阅读说明：本技术 一种靶向树突状细胞的纳米抗体分子及应用 (Nano antibody molecule of targeted dendritic cell and application ) 是由 徐海 李玲 李睿婷 郭子杰 洪伟鸣 朱善元 于 2021-07-07 设计创作，主要内容包括：本发明提供一种靶向树突状细胞的纳米抗体分子及应用。所述的靶向树突状细胞纳米抗体分子的氨基酸序列如SEQ ID NO.1、SEQ ID NO.2所示。本发明构建羊驼源天然纳米抗体T7噬菌体展示文库,利用亲和筛选技术淘选出能够与鸡骨髓DCs高亲和力结合的纳米抗体分子。特异性检测显示筛选到的两个纳米抗体分子不仅跟鸡的DCs有结合能力,跟鸭、鹅等禽类DCs也能结合,而不跟DCs以外的骨髓单核细胞、鸡(鸭、鹅)成纤维细胞结合,具有较好DCs结合特异性。应用该纳米抗体分子作为亚单位抗原载体,能够显著提高抗原分子激发的免疫反应,提高抗体水平。因此,本发明筛选到纳米抗体分子可以作为抗原分子的靶向载体,提高抗原递呈效率,为新型禽类疫苗的开发提供支撑。(The invention provides a dendritic cell-targeted nano antibody molecule and application thereof. The amino acid sequence of the targeted dendritic cell nano antibody molecule is shown in SEQ ID NO.1 and SEQ ID NO. 2. The method constructs an alpaca natural nano antibody T7 phage display library, and elutriates nano antibody molecules capable of being combined with chicken bone marrow DCs with high affinity by utilizing an affinity screening technology. The specificity detection shows that the two screened nano antibody molecules not only have the binding capacity with DCs of chickens, but also can be bound with DCs of poultry such as ducks, geese and the like, but also are not bound with bone marrow mononuclear cells and chicken (duck, goose) fibroblasts except the DCs, and have better DCs binding specificity. The nanometer antibody molecule is used as a subunit antigen carrier, so that the immune reaction excited by the antigen molecule can be obviously improved, and the antibody level is improved. Therefore, the nano antibody molecules screened by the method can be used as a targeting carrier of the antigen molecules, so that the antigen presentation efficiency is improved, and support is provided for the development of novel poultry vaccines.)

1. A nanobody molecule targeting dendritic cells, characterized in that the amino acid sequence of the nanobody molecule is SEQ ID NO: 1 or SEQ ID NO: 2.

2. the dendritic cell-targeting nanobody molecule of claim 1, wherein the nanobody molecule is obtained by affinity screening using a phage display library.

3. The dendritic cell-targeting nanobody molecule of claim 2, wherein the phage display library is an alpaca nanobody T7 phage display library.

4. The dendritic cell-targeting nanobody molecule according to claim 1, wherein the dendritic cells are chicken, duck, goose dendritic cells.

5. Use of the dendritic cell-targeting nanobody molecule of any one of claims 1 to 4 for the preparation of a vaccine.

6. The use of claim 5, wherein the nanobody molecule acts as an antigen carrier.

7. The use according to claim 5, wherein the vaccine is a dendritic cell targeted vaccine.

8. A dendritic cell targeted subunit vaccine, which is obtained by fusion expression of the dendritic cell targeted nanobody molecule of any one of claims 1 to 4 and subunit protein.

Technical Field

The invention belongs to the technical field of biological medicines, relates to a dendritic cell-targeted nano antibody molecule and application thereof, and particularly relates to screening of the nano antibody molecule and preparation of a vaccine by using the nano antibody molecule as an antigen carrier.

Background

Dendritic Cells (DCs) are a class of Antigen Presenting Cells (APCs) in the innate immune system of mammals, and mainly play a role in capturing, processing and presenting antigens. Once activated, DCs migrate to the lymph nodes and, in combination with the T cells and B cells present therein, activate the adaptive immune system. Meanwhile, the DC can also interact with cells of the innate immune system such as natural killer cells, phagocytes and mast cells, and is a bridge for connecting innate immunity and adaptive immunity. DC are considered to be the most efficient antigen presenting cells capable of activating both the initial and memory immune responses, and improving the immunogenicity of vaccines against DC has become one of the important strategies for novel vaccine design. The DC targeted vaccine is to directly deliver antigen, DNA molecules or drugs to DC by identifying DC surface specific receptor molecules, thereby improving antigen presentation efficiency and quickly starting corresponding cellular and humoral immune responses. Therefore, ligand molecules targeting DC are developed to improve the immunogenicity of the antigen, and the ligand molecules have potential application values in the aspects of vaccine development, immunotherapy and the like.

With the continuous and deep research of DC biology, the technology for separating and culturing DC from peripheral blood and bone marrow is mature, which lays a good foundation for the research and development of DC targeted vaccines. The excellent vaccine can generate protective and therapeutic humoral and cellular immune responses at the same time, and the DC can present the targeted vaccine to CD4 in an MHC (major histocompatibility complex) limited mode⁺And CD8⁺T lymphocytes, thereby inducing the body to produce both immune responses simultaneously. Thus, combining an antigenic protein with a ligand for a pattern recognition receptor to prepare an antigen-pattern recognition receptor-ligand patterned targeted vaccine, elicited by targeting DCs to high levelsAn effective and durable immune response becomes one of the main strategies for developing new vaccines. The Gag, Nef and Pol proteins of HIV were fused to anti-CD 40 monoclonal antibodies targeting DCIR from CD40 and dendritic cells, which specifically induced CD4⁺T、CD8⁺T cell responses. The single-chain antibody of the targeted murine DC is fused with the adenovirus fiber knob to replace the original adenovirus fiber knob, and the constructed chimeric virus can improve the gene transduction efficiency of the targeted DC. Despite the advantages of targeting antigens directly to DCs for vaccine development, researchers still face a number of difficulties and challenges. The receptors targeted by different ligands are different, and the ligands targeting the same receptor have different binding sites, targeting properties and affinity, so that the activated immune response strength is different. Therefore, it is important to further explore valuable ligand molecules, evaluate the efficiency of the ligand molecules to activate DCs, and to use different ligand molecules in concert to induce strong and durable immune responses.

The phage display library presents polypeptide or antibody hypervariable regions composed of hundreds of millions of different amino acids on the surface of phage capsid protein in the form of fusion protein. The phage used to construct the library include filamentous phage, T7 phage, T4 phage, lambda phage, and MS phage, among others, with differences in display copy number, polypeptide length, display ends, and amino acid preference among the different phage. At present, filamentous bacteriophage displayed by an N end and T7 bacteriophage displayed by a C end are widely applied, and can display a random amino acid polypeptide library and construct an antibody hypervariable region display library. Through biological panning, i.e. incubation of phage library and target molecule, washing away phage not combined with target, collecting and amplifying combined phage for new panning, through repeated panning finally obtaining phage specifically combined with target molecule, sequencing and analyzing displayed amino acid for further research. The phage display library is a favorable tool for exploring an interaction binding site between a receptor and a ligand, exploring a high-affinity bioactive ligand molecule and detecting an epitope of an unknown protein spatial structure, and is widely applied to the fields of research on mutual recognition of protein molecules, novel vaccines and drug research and development. And (3) performing ligand molecule panning on the complete DC cells by using a phage display library, and exploring valuable ligand molecules to provide technical support for the development of a targeted vaccine.

The information disclosed in this background is intended to enhance an understanding of the invention and should not be taken as an acknowledgement or any form of suggestion that the information forms prior art that is already known to a person skilled in the art.

Disclosure of Invention

The invention aims to solve the technical problem of providing a dendritic cell-targeted nano antibody molecule and application thereof.

The invention provides a nano antibody molecule of a targeted dendritic cell, wherein the amino acid sequence of the nano antibody molecule is SEQ ID NO.1 or SEQ ID NO. 2.

MLGDPNSGGQVQLVESGGGLVQPGGSLRLSCAASGIAFGRAAVGWYRQAAGNERDMVATMTSGGRTSYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNVENVDRVLTLEPYDYWGQGTQVTVSSEPKTPKPQ(SEQ ID NO.1)。

MLGDPNSGGQVQLVESGGGLVQEGGSLRLSCVVSGFRDSKYPMTWVRQAPGKGLEWVSTITSGGTTTYADSVKGRFTISRDNAKDTLYLQMNSLKPEDTAIYYCAELGRGSDRWLWAVTVSSEPKTPKPQ(SEQ ID NO.2)。

The nano antibody molecules are obtained by affinity screening through a phage display library, specifically, SPF chicken bone marrow cells are separated, dendritic cells are induced and differentiated, a T7 phage library of a surface display alpaca nano antibody is constructed for screening target molecules, two high-affinity nano antibody molecules are screened from a T7 phage display library and are named as DC54 and DC74 respectively, and the amino acid sequences of the nano antibody molecules are shown as SEQ ID No.1 and SEQ ID No. 2.

The nano antibody molecule can be combined with not only chicken dendritic cells but also duck dendritic cells and goose dendritic cells, but also bone marrow mononuclear cells, peripheral blood lymphocytes and embryonic fibroblasts derived from chicken, duck and goose.

The invention relates to application of a nano antibody molecule of a targeted chicken dendritic cell in preparation of a vaccine.

The nano antibody molecule is used as an antigen carrier, so that the antigen presentation efficiency can be effectively improved, and the antibody level can be improved. The vaccine is a dendritic cell targeted vaccine.

The invention also provides a dendritic cell targeted subunit vaccine which is obtained by fusion expression of the dendritic cell targeted nano antibody molecule and subunit protein.

Compared with the prior art, the invention has the following effective effects:

1. the alpaca nano antibody T7 phage display library is constructed, affinity screening is carried out on chicken dendritic cells, and the nano antibody molecules with high affinity binding of the chicken dendritic cells are obtained, so that the material basis is improved for researching and developing dendritic cell targeted vaccines. The invention provides two nano antibody molecules, which can be combined with chicken-derived dendritic cells, duck-derived dendritic cells and goose-derived dendritic cells, and have the cell specificity combined with the dendritic cells and the species broad spectrum of different avian-derived dendritic cells.

2. The two nano antibody molecules screened from the alpaca nano antibody T7 phage display library have small relative molecular weight, low autoimmunity, high affinity and strong binding capacity with dendritic cell surface receptors, and can enter cells through receptor-mediated endocytosis. The nano antibody molecule can be used together with various drug carriers to achieve the target transport effect.

3. The invention prepares the chicken dendritic cell targeted subunit vaccine by fusing and expressing the two screened nano antibody molecules and subunit proteins, accelerates the processing and presenting efficiency of antigens, improves the antibody level of vaccine immunity and promotes the body to stimulate immune reaction earlier.

Additional features and advantages of the invention will be set forth in the detailed description which follows.

Drawings

The above and other objects, features and advantages of the present invention will become more apparent by describing in more detail exemplary embodiments thereof with reference to the attached drawings.

FIG. 1 shows the relevant electrophoretic validation results of VHH gene amplification in alpaca nanobody T7 phage display library construction.

FIG. 2 shows the results of sequencing of random clones from the T7 phage library.

FIG. 3 shows the detection results of nanobody molecules in the T7 phage library.

FIG. 4 shows cell-induced differentiation of dendritic cells under microscopic observation.

Figure 5 shows flow detection of dendritic cell surface markers.

Figure 6 shows the results of 3 rounds of screening of nanobody libraries.

FIG. 7 shows the results of monoclonal phage identification in the third round of screening products.

Fig. 8 shows the results of detection of binding affinity and specificity of nanobody molecules.

Fig. 9 shows laser confocal detection of nanobody molecular cellular localization.

Figure 10 shows the dendritic cell targeting subunit vaccine antibody level detection results.

Detailed Description

Preferred embodiments of the present invention will be described in more detail below. While the following describes preferred embodiments of the present invention, it should be understood that the present invention may be embodied in various forms and should not be limited by the embodiments set forth herein.

Example 1 construction of a llama Nanobody T7 phage display library

1. Separation of alpaca peripheral blood lymphocyte and total RNA extraction

The vacuum anticoagulation tube collects 5mL of peripheral blood of 6 alpacas respectively, and the blood and the anticoagulant are fully mixed by shaking up gently. The fresh blood was brought back to the laboratory and operated according to the instructions of the tianjin-third-class peripheral blood lymphocyte separation kit, the obtained lymphocytes were washed with 10mL of cell washing solution, 250 Xg, centrifuged for 10 minutes, the supernatant was discarded, washing was repeated 2 times, and the cells were then resuspended in 2mL of PBS buffer solution for use. The total RNA of the lymphocyte cells was extracted according to the instructions of the Solebao total RNA extraction kit (R1200). Taking total RNA as a template, and respectively carrying out reverse transcription by two primers of Random 6 and Oligo dT (Takara Shuzo) to obtain cDNA. The reaction procedure is as follows: 40min at 40 ℃; 15min at 70 ℃; stopping at 12 deg.C, and storing at-80 deg.C.

2. Total RNA extraction and VHH gene amplification

Using cDNA as a template and F1 and R1 as primers (Table 1), carrying out PCR amplification on the alpaca heavy chain antibody variable region coding gene by the following reaction procedures: 3min at 94 ℃; 30s at 94 ℃; 30s at 52 ℃; 1min at 72 ℃ and 30 cycles; after 10min at 72 ℃, the gel was cut to recover a fragment of about 700bp, as shown in A of FIG. 1. The recovered fragment was used as a template, and F2, R2-1 and R2-2 were used as primers (Table 1), and the reaction procedure was: 3min at 94 ℃; 30s at 94 ℃; 30s at 50 ℃; 30 cycles at 72 ℃ for 30 s; PCR amplification of VHH gene of about 400bp (B in FIG. 1) at 72 ℃ for 10min, recovery of target band with gel, Nanodrop quantification, and storage at-80 ℃ for future use. The recovered VHH gene was ligated to pMD18-T simple at a 3:1 molar ratio to transform E.coli DH 5. alpha. competent cells. The conversion product is coated on a substrate containing Amp⁺A petri dish of/IPTG/X-gal LB solid medium was incubated overnight at 37 ℃. The total number of colonies and the number of blue colonies were counted, and the capacity and recombination rate of the plasmid library were calculated. Removing blue colonies, collecting all colonies in the culture dish, re-suspending the colonies in LB liquid culture medium, extracting plasmids, carrying out double enzyme digestion identification on EcoRI and Hind III, and recovering a 400bp target fragment (C in figure 1).

TABLE 1 library construction and identification primers

Note: k ═ G, T; y ═ C, T; n ═ a, G, C, T; underlined are restriction sites

3. Construction and identification of T7 phage display library

The VHH fragment is subjected to double enzyme digestion treatment by EcoRI and Hind III I and is connected with T7 select EcoRI/Hind III carrier treated by the same enzyme in a molar ratio of 1:1.5, 5 mu l of the connection product is mixed with 25 mu l of packaging protein, the mixture reacts for 2h at 25 ℃, 270 mu l of LB liquid culture medium is added into the reaction system to terminate the reaction, and the rescued phage library is obtained. Taking 5 mul of the packaged product, diluting the packaged product by a 10-fold gradient, mixing the diluted packaged product with 200 mul of overnight-cultured T7 phage host bacteria BLT5403, determining the titer of the phage by a double-layer agar sandwich method, and calculating the library capacity; picking single plaques, detecting the insertion condition of the target gene by PCR, and calculating the recombination rate. Phage library capacity ═ plaque number × dilution multiple × 300; the recombination rate is equal to the number of PCR positive plaques/total number of detected plaques × 100%. Randomly selecting 20 plaque PCR positive products for sequence determination, and adopting DNAStar sequence analysis software to carry out comparison analysis on a sequencing result, wherein the sequencing result is shown in figure 2, and the randomly selected phage monoclonals are all correctly inserted into the VHH gene fragment. Transferring the remaining 295 mul of packaged product to 100ml of log-growth-phase BLT5403, carrying out shake culture at 37 ℃ for 3h until host bacteria are completely cracked, recycling the phage by a PEG-NaCl precipitation method, determining the titer of the library by a double-layer agar sandwich method, and detecting the nano antibody displayed on the surface of the phage by Western-blot, wherein the p10b-VHH protein formed by fusing the nano antibody molecule and T7 phage capsid protein is detected as shown in figure 3.

Example 2 isolated culture and characterization of chicken bone marrow-derived dendritic cells

1. Isolated culture of chicken bone marrow-derived cells

After being injected with an air needle, SPF chicks of about 15 days old die, the SPF chicks are soaked in 75% ethanol for 10min, thighbone and shinbone of the chicks are taken out under aseptic conditions, bone marrow is washed out by PBS containing 2% double antibody, bone marrow cells are collected, PBS buffer solution is used for washing for 2 times, each time is 150 Xg, centrifugation is carried out for 10min, and supernate is discarded. PBS resuspended and pelleted cells, then bone marrow cells were slowly added to centrifuge tubes containing Histopaque-1119(Sigma) at a volume ratio of 1:1 at 150 Xg for 30 minutes, white cell layers at the level of the supernatant were collected, PBS resuspended and washed 2 times at 150 Xg each time for 10 minutes. The cell pellet was collected, the cells were resuspended in 2mL 1640 medium containing 10% FBS and 1% double antibody, and cell counting was performed. Adjusting the cell concentration to 1X 10⁶cells/mL, then 3 mL/well in six-well cell culture plate, each well added working concentration of 30ng/mL rGM-CSF and rIL-4, induced cell differentiation. Half-volume change of the culture medium is carried out on the 3 rd day and the 6 th day after the culture, the mixed cells and the suspension dead cells are removed, meanwhile rGM-CSF and rIL-4 with working concentration are replenished, and the dendritic cells before maturation are harvested on the 8 th day of the culture.

2. Morphology observation of chicken dendritic cells

During the cell culture period, the differentiation induction of the cells was observed by using a light microscope, and the results are shown in FIG. 4. Cells are inoculated into a six-hole plate and gradually attached to the wall after being cultured for 2 to 3 hours, and the cells are bright in color and small in volume. After 24 hours of culture, the cell plate was gently shaken, and it was found that the number of adherent cells was slightly increased and the number of cell colonies was still small. On day 3 of culture, the cell plate was gently shaken to remove the suspended cells, and the culture medium was changed, at which time the number of adherent cells was further increased and the number of cell colonies was increased. When the cells are cultured to the 6 th day, cell colonies are increased and are in a loose adherent state, induced differentiation of the cells can be obviously tentatively performed, at the moment, the cells are dendritic cells before maturation, and LPS (lipopolysaccharide) is added for stimulation, so that the cells can be further promoted.

3. Chicken dendritic cell flow assay

Dendritic cells cultured to day 8 were collected, centrifuged at 150 Xg for 10 minutes, the supernatant was discarded, the cells were resuspended in PBS and adjusted to 1X 10⁶cells/mL. PE-labeled human anti-CD 11c, FITC-labeled chicken anti-CD 86, and FITC-labeled chicken anti-MHC II antibodies at working concentrations were added, incubated at room temperature in the dark for 30 minutes, washed 3 times with PBS (150 Xg each), centrifuged for 10 minutes, resuspended in 200. mu.l PBS, and measured by flow cytometry. As can be seen from FIG. 5, the expression level of CD11c molecules on the surface of the dendritic cells induced to differentiate is 71.26%, the expression level of MHC II molecules is 78.88%, the expression level of CD86 molecules is 73.78%, and the purity of the cultured dendritic cells is high, so that the requirement of differential screening of a nano antibody library can be met.

Example 3 affinity panning of an alpaca Nanobody T7 phage display library

1. Differential screening chicken dendritic cell targeted nano antibody molecule

The chicken dendritic cells prepared in example 2 were subjected to 3 rounds of affinity panning using the alpaca nanobody T7 phage display library constructed in example 1. Nanobody libraries were adjusted to concentrations of 1 × 10¹¹PFU/mL, chicken bone marrow cells resuspended in 2mL 1640 medium containing 10% FBS (5X 10)⁵cells/mL), incubated at 37 ℃ for 30 minutes, centrifuged at 150 Xg for 10 minutes, and the supernatant collected and labeled Input. The collected supernatant was used to resuspend the chicken dendritic cells (1X 10) cultured to day 8 in example 2⁶cells), incubated at 37 ℃ for 30 minutes. 150Xg for 10min, remove the supernatant, labeled Unbound. The cells were resuspended in 1640 medium containing 0.05% Tween-20 and 10% FBS, centrifuged at 150 Xg for 10min, the supernatant was removed, labeled Wash 1, and the washing procedure was repeated 5 times. After the 5 th wash, the cells were resuspended with 200. mu.l of eluent (1% SDS) to completely release the cell-bound phage, labeled R1. Mu.l of the eluted product was used for the double-layer agar assay to determine the product titer, and the remaining product was inoculated with 200mL of OD_600nmBL5405 E.coli host, 1.0, was shake-cultured at 37 ℃ until the host was completely lysed, and the purified amplification product, labeled R1A, was recovered by the PEG-NaCl method. According to the screening process, the amplification products of the first round of screening eluent are used for 2 nd round of screening, and the amplification products of the 2 nd round of screening eluent are used for 3 rd round of screening. As can be seen in FIG. 6, the unbound phage and low-affinity bound phage were rinsed out in each round of screening, the high-affinity bound phage in the eluate increased with the increase of the number of screening, and the phage bound to the chicken dendritic cells was enriched by affinity screening, thereby increasing the efficiency of screening monoclonal phage from the 3 rd round of eluate.

2. Identification of monoclonal phage in screened products

200 phage monoclonals are selected from the 3 rd round screening products to carry out VHH nano antibody gene sequence analysis, and the sequences are counted, synthesized and repeated. Selecting 50 repeated phage clones from the above-mentioned material, making them implement monoclonal culture, and regulating phage concentration to 1X 10¹⁰PFU/mL. 1X 10 wells in 96-well plates⁵cells Bone marrow cells (Bone marrow cells), Dendritic cells (Dendritic cells), and culture medium (Serum) control wells. Each titer adjusted monoclonal phage was inoculated in 100. mu.l, each phage was plated in 3 wells in a single batch, incubated at 37 ℃ for 30 minutes in an incubator, the cell plates were centrifuged at 150 Xg for 10 minutes, and the supernatant in each well was carefully removed. The washing was repeated 5 times at 200. mu.l/well of 1640 culture medium containing 0.05% Tween-20 and 10% FBS, the supernatant was removed, 1% SDS eluent was added at 50. mu.l/well, phages were recovered from each well, and the titer of phages in the recovered solution was determined by a double-agar sandwich method. Calculating the monoclonal antibody and the monoclonal antibody of different phages according to the formula that the recovery rate is recovery Phage (PFU)/input Phage (PFU) x%Affinity and specificity of dendritic cell binding. As shown in FIG. 7, phage monoclonals 16, 54 and 74 have higher affinity with dendritic cells, but do not bind with bone marrow cells and serum or cell plates in cell culture systems, and have better specificity. Selecting phage monoclonals No. 54 and No. 74, and respectively comparing the binding capacities of the selected phage monoclonals with chicken bone marrow cells (C.BMC), chicken dendritic cells (C, DC), chicken embryo layer fibroblasts (CEF), duck bone marrow cells (D.BMC), duck dendritic cells (D, DC), duck embryo layer fibroblasts (DEF), goose bone marrow cells (G.BMC), goose dendritic cells (G, DC) and goose germ layer fibroblasts (GEF) by adopting the detection method. As can be seen from FIG. 8, phage monoclonals 54 and 74 can specifically bind to dendritic cells of chicken, duck and goose, but not to bone marrow cells and lamina fiber cells.

3. Localization of Nanobody molecules on dendritic cells

The sterile slide was placed in a 24-well plate, 200. mu.l of polylysine (0.03mg/mL) was added per well, allowed to act at room temperature for 30 minutes, the solution was spun off, and washed twice with PBS. 1X 10 additions per well⁶cells (chicken dendritic cells) were cultured up to day 6 and continued for 36 hours. Inoculation of 1X 10 per well¹⁰PFU phage monoclonal (54, 74), and set T7-wt blank, 37 degrees C were incubated for 30 minutes, containing 0.05% Tween-20 and 10% FBS 1640 culture solution washing 5 times. Mu.l of 1% paraformaldehyde fixative was added to each well, allowed to react for 15 minutes at room temperature, and washed once with PBS. Then, 100. mu.l of 0.2% Trition-100 was added to each well to permeabilize the cell membrane, and washed once with PBS. DyLight-labeled T7 tag mab (Abcam) and Phalloidin-iFluor 594conjugate (Absin) dye were added at working concentrations, incubated at 37 ℃ for 30 min in the dark, and the slide was washed 4 times with PBST. And (3) dropwise adding an anti-fluorescence quenching blocking tablet (containing DAPI) on the climbing sheet, covering a cover glass, and observing the positioning of the screened targeted phage in the dendritic cells by using a laser confocal microscope. As can be seen from FIG. 9, there are a large number of green fluorescence signal spots on the surface and in the cytoplasm of the dendritic cells, while the wild T7 phage of the control did not bind to the cells, indicating that the nanobody molecules displayed on the surface of the No. 54, 74 phage selected were able to efficiently bind to the dendritic cells andand mediates entry of the phage into the cell.

Example 4 evaluation of the efficiency of a Targeted dendritic cell subunit vaccine

PCR amplification is carried out by taking T7-wt and No. 54, 74T 7 phages as templates to obtain p10B, p10B-VHH fusion genes, and respectively inserted into pET-28a expression vectors to induce and express p10B, p10B-VHH₅₄、p10B-VHH₇₄Three subunit proteins. The three proteins were adjusted to the same concentration and mixed with ISA206 adjuvant to prepare subunit vaccine with antigen content of 50 μ g/0.1 mL. SPF (specific pathogen free) chickens of 20 days are immunized in groups, and blood sampling and serum separation are started after 2 weeks of immunization. Coating p10B protein, establishing indirect ELISA method, detecting the level of p10B antibody of immune chicken. As can be seen from the results of antibody level monitoring in FIG. 10, the fusion protein group of the fused chicken dendritic cell targeting nanobody molecule can stimulate the generation of antibody level earlier than the pure p10B group, and the titer of the antibody level is obviously increased. The result shows that the nanometer antibody of the targeted chicken dendritic cells can promote the antigen presentation of subunit protein and promote the organism to generate immune response more quickly and better.

Having described embodiments of the present invention, the foregoing description is intended to be exemplary, not exhaustive, and not limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments.

Sequence listing

<110> Jiangsu agriculture and animal husbandry science and technology occupational academy

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