A method for preparing a universal all-pig monoclonal antibody

Abstract

The present invention belongs to the technical field of monoclonal antibody preparation, and specifically relates to a method for preparing a universal whole-pig monoclonal antibody. The present invention is based on single B cell antibody preparation technology, and antigen-specific single B lymphocytes are separated from the peripheral blood of immune pigs or clinically recovered pigs by flow cytometry sorting technology; according to the pig immunoglobulin IgG antibody gene sequence in the database, primers are designed to specifically amplify the complete coding genes of antibody heavy and light chains in pig single B cells and add His tags, and pCDNA3.1-Leader is used as a eukaryotic expression vector to construct a recombinant vector of complete coding genes for antibody light and heavy chains, and natural whole-pig monoclonal antibodies are expressed by mammalian cells. The present invention establishes a universal whole-pig monoclonal antibody preparation method for the first time, which can be used for the preparation and screening of pig monoclonal antibody libraries of different pathogens. Compared with other antibody preparation methods, it has the advantages of high throughput, whole pig source, good diversity, complete antibodies and natural pairing, etc., which provides technical support for the rapid preparation and screening of pig monoclonal antibodies and has broad application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of monoclonal antibody preparation, and specifically is a method for preparing a universal all-pig source monoclonal antibody. Background Art

[0002] Monoclonal antibodies (mAbs, hereinafter referred to as "mAbs") are antibodies that are produced and secreted by a single specific B cell after the body is stimulated by a specific antigen and are only targeted at the specific antigenic determinant cluster. Compared with polyclonal antibodies, mAbs have obvious advantages in specificity and homogeneity. They can be used as clinical treatment and detection and diagnosis tools for tumors, autoimmune diseases, transplantation, viral infections, etc. Some mAbs with neutralizing activity play an important role in the control and treatment of infectious diseases. In addition, mAbs can also be used for structural and functional studies of specific antigens.

[0003] The single B cell antibody preparation technology utilizes the characteristics of the corresponding receptors on the surface of memory B cells to sort out antigen-specific single B lymphocytes from peripheral blood or spleen through flow cytometry sorting technology. Subsequently, the antibody light chain and heavy chain encoding genes in a single B cell are obtained by PCR amplification, and then the antibody light chain and heavy chain encoding genes in the same cell are cloned into eukaryotic expression vectors respectively, and co-transfected into mammalian cells for expression to obtain the monoclonal antibody expressed by the single B cell. As a new generation of rapid antibody preparation method, this method maintains the natural combination and conformation of antibodies, and has the advantages of rapidity, high efficiency, and high yield. At present, this method has been widely used in the establishment of pathogen antibody libraries for various diseases such as AIDS, tetanus, hepatitis B, influenza, etc., and has played an important role in the preparation of monoclonal antibodies for the new coronavirus.

[0004] Among the current methods for preparing monoclonal antibodies based on single B cell technology, most of them adopt PCR to amplify the antibody light / heavy chain variable region gene sequence in B cells, and then connect it with the fixed light / heavy chain constant region gene sequence to obtain a complete and expressible antibody gene sequence. Since this strategy cannot guarantee the natural structure between the variable region and the constant region of the antibody, it may affect the expression level and natural activity of the antibody, and the fixed constant region gene destroys the diversity of the antibody. The prepared antibody does not belong to the monoclonal antibody naturally expressed in a single B cell, thus limiting the application of the antibody. In addition, among the existing single B cell antibody preparation technologies, most methods are used to prepare mouse monoclonal clones, and there are also technologies for preparing monoclonal antibodies from other species such as human, bovine, rabbit, and cat, while there are fewer methods for preparing monoclonal antibodies from all pigs. Compared with monoclonal antibodies from other species, monoclonal antibodies from all pigs have a more natural affinity for pathogens of swine infectious diseases. Therefore, establishing a universal whole-swine monoclonal antibody preparation method based on single B cell antibody preparation technology is of great significance for the preparation of neutralizing antibodies or identification antibodies for important swine infectious diseases such as African swine fever, foot-and-mouth disease, and classical swine fever, as well as the analysis of pathogenic antigen structure and antigen detection. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and to provide a universal method for preparing whole-swine monoclonal antibodies.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention is to provide a universal primer set for specifically amplifying the complete coding genes of antibody light and heavy chains in a single B cell of pig origin, and the universal primer set includes the following three groups:

[0008] The first group: primers for amplifying the complete gene of the heavy chain of IgG antibody in a single porcine B cell, the primers are shown in SEQ ID NOs: 1-4, wherein the primers shown in SEQ ID NOs: 1-2 are used for the first round of amplification, and the primers shown in SEQ ID NOs: 3-4 are used for the second round of amplification;

[0009] The second group: primers for amplifying the complete gene of the IgG antibody light chain λ chain in a single porcine B cell, the primers are shown in SEQ ID NOs:5-8, wherein the primers shown in SEQ ID NOs:5-6 are used for the first round of amplification, and the primers shown in SEQ ID NOs:7-8 are used for the second round of amplification;

[0010] The third group: primers used for amplifying the complete gene of the IgG antibody light chain κ chain in a single B cell of pig origin, the primers are shown in SEQ ID NOs: 9-12, wherein the primers shown in SEQ ID NOs: 9-10 are used for the first round of amplification, and the primers shown in SEQ ID NOs: 11-12 are used for the second round of amplification.

[0011] Furthermore, the product of the first round of amplification includes the antibody variable region and constant region, and the product of the second round of amplification includes the expression vector homologous sequence, the antibody variable region and constant region.

[0012] The three sets of universal primers provided by the present invention can realize the amplification of the full length of natural antibody light chain and heavy chain genes in a single porcine B cell, each requiring only one pair of primers, and having high amplification efficiency. Therefore, it can be used to prepare all-porcine monoclonal antibodies, showing its good application in the preparation of all-porcine monoclonal antibodies.

[0013] Furthermore, the preparation of whole-swine monoclonal antibodies is based on the preparation of single B cell antibodies.

[0014] Another aspect of the present invention is to provide a method for preparing a universal whole pig source single B cell complete antibody, comprising the following steps:

[0015] S1: Isolation and screening of porcine B lymphocytes;

[0016] S2: Amplification of pig single B cell cDNA;

[0017] S3: using the universal primer set to amplify the complete antibody coding gene in a single pig B cell;

[0018] S4: Construction of a recombinant expression vector of a whole pig single B cell antibody: The antibody heavy chain or light chain complete encoding gene fragment amplified in step S3 is connected to a eukaryotic expression vector by homologous recombination to obtain a recombinant expression vector containing the antibody complete heavy chain or light chain encoding gene;

[0019] S5: Paired expression of heavy and light chains of whole porcine single B cell antibody: The complete heavy and light chains of the single B cell antibody obtained in step S4 are co-transfected into mammalian expression cells, and the supernatant is collected after transfection to obtain the complete porcine monoclonal antibody expressed by the single B cell.

[0020] Preferably, the step S1 comprises isolating peripheral blood mononuclear cells from the peripheral blood of immunized or clinically recovered pigs by density gradient centrifugation, and sorting antigen-specific single B lymphocytes into the wells of the PCR 8-tube by flow cytometry according to the characteristics of the B cell surface receptors; the step S2 amplifies the pig single B cell cDNA using the pig single B cell isolated in step S1 as a template, and reversely transcribing the mRNA in the single B cell into cDNA according to the routine operation of the single cell cDNA amplification kit.

[0021] Preferably, the step S3 specifically includes the following steps:

[0022] S31: Amplification of the complete antibody heavy chain coding gene

[0023] Using the single cell cDNA amplified in step S2 as a template, using the primers shown in SEQ ID NOs: 1-2 to perform a first round of amplification, using the product of the first round of amplification as a template, using the primers shown in SEQ ID NOs: 3-4 to perform a second round of amplification;

[0024] S32: Amplification of the complete gene encoding the antibody light chain λ chain

[0025] Using the single cell cDNA amplified in step S2 as a template, using the primers shown in SEQ ID NOs: 5-6 to perform a first round of amplification, and using the product of the first round of amplification as a template, using the primers shown in SEQ ID NOs: 7-8 to perform a second round of amplification;

[0026] S33: Amplification of the complete gene encoding the antibody light chain κ chain

[0027] The single cell cDNA amplified in step S2 is used as a template, and the primers shown in SEQ ID NOs: 9-10 are used for the first round of amplification. The product of the first round of amplification is used as a template, and the primers shown in SEQ ID NOs: 11-12 are used for the second round of amplification.

[0028] Preferably, the eukaryotic expression vector in step S4 is pCDNA3.1-Leader, and the eukaryotic expression vector contains a porcine antibody leader sequence.

[0029] Preferably, the PCR program for the first and second rounds of amplification of the complete antibody heavy chain coding gene is: 98°C 30s; 98°C 10s, 60°C 10s, 72°C 50s, 35 cycles; 72°C 5min; the PCR program for the first and second rounds of amplification of the complete antibody light chain λ chain coding gene is: 98°C 30s; 98°C 10s, 60°C 10s, 72°C 30s, 35 cycles; 72°C 5min; the PCR program for the first and second rounds of amplification of the complete antibody light chain κ chain coding gene is: 98°C 30s; 98°C 10s, 60°C 10s, 72°C 20s, 35 cycles; 72°C 5min.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The present invention designs primers based on the complete gene sequences of the heavy and light chains of porcine IgG antibodies published on IMGT and NCBI, and uses the cDNA of a single porcine B cell as a template. Through PCR amplification, the complete antibody gene encoded by the single B cell can be obtained, which includes the natural variable region and constant region of the antibody and the natural pairing of the heavy and light chains of the antibody; after the complete antibody coding gene is cloned into a eukaryotic expression vector, porcine monoclonal antibodies can be produced stably and continuously for a long time. Compared with traditional monoclonal antibody preparation methods, the present invention has the advantages of high throughput, all-porcine source, good diversity, high efficiency, etc., and can be used for the preparation of monoclonal antibodies for any porcine virus, providing technical support for the rapid preparation and screening of porcine monoclonal antibodies, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The PCR amplification results of the complete coding gene of the heavy chain of the swine IgG antibody of the classical swine fever E2 protein are shown, where M is the standard mass of 2000 DNA molecules; 1-7 are the PCR amplification results of the complete coding gene of the heavy chain of the antibody (about 1400 bp) of single B cells from different swine sources;

[0033] Figure 2 The PCR amplification results of the complete coding gene of the light chain λ chain of some swine IgG antibodies of classical swine fever E2 protein are shown, where M is the standard mass of 2000 DNA molecules; 1-7 are the PCR amplification results of the complete coding gene of the antibody light chain λ chain (about 650bp) of different swine single B cells;

[0034] Figure 3 The PCR amplification results of the complete coding gene of the light chain κ chain of the swine IgG antibody of the classical swine fever E2 protein are shown, where M is the standard mass of 2000 DNA molecules; 1-5 are the PCR amplification results of the complete coding gene of the antibody light chain κ chain (about 650bp) of single B cells from different swine sources; NC is the negative control;

[0035] Figure 4 The results of WB detection of the expression product of the classical swine fever E2 protein antibody are shown, where M is the 10-180KDa standard molecular weight protein Maker; 1-4 are the WB results of the expression supernatant after the heavy and light chain plasmids of different porcine antibodies were co-transfected into HEK293 cells; NC is the negative cell supernatant control;

[0036] Figure 5 The indirect immunofluorescence results of the classical swine fever E2 protein antibody are shown (100X), where 1-4 are the indirect immunofluorescence results of different whole-swine monoclonal antibodies; PC is the positive control; NC is the negative control.

[0037] Figure 6Figure 1 shows the PCR amplification results of the complete coding gene of the heavy chain of the swine IgG antibody of the O-type FMDV Mya98146S antigen, where M is the standard mass of 2000 DNA molecules; 1-11 are the PCR amplification results of the complete coding gene of the heavy chain of the antibody (about 1400 bp) of single B cells from different swine sources;

[0038] Figure 7 The PCR amplification results of the complete coding gene of the light chain λ chain of the pig IgG antibody of the O-type FMDV Mya98146S antigen are shown, where M is the standard mass of 2000 DNA molecules; 1-12 are the PCR amplification results of the complete coding gene of the light chain λ chain of the antibody of different pig-derived single B cells (about 650bp);

[0039] Figure 8 The PCR amplification results of the complete coding gene of the light chain κ chain of the porcine IgG antibody of the O-type FMDV Mya98146S antigen are shown, where M is the standard mass of 2000 DNA molecules; 1-9 are the PCR amplification results of the complete coding gene of the light chain κ chain of the antibody of different porcine single B cells (about 650bp);

[0040] Fig. 9 The indirect immunofluorescence results of O-type FMDV Mya98146S antigen antibody are shown (100X), where 1-3 are the indirect immunofluorescence results of different whole-swine monoclonal antibodies; NC is the negative control. DETAILED DESCRIPTION

[0041] The content of the present invention is further elaborated in detail below in conjunction with the accompanying drawings and specific examples of the specification, which are only used to explain the present invention and are not used to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The materials, reagents, etc. used in the present examples are reagents and materials available from commercial sources unless otherwise specified.

[0042] Example 1 Preparation of a swine monoclonal antibody against Classical Swine Fever Virus (CSFV) E2 protein using a universal whole-swine monoclonal antibody preparation method 1 Animal immunization

[0043] Two healthy negative Landrace piglets of about 3 weeks old were selected, and the first vaccination was carried out by immunizing the neck muscle with live Classical Swine Fever C strain vaccine at a dose of 30 doses per pig; after an interval of 21 days, the Classical Swine Fever C strain vaccine was boosted with the dose and route of the first vaccination; 21 days later, the neck muscle was boosted with CSFV-E2 antigen once; blood was collected from the neck vein every 2 days to monitor the serum antibody titer, and when the antibody titer reached 1:1024 or above, 50 ml of peripheral blood was collected from the neck vein in an anticoagulant tube for subsequent single B cell sorting.

[0044] Isolation of single B cells specific for 2CSFV-E2 antigen

[0045] 2.1 Isolation of porcine peripheral blood mononuclear cells

[0046] The collected porcine peripheral anticoagulated blood was separated by gradient density centrifugation to obtain peripheral blood mononuclear cells (PBMCs), and the PBMCs were resuspended in 1 mL of cell staining solution for cell counting.

[0047] 2.2 Cell staining

[0048] CSFV-E2 antigen with His tag (1.5 μg / 10 7 cells), AlexaFluor 647 fluorescently labeled anti-CD21 antibody (4 μL / 10 7 cells) and FITC fluorescently labeled anti-swine IgM antibody (1 μL / 10 7 cells), incubated at 4°C in the dark for 30 min; the same cells were used to set up isotype controls and ISO (CSFV-E2 antigen without His tag) controls. At the same time, single positive tubes were set up to adjust the compensation between channels. After washing the cells with cell staining buffer, the cells were resuspended in 1 mL of cell staining buffer and PE fluorescently labeled anti-His tag secondary antibody (0.3 μL / 10 7 Cells were collected and incubated at 4°C in the dark for 20 min. After rinsing the cells with cell sorting buffer, centrifuged at 1000 r / min at 4°C for 5 min and the supernatant was discarded. The cells were resuspended in 500 μL cell staining buffer and CSFV-E2 antigen-specific single B cells were isolated using a BD FACSAria II flow cytometer.

[0049] 2.3 Flow cytometry sorting of antigen-specific single B cells

[0050] Among all PBMCs, the mononuclear cell population was selected by screening the cell size and cell granularity, the B cell population was identified by the CD21 index (positive selection), and the CD21+ / IgM- / Ag(CSFV-E2)+ single B cells were sorted by the IgM index (negative selection) and the CSFV-E2 antigen index (positive selection). The cells were sorted into the PCR 8 strip tube wells containing 5uL sterile enzyme-free PBS. After collecting a row of cells, they were immediately placed on dry ice for freezing. After the required number of cells (88 cells) were sorted, the cells were transferred to -80°C for temporary storage until subsequent experiments.

[0051] Preparation of single B cell antibodies specific to 3CSFV-E2 antigen

[0052] (1) Reverse transcription of antibody-encoding mRNA in sorted CSFV-E2 antigen-specific single B cells into cDNA.

[0053] (2) Using cDNA as a template, the designed primers were used to amplify the complete coding gene fragments of the antibody heavy chain and light chain, respectively, and cloned into the pCDNA3.1-Leader eukaryotic expression vector containing the porcine antibody leader sequence.

[0054] (3) The antibody heavy chain and light chain recombinant expression vectors are co-transfected into HEK293T cells. After transfection, the supernatant is collected to obtain the monoclonal antibody encoded and expressed by the single B cell, which can be used for subsequent monoclonal antibody identification and screening.

[0055] 3.1 Amplification of porcine single B cell cDNA

[0056] According to SuperScript TM The instructions for the IV Single Cell / Low-Input cDNAPreAmp Kit require that the mRNA (including antibody-encoding genes) in the sorted single B cells be reverse transcribed into cDNA, and the cDNA be further amplified to increase its copy quantity.

[0057] 3.2 Primer design for the complete gene encoding porcine antibodies

[0058] According to the complete gene sequences of the heavy and light chains of the porcine antibodies published on IMGT and NCBI, primers were designed using Oligo 7 software. The upstream primer was located at the head of the variable region of the antibody, the downstream primer was located at the end of the constant region, and a 6×His tag was added to the 3' end. Therefore, the amplified product contains the complete coding gene of the antibody (antibody variable region + constant region). The primer sequences are shown in Table 1. The primers for amplifying the complete coding gene of the heavy chain are shown in SEQ ID NO: 1-2; the primers for amplifying the complete coding gene of the light chain λ chain are shown in SEQ ID NO: 5-6; the primers for amplifying the complete coding gene of the light chain κ are shown in SEQ ID NO: 9-10.

[0059] Table 1 Primers for amplifying complete antibody coding genes

[0060]

[0061] 3.3 Amplification and sequencing of complete coding genes of porcine single B cell antibodies

[0062] 3.3.1 Amplification of the complete coding gene for antibody heavy chain in single porcine B cells

[0063] The single B cell cDNA obtained by amplification in 3.1 was used as a template and amplified using primers Sus-WH-F and Sus-WH-HisR (shown in SEQ ID NO: 1-2 in Table 1, respectively). The amplification system is shown in Table 2, and the PCR amplification program is: 98°C 30s; 98°C 10s, 60°C 10s, 72°C 50s, 35 cycles; 72°C 5min. After the amplified product was identified by 1.5% agarose gel electrophoresis, the band that met the target fragment size (about 1400bp) was recovered, purified and sequenced. The sequencing results were analyzed, and the complete coding gene for the heavy chain of the porcine IgG antibody was amplified from 19 single B cells. Some of the amplification results are shown in Figure 2. Figure 1 shown.

[0064] Table 2 Amplification system of complete coding gene for antibody heavy chain of single B cell in pig

[0065]

[0066] 3.3.2 Amplification of the complete coding gene for the light chain λ chain of porcine single B cell antibody

[0067] The single B cell cDNA obtained by amplification in 3.1 was used as a template and amplified using primers Sus-WL-F and Sus-WL-HisR (shown as SEQ ID NO: 5-6 in Table 1). The amplification system is shown in Table 3, and the PCR amplification program is: 98°C for 30s; 98°C for 10s, 60°C for 10s, 72°C for 20s, 35 cycles; 72°C for 5min. After the amplified product was identified by 1.5% agarose gel electrophoresis, the band that met the target fragment size (about 650bp) was recovered, purified and sequenced. The sequencing results were analyzed, and finally 14 complete coding genes for the light chain λ of porcine IgG antibodies that naturally matched the heavy chain (the heavy chain and light chain λ chains were amplified simultaneously in the same B cell) were obtained. Some of the amplification results are shown in Figure 3. Figure 2 shown.

[0068] Table 3 Amplification system of complete coding gene of light chain λ chain of single B cell antibody in pig

[0069]

[0070] 3.3.3 Amplification of the complete coding gene for the light chain κ chain of porcine single B cell antibody

[0071] The single B cell cDNA obtained by amplification in 3.1 was used as a template and amplified using primers Sus-WK-F and Sus-WK-HisR (shown as SEQ ID NO: 9-10 in Table 1). The amplification system is shown in Table 4, and the PCR amplification program is: 98°C 30s; 98°C 10s, 60°C 10s, 72°C 20s, 35 cycles; 72°C 5min. After the amplified product was identified by 1.5% agarose gel electrophoresis, the band that met the target fragment size (about 650bp) was recovered, purified and sequenced. The sequencing results were analyzed, and finally 5 complete coding genes for the light chain κ of porcine IgG antibodies that naturally matched the heavy chain (the heavy chain and light chain κ chain were amplified simultaneously in the same B cell) were obtained. Some of the amplification results are shown in Figure 4. Figure 3 shown.

[0072] Table 4 Amplification system of complete coding gene of light chain κ chain of single B cell antibody in pig

[0073]

[0074] 3.4 Construction of recombinant plasmid encoding complete gene of porcine single B cell antibody

[0075] Primers were designed based on the sequences of the complete coding genes of the heavy and light chains of the porcine IgG antibody verified by sequencing analysis in 3.3, and the homologous sequences of the pCDNA3.1-Leader expression vector were added to the 5' end of the upstream primer and the 3' end of the downstream primer, respectively. The primer sequences are shown in Table 1 (the heavy chain, light chain λ chain, and light chain κ chain amplification primers are shown in SEQ ID NO: 3-4, 7-8, and 10-12 in Table 1, respectively). The PCR recovered product in 3.3 was used as a template for amplification, and the PCR program was: 98°C 30s; 98°C 10s, 60°C 10s, 72°C 50s, 35 cycles; 72°C 5min. After the amplified product was identified by 1.5% agarose gel electrophoresis, the bands that met the target fragment size were recovered and purified. By homologous recombination, the recovered and purified target fragment was connected to the pCDNA3.1-Leader linearized vector obtained after double digestion with HindIII and BamHI, and the reaction conditions were 50°C for 15 min. The reaction system is shown in Table 5. The homologous recombination product was transformed into Escherichia coli DH5α competent cells, and after successful connection was identified by bacterial liquid PCR, the recombinant expression plasmid of the complete coding gene of the porcine single B cell antibody was extracted.

[0076] Table 5 Homologous recombination reaction system

[0077]

[0078] 3.5 Expression of antibodies in porcine single B cells

[0079] HEK-293T cells were subcultured into six-well plates and transfected when the cell density reached 60%-80%. Taking a single well in a six-well plate as an example, the antibody heavy chain and light chain recombinant expression plasmids cloned from the same pig B cell were co-transfected. Prepare solution A: Add 2μg heavy chain plasmid, 2μg light chain plasmid and 125μL Opti-MEM to a 1.5mL centrifuge tube, flick to mix, and let stand for 5min. Prepare solution B: Add 10μL PEI transfection reagent and 125μL Opti-MEM to another 1.5mL EP tube, flick to mix, and let stand for 5min. Then slowly add solution A to solution B in a dropwise manner, flick to mix, let stand for 20min, and then evenly add the transfection complex to the cells, incubate in the incubator for 4h, and then change to normal culture medium. After transfection for 48h, collect the supernatant.

[0080] 3.6 Western-blot verification of expressed antibodies

[0081] Take 80ul of the collected supernatant and add 20ul of SDS-PAGE protein loading buffer (5×), vortex to mix, and place in a 100℃ water bath for 10min. Load the above protein samples into the corresponding wells according to the instructions for protein precast gels, and perform electrophoresis at 120V constant voltage for 90min. After the electrophoresis, transfer the samples to the PVDF membrane according to the instructions, block with 10% skim milk powder on a shaker at room temperature for 2h, and wash the membrane. Add the primary mouse anti-his tag (1:5000 diluted in PBS) antibody, incubate on a shaker at room temperature for 1h, and wash the membrane. Add the secondary HRP-labeled goat anti-mouse IgG antibody (1:5000 diluted in PBS), incubate on a shaker at room temperature for 1h, and wash the membrane and develop color. The results are as follows: Figure 4 As shown, there are bands at 55 kDa and 25 kDa, respectively, which verifies that porcine monoclonal antibodies are expressed and can be used for subsequent monoclonal antibody identification and screening.

[0082] 4. Indirect immunofluorescence assay (IFA) to detect antibody and CSFV reactivity

[0083] PK-15 cells were cultured in a 96-well plate. When the cells grew to about 80%, the culture medium was discarded, the cells were rinsed once with PBS, and 100 μL of CSFV C strain (virus titer 100-200 TCID 50 / ml), adsorbed in a 37°C incubator for 1h, discarded the virus solution, rinsed once with PBS, added 100μL of MEM culture medium containing 1% FBS to each well, and cultured in a 37°C incubator. After 78h, discarded the culture medium, added 100μL of pre-cooled fixative (acetone: methanol = 1:1) to each well, and fixed at -20°C for 30min. Discarded the fixative, rinsed 3 times with PBS, added 100μL of expressed antibody supernatant to each well, added negative expression supernatant to cell control, and added rabbit anti-CSFV E2 protein polyclonal antibody to positive control, and incubated in a 37°C incubator for 1h. The primary antibody was discarded, and the cells were rinsed three times with PBS. 100 μL of FITC-labeled rabbit anti-swine IgG antibody (1:200 dilution in PBS) was added to each well. FITC-labeled goat anti-rabbit IgG antibody was added as a positive control. The cells were incubated in a 37°C incubator for 1 h. After rinsing three times with PBS, 100 μL of PBS was added to each well. The cells were observed under an inverted fluorescence microscope. Some of the results are shown in Figure 2. Figure 5 As shown, it is demonstrated that the present invention successfully prepared a whole porcine monoclonal antibody that binds to CSFV.

[0084] Example 2 Preparation of pig-derived monoclonal antibodies against type O foot-and-mouth disease virus (FMDV) using a universal whole-swine monoclonal antibody preparation method

[0085] 1 Animal immunization

[0086] Two healthy Landrace piglets around 3 months old and free of specific pathogens were selected and immunized with FMDV Mya98 inactivated vaccine multiple times. When the antibody titer of liquid-phase blocking ELISA was greater than 1:1024, 50 mL of peripheral blood was collected and placed in anticoagulant tubes for subsequent single B cell sorting.

[0087] Isolation of single B cells specific for FMDV type 2O antigen

[0088] 2.1 Isolation of porcine peripheral blood mononuclear cells

[0089] Proceed as described in Example 1, step 2.1.

[0090] 2.2 Cell staining

[0091] According to the number of cells, add biotin-labeled O-type FMDV Mya98146S antigen (1.5 μg / 10 7 cells), AlexaFluor 647 fluorescently labeled anti-CD21 antibody (4 μL / 10 7 cells) and FITC fluorescently labeled anti-swine IgM antibody (1 μL / 10 7cells), incubated at 4°C in the dark for 30 min; the same cells were used to set up isotype controls and ISO (O-type FMDV Mya98146S antigen without biotin labeling) controls. At the same time, single positive tubes were set up to adjust the compensation between channels. After washing the cells with cell staining buffer, the cells were resuspended in 1 mL of cell staining buffer and PE fluorescently labeled anti-Biotin label secondary antibody (0.3 μL / 10 7 Cells were collected and incubated at 4°C in the dark for 20 min. The cells were washed with cell sorting buffer and centrifuged at 1000 r / min at 4°C for 5 min. The cells were resuspended in 500 μL cell staining buffer and sorted using BD FACSAria II flow cytometer for O-type FMDVMya98146S antigen-specific single B cells.

[0092] 2.3 Flow cytometry sorting of antigen-specific single B cells

[0093] Among all PBMCs, the mononuclear cell population was selected by screening the cell size and cell granularity, the B cell population was identified by the CD21 index (positive selection), and the CD21+ / IgM- / Ag(O-type FMDV Mya98146S antigen)+ single cells were sorted by the IgM index (negative selection) and the O-type FMDV 146S antigen index (positive selection). The cells were sorted into the PCR 8 strip tube wells containing 5uL sterile enzyme-free PBS. After collecting a row of cells, they were immediately placed on dry ice for freezing. After the required number of cells were sorted, the cells were transferred to -80°C for temporary storage until subsequent experiments.

[0094] Preparation of single B cell antibodies specific to 146S antigen of 3O FMDV

[0095] The preparation process is the same as described in step 3 of Example 1.

[0096] 3.1 Amplification of porcine single B cell cDNA

[0097] Proceed as described in Example 1, step 3.1.

[0098] 3.2 Primers for the complete coding gene of porcine antibodies

[0099] Use the primer set described in step 3.2 of Example 1.

[0100] 3.3 Amplification and sequencing of complete coding genes of porcine single B cell antibodies

[0101] 3.3.1 Amplification of the complete coding gene for antibody heavy chain in single porcine B cells

[0102] The single B cell cDNA obtained by amplification in 3.1 was used as a template and primers Sus-WH-F and Sus-WH-HisR (shown in SEQ ID NO: 1-2 in Table 1, respectively) were used for amplification. After sequencing analysis, the amplified products were amplified from 29 single B cells (48 B cells were obtained by sorting) to obtain the complete coding gene of the heavy chain of the O-type FMDV Mya98146S antigen swine-derived IgG antibody. Some of the amplification results are shown in Figure 6 As shown, M is the standard mass of 2000 DNA molecules; 1-11 are the PCR amplification results of the complete antibody heavy chain coding gene (about 1400 bp) of single B cells from different pig sources.

[0103] 3.3.2 Amplification of the complete coding gene for the light chain λ chain of porcine single B cell antibody

[0104] The single B cell cDNA obtained by amplification in 3.1 was used as a template and amplified using primers Sus-WL-F and Sus-WL-HisR (shown as SEQ ID NO: 5-6 in Table 1). After sequencing and analysis of the amplified products, 25 (48 B cells were sorted) complete coding genes for the light chain λ of the O-type FMDV Mya98146S antigen pig-derived IgG antibody that naturally matched the heavy chain were finally obtained. Some amplification results are shown in Figure 7 As shown, M is the standard mass of 2000 DNA molecules; 1-12 are the PCR amplification results of the complete coding gene (about 650 bp) of the antibody light chain λ chain of single B cells from different pig sources.

[0105] 3.3.3 Amplification of the complete coding gene for the light chain κ chain of porcine single B cell antibody

[0106] The single B cell cDNA obtained by amplification in 3.1 was used as a template and amplified using primers Sus-WL-F and Sus-WL-HisR (shown as SEQ ID NO: 9-10 in Table 1). After sequencing and analysis of the amplified products, 5 (48 B cells were sorted) complete coding genes for the light chain κ of the O-type FMDV Mya98146S antigen pig-derived IgG antibody that naturally matched the heavy chain were finally obtained. Some amplification results are shown in Figure 8 As shown, M is the standard mass of 2000 DNA molecules; 1-9 are the PCR amplification results of the complete coding gene (about 650 bp) of the antibody light chain κ chain of single B cells from different pig sources.

[0107] 3.4 Construction of recombinant plasmid encoding complete gene of porcine single B cell antibody

[0108] Proceed as described in step 3.4 of Example 1.

[0109] 3.5 Expression of antibodies in porcine single B cells

[0110] Proceed as described in step 3.5 of Example 1.

[0111] 4. Indirect immunofluorescence test to detect the reactivity of antibodies with O-type FMDV Mya98:

[0112] BHK-21 cells were cultured in a 96-well plate. When the cells grew to about 80%, the culture medium was discarded, and the cells were rinsed once with PBS. The FMDV Mya98 strain was inoculated at a 1:10 inoculum. After adsorption in a 37°C incubator for 4 hours, the virus solution was discarded, and the cells were rinsed once with PBS. 100 μL of pre-cooled fixative (acetone: methanol = 1:1) was added to each well, and fixed at -20°C for 30 minutes. The fixative was discarded, and the cells were rinsed 3 times with PBS. 100 μL of expressed antibody supernatant was added to each well, and negative expression supernatant was added to the cell control, and the cells were incubated in a 37°C incubator for 1 hour. The primary antibody was discarded, and the cells were rinsed 3 times with PBS. 100 μL of FITC-labeled rabbit anti-swine IgG antibody (1:200 dilution in PBS) was added to each well, and the cells were incubated in a 37°C incubator for 1 hour. After rinsing 3 times with PBS, 100 μL of PBS was added to each well, and the cells were observed under an inverted fluorescence microscope. Some of the results are shown in the figure. Fig. 9 As shown, 1-3 are the indirect immunofluorescence results of different whole pig monoclonal antibodies, and NC is a negative control, indicating that the present invention successfully prepared a whole pig monoclonal antibody that binds to O-type FMDV Mya98.

[0113] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A primer set for specifically amplifying the complete coding genes of antibody light and heavy chains in a single B cell of pig origin, characterized in that: The primer set includes the following three groups: The first group: primers for amplifying the complete gene of the heavy chain of IgG antibody in a single porcine B cell, the primers are shown in SEQ ID NOs: 1-4, wherein the primers shown in SEQ ID NOs: 1-2 are used for the first round of amplification, and the primers shown in SEQ ID NOs: 3-4 are used for the second round of amplification; The second group: primers for amplifying the complete gene of the IgG antibody light chain λ chain in a single porcine B cell, the primers are shown in SEQ ID NOs: 5-8, wherein the primers shown in SEQ ID NOs: 5-6 are used for the first round of amplification, and the primers shown in SEQ ID NOs: 7-8 are used for the second round of amplification; The third group: primers for amplifying the complete gene of the IgG antibody light chain κ chain in a single porcine B cell, the primers are shown in SEQ ID NOs: 9-12, wherein the primers shown in SEQ ID NOs: 9-10 are used for the first round of amplification, and the primers shown in SEQ ID NOs: 11-12 are used for the second round of amplification; The antibody light and heavy chain encoding genes in the specific amplification of antibody light and heavy chain encoding genes in a single porcine B cell refer to the antibody light and heavy chain encoding genes of porcine IgG.

2. The primer set for specifically amplifying the complete coding genes of antibody light and heavy chains in a single B cell of pig origin according to claim 1, characterized in that: The product of the first round of amplification includes the antibody variable region and constant region, and the product of the second round of amplification includes the expression vector homologous sequence, the antibody variable region and constant region.

3. Use of the primer set according to claim 1 in preparing porcine monoclonal antibodies, characterized in that: The porcine monoclonal antibody is a porcine monoclonal antibody against E2 protein of classical swine fever virus or a porcine monoclonal antibody against type O foot-and-mouth disease virus.

4. The use according to claim 3, characterized in that: The preparation of porcine monoclonal antibodies is based on the preparation of antibodies from single B cells.

5. A method for preparing a complete antibody from a single porcine B cell, characterized in that: The following steps are involved: S1: Isolation and screening of porcine B lymphocytes; S2: Amplification of pig single B cell cDNA; S3: using the primer set described in claim 1 to amplify the complete antibody coding gene in a single pig B cell; S4: Construction of a recombinant expression vector of a porcine single B cell antibody: Connecting the complete heavy chain or light chain encoding gene fragment amplified in step S3 to a eukaryotic expression vector using homologous recombination to obtain a recombinant expression vector containing the complete heavy chain or light chain encoding gene of the antibody; S5: Paired expression of heavy and light chains of porcine single B cell antibodies: co-transfecting the complete heavy and light chains of the single B cell antibodies obtained in step S4 into mammalian expression cells, collecting the supernatant after transfection, and obtaining the complete porcine monoclonal antibody expressed by the single B cell; The step S1 comprises separating peripheral blood mononuclear cells from the peripheral blood of immunized or clinically recovered pigs by density gradient centrifugation, and sorting antigen-specific single B lymphocytes into the wells of the PCR 8-tube by flow cytometry sorting technology according to the characteristics of B cell surface receptors; the step S2 amplifies the pig single B cell cDNA by using the pig single B cell separated in step S1 as a template, and reversely transcribing the mRNA in the single B cell into cDNA according to the conventional operation of the single cell cDNA amplification kit; The step S3 specifically comprises the following steps: S31: Amplification of the complete coding gene for the antibody heavy chain: Using the single cell cDNA amplified in step S2 as a template, using the primers shown in SEQ ID NOs: 1-2 in claim 1 to perform a first round of amplification, and using the product of the first round of amplification as a template, using the primers shown in SEQ ID NOs: 3-4 in claim 1 to perform a second round of amplification; S32: Amplification of the complete coding gene for the antibody light chain λ chain: Using the single cell cDNA amplified in step S2 as a template, using the primers shown in SEQ ID NOs: 5-6 in claim 1 to perform a first round of amplification, and using the product of the first round of amplification as a template, using the primers shown in SEQ ID NOs: 7-8 in claim 1 to perform a second round of amplification; S33: Amplification of the complete coding gene for the antibody light chain κ chain: Using the single cell cDNA amplified in step S2 as a template, using the primers shown in SEQ ID NOs: 9-10 in claim 1 to perform a first round of amplification, and using the product of the first round of amplification as a template, using the primers shown in SEQ ID NOs: 11-12 in claim 1 to perform a second round of amplification; The porcine monoclonal antibody is a porcine monoclonal antibody against E2 protein of classical swine fever virus or a porcine monoclonal antibody against type O foot-and-mouth disease virus.

6. The method for preparing a complete antibody from a single porcine B cell according to claim 5, characterized in that: The eukaryotic expression vector in step S4 is pCDNA3.1-Leader, and the eukaryotic expression vector contains a porcine antibody leader sequence.

7. The method for preparing a complete antibody from a single porcine B cell according to claim 5, characterized in that: The PCR program for the first and second rounds of amplification of the complete antibody heavy chain coding gene was: 98°C 30s; 98°C 10s, 60°C 10s, 72°C 50s, 35 cycles; 72°C 5min; the PCR program for the first and second rounds of amplification of the complete antibody light chain λ chain coding gene was: 98°C 30s; 98°C 10s, 60°C 10s, 72°C 30s, 35 cycles; 72°C 5min; the PCR program for the first and second rounds of amplification of the complete antibody light chain κ chain coding gene was: 98°C 30s; 98°C 10s, 60°C 10s, 72°C 20s, 35 cycles; 72°C 5min.

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