Anti-goat cd207 single-chain antibody and preparation method and application thereof

Abstract

The application discloses an anti-goat CD207 single-chain antibody and a preparation method and application thereof. Based on nucleotide sequences of heavy chain and light chain variable regions of obtained anti-goat CD207 monoclonal antibodies, a single-chain antibody capable of specifically combining with goat CD207 protein is obtained through single-chain antibody gene construction and prokaryotic expression, a heavy chain variable region amino acid sequence of the single-chain antibody is shown as SEQ.ID.NO.1, and a light chain variable region amino acid sequence is shown as SEQ.ID.NO.2. The anti-goat CD207 single-chain antibody of the application has good reactivity with goat CD207 protein.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and molecular detection, and relates to the construction of a prokaryotic expression system for anti-goat CD207 single-chain antibody and the identification of its reactivity. Background Technology

[0002] Immunization is a key measure in the prevention and control of goat diseases. However, in actual animal production, conventional vaccine immunization still faces problems such as uncertain immunization effects and low immunogenicity. The development of novel vaccines based on targeting antigen-presenting cells is an important means to effectively enhance vaccine immunogenicity, and the preparation of specific antibodies targeting antigen-presenting cells is the key and prerequisite for developing such novel vaccines. CD207 is an important cell surface marker molecule of dendritic cells (DCs) and Langerhans cells (LCs). As an antigen receptor, it participates in the capture and presentation of antigens by DCs and other cells, and also has the function of regulating DC migration and DC-lymphocyte interactions.

[0003] Single-chain variable fragments (scFvs) are recombinant antibodies formed by linking the heavy chain variable region (VH) and light chain variable region (VL) of a complete antibody using genetic engineering methods through a flexible short peptide (i.e., a linker). Due to their small molecular weight, lack of Fc terminus and complement binding sites, and low immunogenicity, scFvs have high research and application value in targeted vaccines, targeted anti-tumor drugs, and molecular detection.

[0004] Although single-chain antibodies have been prepared using various expression systems, certain limitations remain. For example, in the study "Prokaryotic Expression, Purification, and Biological Function of DNA-PKcs Single-Chain Antibody DPK3-scFv," the specific humanized single-chain antibody DPK3-scFv gene, previously screened from a phage antibody library, was codon-dehumanized and then re-expressed in prokaryotes, ultimately verifying the transmembrane transport and inhibitory activity of DPK3-scFv on tumor cells. In the study "Preliminary Study on Screening for Targeted CD32ascFv and its Killing Ability in CAR-T Cells," the CD32ascFv gene obtained through phage display technology was introduced into human T cells, and target cell killing experiments were performed on recombinant CD32a-CAR-T cells, but the results showed insufficient specificity for killing. Summary of the Invention

[0005] The purpose of this invention is to provide an anti-goat CD207 single-chain antibody, its preparation method, and its application, which can provide a novel single-chain antibody tool material (specifically binds to goat CD207 protein and has good reactivity) for the development of targeted goat dendritic cell vaccines.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, an anti-goat CD207 single-chain antibody is provided, the single-chain antibody comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ.ID.NO.1 and the amino acid sequence of the light chain variable region is shown in SEQ.ID.NO.2.

[0008] Preferably, in the single-chain antibody, the heavy chain variable region and the light chain variable region are linked by a linker (e.g., a 15-peptide "(Gly4Ser)3").

[0009] Preferably, the amino acid sequence of the single-chain antibody is shown in SEQ.ID.NO.3.

[0010] Preferably, the single-chain antibody is prepared using a prokaryotic expression system (e.g., using prokaryotic cells such as Escherichia coli as a host for expressing the single-chain antibody and introducing the corresponding target gene), and the target expression product obtained using this system is a fusion protein; the fusion protein includes the single-chain antibody, or the fusion protein is composed of the single-chain antibody.

[0011] Preferably, the fusion protein further includes an expression tag (e.g., an HA tag, a His tag), meaning that the fusion protein can be a recombinant protein that fuses the expression tag and the single-chain antibody.

[0012] Preferably, the sequence information of the heavy chain variable region and light chain variable region of the single-chain antibody is obtained through experimental analysis of the transcripts of anti-goat CD207 monoclonal antibody hybridoma cells. Specifically, total RNA is extracted from anti-goat CD207 monoclonal antibody hybridoma cells and reverse transcribed. Using designed primers, gene fragments of the heavy chain and light chain variable regions are cloned from the cDNA obtained by reverse transcription, and then sequenced.

[0013] Preferably, the method for preparing the hybridoma cells includes the following steps:

[0014] 1) The extracellular region of goat CD207 was expressed in prokaryotes to obtain a truncated goat CD207 protein (amino acid sequence as shown in SEQ.ID.NO.7);

[0015] 2) Mice were immunized with the truncated goat CD207 protein prepared in step 1;

[0016] 3) After step 2, mouse spleen cells are fused with myeloma cells, and the resulting fused cells are used to screen for hybridoma cells (strains) against goat CD207 monoclonal antibodies.

[0017] Preferably, in step 3, a number of positive hybridoma cells (strains) are collected by expanding the culture to obtain the cell supernatant type anti-goat CD207 monoclonal antibody.

[0018] Preferably, the heavy chain subtype of the monoclonal antibody is IgG2b, and the light chain subtype is Kappa.

[0019] In a second aspect, an anti-goat CD207 single-chain antibody gene is provided, the gene comprising a nucleotide sequence encoding the aforementioned anti-goat CD207 single-chain antibody.

[0020] Preferably, the gene specifically includes a nucleotide sequence encoding the heavy chain variable region, a linker sequence (i.e., a nucleotide sequence encoding the linker), and a nucleotide sequence encoding the light chain variable region, wherein the nucleotide sequence encoding the heavy chain variable region is shown in SEQ.ID.NO.4, and the nucleotide sequence encoding the light chain variable region is shown in SEQ.ID.NO.5.

[0021] Preferably, the nucleotide sequence of the gene is shown in SEQ.ID.NO.6.

[0022] Thirdly, a recombinant plasmid expressing an anti-goat CD207 single-chain antibody is provided, the recombinant plasmid comprising an initial plasmid backbone and the aforementioned anti-goat CD207 single-chain antibody gene linked to the backbone.

[0023] Preferably, the initial plasmid is a prokaryotic expression plasmid, and the specific type can be selected according to actual needs, such as pET-28a or pET-32a.

[0024] Fourthly, a recombinant cell expressing an anti-goat CD207 single-chain antibody is provided, which is obtained by transforming the aforementioned recombinant plasmid expressing the anti-goat CD207 single-chain antibody into a host cell.

[0025] Preferably, the host cell is Escherichia coli.

[0026] Fifthly, a method for preparing an anti-goat CD207 single-chain antibody is provided, comprising the following steps:

[0027] The above-mentioned anti-goat CD207 single-chain antibody gene (e.g., constructed by overlap extension PCR) is ligated into an initial plasmid to construct a recombinant plasmid; the recombinant plasmid is transformed into host cells to construct recombinant cells; the recombinant cells are cultured and the single-chain antibody is induced to be expressed during the culture process, and then the single-chain antibody is purified from the expression product.

[0028] Sixthly, the application of the aforementioned anti-goat CD207 single-chain antibody in recognizing goat CD207 is provided.

[0029] Preferably, the identification is performed by utilizing the reactivity of the single-chain antibody with goat CD207 protein and detecting goat CD207 protein using ELISA or Western Blot.

[0030] Seventhly, the application of the above-mentioned anti-goat CD207 single-chain antibody in the preparation of goat CD207 protein detection reagents (kits) is provided.

[0031] Preferably, the detection reagent (kit) is specifically an ELISA reagent (kit) or a Western Blot reagent (kit).

[0032] The beneficial effects of this invention are reflected in:

[0033] This invention is the first to propose an anti-goat CD207 single-chain antibody gene that can be directly expressed in prokaryotes. Furthermore, ELISA and Western Blot experiments show that the expressed anti-goat CD207 single-chain antibody has the ability to specifically recognize goat CD207 protein and exhibits good reactivity with goat CD207 protein. It can serve as a tool for developing vaccines targeting goat dendritic cells and is of great significance for improving the prevention and control of diseases in goats.

[0034] Furthermore, the anti-goat CD207 single-chain antibody of the present invention has the structural characteristics of having no Fc terminus and no complement binding site, which can avoid ADCC effect and complement binding effect in vivo.

[0035] Furthermore, this invention utilizes the screened positive hybridoma cells to clone gene fragments of the heavy and light chain variable regions, which not only provides a template for the construction of anti-goat CD207 single-chain antibody genes, but also allows the anti-goat CD207 single-chain antibody genes to be used for efficient expression of anti-goat CD207 single-chain antibodies after being introduced into a prokaryotic host (e.g., Escherichia coli). Attached Figure Description

[0036] Figure 1 goat CD207 protein （69-329） Expression and purification results; where: A is the SDS-PAGE image of the expressed protein induced by IPTG at 37 ℃, 220 rpm, and 0.25 mM for 6 h; B is the His tag (M: protein marker; lane 1: pET-28a empty vector; lane 2: recombinant vector pET-28a-goat CD207 protein) detected by Western Blot after 6 h of induction. （69-329）Uninduced; Lane 3: Whole cells after induction; Lane 4: Supernatant after induction; Lanes 5 and 6 after induction: Precipitate (35.6 kDa); C represents the SDS-PAGE image of the expressed protein and the His tag of the expressed protein detected by Western Blot under the conditions of IPTG induction at 16 ℃, 100 rpm, and 0.25 mM for 20 h (M: Protein Marker; Lane 1: pET-28a empty vector; Lane 2: Recombinant vector pET-28a-goat CD207 protein). （69-329） Uninduced; Lane 3: Whole cells after induced expression; Lane 4: Supernatant after induced expression; Lane 5: Precipitate after induced expression (35.6 kDa); D is the SDS-PAGE image of purified expressed protein (M: Protein Marker; Lane 1: Goat CD207 protein). （69-329） Stock solution; Lane 2: Flow-through buffer after protein loading; Lane 3: 100 mM imidazole flow-through buffer; Lanes 4, 5, 6, 7, and 8: 500 mM imidazole elution buffer.

[0037] Figure 2 The images show the fusion of Balb / c mouse spleen cells and myeloma cells (SP2 / 0) under a microscope (10×10 magnification). Among them: A is the cells under a microscope after 5 days of SP2 / 0 culture expansion (on the day of fusion); B is the cells under a microscope on the 1st day after fusion; C is the cells under a microscope on the 4th day after fusion; and D is the cells under a microscope on the 7th day after fusion.

[0038] Figure 3 The monoclonal antibody subtypes in the supernatant of four positive hybridoma cells were identified using a mouse monoclonal antibody subtype identification kit (PK20002); where: A is the ELISA colorimetric result of the kit; B is the OD450 value of the kit.

[0039] Figure 4 The reactivity of monoclonal antibodies produced from four hybridoma cell lines (B10, F7, E3, and G4) with prokaryotically expressed proteins was determined (M: protein marker; lane 1: prokaryotic expression of truncated bovine CD207 protein; lanes 2, 3, and 4: prokaryotic expression of truncated goat CD207 protein using pET-28a as the initial vector, diluted 2-fold, 5-fold, and 10-fold, respectively; lane 5: prokaryotic expression of truncated goat CD207 protein using pET-32a as the initial vector).

[0040] Figure 5The reactivity of monoclonal antibodies produced from four hybridoma cell lines (B10, F7, E3, and G4) with eukaryotic expressed proteins was identified. Specifically: A represents electrophoresis detection using recombinant plasmids with pcDNA3.1(+) as the initial vector (lane 1: empty pcDNA3.1(+) plasmid; M: DNA Marker; lane 2: recombinant plasmid double digestion); B represents Western blotting detection of the reactivity of the four monoclonal antibodies with eukaryotic expressed goat CD207 protein (FLAG tag indicates successful expression of goat CD207 protein; M: protein Marker; lane 1: untransfected cell sample; lane 2: cell sample transfected with empty pcDNA3.1(+) vector; lane 3: cell sample transfected with recombinant plasmid).

[0041] Figure 6 The reaction of monoclonal antibodies produced by four hybridoma cell lines (B10, F7, E3, and G4) with eukaryotic goat CD207 protein was detected by IFA assay (MOCK was the untransfected control).

[0042] Figure 7 Agarose gel electrophoresis image of total RNA from hybridoma cell line F7 (blank: blank control).

[0043] Figure 8 To amplify the variable region gene fragments of the heavy and light chains of monoclonal antibodies using degenerate primers; where: A is the amplification of the heavy chain variable region using primers HF-3 and HB-1 ( VH Gene fragment electrophoresis detection (M: DNA Marker; Lane 1: Blank control; Lane 2: Heavy chain variable region gene fragment); B is the heavy chain variable region ( VH Colony PCR identification was performed after the gene fragment was ligated into the pMD-19T vector; C represents the amplification of the light chain variable region using primers LF-4 and LB-1. VL Gene fragment electrophoresis detection (M: DNA Marker; Lane 1: Blank control; Lane 2: Light chain variable region gene fragment); D: Light chain variable region ( VL The gene fragment was ligated into the pMD-19T vector and then identified by colony PCR.

[0044] Figure 9 To utilize the IMGT and VBASE2 websites for VH The gene sequences were divided into FR and CDR.

[0045] Figure 10 To utilize the IMGT and VBASE2 websites for VL The gene sequences were divided into FR and CDR.

[0046] Figure 11 for scFv Gene construction; where: A is a sequence with a linker sequence (specifically...) Linker )of VH , VL gene fragments VH-Linker , Linker-VL Amplification (M: DNA Marker; Lane 1: Blank Control; Lane 2: ...) VH-Linker Amplification; Lane 3: Linker-VL (Amplification); B is scFv Genes (through) VH gene sequence and VL It is assembled by introducing linker sequences between gene sequences, that is... VH-Linker-VL Amplification (lane 1: blank control; M: DNA Marker; lane 2: ...) VH-Linker-VL (Amplification).

[0047] Figure 12 Gene sequence analysis for anti-goat CD207 single-chain antibody.

[0048] Figure 13 The results show the prokaryotic expression of the anti-goat CD207 single-chain antibody; A is the SDS-PAGE image of the single-chain antibody expressed using pET-28a as the initial vector under IPTG induction at 37 ℃, 220 rpm, and 0.25 mM for 6 h (M: protein standard molecule; lane 1: pET-28a empty vector; lane 2: recombinant vector pET-28a-anti-goat CD207 scFv-HA uninduced; lane 3: whole bacteria after induction; lane 4: supernatant after induction; lane 5: precipitate after induction (32 kDa)); B is the Western blot detection of the HA tag of the single-chain antibody expressed by pET-28a-anti-goat CD207 scFv-HA; C is the result of the expression of the single-chain antibody expressed using pET-32a as the initial vector under IPTG induction at 37 ℃, 220 rpm, and 0.25 mM for 6 h. SDS-PAGE images of single-chain antibody expression under h conditions (M: protein standard molecule; lane 1: pET-32a empty vector; lane 2: recombinant vector pET-32a-anti-goat CD207 scFv-HA uninduced; lane 3: whole bacteria after induction; lane 4: supernatant after induction; lane 5: precipitate after induction (46.2 kDa)); D is the HA tag of single-chain antibody expressed by pET-32a-anti-goat CD207 scFv-HA detected by Western Blot.

[0049] Figure 14The results of affinity chromatography purification of anti-goat CD207 single-chain antibody are shown below (M: protein standard molecule; lanes 1 and 14: anti-goat CD207 single-chain antibody stock solution; lanes 2, 3, and 4: flow-through buffer; lanes 5, 6, 7, and 8: 50 mM imidazole elution buffer; lanes 9, 10, 11, 12, and 13: 150 mM imidazole elution buffer; lanes 15, 16, 17, and 18: 250 mM imidazole elution buffer; lanes 19, 20, 21, 22, 23, and 24: 500 mM imidazole elution buffer).

[0050] Figure 15 The reactivity of anti-goat CD207 single-chain antibody was detected by indirect ELISA; where: A is the ELISA colorimetric result; B is the OD450 value.

[0051] Figure 16 The reactivity of anti-goat CD207 single-chain antibody was detected by Western blotting. In lane A, the primary antibody was a monoclonal antibody produced using hybridoma cell line F7 (M: protein standard molecule; lane 1: pET-28a empty vector control; lane 2: prokaryotic expression of truncated bovine CD207 protein control; lanes 3 and 4: prokaryotic expression of truncated goat CD207 protein using pET-28a as the initial vector); in lane B, the primary antibody was an anti-goat CD207 single-chain antibody (M: protein standard molecule; lane 1: pET-28a empty vector control; lane 2: prokaryotic expression of truncated bovine CD207 protein control; lanes 3 and 4: prokaryotic expression of truncated goat CD207 protein using pET-28a as the initial vector). Detailed Implementation

[0052] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are only used to explain the present invention and are not intended to limit the scope of protection of the present invention.

[0053] (I) Preparation of anti-goat CD207 monoclonal antibody

[0054] 1. Preparation of goat CD207 immunogen

[0055] 1.1 Recombinant vector pET-28a-goat CD207 protein （69-329） Build

[0056] Search for goats on NCBI CD207 The full-length gene sequence (GenBank accession number: XM_005686383.3) was sent to Xi'an Qingke Biotechnology Co., Ltd. for codon optimization and full-length synthesis. Synthesis was performed in goats. CD207 BamHI and HindIII restriction sites were introduced at the 5' and 3' ends of the gene, ultimately yielding a product containing goat protein from the company. CD207 Cloning plasmids of the full-length gene sequence.

[0057] The amino acid sequence of the extracellular region of goat CD207 was determined using analysis software to predict the transmembrane region and signal peptide (i.e., SEQ.ID.NO.7, a total of 261 amino acids):

[0058] MGTISDVKTNAQLLKGRVDNISSLSSEIKRNRGALVAVGFQVRMVNASLDRISPQIRRLETGLKEASAQLQVLTSSWEAVDELNAQIPGLKQDLDRASALNAKVRELQSGLESISKLLQQQNDILQVVSQ GWKYFRGHFYYFSQISKTWYSAQQMCISRDSHLTSVTSEREQEFLYRTAGGLPYWIGLTKAGSEGDWHWVDGTPFNKVQSEKFWIPGEPNNYGNNEHCVNLKMSSLRSWNDASCDNTFPFICKRPYKPSEP

[0059] Expanding goats CD207 Extracellular gene sequence, using homologous recombinase to sequence goat genes. CD207 The extracellular gene sequence was ligated into the pET-28a vector (preserved in the laboratory or purchased from the manufacturer), transformed into DH5α competent cells, and screened using solid LB plates containing 50 μg / mL kanamycin. Single colonies were picked for colony PCR, plasmid double enzyme digestion identification, and sequencing analysis to obtain the recombinant plasmid for expressing the truncated goat CD207 protein, named pET-28a-goat CD207 protein. （69-329） .

[0060] 1.2 Goat CD207 protein （69-329） Expression and purification

[0061] (1) The constructed recombinant plasmid was transformed into BL21 competent cells, and then evenly spread onto a solid LB plate containing 50 μg / mL kanamycin and inverted in a 37 ℃ incubator overnight.

[0062] (2) Select single clones from the plate and inoculate them into 20 mL of LB liquid medium containing 50 μg / mL kanamycin. Incubate at 37 ℃ and 220 rpm until the OD600 is 0.6~0.8. Then add IPTG to a final concentration of 0.25 mM. Induce expression at 37 ℃ and 220 rpm for 6 h, or at 16 ℃ and 100 rpm for 20 h. At the same time, set up empty vector and uninduced control groups.

[0063] (3) Centrifuge the culture medium at 5000 rpm for 10 min. Resuspend the bacterial pellet in lysis buffer (containing 100 mM NaH2PO4 and 100 mM Tris-HCl). Disrupt the pellet using an ultrasonic cell disruptor for 10 min. Take the disrupted whole cells, supernatant, and pellet (resuspended in lysis buffer containing 8 M Urea) and add them to SDS-PAGE protein loading buffer. Boil at 100 ℃ for 10 min and then perform electrophoresis. After electrophoresis, stain the protein gel with Coomassie Brilliant Blue, then destain. The results are shown in [Figure 1]. Figure 1 A, Figure 1 C. Additionally, using the His antibody (Bio-Long Biotech) as the primary antibody, Western blotting was performed on the expressed goat CD207 protein. （69-329） The test was conducted, and the results are shown below. Figure 1 B Figure 1 C.

[0064] (4) On goat CD207 protein （69-329） Large-scale expression was performed (cultured in a larger volume of medium until OD600 reached 0.6–0.8, followed by induction with 0.25 mM IPTG at 37 ℃, 220 rpm, for 6 h). The bacterial pellet after centrifugation was resuspended in lysis buffer and sonicated. After lysis, the pellet was centrifuged again, and dissolved in lysis buffer containing 8 M Urea. Protein purification was performed using a Ni-NTA affinity chromatography column (Tiandi Renhe Ni NTA Beads 6FF SA005005 5 mL). Different concentrations of imidazole buffer were used to elute the target protein. Each eluted fraction was collected for SDS-PAGE protein electrophoresis. The results are shown in [Figure number missing]. Figure 1 D.

[0065] (5) After purification by Ni-NTA affinity chromatography, the bands with relatively high purity concentration were collected, dialyzed into 1×PBS (pH 8.0), and aliquoted and stored at -80 ℃.

[0066] 2. Immunization of Balb / c mice

[0067] Goat CD207 protein required for immunization （69-329） The antigen was emulsified with Freund's adjuvant at a 1:1 ratio for 2 hours and then used to immunize four Balb / c mice (purchased from Shaanxi Pharmaceutical Holding Group Co., Ltd.). The total amount of antigen per mouse per immunization was 25-50 μg. Immunization was performed every two weeks. After four immunizations, serum titers were measured by indirect ELISA. Cell fusion was performed after a fifth booster immunization. The specific immunization program is shown in Table 1, and the serum antibody titers after four immunizations are shown in Table 2.

[0068]

[0069]

[0070] The above results indicate that Balb / c mice numbered "-2" (i.e., Balb / c-2) had a high serum antibody titer after the fourth immunization, reaching 2.04 × 10⁻⁶. 5 Cell fusion can occur after shock immunization.

[0071] 3. Hybridoma cell preparation

[0072] 3.1 Resuscitation and Expanded Culture of Myeloma Cells (SP2 / 0)

[0073] One week prior to fusion, SP2 / 0 cell cryopreservation tubes were removed from the liquid nitrogen tank and thawed in a 37°C water bath. After complete thawing, the cells were centrifuged at 800 rpm for 10 min at room temperature using an intercellular centrifuge. After centrifugation, the cells were transferred to a UV-sterilized laminar flow hood, the supernatant was discarded, and the pellet was gently resuspended in 1 mL of complete RPMI-1640 medium containing 20% ​​fetal bovine serum and 1% penicillin-dextrose antibiotics. The pellet was then transferred to a 60 mm cell culture dish, and complete RPMI-1640 medium was added to a final volume of 3 mL. The dish was then incubated overnight at 37°C with 5% CO2.

[0074] After overnight culture, the cells were observed to be in good condition under an inverted microscope, with some cell debris floating. The SP2 / 0 cells were then cultured in a new medium. The cells were resuspended in the culture dish and collected into autoclaved 10 mL centrifuge tubes, sealed with film, and centrifuged at 800 rpm for 10 min at room temperature. The tubes were then resuspended in complete RPMI-1640 medium in a clean bench and cultured in new culture dishes. The medium was changed every 12 h depending on the cell growth status. When the cells reached 90%–100% confluence, they were transferred to T-75 cell culture flasks for further culture. Two confluent T-75 cell culture flasks were required to fuse one mouse spleen cell. Figure 2 A).

[0075] On the day of fusion, the cells were resuspended in 30 mL of incomplete RPMI-1640 medium and collected in a 50 mL centrifuge tube. The tube was centrifuged at 800 rpm for 10 min at room temperature. The supernatant was discarded, and 10 mL of incomplete RPMI-1640 medium was added. The mixture was thoroughly mixed and ready for use.

[0076] 3.2 Pretreatment of mouse spleen cells

[0077] (1) After autoclaving the required experimental equipment, place it in a clean bench for ultraviolet sterilization. Remove the eyeballs of mice (specifically Balb / c-2) to expel blood, collect the blood in 1.5 mL EP tubes to prepare positive control serum, then euthanize the mice by dislocation of the neck and immerse them in 75% alcohol for 10 min.

[0078] (2) Place the mouse abdomen up in the cell plate groove, cut open the mouse abdomen skin, use forceps to bluntly separate the skin to expose the peritoneum, find the approximate location of the spleen and disinfect with an alcohol swab.

[0079] (3) Take a culture dish and add 10 mL of incomplete RPMI-1640 medium. Use forceps to pick up the peritoneum covering the spleen, cut open the peritoneum, separate the spleen and place it in the culture dish. Trim the connective tissue that is attached to the spleen. Place the spleen on a 200-mesh filter and place the filter in a culture dish containing incomplete RPMI-1640 medium. Squeeze the spleen to allow the spleen cells to pass through the filter.

[0080] (4) Collect the obtained spleen cell suspension in a 50 mL centrifuge tube and add incomplete RPMI-1640 medium to 30 mL. Centrifuge at 1000 r / min for 10 min at room temperature, discard the supernatant, and resuspend the spleen cells in 10 mL of incomplete RPMI-1640 medium for later use.

[0081] 3.3 Fusion of mouse spleen cells with myeloma cells (SP2 / 0)

[0082] (1) Adjust the water bath temperature to 37 ℃ and place it in the clean bench to provide a suitable temperature for cell fusion.

[0083] (2) Mix 10 mL of mouse spleen cell suspension obtained after pretreatment with 10 mL of myeloma cell suspension thoroughly. Centrifuge at 1000 r / min for 10 min at room temperature, discard the supernatant, and gently tap the bottom of the centrifuge tube with your finger to loosen the cells on the bottom wall of the tube.

[0084] (3) Place the centrifuge tube in a water bath, take 1 mL of PEG 1500 and slowly add it along the wall of the centrifuge tube while rotating the centrifuge tube continuously and evenly. The addition should be completed within 60 seconds.

[0085] (4) Gently shake the bottom continuously, add 1 mL of incomplete RPMI-1640 medium over approximately 1 min, add 3 mL of medium over 3 min, and finally slowly add 10 mL of medium all at once to terminate the fusion. Mix thoroughly and let stand in a water bath for 5 min. Centrifuge at 700 r / min for 8 min at room temperature, discard the supernatant, and add HAT medium. Suspend the fused cells in a 96-well plate containing feeder cells and incubate at 37 ℃ in a 5% CO2 incubator.

[0086] (5) See Figure 2 B. Figure 2 C Figure 2D. On day 1 after cell fusion, observe whether the cells are contaminated; on day 3 after fusion, observe the cell state (grape-like cell clusters); on days 3 and 6 after fusion, change the culture medium, discarding 100 μL of culture supernatant each time and adding 100 μL of complete RPMI-1640 medium containing HAT. When the fused cells grow to about 90% of the cell pores, identify and screen hybridoma cells.

[0087] 3.4 Identification of hybridoma cells

[0088] (1) Antigen coating: Add 100 μL of diluted goat CD207 protein to the sample wells of the ELISA plate. （69-329） (Total amount per well: 400 ng), overnight coating at 4 °C.

[0089] (2) Sealing: Discard the coating solution in the well, add 200 μL of 2.5% skim milk powder to each well, and seal at 37 ℃ for 1 h.

[0090] (3) Incubation of primary antibody: Add 100 μL of fusion cell culture supernatant to each well in sequence, and set up negative and positive control wells. Add 100 μL of Balb / c mouse positive and negative serum diluted 1:1000 to each well, and incubate at 37 ℃ for 1 h.

[0091] (4) Incubation of secondary antibody: Discard the liquid in the well, wash the well with PBST 4-5 times, and forcefully drain the liquid in the well for the last time. Add 100 μL of HRP-goat anti-mouse IgG (Biolong Biotechnology Co., Ltd.) diluted 1:5000 and incubate at 37 ℃ for 1 h.

[0092] (5) Color development: Discard the liquid in the well, wash the well with PBST 4-5 times, and forcefully drain the liquid in the well on the last time. Add 100 μL of TMB in the dark and develop the color at room temperature for 10 min.

[0093] (6) Termination: After the color development is complete, add 50 μL of 2M sulfuric acid stop solution.

[0094] (7) Reading: The absorbance at OD450 nm is detected by the microplate reader.

[0095] Hybridoma cells with the highest OD values ​​and good cell growth status (see row E, column 3 in Table 3) were subjected to subcloning experiments.

[0096]

[0097] 3.5 Limiting dilution method for subcloning screening

[0098] (1) Mark the wells for preparing cloning positive cells and gently pipette to mix them. Take 2 μL of the mixed cells and count them under an inverted microscope.

[0099] (2) Calculate the amount of cells required for subcloning based on the number of cells, and dilute the cells 100 times.

[0100] (3) Add the mixed cells to a new 96-well plate at a rate of 200 µL per well and place it in a CO2 incubator for further culture.

[0101] Based on the growth status of hybridoma cells, subcloning was performed every 7 days using limiting dilution. Subcloning was repeated three times until a hybridoma cell line stably secreting anti-goat CD207 monoclonal antibody appeared in the well. This hybridoma cell line was then expanded and cultured until it reached a confluence of 60 mm in a cell culture dish, at which point it was cryopreserved. Four positive hybridoma cell lines were ultimately obtained and named B10, F7, E3, and G4, respectively. The results of the three rounds of subcloning are shown in Tables 4 (see row F, column 1), 5 (see row C, column 2), 6 (see row E, column 3 and row B, column 10), and 7 (see row G, column 4 and row F, column 7).

[0102]

[0103]

[0104]

[0105]

[0106] 4. Monoclonal antibody subtype identification

[0107] The mouse monoclonal antibody subtype identification kit (PK20002; Proteintech) was used to identify the cell supernatant monoclonal antibodies of positive hybridoma cell lines. The specific steps are as follows:

[0108] (1) Take out the mouse monoclonal antibody subtype identification kit and equilibrate at room temperature for 30 minutes.

[0109] (2) Dilute the cell culture supernatant of the positive hybridoma cell line with 1×PBST at a ratio of 1:100 and add 50 µL / well to the sample well of the strip.

[0110] (3) No incubation is required. Add 50 µL of 1× goat anti-mouse IgM+IgG-HRP to each well and mix gently for 1 min.

[0111] (4) Cover with sealing film and incubate at room temperature for 1 h.

[0112] (5) Discard the liquid in the well, wash the plate 3 times with 1×PBST, and pat dry on absorbent paper.

[0113] (6) Add 100 µL of freshly prepared colorimetric solution to each well.

[0114] (7) Develop color at room temperature in the dark for 15 min.

[0115] (8) Add stop solution to each well, 100 µL / well.

[0116] (9) The hole with the darkest color or the highest OD value after the color development is terminated corresponds to the corresponding subtype.

[0117] The results are as follows Figure 3 As shown, the heavy chain subtype of the monoclonal antibody in hybridoma cell lines B10, F7, E3, and G4 is IgG2b, and the light chain subtype is Kappa.

[0118] 5. Detection of monoclonal antibody titer in cell supernatant

[0119] When the selected positive hybridoma cell lines were expanded, the culture supernatant was centrifuged, and the titer of the monoclonal antibody in the cell supernatant was detected. The specific detection procedure is described in section 3.4, with 10 dilution gradients set for the monoclonal antibody in the cell supernatant. The results are shown in Table 8. The monoclonal antibody titer in the cell supernatant of the positive hybridoma cell lines was good, with a B10 of 1.28 × 10⁻⁶. 4 E3 is 6.40 × 10 3 F7 is 2.56 × 10 4 G4 is 6.40×10 3 .

[0120]

[0121] (II) Identification of reactivity of anti-goat CD207 monoclonal antibody

[0122] 1. Reactivity of monoclonal antibodies with prokaryotic expressed proteins

[0123] Western blotting was used to detect the interaction between monoclonal antibodies produced by selected positive hybridoma cell lines and prokaryotically expressed goat CD207 protein. （69-329） The reactivity was assessed. Simultaneously, a bovine protein with high homology (expressed via pET-28a, specifically expressing a truncated bovine CD207 protein) and a goat CD207 protein with the same truncated protein expressed via pET-32a (to eliminate vector interference, the expression vector was constructed using pET-32a; pET-32a was stored in the laboratory but can also be purchased from the manufacturer) were introduced as controls. The specific detection steps are as follows:

[0124] (1) Gel preparation: Prepare polyacrylamide gel, wherein the stacking gel is 5% and the separating gel is 12%.

[0125] (2) Sample preparation: Use 5×SDS-PAGE Sample Buffe (i.e., SDS-PAGE protein loading buffer, Beijing Dining Biotechnology Co., Ltd.), and load 10 μL (containing 0.25 mg) of prokaryotic protein in each group.

[0126] (4) Electrophoresis: stacking gel 80 V, 30 min; separating gel 120 V, 90 min.

[0127] (5) Transfer membrane: constant current 0.2 A, 1 h.

[0128] (6) Sealing: Add 5% skim milk, shake slowly at 37°C for 2 h; wash 3 times with TBST on a shaker at 200 rpm, 5 min each time.

[0129] (7) Incubation with primary antibody: Add monoclonal antibody produced by hybridoma cell lines B10, F7, E3 or G4, incubate overnight at 4 ℃, and wash 5 times with TBST on a 200 rpm shaker the next day, 5 min each time.

[0130] (8) Incubation of secondary antibody: Add 1:5000 diluted HRP-goat anti-mouse IgG and incubate at 37 ℃ for 1 h; then wash 5 times with TBST on a shaker at 200 rpm, 5 min / time.

[0131] (9) ECL development and exposure.

[0132] The results of the Western blot are shown below. Figure 4 The results showed that only prokaryotic expression of truncated goat CD207 protein showed a specific target band, indicating that the anti-goat CD207 monoclonal antibody could bind to prokaryotic expression of truncated goat CD207 protein.

[0133] 2. Reactivity of monoclonal antibodies with eukaryotic expressed proteins

[0134] A goat CD207 eukaryotic expression vector was constructed, cells were transfected, and cell samples were collected after 48 hours. Western blotting and IFA techniques were used to detect the reactivity of monoclonal antibodies produced by four selected hybridoma cell lines with the naturally folded goat CD207 protein (i.e., the full-length eukaryotically expressed goat CD207 protein).

[0135] 2.1 Construction of eukaryotic expression vectors

[0136] According to NCBI, CD207 The full-length gene sequence (GenBank accession number: XM_005686383.3) was sent to Xi'an Qingke Biotechnology Co., Ltd. for mammalian codon optimization and full-length synthesis. Synthesis required the use of goats. CD207The gene was introduced with restriction enzyme sites BamHI and HindⅢ at the 5' and 3' ends and cloned into the pcDNA3.1(+) vector (Xi'an Qingke Biotechnology Co., Ltd.) to construct the pCDNA3.1-goat CD207-FLAG eukaryotic expression vector (6430 bp in size).

[0137] The synthesized recombinant plasmid was extracted and subjected to BamHI / HindIII (Dalian Takara Bio Co., Ltd.) double enzyme digestion for identification. The results are as follows: Figure 5 As shown in Figure A, two enzyme digestion fragments, one large and one small, are visible, with the smaller fragment being the target gene (approximately 1000 bp).

[0138] 2.2 Recombinant plasmid transfected into HEK-293T cells

[0139] (1) HEK-293T cells were cultured in complete DMEM medium containing 10% fetal bovine serum and 1% penicillin antibodies. The night before transfection, cells cultured to 90% in a 60 mm dish were counted and seeded in 12-well plates to ensure that the cell density in each well was 60%~70%.

[0140] (2) The next day, the culture medium in the 12-well plate was discarded in the clean bench, 1 mL of incomplete DMEM culture medium was added, and the plate was starved for 2 h in a 37℃, 5% CO2 incubator.

[0141] (3) Add 1 μg of recombinant plasmid and 1.5 μL of TurboFect transfection reagent to 50 μL of Optimen high sugar medium, premix and let stand for 5 min; at the same time set up MOCK group and empty vector group.

[0142] (4) After 5 min, the two tubes are mixed evenly and left to stand for 15 min. Then add 100 μL / well to the 12-well plate.

[0143] (5) After 6-8 hours, replace the culture medium with complete DMEM medium containing 5% fetal bovine serum and 1% antibiotics to maintain the culture.

[0144] (6) After culturing for 48 h, discard the culture medium in the 12-well plate and carefully wash with PBS; add 100 μL of RIPA lysis buffer and 1 μL of PMSF to a portion of the cells for cell sample processing, collect in 1.5 mL centrifuge tubes, add 25 μL of SDS-PAGE protein loading buffer, boil at 100 ℃ for 10 min, and use for Western Blot detection. Fix the other portion of cells with 4% paraformaldehyde for IFA experiments.

[0145] 2.3 Western Blot Detection

[0146] The specific Western blotting procedure is the same as for prokaryotic protein expression. Results are as follows: Figure 5 As shown in Figure B, using the FLAG antibody (Bio-Long Biotech) as the primary antibody, a band appeared at the target protein, indicating successful eukaryotic expression of goat CD207 protein. Furthermore, using four monoclonal antibodies (i.e., monoclonal antibodies produced by hybridoma cell lines B10, F7, E3, or G4) as primary antibodies, bands appeared at the target protein in all cases, indicating that the anti-goat CD207 monoclonal antibody can bind to the eukaryotically expressed goat CD207 protein.

[0147] 2.4 Monoclonal Antibody IFA Experiment

[0148] (1) Permeability: After the transfected cells were fixed with 4% paraformaldehyde, 600 μL of PBS containing 0.2% Triton-X-100 was added to each well, and the cells were incubated at room temperature for 15 min. The cells were then washed 3 times with PBS.

[0149] (2) Blocking: Add 600 μL / well of PBST containing 5% BSA, incubate at 37 °C for 90 min, and wash 3 times with PBS.

[0150] (3) Incubation with primary antibody: Add 600 μL / well of cell supernatant monoclonal antibody (monoclonal antibody produced by hybridoma cell lines B10, F7, E3 or G4), incubate overnight at 4 ℃, and wash 5 times with PBST.

[0151] (4) Incubation of secondary antibody: Add 400 μL / well of FITC-goat anti-mouse IgG (Biolong Biotechnology Co., Ltd.) diluted 1:200 with PBST containing 5% BSA, incubate at room temperature in the dark for 2 h, and wash 5 times with PBST.

[0152] (5) Nucleus staining: Add 400 μL / well of Hoest fluorescent dye diluted with PBS at a ratio of 1:100, incubate at room temperature in the dark for 25 min, and wash 5 times with PBST.

[0153] (6) Observe under an inverted fluorescence microscope.

[0154] IFA experimental results are as follows Figure 6 As shown, compared with the MOCK group, all four monoclonal antibody groups showed specific green fluorescence, and the monoclonal antibodies produced by hybridoma cell lines B10 and F7 could make the specific green fluorescence stronger.

[0155] (III) Obtaining anti-goat CD207 single-chain antibody gene based on hybridoma cells

[0156] 1. Total RNA extraction from hybridoma cells

[0157] (1) When the positive hybridoma cells (specifically hybridoma cell line F7) are cultured to 90% of the 60 mm dish, the cells are collected in a 10 mL centrifuge tube, centrifuged at 800 rpm for 10 min, the supernatant is discarded, 900 μL of Trizol is added, and the mixture is vortexed for 20 s.

[0158] (2) Add 200 μL of chloroform pre-cooled at 4 ℃, vortex for 5 s, and let stand at 4 ℃ for 5 min.

[0159] (3) Pre-cool the centrifuge at 4 ℃, centrifuge at 12000 rpm for 15 min, aspirate the supernatant into an EP tube (1.5 mL) without RNase, add an equal volume of isopropanol, vortex for 10 s, and let stand at -20 ℃ for 30 min.

[0160] (4) Centrifuge at 4 ℃ and 12000 rpm for 10 min, discard the supernatant, wash the precipitate twice with 75% ethanol, let it stand at room temperature for 10 min, and add sterile RNase-free water to dissolve the precipitate.

[0161] (5) Take a small amount for agarose gel electrophoresis and RNA concentration determination, and aliquot the rest and store at -80 ℃; the agarose gel electrophoresis results are as follows: Figure 7 As shown, two bands, 28S and 18S, can be observed, with the former being approximately twice as bright as the latter, indicating that the extracted RNA is of high quality.

[0162] 2. Synthesis of first-strand cDNA

[0163] Using the extracted total RNA as a template, a reverse transcription kit was used ( EasyScript First-Strand cDNA Synthesis SuperMix was used to reverse transcribe cDNA. The reverse transcription system was prepared according to the group allocation shown in Table 9. The reverse transcription system was incubated at 42 ℃ for 5 min and then heated at 85 ℃ for 5 s. The reverse transcription product was stored at -80 ℃ for subsequent experiments.

[0164] Table 9. Reverse Transcription System

[0165]

[0166] 3. Antibody heavy chain variable region ( VH ) and light chain variable region ( VL Gene fragment amplification

[0167] 3.1 Design of amplification primers

[0168] Based on the IgG antibody, the highly conserved FR1 and CH1 base sequences of the kappa light chain, amplification was synthesized and amplified respectively. VL , VH The degenerate PCR primers for gene fragments are shown in Tables 10 and 11.

[0169] Table 10. Primers for amplifying the variable region gene of the IgG antibody heavy chain

[0170]

[0171] Table 11. Primers for amplifying the kappa light chain variable region gene

[0172]

[0173] In Tables 10 and 11: Y=C / T; W=A / T; M=A / C; R=A / G; K=G / T; S=G / C; V=A / G / C; all primers were synthesized by Xi'an Qingke Biotechnology Co., Ltd.

[0174] 3.2 Amplification and Electrophoresis Detection

[0175] Using reverse-transcribed cDNA as a template, equal amounts of upstream and downstream primers (both from Table 10 or Table 11) were mixed and subjected to PCR to amplify the DNA. VH or VL Gene fragments, the PCR reaction system is shown in Table 12, and the PCR reaction procedure is shown in Table 13.

[0176] Table 12. Reaction System

[0177]

[0178] Table 13. Reaction Procedure

[0179]

[0180] After the reaction, agarose gel electrophoresis was performed, and the images were observed under a gel imaging system. Images with clear and single bands and a size of 300-500 bp were selected for photographing and storage. The gel was then purified using a gel extraction kit.

[0181] Ultimately, two primer pairs were used: primers HF-3 and HB-1, and primers LF-4 and LB-1. The amplified PCR products showed clear and single bands of 300-500 bp in size on agarose gel electrophoresis (e.g., Figure 8 A and Figure 8 (as shown in C).

[0182] 3.3 Gene Fragment Sequencing and Analysis

[0183] 3.3.1 Constructing Cloning Plasmids

[0184] After being amplified and purified by PCR reaction VH , VL Gene fragments were ligated into the pMD-19T vector (Dalian Takara Bio), and the ligation system is shown in Table 14. The reaction conditions were: ligation overnight at 16 ℃.

[0185] Table 14. Connection System

[0186]

[0187] 3.3.2 Transformation and screening of single clonal colonies

[0188] Remove the DH5α competent bacterial culture from the -80℃ freezer, thaw it on ice, and then add [amount of bacteria]. VH or VL 10 μL of the ligation product was placed on ice for 30 min, then immediately placed in a 42 ℃ water bath for 60 s for heat shock, followed by an ice bath for 5 min. Finally, 100 μL of SOC medium was added, and the mixture was incubated at 37 ℃ and 220 rpm for 1 h on a shaker. All the culture was then evenly spread on a solid plate with ampicillin resistance and incubated overnight at 37 ℃.

[0189] Single colonies were selected and identified by colony PCR using universal M13 primers (M13-F: TGTAAAACGACGGCCAGT; M13-R: CAGGAAACAGCTATGAC). The identification results are as follows: Figure 8 B or Figure 8 As shown in D, select the corresponding colonies with a band size of 300~500 bp and send them to Xi'an Qingke Biotechnology Co., Ltd. for plasmid extraction and sequencing.

[0190] 3.3.3 Regarding VH Gene sequence analysis

[0191] The following is the heavy chain variable region linked into a pMD-19T vector ( VH Sequencing results of the gene fragment (i.e., SEQ.ID.NO.4):

[0192] 5`-GAAGTGATGTTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTATGTCATGGCTTGGGTTCGTCAGTCTCCAGAGAAGAGGCTGGAGTGGGTCGCAGAAATTAGTAGTGGTAGTACTTAC TCCTACTATCCAGACACTGTGACGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAATACCCTGTACCTGGAAATGACCCGTCTGAGGTCTGAAGACACGGCCATGTATTACTGTGCAGTCGGCCCGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCG-3`

[0193] right VH The gene sequence was translated into an amino acid sequence using SnapGene software, as shown below (i.e., SEQ.ID.NO.1):

[0194] EVMLVESGGGLVKPGGSLKLSCAASGFTFSSYVMAWVRQSPEKRLEWVAEISSGSTYSYYPDTVTGRFTISRDNAKNTLYLEMTRLRSEDTAMYYCAVGPFAYWGQGTLVTVSA

[0195] The above sequence, consisting of 114 amino acids, contains no stop codons. Furthermore, it was analyzed using the IMGT and VBASE2 websites. VH The gene sequences were analyzed and FR and CDR were classified, and the results are as follows: Figure 9 As shown, it can be divided into 3 CDR areas and 4 FR areas. VDJ The gene rearrangement is correct and matches the characteristics of murine antibodies; VH The homology of the murine germline antibody sequence that can be classified into IGHV5 is up to 95.49%.

[0196] 3.3.4 (Regarding) VL Gene sequence analysis

[0197] The following is the light chain variable region linked into another pMD-19T vector ( VL Sequencing results of the gene fragment (i.e., SEQ.ID.NO.5):

[0198] 5`-ATGATGACCCAAACTCCACTCTCTTTGTCGGTTTCCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTTTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAGTCTATTTGGTGTCTAAATTG GACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCTCAGACGTTCGGTGGAGGCACCAAGTTGGAAATCAAACGGGCTGATGCT-3`

[0199] right VL The gene sequence was translated into an amino acid sequence using SnapGene software, as shown below (i.e., SEQ.ID.NO.2):

[0200] MMTQTPLSLSVSIGQPASISCKSSQSLLDSDGKTYLNWFLQRPGQSPKRLVYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGGGTKLEIKRADA

[0201] The above sequence, consisting of 114 amino acids, contains no stop codons. Furthermore, the IMGT and VBASE2 websites were used to verify... VL The gene sequences were analyzed and FR and CDR were classified, and the results are as follows: Figure 10 As shown, it can be divided into 3 CDR areas and 4 FR areas. VJ The gene rearrangement is correct and matches the characteristics of murine antibodies; VL The homology of the murine germline antibody sequence that can be classified into IGKV1 is up to 96.60%.

[0202] 4. Single-chain antibodies ( scFv Gene construction

[0203] First, according to VH and VL The gene sequence and the sequence encoding Linker (Gly4Ser)3 were used to design... VH and VL The primer for gene fragment splicing has the following specific sequence:

[0204] HF-3:5`-CGCAGAGACAGTGACCAGAGT-3`

[0205] Linker-B- VH :5`-GCCAGAGCCACCTCCCGCTGAACCGCCTCCACCCGCAGAGACAGTGAC-3`

[0206] Linker-F- VL :5`-CAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGATGATGACCCAAACTC-3`

[0207] LB-1:5`-RCATCAGCMCGTWTGATTYCCW-3`

[0208] Amplification of the amplified material using the overlap extension PCR (SOE-PCR) method VH , Linker and VL The single-chain antibody gene formed by splicing:

[0209] In the first round of amplification, primers HF-3 and Linker-B- were used. VH and containing VH PCR was performed using the cloned plasmid as a template, and primers Linker-F- were used. VL and LB-1, and containing VL PCR was performed using the cloned plasmid as a template, that is, in VH Downstream of the gene fragment (3' end) and VL Introduced upstream (5' end) of the gene fragment Linker (Gly4Ser)3, thereby obtaining the intermediate gene fragments required for splicing. VH-Linker as well as Linker-VL The agarose gel electrophoresis results of these two gene fragments are shown in [the table below]. Figure 11 A. Then, the target band was purified by gel recovery.

[0210] In the second round of amplification, primers HF-3 and LB-1 were used, and the two gel-recovered PCR products from the first round of amplification were used as templates for another PCR to obtain the spliced ​​product. scFv The gene, its agarose gel electrophoresis results are as follows Figure 11 As shown in Figure B, the target band appears at 750 bp. VH-Linker-VL ).

[0211] For the two rounds of amplification mentioned above, the PCR reaction system and reaction procedure are as shown in Tables 12 and 13.

[0212] 5. Single-chain antibodies ( scFvGene sequencing and analysis

[0213] Using an agarose gel extraction kit, the target band appearing at 750 bp was extracted using gel extraction, and then processed according to the methods described in 3.3.1 and 3.3.2. scFv The gene was ligated into the pMD-19T vector and then sequenced. Finally... scFv The gene sequence is shown below (i.e., SEQ.ID.NO.6):

[0214] 5`--3`

[0215] right scFv See gene sequence analysis results Figure 12The amino acid sequence was translated using SnapGene software as shown below (i.e., SEQ.ID.NO.3):

[0216] EVMLVESGGGLVKPGGSLKLSCAASGFTFSSYVMAWVRQSPEKRLEWVAEISSGSTYSYYPDTVTGRFTISRDNAKNTLYLEMTRLRSEDTAMYYCAVGPFAYWGQGTLVTVSAGGGGSGG GGSGGGGSMMTQTPLSLSVSIGQPASISCKSSQSLLDSDGKTYLNWFLQRPGQSPKRLVYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGGGTKLEIKRADA

[0217] The above results indicate that the anti-goat CD207 single-chain antibody gene was successfully constructed.

[0218] (iv) Prokaryotic expression of anti-goat CD207 single-chain antibody

[0219] 4.1 Construction of single-chain antibody prokaryotic expression vector

[0220] To ligate the anti-goat CD207 single-chain antibody gene into two prokaryotic expression vectors, pET-28a and pET-32a (containing a lysis-promoting tag), homologous recombination primers were designed based on the obtained anti-goat CD207 single-chain antibody gene. Simultaneously, an HA tag was added to the single-chain antibody gene using the designed primers, and the restriction enzyme sites BamHI and HindIII were introduced. The specific sequences of the homologous recombination primers are shown below:

[0221] pET-28a-scFv-F:5`-AGCAAATGGGTCGC GGATCC GAAGTGATGTTGGTGGAGTCTGG-3`

[0222] pET-28a-scFv-R:5`-TCGAGTGCGGCCGC AAGCTT AGCGTAATCTGGAACATCGTATGGGTA AGCATCAGCCCGTTTGATTT-3`

[0223] pET-32a-scFv-F:5`-GCCATGGCTGATATC GGATCC GAAGTGATGTTGGTGGAGTCTGG-3`

[0224] pET-32a-scFv-R:5`-TCGAGTGCGGCCGC AAGCTT AGCGTAATCTGGAACATCGTATGGGTA AGCATCAGCCCGTTTGATTT-3`

[0225] In the primer sequences above, the underlined part "GGATCC" is the BamHI restriction site, the underlined part "AAGCTT" is the HindIII restriction site, and the bold italic part "AGCGTAATCTGGAACATCGTATGGGTA" is the HA tag sequence.

[0226] 4.2 Expression and purification of single-chain antibodies

[0227] Referring to sections 1.1 and 1.2 in (I), the recombinant vector pET-28a-anti-goat CD207 scFv-HA was constructed, and the single-chain antibody was expressed in prokaryotes. Since the pET-32a vector contains a TrxA lysis-promoting tag, which increases the solubility of the target protein, this embodiment also constructed a single-chain antibody prokaryote expression vector using pET-32a as the initial vector, namely the recombinant vector pET-32a-anti-goat CD207 scFv-HA.

[0228] The results of prokaryotic expression of single-chain antibodies are as follows: Figure 13 A, Figure 13 As shown in Figure C, after SDS-PAGE, the expressed single-chain antibody was enriched in the precipitate as inclusion bodies. The electrophoretic band size was as expected, and the expression levels of both recombinant vectors were high. Furthermore, Western blotting of the HA tag indicated that the target product was correctly expressed (see Figure C). Figure 13 B Figure 13 D).

[0229] Following the procedure in section 1.2 of part (I), the prokaryotic single-chain antibody was expressed in large quantities. Afterwards, bacterial cells were collected, sonicated, centrifuged, and the precipitate was loaded onto an affinity chromatography column for purification. The purified antibody was then concentrated by dialysis and ultrafiltration (3 kDa ~ 10 kDa ultrafiltration tube; centrifugation at 3000 rpm for 30 min at 4℃). Finally, the concentration of the prokaryotic single-chain antibody was detected using the BCA method, and the antibody was aliquoted and stored at -80℃. The purification results of the single-chain antibody by affinity chromatography are shown below. Figure 14 Furthermore, the purity was high, and the final concentration of the single-chain antibody was 0.5 mg / mL as determined by the BCA method.

[0230] (v) Identification of reactivity of anti-goat CD207 single-chain antibody

[0231] 1. Detection of prokaryotic single-chain antibody reactivity using indirect ELISA.

[0232] Indirect ELISA detection was performed according to section 3.4 of (I). Different coating amounts of prokaryotically expressed truncated goat CD207 protein (800 ng, 400 ng, 200 ng, 50 ng) were used in the detection experiment. Prokaryotically expressed truncated bovine CD207 protein and a negative control were also included. Each coating amount was used in triplicate.

[0233] Because the prepared single-chain antibody lacks an Fc terminus, the enzyme-labeled secondary antibody does not readily bind to it. Therefore, this assay introduced mouse anti-HA IgG (Bio-Long Biotechnology Co., Ltd.) as the secondary antibody, which can bind to the HA tag fused to the single-chain antibody, and used HRP-goat anti-mouse IgG as the third antibody. Readings were taken after TMB colorimetric development and termination. Results are as follows: Figure 15 As shown, the OD value decreases with decreasing coating amount of antigen (i.e., prokaryotically expressed truncated goat CD207 protein). This confirms that the anti-goat CD207 single-chain antibody can bind to prokaryotically expressed truncated goat CD207 protein, and the binding strength is negatively correlated with the dilution of the coating antigen. This result indicates that the anti-goat CD207 single-chain antibody can be used in experiments related to the detection of goat CD207 protein based on ELISA methods.

[0234] 2. Detection of prokaryotic single-chain antibody reactivity using Western blotting.

[0235] Refer to (II) for Western blotting detection. In the detection experiment, an empty vector and prokaryotic expression of truncated bovine CD207 protein were used as sample controls. The primary antibody was an anti-goat CD207 single-chain antibody, the secondary antibody was mouse anti-HA IgG, and the tertiary antibody was HRP-goat anti-mouse IgG. The samples were developed using ECL and then exposed. A control experiment was also set up using a monoclonal antibody produced by hybridoma cell line F7 from (I) as the primary antibody. Results are as follows: Figure 16 As shown, the anti-goat CD207 single-chain antibody can specifically recognize the goat CD207 protein. This result indicates that the anti-goat CD207 single-chain antibody can be applied to experiments based on Western blotting to detect the goat CD207 protein.

[0236] (vi) Detection application of anti-goat CD207 single-chain antibody

[0237] Mature dendritic cells (DCs) are highly differentiated and difficult to culture and proliferate directly in vitro. For mature DCs obtained through in vitro induced differentiation, antibodies against DC surface marker molecules are required for identification.

[0238] In the above experiments, prokaryotically expressed goat CD207 protein (specifically a truncated protein) was used as an immunogenic antigen. Hybridoma cell lines capable of stably secreting anti-goat CD207 monoclonal antibodies were screened using cell fusion technology. Based on the screened positive hybridoma cells that secreted anti-goat CD207 monoclonal antibodies, specific anti-goat CD207 single-chain antibodies were obtained. This single-chain antibody can serve as a key reagent for detecting goat CD207, thereby determining whether goat dendritic cells differentiated in vitro are mature, providing an important tool for developing vaccines targeting goat dendritic cells.

Claims

1. A single-chain antibody against goat CD207, characterized in that: The single-chain antibody includes a heavy chain variable region and a light chain variable region. The amino acid sequence of the heavy chain variable region is shown in SEQ.ID.NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ.ID.NO.

2.

2. The anti-goat CD207 single-chain antibody according to claim 1, characterized in that: The amino acid sequence of the single-chain antibody is shown in SEQ.ID.NO.

3.

3. A single-chain antibody gene against goat CD207, characterized in that: The gene includes a nucleotide sequence encoding the anti-goat CD207 single-chain antibody as described in claim 1 or 2.

4. The anti-goat CD207 single-chain antibody gene according to claim 3, characterized in that: The gene specifically includes, in sequence, a nucleotide sequence encoding the heavy chain variable region, a linker sequence, and a nucleotide sequence encoding the light chain variable region. The nucleotide sequence encoding the heavy chain variable region is shown in SEQ.ID.NO.4, and the nucleotide sequence encoding the light chain variable region is shown in SEQ.ID.NO.

5.

5. The anti-goat CD207 single-chain antibody gene according to claim 3 or 4, characterized in that: The nucleotide sequence of the gene is shown in SEQ.ID.NO.

6.

6. A recombinant plasmid expressing an anti-goat CD207 single-chain antibody, characterized in that: The recombinant plasmid includes the anti-goat CD207 single-chain antibody gene as described in claim 3.

7. A recombinant cell expressing an anti-goat CD207 single-chain antibody, characterized in that: The recombinant cells were obtained by transforming a recombinant plasmid expressing an anti-goat CD207 single-chain antibody as described in claim 6 into host cells.

8. The recombinant cell expressing an anti-goat CD207 single-chain antibody according to claim 7, characterized in that: The host cell is a prokaryotic cell.

9. The application of the anti-goat CD207 single-chain antibody as described in claim 1 or 2 in the preparation of a goat CD207 protein detection reagent.

Images