by APCs, or present captured exogenous antigens through the unique cross-presentation function of special dendritic cells (DCs) in APCs through MHC-I class molecules. Unactivated CD8 + T cells recognize the "MHC-I-antigen peptide" complex to activate CD8 + T cells with cytotoxicity, thereby killing infected cells and producing corresponding cytokines to enhance anti-pathogen response and participate in cellular immune response. MHC-II class molecules usually present exogenous antigen peptides processed by APCs, which are recognized by initial CD4 + T cells to activate and differentiate into different types of helper T cell subsets to assist B cells and CD8 + T cell activation. B lymphocytes recognize antigens through B cell receptors, receive co-stimulatory signals from activated helper CD4 + T cells to drive the proliferation of B cells, the maturation of antibody affinity, and the switching of antibody subtypes to initiate humoral immune responses.

[0004] APCs play a key role in immune responses. Genetic engineering of subunit vaccine antigens to target a specific receptor on the surface of APCs can enable APCs to capture antigen efficiently to promote antigen uptake, processing or presentation by APCs, and enhance the strength and range of immune responses activated by antigenic material. A variety of receptors have been found to be expressed on APCs, among which Fc receptors (FcR) of immunoglobulins are often used as target receptors for targeting APCs. FcR is expressed by many different cell types in the immune system, and their interaction with antibodies can initiate a wide range of immunological effects, which are very important in host defense, including phagocytosis of antibody-coated microorganisms, lysosomal degradation of internalized immune complexes, antibody-dependent cell-mediated cytotoxicity, secretion of cytokines and chemokines, release of potent inflammatory mediators, enhanced antigen presentation, regulation of B lymphocyte antibody production and plasma cell survival. A large number of studies have shown that targeting antigens to FcR on the surface of APCs can enhance adaptive humoral and cellular immune responses.

[0005] Immunoglobulin μ Fc Receptor (FcμR / FcMR) is a newly discovered specific receptor that interacts with IgM in recent years. It is a transmembrane glycoprotein with a size of about 60 kDa. FcMR only recognizes IgM, but not IgA, and its binding with IgM has high specificity. Studies in recent years have shown that FcMR acts as an endocytic receptor to mediate the phagocytosis of IgM or IgM-bound immune complexes by target cells, which is important for the capture and internalization of IgM and the antigen recognized by IgM. FcMR is related to immune balance and activation of the innate immune system. FcMR has a key function in B cell development and immune tolerance. FcMR can limit tonic BCR signaling by regulating the surface expression of monomeric IgM-type BCR, and can provide a signal to increase the survival rate of mature B cells when BCR is cross-linked by activating the non-canonical nuclear factor kappa-B (NF-κB) pathway, and can also regulate the differentiation of marginal zone B cells and B1 cells. In addition, FcMR acts as a costimulatory molecule to enhance TCR signaling and T cell activation and proliferation. T cell activation is a key checkpoint of adaptive immune response. The signaling downstream of TCR after antigen stimulation leads to T cell proliferation, differentiation, and release of effector cytokines. Cell signaling is strictly regulated and has a threshold. B cells support T cell activation by secreting IgM to bind to FcMR on T cells. IgM stimulation of naive T cells entering the spleen and lymph nodes will lead to increased surface expression of TCR and costimulatory molecules under the mediation of FcMR. IgM uptake by FcMR of naive T cells will help naive T cells reach the activation threshold, especially under inflammatory conditions and low antigen concentration. FcMR can promote protein transport to the cell surface of human T cells, thereby enhancing cell signaling, proliferation, and cytokine secretion. When IgM is enriched in cells, the binding of FcMR with IgM will increase the expression of surface molecules unrelated to T cell antigen receptors. Therefore, the capture of recombinant antigens by immune cells through FcMR by targeting FcMR or in the form of immune complexes with IgM can significantly enhance the stimulation effect of antigens on the immune system. In existing studies, such as CN202010069365.5, mouse IgM monoclonal antibody 5D9 is used as an immune enhancer. When the immune complex formed by the immune enhancer is combined with an adjuvant to immunize mice, the IFN γ secreting T cells are significantly increased, indicating that in the process of inactivated virus immunization, the combination of PRRSV specific antibody 5D9 and normal oil-in-water adjuvant can enhance the CTL response stimulated by inactivated virus. In CN202110663821.3, a recombinant PRRSV virus-like particle antigen antibody complex is used.Therefore, the IgM RPPSV immune complex or the use of antigen-specific IgM as a special immune enhancer for co-immunization of the host with the antigen can better stimulate the adaptive immunity of the body.

[0006] At present, the research of FcMR mainly focuses on human and mouse, and there is no report on FcMR related research in pigs. There is only a predicted sequence of pig FcMR in GenBank. The inventors successfully obtained the amino acid sequence of pig FcMR by cloning the cDNA of pig FcMR. Starting from the characteristics of pig FcMR receptor, the specific targeting 12 peptide sequence capable of binding to pig FcMR is designed and screened by phage display peptide library technology, and is named as FcMR No. 2 peptide and FcMR No. 10 peptide. Taking zsGreen as an example, after the flexible linker is fused with the FcMR No. 2 peptide and the FcMR No. 10 peptide, it is confirmed that the zsGreen protein fused with the pig FcMR targeting peptide has good affinity and binding capacity for the pig-derived antigen presenting cells represented by porcine alveolar macrophage (PAMs) cells, and after immunizing the host, the antibody level and cellular immune response stimulated by the model antigen zsGreen can be significantly enhanced. Subsequently, the targeting peptide FcMR10 is selected to be fused with the GP3 protein of porcine reproductive and respiratory syndrome virus (PRRSV) and the P22 and P54 of African swine fever virus (ASFV) respectively to prepare recombinant proteins, and it is determined that FcMR10 can effectively improve the immune response stimulated by the immunized body of different pig-derived DNA viruses and RNA antigens. The present application has important significance for the research and development of future pig APC targeting recombinant subunit vaccine. SUMMARY

[0007] In order to solve the technical problems of low immunogenicity, weak immune response and poor immune protection effect of the pig subunit vaccine in the prior art, the present application aims to provide a pig antigen presenting cell targeting peptide FcMR2 and FcMR10 and application thereof.

[0008] In order to solve the above technical problems, the present application adopts the following technical means:

[0009] The pig-derived antigen presenting cell targeting peptide can specifically target the FcMR receptor on the antigen presenting cell. The amino acid sequence of the targeting peptide is shown in SEQ ID No: 1 or SEQ ID No: 6, or has more than 83% homology with the amino acid sequence shown in SEQ ID No: 1.

[0010] Preferably, the amino acid sequence of the targeting peptide is shown in SEQ ID No: 1, and is named as FcMR10.

[0011] Preferably, the amino acid sequence of the targeting peptide is shown as SEQ ID No: 6, named FcMR2.

[0012] The application also claims a nucleotide sequence encoding the FcMR10 targeting peptide, which is shown as SEQ ID No: 2 or SEQ ID No: 7.

[0013] Further, the application also provides the use of the targeting peptide in the preparation of a drug for targeting in vitro induced porcine bone marrow-derived dendritic cells (BM-DCs).

[0014] Preferably, the drug is a vaccine.

[0015] More preferably, the vaccine is a subunit vaccine.

[0016] A recombinant protein comprising the porcine antigen-presenting cell targeting peptide and an antigen protein or peptide segment of a pathogenic microorganism, wherein the porcine antigen-presenting cell targeting peptide is located at the C-terminal or N-terminal of the antigen protein or peptide segment of the pathogenic microorganism.

[0017] Preferably, the pathogenic microorganism is a pathogenic bacterium or a virus, and more preferably, the virus is porcine reproductive and respiratory syndrome virus (PRRSV) and African swine fever virus (ASFV).

[0018] Preferably, the antigen protein is PRRSV GP3, ASFV P22, and ASFV P54.

[0019] A method for preparing a recombinant protein, comprising transforming the expression vector into the host cell, and inducing expression to obtain the recombinant protein.

[0020] A pharmaceutical composition for preventing or treating a pig disease, comprising the above recombinant protein and a pharmaceutically acceptable carrier.

[0021] Preferably, the carrier comprises one or more of a vaccine adjuvant, a buffer, and an emulsifier.

[0022] Based on the above technical solutions, the application has the following beneficial effects:

[0023] First, the application uses phage random peptide library screening technology to screen out porcine antigen-presenting cell dominant binding polypeptides FcMR10 (shown as SEQ ID NO. 1) and FcMR2 (shown as SEQ ID NO. 6), and through detection of the fusion mode antigen zsGreen with the targeting peptide, it is found that the targeting peptide can strongly mediate the binding of the antigen to the antigen-presenting cell, has good targeting property, and effectively enhances the immune response to the model antigen, thereby providing a material basis for the development of a porcine targeting vaccine.

[0024] Second, the FcMR10 obtained by the application is connected and fused with PRRSV GP3, ASFV P22 and ASFV P54 respectively after being connected with different connecting peptides to construct an expression recombinant protein, and the immune response of the prepared recombinant protein to PRRSV GP3, ASFV P22 and ASFV P54 is significantly improved after the piglets are immunized, so the antigen presenting cell targeting peptide FcMR10 prepared by the application has APC targeting property in the pig body, and has important significance for the research and development of future pig APC targeting recombinant subunit vaccine. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 . Indirect ELISA was used to detect the enrichment of phages in the panning process.

[0026] Figure 2 . The results of indirect ELISA for detecting the reactivity of screened phage clones with FcMR: Clone 1: phage 1; Clone 2: phage 2; Clone 3: phage 3; Clone 4: phage 4; Clone 5: phage 5; Clone 6: phage 6; Clone 7: phage 7; Clone 8: phage 8; Clone 9: phage 9; Clone 10: phage 10.

[0027] Figure 3 . Agarose gel electrophoresis was used to identify the single-stranded DNA of the extracted phage clones: 1: phage 1; 2: phage 2; 3: phage 3; 4: phage 4; 5: phage 5; 6: phage 6; 7: phage 7; 8: phage 8; 9: phage 9; 10: phage 10.

[0028] Figure 4 . SDS-PAGE and Western blot were used to identify the purification results of zsGreen-12 peptide fusion proteins: 1-8: corresponding to the purified zsGreen-FcMR-2, 4, 5, 6, 7, 8, 9 and 10 peptide fusion proteins, respectively. (A) SDS-PAGE was used to identify the purified 12 peptide fusion proteins; (B) Western blot was used to identify the purified 12 peptide fusion proteins, and the primary antibody was mAb His; (C) Western blot was used to identify the purified 12 peptide fusion proteins, and the primary antibody was mAb zsGreen.

[0029] Figure 5 . The results of fluorescence of zsGreen-12 peptide fusion proteins adsorbed to APCs: (A): PAMs cell adsorption test; (B): BM-DCs cell adsorption test.

[0030] Figure 6. Detection of lysosome after PAMs extraction of the fusion protein adsorbed zsGreen.

[0031] Figure 7 . Detection of antibody level in the serum after immunization by indirect ELISA: (A) detection of zsGreen antibody level in the serum of piglets in each immunization group after 7 days of immunization; (B) detection of zsGreen antibody titer in the serum of piglets in each immunization group after 14 days of immunization; "*" indicates difference (P < 0.05); "**" indicates significant difference (P < 0.01); "***" indicates extremely significant difference (P < 0.001).

[0032] Figure 8 . Detection of IFN-γ secretion level of PBMC of piglets after 14 days of immunization by ELISpot: (A) representative ELISpot results; (B) difference analysis results. "ns" indicates no significant difference (P > 0.05); "*" indicates difference (P < 0.05).

[0033] Figure 9 . Detection of antibody level in the serum of piglets after 14 days of immunization by indirect ELISA.

[0034] Figure 10 . Identification of the purification results of PRRSV and ASFV derived recombinant antigens using FcMR10 peptide fusion by SDS-PAGE and Western blot: (A) identification of the purified recombinant proteins by SDS-PAGE; (B) identification of the purified recombinant proteins by Western blot, primary antibody mAb His. 1-3: corresponding to the purified NT11-VAD2-P54-LEAEK2LE-FcMR10-LE6His, NT11-VAD2-P22-GGGGS2-LE-FcMR10-LE6His, His-VAD2-GP3ΔIDR-VAD2-FcMR10 recombinant proteins, respectively.

[0035] Figure 11 . Detection of antibody level in the serum after immunization by indirect ELISA: (A) detection of P54 antibody level in the serum of piglets in each immunization group after 21 days of immunization (B) detection of P22 antibody level in the serum of piglets in each immunization group after 21 days of immunization (C) detection of GP34 antibody level in the serum of piglets in each immunization group after 21 days of immunization.

[0036] Figure 12 . Detection of IFN-γ secretion level of PBMC of piglets after 21 days of immunization by ELISpot: (A) representative ELISpot results; (B) difference analysis results. DETAILED DESCRIPTION

[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: Screening of porcine FcMR-specific targeting 12 peptides and expression of model fusion proteins

[0039] 1.1 Related plasmid vectors, bacterial strains, phage display peptide libraries, and antibodies

[0040] Expression vectors and strains: E. coli The ER2738 host bacteriophage and Ph.D.-12 peptide phage display library were purchased from NEB; the pET-28a-zsGreen plasmid vector was preserved in our laboratory.

[0041] Proteins and Antibodies: Anti-M13 Major Coat Protein antibody was purchased from Santa Cruz, zsGreen monoclonal antibody was prepared in our laboratory; pET-28a-FcMR recombinant protein and monomeric zsGreen green fluorescent protein were prepared and preserved in our laboratory.

[0042] 1.2 Screening of phage random 12-peptide libraries for FcMR-specific targeting of 12 peptides

[0043] 1.2.1 Activation and preservation of host bacterium ER2738

[0044] (1) Bacterial activation: Take out the ER2738 glycerol bacteria stored at -80℃ and thaw them on ice. Take the thawed glycerol bacteria from the laminar flow hood and add the bacterial solution 1:100 to a sterile test tube containing 5 mL of Tet-LB liquid medium. Shake overnight at 37℃ and 220 r / min.

[0045] (2) Preservation of bacterial culture: Open the overnight activated bacterial culture in a clean bench, aspirate 600 μL of bacterial culture into a sterile 1.5 mL microcentrifuge tube, add 400 μL of autoclaved 50% glycerol, mix well and store at -80℃.

[0046] (3) Flat plate streaking to isolate monoclonal colonies: Take the Tet-LB plate in the clean bench, light the alcohol lamp, sterilize the inoculation ring with high temperature, and after the inoculation ring cools down, dip the overnight activated ER2738 bacterial solution in the plate, and parallelly streak the plate to make ER2738 grow into monoclonal colonies on the plate. After streaking, the plate is cultured at 37°C overnight, and the next day, the growth of the monoclonal colonies is observed. When the colonies grow to the appropriate size, the plate is taken out and stored at 4°C in the dark for subsequent experiments (the streaked plate can be stored at 4°C for a short period of time, and within 2 weeks, it can be used to pick monoclonal colonies. Before picking the colonies, observe the plate for contamination.

[0047] 1.2.2 Phage random 12-peptide library screening FcMR specific targeting 12-peptide

[0048] Using the previously expressed porcine recombinant FcMR protein as the screening agent, the NEB Ph.D.-12 phage display peptide library is biopanned. The number of phages input in each round of panning is the same, which is fixed at 2 x 1011pfu. After three rounds of panning, the panning results are identified. 11 pfu, and the panning results are identified after three rounds of panning.

[0049] 1.2.2.1 First round of biopanning of phage random 12-peptide library

[0050] (1) Coating target molecules: Take the recombinant FcMR protein at -80°C, melt it at 4°C, and after melting, dilute the recombinant FcMR protein with NaHCO3 coating solution. Add 15 μg of recombinant protein to a high-adsorption 96-well plate and shake slowly on a 4°C shaker overnight to evenly coat the bottom of the wells. At the same time, select an unrelated control protein to coat the plate.

[0051] (2) Shake the bacteria: The next morning, open the ER2738 streak plate in the clean bench, and use a sterilized small white gun head to pick a single colony of appropriate size from the plate. Inoculate the colony into a 250 mL conical flask containing 20 mL of Tet-LB, and incubate at 37°C with vigorous shaking at 250 r / min for 4-5 h. Before use, take 200 μL of bacterial solution into a 96-well adsorption plate, and use an enzyme-labeled instrument to read OD 600 nm, and OD 600 0.5-0.6 can be used.

[0052] (3) Blocking: Take out the overnight-coated plate, discard the coating solution, and invert the plate on a clean absorbent paper to absorb the residual liquid. Add 200 μL of panning blocking solution to the wells, and block at 37°C for more than 1 h to ensure that the bottom and sides of the wells are fully blocked and non-specific adsorption is minimized.

[0053] (4) Wash the plate: Discard the blocking solution, wash the plate with 0.1% TBS'T solution 200 μL / well, and wash the plate quickly for 6 times. Avoid drying the wells during the washing process. Each time the plate needs to be dried on absorbent paper, and a new clean absorbent paper is used each time to avoid contamination.

[0054] (5) Incubation: Take out the stored phage random 12 display peptide library at -20°C, and pipette 10 μL into 100 μL TBS'T for dilution. Add the diluted phage into the blocked plate, and slowly shake the plate on a shaker at room temperature for 1 h to allow the phage to fully contact and bind to the coated proteins in the wells.

[0055] (6) Plate washing: Discard the phage that does not bind to the proteins, and wash the plate with 200 μL / well of 0.1% TBS'T, and quickly wash the plate for 10 times. Each time of washing requires to pat the plate on a water-absorbing paper, and a new clean water-absorbing paper is used each time to avoid cross contamination.

[0056] (7) Elution: Add 100 μL of triethylamine eluent to each well, and stand at room temperature for 10 min. Blow and suck the eluent for multiple times to fully elute the bound phage, and transfer the eluent to a 1.5 mL microcentrifuge tube.

[0057] (8) Neutralization: Quickly add 100 μL of Tris-HCl neutralization solution to the microcentrifuge tube to neutralize the eluent.

[0058] (9) Reserve 20 μL of the eluent for titer determination, and add the remaining eluent to the ER2738 bacterial liquid for amplification and enrichment.

[0059] 1.2.2.2 Phage titer determination

[0060] (1) Before determining the titer, heat the top agar stored at room temperature in a microwave oven, and store in a 50°C oven to avoid solidification.

[0061] (2) Preheat the IPTG-Xgal plate in advance, and use one plate for each dilution gradient.

[0062] (3) Dilute the phage with Tet-LB liquid medium 10 times in a clean bench: according to the needs, place 8-12 sterile 1.5 mL microcentrifuge tubes, and label them. Add 200 μL of Tet-LB liquid medium to each tube, add 20 μL of phage eluent to the first microcentrifuge tube, vortex to mix well, and use a sterilized gun head to pipette 20 μL of the mixture into the next microcentrifuge tube, continue to mix, and dilute 10 times in gradient. A new sterilized gun head is used for each dilution. Generally, the dilution factor of the unamplified eluent is 10 2 -10 5 , and the dilution factor of the amplified eluent is 10 8 -10 12 .

[0063] (4) Pipette OD 600ER2738 bacteria solution with value 0.5-0.6 was added to a sterile 5 mL centrifuge tube, 200 μL bacteria solution per tube.

[0064] (5) 10 μL of the diluted bacteriophage solution was taken and added to the bacteria solution, labeled, and vortexed to mix quickly.

[0065] (6) The melted top layer agar was taken with a Pasteur pipette and added to the 5 mL centrifuge tube containing the mixed bacteria solution, 3 mL per tube. After addition, the tube was inverted to mix, and air bubbles were avoided as much as possible during the mixing process.

[0066] (7) The preheated IPTG-Xgal plate was taken out, and the top layer agar containing the bacteriophage and ER2738 bacteria solution was poured into the plate. The plate was tilted and shaken to evenly spread the agar on the surface of the plate.

[0067] (8) The plate was placed upright at room temperature for 5-10 min. After the agar in the plate solidified, the plate was placed at 37 °C and inverted for overnight culture.

[0068] (9) The next morning, the plate was observed, and the plate with a blue plaque number of less than 100 was selected for counting. The bacteriophage titer was calculated.

[0069] (10) Bacteriophage titer calculation: the number of bacteriophages on the plate was multiplied by the dilution factor of the bacteriophage on the plate to obtain the plaque forming unit (pfu) of the 10 μL bacteriophage determined, which was the bacteriophage titer.

[0070] 1.2.2.3 Amplification and enrichment of the first round of eluate

[0071] (1) The first round of eluate was added to 20 mL of OD 600 ER2738 bacteria solution with a value of 0.5-0.6 in an ultraclean bench, and incubated at 37 °C on a constant temperature shaker at 250 r / min for 4.5 h.

[0072] (2) The bacteria were collected in the ultraclean bench, and the bacteria solution was collected in a clean 50 mL centrifuge tube. Centrifugation was performed at 4 °C and 8000 r / min for 15 min. The supernatant was poured into a new clean centrifuge tube and centrifuged again.

[0073] (3) 80% of the supernatant in the centrifuge tube was carefully taken and transferred to a new 50 mL centrifuge tube. PEG-NaCl was added according to the ratio of supernatant: PEG-NaCl = 6:1, and the bacteriophage was precipitated at 4 °C overnight.

[0074] (4) The next day, the precipitated bacteriophage was centrifuged at 4 °C and 6000 r / min for 30 min with a horizontal rotor.

[0075] (5) Carefully discard the supernatant from the super-clean bench to avoid destroying the white phage precipitate.

[0076] (6) Resuspend the precipitate with 1 mL sterile TBS, mix well by pipetting, and transfer the suspension to a sterile 1.5 mL microcentrifuge tube. Centrifuge at 14000 r / min for 5 min at 4°C to precipitate the residual cells.

[0077] (7) Carefully pipette the supernatant and transfer it to a new sterile 1.5 mL microcentrifuge tube. Add 1 / 6 volume of PEG-NaCl and mix well by pipetting.

[0078] (8) Incubate on ice for 1 h to re-precipitate the phage supernatant.

[0079] (9) Centrifuge at 14000 r / min for 10 min at 4°C. Discard the supernatant from the super-clean bench.

[0080] (10) Centrifuge briefly at 14000 r / min for 2 min at 4°C. Carefully pipette the residual supernatant from the super-clean bench.

[0081] (11) Resuspend the precipitate with 200 μL sterile TBS and mix well by pipetting.

[0082] (12) Centrifuge briefly at 4°C for 30-60 s. Carefully transfer the supernatant to a new sterile centrifuge tube from the super-clean bench, which is the eluate after amplification.

[0083] (13) Pipette 20 μL of the eluate after amplification to determine the phage titer. The remaining eluate is stored at 4°C and used as the input phage for the second round of panning.

[0084] 1.2.2.4 Second and third rounds of biological panning and amplification of the eluate

[0085] The second and third rounds of biological panning follow the procedure of 1.2.2.1, except that the input phage for the second round of panning is the amplified product of the eluate from the first round of panning, and the input phage for the third round of panning is the amplified product of the eluate from the second round of panning. The washing buffer is changed to 0.3% and 0.5% TBS'T buffer in the second and third rounds of panning, respectively, to increase the panning pressure and thus reduce non-specific binding. The phage titer of the eluate is determined after each round of panning, and the eluate after the third round of panning does not need to be amplified and enriched.

[0086] Table 1 Phage recovery titer and recovery rate during the screening process

[0087] ,

[0088] As shown in Table 1, after three rounds of biological panning, the titer of the recovered phage increased from 6.4 x 10 4 pfu in the first round to 6.2 x 107 pfu, while the recovery rate also increased from 0.32 x 10 -6 pfu in the first round to 310 x 10 -6 pfu in the third round. In addition, the binding of phage to the negative control protein decreased from 3.8 x 10 4 pfu in the first round to 1.2 x 10 4 pfu in the third round, indicating that the phage maintained a low level of binding to the negative control protein, excluding non-specific binding of the phage to the FcMR.

[0089] The amplified phage solution in the third round of panning was subjected to indirect ELISA to detect the enrichment of FcMR-specific binding phage after the third round of screening. The results are shown in Figure 1 After the third round of panning, FcMR-specific phage was obviously enriched, consistent with the data in Table 1, indicating that FcMR-specific binding phage was successfully enriched after three rounds of panning.

[0090] 1.3 Identification of positive phage clones

[0091] 1.3.1 Amplification of plaques:

[0092] (1) Store the IPTG-Xgal plate used to determine the titer of the third round eluate at 4°C, and select a plate with less than 100 blue plaques for plaque amplification.

[0093] (2) Open the ER2738 streak plate in the clean bench, and use a sterilized white gun head to pick single colonies of appropriate size and inoculate them into a test tube containing 5 mL Tet-LB, and shake at 37°C, 220 r / min overnight.

[0094] (3) The next day, transfer the ER2738 to 5 mL test tubes at 1:100, add 1 mL Tet-LB to each test tube, and transfer a total of 10 tubes.

[0095] (4) Open the IPTG-Xgal plate in the clean bench, and carefully pick single blue plaques of appropriate size from the plate with a sterilized white gun head and add them to the test tube. Pick 10 blue plaques and label them.

[0096] (5) Incubate in a constant temperature shaker at 37°C, 250 r / min for 4-5 h.

[0097] (6) Collect the bacterial solution in a sterile 1.5 mL microcentrifuge tube in the clean bench, centrifuge at 4°C, 14000 r / min for 5 min.

[0098] (7) Transfer the supernatant to a new centrifuge tube and centrifuge again. Carefully pipette 80% of the supernatant from the top of the centrifuge tube into a new sterile centrifuge tube. This is the plaque amplification stock.

[0099] (8) Divide the plaque amplification stock into two, 500 μL of which is used for ssDNA extraction for sequencing and the rest is used for ELISA detection.

[0100] 1.3.2. ELISA to identify the binding ability of phage clones to FcMR protein:

[0101] (1) Live bacteria: Open the ER2738 streak plate in a clean bench, and pick a single colony of appropriate size with a sterile white gun tip and inoculate it into a test tube containing 10 mL Tet-LB, and shake at 37°C, 220 r / min overnight.

[0102] (2) Expansion: The next day, transfer the overnight activated ER2738 to a conical flask containing 20 mL Tet-LB at 1:100 for expansion.

[0103] (3) Amplification of phage clones: Measure the OD of the bacterial solution after 3-4 h 600 When the OD 600 When the OD is 0.5-0.6, add the plaque amplification stock of 1.3.1 to the ER2738 bacterial solution, and incubate at 37°C, 250 r / min for 4-5 h.

[0104] (4) Enrichment: Enrichment of phage clones is the same as 1.2.2.3.

[0105] (5) ELISA detection:

[0106] Coating: Dilute the recombinant FcMR protein with coating solution to a final concentration of 4 μg / mL, 100 μL / well, and coat the 96-well plate at 4°C overnight.

[0107] Washing: Place the 96-well plate in the plate washer and wash 3 times with 0.05% PBS'T.

[0108] Blocking: Block with 2.5% skim milk powder, 200 μL / well, incubate at 37°C for 2 h, and wash the plate.

[0109] Primary antibody: Dilute the enriched phage clone amplification solution with TBS 1:100, add 100 μL per well, and set PBS negative controls for each group of phage clones, incubate at 37°C for 1 h, and wash the plate.

[0110] Secondary antibody: HRP-labeled anti-M13 phage antibody, diluted 1:1000, 100 μL / well, incubate at 37°C for 1 h, and wash the plate.

[0111] Color development: add 100 μL TMB color developing solution to each well, incubate at room temperature for 15 min in the dark.

[0112] Termination: add 50 μL 3M H2SO4 termination solution to each well to terminate the color development reaction.

[0113] Reading: use an enzyme marker to read the 450 nm absorbance value, and analyze the results.

[0114] (6) Result analysis: analyze the data according to the OD 450 value, and preliminarily determine the binding force of the selected phage clones and FcMR molecules.

[0115] The results are shown in Figure 2 Table 1. The OD 450 values of the 10 phage clones selected for amplification were all above 1.0, indicating that they had good specific binding with the recombinant FcMR protein.

[0116] 1.4 Extraction and sequencing of ssDNA of positive phage clones

[0117] Select phage clones with positive OD 450 values in ELISA results for single-stranded DNA extraction and sequencing.

[0118] 1.4.1 Extraction of ssDNA of positive phage clones:

[0119] (1) In a clean bench, take 500 μL of the phage plaque amplification stock solution of 1.3.1 into a new clean 1.5 mL centrifuge tube.

[0120] (2) Take 200 μL of autoclaved PEG-NaCl solution and add it to the centrifuge tube, mix well by repeatedly blowing, and centrifuge at 4°C, 14000 r / min for 10 min.

[0121] (3) Discard the supernatant and centrifuge again for 2 min. Slowly aspirate the residual supernatant to avoid damaging the precipitate.

[0122] (4) Resuspend the precipitate with 100 μL of NaI buffer and vortex to ensure complete dissolution of the precipitate.

[0123] (5) Take 250 μL of anhydrous ethanol and add it to the buffer, mix well by blowing, and let it stand at room temperature for 10 min. Centrifuge at 4°C, 14000 r / min for 10 min.

[0124] (6) Discard the supernatant and wash the precipitate with 200 μL of 70% ethanol and centrifuge.

[0125] (7) Discard the supernatant, and invert the centrifuge tube on absorbent paper to absorb the residual supernatant, and dry for 10-30 min to completely volatilize the ethanol.

[0126] (8) Resuspend the precipitate with 30 μL dd H2O, which is the extracted single-stranded DNA of the positive phage clone.

[0127] (9) Prepare 1% agarose gel, and take 5 μL of the extract for 1% agarose gel electrophoresis to identify the purity of the extracted ssDNA.

[0128] Extract the single-stranded DNA of the selected 10 phage clones for 1% agarose gel electrophoresis. The genome of M13 phage has 6407 bases, but since it is single-stranded DNA, and the nucleic acid Maker is double-stranded DNA, the band size of M13 ssDNA is between 3000-4000 bp in agarose gel electrophoresis. The results are shown in Figure 3 , and the extracted 10 ssDNA have obvious specific bands at about 3000 bp, which is consistent with the expected size, and the ssDNA of the phage clone is successfully extracted.

[0129] 1.4.2. Sequencing of ssDNA of the positive phage clone:

[0130] Send the extracted ssDNA and the upstream and downstream primers provided in the NEB random 12-peptide phage display library kit to Xi'an Qikexi Biological Company for sequencing to analyze the nucleotide sequences of the selected 10 phage clones. The primer sequences are shown in Table 2.

[0131] Table 2 Primer sequences for sequencing of M13 phage ssDNA

[0132] .

[0133] 1.5 Expression and purification of recombinant zsGreen-12-peptide fusion protein

[0134] 1.5.1 Construction of recombinant zsGreen-12-peptide fusion protein expression plasmid

[0135] Send the sequenced twelve-peptide nucleotide sequence and the pET-28a-zsGreen plasmid vector to Xi'an Qikexi Biological Company, synthesize the short peptide sequence at the C-terminal of the zsGreen sequence, obtain the recombinant zsGreen-12-peptide fusion protein expression plasmid, and number the fusion protein expression plasmid according to the specific binding short peptide number in order as zsGreen-FcMR-1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0136] 1.5.2 Induced expression of recombinant zsGreen-12-peptide fusion protein

[0137] 1.5.2.1 Plasmid transformation

[0138] (1) Take out from -80℃ E. coli BL21(DE3) expression competent cells, melt on ice, after melting, add 1 μL zsGreen-12 peptide recombinant plasmid in the clean bench.

[0139] (2) Ice water bath for 30 min.

[0140] (3) Metal bath 42℃, heat shock 90s.

[0141] (4) Ice water bath for 2 min.

[0142] (5) Add 500 μL of anti-LB in the clean bench, 37℃, 220r / min, shake for 1h.

[0143] (6) Take 100 μL of bacteria solution in the clean bench and evenly spread on K + LB plate, evenly spread with spreader, invert in 37℃ constant temperature incubator, and culture overnight for 12-14h.

[0144] 1.5.2.2 Shake culture, IPTG induced expression

[0145] (1) Select a single colony of appropriate size on the LB plate, carefully pick it up with a sterilized small white gun head and add it to a sterile test tube containing about 10 mL of K + liquid LB medium, 37℃, 220r / min, and place it in a 37℃ shaker for 12-14h.

[0146] (2) The next day, transfer 1:100 of the bacteria solution to a 500 mL conical flask, 37℃, 220r / min.

[0147] (3) After shaking for 1-2h, take 200 μL of bacteria solution, and read the OD 600 value with a microplate reader.

[0148] (4) When the OD value of the bacteria solution is 0.6-0.8, add IPTG at a ratio of 1:2000, 37℃, 220r / min, shake for 6-8h.

[0149] (5) Place the cultured bacteria solution in a centrifuge tube, centrifuge at 6000rpm / 5min / 4℃, discard the supernatant, and resuspend the precipitate (25 mL Buffer A solution to resuspend the bacteria solution).

[0150] (6) Place the resuspended bacteria solution on an ice-water mixture and break it with an ultrasonic disrupter: power 40%, working time 3s, pause 3s, ultrasonic for 40min.

[0151] (7) The ultrasonic bacteria liquid is centrifuged at 6000 rpm / 10 min / 4°C, and the supernatant and inclusion bodies are divided.

[0152] 1.5.3 Purification of recombinant zsGreen-12 peptide fusion protein

[0153] (1) The supernatant is transferred to a new 50 mL centrifuge tube and filtered through a 0.45 μm filter membrane.

[0154] (2) Ni column equilibration: Add 2 times the column volume of ultrapure water to the Ni purification column and slowly drop it out, which can wash away the ethanol in the Ni filler; then add 5 times the column volume of Buffer A solution to equilibrate the Ni filler.

[0155] (3) Incubate the protein: Add the filtered supernatant to the Ni purification column, control the flow rate at 4-5 s / drop, and slowly flow out, collect the effluent, and repeatedly add the effluent to the purification column 3-5 times to fully incubate and combine the fusion protein with the Ni filler.

[0156] (4) Elute the protein: Use 5 mM, 10 mM, 50 mM, 100 mM, and 250 mM imidazole to elute in sequence.

[0157] (5) Dialysis: Add the eluted fusion protein to an 8-14 KDa dialysis bag, use PBS as the dialysis solution, and dialyze overnight to completely remove the imidazole in the eluent.

[0158] (6) BCA method to determine protein concentration: After dialysis, filter the protein through a 0.45 μm filter membrane, dilute the standard with PBS according to the instructions of the BCA protein assay kit, take 20 μL of the protein to be tested and the diluted standard into the ELISA strip, add 200 uL of working solution, react at 37°C for 15 min, and measure the OD 562 value at a spectrophotometer, calculate the protein concentration by the standard curve of protein concentration, and store at -80°C.

[0159] 1.5.4 SDS-PAGE detection of expression protein purification results

[0160] (1) Sample processing: take 40 μL of whole bacteria liquid after induction, supernatant after ultrasonic, inclusion body precipitate after ultrasonic, add 10 μL of 5x SDS loading buffer, boil for 10 min to denature the protein, run the gel, and identify the expression of recombinant FcMR protein; take 40 μL of supernatant before purification, effluent and 5 mM, 10 mM, 50 mM, 100 mM, 250 mM imidazole eluent, add 10 μL of 5x SDS loading buffer, boil for 10 min, run the gel, and identify the Ni column elution condition of the recombinant protein; take 40 μL of recombinant protein solution after purification, add 10 μL of 5x SDS loading buffer, boil for 10 min, run the gel, and identify the purity of the protein.

[0161] (2) Gel preparation: assemble the gel plate in advance, and prepare 12% electrophoresis separation gel and 5% electrophoresis stacking gel in turn, and let the gel stand at room temperature for 1-2 h to completely solidify.

[0162] (3) Electrophoresis: assemble the electrophoresis instrument in advance, pour 1x Running Buffer, carefully remove the gel comb, and add 2 μL of protein Maker and 15 μL of sample to the gel hole respectively. After adding the sample, run the gel, 80V voltage makes the protein migrate in the stacking gel, and then adjust to 100V voltage to make the protein migrate in the separation gel, and determine the end time of electrophoresis according to the size of the protein and the position of the protein Maker band.

[0163] (4) Staining: prepare the staining box, pour 25 mL of Coomassie Brilliant Blue staining solution into the box, put the separation gel into the box, and slowly shake on the shaker to uniformly stain, and stain for 30 min-2 h.

[0164] (5) Decolorization: transfer the stained separation gel to the decolorization box, add about 25 mL of decolorization solution, slowly shake on the shaker to decolorize, and observe that the part of the separation gel without protein is completely decolorized to colorless, which indicates that the decolorization is complete.

[0165] (6) Analysis of results: observe the blue protein band on the decolorized separation gel, and judge the expression of the recombinant protein, the purification condition and the purity of the protein after dialysis.

[0166] The fusion protein is numbered as zsGreen-FcMR-1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 peptide segment fusion proteins in order according to the specific binding of short peptides. The zsGreen protein itself is a soluble expression protein, and after IPTG induction and ultrasonic treatment of the bacteria liquid, zsGreen is released into the supernatant, and after Ni column purification, it can be directly dialyzed into PBS. After the FcMR specific 12 peptides are screened and fused and expressed with zsGreen, the fusion protein is still a soluble expression protein, which is PBS dialyzed after Ni column purification. The purity of the fusion protein after purification is identified by SDS-PAGE, and the results are as follows Figure 4(A), successfully expressed zsGreen-FcMR-2, 4, 5, 6, 7, 8, 9, 10 peptide segment fusion protein, band size in about 30 KDa consistent with expectations, high purity protein.

[0167] 1.5.5 Western blot detection of protein expression specificity

[0168] The sample is the purified recombinant zsGreen-12 peptide fusion protein, the primary antibody is His-tag antibody and zsGreen monoclonal antibody, and the secondary antibody is HRP labeled goat anti-mouse IgG.

[0169] (1) Sample processing: same as 1.5.4.

[0170] (2) Electrophoresis: gel preparation, SDS-PAGE, method same as 1.5.4.

[0171] (3) Membrane transfer: wet transfer, classic "sandwich" method to assemble filter paper, gel, PVDF membrane, after assembly, ice water bath 1h 100V constant voltage transfer membrane.

[0172] (4) Blocking: after membrane transfer, carefully take out the membrane, put it into 5% skim milk blocking solution, slowly shake on the shaker at room temperature for 1-2h.

[0173] (5) Add primary antibody: dilute primary antibody with PBS'T 1:5000, incubate slowly on the incubator, room temperature for 1h.

[0174] (6) Wash the membrane: take out the membrane, put it into a washing box containing 0.05% PBS'T, shake the membrane on the shaker, 5min / time, wash 3 times.

[0175] (7) Secondary antibody: dilute HRP labeled Goat Anti-Mouse secondary antibody with PBS'T 1:5000, incubate slowly on the incubator, room temperature for 1h.

[0176] (8) Take out the membrane, put it into a washing box containing 0.05% PBS'T, shake the membrane on the shaker, 5min / time, wash 3 times.

[0177] (9) Color development: add appropriate amount of H2O2 to ECL luminescent solution, put the membrane into the dark incubator for 1min, use Bio-Rad gel imaging system for color development.

[0178] (10) Analyze the results: observe the color development band of the membrane, judge the purification results of the recombinant protein.

[0179] The fusion protein zsGreen-FcMR is labeled with His tag, and the purified fusion protein can be detected by His antibody and zsGreen monoclonal antibody respectively through Western blot, and the results are as follows Figure 4 (B) and Figure 4 (C), Western blot shows that His antibody and zsGreen monoclonal antibody can detect obvious specific bands at 25-35 KDa, which is consistent with the results of SDS-PAGE.

[0180] The present application uses recombinant FcMR protein as target molecule, and uses phage display technology to screen recombinant FcMR protein, successfully screens FcMR specific phage, and through amplification and sequencing, 10 FcMR specific 12 peptide amino acid sequences are obtained; through fusion expression of zsGreen green fluorescent protein and screened FcMR specific 12 peptide, 8 soluble zsGreen-12 peptide fusion proteins are successfully expressed, which are used for subsequent FcMR specific 12 peptide targeting verification.

[0181] Example two Identification, verification and application of porcine FcMR specific targeting 12 peptide

[0182] 2.1 Related cells, antibodies and proteins

[0183] Cells: Porcine alveolar macrophages PAMs and porcine bone marrow-derived dendritic cells BM-DCs are preserved by the laboratory. Competent cells BL21 (DE3) are purchased from Beijing Transgene Biotechnology Co., Ltd.

[0184] Proteins and antibodies: Rab7 antibody is purchased from Santa Cruz. ZsGreen protein is prepared by the laboratory. P54 monomer protein, P22 monomer protein and GP3 monomer protein are preserved by the laboratory.

[0185] 2.2 Adsorption and internalization of zsGreen-12 peptide fusion protein by APCs

[0186] 2.2.1 Cell preparation

[0187] (1) Take out the frozen PAMs and induced porcine BM-DCs from the liquid nitrogen tank, and place the frozen tube in a 37℃ water bath.

[0188] (2) Gently shake the frozen tube to quickly melt it, centrifuge at 1000 r / min for 10 min at room temperature.

[0189] (3) Open the frozen tube in the clean bench, discard the frozen liquid, resuspend the cell pellet with 10% FBS 1640 medium, and count the cells.

[0190] (4) The cell solution was mixed by blowing, 1 x 10 6 The cell solution was mixed by blowing, 1 x 10

[0191] 2.2.2 Adsorption of the zsGreen-12 peptide fusion protein

[0192] (1) The zsGreen-12 peptide fusion protein and the zsGreen monomer protein prepared in the previous step were taken out at -80°C and thawed on ice.

[0193] (2) The proteins were filtered into sterile 1.5 mL EP tubes using a high-pressure sterilized 0.22 μm filter in a clean bench, and sterilized by filtration.

[0194] (3) The zsGreen-12 peptide fusion protein was added to the cell culture solution, 5 μg / well, 37°C, for 30 min, using the zsGreen monomer protein as a control protein.

[0195] (4) The cell culture plate was taken out, and the cells in each well were washed with PBS to remove excess unbound protein, and washed three times. When washing the cells each time, the PBS was collected into a 1.5 mL EP tube, centrifuged at 1000 r / min for 10 min, and the PAMs in the supernatant were collected. The cells were resuspended in PBS and added back to the cell culture plate.

[0196] 2.3 Identification of the ability of the zsGreen-12 peptide fusion protein to target APCs

[0197] 2.3.1 Fluorescence analysis of the ability of the zsGreen-12 peptide fusion protein to target APCs

[0198] The cell culture plate was placed on an inverted fluorescence microscope, the laser was adjusted to the green fluorescence excitation band, and the green fluorescence of each group of proteins was observed under the microscope. The same parameters were adjusted for photographing, and the pictures were arranged to analyze the adsorption results.

[0199] The results are shown in Figure 5 (A) and (B), the zsGreen-FcMR-2 peptide fusion protein and the zsGreen-FcMR-10 peptide fusion protein adsorbed PAMs and BM-DCs, respectively, and the cells had green fluorescence. The fluorescence of the zsGreen-FcMR-2 peptide fusion protein was stronger than that of the zsGreen-FcMR-10 peptide fusion protein, and it was preliminarily determined that among the 10 FcMR-specific 12 peptides screened, FcMR-2 and FcMR-10 peptides had APC targeting properties.

[0200] 2.3.2 Western blot to verify the ability of zsGreen-12 peptide fusion protein to target APCs

[0201] (1) Adsorption assay on PAMs cells using zsGreen monomer protein and fusion protein, method same as 2.2.

[0202] (2) After adsorption, add proteinase K to the cell supernatant, 50 μg / mL, room temperature for 30 s to remove unbound proteins and proteins adsorbed on the cell surface but not internalized.

[0203] (3) Wash the cells with PBS 3 times, centrifuge the cells at 5000 r / min for 5 min.

[0204] (4) Add 1000x cocktail to Fractionation buffer before use.

[0205] (5) Resuspend the centrifuged cell pellet with 1 mL pre-cooled Fractionation buffer, mix well by blowing.

[0206] (6) Transfer the cell suspension to a cell grinder and grind on ice for 20 min. During the grinding process, take a small amount of grinding liquid and observe the grinding under a microscope. Ensure that more than 90% of the cells are broken.

[0207] (7) Centrifuge at 8000 r / min for 10 min at 4°C to remove broken cell debris and unbroken cells.

[0208] (8) Carefully pipette the supernatant and transfer it to a new 1.5 mL EP tube and centrifuge again.

[0209] (9) Transfer the supernatant to a 13 mL centrifuge tube, add Fractionation buffer to fill the centrifuge tube, and balance the centrifuge tube.

[0210] (10) Ultracentrifuge at 100,000 g for 1 h. The precipitate is the isolated endosome and lysosome.

[0211] (11) Western blot to detect the internalization of the fusion protein, method same as 1.5.5. The extracted endosome and lysosome were lysed with 100 μL 2x Sample buffer, boiled in a water bath for 15-20 min to fully denature the sample, and cooled to room temperature after boiling. Instantly, 35 μL / well was added to the gel and the membrane was transferred. zsGreen monoclonal antibody was diluted 1:1000 as the primary antibody, and HRP-labeled sheep anti-mouse IgG antibody was diluted 1:5000 as the secondary antibody.

[0212] The results of fluorescence analysis of the fusion protein adsorbed to APCs preliminarily determined that the FCMR-2 peptide segment and the FCMR-10 peptide segment have targeting properties for APCs. If the peptide segment targets the zsGreen protein to APCs in the fusion protein, thereby enhancing the phagocytic internalization of the protein by APCs, then the zsGreen protein will be present in the lysosomes of the cells. Compared with PBMCs and DCs, PAMs cells are easier to isolate, so endosomes and lysosomes were extracted from the PAMs treated with the fusion protein adsorption, Rab7 was used as a lysosome marker, and the difference in the content of zsGreen protein in the extracted lysosomes was detected by Western blot using a zsGreen monoclonal antibody. The results are shown in Figure 6 After the PAMs were adsorbed with the zsGreen-FcMR-2 peptide segment fusion protein and the zsGreen-FcMR-10 peptide segment fusion protein, the zsGreen protein could be detected in the lysosomes of the PAMs, and the lysosomes of the PAMs adsorbed with the zsGreen-FcMR-2 peptide segment fusion protein contained more zsGreen protein. Consistent with the fluorescence analysis results, the FCMR-2 peptide segment and the FCMR-10 peptide segment can specifically target the FcMR molecules on the surface of PAMs.

[0213] 2.4 In vivo immunization of zsGreen-12 peptide fusion protein

[0214] (1) Animal selection: 15 Long White piglets aged 5-6 weeks were selected as immunized animals, and the 15 piglets were randomly divided into 3 groups, with 5 piglets in each group. The 3 groups were named as follows: zsGreen immunization group, zsGreen-FcMR-2 peptide segment fusion protein immunization group, and zsGreen-FcMR-10 peptide segment fusion protein immunization group.

[0215] (2) Protein immunization: The zsGreen monomer protein, the zsGreen-FcMR-2 peptide segment fusion protein, and the zsGreen-FcMR-10 peptide segment fusion protein were mixed with the ISA 206 adjuvant at a ratio of 1:1, emulsified in an emulsifier, and then each protein was immunized in piglets according to the immunization groups. Each piglet was immunized with 30 μg of protein.

[0216] 2.5 Indirect ELISA detection of antibody levels in serum of piglets after immunization

[0217] (1) Collection of piglet serum: After 7 days and 14 days of immunization, the venous blood of piglets in each immunization group was collected, 4 mL of whole blood was collected from each pig, and the serum was collected by centrifugation at 4000 r / min for 10 min after overnight standing at 4°C. A pre-immune piglet serum negative control was also set.

[0218] (2) Plate coating: Dilute zsGreen protein with plate coating solution to a final concentration of 4 μg / mL, 100 μL / well, and coat 96-well plates overnight at 4°C.

[0219] (3) Washing: Place the 96-well plate in a plate washer and wash it with 0.05% PBST for 3 times.

[0220] (4) Blocking: 2.5% skim milk powder, 200 μL / well, incubate at 37℃ for 2 h, then wash the plate.

[0221] (5) Add primary antibody: Add 100 μL of diluted serum per well to the well, incubate at 37°C for 1 h, and wash the plate.

[0222] (6) Secondary antibody: Dilute HRP-labeled porcine secondary antibody 1:5000 with PBS, 100 μL / well, incubate at 37℃ for 1 h, and wash the plate.

[0223] (7) Color development: Add 100 μL of TMB color development solution to each well and incubate at room temperature in the dark for 15 min.

[0224] (8) Termination: Add 50 μL of 3M H2SO4 to each well to terminate the colorimetric reaction.

[0225] (9) Reading value: Use an ELISA reader to read the absorbance value at 450 nm and analyze the results.

[0226] Indirect ELISA was used to detect the level of zsGreen antibody in serum. The results are as follows: Figure 7 (A) ELISA OD of two zsGreen-12 peptide fusion protein immunoassays 450 The value was higher than that of the zsGreen monomeric protein immunization group, and the ELISA OD of the zsGreen-FcMR-10 peptide fusion protein immunization group was also higher. 450 The values ​​were significantly higher than those in the monomeric protein immunization group. Serum was collected from piglets 14 days after immunization, serially diluted, and the zsGreen antibody titer in each immunization group was detected by indirect ELISA. The results are as follows: Figure 7 (B) Fourteen days after immunization, the serum antibody titers of piglets immunized with the two zsGreen-12 peptide fusion protein groups were higher than those in the zsGreen monomer protein immunization group, and the serum antibody titer of the zsGreen-FcMR-10 peptide fusion protein immunization group was the highest. This demonstrates that FCMR-2 and FCMR-10 peptides can target antigens to APCs to enhance humoral immune responses, and that FCMR-10 peptide has a better immune-enhancing effect on antigens.

[0227] 2.6 IFN-γ ELISApot assay for the level of effector cellular immune response in piglets after immunization

[0228] The PBMC of the piglets was isolated 14 days after immunization, and the level of T lymphocyte activation after immunization was detected using the Mabtech IFN-γ ELISpot detection kit.

[0229] (1) Add the stimulating protein to the ELISpot plate, 100 μg / mL, 100 μL / well, and set up a positive ConA stimulating protein control, Con A protein 20 μg / mL, 100 μL / well.

[0230] (2) Count the isolated PBMC cells, and plate 2×10 5 / well of cells in the test group, and 5×10 4 / well of cells in the positive stimulating protein group.

[0231] (3) Place the plate in a 37°C cell incubator, and after 48 h of incubation, take out the plate and perform ELISpot spot detection according to the instructions of the detection kit.

[0232] After 14 days of immunization, the peripheral blood of all piglets was collected, and the peripheral blood mononuclear cells (PBMC) of the piglets were isolated for pig IFN-γ ELISpot detection, to analyze the level of Th1 cell immune response after immunization, and the representative ELISpot results are shown in Figure 8 (A), and the difference analysis results are shown in Figure 8 (B), after stimulation of PBMC with zsGreen monomer protein, the IFN-γ cells produced by the PBMC of the piglets immunized with zsGreen-FcMR-2 peptide segment fusion protein were more than those of the zsGreen monomer protein immunized group, but after analysis of the results of 5 piglets, it was found that there was no significant difference compared with the monomer protein immunized group, while the number of IFN-γ cells produced by the PBMC of the piglets immunized with zsGreen-FcMR-10 peptide segment fusion protein was significantly increased compared with the zsGreen monomer protein immunized group. Further proving that FCMR-10 peptide segment can target antigens to APCs to enhance Th1 cell immune response during immunization, so that the body produces a higher level of cellular immune response after immunization.

[0233] 2.7 FcRM-10 homologous peptide segment

[0234] 2.7.1 The interaction relationship between FcRM-10 and FcRM was simulated using AlphaFold Multimer, and the binding site was determined, and under the condition that the binding site was determined, the homologous peptide segment of FcRM-10 was constructed, as shown in Table 3 below:

[0235] Table 3 Homologous peptide segment of FcRM-10

[0236] ,

[0237] The short peptide was added to the C-terminal of zsGreen sequence to obtain a recombinant zsGreen-12 peptide fusion protein expression plasmid according to the method of 1.5.1, and the recombinant zsGreen-12 peptide fusion protein was obtained by recombinant expression and purification according to the method of 1.5.2, 1.5.2.

[0238] 2.7.2 Recombinant zsGreen-12 peptide fusion protein targeting assay

[0239] The zsGreen-12 peptide fusion protein was used to immunize animals according to the method of 2.4, two animals were immunized for each recombinant protein, and the antibody level of piglet serum after 14 days of immunization was detected by indirect ELISA according to the method of 2.5. The specific results are shown in Figure 9 .

[0240] Based on Figure 9 It can be known that the amino acid at the 9th position in the sequence has an important influence on the binding activity of FcRM-10. Under the condition of keeping this site unchanged, the peptide segment with more than 83% homology with FcRM-10 can efficiently induce the body to produce antibodies.

[0241] 2.8 Targeting peptide FCMR10 fusion protein NT11-VAD2-P54-LEAEK2LE-FcMR10-LE6His, NT11-VAD2-P22-GGGGS2-LE-FcMR10-LE6His, His-VAD2-GP3ΔIDR-VAD2-FcMR10 expression application

[0242] 2.8.1 Synthesis of recombinant plasmid

[0243] The recombinant expression plasmid was synthesized by Nanjing Qikeli Biotechnology Co., Ltd. The amino acid sequence of NT11-VAD2-P54-LEAEK2LE-FcMR10-LE6His is shown in SEQ ID NO. 3, the amino acid sequence of NT11-VAD2-P22-GGGGS2-LE-FcMR10-LE6His is shown in SEQ ID NO. 4, and the amino acid sequence of His-VAD2-GP3ΔIDR-VAD2-FcMR10 is shown in SEQ ID NO. 5.

[0244] 2.8.2 Transformation and expression of recombinant protein

[0245] The three synthesized recombinant plasmids were transformed and expressed, and the method is described in steps (5) of 1.5.2, 1.5.2.1. The bacteria were shaken for 20 min. In step (5) of 1.5.2.2, the bacteria were resuspended in PBS; and in step (7), the precipitate was dissolved with 8M urea.

[0246] 2.8.3 Purification and identification of recombinant proteins

[0247] The proteins were purified by nickel column affinity chromatography, see 1.5.3. The purified proteins were identified by SDS-PAGE, see 1.5.4.

[0248] Results are shown in Figure 10 , the recombinant proteins were dialyzed to PBS after Ni column purification, and the purity of the purified recombinant proteins was identified by SDS-PAGE, the results are shown in Figure 10 (A), NT11-VAD2-P54-LEAEK2LE-FcMR10 was successfully expressed

[0249] -LE6His, NT11-VAD2-P22-GGGGS2-LE-FcMR10-LE6His, His-VAD2-GP3ΔIDR

[0250] -VAD2-FcMR10 recombinant proteins, the band size is consistent with the expected, and the protein purity is high. The recombinant proteins have a tag, which can be detected by Western blot with antibodies to detect the purified recombinant proteins, the results are shown in Figure 10 (B), Western blot showed that His antibody detected obvious specific bands, the size was consistent with the SDS-PAGE results. The results of SDS-PAGE and Western blot showed that NT11-VAD2-P54-LEAEK2LE-FcMR10-LE6His, NT11-VAD2-P22-GGGGS2-LE-FcMR10-LE6His, His-VAD2-GP3ΔIDR-VAD2-FcMR10 recombinant proteins were successfully purified with high purity.

[0251] 2.8.4 NT11-VAD2-P54-LEAEK2LE-FcMR10-LE6His, NT11-VAD2-P22-GGGGS2-

[0252] LE-FcMR10-LE6His, His-VAD2-GP3ΔIDR-VAD2-FcMR10 animal in vivo immunization

[0253] Twenty-four 5-6 week-old Landrace piglets were selected as immunized animals, and randomly divided into three groups, named P54 immunization group, P22 immunization group and GP3 immunization group, 8 piglets in each group, and the piglets in each group were randomly divided into two groups, 4 piglets in each group, the grouping situation is shown in Table 4:

[0254] Table 4 Grouping of immunized animals

[0255] ,

[0256] P54 monomer protein, NT11-VAD2-P54-LEAEK2LE-FcMR10-LE6His recombinant protein, P22 monomer protein, NT11-VAD2-P22-GGGGS2-LE-FcMR10-LE6His recombinant protein, GP3 monomer protein, His-VAD2-GP3ΔIDR-VAD2-FcMR10 recombinant protein were mixed with ISA 206 adjuvant 1:1, respectively, and emulsified in an emulsifier. Each protein was immunized in piglets according to the immunization group, with 100 μg of protein per piglet.

[0257] 2.8.5 Indirect ELISA detection of antibody levels in serum of immunized piglets

[0258] After immunization for 7 days, 10 days, 14 days, and 21 days, the venous blood of piglets in each immunization group was collected, 4 mL of whole blood was collected from each pig, and the serum was collected by centrifugation at 4000 r / min for 10 min after standing overnight at 4°C. A pre-immune piglet serum negative control was also set. The indirect ELISA detection method is described in 2.5. The corresponding monomer protein was diluted with coating buffer and coated on the plate.

[0259] The antibody levels in serum were detected by indirect ELISA, and the results are shown in Figure 11 After immunization, the ELISA OD 450 values of the three groups of proteins in the recombinant protein immunization group with the FCMR10 targeting peptide were higher than those in the corresponding monomer protein immunization group. The antibody titers in the serum of piglets immunized with the fusion protein in the P54 immunization group and the GP3 immunization group were significantly higher than those in the corresponding monomer protein immunization group 10 days after immunization, indicating that the FCMR-10 peptide can target APCs to enhance the humoral immune response.

[0260] 2.8.6 ELISpot detection of the level of effector T cell immune response in immunized piglets

[0261] The level of effector T cell immune response in immunized piglets was detected by ELISpot 21 days after immunization, and the method is described in 2.6.

[0262] After immunization for 21 days, the piglets were collected for anticoagulation and PBMC isolation, and then pig IFN-γ ELISpot detection was performed, with ConA-stimulated cells as a positive control. Representative ELISpot results are shown in Figure 12 The difference analysis results of the number of spots are shown in Figure 12The results show that the IFN-γ cells produced by PBMC of piglets immunized with the recombinant protein containing the targeting peptide FcMR10 are significantly more than those of the corresponding monomer protein immunization group in trend, further proving that the targeting peptide FcMR10 can target the antigens of DNA viruses and RNA viruses to APCs, enhance the Th1 type cellular immune response in the immune process, and make the body produce a higher level of cellular immune response after immunization.

[0263] The present application screens a targeting 12-peptide FcMR10 which can specifically bind to the surface FcMR molecule of pig APCs through in vitro screening and animal experiment, and the peptide segment can specifically target pig APCs with model antigen zsGreen to enhance the humoral and cellular immunity of the antigen in piglets. Further, the FcMR10 is fused with PRRSV GP3, ASFV P22, ASFV P54 and other DNA and RNA viral antigens to prepare NT11-VAD2-P54-LEAEK2LE-FcMR10-LE6His, NT11-VAD2-P22-GGGGS2-LE-

[0264] FcMR10-LE6His, His-VAD2-GP3ΔIDR-VAD2-FcMR10 recombinant proteins and immunize piglets, and it is found that the DNA and RNA viral antigens fused with the targeting 12-peptide FcMR10 produce an earlier and stronger immune response reaction in piglets.

Claims

1. A porcine antigen-presenting cell targeting peptide, characterized in that: The targeting peptide can specifically target the FcMR receptor on the antigen-presenting cell, and the amino acid sequence of the targeting peptide is shown in SEQ ID No: 1 or SEQ ID No:

6.

2. A polynucleotide encoding the porcine antigen-presenting cell targeting peptide of claim 1, characterized in that, The polynucleotide sequence is shown in SEQ ID No: 2 or SEQ ID No:

7.

3. A fusion protein, characterized in that, The fusion protein comprises the peptide of claim 1 and a protein fragment of a pathogenic microorganism.

4. An expression vector, characterized by, The polynucleotide of claim 2 or a vector for expressing the fusion protein of claim 3.

5. A host cell, characterized in that, The expression vector of claim 4; the host cell is E. coli, and is used for preparing the fusion protein; or the host cell is lactic acid bacteria or yeast, and is used for preparing an oral vaccine.

6. Use of the porcine antigen-presenting cell targeting peptide of claim 1 in the preparation of a porcine antigen-presenting cell targeting drug, wherein the targeting peptide can specifically target the FcMR receptor on the antigen-presenting cell.

7. Use according to claim 6, characterized in that, The drug is a porcine vaccine.

8. A subunit vaccine comprising a pharmaceutically acceptable carrier, the swine-derived antigen-presenting cell-targeting peptide of claim 1, and a fragment of a swine pathogen antigen, wherein, The porcine antigen-presenting cell targeting peptide is obtained by fusion expression with a porcine pathogen antigen fragment.

Images