CN117088943B - Porcine dendritic cell targeting peptide KC1 and application thereof - Google Patents

Abstract

The present invention discloses a porcine dendritic cell targeting peptide KC1 and its application. The amino acid sequence of the porcine dendritic cell targeting peptide is shown in SEQ ID NO.1. The present invention uses phage random peptide library and whole cell differential screening technology to screen the dominant binding peptide of porcine mononuclear DCs, and obtains the porcine DCs targeting peptide KC1 (KCCYPN). Experiments have shown that the targeting peptide can strongly bind to porcine DCs, showing good specificity. The targeting peptide can be used as a drug lead molecule such as a gene-guided vector to lay the foundation for cell-specific small molecule therapy, or used in combination with a vaccine to improve the immune efficiency of swine disease vaccines and promote the body to produce immunity early. The proposal of the present invention provides a material basis for the research and development of porcine DCs targeted vaccines.

Description

A porcine dendritic cell targeting peptide KC1 and its application

Technical Field

The present invention relates to a porcine dendritic cell targeting peptide and its application. The present invention belongs to the technical field of biomedicine.

Background technique

The rapid development of immunology and molecular biology has promoted the development of new vaccines. Some studies have involved how to utilize the interaction between immune cells and vaccine antigens to optimize the body's response, accelerate the speed of innate immune response, and improve the specificity of acquired immunity.

In recent years, targeted treatment and prevention have become a research hotspot in the field of medical biotechnology and have been applied to the treatment of human tumors. Among them, biological immunotherapy targeting dendritic cells can effectively deliver drugs to the corresponding targets, reduce the dosage and number of administrations, improve the therapeutic effect of drugs, and reduce adverse reactions. Dendritic cells (DCs) are the most powerful professional antigen-presenting cells known to date. They can effectively activate naive T cells and play an important role in the innate and acquired immune systems. Some studies have used phage display linear 12-peptide library to screen human peripheral blood DCs targeting peptides, which were fused with antigens and expressed in lactobacillus delivery vectors to obtain good immune effects. Another study used phage loop 7 peptide library to screen mouse bone marrow DCs targeting peptides, which were coupled with antigens to make chitosan nanoparticle vaccines, which significantly improved the immune efficiency. However, there are few studies on porcine dendritic cell targeting peptides, which affects the research and development of related new drugs and biological products, and the field of targeted treatment and prevention of porcine diseases is even less involved.

Due to the emergence of mutants or strains with enhanced virulence, the efficacy of existing vaccine products is increasingly affected. The focus of new product development has shifted to how to improve vaccine efficacy, such as increasing the intensity of protective immune responses, generating immunity earlier or extending the immune period, etc. Targeted vaccines have become the focus of new vaccine research. Although existing mammalian DCs targeting peptides can bind to DCs from multiple species to varying degrees, there is currently no research on porcine DCs targeting peptides or targeted treatment or prevention programs.

Based on the characteristic that DCs are key antigen presenting cells, the present invention designs and screens targeting peptides with good affinity for porcine DCs, which is of great significance to the research and development of new targeted vaccines for pigs.

Summary of the invention

The technical problem to be solved by the present invention is to provide a porcine dendritic cell targeting peptide and application thereof.

In order to achieve the above object, the present invention adopts the following technical means:

The present invention separates and cultures porcine DCs, obtains porcine mononuclear DCs with high purity through morphological, phenotypical and functional identification, and then uses phage display 12-peptide library technology, with porcine mononuclear DCs as target cells and porcine monocytes as negative selection cells, to screen out linear 12-peptides that bind well to porcine mononuclear DCs, and verifies the binding effect through experiments such as flow cytometry and immunofluorescence, and finally obtains short peptides that can target porcine DCs.

The porcine dendritic cell targeting peptide of the present invention is named KC1, and the amino acid sequence of the porcine dendritic cell targeting peptide is shown in SEQ ID NO.1.

The polynucleotide encoding the porcine dendritic cell targeting peptide, the expression vector containing the polynucleotide, and the host cell containing the expression vector are also within the protection scope of the present invention.

Furthermore, the present invention also proposes the use of the porcine dendritic cell targeting peptide in the preparation of a porcine dendritic cell targeting drug.

Among them, preferably, the drug is a vaccine.

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

1. The molecular basis and mechanism of DCs in antigen recognition, uptake, processing and presentation have been clarified, and the technology of in vitro DCs culture has become increasingly mature, which has led to the rapid development of DCs-based targeted vaccine research. Screening with intact cells can fully simulate the interaction of protein molecules under natural conditions, which is conducive to finding marker protein polypeptides that specifically bind to target cells. The screened polypeptides can be used as vectors for gene therapy or in combination with drug delivery vectors, which is expected to improve the targeting of treatment, help overcome cell membrane barriers, and directly enter cells to play a role. The present invention uses phage random peptide library and whole cell differential screening technology to screen the dominant binding polypeptides of porcine mononuclear DCs, and obtains the porcine DCs targeting peptide KC1 (KCCYPN). Experiments have shown that the targeting peptide can strongly bind to porcine DCs, showing good specificity. The targeting peptide can be used as a drug lead molecule such as a gene-guided vector, laying the foundation for cell-specific small molecule therapy, or used in combination with vaccines to improve the immune efficiency of pig disease vaccines and promote the body to produce immunity early. The proposal of the present invention provides a material basis for the research and development of porcine DCs targeted vaccines.

2. Peptides have the characteristics of low immunogenicity, small relative molecular mass, fast clearance in blood and non-target tissues, strong tissue penetration, high affinity with receptors, and can enter cells through receptor-mediated endocytosis to form phagosomes. They are easy to synthesize and can reduce their degradation rate and side effects in the body through structural modification. Targeted short peptides can be used in combination with a variety of drug delivery carriers to achieve good targeting effects, and the results obtained are unattainable by ordinary vaccines.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is the microscopic observation result of porcine monocyte-derived dendritic cells (100 μm);

Among them, A: Microscopic observation on the 5th day of cell culture; B: Microscopic observation after 1 day of LPS stimulation;

Figure 2 shows the expression of molecular phenotypes of porcine Mo-DCs detected by IFA (50 μm);

FIG3 is the detection of the molecular phenotypes of porcine monocyte-derived DCs, MHC-Ⅱ and CD172a;

Among them, A: immature Mo-DCs; B: mature Mo-DCs;

FIG4 is a test of the phagocytic ability of porcine Mo-DCs;

FIG5 shows the sequencing comparison results and duplicate sequence results after the fourth round of screening;

FIG6 is a fluorescence microscope observation of the binding effect of fluorescent peptides and DCs;

FIG7 is the flow cytometry analysis result of the affinity effect of FITC-labeled peptides with porcine DCs;

FIG8 is a laser confocal microscope observation of the binding position of fluorescent peptides to DCs;

FIG9 shows the experimental results of competitive binding of alanine-mutated KC peptide to DCs;

Among them, A: KC three-dimensional structure diagram; B, C, D: inhibition binding percentage calculated according to the detected fluorescence intensity;

FIG10 is a fluorescence microscope observation of the binding effect of fluorescent peptides and DCs;

FIG11 is the flow cytometry analysis result of the affinity effect of FITC-labeled peptides with porcine DCs;

FIG. 12 shows the binding position of fluorescent peptides to DCs observed by laser confocal microscopy.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, the embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

Example 1 Isolation, culture and identification of porcine monocyte-derived dendritic cells

1. Isolation and culture of porcine mononuclear dendritic cells

Blood from 5-8 week old piglets was collected aseptically from the anterior vena cava. Heparin sodium anticoagulation tubes were used to dilute the venous blood with sterile PBS plus double antibody at a ratio of 1:1. A sterile centrifuge tube was selected. First, Histopaque-1077 lymphocyte separation solution at room temperature was added to each tube along the tube wall. Then, an equal amount of diluted blood was slowly added along the tube wall to ensure that there was a clear boundary between the separation solution and the blood. Centrifuge at 2500rpm for 25min. Slowly remove the centrifuge tube, slowly aspirate the middle white mist mononuclear cell layer with a pipette and divide it into a sterile centrifuge tube, centrifuge at 1800rpm for 10min, discard the supernatant, add red blood cell lysis solution to each tube to resuspend the cell pellet, lyse at room temperature for 5min, centrifuge at 1800rpm for 8min, and discard the supernatant. Add sterile PBS to each tube to resuspend the cell pellet, centrifuge at 1800rpm for 8min, and discard the supernatant. Obtain peripheral blood mononuclear cells (PBMCs). Resuspend the cell pellet with the pre-prepared RPMI-1640 complete culture medium, inoculate in a six-well plate at a density of 2×10 ⁶ /mL, shake the cells and place them in a 37°C, 5% CO ₂ constant temperature incubator for static culture. After 6 hours of culture, observe under an optical microscope. At this time, the cells attached to the wall are monocytes. Use a pipette to slowly aspirate the non-attached cells along the wall of the well, then slowly add 2mL of RPMI-1640 complete culture medium, add rpGM-CSF and rpIL-4 at a concentration of 20ng/mL to each well, and culture in a 37°C, 5% CO ₂ constant temperature incubator. Change the medium by half every 1d as described above, and induce immature porcine MoDCs after 5d of culture. After 5d of induction and culture of porcine MoDCs, mature DCs were obtained after stimulation with LPS at a final mass concentration of 200ng/mL.

2. Morphological observation of porcine monocyte-derived DCs

During the cell culture period, an optical inverted microscope was used to observe the morphological changes of the cultured DCs, and the results are shown in Figure 1. The observation results showed that the monocytes just isolated were round in shape, small in size, and all suspended in the culture medium; after 1 day of culture, the cells suspended in the culture medium began to grow adherently, and a small number of cell colonies appeared. After 2 days of culture, the cell volume began to increase, and the number of cell colonies increased; after 4 days of culture, the cell volume in the culture medium showed different sizes, and some cells had radial irregular dendrites on the surface, with different shapes, and the cells were semi-adherent; after 5 days of culture, most cells had protrusions on the surface all around, and the cells were irregular and semi-suspended. At this time, the cells showed typical morphological characteristics of immature DCs. After LPS was added to the cells and induced for another 1 day, the surface protrusions of the cells became more obvious.

3. Detection of the phenotype of porcine mononuclear DCs by fluorescence microscopy

Slowly aspirate the culture medium of monocyte-derived DCs in the six-well plate to avoid cells falling off the well plate, slowly add 0.01M PBS along the wall of the well plate, wash twice repeatedly, and add RPMI-1640 basic culture medium. Add PE-mouse anti-swine CD172a and FITC-mouse anti-swine MHC-Ⅱ antibodies to the six-well plate in turn and incubate at room temperature in the dark for 30 minutes. Slowly aspirate the antibody with a pipette and fix it with 6% formaldehyde for 5 minutes. Wash twice with 0.01M PBS to remove non-specifically bound fluorescent antibodies, incubate with DAPI at room temperature for 10 minutes, wash twice with 0.01M PBS, observe and take pictures with a fluorescence microscope. The results are shown in Figure 2. It can be found by fluorescence microscopy that monocytes induced by rPGM-CSF and rPIL-4 for 5 days can express CD172a and MHC-Ⅱ molecular phenotypes.

4. Detection of Purity of Porcine Mononuclear Dendritic Cells by Flow Cytometry

Sterile 0.01M PBS was added to the cell wells of immature and mature monocyte-derived DCs, and the cells were blown up from the wells and suspended in PBS. After centrifugation at 2000rpm for 5min, the supernatant was discarded after repeated washing twice, and the immature and mature monocyte-derived DCs were evenly divided into 1.5mL EP tubes. PE-mouse anti-swine CD172a and FITC-mouse anti-swine MHC-Ⅱ antibodies were added to the immature and mature monocyte-derived DCs, respectively. Finally, only 0.01M PBS was added to the last tube as a negative control. After adding, resuspend, incubate with tin foil at room temperature for 30min in the dark, and centrifuge at 2000rpm for 5min. Wash the cells 3 times with 0.01M PBS to remove nonspecific fluorescent antibodies. After centrifugation and discarding the supernatant, 500μL 0.01M PBS was added to each tube to resuspend the cells and detect them by flow cytometry. Before sending them for inspection, ensure that there are at least 1×10 ⁵ cells in each tube.

Flow cytometry was used to detect the expression of surface molecules (CD172a and MHC-Ⅱ) on induced DCs. The cells induced by pGM-CSF and pIL-4 were incubated with CD172a and MHC-Ⅱ monoclonal antibodies, and MoDCs without fluorescent antibody incubation were used as negative controls. The flow cytometry results showed that the expression levels of CD172a and MHC-Ⅱ molecules could reach 92.5% and 69.5%, respectively, indicating that PGM-CSF and PIL-4 can induce PBMCs to differentiate into immature mononuclear DCs. After immature mononuclear DCs were matured by LPS stimulation, the expression levels of surface molecules CD172a and MHC-Ⅱ on their surface could reach 98.1% and 78.3%, respectively, as shown in Figure 3.

5. Detection of phagocytic function of porcine mononuclear DCs

0.01M PBS was added to the cultured immature and mature mononuclear DCs, centrifuged at 2000rpm for 5min, the supernatant was discarded, and the cells were washed repeatedly for 3 times according to the number of cells, and the cell concentration was adjusted to 2×10 ^5. Five gradients were set up, and three replicates of each gradient were connected to a 96-well plate, 100μL per well, and 100μL of 0.1% neutral red was added to each well. One group was added with 0.01M PBS as a negative control, and the cells were cultured in a 37°C, 5% CO ₂ incubator for 60min, 90min, 120min and 150min. After incubation, the remaining neutral red that was not phagocytosed was discarded, and the cells were washed repeatedly twice with 0.01M PBS (preheated), and 100μL of 1% SDS lysis buffer was added to each well. The cells were incubated at room temperature for 2h and the lysed cells were read on an OD540 instrument, where the OD value is proportional to the phagocytic function of the cells.

To further determine whether PBMCs were successfully differentiated into monocyte-derived DCs after induction by the induction agent, this study used neutral red to detect the phagocytic ability of immature and mature monocyte-derived DCs based on the fact that immature monocyte-derived DCs have strong processing, uptake and phagocytic functions for antigens, while mature monocyte-derived DCs have gradually reduced their processing, uptake and phagocytic abilities. The results are shown in Figure 4. The amount of neutral red phagocytosis by immature monocyte-derived DCs increased significantly with time, reaching the maximum at 120 minutes; however, the phagocytic ability of mature monocyte-derived DCs induced by LPS did not change significantly with time, and showed a slightly downward trend. The above results show that this experiment successfully isolated high-purity porcine monocyte-derived DCs.

Example 2 Phage display 12 peptide library subtractive screening of porcine dendritic cell targeting peptides

1. Screening of targeting peptides

The screening process is divided into four rounds, as follows:

First round: 2×10 ¹¹ pfu of PhD-12 library was added to porcine mononuclear DCs (5×10 ⁵ /mL) and incubated at 4°C for 30 min. After centrifugation at 2000 rpm for 3 min, the cell pellet was resuspended in PBS containing 1% bovine serum albumin (BSA) and 0.05% Tween 20 and washed three times. 1 mL of buffer (0.2 mol/L glycine-hydrochloric acid, pH=2.2, 1 mg/mL bovine serum albumin) was added, and 50 μl of Tris-HCl (pH 9.1) was added for neutralization after gentle mixing at room temperature for 10 min. Centrifugation was performed and the supernatant was the membrane surface bound phage eluate. The eluate was mixed with E. coli ER2738 culture medium (A600 nm≈0.5) and shaken vigorously in a conical flask for 4.5 h. The phage amplification solution was prepared by the polyethylene glycol-NaCl secondary precipitation method.

The second to fourth rounds of screening: the first round of amplification solution was added to 5×10 ⁵ /mL PBMC (negative selection cells) and incubated at 4°C for 30 minutes. After centrifugation at 2000rpm for 3 minutes, the supernatant was transferred to porcine mononuclear DCs and the first round of steps was repeated; the incubation time was shortened to 20 minutes in the third round and to 10 minutes in the fourth round. The eluate and amplification solution of each round of screening were cultured in LB isopropyl-thio-β-D-galactoside (IPTG) 5-bromo-4-chloro-3-indole-β-thiogalactoside (Xgal) Tet plate to determine the phage titer; after the fourth round of biological elimination, 204 plaques were randomly selected, and the M13 phage single-stranded DNA was extracted using a kit for sequencing.

The number of screening rounds, input amount, and recovery amount are shown in Table 1. After each round of screening, non-specifically bound phages were rinsed off, and the phages bound to the surface were eluted and amplified. Four rounds of selection were performed continuously. The recovery rate increased in the second round. After reducing the incubation time in the third and fourth rounds, the recovery rate tended to be stable. In the fourth round of plaque sequencing results, a total of 161 were sequenced. The repetition rate and sequence results are shown in Figure 5. After four rounds of selection, phages displaying short peptides of HS (HSLRHDYGYPGH), KC (KCCYPNMAAFA), and SF (SFLTNFVQPHAS) appeared repeatedly, indicating that they may be peptide sequences with good affinity for porcine mononuclear DCs.

Table 1 Screening rounds, input amount and recovery amount

2. Synthesis of peptides

The peptides HS (HSLRHDYGYPGH), KC (KCCYPNMAAFA) and SF (SFLTNFVQPHAS) that appeared repeatedly in the fourth round of screening were selected and synthesized by Fmoc method, and then purified by HPLC (purity>95%) and analyzed by mass spectrometry (completed by Genscript). The specific sequences are shown in Table 2. The fluorescent peptide powder was pre-dissolved in PBS and stored at -20°C for later use.

Table 2 Peptide sequences

Peptides sequence KC-FITC KCCYPNOMAAFA-FITC HS-FITC HSLRHDYGYPGH-FITC SF-FITC SFLTNFVOPHAS-FITC KC KCCYPNOMAAFA KC-K1A ACCYPNOMAAFA KC-C2A KACYPNOMAAFA KC-C3A KCAYPNOMAAFA KY4A KCCAPNOMAAFA KC-P5A KCCYANOMAAFA KC-N6A KCCYPAQMAAFA KC-Q7A KCCYPNAMAAFA KC-M8A KC YPNQAAAFA KC-F11A KCCYPNOMAAAA KC-C2AC3A KAAYPNOMAAFA KC-Q7AM8AF11A KCCYPNAAAAAA KC-10 KYPNOMAAFA KC1 KC Y KC1-FITC KCCYPN-FITC KC2-FITC OMAAFA-FITC KC-Biotin KCCYPNOMAAFAGGGK-Biotin

3. Detection of short peptide affinity

3.1 Observation of the binding effect of fluorescent peptides on porcine mononuclear DCs using fluorescence microscopy

After washing twice with PBS, the monocyte-derived DCs (cell number is about 10 ⁵ /mL) on the 6th day of culture were added with 50 μg of FITC-labeled 12-peptide HS, KC and SF respectively to each well, and the cells were allowed to react for 30 minutes at room temperature, and then rinsed with PBS three times. The fluorescent peptide binding effect was observed under a fluorescence microscope. The results are shown in Figure 6. The fluorescence intensity of the KC short peptide binding to the cells is higher than that of the HS short peptide, indicating that the KC fluorescent peptide can bind well to the DCs cells.

3.2 Flow cytometry to detect the affinity of peptides to porcine mononuclear DCs

25 μg of FITC-labeled 12-peptide HS, KC and SF were added to DCs on the 6th day of culture (cell volume was about 10 ⁶ /mL), respectively, at 4°C for 30 minutes, centrifuged at 1800 r/min, the precipitate was washed twice with PBS, the cells were resuspended with 500 μl PBS, and the fluorescence signal was detected by flow cytometry, and each sample was repeated three times. The test results are shown in Figure 7. Under the same conditions, the fluorescence signal intensity of KC and SF short peptides was significantly higher than that of HS short peptide, indicating that they can specifically bind to porcine dendritic cells well.

3.3 Laser confocal microscopy to observe the binding of fluorescent peptides to porcine intestinal DCs

Take frozen sections of porcine small intestine and place them at room temperature for 10 minutes. Block with 5% goat serum at room temperature for 30 minutes, add 400μM HC-Ⅱ-FITC, CD172a-PE, and incubate at 37℃ for 30 minutes. Wash the sections 3 times with 0.01M PBS to remove nonspecific fluorescence. Add 50μg of FITC-labeled 12 peptides HS, KC and SF to the sections, and incubate at 4℃ for 30 minutes in the dark. Wash the sections twice with 0.01M PBS to remove nonspecific fluorescence. Add 100μL of DAPI to the sections, and incubate at room temperature for 10 minutes in the dark. Wash the sections twice with 0.01M PBS to remove nonspecific fluorescence. The results of laser confocal observation of the sections are shown in Figure 8. The KC short peptide can bind well to DCs in the porcine small intestine.

3.4 Effect of alanine mutation on the binding of peptides to porcine mononuclear DCs

After washing twice with PBS, monocyte-derived DCs (cell number is about 10 ⁴ /mL) cultured on day 6 were added with 5 μg/mL, 10 μg/mL, 20 μg/mL, and 40 μg/mL of KC and amino acid mutated 12 peptides, respectively, and incubated at 37°C for 1 hour, rinsed three times with PBS, incubated biotin-labeled KC with monocyte-derived DCs at 37°C for 1 hour, rinsed three times with PBS, added HRP-streptavidin, incubated at 37°C for 1 hour, rinsed three times with PBS, added 100 μL/well TMB for color development for 10 minutes, added 2M H ₂ SO ₄ to terminate the reaction, and detected the OD ₄₅₀ absorbance with a microplate reader. Figure 9 (B) shows the inhibition percentage of peptide binding at different concentrations of competing peptides. The reference KC peptide effectively competes with the biotinylated KC peptide for porcine monocyte-derived DCs, and loses this inhibitory binding ability after the 1st, 4th, 5th, and 6th amino acids are replaced by alanine. This indicates that the 1st, 4th, 5th and 6th amino acids are essential for KC to bind to the targeting protein on the surface of porcine mononuclear DCs. The results of Figure 9 (A, C) show that after the 2nd and 3rd amino acids are replaced by alanine, the double mutant KC still has the ability to inhibit binding, but after the 2nd and 3rd amino acids are deleted, the inhibitory binding ability of the short peptide is significantly reduced, which indicates that when KC binds to the targeting protein on the surface of porcine mononuclear DCs, a certain space is required between the 1st and 4th amino acids. The results of Figure 9 (D, E) show that after the last six amino acids of KC are deleted, not only does KC1 (KCCYPN, shown in SEQ ID NO.1) not lose its inhibitory binding ability, but its binding ability is also higher than that of KC.

3.5 Observation of the binding effect of fluorescent peptides on porcine mononuclear DCs using fluorescence microscopy

After washing twice with PBS, the monocyte-derived DCs (cell number of about 10 ⁵ /mL) on the 6th day of culture were added with 50 μg of FITC-labeled 12-peptide HS, KC and SF and hexapeptide KC1 and KC2 respectively to each well, and allowed to act at room temperature for 30 minutes, then rinsed three times with PBS, and the fluorescent peptide binding effect was observed under a fluorescence microscope. The results are shown in Figure 10, and the fluorescence intensity of the KC1 short peptide and the KC2 short peptide indicates that the KC1 fluorescent peptide can bind well to the DCs cells.

3.6 Flow cytometry to detect the affinity of peptides to porcine mononuclear DCs

25 μg of FITC-labeled 12-peptide KC and hexapeptides KC1 and KC2 were added to DCs on the 6th day of culture (cell volume of about 10 ⁶ /mL), incubated at 4°C for 30 minutes, centrifuged at 1800 r/min, washed the precipitate twice with PBS, resuspended the cells with 500 μl PBS, and detected the fluorescence signal by flow cytometry. Each sample was repeated three times. The test results are shown in Figure 11. Under the same conditions, the fluorescence signal intensity of the KC1 short peptide was significantly higher than that of the KC and KC2 short peptides, indicating that the KC1 short peptide can specifically bind to porcine dendritic cells.

3.7 Laser confocal microscopy to observe the binding of fluorescent peptides to porcine small intestinal DCs

Take the frozen sections of porcine small intestine and place them at room temperature for 10 minutes. Block with 5% goat serum at room temperature for 30 minutes, add 400μMHC-Ⅱ-FITC, CD172a-PE, and incubate at 37℃ for 30 minutes. Wash the sections 3 times with 0.01M PBS to remove nonspecific fluorescence. Add 50μg FITC-labeled 12-peptide KC and hexapeptide KC1, KC2 to the sections, and incubate at 4℃ for 30 minutes in the dark. Wash the sections twice with 0.01M PBS to remove nonspecific fluorescence. Add 100μL DAPI to the sections, and incubate at room temperature for 10 minutes in the dark. Wash the sections twice with 0.01M PBS to remove nonspecific fluorescence. The results of laser confocal observation of the sections are shown in Figure 12. The KC1 short peptide can bind well to DCs in the porcine small intestine.

Claims (6)

1. The pig-derived dendritic cell targeting peptide is named KC1, and is characterized in that the amino acid sequence of the pig-derived dendritic cell targeting peptide is shown as SEQ ID NO. 1.

2. A polynucleotide encoding the swine dendritic cell targeting peptide of claim 1.

3. An expression vector comprising the polynucleotide of claim 2.

4. A host cell comprising the expression vector of claim 3.

5. Use of the swine dendritic cell targeting peptide of claim 1 in the preparation of swine dendritic cell targeting drugs.

6. The use according to claim 5, wherein the medicament is a vaccine.