Antibody or antigen binding fragment aiming at porcine epidemic diarrhea virus and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to an antibody or an antigen binding fragment aiming at porcine epidemic diarrhea virus and application thereof. The antibody or the antigen binding fragment has excellent specificity and affinity to porcine epidemic diarrhea virus PEDV S1 protein.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an antibody or antigen-binding fragment against porcine epidemic diarrhea virus and an application thereof. Background Art

[0002] Porcine epidemic diarrhea (PED) is an acute, contact-transmitted enteric infectious disease of pigs caused by porcine epidemic diarrhea virus (PEDV). Clinical symptoms include watery diarrhea, vomiting, dehydration, metabolic acidosis, etc., and a high mortality rate. Pigs of all ages and breeds are susceptible, but there are large differences in clinical symptoms and mortality rates after infection among pigs of different ages. The morbidity and mortality rates are inversely proportional to the age of the pigs. As the age of the pigs increases, the morbidity and mortality rates gradually decrease. The disease is particularly harmful to suckling piglets, with an morbidity rate of 80% to 100% and a mortality rate of more than 90% for newborn piglets.

[0003] Sick pigs and infected pigs are the main sources of infection for the spread of the disease. The main transmission routes are oral and digestive tract transmission. The virus is directly transmitted or indirectly infected through direct contact or fecal-oral contact between healthy pigs and sick pigs. Semen and milk transmission are potential routes. The respiratory secretions discharged by sick pigs also have high levels of viruses. In farms with repeated epidemics and inadequate ventilation facilities, air transmission is also an influencing factor. Therefore, it is necessary to develop a product to prevent, diagnose or treat related diseases caused by porcine epidemic diarrhea virus infection.

[0004] Antibodies are proteins that can specifically recognize antigens. They can recognize and neutralize viruses by specifically recognizing viral envelope proteins, structural proteins or enzymes. They are currently one of the technical means for treating virus-related diseases. Therefore, the development of an antibody against porcine epidemic diarrhea virus is of great significance for the prevention, diagnosis or treatment of related diseases caused by porcine epidemic diarrhea virus infection. Summary of the invention

[0005] The present invention screens out an antibody or antigen-binding fragment against porcine epidemic diarrhea virus (PEDV), which has excellent specificity and affinity for porcine epidemic diarrhea virus PEDV S1 protein.

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

[0007] On the one hand, the present invention provides an antibody or antigen-binding fragment against porcine epidemic diarrhea virus (PEDV), the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, the amino acid sequence of HCDR1 is shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is shown in SEQ ID NO:2, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:3; the light chain variable region comprises LCDR1, LCDR2 and LCDR3, the amino acid sequence of LCDR1 is shown in SEQ ID NO:10, the amino acid sequence of LCDR2 is shown in SEQ ID NO:11, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:12.

[0008] Preferably, the above-mentioned heavy chain variable region also includes: HFR1 having at least 80% identity with the sequence shown in SEQ ID NO:4 and / or HFR2 having at least 80% identity with the sequence shown in SEQ ID NO:5 and / or HFR3 having at least 80% identity with the sequence shown in SEQ ID NO:6 and / or HFR4 having at least 80% identity with the sequence shown in SEQ ID NO:7; and / or the light chain variable region also includes: LFR1 having at least 80% identity with the sequence shown in SEQ ID NO:13 and / or LFR2 having at least 80% identity with the sequence shown in SEQ ID NO:14 and / or LFR3 having at least 80% identity with the sequence shown in SEQ ID NO:15 and / or LFR4 having at least 80% identity with the sequence shown in SEQ ID NO:16.

[0009] Preferably, the above antibody comprises a heavy chain variable region having at least 70% identity with the sequence shown in SEQ ID NO:8 and a light chain variable region having at least 70% identity with the sequence shown in SEQ ID NO:17.

[0010] Preferably, the above antibody or antigen-binding fragment further comprises a heavy chain constant region and / or a light chain constant region, and the heavy chain constant region and the light chain constant region are both derived from human or mouse IgG antibodies or mutants thereof.

[0011] Preferably, the heavy chain constant region comprises a sequence that is at least 70% identical to the sequence shown in SEQ ID NO:9, and the light chain constant region comprises a sequence that is at least 70% identical to the sequence shown in SEQ ID NO:18.

[0012] Another aspect of the present invention also provides any one of the following substances:

[0013] (i) a polynucleotide molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment of the present invention;

[0014] (ii) an expression vector comprising the polynucleotide molecule described in (i);

[0015] (iii) an engineered bacterium, comprising the expression vector in (ii);

[0016] (iv) a host cell, comprising the expression vector in (ii);

[0017] (v) A product for detecting porcine epidemic diarrhea virus, comprising the antibody or antigen-binding fragment of the present invention, or the polynucleotide molecule in (i), or the expression vector in (ii), or the engineered bacteria in (iii), or the engineered cells in (iv);

[0018] (vi) a pharmaceutical composition comprising the antibody and / or antigen-binding fragment of the present invention and / or the polynucleotide molecule in (i) or the expression vector in (ii) and / or the engineered bacteria in (iii) and / or the engineered cells in (iv);

[0019] (vii) a pharmaceutical preparation comprising the antibody and / or antigen-binding fragment of the present invention and / or the polynucleotide molecule in (i) and / or the expression vector in (ii) and / or the engineered bacteria in (iii) and / or the engineered cells in (iv) and / or the pharmaceutical composition in (vi).

[0020] Preferably, the pharmaceutical composition in the above substances is a bispecific antibody, a polyspecific antibody, an ADC or a fusion protein.

[0021] In another aspect, the present invention provides the use of the antibody and / or antigen-binding fragment and / or the polynucleotide molecule in (i) and / or the expression vector in (ii) and / or the engineered bacteria in (iii) and / or the engineered cells in (iv) of the present invention in the preparation of a product for diagnosing porcine epidemic diarrhea virus infection.

[0022] In another aspect, the present invention provides the use of the antibody and / or antigen-binding fragment and / or the polynucleotide molecule in (i) and / or the expression vector in (ii) and / or the engineered bacteria in (iii) and / or the engineered cells in (iv) of the present invention in the preparation of a medicament for preventing or treating porcine epidemic diarrhea virus infection.

[0023] Preferably, in the above application, the porcine epidemic diarrhea virus infection disease is porcine epidemic diarrhea.

[0024] The beneficial effects of the present invention include: the antibody or antigen-binding fragment against porcine epidemic diarrhea virus (PEDV) provided by the present invention has excellent specificity and affinity for porcine epidemic diarrhea virus PEDV S1 protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The conditions for PCR amplification of PEDV S1 protein;

[0026] Figure 2 The purification of PEDV S1 protein by HisTrap Excel column;

[0027] Figure 3 The purification of PEDV S1 protein by Superdex 200 10 / 300 gel chromatography column;

[0028] Figure 4 The SDS-PAGE protein identification gel was stained with Coomassie Brilliant Blue;

[0029] Figure 5 To identify the purified PEDV S1 protein by Western Blot;

[0030] Figure 6 Flow cytometry was used to sort specific B cells;

[0031] Figure 7 The nested PCR amplification of the light and heavy chain variable region fragments of the antibody;

[0032] Figure 8 The expression of PEDV antibodies was detected by Western Blot method;

[0033] Fig. 9 To detect the binding of PEDV antibodies using ELISA;

[0034] Fig.10 Determine the epitope recognition of the antibody for indirect immunofluorescence assay;

[0035] Fig.11 This is the purification status of PEDV S-A9 antibody;

[0036] Fig.12 The results of microneutralization tests with different antibodies;

[0037] Fig.13 This is the specific identification of PEDV S-A9 antibodies;

[0038] Fig.14 The affinity of PEDV S-A9 antibody to PEDV S1 protein. DETAILED DESCRIPTION

[0039] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0040] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0041] In the present invention, the term "antigen-binding fragment" refers to antigen-binding fragments of antibodies and antibody analogs, which generally include at least part of the antigen-binding region or variable region of the parent antibody, such as one or more CDRs; antibody fragments retain at least some of the binding specificity of the parent antibody.

[0042] An embodiment of the present invention provides an antibody or antigen-binding fragment against porcine epidemic diarrhea virus (PEDV), the antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, the amino acid sequence of HCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 3; the light chain variable region comprises LCDR1, LCDR2 and LCDR3, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 10, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 11, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 12.

[0043] It should be noted that the antibodies in the present invention can be any antibody form such as monoclonal antibodies or recombinant antibodies; in terms of source, they can be humanized antibodies or animal antibodies, such as mouse, rabbit or camel sources; in addition, HCDR3 and LCDR3 in the above antibodies belong to the hypervariable regions of the heavy chain variable region and the light chain variable region, and their stability is weaker than HCDR1 and HCDR2 and LCDR1 and LCDR2.

[0044] In some specific examples, the heavy chain variable region further includes: HFR1 having at least 80% identity with the sequence shown in SEQ ID NO:4 and / or HFR2 having at least 80% identity with the sequence shown in SEQ ID NO:5 and / or HFR3 having at least 80% identity with the sequence shown in SEQ ID NO:6 and / or HFR4 having at least 80% identity with the sequence shown in SEQ ID NO:7; and / or the light chain variable region further includes: LFR1 having at least 80% identity with the sequence shown in SEQ ID NO:13 and / or LFR2 having at least 80% identity with the sequence shown in SEQ ID NO:14 and / or LFR3 having at least 80% identity with the sequence shown in SEQ ID NO:15 and / or LFR4 having at least 80% identity with the sequence shown in SEQ ID NO:16.

[0045] It should be noted that the FR (HFR1, HFR2, HFR3, HFR4 and LFR1, LFR2, LFR3, LFR4) of the above-mentioned antibodies is a framework region, which is used to connect the CDR region and is relatively stable; in addition, the sequences of the FR regions of the heavy chain and light chain in the above-mentioned antibodies and the sequences of the CDR regions can be arranged according to FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, respectively constituting the heavy chain variable region and light chain variable region of the above-mentioned two antibodies; in addition, the at least 80% identity in the present invention includes ≥80%, ≥85%, ≥90%, ≥95% or 100% identity, etc.

[0046] In some specific examples, the above-mentioned antibody includes a heavy chain variable region that is at least 70% identical to the sequence shown in SEQ ID NO:8 and a light chain variable region that is at least 70% identical to the sequence shown in SEQ ID NO:17.

[0047] It should be noted that the above-mentioned at least 70% identity refers to an identity ≥ 70%, such as 75%, 80%, 85%, 90%, 95% or 100%; of course, it should be understood that sequences with at least 70% identity have similar functions to the above-mentioned sequences.

[0048] In some specific examples, the above-mentioned antibody or antigen-binding fragment further comprises a heavy chain constant region and / or a light chain constant region, and the heavy chain constant region and the light chain constant region are both derived from human or mouse IgG antibodies or mutants thereof.

[0049] In some specific examples, the heavy chain constant region comprises a sequence that is at least 70% identical to the sequence shown in SEQ ID NO:9, and the light chain constant region comprises a sequence that is at least 70% identical to the sequence shown in SEQ ID NO:18.

[0050] It should be noted that, in addition to the heavy chain variable region and light chain variable region mentioned above, the antibody or antigen-binding fragment of the present invention also includes a heavy chain constant region, and the light chain also includes a light chain constant region. The sources of the heavy chain constant region and the light chain constant region can be human or other animal sources (such as rabbit, pig, etc.), which are almost non-mutation regions. In addition, the heavy chain and light chain of the antibody can also include a signal peptide, which can help the antibody to penetrate the membrane; the signal peptide can be a signal peptide known in the art.

[0051] It should be noted that, as mentioned above, the above-mentioned at least 70% identity refers to an identity ≥ 70%, such as 75%, 80%, 85%, 90%, 95% or 100%; of course, it should be understood that sequences with at least 70% identity have similar functions to the above-mentioned sequences.

[0052] The present invention also provides any one of the following substances:

[0053] (i) a polynucleotide molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment of the present invention; specifically, the polynucleotide molecule of the present invention is obtained by translating the antibody or antigen-binding fragment of the present invention according to a conventional method; in addition, it can also be a nucleotide sequence obtained by translating the above amino acid sequence and further modifying the sequence; the modification method is a nucleotide modification method known in the art to increase expression efficiency or for other purposes;

[0054] (ii) an expression vector, comprising the polynucleotide molecule in (i); specifically, the expression vector in the present invention can be selected from any one of a lentiviral expression vector, a retroviral expression vector, an adenoviral expression vector, an adeno-associated viral expression vector, a DNA vector, an RNA vector and a plasmid. The lentiviral vector can be selected from the following groups: human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), visna-maedivirus (VMV), caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV) and simian immunodeficiency virus (SIV);

[0055] (iii) engineered bacteria, including the expression vector in (ii); specifically, the engineered bacteria in the present invention refers to bacteria that can assist the above-mentioned expression vector in expression, such as Escherichia coli, and the Escherichia coli including the expression vector here refers to the Escherichia coli after the expression vector is transferred;

[0056] (iv) a host cell, including the expression vector in (ii); specifically, the host cell in the present invention refers to a cell that can assist the expression vector mentioned above in expression, such as a yeast cell;

[0057] (v) A product for detecting porcine epidemic diarrhea virus, including the antibody or antigen-binding fragment of the present invention, or the polynucleotide molecule of (i), or the expression vector of (ii), or the engineered bacteria of (iii), or the engineered cells of (iv); specifically, the antibody or antigen-binding fragment of the present invention has a strong binding effect with the surface protein of porcine epidemic diarrhea virus, and a product for detecting porcine epidemic diarrhea virus can be prepared based on this; the product can be a detection reagent or a detection kit, such as a reagent or kit based on immunoblotting, or a reagent or kit based on immunoenzyme technology, etc.;

[0058] (vi) a pharmaceutical composition, comprising the antibody and / or antigen-binding fragment of the present invention and / or the polynucleotide molecule of (i) or the expression vector of (ii) and / or the engineered bacteria of (iii) and / or the engineered cells of (iv); specifically, as described above, based on the strong binding effect between the antibody or antigen-binding fragment of the present invention and the surface protein of porcine epidemic diarrhea virus, a pharmaceutical composition for preventing or treating porcine epidemic diarrhea virus infection can be prepared; the pharmaceutical composition herein refers to a pharmaceutical composition composed of the antibody or antigen-binding fragment of the present invention in combination with other small molecule compounds, polypeptides, antibodies or proteins that can be effectively used to prevent or treat porcine epidemic diarrhea virus infection; such as a bispecific antibody or polyspecific antibody combined with other antibodies, an ADC combined with a small molecule compound, or a fusion protein combined with other proteins; it can be selected and used according to the specific situation;

[0059] (vii) Pharmaceutical preparations, including the antibodies and / or antigen-binding fragments of the present invention and / or the polynucleotide molecules in (i) and / or the expression vectors in (ii) and / or the engineered bacteria in (iii) and / or the engineered cells in (iv) and / or the pharmaceutical composition in (vi); specifically, the antibodies and / or antigen-binding fragments and the pharmaceutical composition can be added with pharmaceutically acceptable carriers to prepare different dosage forms suitable for different clinical needs; the dosage forms can include sprays, oral liquids, tablets, atomizers, granules, capsules or ointments; of course, the choice of pharmaceutically acceptable carriers varies according to different dosage forms, and can be prepared according to methods known in the art.

[0060] The embodiments of the present invention also provide an application of the antibody and / or antigen-binding fragment and / or the polynucleotide molecule in (i) and / or the expression vector in (ii) and / or the engineered bacteria in (iii) and / or the engineered cells in (iv) in the present invention in the preparation of a product for diagnosing porcine epidemic diarrhea virus infection.

[0061] It should be noted that it should be understood that the detection of porcine epidemic diarrhea virus can be used for the diagnosis of porcine epidemic diarrhea virus infection diseases, that is, the product for detecting porcine epidemic diarrhea virus in the present invention is also suitable for the diagnosis of porcine epidemic diarrhea virus infection diseases, such as the diagnosis of porcine epidemic diarrhea; in addition, the product for detecting porcine epidemic diarrhea virus includes but is not limited to detection reagents, detection kits, reagent cards or microfluidic chips, etc.

[0062] The embodiments of the present invention also provide the use of the antibody and / or antigen-binding fragment and / or the polynucleotide molecule in (i) and / or the expression vector in (ii) and / or the engineered bacteria in (iii) and / or the engineered cells in (iv) in the present invention in the preparation of a drug for preventing or treating porcine epidemic diarrhea virus infection.

[0063] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0064] 1. Mouse Immunization

[0065] (I) Preparation and purification of PEDV S1 protein

[0066] (1) Construction and extraction of pCAGGS-PEDV S1 gene recombinant plasmid

[0067] 1) Primer design: Based on the PEDV CH-HB2-2018 strain (GenBank No. MK606369) published on NCBI as a template, the PEDV S1 gene sequence was codon optimized, the Kozak sequence and human signal peptide were added to the N segment, the 8×His tag was added to the C segment, and EcoRⅠ and XhoⅠ restriction sites were added to the two segments. The gene was sent to the company for gene synthesis, and the pCAGGS-PEDV-S1 eukaryotic expression plasmid was constructed.

[0068] 2) PCR identification of plasmid: pCAGGS-PEDV-S1 plasmid was used as a template and PCR identification was performed using pCAGGS universal primers. The reaction system and amplification conditions are shown in Tables 1 and 2.

[0069] Table 1 PCR amplification reaction system

[0070] Components volume pCAGGS-PEDV-S1 Plasmid 10ng Upstream primer 20pmol Downstream primer 20pmol MCLAB enzyme 10μL Deionized water Make up to 20 μL

[0071] Table 2 PCR amplification reaction conditions

[0072]

[0073] The size of the amplified gene fragment is 1028 bp. Figure 1As shown, the size of the amplified band is consistent with the expected target band size. Take 60ng of the vector and perform ligation transformation according to the pmol ratio of vector to target fragment of 1:3. Pick a single colony for sequencing, compare and analyze the sequencing results, and there is no base mutation. Glycerol bacteria are preserved and plasmids are extracted.

[0074] 3) Plasmid extraction: Inoculate the pCAGGS-PEDV S1 cloned bacterial solution into 200 mL of LB liquid medium containing Amp resistance at a ratio of 1:1000, and culture it in a constant temperature shaker at 37°C for 12-16 hours. Use an endotoxin-free plasmid extraction kit to extract the plasmid. Improve the experimental steps based on the instructions, pre-cool isopropanol at -80°C for 30 minutes, mix it with the plasmid extract, and place it at -20°C for 1 hour to accelerate plasmid precipitation, which can significantly increase the amount of plasmid extraction. After measuring the plasmid concentration, freeze it at -20°C.

[0075] (2) Expression of PEDV S1 protein

[0076] Place the HEK-293F cell culture flask at 37°C and 5% CO 2 , cultured in a constant temperature shaker at 150-175 rpm; counted after 12 h, the cell concentration was between 2.5 and 3.0 × 10 6 / mL for transfection; transfect 400mL of HEK-293F cells, dilute 400μg DNA with 150mM NaCl to a total volume of 10mL, and mix gently; dilute 2mL Sinofection transfection reagent with 150mM NaCl to a total volume of 10mL, and mix gently; let the diluted DNA and transfection reagent stand alone for about 5min; gently mix the plasmid and transfection reagent, the total volume is 20mL, and stand at room temperature for 20min. Add the mixture drop by drop to the cell culture medium, gently shake the culture bottle while adding, and put it back on the shaker to continue culturing after shaking, and tie the bottle mouth (that is, no longer introduce CO into the cell bottle) 2 ). After 24 hours of transfection, loosen the bottle cap to ensure that the dissolved oxygen and CO 2 Emission requirements to prevent CO 2 Accumulation leads to too low pH value of culture medium (culture medium turns yellow), which affects cell growth. 14 mL of SMS293-SUPI feed solution was added 24 h after transfection, and feed solution (14 mL) was added every 48 h thereafter, and samples were collected 7 days after transfection.

[0077] (3) Purification of PEDV S1 recombinant protein

[0078] The PEDV S1 recombinant protein was expressed using the HEK-293F mammalian cell expression system and purified using a HisTrap Excel column. Figure 2The PEDV S1 protein was further purified using a Superdex 200 10 / 300 gel chromatography column. Figure 3 The sample purified by molecular sieve was identified by SDS-PAGE protein identification gel. The monomer size of the target protein was estimated to be 83kDa, and the polymer size was about 100kDa. The SDS-PAGE protein identification gel was stained with Coomassie brilliant blue. After decolorization, a clear protein band was seen at 100kDa, as shown in Figure 1. Figure 4 As shown, protein samples 6 to 9 were concentrated and replaced with 30 kDa protein concentrators. Figure 5 As shown, the purified PEDV S1 protein was identified by Western Blot using mouse Anti-His tag antibody, and the His tag was detected at 83 kDa, which is consistent with the expected size of the target protein; the details are as follows:

[0079] The HEK-293F cell culture fluid was collected 7 days after transfection, and centrifuged at 8000rpm for 1h in a vertical high-speed low-temperature centrifuge at 4℃, then the HEK-293F suspended cell expression supernatant was collected, and the supernatant was filtered by negative pressure suction through a 0.22μm filter membrane to remove cell debris; the column was first rinsed with deionized water filtered by negative pressure suction through a 0.22μm filter membrane at a flow rate of 2mL / min, and then equilibrated with buffer A (20mmol / L Tris-HCl, 150mmol / L NaCl, pH 8.0) at the same flow rate for 3 to 5 column volumes; the expression supernatant of the PDV-S1 recombinant protein was transferred to the his column after negative pressure suction filtration, and the supernatant was passed through at 2mL / min. Be careful not to let bubbles enter during this process; the A pump and B pump of the protein purification instrument were quickly rinsed with water and buffer A at a flow rate of 10mL / min in turn, and then adjusted to a flow rate of 2mL / min, and the HisTrap was connected. Excel column, rinse the protein purification instrument with 100% buffer A until the UV value is flat; elute with buffer B containing 50mmol / L, 100mmol / L, 250mmol / L, and 500mmol / L imidazole concentrations in sequence, collect the eluate for SDS-PAGE detection, transfer the eluate to a protein concentration tube, concentrate and wash it with sterile PBS buffer three times, and after elution, select a suitable molecular sieve according to the molecular weight and protein amount to purify the PEDV S1 protein; Use of superdex 200 molecular sieve: Before installing the column, first set the maximum alarm pressure and flow rate on Akta, the column pressure of the large column is: column front pressure (ie system pressure) + 0.15MPa, flow rate: 0.5-1mL / min, column volume is 120mL.

[0080] Before purifying the protein, 40 mL of water should be passed through, and then 80 mL of buffer (PBS with pH 7.4) should be passed through. After the UV value curve is stable, the loop should be loaded. According to the volume of the concentrated protein, the appropriate loop (1 / 2 / 5 mL) should be selected. After 3 volumes of PBS are rinsed with a syringe, the protein should be sucked into the loop, and inject should be clicked. After PBS passes through 3 volumes of the loop, click load. The corresponding volume can be found in the molecular sieve list according to the molecular weight of the protein. Set peak collection: 5mAU 0.8 mL per tube. Put the collected protein on ice, select the appropriate concentration tube for liquid change after identification with SDS-PAGE protein gel, and identify the purified PEDV S1 protein with anti-his tag antibody by protein immunoblotting.

[0081] (II) Animal Immunization

[0082] Immunization procedure: The mice were immunized three times with purified PEDV S1 protein, each time with an interval of 15 days. One week before cell sorting, a booster immunization was performed, and the mice were immunized a total of 4 times (as shown in Table 3).

[0083] Table 3 Mouse immunization program

[0084] Immunization times Immunization dose (μg / mouse) Immunization methods Adjuvant One Free 50 Subcutaneous injection at the back Freund's complete adjuvant Second exemption 50 Subcutaneous injection at the back Freund's incomplete adjuvant Three exemptions 50 Subcutaneous injection at the back Freund's incomplete adjuvant Strengthen immunity 100 Subcutaneous injection at the back Freund's incomplete adjuvant

[0085] Before flow cytometry sorting of specific B lymphocytes, mice were anesthetized by isoflurane breathing, and blood was collected from the orbital venous plexus. 500 μL of blood was collected; it was placed in a 4°C refrigerator overnight, centrifuged at 12000 rpm for 1 min, and serum was collected. ELISA test was performed to test the immune effect, and 10-fold dilution was used to verify the neutralization effect of the serum. The results are shown in Table 4. After the serum of the first three mice was diluted 768,000 times, its OD 450nm > cut-off value and P / N≥2; After the serum of the fourth mouse was diluted 576000 times, its OD 450nm >cut-off value and P / N≥2; the results of virus microneutralization test showed that the serum still had obvious neutralizing effect after being diluted 512 times.

[0086] Table 4 Mouse serum antibody titer detection

[0087]

[0088] 2. Flow cytometry sorting of mouse antibody secreting cells and memory B cells

[0089] (I) Obtaining B lymphocytes from mouse lymph nodes

[0090] (1) Take mice immunized with PEDV S1 protein, kill them by short neck, and immediately take out lymph nodes and grind them in a cell sieve, which is pre-infiltrated with 0.5% FBS;

[0091] (2) After grinding, the cell suspension was centrifuged to remove the supernatant and centrifuged at 500 g for 5 min at 4°C;

[0092] (3) Add 3 to 5 times the volume of cell lysate, gently pipette to mix, lyse at room temperature for 1 to 2 min, centrifuge at 4°C, 500 g for 5 min, and discard the red supernatant;

[0093] (4) Add an appropriate amount of 0.5% FBS, resuspend the precipitate, centrifuge at 400-500 g for 2-3 min, and discard the supernatant;

[0094] (5) Repeat once, washing twice in total (the amount of 0.5% FBS used should be at least 5 times the volume of the cell pellet).

[0095] (II) Mouse-specific B cell sorting

[0096] First, prepare the cell lysis solution: 1 μL Recombinant RNase inhibitor plus 19 μL 0.2% TritonX-100; add the following solution to each well of the 96-well plate: 2 μL cell lysis solution; 1 μL 3'-RACE CDSPrimer A; 1 μL dNTP mix (10 mM each); 4 μL / well, pre-cool on ice; use a flow cytometer (BD FACSAriaIII flow sorter) to sort specific B cells into a 96-well plate and collect CD138 - CD93 - CD38 low IgD - CD45R + GL7 + PEDVS1 + Positive cells were sorted into 96-well PCR Multiplate TM 96-Well PCR Plates, set each well to sort one cell; the markers used for flow cytometry sorting are shown in Table 5 below.

[0097] Table 5 Markers used for flow cytometry

[0098] Molecular Marker Cellular Expression GL7 Activated B cells, activated T cells CD45R B cells, activated T cells, activated NK cells IgD B cells CD138 Plasma, pre-B cells, epithelial cells, neural cells CD38 B, T and NK cells, germinal center B and plasma cells are downregulated CD93 Immature B, HSC and progenitor cells

[0099] Then, lymphocytes were circled, negatively selected with CD93 and CD138 to remove epithelial cells, neural cells, pre-B cells, and immature B cells, negatively selected with CD38 and IgD to remove T cells, NK cells, etc., and then positively selected with GL7, CD45R, and Strep tag antibodies to select PEDV-specific memory B cells; Figure 6 As shown, a total of 192 wells of positive cells were sorted; the details are as follows:

[0100] (1) Resuspend the cells obtained in the above steps with 1 mL of FACS buffer (FACS buffer is a PBS solution with 0.5% FBS added); transfer the resuspended cells to the imported 1.5 mL EP tube, centrifuge at 4°C and 400 g for 10 min, and discard the supernatant (about 10 cells after cell counting). 7 / ml level); Cell division: resuspend with the liquid at the bottom of the tube, take out 4μL, add 400μL FACS buffer, mix well and divide evenly into 8 EP tubes, 50μL per tube, 7 single positive tubes and 1 negative tube, and the rest of the cells are used as sample tubes (resuspend the sample tube with 400μL FACS buffer, generally the total number of cells is 10 7 If the total number of cells is large, the volume needs to be increased, otherwise there will be too little antigen to label all cells).

[0101] (2) Antigen staining: The initial concentration of biotin-labeled PEDV S1 protein is 4 μg / μL. Sample tube staining: Add 1.44 μL of biotin-labeled PEDV S1 protein to every 200 μL and incubate at 4°C in the dark for 30 min. BV711 single positive tube: Add 0.36 μL of biotin-labeled PEDV S1 protein and incubate at 4°C in the dark for 30 min; add 1 mL of FACS buffer, mix well, and centrifuge at 4°C to precipitate the cells. Add another 1 mL of FACS buffer, mix well, and centrifuge at 4°C to precipitate the cells;

[0102] (3) Secondary antibody staining: Sample tube staining: 200 μL of antibody system: FITC-GL7 (2 μL); PE-CD138 (4 μL); PE / CY7-CD38 (4 μL); APC-CD93 (4 μL); BV421-CD45R (16 μL); BV510-IgD (2 μL); BV711 (4 μL); incubate at 4°C in the dark for 30 min; Single positive tube: add 0.2 μL of the corresponding antibody to each EP tube; incubate at 4°C in the dark for 30 min; add 1 ml of FACS buffer, mix well, and centrifuge at 4°C to precipitate cells; add 1 mL of FACS buffer, mix well, and centrifuge at 4°C to precipitate cells. Add 2 mL of FCAS buffer, suspend the cells, filter with a 0.45 μm filter, and transfer to a flow tube.

[0103] 3. Antibody Screening

[0104] In the following experiments, the sequence of S-A9 is shown in Table 6 below.

[0105] Table 6S-A9 antibody sequence

[0106]

[0107]

[0108] (I) Single-cell PCR amplification of antibody variable region genes

[0109] The present invention sorted out 192 positive cells, and amplified the light and heavy chain variable region fragments of the antibody by nested PCR. Gene linkers were added to the two segments of the fragment. The variable region fragment was about 400 bp long. A total of 114 heavy chains and 176 light chains were amplified. The heavy chain PCR amplification rate was 54.5%, and the light chain amplification rate was 73.7% (such as Figure 7 ); specifically as follows:

[0110] (1) RT (reverse transcription)

[0111] The sorted 96-well plate was centrifuged at 72°C for 3 min and 42°C for 2 min. RT Mix was prepared according to the results of the test positive cell wells and then dispensed into each well of each 96-well PCR plate. The RT Mix system is shown in Table 7 below.

[0112] Table 7 RT Mix system

[0113]

[0114] Then, the RT Mix was added to the 96-well plate, mixed, shaken and centrifuged, and operated in a PCR instrument according to the following conditions (Table 8).

[0115] Table 8 RT-PCR reaction procedure

[0116]

[0117] (2) Amplification of ds cDNA

[0118] The above PCR products were added with reagents according to the following method to amplify ds cDNA. The cDNA PCR reaction system and reaction program are shown in Tables 9 and 10 below, respectively.

[0119] Table 9ds cDNA PCR reaction system

[0120] Element Volume (μL) <![CDATA[H 2 The]]> 27.5 5×Phusion HF Buffer 10 dNTP mix (10mM each) 1 5′PCR primer II A 1 ss cDNA 10 Phusion DNA Polymerase(2U / μL) 0.5

[0121] 5'PCR Primer II A (12 μM) or Nested Universal Primer A (NUP; 10 μM)

[0122] Table 10ds cDNA reaction program

[0123]

[0124] (3) Nested PCR amplification of antibody light and heavy chain genes

[0125] 1) Nested PCRa

[0126] The above RT-PCR products were subjected to three rounds of nested PCR. The first round of PCR (PCRa) was performed first. The primers of PCRa were divided into the following types: a. antibody heavy chain upstream primers (1hVH1-6, 6 in total), antibody heavy chain downstream primers (1hCH); b. antibody light chain upstream primers 1hVk1-4 (4 in total), antibody light chain downstream primers (1hCk); the heavy chain primers and light chain primers of this round can be mixed and added separately; the PCRa heavy chain and light chain amplification systems and amplification procedures are shown in Tables 11, 12 and 13, respectively.

[0127] Table 11PCRa heavy chain amplification system

[0128] Element Volume (μL) 1hVH(1-6)(100μm) 0.6 1hCH 0.1 dNTP 1 HotstarTaq 0.4 Q(5×) 4 Buffer (10×) 2 cDNA 2 <![CDATA[25mM MgCL 2 ]]> 1.2 <![CDATA[H 2 The]]> 8.7 Total volume 20

[0129] Table 12PCRa light chain amplification system

[0130]

[0131]

[0132] Table 13 Heavy chain amplification and light chain amplification PCR amplification reaction program

[0133]

[0134] 2) Nested PCR

[0135] The above PCRa product is used as a template to perform PCRb. In this step, the light and heavy chain PCR primers are not mixed, and each primer is used for separate PCR. Compared with the light chain, the heavy chain PCR positive rate is lower. According to preliminary test experience, the heavy chain uses the PCRa product as a template and uses the VH upstream primer to perform PCRb. The primers used are divided into the following categories: a. antibody heavy chain upstream primer (2hVH1-5), antibody heavy chain downstream primer (2hCH); b. antibody light chain upstream primer (2hVK1-4), antibody light chain downstream primer (2hCK); PCRa heavy chain, light chain amplification system and amplification program are shown in Table 14, Table 15 and Table 16, respectively.

[0136] Table 14 Antibody heavy chain PCR b amplification system

[0137] Element Volume (μL) 2hVH1 / 5(10μm) 1 2hCH(10μm) 1 dNTP 1 HotstarTaq 0.4 Q(5×) 4 Buffer (10×) 2 PCRa products 2 <![CDATA[25mM MgCl 2 ]]> 1.2 <![CDATA[H 2 The]]> 7.4 Total volume 20

[0138] Table 15 Antibody light chain amplification system

[0139]

[0140]

[0141] Table 16 Reaction conditions for antibody heavy chain and light chain PCRb

[0142]

[0143] 3) Construction of antibody light and heavy chain eukaryotic expression plasmids

[0144] The amplified antibody light and heavy chain PCRb products were subjected to agarose electrophoresis, the gel was cut, and the PCRb light and heavy chain positive fragments were recovered using a universal gel recovery kit. The recovered antibody light and heavy chain fragments were homologously recombined into pCAGGS-IgK and pCAGGS-IgH vectors, respectively, and transformed into DH5α competent cells. After plating, a single colony was picked for identification and sent for sequencing; the sequencing results of the amplified antibody light and heavy chain variable region genes were compared on NCBI Ig Blast to analyze the gene pedigree and mutation rate of the antibody, and then the antibody light and heavy chain recombinant bacteria with correct sequencing results were extracted using an endotoxin-free plasmid extraction kit.

[0145] (II) Western Blot method to detect the expression of PEDV antibodies

[0146] One day before the experiment, HEK-293T cells were plated on a 6-well cell plate. When the cells were fully grown on the next day, each well was transfected with a vector plasmid containing antibody light and heavy chain genes through PEI. Each well of the 6-well plate was transfected with 2μg of light and heavy chain plasmids, and the transfection ratio of light and heavy chain plasmids was 3:2, that is, 1.2μg and 0.8μg of light and heavy chains were transfected in each well. The cell supernatant was collected once 3 days after transfection and once after 7 days; 6% SDS-PAGE reducing gel was prepared, and the antibody supernatant to be expressed was spotted, and the electrophoresis instrument was kept at a constant voltage of 150V for 1h; after cutting the NC membrane of appropriate size and soaking it in the transfer buffer for 5min, the gel was tightly attached to the NC membrane, and 200mA was continued for 1h 20min. 5% skim milk powder was blocked for 1h, the blocking solution was discarded, and PBST was washed twice, each time for 5min. Add 40 ml of diluted goat anti-human IgG secondary antibody (1:3000 diluted with PBST) and incubate at room temperature for 1 hour. Discard the secondary antibody, wash 3 times with PBST, develop with Western Blot colorimetric solution, and photograph.

[0147] The results are as follows Figure 8 As shown, most of the antibodies transfected into HEK-293T cells were expressed. According to the comparison results of the antibody light and heavy chain sequence information returned by the sequencing company, 66 different antibodies were screened and expressed from 100 pairs of paired antibody light and heavy chains, with an antibody expression rate of 66%.

[0148] (III) Detection of PEDV antibody binding using ELISA

[0149] The specific binding activity of the cell supernatant to the antigen was determined by ELISA test, and antibodies with specific antigen binding activity were preliminarily screened. Binding ability was determined by using 1μg / mL PEDV S1 protein as ELISA coated antigen and goat anti-human HRP as secondary antibody to perform ELISA binding test on the purified antibody. For antibodies with binding activity, HEK-293F cells were transfected with PEI for large-scale expression. After counting the HEK-293F cells, they were diluted to 3 units and placed on a shaker for transfection. 1mL HEK-293F cells were transfected with 1μg of plasmid. Transfection reagent was prepared, and the plasmid concentration used for transfection was 1mg / mL, and PEI was 3 to 4 times the plasmid concentration. Transfection reagent volume: 50mL transfection reagent / L (too small volume affects transfection efficiency). Mix 25mL of HEPES / 150mM NaCl with plasmid / PEI respectively, let it stand for 5 minutes, then slowly add PEI to the plasmid and let it stand for 15 minutes after mixing. Do not exceed 30 minutes, otherwise precipitation will easily occur. To transfect HEK-293F cells, add the mixed PEI and plasmid to the culture medium and place it in a CO2-free medium. 2 Culture in an incubator (with or without CO 2 The feed solution was added 24 hours after transfection and every two days thereafter.

[0150] The results are as follows Fig. 9 As shown, among the 66 pairs of antibodies expressing different light and heavy chains, 21 pairs of positive clones had the ability to bind to PEDV-S1 with a binding rate of 31.8%. The specific antibody names were named S-G1, S-B3, S-H3, S-B5, S-C5, S-B6, S-D6, S-B7-3, S-D7-3, S-D8, S-H8, S-D9, S-D12, S-E12, S-H12, S-G4-1, S-G4-3, S-A9, S-F9 and S-B11.

[0151] (IV) Determine the antibody recognition epitope based on indirect immunofluorescence test

[0152] The 21 PEDV antibodies were used as primary antibodies to perform indirect immunofluorescence tests, including: placing an appropriate amount of Vero cells in a 24-well plate, and then infecting PEDV after the cells adhered to the wall, and setting up a negative control. 37°C, 5% CO 2 After 24 hours of culture in the incubator, the cells were harvested, the culture medium was discarded, and the cells were washed twice with PBS. 400 μL of 4% paraformaldehyde was added to each well and fixed at room temperature for 15 minutes. The paraformaldehyde was discarded and 1 mL of pre-cooled (100% methanol should be placed in a -20°C refrigerator 20 minutes in advance) methanol was immediately added for permeabilization at room temperature for 10 minutes. Washed three times with PBS, 5% BSA was added, and the cells were blocked at room temperature for at least 45 minutes (or overnight at 4°C). The blocking solution was discarded, and 300 μL of the diluted PEDV antibody (1:200 diluted with 5% BSA) was added to each well, and incubated at room temperature for 1 hour (or overnight at 4°C). The primary antibody was recovered, washed three times with PBS, and 300 μL of the diluted 488 or 594 goat anti-human IgG secondary antibody (1:300 diluted with PBS) was added to each well, and incubated in the dark for 1 hour. Discard the secondary antibody, add 500 μL of diluted DAPI dye (1:5000 diluted with PBS) to each well, and protect from light for 15 minutes (DAPI concentration is 1 μg / mL). Discard DAPI, wash three times with PBS, and finally add less PBS to observe the cells with a fluorescence microscope.

[0153] The results show that Fig.10 ), 12 antibodies, S-B3, S-H3, S-B5, S-D6, S-D7-3, S-H8, S-D9, S-E12, S-G4-1, S-A9, S-F9, and S-F10, cross-reacted with PEDV, while 9 antibodies, S-G1, S-C5, S-B6, S-B7-3, S-D8, S-D12, S-H12, S-G4-3, and S-B11, could not react with the virus.

[0154] (V) Preparation of PEDV-specific antibodies

[0155] Preparation of PEDV antibody using HEK-293F mammalian cell expression system:

[0156] (1) Count HEK-293F cells and calculate cell density and cell viability; the cell count was controlled to 2.0×10 6 The cell culture flask was placed at 37°C and 5% CO 2 , cultured in a constant temperature shaker at 150-175 rpm; counted after 12 h, the cell concentration was between 2.5 and 3.0 × 10 6 Transfection was performed at 100 μg / mL;

[0157] (2) Preparation of transfection solution: dilute 400 μg DNA (PEDV antibody light and heavy chain plasmid transfection ratio is 3:2, i.e. 240 μg light chain plasmid transfection, 160 ug heavy chain plasmid transfection) with 150 mM NaCl to a total volume of 10 mL, and mix gently; dilute 2 mL Sinofection transfection reagent with 150 mM NaCl to a total volume of 10 mL, and mix gently;

[0158] (3) The diluted DNA and transfection reagent were allowed to stand for about 5 minutes and then gently mixed to a total volume of 20 mL. The total volume was then allowed to stand at room temperature for 20 minutes. The transfection solution was added dropwise to the cell culture medium. While adding, the culture bottle was gently shaken. After shaking, the culture bottle was returned to the shaker for continued culture. The bottle mouth was tied tightly (i.e., no more CO was introduced into the cell bottle). 2 );

[0159] (4) 24 hours after transfection, loosen the bottle cap to ensure the dissolved oxygen and CO for subsequent high-density cell growth. 2 Emission requirements to prevent CO 2 The accumulation caused the pH value of the culture medium to be too low (the culture medium was yellow), which affected cell growth; 14 mL of SMS293-SUPI feed solution was added 24 hours after transfection, and thereafter, feed solution (14 mL) was added every 48 hours, and samples were collected 7 days after transfection.

[0160] (VI) PEDV antibody purification

[0161] (1) Centrifuge at 8000 rpm at 4°C for 30-60 min in a vertical high-speed low-temperature centrifuge, and filter the supernatant with a 0.22 μm filter membrane to remove cell debris;

[0162] (2) Protein A column: First, filter deionized water with a 0.22 μm filter membrane, quickly flush the peristaltic pump, adjust the flow rate to 2 mL / min, and hang the Protein A column. Pass buffer A (20 mM Na 3 PO 4 ; pH 7.0) 15 mL (i.e., 3 column volumes), and the protein supernatant after suction filtration;

[0163] (3) Operate on the machine, quickly flush the A and B pumps of the Akta instrument with water, pause the instrument, replace the A and B solutions used for purifying Protein A column, first flush the B pump with 100% B solution (0.1M Glycin-HCl; pH 3.0), and then flush the A pump with 0% B solution (i.e. 100% A solution). Adjust the flow rate to 2mL / min, apply the column, pass A solution, until the UV value is flat, elute with 100% B solution, set peak collection, 3.2mL per tube, and add 0.8mL 1M Tris (pH 9.0) at the bottom of the collection tube to neutralize the acidity of B solution;

[0164] (4) Use PBS to replace the purified antibody with a 100kDa concentrator tube for 3 times. After the antibody concentration is determined, dilute it to 1mg / mL for later use. Prepare 6% SDS-PAGE reducing gel, take 20μL of antibody, add 5μL 5×SDS-PAGE loading buffer (add 0.1M DTT), and treat the sample at 100℃ for 5min. Take 7μL of sample, 150V, 50min; transfer, 200mA, 1h20min; block with 5% skim milk for 1h, wash 3 times with PBST, add goat anti-human HRP secondary antibody (1:3000 dilution), incubate at room temperature for 1h, discard the secondary antibody, wash 3 times with PBST, develop with Western Blot colorimetric solution, and photograph.

[0165] The PEDV S-A9 antibody was reduced to a heavy chain of 50 kDa and a light chain of 25 kDa (eg, Fig.11 As shown); by protein concentration determination, the concentration of the purified antibody expressed by 400 mL of HEK-293F cells was 2.1 mg / mL, with a total of 16 mg of protein.

[0166] (VII) Neutralization test identification

[0167] Resuscitated Vero cells and passaged to 15 cm 2 After the cells are fully spread, wash the cell plate with PBS three times, take 100 μL of PEDV CH-HB2-2018 virus seed and dilute it into 20 mL DMEM, add it to the cell plate, and incubate at 37°C and 5% CO 2 After incubation in the incubator for 1 hour, wash with PBS three times. Add 10μg / mL trypsin in DMEM maintenance solution, collect samples after 48 hours, freeze and thaw repeatedly three times in a -80℃ refrigerator, and collect viruses. Virus TCID 50 For the assay, place an appropriate amount of Vero cells in a 96-well plate, wait until the cells are fully grown, dilute the virus 10-fold in an EP tube, wash the 96-well plate cells three times, and add the diluted virus solution to the 96-well plate cells in sequence. Observe the cytopathic effect after 48 hours and calculate the TCID of the virus 50 . Inoculate an appropriate amount of Vero cells in a 96-well plate and wait for the cells to grow completely. Add purified PEDV antibodies and virus solution (200 TCID 50 ) and mix well, 37°C, 5% CO 2 Incubate in the incubator for 1 hour. Wash the 96-well plate cells with PBS three times, add the antibody-virus mixture to the 96-well plate, and incubate at 37°C and 5% CO 2 Incubate in the incubator for 1 hour. After washing with PBS three times, add 10 μg / mL pancreatin in DMEM and observe the neutralization results after 48 hours. Fig.12 As shown in the figure, 7 antibodies, including S-B3, S-D6, S-D7-3, S-D9, S-A9, S-F9, and S-F10, can neutralize PEDV, among which S-A9 has the highest neutralization titer, IC 50 Up to 0.03286μg / mL.

[0168] IV. Antibody Validation

[0169] (I) Specificity identification of antibodies

[0170] PEDV, PDCoV, and TGEV stored in the laboratory were used as antigens to coat 96-well plates. The antigen coated in each well was 200 ng. PBS was used as a control. After blocking with 10% skim milk powder for 1 hour, 500 ng of purified PEDV-specific antibody (S-A9) was added as the primary antibody. The plates were incubated at room temperature for 30 minutes. After washing three times, the plates were incubated with goat anti-mouse HRP-labeled secondary antibodies at room temperature for 30 minutes. After washing three times, TMB colorimetric solution was added. After incubation in dark for 30 minutes, OD was read using an enzyme immunochromatography instrument. 450 nm numerical value; experimental results show that ( Fig.13 ), the S-A9 antibody was able to specifically bind to purified PEDV and had no cross-reaction with PDCoV and TGEV.

[0171] (II) Affinity determination of PEDV S1 and its antibodies

[0172] The binding kinetics of PEDV S1 and S-A9 antibody were analyzed by surface plasmon resonance (SPR), as follows:

[0173] (1) The two proteins to be studied for interaction were replaced with the HEPES buffer used in the SPR experiment (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v / v surfactant P20). The HEPES buffer needed to be ultrasonically degassed before the experiment.

[0174] (2) The concentrations of the stationary phase and mobile phase were set to 10 μg / mL and 1-100 μM, respectively;

[0175] (3) The stationary phase is coupled to one channel in the chip, and bovine serum albumin (BSA) is coupled to another channel as a background control. In some cases, a blank channel can also be used as a background control. When coupling proteins, the machine flow rate is generally set to 5 μL / min.

[0176] (4) Estimate the affinity range of the research object, which can generally be divided into nanomolar and micromolar levels. Binding experiments can be performed using 1nM and 1μM mobile phase proteins to determine the affinity range;

[0177] (5) Within the affinity range obtained in the previous step, design a series of concentration gradients, dilute the mobile phase protein in multiples, and conduct a complete on-machine binding test; we generally select the KINJECT mode for kinetic parameter determination, the machine flow rate is 30 μL / min, the sample injection binding time is generally 1 to 2 minutes (the required time is determined based on whether the binding curve is saturated), and the dissociation curve acquisition time is generally 1 to 5 minutes (the specific time setting is adjusted based on the speed of dissociation);

[0178] (6) Use BIAevaluation software to analyze the kinetic parameters of the research subjects and draw graphs.

[0179] SPR results are as follows Fig.14 As shown, the results show that PEDV S1 and S-A9 antibody have a very high affinity, which is close to the order of magnitude of the affinity of the antigen and antibody, and is a relatively high affinity.

[0180] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. An antibody or antigen-binding fragment against porcine epidemic diarrhea virus (PEDV), the antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, the amino acid sequence of HCDR1 being shown in SEQ ID NO: 1, the amino acid sequence of HCDR2 being shown in SEQ ID NO: 2, and the amino acid sequence of HCDR3 being shown in SEQ ID NO: 3; the light chain variable region comprising LCDR1, LCDR2 and LCDR3, the amino acid sequence of LCDR1 being shown in SEQ ID NO: 10, the amino acid sequence of LCDR2 being shown in SEQ ID NO: 11, and the amino acid sequence of LCDR3 being shown in SEQ ID NO:

12.

2. The antibody or antigen-binding fragment according to claim 1, characterized in that: The heavy chain variable region also includes: NO:4 has at least 80% identity with the sequence shown in HFR1 and / or has at least 80% identity with the sequence shown in SEQ ID NO:5 HFR2 having at least 80% identity to the sequence shown in SEQ ID NO: 6 and / or HFR3 having at least 80% identity to the sequence shown in SEQ ID NO: 6 and / or ID NO: 7 has at least 80% identity with the sequence shown in HFR4; and / or the light chain variable region also includes: LFR1 having at least 80% identity with the sequence shown in SEQ ID NO:13 and / or LFR2 having at least 80% identity with the sequence shown in SEQ ID NO:14 and / or LFR3 having at least 80% identity with the sequence shown in SEQ ID NO:15 and / or LFR4 having at least 80% identity with the sequence shown in SEQ ID NO:

16. The sequence shown in ID NO: 16 has at least 80% identity to LFR4.

3. The antibody or antigen-binding fragment according to claim 1, characterized in that: The antibody comprises a heavy chain variable region that is at least 70% identical to the sequence shown in SEQ ID NO:8 and a light chain variable region that is at least 70% identical to the sequence shown in SEQ ID NO:

17.

4. The antibody or antigen-binding fragment according to any one of claims 1 to 3, characterized in that: The antibody or antigen-binding fragment further comprises a heavy chain constant region and / or a light chain constant region, and the heavy chain constant region and the light chain constant region are both derived from human or mouse IgG antibodies or mutants thereof.

5. The antibody or antigen-binding fragment according to claim 4, characterized in that: The heavy chain constant region comprises SEQ ID The sequence shown in NO:9 has at least 70% identity, and the light chain constant region comprises a sequence having at least 70% identity with the sequence shown in SEQ ID NO:

18.

6. Any of the following substances: (i) a polynucleotide molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment of any one of claims 1 to 5; (ii) an expression vector comprising the polynucleotide molecule described in (i); (iii) an engineered bacterium, comprising the expression vector in (ii); (iv) a host cell, comprising the expression vector in (ii); (v) A product for detecting porcine epidemic diarrhea virus, comprising the antibody or antigen-binding fragment according to any one of claims 1 to 5, or the polynucleotide molecule in (i), or the expression vector in (ii), or the engineered bacteria in (iii), or the engineered cells in (iv); (vi) a pharmaceutical composition comprising the antibody and / or antigen-binding fragment according to any one of claims 1 to 7 and / or the polynucleotide molecule in (i) or the expression vector in (ii) and / or the engineered bacteria in (iii) and / or the engineered cells in (iv); (vii) a pharmaceutical preparation comprising the antibody and / or antigen-binding fragment according to any one of claims 1 to 7 and / or the polynucleotide molecule in (i) and / or the expression vector in (ii) and / or the engineered bacteria in (iii) and / or the engineered cells in (iv) and / or the pharmaceutical composition in (vi).

7. The substance according to claim 6, characterized in that: The pharmaceutical composition is a bispecific antibody, a polyspecific antibody, an ADC or a fusion protein.

8. Use of the antibody and / or antigen-binding fragment according to any one of claims 1 to 5 and / or the polynucleotide molecule in (i) and / or the expression vector in (ii) and / or the engineered bacteria in (iii) and / or the engineered cells in (iv) in the preparation of a product for diagnosing porcine epidemic diarrhea virus infection.

9. Use of the antibody and / or antigen-binding fragment according to any one of claims 1 to 5 and / or the polynucleotide molecule in (i) and / or the expression vector in (ii) and / or the engineered bacteria in (iii) and / or the engineered cells in (iv) in the preparation of a medicament for preventing or treating porcine epidemic diarrhea virus infection.

10. The use according to claim 8 or 9, characterized in that: The disease caused by porcine epidemic diarrhea virus infection is porcine epidemic diarrhea.

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