A monoclonal antibody against porcine epidemic diarrhea virus S protein, its preparation method and application

Abstract

This invention belongs to the fields of immunology and in vitro diagnostics, specifically relating to a monoclonal antibody against the S protein of porcine epidemic diarrhea virus (PEDV), its preparation method, and its applications. This invention utilizes a prokaryotic expression system to express and purify the full-length PEDV S protein, using it as an immunogen to immunize mice. Through cell fusion and subcellular screening, a monoclonal antibody targeting the S protein was successfully obtained. The monoclonal antibody provided by this invention can be obtained using conventional genetic engineering or protein engineering methods, avoiding antibody loss during long-term cryopreservation of hybridoma cells. It also facilitates antibody optimization at the gene and protein levels, thereby improving antibody specificity and affinity. The monoclonal antibody prepared by this invention exhibits good conservation, providing materials for the study of PEDV pathogenic mechanisms and the development of PEDV diagnostic kits.

Description

Technical Field

[0001] This invention belongs to the fields of immunology and in vitro diagnostic technology, specifically relating to a monoclonal antibody against the S protein of porcine epidemic diarrhea virus, its preparation method, and its application. Background Technology

[0002] Porcine epidemic diarrhea (PED) porcine epidemic diarrhea PED is caused by porcine epidemic diarrhea virus (PED). Porcine epidemic diarrhea virus Pigs are susceptible to an acute, highly contagious disease caused by Pediatric Edema Virus (PEDV). The main symptoms are vomiting, watery diarrhea, and dehydration. PEDV affects pigs of all ages. Adult pigs infected with PEDV mainly exhibit weight loss, malnutrition, and impaired reproductive performance. The mortality rate in piglets under one week old can be as high as 80%–100%, causing serious economic losses to the global pig industry.

[0003] PEDV belongs to the genus Coronavirus in the family Coronaviridae. It is an enveloped, single-stranded, positive-sense RNA virus with a genome length of approximately 28 kb. It contains a 5' cap structure and a 3' polyadenylated tail, encoding seven open reading frames (ORFs), corresponding to ORF1a, ORF1b, and structural and non-structural proteins such as nucleocapsid protein (N), membrane protein (M), spike glycoprotein (S), envelope protein (E), and accessory protein (ORF3). The S protein, as the most critical structural and immunogenic protein of PEDV, is approximately 1383 amino acids in length. It can be cleaved by host proteases into two subunits: S1 (1-789 aa) and S2 (790-1383 aa). The S1 subunit contains an N-terminal domain (NTD) and a C-terminal domain (CTD), responsible for recognizing host cell receptors (such as porcine aminopeptidase N pAPN) and mediating viral adsorption. Its NTD region has a high mutation rate, which is the main cause of antigenic drift in the strain. The S2 subunit contains conserved structures such as fusion peptides and heptapeptide repeat regions (HR1 / HR2), responsible for mediating the fusion of the viral envelope with the host cell membrane. The S protein carries multiple B-cell epitopes, among which the core neutralizing epitope region (COE, 499-638 aa) has become a core target for the development of PEDV subunit vaccines and diagnostic reagents due to its high sequence conservation.

[0004] Given the central role of the S protein in PEDV infection and immune response, and the shortcomings of existing monoclonal antibodies in terms of broad spectrum, affinity, and epitope conservation, developing a monoclonal antibody that can specifically recognize the conserved epitopes of the PEDV S protein is of great significance for improving rapid and accurate detection technology for PEDV, optimizing vaccine quality evaluation system, and exploring the functional mechanism of the S protein. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a monoclonal antibody against the S protein of porcine epidemic diarrhea virus. The monoclonal antibody comprises a heavy chain constant region and a light chain constant region, a heavy chain variable region and a light chain variable region; the CDR of the heavy chain variable region comprises the CDR1 amino acid sequence, the CDR2 amino acid sequence and the CDR3 amino acid sequence; the CDR of the light chain variable region comprises the CDR1 amino acid sequence, the CDR2 amino acid sequence and the CDR3 amino acid sequence.

[0006] Furthermore, the CDR1 amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.1; the CDR2 amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.2; and the CDR3 amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.3.

[0007] Furthermore, the CDR1 amino acid sequence of the light chain variable region is shown in SEQ ID NO.4; the CDR2 amino acid sequence of the light chain variable region is shown in SEQ ID NO.5; and the CDR3 amino acid sequence of the light chain variable region is shown in SEQ ID NO.6.

[0008] The present invention also provides a gene fragment encoding the monoclonal antibody, wherein the heavy chain constant region of the monoclonal antibody against the first half of the S protein of porcine epidemic diarrhea virus is of type IgG1 and the light chain constant region is of type Kappa.

[0009] This invention also provides a method for preparing a monoclonal antibody against the S protein of porcine epidemic diarrhea virus (PEDV). The method involves transfecting a plasmid containing the S gene of PEDV into CHO cells using liposomes for eukaryotic expression, collecting the supernatant, purifying the protein using different concentrations of imidazole elution, and using the purified recombinant S protein as an antigen to immunize Balb / c mice. Mouse spleen cells are then fused with myeloma cells SP2 / 0 to prepare hybridoma cells. The cell supernatant is verified by indirect ELISA, and positive clones are screened. After three subcloning processes, the hybridoma cells are injected into mice to prepare ascites. Finally, the obtained ascites is purified to obtain the monoclonal antibody against the S protein of PEDV.

[0010] The present invention has the following beneficial effects: This invention utilizes a prokaryotic expression system to express and purify the full-length PEDV S protein, which is then used as an immunogen to immunize mice. Through cell fusion and subcellular screening, a monoclonal antibody targeting the S protein was successfully obtained.

[0011] The monoclonal antibody provided by this invention can be obtained using conventional genetic engineering or protein engineering methods, avoiding antibody loss during long-term cryopreservation of hybridoma cells. It also facilitates antibody optimization at the gene and protein levels, thereby improving antibody specificity and affinity. The monoclonal antibody prepared by this invention exhibits good conservation, providing materials for research on the pathogenesis of PEDV and for the development of PEDV diagnostic kits. Attached Figure Description

[0012] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0013] Figure 1 Results of eukaryotic expression and purification of PEDV S protein; Note: M: Protein Marker; 1: Purified S protein; Figure 2 Serum titer of S protein in mice after triple immunization; Figure 3 Hybridoma supernatant detection; Figure 4 Western blot is used to identify the reactivity of monoclonal antibodies; Note: In Figure A, M: Protein Marker; 1: Purified full-length PEDV S protein; in Figure B, M: Protein Marker; 1: Purified recombinant PEDV S1 protein; Figure 5 Indirect immunofluorescence (IFA) was used to identify the cellular-level binding activity of monoclonal antibodies. Note: A: Vero cells infected with PEDV (incubated with the monoclonal antibody of this invention); B: Vero cells infected with PEDV (incubated with isotype negative control antibody); green fluorescence is the antibody binding signal, and blue is DAPI staining of cell nuclei; Figure 6 Antibody heavy chain variable region structure diagram; Figure 7 Structure diagram of the variable region of the antibody light chain. Detailed Implementation

[0014] Various exemplary embodiments of the present invention are now described in detail. Unless otherwise specified, the methods used in the embodiments are conventional methods, and the reagents used are commercially available reagents or reagents prepared using conventional methods. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and embodiments of the present invention.

[0015] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0016] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0017] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0018] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0019] Example: Preparation of a monoclonal antibody against the S protein of porcine epidemic diarrhea virus 1. Eukaryotic expression and purification of full-length PEDV S protein The PEDV-S plasmid preserved in our laboratory was introduced into CHO cells using liposome transfection. After culturing for 4-7 days, the cell culture was collected, centrifuged at 3000×g for 30 min at 4℃ to remove the cell pellet, and the supernatant was filtered through 0.45μm and 0.22μm filter membranes to obtain a clear crude protein extract.

[0020] a. Column packing and equilibration: Pack 2 mL of NTA nickel column resin, rinse with 5 column volumes of distilled water, and equilibrate with equilibration buffer (50 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH 8.0) until baseline stability.

[0021] b. Sample loading and binding: Mix the crude protein extract with nickel column resin, incubate overnight at 4°C, then load onto the column and collect the permeate.

[0022] c. Washing and elution: Wash with equilibration buffer to remove impurities, then elute with a gradient of elution buffers containing 10mM, 50mM, 100mM, 250mM, and 500mM imidazole, eluting 10mL of each concentration, and collect each fraction.

[0023] d. Purification and identification: Each elution fraction was analyzed by SDS-PAGE electrophoresis to determine the elution fraction corresponding to the high-purity target protein.

[0024] e. Pack the high-purity elution fraction into a 10 kDa molecular weight cutoff dialysis bag, dialyze to PBS buffer (pH 7.4) at 4°C to desalt, change the buffer 3 times, and dialyze for 12 h at each gradient; after dialysis, filter to sterilize, determine the concentration by BCA method, and aliquot and freeze at -80°C.

[0025] 2. Serum titer determination ELISA method for detecting polyclonal antibody titers in mouse serum Protein was coated onto an ELISA plate at a concentration of 200 ng / well and incubated overnight at 4°C. The plate was then washed four times with PBST, patted dry, and blocked with 5% skim milk at 37°C for 2 h. Positive and negative sera were serially diluted with PBST at a gradient of 1:1000 to 1:128000, with eight gradients set. The plates were incubated at 37°C for 1 h, washed four times with PBST, and HRP-labeled goat anti-mouse IgG (1:10000 dilution) was added. The plates were washed four times with PBST, patted dry, and incubated with TMB for 15 min. Stop solution was then added, and the plate was used to measure OD at 450 nm using a microplate reader.

[0026] 3. Preparation, reactivity, and variable region sequencing of monoclonal antibodies. 3.1 Animal Immunization Two 8-week-old female BALB / c mice were selected, and the purified S recombinant protein was immunized at a dose of 50 μg / mouse. For the first immunization, the S protein was mixed with an equal volume of Freund's adjuvant (CFA for the first immunization and IFA for the second immunization), emulsified, and then injected subcutaneously into multiple sites on the back of the mice. A booster immunization was given 21 days after the first immunization, and a total of three immunizations were performed. Blood samples were collected from the eyeballs 14 days after the third immunization to measure antibody titers. Before fusion, a booster immunization was performed, with the S recombinant protein at a dose of 100 μg / mouse injected intraperitoneally into the mice to be subjected to cell fusion.

[0027] 3.2 Preparation of Monoclonal Antibodies The specific operating steps are as follows: (1) Preparation of feeder cells The day before fusion, a healthy, unvaccinated Kunming mouse was euthanized by cervical indwelling, and feeder cells were added to a 96-well plate. Buffed mice were euthanized by enucleation to remove blood, and serum was collected as a positive control. The mouse spleen was removed and placed on a sterile dish. An appropriate amount of DMEM medium was drawn up using a syringe, and the spleen was repeatedly defrosted to prepare a single-cell suspension. The spleen cell suspension and SP2 / 0 cells were then fused at a 5:1 ratio. The cells were gently resuspended in HAT selective medium and added at 100 μL / well to the feeder cells prepared the day before. The wells were then incubated at 37 ℃ in a 5% CO2 incubator. Cell status was observed approximately 4 days later.

[0028] (2) Cell fusion Spleen cells and an appropriate amount of SP2 / 0 cells were fused using the fusion agent PEG. The fused cells were then seeded in 96-well plates.

[0029] (3) Screening of positive clones: a) Indirect ELISA detection: The purified S-fraction recombinant protein was coated onto an ELISA plate to detect the antibody secretion of fusion cells.

[0030] (4) Subcloning of positive hybridoma cells Wells showing ELISA positivity were selected, and the selected positive hybridoma cells were subcloned using a limiting dilution method. After 10 days, the cells were observed under an inverted microscope, and wells showing only a single clone were marked. The supernatant was collected, and antibody detection was performed using the ELISA method described above. Positive cells were then used in the next round of subcloning, for a total of three cycles.

[0031] (5) Preparation of ascites Take 10-12 week old BALB / c female mice, inject 0.5 mL of ascites fluid into each mouse to prepare a special adjuvant, and inject 5 × 10⁵ hybridoma cells (0.5 mL) into each mouse intraperitoneally 10 days later. After 7-10 days, the mouse's peritoneum will be obviously distended. Collect the ascites fluid and store it at -80℃.

[0032] (6) Purification of ascites The antibody was purified by affinity chromatography using Protein G Resin from TransGen, and its purity was determined by SDS-PAGE electrophoresis.

[0033] 3.3 Identification of Monoclonal Antibody Reactivity (1) Reactivity verification: including Western blot and IFA verification.

[0034] a) Western blot verification of antibody reactivity: The purified S protein and S1 recombinant protein were run on SDS-PAGE, and then the protein gel was transferred to an NC membrane for Western blot verification. The primary antibody was diluted 4-2H monoclonal antibody, and the secondary antibody was HRP-labeled anti-mouse IgG.

[0035] b) IFA verification of antibody reactivity: To further determine the specificity of the obtained antibody, Vero cells infected with PEDV for 48 h were fixed and permeabilized, and IFA verification was performed using 4-2H monoclonal antibody as the primary antibody and FITC-labeled goat anti-mouse IgG as the secondary antibody. A negative control group without Vero cell infection was set up, and the antibody specificity was determined by observing the fluorescence signal.

[0036] 3.4 Sequencing of the variable region of monoclonal antibodies Monoclonal antibody hybridoma cell samples were collected, and Detai Biotechnology Co., Ltd. was commissioned to sequence the variable region gene of the antibody. The sequencing results were then analyzed and compared.

[0037] 4. Results 4.1 Eukaryotic expression and purification of S protein and determination of serum titer The recombinant plasmid PEDV-S was transfected into CHO cells, and the protein was purified using a nickel column purification method after collecting the cell supernatant. Figure 1 It is evident that a high-purity full-length S protein was obtained. The purified full-length S protein was used to immunize Balb / c mice three times, followed by antibody titer testing. An ELISA plate was coated with the full-length S protein, and antibody titers were detected. Serum from unimmunized mice was used as a negative control (NC). The results showed that at a serum dilution of 1:64000, the OD450nm / NC ratio of immunized mice 1 and 2 was ≥2.1, indicating that the antibody titer could reach above 1:64000. Figure 2 ).

[0038] 4.2 Screening for positive hybridomas To obtain monoclonal hybridoma cells that stably secrete anti-S protein antibodies, the antibody secretion capacity of the hybridoma cells was detected by indirect ELISA. The results are as follows: Figure 3 As shown, wells with high positive values ​​were selected for three subcloning operations.

[0039] 4.3 Monoclonal antibody reactivity detection A monoclonal antibody that specifically recognizes the PEDV S protein was obtained through cell fusion technology and named 4-2H. Western blot validation results are as follows... Figure 4 As shown, 4-2H specifically binds to both the purified full-length recombinant S protein and the S1 protein. IFA results are as follows. Figure 5 As shown, by Figure 5 As shown in A, 4-2H acts on Vero cells infected with PEDV, resulting in a specific green fluorescent signal that can be detected. Figure 5 B indicates that uninfected Vero cells do not react with 4-2H.

[0040] 5. Monoclonal antibody variable region sequence The sequencing results were compared with the antibody gene library. The heavy chain constant region of the 4-2H monoclonal antibody was of the IgG1 type, and its light chain constant region was of the Kappa type. The sequencing results confirmed that the amplified sequences were the complementarity determining regions (CDRs) of the heavy chain and light chain variable regions of the monoclonal antibody, as shown in the table.

[0041] Table 1 Antibody variable region sequence

[0042] The amino acid sequence of the heavy chain variable region is as follows: QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGHIYPGDGDTNYNGKFKYKATLTVDKSSSTAYIQFSSLTSEDSAVYFCARGYYGSRAYGMDYWGQGTSVTVSS The amino acid sequence of the light chain variable region is as follows: DIILTQSPASLAVSLGQRATISCRASESVDNFGVSFMNWFQQKPGQPPKLLIYAASNQGSGVPARFSGSGSGTAFSLNIHPMEEDDAAMYFCQQTKDASWTFGGGTKLEIK The gene sequence encoding the variable region of the heavy chain is as follows: CAGGTTCAGCTGCAGCAGTCTGGGGCTGAGTTGGTGAGGCCTGGGTCCTCAGTGAAGATTTCCTGCAAGGCTTCTGGCTATGCATTCAGTAGCTACTGGATGAACTGGGTGAAGCAGAGGCCTGGACAGGGTCTTGAGTGGATTGGACACATTTATCCTGGAGATGGTGATACTAACTACA ATGGAAAGTTCAAGTATAAAGCCACACTGACTGTTGACAAATCCTCCAGCACAGCCTACATCCAGTTCAGCAGCCTAACATCTGAGGACTCTGCGGTCTATTTCTGTGCAAGAGGATACTACGGTAGTAGGGCCTATGGTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA The gene sequence encoding the variable region of the light chain is as follows: GACATTATACTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAGAGCCAGCGAAAGTGTTGATAATTTTGGCGTTAGTTTTATGAACTGGTTCCAACAGAAGCCAGGACAGCCACCCAAACTCCTCATCTATGCTGCAT CCAACCAAGGATCCGGGGTCCCTGCCAGATTTAGTGGCAGTGGGTCTGGGACAGCCTTCAGCCTCAACATCCATCCTATGGAGGAGGATGATGCTGCAATGTATTTCTGTCAGCAAACTAAGGACGCTTCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA In summary, this invention provides a monoclonal antibody against the S protein of porcine epidemic diarrhea virus (PEDV), offering a reliable tool for exploring the function of the S protein. The monoclonal antibody provided by this invention can be obtained using conventional genetic engineering or protein engineering methods, avoiding antibody loss during long-term cryopreservation of hybridoma cells. It also facilitates antibody optimization at the gene and protein levels, thereby improving antibody specificity and affinity. The monoclonal antibody prepared by this invention provides materials for studying the pathogenic mechanism of PEDV and for developing PEDV diagnostic kits.

[0043] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A monoclonal antibody against S protein of porcine epidemic diarrhea virus, characterized in that, The monoclonal antibody includes a heavy chain constant region and a light chain constant region, a heavy chain variable region and a light chain variable region; the CDR of the heavy chain variable region includes the CDR1 amino acid sequence, the CDR2 amino acid sequence and the CDR3 amino acid sequence; the CDR of the light chain variable region includes the CDR1 amino acid sequence, the CDR2 amino acid sequence and the CDR3 amino acid sequence.

2. The monoclonal antibody according to claim 1, characterized in that, The CDR1 amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.1; the CDR2 amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.2; and the CDR3 amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

3.

3. The monoclonal antibody according to claim 1, characterized in that, The CDR1 amino acid sequence of the light chain variable region is shown in SEQ ID NO.4; the CDR2 amino acid sequence of the light chain variable region is shown in SEQ ID NO.5; and the CDR3 amino acid sequence of the light chain variable region is shown in SEQ ID NO.

6.

4. A gene fragment encoding the monoclonal antibody, characterized in that, The monoclonal antibody against the first half of the S protein of porcine epidemic diarrhea virus has a heavy chain constant region of IgG1 type and a light chain constant region of Kappa type.

5. A method for preparing a monoclonal antibody against the S protein of porcine epidemic diarrhea virus, characterized in that, Plasmids containing the S gene of porcine epidemic diarrhea virus (PEDV) preserved in the laboratory were transfected into CHO cells for eukaryotic expression using liposomes. The supernatant was collected, and the protein was purified by elution with different concentrations of imidazole. The purified recombinant S protein was used as an antigen to immunize Balb / c mice. Mouse spleen cells were fused with myeloma cells SP2 / 0 to prepare hybridoma cells. The cell supernatant was verified by indirect ELISA, and positive clones were screened. After three subcloning processes, the hybridoma cells were injected into mice to prepare ascites fluid. Finally, the obtained ascites fluid was purified to obtain a monoclonal antibody against porcine epidemic diarrhea virus (PEDV) S protein.

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