Establishment method of porcine nasal mucosa M cell differentiation model

Abstract

The invention is applicable to the technical field of cell models, and provides a method for establishing a porcine nasal mucosa M cell differentiation model, which comprises the following steps: preparing porcine nasal mucosa epithelial cells; construction of a pGEX-6p-1-RANKL recombinant expression vector, and prokaryotic expression of the recombinant expression vector; inducing the porcine nasal mucosa epithelial cells to differentiate into M cells by the porcine RANKL; and RT-qPCR (real-time quantitative polymerase chain reaction) is used for detecting the expression conditions of M cell differentiation related genes Spi-B, Tnfaip2 and GP2. According to the invention, the functional pig source RANKL recombinant protein is successfully constructed, and pig nasal mucosa epithelial cells are successfully induced to be differentiated into M cells by using the functional pig source RANKL recombinant protein. Through an RT-qPCR experiment, the expression level of M cell related genes is accurately detected, and the differentiation efficiency is verified. The achievement not only provides a tool for research on porcine respiratory diseases and development of drug vaccines, but also promotes research on cross-species mucosal immunity, and provides a new perspective and an experimental tool for the field of mucosal immunity.

Description

Technical Field

[0001] The invention belongs to the technical field of cell models, and in particular relates to a method for establishing a pig nasal mucosa M cell differentiation model. Background Art

[0002] As an important part of the body's immune network, the mucosal immune system (MIS) includes exocrine glands such as the gastrointestinal mucosa, respiratory mucosa, genital mucosa, and conjunctiva, and plays an active and important role in resisting infection. Against the background of the growing scale of pig farming, the severe situation of pig respiratory diseases highlights the importance of mucosal immune research. Microfold cells (M cells) in the respiratory mucosa, as an important part of the respiratory tract-associated lymphoid tissue, play an important role in the mucosal immune process. M cells are specialized epithelial cells found in the follicle-associated epithelium of Peyer's patches. They have unique functions and can efficiently take up exogenous antigens and deliver them to mucosa-associated lymphoid tissue (MALT), thereby activating immune responses. M cells can deliver microorganisms, particles or vaccine antigens to immune cells (such as macrophages and dendritic cells) on the basal surface through phagocytosis and transport mechanisms, thereby promoting the activation of T cells and B cells and the secretion of specific antibodies such as IgA. Therefore, the efficient delivery function of M cells makes them a core target for the development of nasal mucosal vaccines.

[0003] However, the differentiation mechanism of M cells is complex, involving the regulation of specific signaling pathways and the intercellular microenvironment. This makes the establishment of in vitro models face many challenges, especially considering the scarcity of M cells and the harsh differentiation conditions. Although some in vitro models have been used to study the biological characteristics of M cells, such as the co-culture model of human Caco-2 colon adenocarcinoma cell line with B lymphocyte origin in Transwell culture, adding human Raji B lymphoblasts to Caco-2 cells can induce them to differentiate into a monolayer of M cells. However, this model is mainly based on intestinal M cells and cannot fully reflect the characteristics of respiratory M cells. Given that the nasal cavity of pigs and humans has a high similarity in anatomical and immunological characteristics, the use of pig nasal mucosa as a model is closer to human immune response than the traditional mouse model. However, there are significant differences between species, and most of the genes selectively expressed by pig natural respiratory M cells are not induced in the above in vitro models. In addition, the expression levels of M cell-specific markers (such as GP2, Tnfaip2, Spi-B, etc.) in M ​​cell differentiation models of different species vary greatly, limiting the credibility of the research results and the versatility of the model. Trypsin-sensitive glycoprotein 2 (GP2) is involved in antigen capture and presentation and is an important marker of M cell differentiation and function. Tumor Necrosis Factor-Alpha Induced Protein 2 (Tnfaip2) is involved in regulating the morphological and functional differentiation of M cells, promoting the development and maturation of M cells, and enhancing their antigen transport capacity. The expression of Spi-1 / PU.1Related Transcription Factor B (Spi-B) is a necessary condition for the development of M cells. It activates a series of gene expressions, including GP2 and other M cell-related functional proteins. Its lack of expression will lead to developmental defects of M cells.

[0004] Existing studies have shown that RANKL activates the transcription factor Spi-B by binding to RANK, inducing epithelial cells to differentiate into M cells. Spi-B promotes the expression of genes related to M cell function (such as GP2 and Tnfaip2), thereby completing the differentiation of M cells. In different tissue microenvironments, the expression level of RANKL can regulate the number and function of M cells. For example, exogenous supplementation of RANKL (such as experimental injection of RANKL) can induce the differentiation of M cells in non-Peyer's patch areas. Although some models can induce some characteristics of M cells, such as GP2 expression, their functions are imperfect, the efficiency of transcellular transport is low, and they cannot completely simulate functions such as antigen delivery and immune cell activation. There is a lack of means to monitor M cell functions (such as antigen uptake and delivery) in real time. In addition, existing models are usually cumbersome to operate, have a long culture cycle, and have high technical requirements. The differentiation results are affected by multiple factors such as culture conditions and experimental techniques, and the experimental reproducibility is poor. This limits the promotion and use of this model and increases the technical and cost burden of researchers.

[0005] In view of this, the present invention proposes a method for establishing a porcine nasal mucosal M cell differentiation model. Summary of the invention

[0006] The purpose of the present invention is to provide a method for establishing a porcine nasal mucosal M cell differentiation model, aiming to solve the problems raised in the above background technology.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A method for establishing a porcine nasal mucosal M cell differentiation model comprises the following steps:

[0009] Step 1: Preparation of porcine nasal mucosal epithelial cells;

[0010] Step 2: Construction of pGEX-6p-1-RANKL recombinant expression vector and prokaryotic expression;

[0011] Step 3: Porcine RANKL induces porcine nasal mucosal epithelial cells to differentiate into M cells;

[0012] Step 4: RT-qPCR was used to detect the expression of M cell differentiation-related genes Spi-B, Tnfaip2 and GP2.

[0013] Furthermore, the specific steps of step 2 are as follows:

[0014] Step 21: Construction of pGEX-6p-1-RANKL;

[0015] The sequence of porcine RANKL protein was obtained from Ncbi, and the domain and active domain of porcine RANKL protein were analyzed using SignalP and TMHMM V2.0c online analysis websites, confirming that porcine RANKL protein had no signal peptide and had a transmembrane domain (49-71aa); the amino acid sequence from position 72 to position 316 of the extracellular segment of porcine RANKL protein was selected as the target amplification region and specific PCR primers were designed, including forward primer RANKL-f and reverse primer RANKL-r, the sequences of which are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively;

[0016] The pig pancreas was taken, and the total RNA of the cells was extracted by the TRizol method, and the mRNAs were reverse transcribed into cDNAs by using a reverse transcription kit; the cDNAs were used as templates, and RANKL-f and RANKL-r primers and DNA polymerase were added to perform PCR amplification to obtain the DNA fragment of the protein coding region of the RANKL gene; the PCR product was subjected to 1% agarose gel electrophoresis to recover the target fragment, and the recovered target fragment was homologously recombined with the pGEX-6P-1 vector that had been linearized by double restriction enzymes (EcoRI / XhoI), and the ligation product was transformed into competent bacteria DH5α, and was coated on a Luria-Bertani (LB) solid culture medium plate containing ampicillin for screening and culture; the positive clones were picked and expanded the next day, and the plasmids were extracted according to the instructions of the plasmid small-scale extraction kit, and DNA sequencing was performed for identification, and the recombinant plasmid with the correct sequence was named GST-pGEX-6P-1pigRANKL;

[0017] Step 22: Induced expression of porcine RANKL;

[0018] The recombinant prokaryotic expression plasmid GST-pGEX-6P-1pig RANKL was transformed into competent cells of Escherichia coli BL21 (DE3), and spread on LB solid medium containing ampicillin for screening and culture. A single clone was picked and expanded, and then 1 mL of bacterial solution was added to 200 mL LB medium; the bacterial solution was shaken at 200 r / min and 37°C until OD600 was 0.5, the temperature was adjusted to 16°C, and isopropyl-β-D-thiogalactoside (IPTG) was added to the bacterial solution at a final concentration of 0.1 mM, and the culture was continued for 2 hours to induce the expression of the target protein; the bacterial solution before and after induction was collected and samples were prepared, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sodium dodecyl sulfatepolyacrylamide The expression of fusion protein (GST-RANKL) induced by IPTG was detected by SDS-PAGE and Coomassie Brilliant Blue staining.

[0019] Step 23: Purification of porcine RANKL;

[0020] The induced bacterial solution was centrifuged at 12000 r / min for 10 min at 4°C, and the precipitated bacteria were collected and resuspended in PBS. The bacteria were broken by ultrasonication, and the supernatant and precipitate were subjected to SDS-PAGE, Coomassie Brilliant Blue staining and Western blot to detect the solubility of the fusion protein. The soluble protein supernatant was purified by GST purification column at 4°C. The specific steps are as follows: (1) equilibrate the adsorption column by adding 5 column volumes of PBS for equilibration; (2) load the sample by taking 2 column volumes of sample for loading and After repeating the column for 3 times, discard the unbound sample; (3) Wash, wash the column with 10 times the column volume of PBS to remove non-specific impurities; (4) Elute the target protein, add 2 times the column volume of 10 mmol / L glutathione eluent, incubate for 10 to 15 minutes, repeat the column for 3 times, collect the eluate and store it at -80°C; (5) Use 20% ethanol to seal the GST purification column to purify the protein, and store it at 4°C for next use; Take the eluate for sample preparation, perform SDS-PAGE, Coomassie Brilliant Blue staining and Western blot to detect the purification effect of the protein.

[0021] Furthermore, in step 21, the PCR reaction conditions are set as: 95°C pre-denaturation for 5 min, 95°C denaturation for 30 s, 60°C annealing for 30 s, 72°C extension for 45 s, 35 cycles, and 72°C stabilization for 10 min.

[0022] Furthermore, in step 23, when resuspending with PBS, 5 mL of PBS is added to every 100 mL of bacterial liquid precipitation.

[0023] Furthermore, the specific steps of step 3 are as follows:

[0024] Step 31: When the confluence of the porcine nasal mucosal epithelial cells reaches 80% to 90%, first discard the culture medium and wash the cell surface twice with 1 mL PBS;

[0025] Step 32: Add porcine RANKL protein to fresh complete medium to a final concentration of 10 μg / mL;

[0026] Step 33: After culturing for 24 hours, discard the waste liquid, wash the cell surface twice with 1 mL of PBS, and replace with fresh culture medium supplemented with porcine RANKL protein;

[0027] Step 34: After 6 days of continuous culture, the differentiation ratio of M cells is detected.

[0028] Furthermore, the specific steps of step 4 are as follows:

[0029] Step 41: extracting cellular RNA;

[0030] Extract total RNA from cells and test the quality of RNA using a spectrophotometer (A260 / A280 ratio should be between 1.8-2.0) or a fluorometer;

[0031] Step 42: synthesizing cDNA;

[0032] The first strand cDNA was synthesized using the Perfect Real Time kit;

[0033] Step 43: Setting up the RT-qPCR reaction system;

[0034] The total volume of the reaction system is 20 μL, and the specific components are as follows:

[0035] Template: 1-2 μL cDNA;

[0036] Primers: forward and reverse primers designed for target genes GP2, Spi-B and Tnfaip2, the concentration of each forward and reverse primer is 0.2-0.5 μM;

[0037] qPCR reagents: 10 μL of 2×SYBR Green or TaqMan reagent;

[0038] ddH2O: make up the system to 20μL;

[0039] Step 44: Setting RT-qPCR reaction conditions;

[0040] The thermal cycling program included:

[0041] First, an initial denaturation is performed at 95°C for 2-5 min to activate the DNA polymerase and denature the template;

[0042] Then a cycle reaction is carried out, including:

[0043] Denaturation: 95°C, 15-30s; annealing: 50-65°C, 15-30s; extension: 72°C, 30s; 40 cycles;

[0044] Finally, melting curve analysis was performed, with the temperature rising from 65°C to 95°C and the fluorescence signal read every 0.5°C to verify the specificity of amplification.

[0045] Furthermore, in step 43, the forward and reverse primer sequences of GP2 are shown as SEQ ID NO.3 and SEQ ID NO.4; the forward and reverse primer sequences of Spi-B are shown as SEQ ID NO.5 and SEQ ID NO.6; and the forward and reverse primer sequences of Tnfaip2 are shown as SEQ ID NO.7 and SEQ ID NO.8.

[0046] Furthermore, the specific steps of step 1 are as follows:

[0047] Step 11: Sampling;

[0048] After the pig maxilla was separated, the tissue material was disinfected with 75% alcohol, and then the pig nasal mucosa was removed within 15 minutes in a sterile environment. The removed nasal mucosa was washed several times with sterile PBS containing 3 antibodies precooled at 4°C, and stored in DMEM / F12 culture medium containing 3 antibodies; the 3 antibodies included penicillin, streptomycin and amphotericin B;

[0049] Step 12: Isolation of porcine nasal mucosal epithelial cells;

[0050] Use sterile ophthalmic scissors to cut the nasal mucosal epithelial tissue into 2-3 mm 3The tissue blocks of different sizes were washed several times with sterile PBS containing 3 antibodies and placed in sterile centrifuge tubes; mixed digestion enzyme solution was added to the tissue blocks for enzymatic digestion, the mixed digestion enzyme solution was composed of collagenase I, collagenase IV and hyaluronidase; the specimens were completely immersed in the mixed digestion enzyme and digested overnight at 4°C; the next day, the digestion was terminated with preheated DMEM / F12 culture medium containing 10% fetal bovine serum, filtered with a 100μm mesh, and centrifuged at 1000rpm / min at 4°C for 5min to collect cells; Add 5 times the volume of complete medium to the cell pellet and mix it gently with a pipette; filter it with a 70 μm mesh, centrifuge it at 1000 rpm / min for 5 min at 4°C to collect the filtered cells, transfer the cell suspension to a new culture dish, incubate it at 37°C for 1 h, and remove the fibroblasts; collect the upper cell suspension, centrifuge it at 1000 rpm / min for 5 min at 4°C, and then add 3 times the volume of complete medium to the cell pellet again, and mix it gently with a pipette;

[0051] Step 13: Cultivation of porcine nasal mucosal epithelial cells;

[0052] The cells were counted under a microscope, with 5×10 5 / ml of cells were inoculated into a new sterile cell culture dish and cultured in a 37°C, 5% CO2 incubator for 48 hours. The culture medium was then replaced with a complete culture medium containing epidermal growth factor (EGF) and vitamin A (VA), and the medium was changed every 2 days. After 8-10 days of culture, the cells grew into a single layer of epithelial cells with a density of 80%-90%.

[0053] Compared with the prior art, the present invention has the following beneficial effects:

[0054] 1. The present invention achieves efficient separation and purification of primary porcine nasal mucosal epithelial cells by optimizing enzymatic hydrolysis and culture conditions, ensuring the activity and purity of the cells. At the same time, the interference of fibroblasts is cleverly eliminated, making the cultured epithelial cells more uniform, providing favorable conditions for subsequent experiments.

[0055] 2. The present invention successfully constructed a functional porcine RANKL recombinant protein. The present invention uses bioinformatics analysis and molecular biology methods to accurately design and amplify the key functional regions of porcine RANKL, avoiding the effects of signal peptides and transmembrane regions on protein expression. By using the pGEX-6p-1 vector and GST purification system, efficient protein expression and purification are achieved, ensuring the high solubility and functionality of the target protein.

[0056] 3. The present invention utilizes the functionality of porcine RANKL to successfully induce the differentiation of porcine nasal mucosal epithelial cells into M cells. This achievement provides an in vitro model for studying the porcine nasal mucosal immune system, which helps to reveal the mechanism of action of M cells in anti-infection and the development of mucosal immune vaccines. Through RT-qPCR experiments, the present invention accurately detected the expression levels of M cell-related genes (such as Spi-B, Tnfaip2, GP2), verified the differentiation efficiency, and provided reliable molecular biological evidence for the experimental conclusions.

[0057] 4. The application potential of the present invention is huge. It can not only provide a powerful tool for studying the pathogenic mechanism of porcine respiratory diseases, drug and vaccine development, but also can be used as an in vitro infection model or drug and vaccine target analysis platform. In addition, the present invention also lays a solid foundation for cross-species mucosal immunity research (such as comparison with human M cell differentiation mechanism), and provides a new perspective and experimental tools for mucosal immune disease research and mucosal immunology.

[0058] In summary, the present invention has made important breakthroughs in the isolation and culture of porcine nasal mucosal epithelial cells, the construction and application of porcine RANKL recombinant protein, and the induction of porcine M cell differentiation, opening up a new path for research and application in related fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 This is a schematic diagram of inducing pig nasal mucosal epithelial cells to differentiate into M cells.

[0060] Figure 2 The figure shows the construction of the pGEX-6p-1-RANKL recombinant plasmid; A is the sequence of the porcine RANKL protein; B is the analysis result of whether the porcine RANKL has a signal peptide; C is the analysis result of whether the porcine RANKL has a transmembrane domain;

[0061] Figure 3 The figure shows the construction of pGEX-6p-1-RANKL recombinant plasmid; A is the RANKL DNA amplification diagram; B is the pGEX-6p-1 plasmid restriction enzyme digestion diagram, where 1 indicates before restriction enzyme digestion and 2 indicates after double restriction enzyme digestion (EcoRI and XhoI); C is the screening of pGEX-6p-1-RANKL positive clones using RANKL-f and RANKL-r; D is the pGEX-6p-1-RANKL recombinant plasmid map.

[0062] Figure 4 The inducible expression of GST-RANKL.

[0063] Figure 5The figure shows the purification of GST-RANKL; wherein M: Marker; FT: flow through; W: wash; E1-E5: elutions (elution 1-5).

[0064] Figure 6 This is an immunofluorescence identification image of Tnfaip2 and GP2 in pig M cells (40×).

[0065] Figure 7 RT-qPCR was used to monitor the process of RANKL-induced differentiation of nasal epithelial cells.

[0066] Figure 8 Flow cytometry was used to detect the differentiation ratio of M cells in porcine nasal mucosal epithelium. DETAILED DESCRIPTION

[0067] In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below, but it should not be construed as limiting the applicable scope of the present invention.

[0068] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0069] Example 1: Analysis of the structural domain, active domain and signal peptide of porcine RANKL protein;

[0070] First, the sequence of porcine RANKL protein was obtained from Ncbi ( Figure 2 A in (see SEQ ID NO.1). SignalP (https: / / services.healthtech.dtu.dk / services / SignalP-6.0 / ) and TMHMMV2.0c (https: / / services.healthtech.dtu.dk / services / TMHMM-2.0 / ) were used to analyze whether porcine RANKL has a signal peptide and a transmembrane domain. The analysis results showed that the porcine RANKL protein has no signal peptide and a transmembrane domain (49-71aa) ( Figure 2 B and C), the crystal structure of porcine RANKL protein has not been reported in PDB (Protein Data Bank). Therefore, the amino acid sequence from position 72 to 316 of the extracellular segment of porcine RANKL protein was selected as the target amplification region and specific PCR primers (forward primer RANKL-f and reverse primer RANKL-r) were designed. The primer sequences are shown in Table 1.

[0071] Table 1 Primer sequences

[0072]

[0073] Example 2: Construction of pGEX-6p-1-RANKL;

[0074] First, pig pancreas was taken, total cellular RNA was extracted using the TRizol method, and mRNAs were reverse transcribed into cDNAs using a reverse transcription kit. Using cDNAs as templates, RANKL-f and RANKL-r primers and high-fidelity DNA polymerase were added to perform PCR amplification to obtain a DNA fragment of the RANKL gene protein coding region with a length of 789 bp ( Figure 3 The PCR reaction conditions were set as follows: 95°C pre-denaturation for 5 min, 95°C denaturation for 30 s, 60°C annealing for 30 s, 72°C extension for 45 s, 35 cycles, and 72°C stabilization for 10 min. The PCR product was subjected to 1% agarose gel electrophoresis, and the target fragment was recovered and homologously recombined with the pGEX-6P-1 vector that had been linearized by double restriction enzymes (EcoRI / XhoI) ( Figure 3 The ligation product was transformed into competent bacteria DH5α and plated on Luria-Bertani (LB) solid medium containing ampicillin for screening and culture. The next day, positive clones were picked and expanded, and PCR detection of the bacterial solution was performed using RANKL-f and RANKL-r primers to preliminarily screen positive clones ( Figure 3 C). For the positive clones obtained by screening, the plasmids were extracted according to the instructions of the plasmid mini-preparation kit and DNA sequencing was performed. The recombinant plasmid with the correct sequence was named GST-pGEX-6P-1pig RANKL. The recombinant plasmid map is shown in Figure 3 As shown in D.

[0075] Example 3: Inducible expression of porcine RANKL;

[0076] First, the recombinant prokaryotic expression plasmid GST-pGEX-6P-1pig RANKL was transformed into competent cells of Escherichia coli BL21 (DE3) and spread on LB solid medium containing ampicillin for screening and culture. After a single clone grows out, pick the single clone and expand the culture, then take 1mL of bacterial solution and add it to 200mL LB medium. At 200r / min and 37℃, shake the bacterial solution to OD600 of 0.5, adjust the temperature to 16℃, add isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.1mM to the bacterial solution, and continue to culture for 2h to induce the expression of the target protein. The bacterial solution before / after induction was collected and samples were prepared, and the expression of the fusion protein (GST-RANKL) under IPTG induction was detected by SDS-PAGE and Coomassie Brilliant Blue staining.

[0077] The experimental results are as follows Figure 4 As shown in the figure, after IPTG induction, the fusion protein was successfully expressed in the supernatant, and its size was consistent with the expected value (about 53 kDa). This result fully proves the successful performance of the fusion expression.

[0078] Example 4: Purification of porcine RANKL;

[0079] First, the induced bacterial solution was centrifuged at 12000r / min for 10min at 4°C, the centrifuged precipitated bacteria were collected and resuspended with PBS (5mL PBS was added for every 100ml bacterial solution precipitate). Next, the bacteria were broken using an ultrasonic disruptor. In order to detect the solubility of the fusion protein, the supernatant and precipitate were taken for SDS-PAGE, Coomassie Brilliant Blue staining and Western blot experiments respectively. The soluble protein supernatant was used for protein purification steps using a GST purification column (performed at 4°C): (1) Equilibrate the adsorption column by adding 5 column volumes of PBS for equilibrium treatment; (2) Load the sample by loading 2 column volumes of the sample, and repeat the column washing process 3 times before discarding the unbound sample; (3) Wash the column by washing the column with 10 column volumes of PBS to remove non-specific impurities; (4) Elute the target protein by adding 2 column volumes of 10 mmol / L glutathione eluent, incubate for 10 to 15 minutes, repeat the column washing process 3 times, and collect the eluent and store it at -80°C; (5) Seal the GST purification column with 20% ethanol to purify the protein and store it at 4°C for next use.

[0080] In order to detect the purification effect of the protein, the eluate was sampled and subjected to SDS-PAGE, Coomassie Brilliant Blue staining and Western blot experiments. Figure 5 The protein purified by glutathione affinity chromatography has a high purity and the size of the target protein is correct. The specific amino acid sequence is (without GST tag, see SEQ ID NO.12): FRAQMDPNRISEDDTHCINRIFQLHENTDLQDTTLESQDSKLIPDSCKRIKQAFQAAVQKEMQHIVKSQHIRAEKAMVEGSWLDLSRRNKPETQPFAHLTINATDIPSGSHKVSLSCWYHDRGWAKISNMTFSNGKLIVNQDGFYYLYANICFRHHETSGNLDAKYLQLMVYVTKTSIKIPSSHTLMKGGSTKYWSGNSEFHFYSINVGGFFKLRSGEEISIEVSNHSLLDPDQDATYFGAFKVRDID*

[0081] Example 5: Immunofluorescence identification of pig M cell differentiation;

[0082] First, the coverslips containing primary porcine nasal epithelial cells were rinsed with PBS for 5 minutes, repeated 3 times, and then fixed with 4% paraformaldehyde for 30 minutes. The cells were permeabilized with 0.1% Triton X-100 for 10 minutes at room temperature; then blocked with 4% fetal bovine serum for 30 minutes; then incubated with Fitc direct-labeled Mouse anti-Tnfaip2 antibody and Rabbit anti-GP2 antibody (both diluted at 1:200) at 4°C overnight. After the incubation, PBS was used for 5 minutes, rinsed 3 times in total, PE-labeled rabbit secondary antibody (dilution ratio of 1:500) was added, and incubated at 37°C in the dark for 2 hours; DAPI was used to stain the cell nucleus. Finally, after rinsing with PBS for 5 minutes, rinsed 3 times in total, the sample was placed under a confocal microscope for observation, and fluorescence color development indicated positivity.

[0083] Epithelial cells were induced with 2 μg / ml porcine RANKL protein for 5 days. Immunofluorescence was used to observe green fluorescence in the cytoplasm and red fluorescence in the cell membrane. After merging the images, we found that the two fluorescences were displayed on the same cell, so we identified it as M cells ( Figure 6 ).

[0084] Example 6: RT-qPCR detection of the expression of genes related to M cell differentiation;

[0085] First, primary nasal epithelial cells were cultured with GST-RANKL protein for 6 consecutive days. To explore the effect of different concentrations of RANKL protein on the expression of genes related to M cell differentiation, TRIzol reagent was used to extract RNA from cell samples treated with 0, 2.5, 5, 10, and 20 μg / ml RANKL protein. The extracted RNA was purified by PrimeScript TM RT reagent Kit with gDNA Eraser (Perfect Real Time) kit was used for reverse transcription to synthesize the first strand of cDNA. Next, the expression of M cell differentiation-related genes Spi-B, Tnfaip2 and GP2 was detected by qPCR. The sequences of primers (Gp2-f, Gp2-r, Spib-f, Spib-r, Tnfaip2-f, Tnfaip2-r) are shown in Table 1.

[0086] The experimental results showed that 10 μg / ml RANKL protein could induce the optimal M cell differentiation ratio ( Figure 7 ).

[0087] Example 7: Flow cytometry detection of pig nasal mucosal epithelial cell differentiation;

[0088] First, wash the cells once with PBS, then add 100μL of trypsin to the cells and digest for 5min. Next, add serum-containing culture medium to terminate digestion, collect the cells of the same group into the same 1.5mL centrifuge tube, centrifuge, wash the cells once with 150μL of washing solution, and put the blank cells in a 4℃ refrigerator for later use. Resuspend the remaining cells with 100μL of washing solution and add 2μL of Rabbit anti-GP2 antibody to each, incubate at room temperature for 30min, and wash the cells once with 150μL of washing solution. Then add 100μL of washing solution and 1μL of anti-rabbit PE secondary antibody, and incubate in the dark for 15min. Wash once with washing solution and then permeabilize. Suspend the cells with 100μL of washing solution.

[0089] The specific steps of cell membrane permeabilization are as follows: Cyto-Fast TM Perm Washsolution (10×) was diluted to 1× (prepared in advance). 100 μL of cells (2×10 5 -1×10 6 ) into a 12×75 mm tube and add 150 μL Cyto-Fast TM Fix / PermBuffer was mixed and incubated at room temperature for 20 min. Then, 150 μL 1×Cyto-Fast TM PermWash solution, centrifuge at 350xg for 5 min, discard the supernatant, and repeat the washing once. After the membrane is permeabilized, stain the cells with the optimal concentration of intracellular antibody. TM Prepare antibodies in PermWash solution with a total volume of 100 μL, add 2 μL of FiTC-labeled Mouse anti-Tnfaip2 antibody, and incubate at room temperature in the dark for 20 min. Use 150 μL of 1×Cyto-Fast TM Wash the cells with Perm Wash solution and centrifuge at 350xg for 5 min, discard the supernatant. Add 150 μL of cell wash buffer (PBS containing 10% serum), centrifuge at 350xg for 5 min, discard the supernatant. Resuspend the cells with 200 μL of cell wash buffer and collect samples using flow cytometry.

[0090] In order to explore the effect of RANKL protein on the differentiation of porcine nasal epithelial cells into M cells, porcine RANKL protein was used to act on porcine nasal epithelial cells for 0, 2, 4, and 6 days. GP2 is expressed on the surface of mature M cells, and Tnfaip is expressed in the cytoplasm of mature / immature M cells. Therefore, we used GP2 + Tnfaip + The cells were identified as mature M cells. The experimental results showed that when RANKL protein was activated for about 6 days, the proportion of mature M cells was the largest, reaching 4.31% of epithelial cells, which was 9 times that of cells without RANKL protein induction (P<0.05) ( Figure 8 ).

[0091] The above are only preferred embodiments of the present invention. It should be pointed out that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention. These should also be regarded as the protection scope of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. A method for establishing a porcine nasal mucosal M cell differentiation model, characterized in that: The following steps are involved: Step 1: Preparation of porcine nasal mucosal epithelial cells; Step 2: Construction of pGEX-6p-1-RANKL recombinant expression vector and prokaryotic expression; Step 3: Porcine RANKL induces porcine nasal mucosal epithelial cells to differentiate into M cells; Step 4: RT-qPCR was used to detect the expression of M cell differentiation-related genes Spi-B, Tnfaip2 and GP2.

2. The method for establishing a porcine nasal mucosal M cell differentiation model according to claim 1, characterized in that: The specific steps of step 2 are as follows: Step 21: Construction of pGEX-6p-1-RANKL; The sequence of porcine RANKL protein was obtained from Ncbi, and the structural domain and active domain of porcine RANKL protein were analyzed using SignalP and TMHMM V2.0c online analysis websites, confirming that the porcine RANKL protein had no signal peptide and had a transmembrane domain; the amino acid sequence from position 72 to position 316 of the extracellular segment of the porcine RANKL protein was selected as the target amplification region and specific PCR primers were designed, including forward primer RANKL-f and reverse primer RANKL-r, and the sequences were shown in SEQ ID NO.1 and SEQ ID NO.2, respectively; The pig pancreas was taken, and the total cell RNA was extracted using the TRizol method, and the mRNAs were reverse transcribed into cDNAs using a reverse transcription kit; the cDNAs were used as templates, RANKL-f and RANKL-r primers and DNA polymerase were added, and PCR amplification was performed to obtain the DNA fragment of the RANKL gene protein coding region; The PCR product was subjected to 1% agarose gel electrophoresis to recover the target fragment, and the recovered target fragment was homologously recombined with the pGEX-6P-1 vector that had been linearized by double enzyme digestion. The ligation product was transformed into competent bacteria DH5α and spread on LB solid culture medium plates containing ampicillin for screening and culture. The next day, positive clones were picked and expanded, and plasmids were extracted according to the instructions of the plasmid small-scale extraction kit, and DNA sequencing was performed for identification. The recombinant plasmid with the correct sequence was named GST-pGEX-6P-1pig RANKL. Step 22: Induced expression of porcine RANKL; The recombinant prokaryotic expression plasmid GST-pGEX-6P-1pig RANKL was transformed into competent cells of Escherichia coli BL21 (DE3), and spread on LB solid medium containing ampicillin for screening and culture, and a single clone was picked and expanded, and then 1 mL of bacterial solution was added to 200 mL of LB medium; the bacterial solution was shaken at 200 r / min and 37°C until OD600 was 0.5, the temperature was adjusted to 16°C, and IPTG with a final concentration of 0.1 mM was added to the bacterial solution, and the culture was continued for 2 hours to induce the expression of the target protein; the bacterial solution before / after induction was collected and samples were prepared, and the expression of the fusion protein under IPTG induction was detected by SDS-PAGE and Coomassie Brilliant Blue staining respectively; Step 23: Purification of porcine RANKL; The induced bacterial solution was centrifuged at 12000r / min for 10min at 4℃, the centrifuged precipitated bacteria were collected and resuspended with PBS; the bacteria were broken by ultrasonic disruptor, and the supernatant and precipitate were respectively subjected to SDS-PAGE, Coomassie Brilliant Blue staining and Western blot to detect the solubility of the fusion protein; the soluble protein supernatant was taken to purify the protein with GST purification column at 4℃, and the specific steps were as follows: balance the adsorption column, add 5 times column volume of PBS for balance treatment; load the sample, take 2 times column volume of sample for sample loading, repeat the column for 3 times and discard the unbound sample; wash, wash the column with 10 times column volume of PBS to remove non-specific impurities; To elute the target protein, add 2 times column volume of 10mmol / L glutathione eluent, incubate for 10-15min, repeat the column for 3 times, collect the eluent and store it at -80℃; seal the GST purification column with 20% ethanol to purify the protein, and store it at 4℃ for next use; prepare samples from the eluent, perform SDS-PAGE, Coomassie Brilliant Blue staining and Western blot to detect the purification effect of the protein.

3. The method for establishing a porcine nasal mucosal M cell differentiation model according to claim 2, characterized in that: In step 21, the PCR reaction conditions are set as: 95° C. pre-denaturation for 5 min, 95° C. denaturation for 30 s, 60° C. annealing for 30 s, 72° C. extension for 45 s, 35 cycles, and 72° C. stabilization for 10 min.

4. The method for establishing a porcine nasal mucosal M cell differentiation model according to claim 2, characterized in that: In step 23, when resuspending with PBS, 5 mL of PBS was added for every 100 mL of bacterial liquid sediment.

5. The method for establishing a porcine nasal mucosal M cell differentiation model according to claim 1, characterized in that: The specific steps of step 3 are as follows: Step 31: When the confluence of the porcine nasal mucosal epithelial cells reaches 80% to 90%, first discard the culture medium and wash the cell surface twice with 1 mL PBS; Step 32: Add porcine RANKL protein to fresh complete medium to a final concentration of 10 μg / mL; Step 33: After culturing for 24 hours, discard the waste liquid, wash the cell surface twice with 1 mL of PBS, and replace with fresh culture medium supplemented with porcine RANKL protein; Step 34: After 6 days of continuous culture, the differentiation ratio of M cells is detected.

6. The method for establishing a porcine nasal mucosal M cell differentiation model according to claim 1, characterized in that: The specific steps of step 4 are as follows: Step 41: extracting cellular RNA; Extract total RNA from cells and test the quality of RNA using a spectrophotometer or fluorometer; Step 42: synthesizing cDNA; The first strand cDNA was synthesized using the Perfect Real Time kit; Step 43: Setting up the RT-qPCR reaction system; The total volume of the reaction system is 20 μL, and the specific components are as follows: Template: 1-2 μL cDNA; Primers: forward and reverse primers designed for target genes GP2, Spi-B and Tnfaip2, the concentration of each forward and reverse primer is 0.2-0.5 μM; qPCR reagents: 10 μL of 2×SYBR Green or TaqMan reagent; ddH2O: make up the system to 20μL; Step 44: Setting RT-qPCR reaction conditions; The thermal cycling program included: First, an initial denaturation is performed at 95°C for 2-5 min to activate the DNA polymerase and denature the template; Then a cycle reaction is carried out, including: Denaturation: 95°C, 15-30s; annealing: 50-65°C, 15-30s; extension: 72°C, 30s; 40 cycles; Finally, melting curve analysis was performed, with the temperature rising from 65°C to 95°C and the fluorescence signal read every 0.5°C to verify the specificity of amplification.

7. The method for establishing a porcine nasal mucosa M cell differentiation model according to claim 6, characterized in that: In step 43, the forward and reverse primer sequences of GP2 are shown in SEQ ID NO.3 and SEQ ID NO.4; the forward and reverse primer sequences of Spi-B are shown in SEQ ID NO.5 and SEQ ID NO.6; the forward and reverse primer sequences of Tnfaip2 are shown in SEQ ID NO.7 and SEQ ID NO.8.

Images