Antibodies or antigen-binding fragments against porcine epidemic diarrhea virus and uses thereof

Abstract

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

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to an antibody or antigen binding fragment for porcine epidemic diarrhea virus and application thereof. BACKGROUND

[0002] Porcine epidemic diarrhea (PED) is an acute and contagious enteric disease of pigs caused by porcine epidemic diarrhea virus (PEDV), and the clinical symptoms are watery diarrhea, vomiting, dehydration, metabolic acidosis and high mortality. Pigs of all ages and breeds are susceptible, but there are great differences in clinical symptoms and mortality after infection in pigs of different ages. The morbidity and mortality are inversely proportional to the age of pigs, and the morbidity and mortality gradually decrease with the increase of the age of pigs. The disease is particularly harmful to nursing piglets, and the morbidity is as high as 80% to 100%, and the mortality of newborn piglets is more than 90%.

[0003] Diseased pigs and virus-carrying pigs are the main sources of transmission and spread of the disease, and the main transmission routes are oral and digestive tract transmission, direct transmission or indirect infection of the virus through direct contact or fecal-oral contact of healthy pigs and diseased pigs, and semen and emulsion transmission is a potential route. There is also a high content of virus in the respiratory tract secretions discharged by diseased pigs, and air transmission is also one of the influencing factors in breeding farms with repeated outbreaks and poor ventilation facilities. Therefore, it is necessary to develop a product for preventing, diagnosing or treating diseases related to porcine epidemic diarrhea virus infection.

[0004] Antibody is a protein that can specifically recognize antigen, and it can recognize and neutralize viruses by specifically recognizing viral envelope proteins, structural proteins or enzymes, and it is one of the technical means for diseases related to viruses. Therefore, it is of great significance to develop an antibody against porcine epidemic diarrhea virus for preventing, diagnosing or treating diseases related to porcine epidemic diarrhea virus infection. SUMMARY

[0005] The application is to screen an antibody or antigen binding fragment against porcine epidemic diarrhea virus (PEDV), which has excellent specificity and affinity for PEDV S1 protein.

[0006] In order to achieve the above purpose, the application can adopt the following technical scheme:

[0007] In one aspect, the present application 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 being shown as SEQ ID NO: 1, the amino acid sequence of HCDR2 being shown as SEQ ID NO: 2, and the amino acid sequence of HCDR3 being shown as SEQ ID NO: 3; the light chain variable region comprising LCDR1, LCDR2 and LCDR3, the amino acid sequence of LCDR1 being shown as SEQ ID NO: 10, the amino acid sequence of LCDR2 being shown as SEQ ID NO: 11, and the amino acid sequence of LCDR3 being shown as SEQ ID NO: 12.

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

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

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

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

[0012] In another aspect, the present application also provides any one of the following:

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

[0014] (ii) an expression vector comprising the polynucleotide molecule 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 in the present application or the polynucleotide molecule in (i) or the expression vector in (ii) or the engineered bacterium in (iii) or the engineered cell in (iv);

[0018] (vi) a pharmaceutical composition, comprising the antibody and / or antigen-binding fragment in the present application and / or the polynucleotide molecule in (i) or the expression vector in (ii) or the engineered bacterium in (iii) or the engineered cell in (iv);

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

[0020] Preferably, the pharmaceutical composition in the above-mentioned substances is a diabody, a multibody, an ADC or a fusion protein.

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

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

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

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

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

[0026] Figure 2 Purification of PEDV S1 protein by HisTrap Excel column

[0027] Figure 3 Purification of PEDV S1 protein by Superdex 200 10 / 300 gel filtration column

[0028] Figure 4 SDS-PAGE protein identification gel staining by Coomassie blue

[0029] Figure 5 Western Blot identification of purified PEDV S1 protein

[0030] Figure 6 Flow cytometry sorting of specific B cells

[0031] Figure 7 Nested PCR amplification of light and heavy chain variable region fragments of antibodies

[0032] Figure 8 Western Blot method for detecting the expression of PEDV antibodies

[0033] Figure 9 ELISA method for detecting the binding of PEDV antibodies

[0034] Figure 10 Indirect immunofluorescence test to determine the recognition epitope of the antibody

[0035] Figure 11 Purification of PEDV S-A9 antibody

[0036] Figure 12 Results of different antibody micro-neutralization test

[0037] Figure 13 Specificity identification of PEDV S-A9 antibody

[0038] Figure 14 Affinity of PEDV S-A9 antibody to PEDV S1 protein DETAILED DESCRIPTION

[0039] The examples are given to better illustrate the present application, but are not intended to limit the present application to only the examples. Therefore, those skilled in the art can make non-essential improvements and adjustments to the embodiments according to the above disclosure, which still belong to the protection scope of the present application.

[0040] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. As used herein, including in the appended claims: "or" as used herein, including the conjunction "or," as used herein, is used in the inclusive sense and not the exclusive sense. That is, unless the context clearly indicates otherwise, the word "or" means "and / or." As used herein, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. As used herein, the term "includes" and / or "including" shall mean, and are used herein in the sense of "including but not limited to." As used herein, the term "consisting essentially of shall mean, and is used herein in the sense of "including normal variances and / or variations that can be expected by one of ordinary skill in the art in the field of the disclosure, such as changes that do not significantly affect the nature of the sequence being described herein." As used herein, the term "consisting of shall mean, and is used herein in the sense of "including the exact components listed and nothing else." As used herein, the term "and / or" shall mean, and is used herein in the sense of "and" or "or," depending on the context.

[0041] In the present application, the term "antigen binding fragment" refers to an antigen binding fragment of an antibody and an antibody analog, which generally includes at least part of the antigen binding region or variable region of the parent antibody, such as one or more CDRs; the fragment of the antibody retains at least certain binding specificity of the parent antibody.

[0042] The embodiment of the present application 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 being as shown in SEQ ID NO:1, the amino acid sequence of HCDR2 being as shown in SEQ ID NO:2, and the amino acid sequence of HCDR3 being as shown in SEQ ID NO:3; the light chain variable region comprising LCDR1, LCDR2 and LCDR3, the amino acid sequence of LCDR1 being as shown in SEQ ID NO:10, the amino acid sequence of LCDR2 being as shown in SEQ ID NO:11, and the amino acid sequence of LCDR3 being as shown in SEQ ID NO:12.

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

[0044] In some specific examples, the heavy chain variable region further comprises HFR1 having at least 80% identity to the sequence shown in SEQ ID NO: 4 and / or HFR2 having at least 80% identity to the sequence shown in SEQ ID NO: 5 and / or HFR3 having at least 80% identity to the sequence shown in SEQ ID NO: 6 and / or HFR4 having at least 80% identity to the sequence shown in SEQ ID NO: 7; and / or the light chain variable region further comprises LFR1 having at least 80% identity to the sequence shown in SEQ ID NO: 13 and / or LFR2 having at least 80% identity to the sequence shown in SEQ ID NO: 14 and / or LFR3 having at least 80% identity to the sequence shown in SEQ ID NO: 15 and / or LFR4 having at least 80% identity to 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 antibody is a framework region, which is used to connect the CDR region and is relatively stable; in addition, the sequence of the FR region of the heavy chain and the light chain in the above-mentioned antibody and the sequence of the CDR region can be arranged in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, respectively, to form the heavy chain variable region and the light chain variable region of the above-mentioned two antibodies; in addition, the at least 80% identity in the present application includes ≥80%, ≥85%, ≥90%, ≥95% or 100% identity, etc.

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

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

[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 from a human or murine IgG antibody or a mutant thereof.

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

[0050] It should be noted that the antibody or antigen binding fragment in the present application further comprises a heavy chain constant region and a light chain constant region in addition to the above-mentioned heavy chain variable region and light chain variable region, and the heavy chain constant region and the light chain constant region can be of human origin or other animal origin (such as rabbit origin, pig origin, etc.), and are almost regions that do not mutate. In addition, the heavy chain and the 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 described above, the above-mentioned at least 70% identity refers to an identity of ≥ 70%, such as 75%, 80%, 85%, 90%, 95% or 100%; of course, it should be understood that the sequence with at least 70% identity has a similar function to the above-mentioned sequence.

[0052] The embodiments of the present application also provide any one of the following substances:

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

[0054] (ii) an expression vector comprising the polynucleotide molecule in (i); specifically, the expression vector in the present application 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 group: human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), visna-maedivirus (VMV) virus, 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) an engineered bacterium comprising the expression vector in (ii); specifically, the engineered bacterium in the present application refers to a bacterium that can assist the expression of the above-mentioned expression vector, such as Escherichia coli, and the Escherichia coli comprising the expression vector herein is the Escherichia coli into which the expression vector is transferred;

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

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

[0058] (vi) a pharmaceutical composition, comprising the antibody and / or antigen-binding fragment of the present application or the polynucleotide molecule of (i) or the expression vector of (ii) or the engineered bacteria of (iii) or the engineered cell of (iv); in particular, as described above, based on the strong binding of the antibody or antigen-binding fragment of the present application with 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 here refers to a pharmaceutical composition in which the antibody or antigen-binding fragment of the present application is combined with other small molecule compounds, polypeptides, antibodies or proteins that can be effectively used for preventing or treating porcine epidemic diarrhea virus infection; for example, a bi- or multi-antibody combined with other antibodies, an ADC combined with a small molecule compound, a fusion protein combined with other proteins; the selection and use can be made according to specific conditions.

[0059] (vii) a pharmaceutical preparation, comprising the antibody and / or antigen-binding fragment of the present application or the polynucleotide molecule of (i) or the expression vector of (ii) or the engineered bacteria of (iii) or the engineered cell of (iv) or the pharmaceutical composition of (vi); in particular, the above-mentioned antibody and / or antigen-binding fragment, etc. and the above-mentioned pharmaceutical composition can be added to a pharmaceutically acceptable carrier to prepare different dosage forms for different clinical needs; the dosage forms can include sprays, oral liquids, tablets, atomizing agents, granules, capsules or ointments; of course, according to different dosage forms, the selection of the pharmaceutically acceptable carrier is different, and the preparation can be made according to the methods known in the art.

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

[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 disease, that is, the product for detecting porcine epidemic diarrhea virus in the present application is also applicable to the diagnosis of porcine epidemic diarrhea virus infection disease, 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 present application also provides a use of the antibody and / or antigen binding fragment in the present application 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 cell in (iv) in the preparation of a medicine for preventing or treating porcine epidemic diarrhea virus infection disease.

[0063] In order to better understand the present application, the content of the present application will be further illustrated below in combination with specific examples, but the content of the present application is not limited to the following examples only.

[0064] I. Mouse immunization

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

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

[0067] 1) Primer design: According to the PEDV CH-HB2-2018 strain (GenBank No. MK606369) published on NCBI as a template, the PEDV S1 gene sequence was codon-optimized, Kozak sequence and human signal peptide were added in N segment, 8×His tag was added in C segment, and EcoR I and Xho I enzyme digestion sites were added in both segments. The gene was synthesized by a company and a pCAGGS-PEDV-S1 eukaryotic expression plasmid was constructed.

[0068] 2) PCR identification of plasmid: pCAGGS universal primers were used for PCR identification of pCAGGS-PEDV-S1 plasmid, and the reaction system and amplification conditions are shown in Table 1 and Table 2.

[0069] Table 1 PCR amplification reaction system

[0070] Component Volume pCAGGS-PEDV-S1 plasmid 10 ng Upstream primer 20 pmol Downstream primer 20 pmol MCLAB enzyme 10 μL 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, as shown in Figure 1As shown, the amplified band size is consistent with the expected target band size. Take 60 ng of the vector, and perform ligation transformation according to the pmol ratio of the vector to the target fragment of 1:3, pick a single colony for sequencing, and perform alignment analysis on the sequencing results. No base mutation, glycerol preservation, and plasmid extraction.

[0074] 3) Plasmid extraction: inoculate the pCAGGS-PEDV S1 clone bacteria liquid into 200 mL of LB liquid medium containing Amp resistance at a ratio of 1:1000, and incubate at 37°C in a constant temperature shaker for 12-16 h. Use an endotoxin-free plasmid maxi kit to extract the plasmid, and improve the experimental steps based on the operation according to the instructions. Mix isopropanol pre-cooled at -80°C for 30 min with the plasmid extract, and place it at -20°C for 1 h to accelerate plasmid precipitation, which can significantly improve the plasmid extraction amount. After determining the plasmid concentration, store it at -20°C.

[0075] (2) Expression of PEDV S1 protein

[0076] Place the HEK-293F cell culture bottle in a constant temperature shaker at 37°C, 5% CO2, and a rotation speed of 150-175 rpm; count after 12 h, and perform transfection when the cell concentration is 2.5-3.0 x 10 6 / mL; dilute 400 g of DNA to a total volume of 10 mL with 150 mM NaCl, and mix gently; dilute 2 mL of Sinofection transfection reagent to a total volume of 10 mL with 150 mM NaCl, and mix gently; let the diluted DNA and transfection reagent stand separately for about 5 min; gently mix the plasmid and transfection reagent to a total volume of 20 mL, and let it stand at room temperature for 20 min. Add the mixture dropwise to the cell culture liquid, and gently shake the culture bottle while adding. After shaking, return the culture bottle to the shaker for continued culture, and tighten the bottle cap (i.e., do not introduce CO2 into the cell bottle anymore). Loosen the bottle cap 24 h after transfection to meet the oxygen dissolution and CO2 discharge requirements for subsequent high-density cell growth, and prevent the accumulation of CO2, which can cause the pH value of the culture liquid to be too low (the culture liquid turns yellow), affecting cell growth. Add 14 mL of SMS293-SUPI feed liquid 24 h after transfection, and add the feed liquid (14 mL) every 48 h thereafter. Collect the sample 7 d after transfection.

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

[0078] Express PEDV S1 recombinant protein using the HEK-293F mammalian cell expression system, and first purify it using a HisTrap Excel column, as shown in Figure 2 . Further purify the PEDV S1 protein using a Superdex 200 10 / 300 gel chromatography column, as shown in Figure 3The purified sample was identified by SDS-PAGE protein identification gel. The expected monomer size of the target protein was 83 kDa, and the size of the aggregate was about 100 kDa. After staining with Coomassie brilliant blue and decolorizing, an obvious protein band was observed at 100 kDa, as shown in Fig. 4. Figure 4 The protein samples 6-9 were concentrated and exchanged by using a 30 kDa protein concentration tube. As shown in Fig. 5, the purified PEDV S1 protein was identified by Western Blot using a mouse Anti-His tag antibody. The His tag was detected at 83 kDa, which was consistent with the expected size of the target protein. Figure 5 The purified PEDV S1 protein was identified by Western Blot using a mouse Anti-His tag antibody. The His tag was detected at 83 kDa, which was consistent with the expected size of the target protein. The specific process was as follows:

[0079] The culture solution of HEK-293F cells after 7 days of transfection was collected and centrifuged at 8000 rpm for 1 h at 4°C using a vertical high-speed low-temperature centrifuge. The supernatant of the HEK-293F suspension cell expression was collected by filtering the supernatant through a 0.22 μm filter membrane under negative pressure to remove cell debris. The deionized water filtered by the 0.22 μm filter membrane was first used to flush the column at a flow rate of 2 mL / min, and then the same flow rate was used to balance the buffer A (20 mmol / L Tris-HCl, 150 mmol / L NaCl, pH 8.0) for 3-5 column volumes. The expression supernatant of PDV-S1 recombinant protein was filtered under negative pressure and transferred to the his column, and the supernatant was passed through at a flow rate of 2 mL / min. Attention should be paid to prevent air bubbles from entering during this process. The A pump and B pump of the protein purification instrument were flushed with water and buffer A at a flow rate of 10 mL / min, and then the flow rate was adjusted to 2 mL / min. The HisTrap Excel column was connected, and the protein purification instrument was flushed with 100% buffer A until the UV value was flat. Buffer B containing 50 mmol / L, 100 mmol / L, 250 mmol / L, and 500 mmol / L imidazole was used for elution, and the eluate was collected for SDS-PAGE detection. The eluate was transferred to a protein concentration tube and concentrated and exchanged with sterile PBS buffer for 3 times. After elution, the appropriate molecular sieve was selected for purification of PEDV S1 protein according to the molecular weight and protein size. The use of superdex 200 molecular sieve: before installing the column, the maximum alarm pressure and flow rate were set on the Akta. The column pressure of the large column was: pre-column pressure (i.e. system pressure) + 0.15 MPa, and the flow rate was 0.5-1 mL / min. The column volume was 120 mL.

[0080] Before purifying the protein, 40 mL of water and 80 mL of buffer (PBS with pH 7.4) were passed through. After the UV value curve was stable, the sample was loaded on the loop ring. According to the volume of the concentrated protein, the appropriate loop ring (1 / 2 / 5 mL) was selected. After the PBS was flushed through the loop ring for 3 volumes, the protein was absorbed into the loop ring. Click inject, and after the PBS passed through the loop ring for 3 volumes, click load. The volume can be found in the molecular sieve list according to the molecular weight of the protein. Set the peak collection: 5 mAU 0.8 mL per tube. The collected protein was placed on ice, and after identification by SDS-PAGE protein gel, the appropriate concentration tube was replaced with liquid. The purified PEDV S1 protein was identified by Western blotting using an anti-his tag antibody.

[0081] (ii) Animal immunization

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

[0083] Table 3 Mouse immunization procedure

[0084] Immunization times Immunization dose (μg / each) Immunization method Adjuvant First immunization 50 Back subcutaneous, point injection Freund's complete adjuvant Second immunization 50 Back subcutaneous, point injection Freund's incomplete adjuvant Third immunization 50 Back subcutaneous, point injection Freund's incomplete adjuvant Reinforcement 100 Back subcutaneous, point injection Freund's incomplete adjuvant

[0085] Before flow sorting specific B lymphocytes, the mice were anesthetized with isoflurane, and blood was collected from the orbital venous plexus. 500 μL of blood was collected, and the serum was placed in a 4°C refrigerator overnight and centrifuged at 12000 rpm for 1 min. ELISA test was performed to test the immunization 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 by 768000 times, the OD 450nm >cut-off value and P / N≥2; the serum of the fourth mouse was diluted by 576000 times, and the OD 450nm >cut-off value and P / N≥2; the virus micro-neutralization test results showed that the serum still had obvious neutralization effect after dilution by 512 times.

[0086] Table 4 Detection of antibody titer of mouse serum

[0087]

[0088] II. Flow sorting of mouse antibody-secreting cells and memory B cells

[0089] (i) Acquisition of mouse lymph node B lymphocytes

[0090] (1) Take the mouse immunized with PEDV S1 protein, immediately take the lymph node after short neck death, grind in the cell screen, and soak the cell screen in 0.5% FBS in advance;

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

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

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

[0094] (5) Repeat once, a total of 2 times (0.5% FBS is at least 5 times the volume of cell precipitate).

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

[0096] First, prepare the cell lysis solution: 1 μL Recombinant RNase inhibitor + 19 μL 0.2% TritonX-100; 2 μL cell lysis solution, 1 μL 3'-RACE CDSPrimer A, 1 μL dNTP mix (10 mM each) are added to each well of the 96-well plate in advance, and pre-cooled on ice; specific B cells are sorted into 96-well plates using a flow cytometer (BD FACSAriaIII flow cytometer), and CD138 - CD93 - CD38 low IgD - CD45R + GL7 + PEDVS1 + positive cells are sorted into 96-well PCR Multiplates TM 96-Well PCR Plates, set one cell per cell well; the markers used for flow sorting are shown in Table 5.

[0097] Table 5 Markers used for flow sorting

[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 cell down-regulation CD93 Immature B, HSC and progenitor cells

[0099] Then circle the lymphocytes, use CD93 and CD138 negative selection to remove epithelial cells, nerve cells, pre-B cells and immature B cells, and then use CD38 and IgD negative selection to remove T cells, NK cells, etc., and then use GL7, CD45R and Strep tag antibody positive selection to obtain specific memory B cells of PEDV; as shown in Figure 6 192 positive cells were sorted in total; the details are as follows:

[0100] (1) Resuspend the cells obtained in the above step with 1 mL of FACS buffer (FACS buffer is a PBS solution with 0.5% FBS added); after resuspending the cells, transfer them to an imported 1.5 mL EP tube, centrifuge at 400g for 10 min at 4°C, and discard the supernatant (the cell count is about 10 7 / ml level); cell distribution: resuspend with the liquid at the bottom of the tube, take out 4 μL, add 400 μL of FACS buffer, mix well, and then evenly divide into 8 EP tubes, 50 μL per tube, 7 single positive tubes and 1 negative tube, and the rest of the cells are used as sample tubes (the sample tube is resuspended with 400 μL of FACS buffer, and the total number of cells is about 10 7 ; if the total number of cells is large, the volume needs to be increased, otherwise the antigen will be too low and not all cells can be labeled);

[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 per 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 the cells at 4°C. Add 1 mL of FACS buffer, mix well, and centrifuge the cells at 4°C.

[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 the cells at 4°C; add 1 mL of FACS buffer, mix well, and centrifuge the cells at 4°C. Add 2 mL of FCAS buffer, suspend the cells, filter them using a 0.45 μm filter, and transfer them to a flow tube.

[0103] III. Antibody screening

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

[0105] Table 6 Sequence of S-A9 antibody

[0106]

[0107]

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

[0109] The present application has selected 192 positive cells, and the light and heavy chain variable region fragments of the antibody were amplified by nested PCR. A gene linker was added to both fragments, and the variable region fragment was about 400 bp long. A total of 114 heavy chains and 176 light chains were amplified, with an amplification rate of 54.5% for heavy chains and 73.7% for light chains (as shown in Table 11 below). Figure 7 ) The details are as follows:

[0110] (1) RT (reverse transcription)

[0111] The sorted 96-well plate was centrifuged at 72°C for 3 min and at 42°C for 2 min. According to the results of the positive cell wells in the experiment, RT Mix was prepared and then dispensed into each well of the 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. The following conditions (Table 8) were used in the PCR instrument.

[0115] Table 8 RT-PCR reaction program

[0116]

[0117] (2) Amplification of ds cDNA

[0118] The PCR product was taken and subjected to ds cDNA amplification by adding reagents according to the following method. The cDNA PCR reaction system and reaction program are shown in Tables 9 and 10, respectively.

[0119] Table 9 ds cDNA PCR reaction system

[0120] Component Volume (μL) H2O 27.5 5x Phusion HF Buffer 10 dNTP mix (10 mM each) 1 5' PCR primer II A 1 The above ss cDNA 10 Phusion DNA Polymerase (2 U / μL) 0.5

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

[0122] Table 10 ds cDNA reaction procedure

[0123]

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

[0125] 1) Nested PCR a

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

[0127] Table 11 PCRa heavy chain amplification system

[0128] Component Volume (μL) 1 hVH (1-6) (100 μm) 0.6 1 hCH 0.1 dNTP 1 HotstarTaq 0.4 Q(5×) 4 Buffer (10x) 2 cDNA 2 25 mM MgCL2 1.2 H2O 8.7 Total volume 20

[0129] Table 12 PCRa light chain amplification system

[0130]

[0131]

[0132] Table 13 PCRa amplification reaction procedure for heavy chain amplification and light chain amplification

[0133]

[0134] 2) Nested PCR b

[0135] The above PCRa products were used as templates for PCRb, in this step, the light chain and heavy chain primers were not mixed, and each primer was subjected to separate PCR, the positive rate of heavy chain PCR was relatively low compared to light chain, according to the experience of pre-experiments, the heavy chain was used as a template for PCRa, and the VH upstream primer was used for PCRb, the primers used were divided into the following types: a, antibody heavy chain upstream primers (2hVH1-5), antibody heavy chain downstream primers (2hCH); b, antibody light chain upstream primers (2hVK1-4), antibody light chain downstream primers (2hCK); the PCRa heavy chain and light chain amplification system and the amplification procedure are shown in Tables 14, 15 and 16, respectively.

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

[0137] Component Volume (μL) 2 hVH 1 / 5 (10 μm) 1 2 hCH (10 μm) 1 dNTP 1 HotstarTaq 0.4 Q(5×) 4 Buffer (10x) 2 PCR a product 2 25 mM MgCl2 1.2 H2O 7.4 Total volume 20

[0138] Table 15 Antibody light chain amplification system

[0139]

[0140]

[0141] Table 16 Reaction conditions of antibody heavy chain and light chain PCR b

[0142]

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

[0144] The amplified antibody light and heavy chain PCR b products were subjected to agarose electrophoresis, the gel was cut and the universal gel recovery kit was used to recover the positive fragments of the light and heavy chain PCR b. The recovered antibody light and heavy chain fragments were homologously recombined into pCAGGS-IgK and pCAGGS-IgH vectors respectively, the DH5a competent cells were transformed, and after plating, single colonies were picked for identification, then sequencing was performed. The sequencing results of the amplified antibody light and heavy chain variable region genes were compared on NCBI Ig Blast, the gene lineage and mutation rate of the antibody were analyzed, and then the no-endotoxin plasmid large extraction kit was used to extract the antibody light and heavy chain recombinant bacteria with correct sequencing results.

[0145] (ii) Western Blot method for detecting the expression of PEDV antibody

[0146] The day before the experiment, HEK-293T cells were plated in a 6-well cell plate. The next day, when the cells were fully grown, the vector plasmid with the antibody light and heavy chain genes was transfected into each well of cells by PEI. 2 μg of plasmid was transfected into each well of the 6-well plate for the light and heavy chain plasmids, and the ratio of the light and heavy chain plasmids was 3:2, i.e. 1.2 μg of light chain and 0.8 μg of heavy chain were transfected into each well. After 3 days of transfection, the cell supernatant was collected once, and after 7 days, the cell supernatant was collected once. 6% SDS-PAGE reducing gel was prepared, and the expressed antibody supernatant was spotted and electrophoresed on an electrophoresis instrument at 150 V for 1 h. An appropriate size of NC membrane was cut and soaked in transfer buffer for 5 min, then the gel and the NC membrane were tightly attached together, and 200 mA was maintained for 1 h 20 min. 5% skimmed milk was blocked for 1 h, the blocking solution was discarded, and PBST was washed twice for 5 min each time. 40 ml of diluted goat anti-human IgG secondary antibody (1:3000 diluted with PBST) was added, and incubated at room temperature for 1 h. The secondary antibody was discarded, washed with PBST for 3 times, developed with Western Blot developing solution, and developed.

[0147] Results are shown in Figure 8 Table 1. As shown, most of the transfected HEK-293T cells expressed antibodies. According to the sequencing company's return of antibody light and heavy chain sequence information comparison results, 66 different antibodies were screened from 100 pairs of antibody light and heavy chains, and the antibody expression rate was 66%.

[0148] (III) Detection of the binding of PEDV antibodies by ELISA

[0149] The specific binding activity of the cell supernatant to the antigen was determined by ELISA, and antibodies with specific binding activity to the antigen were preliminarily screened. The binding capacity was determined by using 1 μg / mL PEDV S1 protein as the ELISA coating antigen and goat anti-human HRP as the secondary antibody. The purified antibodies were subjected to ELISA binding test. Antibodies with binding activity were expressed in large quantities by PEI transfection of HEK-293F cells. After counting the HEK-293F cells, they were diluted to 3 units, and the diluted HEK-293F cells were placed on a shaker for transfection. 1 μg of plasmid was used to transfect 1 mL of HEK-293F cells. The transfection reagent was prepared, and the concentration of the plasmid used for transfection was 1 mg / mL, and the concentration of PEI was 3-4 times that of the plasmid. The volume of the transfection reagent was 50 mL transfection reagent / L (too small volume would affect the transfection efficiency). 25 mL of HEPES / 150 mM NaCl was mixed with the plasmid / PEI, and the mixture was incubated for 5 min. PEI was slowly added to the plasmid, and the mixture was incubated for 15 min. Do not exceed 30 min, otherwise it is easy to produce precipitation. The transfected HEK-293F cells were added to the culture medium, and the mixture was incubated in a CO2-free incubator (CO2 has little effect). After 24 h of transfection, the medium was supplemented, and then supplemented every two days.

[0150] Results are shown in Figure 9 of the 66 pairs of different light and heavy chain expressed antibodies, 21 pairs of positive clones had binding ability with PEDV-S1, and the binding rate was 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) Determination of the recognition epitope of the antibody by indirect immunofluorescence test

[0152] Using these 21 PEDV antibodies as primary antibodies, an indirect immunofluorescence assay was performed. The specific steps included: Inoculating a suitable amount of Vero cells into 24-well plates, and after cell adhesion, infecting them with PEDV, with a negative control included. After culturing at 37°C and 5% CO2 for 24 hours, 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 the cells were fixed at room temperature for 15 minutes. The paraformaldehyde was discarded, and immediately 1 mL of pre-chilled methanol (100% methanol should be placed in a -20°C freezer 20 minutes prior) was added for permeabilization at room temperature for 10 minutes. The cells were washed three times with PBS, and 5% BSA was added. Blocking was performed at room temperature for at least 45 minutes (or overnight at 4°C). The blocking solution was discarded, and 300 μL of diluted PEDV antibody (1:200 dilution with 5% BSA) was added to each well. The cells were incubated at room temperature for 1 hour (or overnight at 4°C). Recover the primary antibody, wash three times with PBS, and add 300 μL of diluted 488 or 594 goat anti-human IgG secondary antibody (1:300 diluted with PBS) to each well. Incubate in the dark for 1 h. Discard the secondary antibody, add 500 μL of diluted DAPI dye (1:5000 diluted with PBS) to each well, and incubate in the dark for 15 min (DAPI concentration used is 1 μg / mL). Discard the DAPI, wash three times with PBS, and finally add a small amount of PBS. Observe the cells using a fluorescence microscope.

[0153] The results show ( Figure 10 Of the 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-react with PEDV, while the 9 antibodies, S-G1, S-C5, S-B6, S-B7-3, S-D8, S-D12, S-H12, S-G4-3, and S-B11, do not react with the virus.

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

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

[0156] (1) Count HEK-293F cells, calculate cell density and cell viability; control cell count to 2.0 × 10⁻⁶. 6 Cells / mL, 400mL per flask. Incubate the cell culture flasks at 37℃, 5% CO2, and 150–175 rpm in a constant temperature shaker; count the cells after 12 hours, with a cell concentration of 2.5–3.0 × 10⁻⁶ cells / mL. 6 Transfection was performed at a concentration of [number] cells / mL;

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

[0158] (3) After the diluted DNA and transfection reagent are separately placed for about 5 min, mix gently, the total volume is 20 mL, and then place at room temperature for 20 min. Add the transfection solution dropwise to the cell culture solution, shake the culture bottle gently while adding, shake well, and then place back on the shaker for continued culture. Tighten the bottle neck (i.e. do not introduce CO2 into the cell bottle any more);

[0159] (4) After 24 h of transfection, loosen the bottle neck to meet the oxygen dissolution and CO2 discharge requirements for subsequent high-density cell growth, so as to prevent the pH value of the culture solution from being too low (the culture solution is yellow) due to CO2 accumulation, which affects cell growth. Add 14 mL of SMS293-SUPI feeding solution at 24 h after transfection, and then add the feeding solution (14 mL) every 48 h. Collect the sample at 7 days after transfection.

[0160] (Six) PEDV antibody purification

[0161] (1) Use a vertical high-speed low-temperature centrifuge to centrifuge at 8000 rpm for 30-60 min at 4°C. Suck filter the supernatant through a 0.22 μm filter membrane to filter out cell debris;

[0162] (2) Hang the Protein A column: first, filter deionized water through a 0.22 μm filter membrane, quickly rinse the peristaltic pump, adjust the flow rate to 2 mL / min, hang the Protein A column, and then pass 15 mL (i.e. 3 column volumes) of buffer A (20 mM Na3PO4; pH 7.0) through the column, and then pass the filtered protein supernatant through the column;

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

[0164] (4) The purified antibody was exchanged with a 100 kDa concentration tube with PBS three times. After the antibody concentration was determined, the concentration was diluted to 1 mg / mL for standby. Prepare 6% SDS-PAGE reducing gel, take 20 μL of antibody, add 5 μL of 5x SDS-PAGE loading buffer (add 0.1 M DTT), and heat the sample at 100°C for 5 min. Take 7 μL of sample and run at 150 V for 50 min; transfer the membrane at 200 mA for 1 h 20 min; block with 5% skim milk for 1 h, wash with PBST three times, add goat anti-human HRP secondary antibody (1:3000 dilution), incubate at room temperature for 1 h, then discard the secondary antibody, wash with PBST three times, and develop with Western Blot developing solution. Take a photo.

[0165] Under the reducing effect of DTT and SDS-PAGE reducing gel, the PEDV S-A9 antibody was reduced to a 50 kDa heavy chain and a 25 kDa light chain (as shown in Figure 11 After protein concentration determination, the concentration of 400 mL of HEK-293F cell-expressed and purified antibody was 2.1 mg / mL, a total of 16 mg of protein.

[0166] (Seven) Neutralization test identification

[0167] Recover Vero cells and pass them to a 15 cm 2 cell dish. After the cells are fully confluent, wash them with PBS three times, dilute 100 μL of PEDV CH-HB2-2018 strain in 20 mL of DMEM, and add it to the cell dish. Incubate in a 37°C, 5% CO2 incubator for 1 h, then wash with PBS three times. Add 10 μg / mL trypsin in DMEM maintenance solution, and after 48 h, collect the sample, freeze and thaw it three times in a -80°C refrigerator, and collect the virus. Determine the TCID 50 of the virus in a 96-well plate with an appropriate amount of Vero cells. After the cells are fully confluent, dilute the virus in an EP tube by 10-fold, wash the 96-well plate cells three times, and add the diluted virus solution to the 96-well plate cells in order. After 48 h, observe the cytopathic effect and calculate the TCID 50 of the virus. In a 96-well plate, inoculate an appropriate amount of Vero cells, and after the cells are fully confluent, mix the purified PEDV antibody and virus solution (200 TCID 50 ) uniformly, and incubate in a 37°C, 5% CO2 incubator for 1 h. Wash the 96-well plate cells with PBS three times, add the antibody-virus mixture to the 96-well plate, and incubate in a 37°C, 5% CO2 incubator for 1 h. After washing with PBS three times, add 10 μg / mL trypsin in DMEM maintenance solution, and after 48 h, observe the neutralization results. The results of the micro-neutralization test are as follows Figure 12As shown, 7 antibodies, S-B3, S-D6, S-D7-3, S-D9, S-A9, S-F9, S-F10, etc., can neutralize PEDV, in which S-A9 has the highest neutralization titer, IC 50 up to 0.03286 μg / mL.

[0168] IV. Antibody verification

[0169] (I) Specificity identification of the antibody

[0170] Take the laboratory-preserved PEDV, PDCoV and TGEV as antigens to coat 96-well plates, 200 ng of antigen is coated in each well, PBS is used as a control, 10% skim milk is blocked for 1 h, 500 ng of purified PEDV-specific antibody (S-A9) is added as a primary antibody, incubated at room temperature for 30 min, washed three times, and then incubated with a goat anti-mouse HRP-labeled secondary antibody at room temperature for 30 min, washed three times, and then added with TMB color developing solution, incubated in the dark for 30 min, and then read OD 450 nm values; the experimental results show that Figure 13 ), S-A9 antibody can specifically bind to purified PEDV and has no cross-reaction with PDCoV and TGEV.

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

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

[0173] (1) Both of the two proteins for interaction were replaced into HEPES buffer (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v / v surfactant P20) for SPR experiments, and the HEPES buffer was degassed by ultrasonic before the experiment;

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

[0175] (3) The fixed phase was coupled to one channel in the chip, and bovine serum albumin (BSA) was coupled to another channel as a background control, and in some cases, a blank channel can also be used as a background control; when the protein is coupled, the machine flow rate is generally set to 5 μL / min;

[0176] (4) Estimate the affinity range of the research object, which can be divided into nanomolar level and micromolar level, and 1 nM and 1 μM mobile phase proteins can be used to perform the binding test, so as to determine the affinity range thereof;

[0177] (5) In the affinity range obtained in the above step, a series of concentration gradient is designed, the mobile phase protein is diluted by ratio, and the complete on-machine binding test is performed; we generally select KINJECT mode to perform the kinetic parameter determination, the machine flow rate is 30 μL / min, the injection binding time is generally 1-2 min (the required time is determined according to whether the binding curve is saturated), and the dissociation curve collection time is generally 1-5 min (the specific time setting is adjusted according to the fast or slow dissociation);

[0178] (6) The kinetic parameters of the research object are analyzed by using BIAevaluation software, and the graph is drawn.

[0179] The SPR result is shown in Table 1. Figure 14 The result shows that the PEDV S1 and the S-A9 antibody have high affinity, and the affinity is close to the order of magnitude of the antigen-antibody affinity, which belongs to high affinity.

[0180] Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present application but not limit the same, although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced by the equivalent, without departing from the purpose and scope of the technical solutions of the present application, which should be covered in the scope of the claims of the present application.

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 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.

3. The antibody or antigen-binding fragment according to claim 1, characterized in that: The antibody includes 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.

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 also includes a heavy chain constant region and / or a light chain constant region, both of which are derived from human or murine IgG antibodies or their mutants.

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

18.

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

7. The use of the antibody and / or antigen-binding fragment of any one of claims 1 to 5 and / or the polynucleotide molecule and / or expression vector of claim 6 and / or engineered bacteria and / or engineered cells in the preparation of a product for diagnosing porcine epidemic diarrhea virus infection.

8. The use of the antibody and / or antigen-binding fragment of any one of claims 1 to 5 and / or the polynucleotide molecule and / or expression vector of claim 6 and / or engineered bacteria and / or engineered cells in the preparation of a medicament for treating porcine epidemic diarrhea virus infection.

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