Porcine epidemic diarrhea virus monoclonal neutralizing antibody and application thereof

Abstract

The application discloses a porcine epidemic diarrhea virus (PEDV) monoclonal neutralizing antibody and application, and can be used for detecting PEDV S1 protein, expression PEDV-S1 full-length protein expression plasmid and soluble PEDV-S1 protein, and exhibits good neutralization effect on Huh7 cells infected by PEDV, and can be based on the preparation of specific monoclonal antibody, and has important significance for providing effective protection for PEDV infection, and can widely control PEDV infection, and improve the prevention strength of domestic porcine epidemic diarrhea disease.

Description

Technical Field

[0001] This invention relates to veterinary medical technology and drug development, particularly a monoclonal neutralizing antibody against porcine epidemic diarrhea virus (PEDV) that serves to detect and treat the disease. Background Technology

[0002] Porcine epidemic diarrhea virus (PEDV) is a serious and highly contagious intestinal infectious disease in pigs, with main symptoms including anorexia, vomiting, and watery diarrhea. PEDV was first discovered in the UK in 1971, and the classic strain CV777 was identified as the pathogen in Belgium in 1977. Porcine epidemic diarrhea was first reported in China in 1973, and PEDV was identified as the PED pathogen in 1984. Since 2010, PEDV has recurred in Europe, Asia, and the Americas. In China, the variant strains causing PED outbreaks are particularly severe, with mortality rates in newborn piglets reaching as high as 90%, resulting in huge economic losses to the pig farming industry.

[0003] PEDV belongs to the genus Alphacoronavirus in the family Coronaviridae. It is a single-stranded positive-sense RNA genome, 28 kb in length, containing at least seven open reading frames (ORF1a, ORF1b, ORF2-ORF6). ORF1a and ORF1b encode non-structural proteins (nsps), which are primarily responsible for regulating transcription, translation, and viral RNA synthesis within the host cell. The remaining ORFs are located at the 3' end of the viral genome, encoding four structural proteins: a 150-220 kDa glycosylated spike protein (S), a 7 kDa envelope protein (E), a 20-30 kDa membrane protein (M), a 58 kDa nucleocapsid protein (N), and one accessory protein. The S protein, located on the viral surface, forms a homotrimeric complex responsible for binding to specific cellular receptors, making it a key factor in viral entry into cells and a primary target for neutralizing antibodies. The S protein consists of S1 and S2 domains, with the S1 subunit proven to be the determining factor in viral virulence. Multiple antibody epitopes are present on S1, and polyclonal sera containing amino acid residues 499–638 of the S1 domain or downstream of the S1–S2 boundary (residues 636–789) have been shown to possess neutralizing activity. Currently, only one neutralizing monoclonal antibody (MAb 8A3A10) has an epitope located at amino acid residues 390–789 of the S protein. Furthermore, two B-cell epitopes (SS2: residues 748–755 and SS6: 764–771) downstream of the predicted S1–S2 junction have been identified as targets for non-neutralizing antibodies. The N-terminus of S1 exhibits high genetic diversity among different PEDV strains, which may also contribute to differences in sialic acid binding activity among different PEDV strains.

[0004] In 1995, my country began using bivalent inactivated or attenuated vaccines for the combined prevention of PEDV and TGEV. However, since the beginning of 2006, PED outbreaks have still occurred in pig herds immunized with PEDV vaccines. Furthermore, PED remains prevalent in pig farms in some parts of my country in recent years, and traditional CV777-based vaccines no longer provide effective protection against PEDV infection. Although existing vaccines cannot provide effective long-term protection, vaccination remains a realistic and feasible method for controlling PEDV infection. Summary of the Invention

[0005] Given the limited protective efficacy of current classic vaccines, attenuated vaccines, and inactivated viruses against porcine epidemic diarrhea (PED) in China, the purpose of this invention is to provide a monoclonal neutralizing antibody against PED virus and its application.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] The inventors previously obtained the S1 protein gene sequence of a prevalent PEDV strain from Henan Muyuan Group Co., Ltd. Based on this sequence, a monoclonal neutralizing antibody against porcine epidemic diarrhea virus was screened and obtained. This monoclonal neutralizing antibody is one or more of Mab-S1-1D12-7, Mab-S1-2D8-8, and Mab-S1-2C10-1. Specifically, the amino acid sequence of the heavy chain variable region of Mab-S1-1D12-7 is shown in SEQ ID NO. 25, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 29; the amino acid sequence of the heavy chain variable region of Mab-S1-2D8-8 is shown in SEQ ID NO. 26, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 30; the amino acid sequence of the heavy chain variable region of Mab-S1-2C10-1 is shown in SEQ ID NO. 25, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 29.

[0008] Furthermore, the monoclonal antibody specifically recognizes the S1 region of the PEDV strain's S protein.

[0009] The encoding gene of the porcine epidemic diarrhea virus monoclonal neutralizing antibody.

[0010] Furthermore, the coding genes for the heavy and light chains of Mab-S1-1D12-7 are shown in SEQ ID NO.31 and SEQ ID NO.34, respectively; the coding genes for the heavy and light chains of Mab-S1-2D8-8 are shown in SEQ ID NO.32 and SEQ ID NO.35, respectively; and the coding genes for the heavy and light chains of Mab-S1-2C10-1 are shown in SEQ ID NO.33 and SEQ ID NO.36, respectively.

[0011] The application of the monoclonal neutralizing antibody against porcine epidemic diarrhea virus in the preparation of porcine epidemic diarrhea virus detection reagents.

[0012] The application of the porcine epidemic diarrhea virus monoclonal neutralizing antibody in the preparation of reagents for treating porcine epidemic diarrhea.

[0013] The beneficial effects of this invention are:

[0014] 1. This invention utilizes virology and immunology technologies to prepare three monoclonal antibodies that specifically recognize circulating PEDV strains.

[0015] 2. All three monoclonal antibodies prepared in this invention can be used for the detection of PEDV S1 protein.

[0016] 3. The three monoclonal antibodies of this invention exhibit good neutralizing effects on PEDV-infected human liver cancer cell line Huh7 cells. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the pHLsec-PEDV S1-His protein fragment construction.

[0018] Figure 2 This is a Western blot result of the expression and purification of PEDV S1 protein;

[0019] Figure 3 The graphs show the serum titer validation results of mice after immunization using indirect ELISA and Western blot. A is a line graph showing the serum titer detection results of 5 mice immunized with pHLsec-S1-noHis at different dilutions; B is a line graph showing the serum titer detection results of 5 mice immunized with pHLsec-S1-His at different dilutions; and C is a graph showing the antibody Wb screening results in the serum of 10 mice. In graphs A and B, the corresponding numbers need to be followed by "C" to indicate the control group (mice immunized with irrelevant proteins), "neg" indicates the negative control, and "WT" indicates the blank control.

[0020] Figure 4 This image shows the immunofluorescence and Western blotting results of 16 positive tumor cell lines selected from the first amplification using positive monoclonal antibodies. The cell lines are: 1. 1A4-2; 2. 1C3-8; 3. 1C2-2; 4. 1D12-7; 5. 2D8-8; 6. 2C10-1; 7. 1F8-3; 8. 2A10-8; 9. 2B10-4; 10. 2F3-7; 11. 2D2-1; 12. 2F1-3; 13. 2E7-2; 14. 2E1-1; 15. 2D6-8; 16. 2D1-2; positive. (Image also shows 13# mice serum.)

[0021] Figure 5 This is a graph showing the results of three monoclonal antibodies after identification and purification using 15% SDS-PAGE.

[0022] Figure 6 This is a graph showing the binding / dissociation curves of anti-S1 protein monoclonal antibodies in the supernatant of three hybridoma cell lines determined using the BLI method.

[0023] Figure 7 The results are 1% agarose gel electrophoresis (A) and sequencing results (B) of the heavy chains of three monoclonal antibodies.

[0024] Figure 8 The results are 1% agarose gel electrophoresis (A) and sequencing results (B) of the light chains of three monoclonal antibodies.

[0025] Figure 9 The graph shows the inhibition curves and inhibition rates of three PEDV-S1 neutralizing antibodies. Detailed Implementation

[0026] The specific embodiments of the present invention will be further described in detail below with reference to examples. Unless otherwise specified, the instruments, equipment, and reagents involved in this embodiment, unless otherwise specified, are all conventional products. The experimental methods involved are all conventional methods.

[0027] The present invention will be further illustrated below with reference to the embodiments;

[0028] Example 1: Construction of expression vector

[0029] Construct the PEDV S1 protein expression vector. The specific steps are as follows:

[0030] 1. After sequencing the PEDV isolated from a pig farm (Henan Muyuan Group Co., Ltd.) to obtain the full sequence of the S1 protein and the partial sequence downstream of the S1-S2 linker (AA: 751-793), the original sequence was optimized to promote better translation and expression of the protein at the mRNA level.

[0031] 2. Design primers based on the full sequence of the S1 protein and the partial sequence of the downstream linker of S1-S2:

[0032] AgeI-S1-F: ATGCACCGGTATGAAGTCCCTGACATACTTCTG (SEQ ID NO.37); XhoI-S1-6xhis-R: ATGCCTCGAGTTAGTGATGATGATGATGATGCCGGATGCTCATGCTGAAG (SEQ ID NO.38); XhoI-S1-nohis-R: CCCTCGAGTAATTACCGGATGCTCATGCTGAAG (SEQ ID NO.39). The target gene fragments with and without His-tags were amplified separately. For fragment 1 (with His-tag), the primers were designed with an AgeI restriction site added upstream of the N-terminus, and a 6×His-tag, a terminator, and an XhoI restriction site added downstream of the C-terminus. For fragment 2 (without His-tag), the upstream N-terminus was identical to fragment 1, but only a terminator and an XhoI restriction site were added downstream of the C-terminus.

[0033] 3. The two PCR products and the pHLsec vector were digested with AgeI and XhoI restriction endonucleases, respectively. The gene fragments were separated by agarose gel electrophoresis and recovered.

[0034] 4. The PCR product digested with the enzymes was ligated with the vector pHLsec using T4 ligase at 16°C for 12-16 hours.

[0035] 5. The ligation product was transformed into BL21 competent cells and ampicillin-resistant cells were screened.

[0036] 6. After 14-16 hours, use the following primers to perform PCR identification of single colonies:

[0037] PEDV S1-CX-F: ACCGGTATGAAGTCCCTGAC (SEQ ID NO. 40);

[0038] PEDV S1-CX-R: TGAGGAGTGAATTATCGCGA (SEQ ID NO. 41);

[0039] 7. Extract plasmids from correctly identified monoclonal strains and send them for sequencing, while preserving the bacterial strain.

[0040] 8. Name and save the correctly identified plasmids and strains. Name them pHLsec-S1-His and pHLsec-S1-noHis. The construction diagram of the pHLsec-PEDV S1-His protein fragment is shown below. Figure 1 As shown.

[0041] 9. To obtain sufficient expression plasmids, after obtaining single clones of the preserved strain by streaking on plates, they were cultured overnight in LB liquid medium at 37°C and 220 rpm / min on a shaker to extract a large amount of plasmids (CWBIO / Kangwei Century, CW2104M).

[0042] Example 2 Protein expression of PEDV-S1

[0043] 1. Seed 293-F cells in dedicated culture medium (HEK293 Cell Complete Medium, M293TII), adding 1×PS penicillin antibody. The cell density before transfection should reach 1.5-2×10⁶ cells / year. 6 cell / mL.

[0044] 2. The pHLsec-S1-His plasmid obtained in Example 1 was transiently transfected into 293-F cells using PEI (MW40000). (When the total culture medium volume is 1000 mL, the plasmid DNA is 2 mg, and the PEI addition is 6 mL). The transfection steps are as follows:

[0045] (1) Prepare DNA-PEI complex: Dilute 2 mg plasmid DNA with 100 mL serum-free and antibiotic-free culture medium and mix thoroughly to form plasmid DNA dilution solution.

[0046] (2) Immediately add 6 mL of PEI 40,000 transfection reagent to the plasmid DNA dilution solution and mix gently. Incubate at room temperature for 10-15 min to allow the DNA-PEI complex to form.

[0047] (3) Add the DNA-PEI complex directly to 293-F cells and mix gently.

[0048] (4) Cells were cultured in a shaker at 37 °C and 5 vol% CO2 for 96 h. The cell culture medium was collected and PEDV-S1 recombinant protein was obtained using the His tag.

[0049] (5) Western blot identification. The purified PEDV-S1 protein and remaining cell pellet were subjected to SDS-PAGE electrophoresis, transferred to an NC membrane, blocked with 5 vol% BSA (dissolved in 1×TBST), and tested with His anti-mouse monoclonal antibody as the primary antibody and goat anti-mouse HRP as the secondary antibody. The results are as follows: Figure 2 As shown.

[0050] Example 3: Preparation of PEDV Monoclonal Antibody

[0051] 1. Animal immunization

[0052] Five 6-week-old BALB / c mice were immunized with expression plasmids pHLsec-PEDV-S1-noHis and pHLsec-PEDV-S1-His, respectively, via intramuscular injection in each limb, with 100 μg per mouse. A total of three immunizations were administered, with each immunization spaced 21 days apart. Serum was collected 9 days after the third immunization to determine its titer. Mice with the highest titers were selected and administered an intraperitoneal injection of PEDV-S1 protein, followed by a second immunization at 50 μg per mouse. The mice were sacrificed one week later.

[0053] 2. Detection of serum titers in immunized mice using indirect ELISA and Western blot.

[0054] 96-well microplates coated with PEDV-S1 recombinant protein and irrelevant protein were used, with a protein coating amount of 1 μg / mL, incubated overnight at 4 ℃, 50 μL / well. The liquid in the plate was discarded, and the plate was washed with PBST, 100 μL / well, 5 times. The plate was blocked with 1 vol% BSA at 37 ℃ for 2 h. The liquid in the plate was discarded, and the plate was washed with PBST, 100 μL / well, 5 times. The test sample was added, and serum from immunized or pre-immunized mice was diluted 1:300 with PBS buffer, then serially diluted 3-fold to 1:218700 as the primary antibody. PBS was used as the blank control, and negative mouse serum was used as the negative control, 50 μL / well. The plates were incubated at 37 ℃. Incubate for 45 min; discard the liquid in the plate, wash the plate with PBST 100 μL / well, 5 times; add GAM-HRP as secondary antibody, diluted 1:2000 with washing buffer, 100 μL / well; discard the liquid in the plate, wash the plate with PBST 100 μL / well, 5 times; add TMB chromogenic solution, 50 μL / well, incubate at 37℃ in the dark for 10 min; add stop solution, 50 μL / well; adjust the microplate reader parameters (450, 630) and measure the absorbance. The acceptable standard is 1:8100 dilution, OD value >1.0, and titer >72900.

[0055] PEDV-S1 recombinant protein was transferred onto an NC membrane. Serum from immunized mice was diluted 1:1000 as the primary antibody, and goat anti-mouse HRP was used as the secondary antibody. Western blot analysis was performed. The titer was then determined (results are shown below). Figure 3 ), and selected mouse #13 for cell fusion experiment.

[0056] 3. Hybridoma cell fusion

[0057] (1) The mice were sacrificed, and the spleen was removed under aseptic conditions. The spleen cells were squeezed and ground in a culture dish to prepare a spleen cell suspension. The myeloma cell SP2 / 0 suspension was mixed with the spleen cells and transferred to a 50ml centrifuge tube. The cells were collected by centrifugation (1000rpm, 10min). The remaining liquid was aspirated as much as possible, and the cells were gently shaken to make the cell precipitate appear as quicksand.

[0058] (2) Quickly mix sterilized PEG2000 (Sigma, St. Louis, MO, USA) (1.0 g) with 1.0 ml DMEM, and adjust the pH to about 7.8 with NaHCO3 solution. Detect the PEG solution with pH test paper.

[0059] (3) Slowly add the PEG solution to the cells, gently aspirate the cells with a pipette, let stand, and then blow it out. Control the time of the four processes to 60s, 30s, 30s, and 30s respectively.

[0060] (4) To counteract the fusion-promoting effect of PEG, DMEM culture medium was slowly added to the cell suspension. 1.0 mL was added over 60 seconds, followed by 1.0 mL every 30 seconds (4 times), and then 20 mL of DMEM culture medium was added dropwise over the next 3 minutes. The centrifuge tube was rotated rapidly throughout the process.

[0061] (5) Centrifuge at 1000 rpm for 5 min and collect the cells. Resuspend the cells in 50 mL of HAT (Sigma, St. Louis, MO, USA) culture medium.

[0062] (6) Spread the cells evenly in a 96-well culture plate containing feeder cells, 100 μL / well, and incubate at 37°C.

[0063] (7) Collect the supernatant of fused cells and screen them by indirect ELISA. Use serum from immunized mice as a positive control and serum from normal mice as a negative control to preliminarily screen for hybridoma cells with positive staining.

[0064] 4. Hybridoma cell screening

[0065] (1) After initial screening, 155 positive hybridomas were selected, and cell culture supernatant was used for ELISA detection.

[0066] (2) The protein concentration of PEDV S1 protein preparation plate is 1 μg / mL, 50 μL / well, overnight at 4℃; wash 3 times with washing solution.

[0067] (3) Block 100 μL / well with 1% BSA and place at 4°C; wash 3 times with washing solution.

[0068] (4) The supernatant of hybridoma cells was used as the primary antibody, the positive control was a 1 / 500 dilution of positive serum, and the blank control was HATmedium, all at 50 μL / well; incubated at 37℃ for 45 min; washed 3 times with washing solution.

[0069] (5) GAM-HRP diluted 1 / 10000 as secondary antibody, 50 μL / well. Incubate at 37℃ for 45 min; wash 3 times with washing buffer.

[0070] (6) Add 100 μL / well of colorimetric solution, 37℃, 10 min.

[0071] (7) Add 50 μL / well of stop solution.

[0072] (8) Measure absorbance at dual wavelengths (450, 630). OD > 1.4 is considered positive and further screening is performed.

[0073] (9) The results of hybridoma cell screening are shown in Table 1 and Table 2. The 20 positive hybridomas were further subcloned.

[0074] Table 1 Results of secondary screening for positive hybridoma cell fusion

[0075]

[0076] Note: The black boxes indicate positive hybridomas. The dark gray wells (91H, 101H, 92F, 102F) are positive control wells containing the corresponding 13# mouse serum at a dilution of 1 / 500. The light gray wells (111H, 121H, 112F, 122F) are negative control wells containing the stock solution of HAT medium.

[0077] Table 2 Results of positive hybridoma cell fusion rescreening - negative control for irrelevant proteins

[0078]

[0079] Note: Dark gray wells are positive control wells, containing serum from mouse #13 at a dilution of 1 / 500. Light gray wells are negative control wells, containing undiluted HAT medium.

[0080] 5. Monoclonalization of positive hybridomas

[0081] (1) First subcloning

[0082] From each of the 20 positive hybridomas, 1-8 subclones were selected and subjected to ELISA testing. The ELISA conditions and procedures were the same as in step 4 of Example 3, and the results are shown in Table 3.

[0083] Table 3 Results of the first subcloning of positive hybridoma cells

[0084]

[0085] Note: The horizontal axis represents the well number of the hybridoma cell lines screened in the previous round, and the vertical axis represents the general numbering. Positive hybridomas marked with black boxes are monoclonal antibodies and will be directly enlarged to 24-well plates. Dark gray wells (Serum 1:500) are positive control wells, containing the corresponding #13 mouse serum at a dilution ratio of 1 / 500. (+): Wells with an OD value > 3.4 are labeled +.

[0086] (2) First subcloning amplification

[0087] One to four positive monoclonal antibodies from the first subcloning were selected and amplified into 24-well plates, with two replicates per group. ELISA was performed, as shown in Tables 4 and 5. A total of 16 positive tumor cell lines were obtained.

[0088] Table 4. Results of positive monoclonal antibody amplification from the first subclone.

[0089]

[0090] Note: Positive hybridomas marked in black boxes are monoclonal antibodies, directly amplified to T25 culture flasks, with two replicates per group. Dark gray wells (Serum (1 / 500)) are positive control wells, containing the corresponding 13# mouse serum at a dilution of 1 / 500. Light gray wells are negative control wells (HAT medium), containing the original HAT medium solution. (+): Wells with an OD value > 3.4 are marked +.

[0091]

[0092] Note: Each group has two replicates. The dark gray wells are positive control wells, containing serum from mouse #13 at a dilution of 1 / 500. The light gray wells are negative control wells, containing undiluted HAT medium.

[0093] Example 4: Screening of positive hybridoma cell lines

[0094] Three hybridoma cell lines with the strongest affinity were screened using immunofluorescence (IF) and Western blot experiments, and their affinity was detected by BLI.

[0095] 1. Western blot identification of monoclonal antibodies

[0096] S1 protein was transferred to an NC membrane after 11.9% SDS-PAGE, blocked with 5% BSA at room temperature for 1 h, and washed three times with PBST. The supernatant from 16 hybridoma cell lines was diluted 1:100 as the primary antibody, and goat anti-mouse HRP was used as the secondary antibody (1:10000). The positive group was tested by Western blotting using serum from mouse #13 at a dilution of 1:500. The results showed that cells #2, #4, #5, #6, #9, #14, #15, and #16 had better Western blotting results (see...). Figure 4 ).

[0097] 2. Immunofluorescence (IFA) screening of monoclonal antibodies

[0098] Transfection: 293T cells were seeded into 24-well cell culture plates. When the cell density reached 70-80%, the PEDV-S1 plasmid was transfected into the cells. 0.5 μg of pHLsec-S1-His plasmid / well was added to 30 μL of DMEM (antibiotic-free / serum-free) and mixed well; 1.5 μL of PEDV-S1 plasmid was added to another 30 μL of DMEM (antibiotic-free / serum-free). Polyjet, mix well; quickly add DMEM containing polyjet to DMEM containing DNA, mix well, and incubate at room temperature for ≤10 min; then add the above Polyjet-DNA mixture to a 24-well plate containing cells, culture for 12 h, then replace the culture medium with DMEM complete medium and observe the cell status; after 3 days of culture, fix with 4% paraformaldehyde at room temperature for 30 min, 500 μL / well, and let stand; wash very gently with PBS 1-2 times; let stand with PBS-T (containing 0.2% / 0.5% Triton), 500 μL / well, soak for 10-30 min; block with 5% BSA for 30 min; use the hybridoma cell supernatant as primary antibody (dilution ratio 1:100), incubate overnight at 4℃; wash gently with PBS-T 3 times; use goat anti-mouse-GFP as secondary antibody (dilution ratio 1:1000), incubate at room temperature for 1 h; wash 3 times with PBS-T; mount with DAPI and observe under a microscope (see...). Figure 4 ).

[0099] Based on the combined results of Western blot (WB) and in vitro anatomical (IFA), monoclonal cells numbered 4 (1D12-7), 5 (2D8-8), and 6 (2C10-1) were selected as candidate monoclonal antibody purification cell lines.

[0100] 3. BLI test for the affinity of three monoclonal antibodies.

[0101] The affinity of monoclonal antibodies was determined using Biolayer Interferometry (BLI) and a Fortebio Octet (RED96e) instrument (see [link to instrument]). Figure 6First, pre-wet the AMC sensor for 10-15 minutes. The pre-wetting solution is 89% DMEM + 10% FBS + 1% double antibody. The baseline and loading program buffers have the same composition as the pre-wetting solution. The buffers for Baseline 2, Association, and Dissociation programs are all 20mM Tris-HCl, 150mM NaCl, and 0.02% Triton. The specific steps are as follows: After pre-wetting the sensor, baseline calibration is performed for 60 seconds. Once the baseline stabilizes, the loading procedure begins. The loading process aims to solidify the antibody. The negative control is wild-type mouse serum at a dilution of 1:200. The positive test groups are monoclonal cell supernatants from cells 4 (1D12-7), 5 (2D8-8), and 6 (2C10-1), with a loading volume of 200 μL / well for 180 seconds. The baseline 2 process aims to remove excess, poorly solidified antibody from the sensor, taking 240 seconds. The association process is the binding process, where S1 protein is serially diluted with buffer at 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM for measurement, retaining at least three effective concentrations, taking 170 seconds. The disassociation process is the dissociation process, taking 600 seconds. The results are shown in Table 6 and... Figure 6 As shown, it can be seen that 4 (1D12-7), 5 (2D8-8), and 6 (2C10-1) all have good affinity.

[0102] Table 6. Affinity test results of three monoclonal antibodies

[0103]

[0104] Example 5: Preparation and purification of monoclonal antibody ascites fluid

[0105] 8-16 week old BALB / c mice were intraperitoneally injected with sterile paraffin oil (0.5 mL / mouse). Seven days later, they were intraperitoneally injected with hybridoma cells in good growth condition, at a concentration of 5 × 10⁻⁶ mcg. 5 / mouse. Mice were continuously monitored for their condition. After approximately 7 days, when the abdomen of the mice became distended, ascites fluid was repeatedly aspirated. The fluid was centrifuged at 3000 rpm for 10 minutes to remove cells and other precipitates, and the supernatant was collected. After purification, 0.02 wt% sodium azide was added, and the solution was aliquoted and stored at -80°C.

[0106] Monoclonal antibodies were purified from ascites fluid. Initial purification was performed using the (NH4)2SO4 method, followed by further purification using a Protein G affinity chromatography column (Thermo Fisher Scientific, USA). The purification results were analyzed by 15% SDS-PAGE. Results are as follows: Figure 5 As shown.

[0107] Example 6: Sequencing of PEDV S1 monoclonal antibody

[0108] Hybridoma cells in good condition that stably secrete PEDV S1 monoclonal antibody were collected, and total RNA was extracted from them. The RNA quality was then assessed by 1% agarose gel electrophoresis (see [link to relevant documentation]). Figure 7-8 cDNA was synthesized via reverse transcription. The heavy and light chain variable region genes were amplified using mouse antibody variable region degenerate primers (primers are shown in Table 7). After separation of the heavy chain Fd fragment and the light chain Fd fragment by agarose gel electrophoresis, a specific band was observed at approximately 700 bp (heavy chain Fd fragment is shown in Table 7). Figure 7 A, see light chain Figure 8 A). After sequencing the PCR products, the sequencing results of the three monoclonal antibodies are as follows: Figure 7-8 (See relink) Figure 7 B, light chain see 8B), sequence as follows:

[0109] (1) Heavy chain

[0110] 1D 12 -7

[0111] Amino acid sequence (SEQ ID NO.25):

[0112] GGTLVKPGGSLKLSCVASGFAFNHYVMSWVRQTPEKRLEWVAYIGSDGGDTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARHGGDGKGYYFDYWGHG TTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPR

[0113] Gene sequence (SEQ ID NO.31):

[0114] GGGGGAACCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGTAGCCTCTGGATTCGCTTTCAATCACTATGTCATGTCTTGGGTTCGCCAGACTCCGGAGAAGAGGCTGGAGTGGGTCGCATATATTGGTAGTGATGGTGGTGACACCTACTATCCAGACACTGTGAAGGGCCGATTCACCATTTCCAGAGACAATGCCAAGAATACCCTGTACCTACAAATGAGTAGTCTGAAGTCTGAGGACACAGCCATGTATTACTGTGCAAGACATGGAGGAGATGGCAAGGGGTACTACTTTGACTATTGGGGCCATGGCACCACTCTCACAGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGG

[0115] 2D8-8

[0116] Amino acid sequence (SEQ ID NO.26):

[0117] GGGLVKPGGSLKLSCVASGFAFNNYVMSWVRQTPEKRLEWVAYIGSVDGDTYYPDTVKGRFTISRDNAKNTLYLQMISLKSEDTSIYYCVRHGGEGKGYYFDYWGHGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRD

[0118] Gene sequence (SEQ ID NO.32):

[0119] GGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGTAGCCTCTGGATTCGCTTTCAACAACTATGTCATGTCTTGGGTTCGCCAGACTCCGGAGAAGCGGCTGGAATGGGTCGCATACATTGGTAGTGTTGATGGTGACACCTACTATCCAGACACTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTGCAAATGATCAGTCTGAAGTCTGAAGACACATCCATTTATTACTGTGTAAGACATGGAGGAGAGGGCAAGGGATACTACTTTGACTATTGGGGCCATGGCACCACTCTCACAGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGAT

[0120] 2C 10 -1

[0121] Amino acid sequence (SEQ ID NO.27):

[0122] GGGLVKPGGSLKLSCVASGFAFNKHVMSWIRQTPEKRLEWVAYIGSVSGDTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTSIYYCVRHGGEGKGYYFDYWGHGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPR

[0123] Gene sequence (SEQ ID NO.33):

[0124] GGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGTAGCCTCTGGATTCGCTTTCAACAAACATGTCATGTCTTGGATTCGCCAGACTCCGGAAAAGAGGCTGGAGTGGGTCGCATACATTGGTAGTGTTAGTGGTGACACCTACTATCCAGACACTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTGCAAATGAGCAGTCTGAAGTCTGAAGACACATCCATTTATTACTGTGTAAGACATGGAGGAGAGGGCAAGGGATACTACTTCGACTATTGGGGCCACGGCACCACTCTCACAGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGG

[0125] (2) Light chain

[0126] 1D 12 -7

[0127] Amino acid sequence (SEQ ID NO.28):

[0128] MTHTPTSLAVSLGQRATISCRASESVDNFGITFMNWFQQKPGQPPKLLIYAASNQGSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYFCQQSKEVPWTFGGGTKLNIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0129] Gene sequence (SEQ ID NO.34):

[0130] ATGACCCACACTCCAACTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAGAGCCAGCGAAAGTGTTGATAATTTTGGCATTACTTTTATGAACTGGTTCCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATGCTGCATCCAACCAAGGATCCGGGGTCCCTGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCAGCCTCAACATCCATCCTATGGAGGAGGATGATACTGCAATGTATTTCTGTCAGCAAAGTAAGGAGGTTCCGTGGACGTTCGGTGGAGGCACCAAACTGAATATCAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT

[0131] 2D8-8

[0132] Amino acid sequence (SEQ ID NO.29):

[0133] VMTQSPTSLAVSLGQRATISCRASESVDNYGISFMNWFQQKPGQPPKLLIYAASNQGSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYFCQQSKEVPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0134] Gene sequence (SEQ ID NO.35):

[0135] GTGATGACCCAGTCTCCAACTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAGAGCCAGCGAAAGTGTTGATAATTATGGCATTAGTTTTATGAACTGGTTCCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATGCTGCATCCAACCAAGGATCCGGGGTCCCTGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCAGCCTCAACATCCATCCTATGGAGGAGGATGATACTGCAATGTATTTCTGTCAGCAAAGTAAGGAGGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT

[0136] 2C 10 -1

[0137] Amino acid sequence (SEQ ID NO.30):

[0138] VMTQSPTSLAVSPGQRATISCRASESVDNYGISFMNWFQQKPGQPPKLLIYAASNQGSGVPARFSGSGSGTDFSLSIHPMEGDDAAMYFCQQSKEVPWTFSGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0139] Gene sequence (SEQ ID NO.36):

[0140] GTGATGACCCAGTCTCCAACTTCTTTGGCTGTGTCTCCAGGGCAGAGGGCCACCATCTCCTGCAGAGCCAGCGAAAGTGTTGATAATTATGGCATTAGTTTTATGAACTGGTTCCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATGCTGCATCC AACCAAGGATCCGGGGTCCCTGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCAGCCTCAGCATCCATCCTATGGAGGGGGATGATGCTGCAATGTATTTCTGTCAGCAAAGTAAGGAGGTTCCGTGGACGTTCAGTGGAGGCACCAAGCTGGAAATC AAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTG AACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATAACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT

[0141] Table 7. Primers for Monoclonal Antibody Sequencing

[0142]

[0143] Among them, the other types of letters are degenerate primers: S=C / G, Y=C / T, M=A / C, W=A / T, K=T / G, R=A / G;

[0144] Antibodies can be synthesized by gene synthesis and expressed exogenously according to the antibody sequence to obtain self-prepared antibodies.

[0145] Example 7: Detection of Neutralizing Activity of Monoclonal Antibodies

[0146] Healthy 2.5 × 10^4 human hepatocellular carcinoma cell line Huh 7 cells were evenly seeded into 48-well cell culture plates and cultured for 12 h until complete adhesion and stability were achieved before experiments. Three monoclonal antibodies, 1D12-7, 2C10-1, and 2D8-8, were initially diluted 320 μg / mL and serially diluted 7 times (3-fold). PEDV-GFP virus solution with a titer of 10^3.833 TCID50 / mL was diluted 80-fold and mixed with equal volumes of the three diluted monoclonal antibodies. The mixture was incubated at 37°C for 30 min, and then the virus solution was removed. Infected cells were cultured in DMEM complete medium at 37°C for 12 h, digested with trypsin for 1 min, and then re-seeded into 48-well cell culture plates for another 35 h. Cells were stained with Hochest stain, and the total cell count (Hochest) and the number of infected cells (GFP-positive) were counted using a high-content imaging analysis system. The inhibition rate was calculated using the following formula: Inhibition rate of neutralizing antibody against PEDV virus infection = (Virus infection rate without neutralizing antibody - Actual infection rate of the test group) / Virus infection rate without neutralizing antibody × 100; Results are shown in [link to results]. Figure 9 As can be seen, the antibody of the present invention has a good inhibition rate and can be used to prepare a reagent for detecting or treating porcine epidemic diarrhea virus.

[0147] Researchers in the art can draw upon the content of this document to appropriately improve the process parameters. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can clearly modify or appropriately alter and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

Claims

1. A porcine epidemic diarrhea virus monoclonal neutralizing antibody, characterized in that, The porcine epidemic diarrhea virus monoclonal neutralizing antibody is one or more of Mab-S1-1D12-7, Mab-S1-2D8-8, and Mab-S1-2C10-1; wherein, the amino acid sequence of the heavy chain variable region of Mab-S1-1D12-7 is shown in SEQ ID NO.25, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.28; the amino acid sequence of the heavy chain variable region of Mab-S1-2D8-8 is shown in SEQ ID NO.26, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.29; the amino acid sequence of the heavy chain variable region of Mab-S1-2C10-1 is shown in SEQ ID NO.27, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

30.

2. The porcine epidemic diarrhea virus monoclonal neutralizing antibody according to claim 1, characterized by, Monoclonal antibodies specifically recognize the S1 region of the S protein of PEDV strains.

3. The encoding gene of the porcine epidemic diarrhea virus monoclonal neutralizing antibody according to any one of claims 1-2.

4. The encoding gene according to claim 3, characterized in that, The coding genes for the heavy chain variable region and the light chain variable region of Mab-S1-1D12-7 are shown in SEQ ID NO.31 and SEQ ID NO.34, respectively; the coding genes for the heavy chain variable region and the light chain variable region of Mab-S1-2D8-8 are shown in SEQ ID NO.32 and SEQ ID NO.35, respectively; and the coding genes for the heavy chain variable region and the light chain variable region of Mab-S1-2C10-1 are shown in SEQ ID NO.33 and SEQ ID NO.36, respectively.

5. The use of the porcine epidemic diarrhea virus monoclonal neutralizing antibody according to claim 1 or 2 in the preparation of porcine epidemic diarrhea virus detection reagent.

6. The use of the porcine epidemic diarrhea virus monoclonal neutralizing antibody according to claim 1 or 2 in the preparation of a drug for treating porcine epidemic diarrhea.

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