Anti-goat CD207 single-chain antibody as well as preparation method and application thereof

Abstract

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

Description

Technical Field

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

[0002] Vaccination 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 immunopotency levels. The research and development of novel vaccines targeting antigen-presenting cells of the body is an important means to effectively enhance the immunopotency of vaccines, and the preparation of specific antibodies targeting antigen-presenting cells of the body is the key and prerequisite for the research and development of 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 antigenic substances by cells such as DCs, and has the functions of regulating the migration of DCs and the interaction between DCs and lymphocytes.

[0003] A single-chain antibody (single chain variable fragment, scFv) is a recombinant antibody formed by connecting the variable region of the heavy chain (VH) and the variable region of the light chain (VL) of a complete antibody through a flexible short peptide (i.e., Linker) using genetic engineering methods. Due to the characteristics of small molecular weight, no Fc end and complement binding site, and low immunogenicity of single-chain antibodies, they have high research and application value in targeted vaccines, targeted anti-tumor drugs, and molecular detection.

[0004] Although the preparation of single-chain antibodies has been achieved through various expression systems, there are still certain limitations. For example, in "Prokaryotic Expression, Purification and Biological Effects of the Single Chain Antibody DPK3-scFv against DNA-PKcs", the codons of the specific humanized single-chain antibody DPK3-scFv gene screened from a phage antibody library in the early stage were dehumanized and then prokaryotically expressed again, and finally the transmembrane transport and inhibitory activity of DPK3-scFv on tumor cells were verified; in "Preliminary Study on the Screening of Targeting CD32a scFv and the Killing Ability of CAR-T Cells", the CD32a scFv gene obtained by phage display technology was introduced into human T cells, and the target cell killing test was carried out on the recombinant cell CD32a-CAR-T, but the test results showed insufficient specificity of killing. Summary of the Invention

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

[0006] To achieve the above object, the present invention adopts the following technical solutions: In the first aspect, an anti-goat CD207 single-chain antibody is provided. The single-chain antibody includes a heavy-chain variable region and a light-chain variable region, wherein the amino acid sequence of the heavy-chain variable region is as shown in SEQ.ID.NO.1, and the amino acid sequence of the light-chain variable region is as shown in SEQ.ID.NO.2.

[0007] Preferably, in the single-chain antibody, the heavy-chain variable region and the light-chain variable region are connected by a Linker (such as the 15-mer peptide "(Gly4Ser)3").

[0008] Preferably, the amino acid sequence of the single-chain antibody is as shown in SEQ.ID.NO.3.

[0009] Preferably, the single-chain antibody is prepared by a prokaryotic expression system (such as using prokaryotic cells of types such as Escherichia coli as the host for expressing the single-chain antibody and into which the corresponding target gene has been introduced), and the target expression product obtained by using this system is a fusion protein; the fusion protein includes the single-chain antibody, or the fusion protein consists of the single-chain antibody.

[0010] Preferably, the fusion protein further includes an expression tag (such as an HA tag, a His tag), that is, the fusion protein can be a recombinant protein that fuses an expression tag and the single-chain antibody.

[0011] Preferably, the sequence information of the heavy-chain variable region and the light-chain variable region of the single-chain antibody is obtained by experimental analysis of the transcription products of anti-goat CD207 monoclonal antibody hybridoma cells. Specifically, total RNA of anti-goat CD207 monoclonal antibody hybridoma cells is extracted and reverse-transcribed, and the heavy-chain and light-chain variable region gene fragments are cloned from the cDNA obtained by reverse transcription using designed primers, and then sequenced.

[0012] Preferably, the preparation method of the hybridoma cells includes the following steps: 1) Prokaryotic expression of the extracellular region of goat CD207 to obtain a truncated goat CD207 protein (the amino acid sequence is as shown in SEQ.ID.NO.7); 2) Immunize mice with the truncated goat CD207 protein prepared in step 1; 3) After step 2, mouse spleen cells are fused with myeloma cells, and the resulting fused cells are used to screen for anti-goat CD207 monoclonal antibody hybridoma cells (strains).

[0013] Preferably, in step 3, cell supernatant anti-goat CD207 monoclonal antibody of several positive hybridoma cells (strains) is collected by large-scale culture.

[0014] Preferably, the heavy chain subtype of the monoclonal antibody is IgG2b, and the light chain subtype is Kappa.

[0015] In a second aspect, there is provided an anti-goat CD207 single-chain antibody gene, which gene comprises a nucleotide sequence encoding the above-mentioned anti-goat CD207 single-chain antibody.

[0016] Preferably, the gene specifically comprises a nucleotide sequence encoding a heavy chain variable region, a linker sequence (i.e., a nucleotide sequence encoding a Linker), and a nucleotide sequence encoding a light chain variable region arranged in sequence, wherein the nucleotide sequence encoding the heavy chain variable region is as shown in SEQ.ID.NO.4, and the nucleotide sequence encoding the light chain variable region is as shown in SEQ.ID.NO.5.

[0017] Preferably, the nucleotide sequence of the gene is as shown in SEQ.ID.NO.6.

[0018] In a third aspect, there is provided a recombinant plasmid expressing an anti-goat CD207 single-chain antibody, which recombinant plasmid comprises an initial plasmid backbone and the above-mentioned anti-goat CD207 single-chain antibody gene connected to the backbone.

[0019] Preferably, the initial plasmid is a prokaryotic expression plasmid, and specific types can be selected according to actual needs, such as pET-28a, pET-32a.

[0020] In a fourth aspect, there is provided a recombinant cell expressing an anti-goat CD207 single-chain antibody, which recombinant cell is obtained by transforming the above-mentioned recombinant plasmid expressing an anti-goat CD207 single-chain antibody into a host cell.

[0021] Preferably, the host cell is Escherichia coli.

[0022] In a fifth aspect, there is provided a method for preparing an anti-goat CD207 single-chain antibody, comprising the following steps: Connect the above-mentioned anti-goat CD207 single-chain antibody gene (for example, constructed by overlap extension PCR) into an initial plasmid to construct a recombinant plasmid; transform the recombinant plasmid into a host cell to construct a recombinant cell; culture the recombinant cell and induce the expression of the single-chain antibody during the culture process, and then purify the single-chain antibody from the expression product.

[0023] Sixth aspect, there is provided an application of the above-mentioned anti-goat CD207 single-chain antibody in recognizing goat CD207.

[0024] Preferably, the recognition is to detect goat CD207 protein by using the reactivity of the single-chain antibody with goat CD207 protein and adopting ELISA and Western Blot.

[0025] Seventh aspect, there is provided an application of the above-mentioned anti-goat CD207 single-chain antibody in preparing a detection reagent (kit) for goat CD207 protein.

[0026] Preferably, the detection reagent (kit) is specifically an ELISA reagent (kit) or a Western Blot reagent (kit).

[0027] The beneficial effects of the present invention are as follows: The present invention firstly proposes an anti-goat CD207 single-chain antibody gene, which can be directly used for prokaryotic expression; and ELISA and Western Blot experiments show that the expressed anti-goat CD207 single-chain antibody has the ability to specifically recognize goat CD207 protein, has good reactivity with goat CD207 protein, can be used as a tool for developing a targeted goat dendritic cell vaccine, and is of great significance for improving the prevention and control level of goat diseases.

[0028] Furthermore, the anti-goat CD207 single-chain antibody of the present invention has a structural feature of no Fc end and complement binding site, which can avoid the occurrence of ADCC effect and complement binding effect in the body.

[0029] Furthermore, the present invention uses the screened positive hybridoma cells to clone the heavy chain and light chain variable region gene fragments, which not only provides a template for the construction of the anti-goat CD207 single-chain antibody gene, but also the anti-goat CD207 single-chain antibody gene can be used for highly expressing the anti-goat CD207 single-chain antibody after being introduced into a prokaryotic host (such as Escherichia coli). Description of the Drawings

[0030] Figure 1 For goat CD207 protein （69-329） Expression and purification results; wherein: A is the SDS-PAGE diagram of the expressed protein under the condition of inducing for 6 h with IPTG at a concentration of 0.25 mM at 37 °C and 220 rpm, and B is the Western Blot detection of the His tag of the expressed protein after inducing for 6 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 bacteria after induced expression; Lane 4: Supernatant after induced expression; Lanes 5 and 6 after induced expression: Precipitate (35.6 kDa)); C is the SDS-PAGE map of the expressed protein and the Western Blot detection of the His tag of the expressed protein under the condition of inducing the expression of protein for 20 h at 16 °C, 100 rpm, and 0.25 mM IPTG concentration (M: Protein Marker; Lane 1: pET-28a empty vector; Lane 2: Recombinant vector pET-28a-goat CD207 protein （69-329） Uninduced; Lane 3: Whole bacteria after induced expression; Lane 4: Supernatant after induced expression; Lane 5: Precipitate (35.6 kDa) after induced expression)); D is the SDS-PAGE map of the purified expressed protein (M: Protein Marker; Lane 1: Goat CD207 protein （69-329） Stock solution; Lane 2: Flow-through after protein loading; Lane 3: 100 mM imidazole flow-through; Lanes 4, 5, 6, 7, 8: 500 mM imidazole eluate).

[0031] Figure 2 Observation of the fusion of Balb / c mouse spleen cells and myeloma cells (SP2 / 0) under the microscope (10×10 magnification); among them: A is the cells under the microscope after 5 days of SP2 / 0 expansion culture (the day of fusion); B is the cells under the microscope on the 1st day after fusion; C is the cells under the microscope on the 4th day after fusion; D is the cells under the microscope on the 7th day after fusion.

[0032] Figure 3 Identification of the monoclonal antibody subtypes in the supernatants of four positive hybridoma cells using a mouse monoclonal antibody subtype identification kit (PK20002); among them: A is the ELISA color development result of the kit; B is the OD450 value of the kit.

[0033] Figure 4 Reactivity identification of the monoclonal antibodies produced by four hybridoma cells B10, F7, E3, and G4 with the prokaryotic expressed protein (M: Protein Marker, Lane 1: Prokaryotic expressed truncated bovine CD207 protein; Lanes 2, 3, 4: Prokaryotic expressed truncated goat CD207 protein using pET-28a as the initial vector and diluted 2-fold, 5-fold, and 10-fold respectively; Lane 5: Prokaryotic expressed truncated goat CD207 protein using pET-32a as the initial vector).

[0034] Figure 5Figure 4: Identification of the reactivity of monoclonal antibodies produced by four hybridoma cell lines, B10, F7, E3, and G4, with eukaryotic expressed proteins. A is the electrophoresis detection of double-enzyme digestion of the recombinant plasmid using pcDNA3.1(+) as the original vector (lane 1: pcDNA3.1(+) empty plasmid; M: DNA Marker; lane 2: double-enzyme digestion of the recombinant plasmid). B is the Western Blot detection of the reactivity of the four monoclonal antibodies with eukaryotic expressed goat CD207 protein (FLAG tag to verify the successful expression of goat CD207 protein; M: protein marker; lane 1: untransfected cell sample; lane 2: empty cell sample transfected with pcDNA3.1(+); lane 3: cell sample transfected with the recombinant plasmid).

[0035] Figure 6 IFA assay was used to detect the reactivity of monoclonal antibodies produced by four hybridoma cell lines, B10, F7, E3, and G4, with eukaryotically expressed goat CD207 protein (MOCK was the untransfected control).

[0036] Figure 7 This is the agarose gel electrophoresis of total RNA of hybridoma cell line F7 (blank: blank control).

[0037] Figure 8 Amplify the heavy chain and light chain variable region gene fragments of monoclonal antibodies using degenerate primers; A is the heavy chain variable region amplified using primers HF-3 and HB-1 ( VH ) gene fragments after 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 ) gene fragment was connected to the pMD-19T vector and then the colony was PCR identified; C was the light chain variable region amplified using primers LF-4 and LB-1 ( VL ) gene fragments after electrophoresis detection (M: DNA Marker; Lane 1: blank control; Lane 2: light chain variable region gene fragment); D is the light chain variable region ( VL ) The gene fragment was ligated into the pMD-19T vector and identified by colony PCR.

[0038] Figure 9 To use IMGT, VBASE2 website VH The gene sequence is divided into FR and CDR.

[0039] Figure 10 To use IMGT, VBASE2 website VL The gene sequence is divided into FR and CDR.

[0040] Figure 11 for scFv The construction of gene; wherein: A is a sequence with a connection (specificallyLinker of VH 、 VL gene fragment 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 gene (spliced by introducing a linker sequence between the gene sequences of VH and VL , that is VH-Linker-VL ), amplification (Lane 1: blank control; M: DNA Marker; Lane 2: VH-Linker-VL amplification). Figure 12 is the gene sequence analysis of anti-goat CD207 single-chain antibody.

[0041] Figure 13 is the prokaryotic expression result of anti-goat CD207 single-chain antibody; A is the SDS-PAGE map of the expression of single-chain antibody induced by IPTG at a concentration of 0.25 mM at 37 °C and 220 rpm for 6 h with pET-28a as the initial vector (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 induced expression; Lane 4: supernatant after induced expression; Lane 5: precipitate after induced expression (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 SDS-PAGE map of the expression of single-chain antibody induced by IPTG at a concentration of 0.25 mM at 37 °C and 220 rpm for 6 h with pET-32a as the initial vector (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 induced expression; Lane 4: supernatant after induced expression; Lane 5: precipitate after induced expression (46.2 kDa)); D is the Western Blot detection of the HA tag of the single-chain antibody expressed by pET-32a-anti-goat CD207 scFv-HA.

[0042] Figure 14Purification results of anti-goat CD207 single-chain antibody by affinity chromatography (M: Protein standard molecule; Lanes 1, 14: Anti-goat CD207 single-chain antibody stock solution; Lanes 2, 3, 4: Flow-through; Lanes 5, 6, 7, 8: 50 mM imidazole eluate; Lanes 9, 10, 11, 12, 13: 150 mM imidazole eluate; Lanes 15, 16, 17, 18: 250 mM imidazole eluate; Lanes 19, 20, 21, 22, 23, 24: 500 mM imidazole eluate).

[0043] Figure 15 To detect the reactivity of anti-goat CD207 single-chain antibody by indirect ELISA; where: A is the ELISA color development result; B is the OD450 value.

[0044] Figure 16 To detect the reactivity of anti-goat CD207 single-chain antibody by Western Blot; where: A is the monoclonal antibody produced by hybridoma cell line F7 used as the primary antibody (M: Protein standard molecule; Lane 1: pET-28a empty vector control; Lane 2: Prokaryotic expression truncated bovine CD207 protein control; Lanes 3, 4: Prokaryotic expression truncated goat CD207 protein with pET-28a as the initial vector); B is the anti-goat CD207 single-chain antibody used as the primary antibody (M: Protein standard molecule; Lane 1: pET-28a empty vector control; Lane 2: Prokaryotic expression truncated bovine CD207 protein control; Lanes 3, 4: Prokaryotic expression truncated goat CD207 protein with pET-28a as the initial vector). Detailed implementation mode

[0045] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are only used to explain the present invention and not to limit the protection scope of the present invention.

[0046] (I) Preparation of anti-goat CD207 monoclonal antibody 1. Preparation of goat CD207 immunogen 1.1 Recombinant vector pET-28a-goat CD207 protein （69-329） Construction Search for goats on NCBI CD207 Full-length gene sequence (GenBank accession number: XM_005686383.3). It was sent to Xi'an Qingke Biotechnology Co., Ltd. for codon optimization and full-length synthesis, and it was required to introduce BamHI and HindⅢ restriction enzyme sites at the 5' end and 3' end of the goat CD207 gene. Finally, a cloning plasmid containing the full-length sequence of the goat CD207 gene was obtained from the company.

[0047] The transmembrane region and signal peptide were predicted using analytical software, and the amino acid sequence of the extracellular region of goat CD207 was determined as follows (i.e., SEQ.ID.NO.7, a total of 261 amino acids): MGTISDVKTNAQLLKGRVDNISSLSSEIKRNRGALVAVGFQVRMVNASLDRISPQIRRLETGLKEASAQLQVLTSSWEAVDELNAQIPGLKQDLDRASALNAKVRELQSGLESISKLLQQQNDILQVVSQGWKYFRGHFYYFSQISKTWYSAQQMCISRDSHLTSVTSEREQEFLYRTAGGLPYWIGLTKAGSEGDWHWVDGTPFNKVQSEKFWIPGEPNNYGNNEHCVNLKMSSLRSWNDASCDNTFPFICKRPYKPSEP Amplify the CD207 extracellular region gene sequence of goat, and use homologous recombinase to ligate the CD207 extracellular region gene sequence into the pET-28a vector (preserved in the laboratory or available for purchase from the manufacturer), transform it into DH5α competent cells, screen with a solid LB plate containing 50 μg / mL kanamycin, pick monoclonal colonies for colony PCR, plasmid double digestion identification, and sequencing analysis to obtain a recombinant plasmid for expressing truncated goat CD207 protein, named pET-28a-goat CD207 protein （69-329） .

[0048] 1.2 Goat CD207 protein （69-329） Expression and purification (1) Transform the constructed recombinant plasmid into BL21 competent cells, then evenly coat it onto a solid LB plate containing 50 μg / mL kanamycin, and incubate it upside down in a 37 °C incubator overnight.

[0049] (2) Pick monoclonal colonies from the plate and inoculate them into 20 mL of LB liquid medium containing 50 μg / mL kanamycin. When the OD600 reaches 0.6 - 0.8 at 37 °C and 220 rpm, add IPTG with a final concentration of 0.25 mM, then induce expression at 37 °C and 220 rpm for 6 h, or induce expression at 16 °C and 100 rpm for 20 h. At the same time, set up an empty vector and an uninduced control group.

[0050] (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), and lyse it using an ultrasonic cell disruptor for 10 min. Take the whole bacteria, supernatant, and pellet (resuspended in lysis buffer containing 8 M Urea) after ultrasonic disruption, add SDS-PAGE protein loading buffer, boil at 100 °C for 10 min, and then perform electrophoresis. After electrophoresis, stain the protein gel with Coomassie Brilliant Blue, and then decolorize it. The results are shown in Figure 1 A, Figure 1 C. In addition, using His antibody (from Beijing Protein Innovation Co., Ltd.) as the primary antibody, detect the expressed goat CD207 protein by Western Blot （69-329） and the results are shown in Figure 1 B, Figure 1 C. (4) Express a large amount of goat CD207 protein （69-329） (culture in a larger volume of medium until OD600 reaches 0.6 - 0.8, then induce expression with 0.25 mM IPTG at 37 °C, 220 rpm for 6 h). Resuspend the centrifuged bacterial pellet in lysis buffer and perform ultrasonic disruption. After disruption, centrifuge, and dissolve the pellet in lysis buffer containing 8 M Urea. Purify the protein using a Ni-NTA affinity chromatography column (TianDiRenHe Ni NTA Beads 6FF SA005005 5 mL), set different concentrations of imidazole buffer to elute the target protein, collect each elution fraction, and perform SDS-PAGE protein electrophoresis. The results are shown in Figure 1 D.

[0051] (5) After purification by Ni-NTA affinity chromatography, collect the bands with relatively high purity and concentration, dialyze them into 1×PBS (pH 8.0), and aliquot and store them at -80 °C.

[0052] 2. Immunize Balb / c mice Emulsify the goat CD207 protein required for immunization （69-329） with Freund's adjuvant at a ratio of 1:1 for 2 h, and immunize 4 Balb / c mice (purchased from: Shaanxi Pharmaceutical Holdings Group Co., Ltd. (Shaanxi Pharmaceutical Holdings)) (the total amount of antigen per mouse per immunization is 25 - 50 μg), immunize once every two weeks. After the fourth immunization, detect the serum titer by indirect ELISA, and perform cell fusion after the fifth booster immunization. The specific immunization procedure is shown in Table 1, and the serum antibody titers after the fourth immunization are shown in Table 2.

[0053]

[0054]

[0055] The above results show that the serum antibody titer level of Balb / c mice numbered "-2" (i.e., Balb / c-2) was relatively high after the fourth immunization, reaching 2.04×10 5 , and cell fusion can be carried out after booster immunization.

[0056] 3. Preparation of hybridoma cells 3.1 Resuscitation and expansion culture of myeloma cells (SP2 / 0) One week before fusion, take out the cryopreservation tube of SP2 / 0 cells from the liquid nitrogen tank and place it in a 37 °C water bath for thawing. After complete thawing, place it in a cell centrifuge at room temperature and centrifuge at 800 rpm for 10 min. After centrifugation, transfer it to a laminar flow hood sterilized by ultraviolet light, discard the centrifugation supernatant, and gently resuspend the precipitate with 1 mL of complete RPMI-1640 medium containing 20% fetal bovine serum and 1% double antibody. Transfer it to a 60 mm cell culture dish, supplement with complete RPMI-1640 medium to 3 mL, and incubate overnight in a 37 °C, 5% CO2 incubator.

[0057] After overnight culture, observe the cell status under an inverted microscope. There are some cell debris floating. Replace the medium of SP2 / 0 cells. Resuspend the cells in the culture dish and collect them into a sterilized 10 mL centrifuge tube, wrap the sealing film, centrifuge at 800 rpm for 10 min at room temperature, resuspend with complete RPMI-1640 medium in the laminar flow hood, and place it in a new culture dish for culture. According to the growth status of the cells, change the medium every 12 h. When the cells grow to 90% - 100%, transfer them to a T-75 cell culture flask for expansion culture. Two full T-75 cell culture flasks of cells are required to fuse the spleen cells of one mouse ( Figure 2 A).

[0058] On the day of fusion, resuspend the cells with 30 mL of incomplete RPMI-1640 medium, collect them into a 50 mL centrifuge tube, centrifuge at 800 rpm for 10 min at room temperature, discard the supernatant, add 10 mL of incomplete RPMI-1640 medium, and mix well for standby.

[0059] 3.2 Pretreatment of mouse spleen cells (1) Autoclave the required experimental equipment and place it in the laminar flow hood for ultraviolet sterilization. Exsanguinate the mouse (specifically Balb / c-2) by removing the eyeball and collect the blood in a 1.5 mL EP tube to prepare positive control serum. Immediately decapitate the mouse and soak it in 75% alcohol for 10 min.

[0060] (2)Place the mouse on its back in the cell plate groove, cut open the abdominal skin of the mouse, bluntly separate the skin with forceps to expose the peritoneum, find the approximate position of the spleen and disinfect it with an alcohol cotton ball.

[0061] (3)Take a petri 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 petri dish, and trim the connective tissue adhered to the spleen. Place the spleen on a 200-mesh filter screen, and place the filter screen in a petri dish containing incomplete RPMI-1640 medium and squeeze the spleen to allow splenocytes to pass through the filter screen.

[0062] (4)Collect the obtained splenocyte suspension in a 50 mL centrifuge tube, and supplement incomplete RPMI-1640 medium to 30 mL. Centrifuge at 1000 r / min for 10 min at room temperature, discard the supernatant, add 10 mL of incomplete RPMI-1640 medium to resuspend the splenocytes for standby.

[0063] 3.3 Fusion of mouse splenocytes and myeloma cells (SP2 / 0) (1)Adjust the temperature of the water bath to 37 °C and place it in the ultra-clean workbench to provide an appropriate temperature for cell fusion.

[0064] (2)Thoroughly mix 10 mL of the mouse splenocyte suspension obtained by pretreatment with 10 mL of the myeloma cell suspension. 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.

[0065] (3)Place the centrifuge tube in the water bath, pipette 1 mL of PEG 1500, and slowly add it along the wall of the centrifuge tube, continuously and evenly rotating the centrifuge tube, and finish adding it within 60 s.

[0066] (4)Keep gently shaking the bottom, add 1 mL of incomplete RPMI-1640 medium in about 1 min, add 3 mL of medium within 3 min, and finally slowly add 10 mL of medium all at once to terminate the fusion, mix well, and let it stand in the 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 and add them to a 96-well plate containing feeder cells, and culture them in an incubator at 37 °C and 5% CO2.

[0067] (5)Refer to Figure 2 B、 Figure 2 C、 Figure 2On the 1st day after cell fusion, observe whether the cells are contaminated; on the 3rd day after fusion, observe the cell status (grape-like cell clusters); on the 3rd and 6th days after fusion, change the culture medium. Each time, discard 100 μL of the culture supernatant and then supplement 100 μL of complete RPMI-1640 medium containing HAT. When the fused cells grow to about 90% of the cell well, identify and screen the hybridoma cells.

[0068] 3.4 Hybridoma cell identification (1)Antigen coating: Add 100 μL of diluted goat CD207 protein （69-329） (400 ng in total per well) to the sample wells of the ELISA plate and coat at 4 °C overnight.

[0069] (2)Blocking: Discard the coating solution in the wells, add 200 μL of 2.5% skim milk powder to each well, and block at 37 °C for 1 h.

[0070] (3)Incubate with the primary antibody: Add 100 μL of the fused 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 sera diluted 1:1000 respectively, and incubate at 37 °C for 1 h.

[0071] (4)Incubate with the secondary antibody: Discard the liquid in the wells, wash the wells 4 - 5 times with PBST. For the last time, forcefully drain the liquid in the wells, and add 100 μL of HRP-goat anti-mouse IgG (BioDragon Biotechnology Co., Ltd.) diluted 1:5000, and incubate at 37 °C for 1 h.

[0072] (5)Color development: Discard the liquid in the wells, wash the wells 4 - 5 times with PBST. For the last time, forcefully drain the liquid in the wells, add 100 μL of TMB in the dark, and develop color at room temperature for 10 min.

[0073] (6)Termination: After color development, add 50 μL of 2M sulfuric acid termination solution.

[0074] (7)Reading: Measure the absorbance at OD450 nm with an ELISA reader.

[0075] Perform subcloning experiments on the hybridoma cells with the highest OD value obtained from the identification and good cell growth status (see the 3rd column in the E row of Table 3).

[0076]

[0077] 3.5 Subcloning and screening by limiting dilution method (1)Label the positive wells to be cloned, gently pipette the cells to mix well. Take 2 μL of the mixed cells and perform cell counting under an inverted microscope.

[0078] (2)Calculate the amount of cells required for subcloning according to the cell count, and dilute the cells 100-fold.

[0079] (3)Add 200 µL of the mixed cells per well to a new 96-well plate, and continue culturing in a CO2 incubator.

[0080] According to the growth status of the hybridoma cells, subclone the hybridoma cells using the limiting dilution method every 7 days. Subclone continuously 3 times until a hybridoma cell line that can stably secrete anti-goat CD207 monoclonal antibody appears in the well, and expand the culture of this hybridoma cell line. When it grows to confluence in a 60 mm cell culture dish, cryopreserve the corresponding hybridoma cells. Finally, 4 positive hybridoma cell lines were obtained, named B10, F7, E3, and G4 respectively. The results of the three rounds of subcloning are shown in Table 4 (specifically see the 1st column of the F row), Table 5 (specifically see the 2nd column of the C row), Table 6 (specifically see the 3rd column of the E row and the 10th column of the B row), and Table 7 (specifically see the 4th column of the G row and the 7th column of the F row).

[0081]

[0082]

[0083]

[0084]

[0085] 4. Identification of monoclonal antibody subtypes Use a mouse monoclonal antibody subtype identification kit (PK20002; Proteintech) to identify the monoclonal antibody in the cell supernatant of the positive hybridoma cell line. The specific steps are as follows: (1)Take out the mouse monoclonal antibody subtype identification kit and equilibrate it at room temperature for 30 minutes.

[0086] (2)Dilute the cell culture supernatant of the positive hybridoma cell line 1:100 with 1×PBST and add 50 µL per well to the sample wells of the strip.

[0087] (3)Without incubation, add 50 µL per well of 1×goat anti-mouse IgM+IgG-HRP to the sample wells and gently mix for 1 min.

[0088] (4)Cover with a sealing film and incubate at room temperature for 1 h.

[0089] (5)Discard the liquid in the wells, wash the plate 3 times with 1×PBST, and pat dry on absorbent paper.

[0090] (6)Add 100 µL per well of the freshly prepared chromogenic solution to the wells.

[0091] (7) Incubate at room temperature in the dark for 15 min for color development.

[0092] (8) Add the stop solution to each well, 100 µL / well.

[0093] (9) The well corresponding to the deepest color or the highest OD value after the color development is terminated is the corresponding subtype.

[0094] The results are as Figure 3 shown. The heavy chain subtypes of the monoclonal antibodies of the hybridoma cell lines B10, F7, E3, and G4 are all IgG2b, and the light chain subtypes are all Kappa.

[0095] 5. Detection of the titer of the monoclonal antibody in the cell supernatant When the selected positive hybridoma cell lines are expanded in culture, the culture supernatant is centrifuged, and the titer of the monoclonal antibody in the cell supernatant is detected. The specific detection steps refer to 3.4, and 10 dilution gradients are set for the monoclonal antibody in the cell supernatant during the detection. The results are shown in Table 8. The titer of the monoclonal antibody in the cell supernatant of the positive hybridoma cell lines is good. Among them, B10 is 1.28×10 4 , E3 is 6.40×10 3 , F7 is 2.56×10 4 , and G4 is 6.40×10 3 .

[0096]

[0097] (II) Identification of the reactivity of the anti-goat CD207 monoclonal antibody 1. Reactivity of the monoclonal antibody with the prokaryotic expression protein Use Western Blot to detect the reactivity of the monoclonal antibody produced by the selected positive hybridoma cell lines with the prokaryotic expression goat CD207 protein （69-329） . At the same time, introduce the prokaryotic expression bovine protein with high homology (the expression vector is constructed using pET-28a, and the specifically expressed is the same truncated bovine CD207 protein), and the prokaryotic expression of the same truncated goat CD207 protein (to exclude vector interference, the expression vector is constructed using pET-32a; among them, pET-32a is stored in the laboratory and can also be purchased from the manufacturer) as a control. The specific detection steps are as follows: (1) Gel preparation: Prepare a polyacrylamide gel, with a stacking gel of 5% and a separating gel of 12%.

[0098] (2) Sample preparation: Use the SDS-PAGE Sample Buffer (i.e., SDS-PAGE protein loading buffer, Beijing Dinning Biotech Co., Ltd.), and the loading amount of each group of prokaryotic expression proteins is 10 μL (containing 0.25 mg).

[0099] (4) Electrophoresis: Stacking gel, 80 V, 30 min; Separating gel, 120 V, 90 min.

[0100] (5) Transfer: Constant current of 0.2 A, 1 h.

[0101] (6) Blocking: Add 5% non-fat milk, gently shake at 37 °C for 2 h; Wash 3 times with TBST on a shaker at 200 rpm, 5 min each time.

[0102] (7) Incubate with primary antibody: Add monoclonal antibodies produced by hybridoma cell lines B10, F7, E3 or G4, incubate overnight at 4 °C, and wash 5 times with TBST on a shaker at 200 rpm the next day, 5 min each time.

[0103] (8) Incubate with secondary antibody: Add HRP-goat anti-mouse IgG diluted 1:5000, incubate at 37 °C for 1 h; Then wash 5 times with TBST on a shaker at 200 rpm, 5 min each time.

[0104] (9) ECL development and exposure. The results of Western Blot are shown in Figure 4 . The results showed that only the specifically targeted band appeared for the prokaryotic-expressed truncated goat CD207 protein, indicating that the anti-goat CD207 monoclonal antibody could bind to the prokaryotic-expressed truncated goat CD207 protein.

[0105] 2. Reactivity of monoclonal antibody with eukaryotic-expressed protein Construct a eukaryotic expression vector of goat CD207, transfect cells, collect cell samples after 48 h, and use Western Blot and IFA techniques to detect the reactivity of the monoclonal antibodies produced by the four screened hybridoma cell lines with the naturally folded goat CD207 protein (i.e., the full-length eukaryotic-expressed goat CD207 protein).

[0106] 2.1 Construction of eukaryotic expression vector According to the full-length gene sequence of goat on NCBI CD207 (GenBank accession number: XM_005686383.3). It was sent to Xi'an Tsingke Biotechnology Co., Ltd. for mammalian codon optimization and full-length synthesis. When synthesizing, it was required to introduce restriction enzyme sites BamHI and HindⅢ at the 5' and 3' ends of the goat CD207 gene, and clone it into the pcDNA3.1(+) vector (Xi'an Tsingke Biotechnology Co., Ltd.) to construct the pCDNA3.1-goat CD207-FLAG eukaryotic expression vector (with a size of 6430 bp).

[0107] The synthesized recombinant plasmid was extracted and identified by double digestion with BamHI / HindⅢ (TaKaRa Biotechnology Dalian Co., Ltd.). The results are as Figure 5 shown in A. Two digestion fragments, a large one and a small one, can be seen. The small fragment is the target gene (about 1000 bp).

[0108] 2.2 Transfection of the recombinant plasmid into HEK-293T cells (1)HEK-293T cells were cultured in complete DMEM medium containing 10% fetal bovine serum and 1% double antibiotics. One night before transfection, cells cultured in a 60 mm dish reaching 90% confluence were counted and seeded into a 12-well plate, ensuring that the cell density in each well was 60% - 70%.

[0109] (2)The next day, the medium in the 12-well plate was discarded in a laminar flow hood, and 1 mL of incomplete DMEM medium was added. The cells were placed in an incubator at 37℃ and 5% CO2 for starvation treatment for 2 h.

[0110] (3)1 μg of the recombinant plasmid and 1.5 μL of TurboFect transfection reagent were respectively added to high-glucose medium containing 50 μL of optimen, pre-mixed and allowed to stand for 5 min. At the same time, MOCK group and empty vector group were set up.

[0111] (4)After 5 min, the two tubes were mixed well and allowed to stand for 15 min, and then added to the 12-well plate at 100 μL / well.

[0112] (5)After 6 - 8 h, the cells were switched to complete DMEM medium containing 5% fetal bovine serum and 1% double antibiotics for maintenance culture.

[0113] (6)After culturing for 48 h, the medium in the 12-well plate was discarded and the cells were carefully washed with PBS. In some cells, 100 μL of RIPA lysis buffer and 1 μL of PMSF were added for cell sample treatment, collected in a 1.5 mL centrifuge tube, and 25 μL of SDS-PAGE protein loading buffer was added. The mixture was boiled at 100℃ for 10 min for Western Blot detection. In other cells, 4% paraformaldehyde was used for fixation for IFA experiment.

[0114] 2.3 Western Blot detection The specific steps of Western Blot detection were the same as those for prokaryotic expressed proteins. The results are as Figure 5As shown in Figure B, when the FLAG antibody (Biolong Biotechnology Co., Ltd.) was used as the primary antibody, a band appeared at the target protein, indicating the successful eukaryotic expression of goat CD207 protein. When four monoclonal antibodies (i.e., monoclonal antibodies produced by hybridoma cell lines B10, F7, E3, or G4) were used as the primary antibody respectively, bands also appeared at the target protein, indicating that the anti-goat CD207 monoclonal antibody could bind to the eukaryotic-expressed goat CD207 protein. 2.4 Monoclonal antibody IFA experiment (1) Permeabilization: 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 incubated at room temperature for 15 min, then washed 3 times with PBS.

[0115] (2) Blocking: 600 μL / well of PBST containing 5% BSA was added and incubated at 37 °C for 90 min, then washed 3 times with PBS.

[0116] (3) Incubation with primary antibody: 600 μL / well of the cell supernatant type monoclonal antibody (monoclonal antibody produced by hybridoma cell lines B10, F7, E3, or G4) was added and incubated overnight at 4 °C, then washed 5 times with PBST.

[0117] (4) Incubation with secondary antibody: 400 μL / well of FITC-goat anti-mouse IgG (Biolong Biotechnology Co., Ltd.) diluted 1:200 with PBST containing 5% BSA was added and incubated in the dark at room temperature for 2 h, then washed 5 times with PBST.

[0118] (5) Nuclear staining: 400 μL / well of the fluorescent dye Hoest diluted 1:100 with PBS was added and incubated in the dark at room temperature for 25 min, then washed 5 times with PBST.

[0119] (6) Observe under an inverted fluorescence microscope.

[0120] The results of the IFA experiment are as Figure 6 shown. Compared with the MOCK group, all four monoclonal antibody groups had specific green fluorescence, and the monoclonal antibodies produced by hybridoma cell lines B10 and F7 could make the specific green fluorescence stronger.

[0121] (III) Obtaining the anti-goat CD207 single-chain antibody gene based on hybridoma cells 1. Total RNA extraction from hybridoma cells (1) When the positive hybridoma cells (specifically, hybridoma cell line F7) were cultured to 9 * 10^5 cells in a 60 mm dish, the cells were collected in a 10 mL centrifuge tube, centrifuged at 800 rpm for 10 min, the supernatant was discarded, and 900 μL of Trizol was added and vortexed for 20 s.

[0122] (2) Add 200 μL of chloroform pre-cooled at 4 °C, vortex for 5 s, and let stand at 4 °C for 5 min.

[0123] (3) Pre-cool the centrifuge at 4 °C, centrifuge at 12,000 rpm for 15 min, aspirate the supernatant and transfer it to a RNase-free EP tube (1.5 mL), add an equal volume of isopropanol, vortex for 10 s, and let stand at -20 °C for 30 min.

[0124] (4) Centrifuge at 12,000 rpm at 4 °C for 10 min, discard the supernatant, wash the precipitate twice with 75% ethanol, let stand at room temperature for 10 min, and dissolve the precipitate with sterile RNase-free water.

[0125] (5) Take a small amount for agarose gel electrophoresis detection and RNA concentration measurement, and aliquot the rest and store at -80 °C; among them, the results of agarose gel electrophoresis detection are as Figure 7 shown, two bands of 28S and 18S can be observed, and the former is about twice as bright as the latter, indicating that the extracted RNA has high quality.

[0126] 2. Synthesis of the first-strand cDNA Using the extracted total RNA as a template, reverse transcribe it into cDNA using a reverse transcription kit ( EasyScript First-Strand cDNA Synthesis SuperMix); among them, prepare the reverse transcription system according to the component ratios shown in Table 9. Incubate the reverse transcription system at 42 °C for 5 min and then heat at 85 °C for 5 s. Store the reverse transcription product at -80 °C for subsequent experiments.

[0127] Table 9. Reverse transcription system

[0128] 3. Amplification of the gene fragments of the variable region of the antibody heavy chain ( VH ) and the variable region of the light chain ( VL ) 3.1 Design amplification primers According to the highly conserved FR1 and constant region CH1 base sequences of IgG antibody and kappa light chain, synthesize degenerate PCR primers for amplifying VL , VH gene fragments respectively, as shown in Table 10 and Table 11 specifically.

[0129] Table 10. Primers for amplifying the variable region gene of IgG antibody heavy chain

[0130] Table 11. Primers for amplifying the variable region gene of kappa light chain

[0131] 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.

[0132] 3.2 Amplification and electrophoresis detection Using reverse transcribed cDNA as template, mix equal amounts of upstream and downstream primers (both from Table 10 or Table 11) and perform PCR reaction to amplify VH or VL gene fragments, wherein the PCR reaction system is shown in Table 12, and the PCR reaction procedure is shown in Table 13.

[0133] Table 12. Reaction system

[0134] Table 13. Reaction Procedure

[0135] After the reaction, perform agarose gel electrophoresis and observe the image under a gel imaging system. Select clear and single bands with a size of 300-500 bp, take pictures and save them, and use a gel recovery kit for gel recovery and purification.

[0136] Finally, there are two pairs of primers, namely primers HF-3 and HB-1 and primers LF-4 and LB-1. The corresponding agarose gel electrophoresis results of the PCR products amplified by them show clear and single bands with a size of 300-500 bp (such as Figure 8 A and Figure 8 C).

[0137] 3.3 Gene fragment sequencing and analysis 3.3.1 Construction of cloning plasmids The PCR-amplified and purified VH 、 VL The gene fragments were ligated into the pMD-19T vector (Dalian Takara Biotech), respectively. The ligation system is shown in Table 14. The reaction conditions were: 16°C overnight ligation.

[0138] Table 14. Connection system

[0139] 3.3.2 Transformation and single clone screening Take out the DH5α competent bacterial solution from the -80 ℃ refrigerator, put it on ice to melt, and then add VH or VL10 μL of the ligation product was incubated on ice for 30 min, immediately heat-shocked in a 42 °C water bath for 60 s, then ice-bathed for 5 min. Finally, 100 μL of SOC medium was added, and the mixture was cultured in a shaker at 37 °C and 220 rpm for 1 h. All the culture was evenly spread on a solid plate with ampicillin resistance and cultured overnight at 37 °C.

[0140] Single colonies were picked and identified by colony PCR using universal M13 primers (M13-F: TGTAAAACGACGGCCAGT; M13-R: CAGGAAACAGCTATGAC). The identification results are as Figure 8 shown in Figure 8 B or

[0141] 3.3.3 For VH gene sequence analysis The following is the sequencing result (i.e., SEQ.ID.NO.4) of the heavy chain variable region ( VH ) gene fragment ligated into a pMD-19T vector: 5`-GAAGTGATGTTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTATGTCATGGCTTGGGTTCGTCAGTCTCCAGAGAAGAGGCTGGAGTGGGTCGCAGAAATTAGTAGTGGTAGTACTTACTCCTACTATCCAGACACTGTGACGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAATACCCTGTACCTGGAAATGACCCGTCTGAGGTCTGAAGACACGGCCATGTATTACTGTGCAGTCGGCCCGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCG-3` For VH the gene sequence, it was translated into an amino acid sequence using SnapGene software as follows (i.e., SEQ.ID.NO.1): EVMLVESGGGLVKPGGSLKLSCAASGFTFSSYVMAWVRQSPEKRLEWVAEISSGSTYSYYPDTVTGRFTISRDNAKNTLYLEMTRLRSEDTAMYYCAVGPFAYWGQGTLVTVSA The sequence consisting of 114 amino acids as above has no stop codon inside. And by using the IMGT and VBASE2 websites to VH analyze the gene sequence of Figure 9 and divide FR and CDR, the results are as V-D-J shown. Three CDR regions and four FR regions can be divided. VH The gene rearrangement is correct, conforming to the characteristics of murine antibodies;

[0142] 3.3.4 Analysis of the gene sequence of VL The following is the sequencing result of the gene fragment of the light chain variable region ( ), which is ligated into another pMD-19T vector (i.e., as SEQ.ID.NO.5): VL 5`-ATGATGACCCAAACTCCACTCTCTTTGTCGGTTTCCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTTTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAGTCTATTTGGTGTCTAAATTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCTCAGACGTTCGGTGGAGGCACCAAGTTGGAAATCAAACGGGCTGATGCT-3` Translating the gene sequence of into an amino acid sequence using SnapGene software, the specific result is as follows (i.e., SEQ.ID.NO.2): VL MMTQTPLSLSVSIGQPASISCKSSQSLLDSDGKTYLNWFLQRPGQSPKRLVYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGGGTKLEIKRADA The sequence consisting of 114 amino acids as above has no stop codon inside. And by using the IMGT and VBASE2 websites to analyze the gene sequence of VL and divide FR and CDR, the results are as Figure 10As shown, three CDR regions and four FR regions can be delineated. V-J The gene rearrangement is correct and conforms to the characteristics of murine antibodies; VL It can be attributed to the murine germline antibody sequence of IGKV1, with a homology of up to 96.60%.

[0143] 4. Construction of single-chain antibody ( scFv ) gene First, according to the VH and VL gene sequences and the sequence encoding Linker (Gly4Ser)3, design VH and VL gene fragment splicing primers, and the specific sequences of the primers are as follows: HF-3: 5`-CGCAGAGACAGTGACCAGAGT-3` Linker-B- VH : 5`-GCCAGAGCCACCTCCGCCTGAACCGCCTCCACCCGCAGAGACAGTGAC-3` Linker-F- VL : 5`-CAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGATGATGACCCAAACTC-3` LB-1: 5`-RCATCAGCMCGTWTGATTYCCW-3` Use the overlap extension PCR method (SOE-PCR) to amplify the single-chain antibody gene formed by splicing VH , Linker and VL : In the first round of amplification, use primers HF-3 and Linker-B- VH and use the cloning plasmid containing VH [[ID= forty-five]] as a template for PCR, and use primers Linker-F- VL and LB-1, and use the cloning plasmid containing VL as a template for PCR, that is, introduce VH (Gly4Ser)3 downstream (3` end) of the VL gene fragment and upstream (5` end) of the Linker gene fragment respectively, so as to obtain the intermediate gene fragments VH-Linker and Linker-VL required for splicing. The agarose gel electrophoresis results of these two gene fragments are shown in Figure 11 A, and then the target band is recovered and purified by gel extraction.

[0144] In the second round of amplification, primers HF-3 and LB-1 were used, and the two gel-extracted PCR products from the first round of amplification were used as templates for PCR again to obtain the spliced scFv gene. The results of agarose gel electrophoresis are shown in Figure 11 Figure B. It can be seen that a target band appears at 750 bp ( VH-Linker-VL ).

[0145] In the above two rounds of amplification, the PCR reaction system and reaction program refer to Table 12 and Table 13.

[0146] 5. Gene sequencing and analysis of single-chain antibody ( scFv ) Using an agarose gel recovery kit, the target band that appears at 750 bp was gel-extracted, and then the scFv gene was ligated to the pMD-19T vector according to the methods in 3.3.1 and 3.3.2, and then sequenced. Finally, the scFv gene sequence is as follows (i.e., SEQ.ID.NO.6): 5`-GAAGTGATGTTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTATGTCATGGCTTGGGTTCGTCAGTCTCCAGAGAAGAGGCTGGAGTGGGTCGCAGAAATTAGTAGTGGTAGTACTTACTCCTACTATCCAGACACTGTGACGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAATACCCTGTACCTGGAAATGACCCGTCTGAGGTCTGAAGACACGGCCATGTATTACTGTGCAGTCGGCCCGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCGGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGATGATGACCCAAACTCCACTCTCTTTGTCGGTTTCCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTTTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAGTCTATTTGGTGTCTAAATTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCTCAGACGTTCGGTGGAGGCACCAAGTTGGAAATCAAACGGGCTGATGCT-3` For scFv the sequence analysis results of the Figure 12 gene, see EVMLVESGGGLVKPGGSLKLSCAASGFTFSSYVMAWVRQSPEKRLEWVAEISSGSTYSYYPDTVTGRFTISRDNAKNTLYLEMTRLRSEDTAMYYCAVGPFAYWGQGTLVTVSAGGGGSGGGGSGGGGSMMTQTPLSLSVSIGQPASISCKSSQSLLDSDGKTYLNWFLQRPGQSPKRLVYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGGGTKLEIKRADA The above results indicate that the anti-goat CD207 single-chain antibody gene was successfully constructed.

[0147] (IV) Prokaryotic expression of anti-goat CD207 single-chain antibody 4.1 Construction of prokaryotic expression vector for single-chain antibody In order to ligate the anti-goat CD207 single-chain antibody gene to two prokaryotic expression vectors, pET-28a and pET-32a (containing solubility-enhancing tags) respectively, homologous recombination primers were designed based on the obtained anti-goat CD207 single-chain antibody gene. At the same time, HA tag was added after the single-chain antibody gene using the designed primers, and restriction enzyme sites BamHI and HindⅢ were introduced. The specific sequences of the homologous recombination primers are as follows: pET-28a-scFv-F: 5`-AGCAAATGGGTCGC GGATCC GAAGTGATGTTGGTGGAGTCTGG-3` pET-28a-scFv-R: 5`-TCGAGTGCGGCCGC AAGCTT AGCGTAATCTGGAACATCGTATGGGTA AGCATCAGCCCGTTTGATTT-3` pET-32a-scFv-F: 5`-GCCATGGCTGATATC GGATCC GAAGTGATGTTGGTGGAGTCTGG-3` pET-32a-scFv-R: 5`-TCGAGTGCGGCCGC AAGCTT AGCGTAATCTGGAACATCGTATGGGTA AGCATCAGCCCGTTTGATTT-3` In the above primer sequences, the underlined part "GGATCC" is the BamHI restriction enzyme site, the underlined part "AAGCTT" is the HindⅢ restriction enzyme site, and the bold and italic part "AGCGTAATCTGGAACATCGTATGGGTA" is the sequence of the HA tag.

[0148] 4.2 Single-chain antibody expression and purification Referring to 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 prokaryotically. Since the pET-32a vector contains a TrxA solubility-enhancing tag that can increase the probability of the target protein being soluble, a prokaryotic expression vector of the single-chain antibody with pET-32a as the initial vector, namely the recombinant vector pET-32a-anti-goat CD207 scFv-HA, was also constructed in this example.

[0149] The results of the prokaryotic expression of the single-chain antibody are as Figure 13 A, Figure 13 shown in C. After SDS-PAGE, it can be seen that the expressed single-chain antibody is enriched in the precipitate in the form of inclusion bodies. The size of the electrophoresis band conforms to the expectation and the expression levels of both recombinant vectors are relatively high. In addition, the results of detecting the HA tag by Western Blot indicate that the target product is correctly expressed (see Figure 13 B, Figure 13 D).

[0150] Subsequently, referring to 1.2 in (I), the prokaryotically expressed single-chain antibody was expressed in large quantities. Then, the bacterial cells were collected, ultrasonically disrupted, centrifuged, and the precipitate was loaded onto an affinity chromatography column for purification. After that, dialysis and ultrafiltration concentration were carried out (3 kDa - 10 kDa ultrafiltration tube; centrifuged at 4 °C and 3000 rpm for 30 min). Finally, the concentration of the prokaryotically expressed single-chain antibody was detected by the BCA method and aliquoted and stored at -80 °C. The purification results of the single-chain antibody affinity chromatography are shown in Figure 14 , and the purity is relatively high. Finally, the concentration of the single-chain antibody was detected by the BCA method to be 0.5 mg / mL.

[0151] (V) Reactivity identification of anti-goat CD207 single-chain antibody 1. Detection of the reactivity of the prokaryotically expressed single-chain antibody by indirect ELISA Indirect ELISA detection was carried out referring to 3.4 in (I). In the detection experiment, prokaryotically expressed truncated goat CD207 proteins with different coating amounts (800 ng, 400 ng, 200 ng, 50 ng) were set, and prokaryotically expressed truncated bovine CD207 protein and negative control were introduced. Three replicates were set for each coating amount.

[0152] Since the prepared single-chain antibody lacks the Fc end and it is not easy for the enzyme-labeled secondary antibody to bind to it, mouse anti-HA IgG (BioDragon Biotech) that can bind to the HA tag fused to the single-chain antibody was introduced as the secondary antibody in this detection experiment, and HRP-goat anti-mouse IgG was used as the tertiary antibody. After TMB color development and termination, readings were taken. The results are as Figure 15As shown in the figure, the OD value decreased with the decrease of the coating amount of the antigen (i.e., the prokaryotic expression truncated goat CD207 protein). Thus, it can be determined that the anti-goat CD207 single-chain antibody can bind to the prokaryotic expression truncated goat CD207 protein, and the binding strength is negatively correlated with the dilution degree of the coated antigen. This result indicates that the anti-goat CD207 single-chain antibody can be applied to the related experiments for detecting goat CD207 protein based on the ELISA method.

[0153] 2. Detection of the reactivity of the prokaryotic expression single-chain antibody by Western Blot The Western Blot detection was carried out with reference to (II). In the detection experiment, the empty vector and the prokaryotic expression truncated bovine CD207 protein were introduced as sample controls. The primary antibody was the anti-goat CD207 single-chain antibody, the secondary antibody was mouse anti-HA IgG, and the tertiary antibody was HRP-goat anti-mouse IgG. After ECL development, it was exposed; at the same time, a control experiment was set with the monoclonal antibody produced by the hybridoma cell line F7 in (I) as the primary antibody. The results are as Figure 16 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 the related experiments for detecting goat CD207 protein based on the Western Blot method.

[0154] (VI) Detection application of the anti-goat CD207 single-chain antibody Mature dendritic cells (DCs) are in a highly differentiated state and are difficult to be directly cultured and proliferated in vitro. For the mature DCs obtained by in vitro induced differentiation, antibodies against the surface marker molecules of DCs need to be used for identification.

[0155] In the above experiments, the prokaryotic expression goat CD207 protein (specifically the truncated protein) was used as the immunizing antigen. The hybridoma cell line that can stably secrete the anti-goat CD207 monoclonal antibody was screened by using the cell fusion technology. Based on the positive hybridoma cells that can secrete the anti-goat CD207 monoclonal antibody screened, the specific anti-goat CD207 single-chain antibody was obtained. And this single-chain antibody can be used as a key reagent for detecting goat CD207, so as to determine whether the dendritic cells differentiated in vitro in goats are mature, providing an important tool for the research and development of vaccines targeting goat dendritic cells.

Claims

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

2.

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

3.

3. An anti-goat CD207 single-chain antibody gene, characterized in that: The gene comprises a nucleotide sequence encoding the anti-goat CD207 single-chain antibody as claimed in claim 1 or 2.

4. The single-chain antibody gene against goat CD207 according to claim 3, characterized in that: Specifically, the gene comprises a nucleotide sequence encoding the heavy-chain variable region, a linker sequence, and a nucleotide sequence encoding the light-chain variable region which are arranged in sequence. The nucleotide sequence encoding the heavy-chain variable region is shown as SEQ.ID.NO.4, and the nucleotide sequence encoding the light-chain variable region is shown as SEQ.ID.NO.

5.

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

6.

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

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

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

9. A fusion protein, characterized in that: Contains the anti-goat CD207 single-chain antibody as claimed in claim 1 or 2.

10. Use of the anti-goat CD207 single-chain antibody as claimed in claim 1 or 2 or a fusion protein containing the single-chain antibody in the preparation of a reagent for detecting goat CD207 protein.

Images