A method for preparing and identifying a PEDV RBD protein polypeptide antibody

Abstract

The present invention discloses a preparation and identification method of a PEDV RBD protein polypeptide antibody, comprising the following steps: step 1: designing PEDV RBD antigen epitopes and synthesizing polypeptides; step 2: immunizing mice with polypeptide antigens to prepare specific antibodies; step 3: transfecting Expi293F cells with eukaryotic expression plasmid pcDNA3.1‑PER‑Strep; step 4: virus infection of host cells; step 5: Western Blot detection of PEDV RBD target protein expression; step 6: IFA detection of specific proteins expressed by virus-infected host cells; step 7: indirect ELISA detection of polypeptide antibody titer. The polypeptide antibody prepared by the present invention has good specificity and reactivity, can be used as an antibody with specific binding effect in an anti-PEDV RBD detection method, and also provides an important research and diagnostic tool for further studying PEDV S protein in the later stage; at the same time, a new detection antibody is also provided for the rapid detection of PEDV in clinical diagnosis.

Description

Technical Field

[0001] The present invention relates to the field of PEDV RBD protein polypeptide antibodies, and in particular to a preparation and identification method of a PEDV RBD protein polypeptide antibody. Background Art

[0002] Porcine epidemic diarrhea virus (PEDV) is a member of the Coronaviridae family and the Alphacoronavirus genus. Porcine epidemic diarrhea is a highly contagious intestinal disease characterized by acute diarrhea, vomiting and dehydration in newborn piglets caused by infection with PEDV, which is mainly transmitted through the fecal-oral route. PEDV mainly infects the small intestine and intestinal villus cells, and spreads rapidly between farms mainly through the digestive tract. Due to the frequent exchange of pork trade, the virus has spread on a large scale, causing huge economic losses to the global pig industry. Therefore, there is an urgent need to continuously develop and improve related drugs or antibodies to prevent and control the infection and spread of PEDV.

[0003] PEDV is a single-stranded positive-sense RNA virus with a genome size of approximately 28kb. It is mainly composed of four structural proteins, namely spike (S), envelope (E), membrane (M) and nucleocapsid (N) proteins. Among them, the S protein interacts with specific host cell receptors to mediate viral infection and induce the body to produce neutralizing antibodies. Therefore, the S protein is the key for PEDV to enter target cells. Through cryo-electron microscopy technology, it was found that the S protein of PEDV contains an S1 subunit involved in receptor binding and an S2 subunit involved in membrane fusion. Its receptor binding domain (RBD) is located in the S1 subunit, and the receptor binding motif (RBM) has only a few amino acids. Almost all coronaviruses rely on the RBD of the S protein to bind to the membrane receptor to enter the host cell. The RBD of PEDV may be located in the S1 CTD (C-terminal domain, CTD) between amino acids 477–629. Blocking the binding between the virus and the host cell by blocking the S protein RBD is an important target for vaccine development, diagnostic reagents and therapeutic antibody development. BROUWER et al. analyzed 19 strains of antibodies with neutralizing activity against pseudoviruses and found that most (74%) targeted RBD and bound to different epitopes on RBD. RBD is highly conserved among different strains. The RBD region determines the host range and cell tropism of the virus, and is also a key factor in determining the pathogenicity of the virus.

[0004] At present, facing the severe situation of repeated outbreaks of PEDV and the emergence of highly pathogenic strains, a rapid and effective detection method is urgently needed. Peptide antibodies have the advantages of being able to recognize highly specific antigenic epitopes, short preparation cycle, and high antibody titer, and are often used as detection tools for clinical diagnosis. A study successfully established an indirect ELISA method for detecting African swine fever virus based on tandem peptide antigens of African swine fever virus p30 and p54 protein epitopes. Usually, a polypeptide with a length of 10-20 amino acids is selected as an immune antigen and immunized after coupling with a carrier. Hemocyanin (KLH) is a chemical antibody conjugate. A study used KLH as a carrier protein to couple with the bovine viral diarrhea virus (BVDV) p7 protein antigen and an artificially synthesized polypeptide to successfully prepare a good rabbit anti-BVDV p7 polypeptide polyclonal antibody with good reactivity and specificity.

[0005] The present invention selects the RBD region without redundant amino acids to synthesize antigen epitopes, and couples them with KLH carriers to ensure that they have high specificity and affinity. After immunizing mice, PEDV RBD polypeptide antibodies are prepared, which not only provides an antibody for rapid detection of PEDV, but also provides ideas for the study of immunogenicity of PEDV subunit vaccines. Summary of the invention

[0006] In view of this, the present invention provides a method for preparing and identifying a PEDV RBD protein polypeptide antibody.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0008] A method for preparing and identifying a PEDV RBD protein polypeptide antibody comprises the following steps:

[0009] Step 1: Design of PEDV RBD antigen epitope and peptide synthesis;

[0010] Step 2: Immunize mice with peptide antigens to prepare specific antibodies

[0011] Freund's complete adjuvant and the prepared PEDV polypeptide are mixed and shaken in proportion, and injected to immunize mice after being fully emulsified. After a period of time after the first immunization, the second and third immunizations are performed with the same dose, and the polypeptide is prepared for booster immunization. Blood is collected under anesthesia, and the serum is separated and stored for testing; the separated serum is appropriately tilted and placed in a test tube to make a slope overnight; the serum is centrifuged the next day, and the supernatant is collected for later use;

[0012] Step 3: Transfect Expi293F cells with the eukaryotic expression plasmid pcDNA3.1-PER-Strep;

[0013] Step 4: The virus infects the host cell;

[0014] Step 5: Western Blot detection of PEDV RBD target protein expression

[0015] The protein samples included PEDV RBD protein expressed by Expi293F cells transfected with pcDNA3.1-PDR-Strep, and PEDV RBD protein expressed by SF9 cells infected with baculovirus; the protein samples were mixed with 5x SDS Loading Buffer in proportion and then boiled, subjected to SDS-PAGE and then transferred to NC membrane; blocked at room temperature with skim milk powder; the protein samples were diluted and Strep tag and mouse gradient dilution serum were used as primary antibodies, incubated and washed; then HRP-labeled goat anti-mouse IgG was used as secondary antibody, incubated at room temperature, and finally observed with ECL luminescent liquid;

[0016] Step 6: IFA detection of specific proteins expressed by virus-infected host cells

[0017] Vero cells in good condition were inoculated, and after the cell growth reached the required level, the culture medium was discarded, washed, and the PEDV infection solution was inoculated into the Vero cells. After culture, the virus infection solution was discarded and washed; paraformaldehyde was used for fixation and washing; TrionX-100 permeabilization was performed, washed, skim milk was blocked, and washed; PEDV polypeptide antibody was diluted and incubated at room temperature and washed; FITC-labeled goat anti-mouse IgG was diluted and incubated at room temperature in the dark and washed; DAPI staining solution was incubated at room temperature in the dark; observation was performed after washing;

[0018] Step 7: Indirect ELISA to detect peptide antibody titer

[0019] The PEDV RBD protein was coated, and the wells were blocked with skim milk and PBST. The mouse serum polyclonal antibody was diluted and added to each well for incubation. The goat anti-mouse IgG was labeled and incubated. The color developing solution was added for color development. The stop solution was added to stop the reaction, and the D450nm value was measured.

[0020] Preferably, in step 1, the specific method is: first, according to the S protein sequence of PEDV, the biological function analysis is performed by software to predict the potential antigenic sites of PEDV; predict its hydrophilicity and hydrophobicity; PEDV RBD amino acid sequence is used for protein secondary structure analysis; the 3D structure of PEDV RBD protein is predicted, and the synthetic antigen epitope site is predicted; amino acid sequence alignment is performed for similarity analysis. Finally, through comprehensive analysis, a polypeptide fragment located in a specific region of PEDV RBD is designed, and it is coupled with the carrier protein hemocyanin (Keyhole limpet hemocyanin, KLH) to prepare short peptide synthesis.

[0021] Preferably, in step 2, the specific method is: 200 μL of Freund's complete adjuvant and 200 μg of the prepared PEDV polypeptide are mixed and shaken at a volume ratio of 1:1, and after sufficient emulsification, 3 female Balb / c mice aged 4-6 weeks are immunized with an immunization dose of 200 μg per mouse by intramuscular injection. On 21 days and 42 days after the first immunization, the second and third immunizations are performed with the same dose, respectively, and on 56 days, a polypeptide prepared with an immunization dose of 500 μg per mouse is used for booster immunization, and blood is collected under anesthesia on 59 days, and the serum is separated and stored for inspection; the separated serum is appropriately tilted and placed in a test tube to make a slope, and stored at 4°C overnight; the serum is centrifuged at 5000 r / min for 10 minutes the next day, and the supernatant is collected after repeated twice, and the serum of mice before immunization is used as a negative control.

[0022] Preferably, in step 3, the specific method is: before transfection, the Expi293F cells are suspended and cultured at 37°C and 8% CO2. When the cell density reaches 4.5×10 6 -5.5×10 6 When the viability is 95%-99%, dilute the cells to 3×10 6 / mL; the transfection system is 50μg of eukaryotic expression vector pcDNA3.1-PER-Strep and 3mL Opti-MEMTM culture medium are mixed separately, and 160μL Expi FectamineTM 293 transfection reagent is mixed separately with 2.8mL Opti-MEMTM culture medium. After standing at room temperature for 5 minutes, the transfection reagent is added to the ep tube of the eukaryotic expression vector and continued to stand for 20 minutes; then the mixed system is slowly transferred to the cell shake flask containing 50mL Expi293F cell culture by dropwise addition, and the shake flask is gently rotated while adding dropwise; after the transfection is completed, the culture is continued in a 37℃ shaker, and the transfection enhancer is added 18h ​​after transfection, and the culture is continued for 5d; after 5d, the cell culture is centrifuged at 3000r / min for 20min, and the supernatant is collected and stored for later use.

[0023] Preferably, in step 4, the specific method is: culturing Vero cells with DMEM complete medium containing 10% fetal bovine serum and 1% double antibody, inoculating them into a 12-well plate so that the cell density reaches 80%, discarding the DMEM complete medium, and washing the cells 3 times with PBS; adding DMEM medium containing 2% FBS, and inoculating 0.5MOI of PEDV infection fluid into Vero cells, TGEV and PDCoV infection fluid into ST cells; 272h after infection, digesting the cells with 1x trypsin, centrifuging at 3500r / min for 5min, and collecting the cell precipitate.

[0024] Preferably, in step 5, the protein sample is mixed with 5x SDS Loading Buffer in a ratio of 4:1 and then boiled for 10 minutes, subjected to SDS-PAGE and then transferred to an NC membrane; blocked with 5% skim milk powder at room temperature for 2 hours; the PEDV RBD protein expressed by the transfected Expi293F cells and the PEDV RBD protein expressed by the SF9 cells are diluted respectively and Strep tags and mouse gradient dilution serum are used as primary antibodies, with concentrations of 1:500, 1:1000, and 1:2000, respectively; for the PEDV S protein expressed by the PEDV virus-infected Vero cells, a 1:1000 diluted mouse serum is used as the primary antibody for Western Blot detection, and the mixture is incubated overnight at 4°C and washed 3 times with TBST.

[0025] Preferably, in step 5, HRP-labeled goat anti-mouse IgG is used as the secondary antibody at a dilution of 1:5000, incubated at room temperature for 40 minutes, and finally developed with ECL luminescent solution for observation.

[0026] Preferably, in step 6, the specific method is: inoculating Vero cells in good condition into a 12-well plate, discarding the culture medium after the cells grow to 90%, washing the cells twice with PBS, and then inoculating 0.5 MOI of PEDV infection solution into the Vero cells in each well, discarding the virus infection solution after culturing for 48 hours, and washing the cells twice with PBS.

[0027] Preferably, in step 6, fixation with 4% paraformaldehyde for 20 minutes and washing with PBS for 3 times; permeabilization with TrionX-100 for 10 minutes, washing with PBS for 3 times, blocking with 5% skim milk for 1 hour, and washing with PBST for 3 times.

[0028] Preferably, in step 6, the PEDV polypeptide antibody is diluted 1:500 and incubated at room temperature for 2 hours, and washed 3 times with PBST; the FITC-labeled goat anti-mouse IgG is diluted 1:1000 and incubated at room temperature in the dark for 30 minutes; washed 3 times with PBST; incubated with DAPI staining solution at room temperature in the dark for 20 minutes; and observed under a fluorescence microscope after washing 3 times with PBST.

[0029] Preferably, in step 7, the specific method is: coating 5 μg / mL PEDV RBD protein on a 96-well plate, overnight at 4°C, blocking with 5% skim milk and PBST at 37°C for 1h; diluting mouse serum polyclonal antibody by multiples of 1:400, 1:800, 1:1600, 1:3200, 1:6400, and 1:12800; adding the diluted antibody to each well at 100 μL per well, and incubating at 37°C for 1h; adding 100 μL HRP-labeled goat anti-mouse IgG to each well at a dilution of 1:5000, and incubating at 37°C for 30min; adding 100 μL TMB colorimetric solution for color development for 5min, adding 100 μL stop solution to terminate the reaction, and measuring the D450nm value.

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

[0031] (1) The polypeptide antibodies prepared by the present invention have good specificity and reactivity;

[0032] (2) The present invention can be used as an antibody with specific binding effect in the anti-PEDV RBD detection method;

[0033] (3) The present invention provides an important research and diagnostic tool for further research on PEDV S protein;

[0034] (4) The present invention provides a new detection antibody for the rapid detection of PEDV in clinical diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the predicted structure diagram of the PEDV RBD protein of the present invention;

[0036] Among them, A. Hydrophilicity analysis; B. Protein secondary structure prediction, the green arrow represents the β-fold structure, and B represents the hydrophobicity of the PEDV RBD amino acid sequence; C. Protein tertiary structure prediction;

[0037] Figure 2 is a diagram comparing the similarity of epitope amino acid sequences between different virus strains of the present invention;

[0038] Among them, A. Comparison of similarity with PEDV virus strain; B. Comparison of similarity with TGEV virus strain; C. Comparison of similarity with PDCoV virus strain;

[0039] Figure 3 This is a graph of the mouse PEDV RBD polypeptide antibody of the present invention detecting the PEDV RBD protein expressed in the transfected Expi293F cells;

[0040] Among them, A. 1:500 PEDV polypeptide antibody as primary antibody; B. 1:1000 PEDV polypeptide antibody as primary antibody; C. 1:2000 PEDV polypeptide antibody as primary antibody; D. Strep tag (1:1000) as primary antibody;

[0041] M. Protein molecular mass standard; 1. Blank cell control; 2. PEDV RBD protein expressed by transfected Expi293F cells;

[0042] Figure 4 This is a diagram of the mouse PEDV RBD polypeptide antibody of the present invention detecting the PEDV RBD protein expressed by the transfected baculovirus system;

[0043] Among them, A. 1:500 PEDV polypeptide antibody as primary antibody; B. 1:1000 PEDV polypeptide antibody as primary antibody; C. 1:2000 PEDV polypeptide antibody as primary antibody; D. Strep tag (1:1000) as primary antibody;

[0044] M. Protein molecular mass standard; 1. Blank cell control; 2. PEDV RBD protein expressed by baculovirus system

[0045] Figure 5 This is a graph showing the specific protein expressed in infected Vero cells detected by the mouse PEDV RBD polypeptide antibody of the present invention;

[0046] Among them, A. 1:500 PEDV polypeptide antibody as primary antibody; B. 1:1000 PEDV polypeptide antibody as primary antibody; C. 1:2000 PEDV polypeptide antibody as primary antibody; D. Strep tag (1:1000) as primary antibody;

[0047] M. Protein molecular mass standard; 1. Blank cell control; 2. PEDV infected Vero cells; 3. TGEV infected ST cells; 4. PDCoV infected ST cells;

[0048] Figure 6 Identification of specific proteins expressed by PEDV infection by IFA (200x);

[0049] Figure 7 This is a graph showing the titer of PEDV RBD polypeptide antibodies detected by ELISA. DETAILED DESCRIPTION

[0050] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0051] Embodiment 1:

[0052] Material:

[0053] The SF9 insect cell line and P3-generation recombinant baculovirus were maintained by our laboratory;

[0054] pFastBac TM The dual vector was purchased from Thermo Fisher Scientific (China) Co., Ltd.;

[0055] The eukaryotic expression vector pcDNA3.1-PEDVRBD-Strep containing the PEDV RBD protein and the PEDV RBD protein expressed by the insect baculovirus system were prepared by our laboratory;

[0056] The short peptide prepared by coupling PEDV RBD protein (PER) with KLH was synthesized by Jiangsu GenScript Biotechnology Co., Ltd.;

[0057] African green monkey kidney cells (Vero cells) and porcine testicular cells (ST cells) were purchased from the American Type Culture Collection (ATCC);

[0058] Human kidney suspension cells (Expi293F) were purchased from Thermo Fisher Scientific (China) Co., Ltd.;

[0059] The PDCoV virus strain (Genbank accession number: MW816149) was kindly donated by Jia Aiqing, a researcher at Guangdong Haida Group Research Institute;

[0060] The PEDV virus strain (Genbank accession number: AKN45969.1) and the TGEV virus strain (Genbank accession number: OM802899) were maintained by our laboratory;

[0061] SF9 cell culture medium, DMEM medium, Expi293F medium, and fetal bovine serum were purchased from Gibco;

[0062] BALB / c mice were purchased from Jintai Medi Biotechnology Co., Ltd.;

[0063] Freund's complete and incomplete adjuvants were purchased from Sigma;

[0064] HRP-labeled goat anti-mouse secondary antibody, antibody diluent, and BCA protein quantification kit were purchased from Biotechnology Co., Ltd.;

[0065] TMB colorimetric solution and stop solution were purchased from Beijing Solebow Technology Co., Ltd.

[0066] method:

[0067] Step 1: Design and peptide synthesis of PEDV RBD antigen epitope

[0068] First, the biological function analysis was performed based on the S protein sequence of PEDV through software to predict the potential antigenic sites of PEDV; the PEDV RBD amino acid sequence was input using the Expasy-ProtScale website to predict its hydrophilicity and hydrophobicity; the PEDV RBD amino acid sequence was analyzed for protein secondary structure using the Jpred4 website; the 3D structure of the PEDV RBD protein was predicted using the SWISS-Model protein 3D structure modeling website, and the antigenic epitope site was synthesized after prediction; then the antigenic epitope sequence of PEDV RBD was compared with the PEDV virus strain, TGEV virus strain and other porcine coronavirus strains in Genbank using MAGA11 software for amino acid sequence alignment and similarity analysis; finally, through comprehensive analysis, a polypeptide fragment located in a specific region of PEDVRBD was designed, which was coupled with the carrier protein hemocyanin (Keyhole limpet hemocyanin, KLH) to prepare a short peptide, which was then synthesized by Jiangsu GenScript Biotechnology Co., Ltd.;

[0069] Step 2: Immunize mice with peptide antigens to prepare specific antibodies

[0070] 200 μL of Freund's complete adjuvant and 200 μg of prepared PEDV polypeptide were mixed and shaken at a volume ratio of 1:1. After sufficient emulsification, 200 μg of each immunization dose was used for intramuscular injection to immunize 3 female Balb / c mice aged 4-6 weeks. The same dose was used for the second and third immunizations at 21d and 42d after the first immunization. On the 56th day, 500 μg of each immunization dose of prepared polypeptide was used for booster immunization. Blood was collected under anesthesia on the 59th day, and the serum was separated and stored for testing. The separated serum was appropriately tilted and placed in a test tube to make a slope, and stored at 4°C overnight; the next day, the serum was centrifuged at 5000 r / min for 10 minutes, and the supernatant was collected after two repetitions. The serum of mice before immunization was used as a negative control;

[0071] Step 3: Transfect Expi293F cells with eukaryotic expression plasmid pcDNA3.1-PER-Strep

[0072] Before transfection, Expi293F cells were cultured in suspension at 37°C and 8% CO2. When the cell density reached 4.5×10 6 -5.5×10 6 When the viability is 95%-99%, dilute the cells to 3×10 6 / mL; the transfection system is 50μg of eukaryotic expression vector pcDNA3.1-PER-Strep and 3mL Opti-MEMTM medium are mixed separately, and 160μLExpi FectamineTM 293 transfection reagent is mixed separately with 2.8mL Opti-MEMTM medium. After standing at room temperature for 5 minutes, the transfection reagent is added to the ep tube of the eukaryotic expression vector and continued to stand for 20 minutes; then the mixed system is slowly transferred to the cell shake flask containing 50mL Expi293F cell culture by dropwise addition, and the shake flask is gently rotated while dropping. After the transfection is completed, continue to culture in a 37℃ shaker, add the transfection enhancer 18h after transfection, and continue to culture for 5d; after 5d, the cell culture is centrifuged at 3000r / min for 20min, and the supernatant is collected and stored for later use;

[0073] Step 4: Virus infects host cells

[0074] Vero cells were cultured with DMEM complete medium containing 10% fetal bovine serum and 1% double antibody, inoculated into 12-well plates to a cell density of 80%, and then the DMEM complete medium was discarded and the cells were washed 3 times with PBS; then, DMEM medium containing 2% FBS was added, and 0.5 MOI of PEDV infection fluid was inoculated into Vero cells, and TGEV and PDCoV infection fluid were inoculated into ST cells respectively; 272 h after infection, the cells were digested with 1x trypsin, centrifuged at 3500 r / min for 5 min, and the cell precipitate was collected; then, protein lysis buffer was added, the cells were broken under ultrasonic conditions, and the supernatant protein was collected;

[0075] Step 5: Western Blot detection of PEDV RBD target protein expression

[0076] Western Blot analysis of target protein expression, including PEDV RBD protein expressed by Expi293F cells transfected with pcDNA3.1-PDR-Strep, and PEDV RBD protein expressed by SF9 cells infected with baculovirus; samples were mixed with 5x SDS Loading Buffer at a ratio of 4:1 and boiled for 10 min, subjected to SDS-PAGE and then transferred to NC membrane; then, 5% skim milk powder was used for blocking at room temperature for 2 h; PEDV RBD protein expressed by Expi293F cells transfected with pcDNA3.1-PDR-Strep and PEDV RBD protein expressed by SF9 cells were diluted and Strep tag and mouse gradient dilution serum were used as primary antibodies at concentrations of 1:500, 1:1000, and 1:2000, respectively; for PEDV S protein expressed by Vero cells infected with PEDV virus, mouse serum diluted 1:1000 was used as primary antibody for Western Blot. Blot detection, after overnight incubation at 4°C, wash with TBST three times; then use HRP-labeled goat anti-mouse IgG (1:5000 dilution) as the secondary antibody, incubate at room temperature for 40 minutes, and finally observe with ECL luminescent solution;

[0077] Step 6: IFA detection of specific proteins expressed by virus-infected host cells

[0078] Vero cells in good condition were inoculated into 12-well plates. After the cells grew to 90%, the culture medium was discarded, and the cells were washed twice with PBS. Then, 0.5 MOI of PEDV infection solution was added to each well of Vero cells. After 48 hours of culture, the virus infection solution was discarded and the cells were washed twice with PBS; fixed with 4% paraformaldehyde for 20 minutes and washed with PBS three times; permeabilized with TrionX-100 for 10 minutes, washed with PBS three times, blocked with 5% skim milk for 1 hour, and washed with PBST three times; incubated with PEDV polypeptide antibody diluted 1:500 at room temperature for 2 hours, and washed with PBST three times; incubated with FITC-labeled goat anti-mouse IgG diluted 1:1000 at room temperature in the dark for 30 minutes; washed with PBST three times; incubated with DAPI staining solution at room temperature in the dark for 20 minutes; and observed under a fluorescence microscope after washing with PBST three times;

[0079] Step 7: Indirect ELISA to detect peptide antibody titer

[0080] 5μg / mL PEDV RBD protein was coated on a 96-well plate at 4℃ overnight, and then blocked with 5% skim milk and PBST at 37℃ for 1h; mouse serum polyclonal antibody was diluted in multiples (1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800); 100μL of the diluted antibody was added to each well and incubated at 37℃ for 1h; 100μL of HRP-labeled goat anti-mouse IgG (dilution 1:5000) was added to each well and incubated at 37℃ for 30min; 100μL of TMB colorimetric solution was added for 5min, and then 100μL of stop solution was added to terminate the reaction and the OD450nm value was measured. The highest dilution factor that can detect a positive signal is the antibody titer.

[0081] Example 2 Results

[0082] 1. Design of PEDV RBD peptide

[0083] According to the amino acid sequence of PEDV RBD protein, the hydrophilicity, secondary structure and tertiary structure of RBD protein were comprehensively analyzed by bioinformatics software; Figure 1 A shows that after the hydrophilicity analysis of the PEDV RBD amino acid sequence, a continuous region with strong hydrophilicity was selected as the candidate region ( Figure 1 A, as shown in the red box); the secondary structure of the PEDV RBD amino acid sequence was predicted using the JPred website, where ( Figure 1 B), avoiding hydrophobic areas as much as possible; using SWISS-Model to complete the 3D modeling of PEDV RBD protein and predict the location of specific synthetic antigen epitopes ( Figure 1 C).

[0084] 2. Sequence alignment of PEDV RBD antigen epitopes

[0085] A short peptide sequence including the antigenic epitope of the RBD of PEDV and sequences of strains from various other sources were selected, and the similarity of the RBD short peptide sequence with those of other strains was compared using MAGA V11 software. Figure 2 The results showed that the selected PEDVRBD short peptide sequence had a sequence similarity of up to 100% with the other 18 PEDV strains, indicating that the epitope sequence was highly conserved. Sequence comparison with TGEV strains and PDCoV strains showed that the similarity between the target sequence and the 11 TGEV strains was 27.8%-29.3% ( Figure 2 B), and the similarity with the 16 PDCoV sequences was 10.5%-13.4% ( Figure 2C). Based on the above results and considering multiple factors such as the hydrophilic and hydrophobic regions of the epitope sequence and the conservation of the designed epitope, the LITGTPKPLEGVTD polypeptide was finally selected and coupled with the KLH carrier protein to prepare the KLH-Cys-LITGTPKPLEGVTD polypeptide.

[0086] 3. Western Blot detection of PEDV RBD protein expression in transfected Expi293F cells

[0087] Different gradients of mouse PEDV RBD polypeptide antibody and Strep antibody were used as primary antibodies for incubation. Western Blot detection showed that the PEDV RBD protein expressed by the transfected Expi293 cells could be detected when the polyclonal antibody was diluted at 1:500, 1:1000, and 1:2000 ( Figure 3 AC), indicating that the polypeptide antibody can detect the 25kDa target protein band at different dilution ratios, indicating that the antibody has high detection sensitivity. At the same time, Strep antibody with a dilution ratio of 1:1000 was incubated as a control antibody, and the specific target band was also detected, and the protein size was consistent with the theoretical value ( Figure 3 D).

[0088] 4. Western Blot detection of PEDV RBD protein expression in SF9 cells infected with baculovirus

[0089] Different gradients of mouse PEDV RBD polypeptide antibody and Strep antibody were used as primary antibodies for overnight incubation. Western Blot detection showed that the polyclonal antibody dilution ratio of 1:500, 1:1000, and 1:2000 could detect the PEDV RBD protein expressed in SF9 cells infected with baculovirus ( Figure 4 AC), indicating that the polypeptide antibody can detect the 25kDa target protein band at different dilution ratios. Similarly, the Strep antibody with a dilution ratio of 1:1000 as the control antibody can also detect the specific target band, which is consistent with the theoretical value ( Figure 4 D).

[0090] 5. Western Blot detection of specific proteins expressed by virus-infected host cells

[0091] PEDV infected Vero cells to prepare protein samples, mouse PEDV RBD polypeptide antibody and commercial PEDV N protein antibody were used as primary antibodies for incubation, and TGEV and PDCoV infected ST cells to prepare protein samples, mouse PEDV RBD polypeptide antibody was used as primary antibody for incubation. Western Blot results showed that mouse PEDV RBD polypeptide antibody could detect PEDV S protein expressed in PEDV infected Vero cells. Since S protein was cleaved into S1 and S2, the detected protein size was about 70Kda ( Figure 5 A), while no specific proteins expressed by TGEV and PDCoV were detected ( Figure 5 BC), indicating that the specificity of the polypeptide antibody is good. The commercial antibody against PEDV N protein can also detect the PEDV N protein expressed by PEDV-infected Vero cells, which also reflects that the protein sample prepared in this experiment has good antigenicity ( Figure 5 D).

[0092] 6. IFA Identification of Specific Proteins Expressed by PEDV Infection

[0093] PEDV RBD polypeptide antibody was used to perform IFA detection on Vero cells infected with PEDV, and Vero cells not infected with PEDV were used as negative control. As shown in the figure, no specific green fluorescence was observed in the blank control group, while obvious specific fluorescence was observed in Vero cells infected with PEDV, which was basically consistent with the positive control group of PEDV-N. The results showed that the polypeptide antibody prepared in this test has good specificity ( Figure 6 ).

[0094] 7. ELISA to detect the titer of mouse PEDV RBD polypeptide antibody

[0095] Mouse serum before the first immunization was collected as a negative control, and the indirect ELISA method was used to detect the titer of PEDV RBD polypeptide antibodies in mice after immunization. Mouse polypeptide antibodies of different dilutions were used as primary antibodies for incubation, and the highest dilution multiple at which a positive signal was detected was used as the antibody titer. The results showed that it had a significant reaction with PEDV RBD antigen, and the polypeptide antibody titer was 1:25600 ( Figure 7 ).

[0096] The above description is only a preferred embodiment of the present invention and does not limit the technical scope of the present invention. Therefore, any slight modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for preparing and identifying a PEDV RBD protein polypeptide antibody, characterized in that: The following steps are involved: Step 1: Design of PEDV RBD antigen epitope and peptide synthesis; Step 2: Immunize mice with peptide antigens to prepare specific antibodies Freund's complete adjuvant and the prepared PEDV polypeptide are mixed and shaken in proportion, and injected to immunize mice after being fully emulsified. After a period of time after the first immunization, the second and third immunizations are performed with the same dose, and the polypeptide is prepared for booster immunization. Blood is collected under anesthesia, and the serum is separated and stored for testing; the separated serum is appropriately tilted and placed in a test tube to make a slope overnight; the serum is centrifuged the next day, and the supernatant is collected for later use; Step 3: Transfect Expi293F cells with the eukaryotic expression plasmid pcDNA3.1-PER-Strep; Step 4: The virus infects the host cell; Step 5: Western Blot detection of PEDV RBD target protein expression The protein samples included PEDV RBD protein expressed by Expi293F cells transfected with pcDNA3.1-PDR-Strep, and PEDV RBD protein expressed by SF9 cells infected with baculovirus; the protein samples were mixed with 5x SDS Loading Buffer in proportion and then boiled, subjected to SDS-PAGE and then transferred to NC membrane; blocked at room temperature with skim milk powder; the protein samples were diluted and Strep tag and mouse gradient dilution serum were used as primary antibodies, incubated and washed; then HRP-labeled goat anti-mouse IgG was used as secondary antibody, incubated at room temperature, and finally observed with ECL luminescent liquid; Step 6: IFA detection of specific proteins expressed by virus-infected host cells Vero cells in good condition were inoculated, and after the cell growth reached the required level, the culture medium was discarded, washed, and the PEDV infection solution was inoculated into the Vero cells. After culture, the virus infection solution was discarded and washed; paraformaldehyde was used for fixation and washing; TrionX-100 permeabilization was performed, washed, skim milk was blocked, and washed; PEDV polypeptide antibody was diluted and incubated at room temperature and washed; FITC-labeled goat anti-mouse IgG was diluted and incubated at room temperature in the dark and washed; DAPI staining solution was incubated at room temperature in the dark; observation was performed after washing; Step 7: Indirect ELISA to detect peptide antibody titer The PEDV RBD protein was coated, and the wells were blocked with skim milk and PBST. The mouse serum polyclonal antibody was diluted and added to each well for incubation. The goat anti-mouse IgG was labeled and incubated. The color developing solution was added for color development. The stop solution was added to stop the reaction, and the D450nm value was measured.

2. The method for preparing and identifying a PEDV RBD protein polypeptide antibody according to claim 1, characterized in that: In the step 1, the specific method is: first, according to the S protein sequence of PEDV, biological function analysis is performed by software to predict the potential antigenic sites of PEDV; its hydrophilicity and hydrophobicity are predicted; the PEDV RBD amino acid sequence is used for protein secondary structure analysis; the 3D structure of the PEDV RBD protein is predicted, and the synthetic antigen epitope site is predicted; amino acid sequence alignment is performed, similarity analysis is performed, and finally, through comprehensive analysis, a polypeptide fragment located in a specific region of PEDV RBD is designed, and it is coupled with the carrier protein hemocyanin to prepare short peptide synthesis.

3. The method for preparing and identifying a PEDV RBD protein polypeptide antibody according to claim 1, characterized in that: In the step 2, the specific method is: 200 μL of Freund's complete adjuvant and 200 μg of the prepared PEDV polypeptide are mixed and shaken at a volume ratio of 1:1, and after sufficient emulsification, 200 μg of each immunization dose is used for intramuscular injection to immunize 3 female Balb / c mice aged 4-6 weeks, and the same dose is used for the second and third immunizations at 21d and 42d after the first immunization, respectively, and the polypeptide prepared at an immunization dose of 500 μg per mouse is used for booster immunization at 56d, and blood is collected under anesthesia at 59d, and the serum is separated and stored for inspection; the separated serum is appropriately tilted and placed in a test tube to make a slope, and stored at 4°C overnight; the serum is centrifuged at 5000 r / min for 10 min the next day, and the supernatant is collected after repeated twice, and the serum of mice before immunization is used as a negative control.

4. The method for preparing and identifying a PEDV RBD protein polypeptide antibody according to claim 1, characterized in that: In step 3, the specific method is as follows: before transfection, the Expi293F cells were suspended and cultured at 37°C and 8% CO2. When the cell density reached 4.5×10 6 -5.5×10 6 When the viability is 95%-99%, dilute the cells to 3×10 6 / mL; the transfection system is 50μg of eukaryotic expression vector pcDNA3.1-PER-Strep and 3mL Opti-MEMTM culture medium are mixed separately, and 160μL Expi FectamineTM 293 transfection reagent is mixed separately with 2.8mL Opti-MEMTM culture medium. After standing at room temperature for 5 minutes, the transfection reagent is added to the ep tube of the eukaryotic expression vector and continued to stand for 20 minutes; then the mixed system is slowly transferred to the cell shake flask containing 50mL Expi293F cell culture by dropwise addition, and the shake flask is gently rotated while adding dropwise; after the transfection is completed, the culture is continued in a 37℃ shaker, and the transfection enhancer is added 18h ​​after transfection, and the culture is continued for 5d; after 5d, the cell culture is centrifuged at 3000r / min for 20min, and the supernatant is collected and stored for later use.

5. The method for preparing and identifying a PEDV RBD protein polypeptide antibody according to claim 1, characterized in that: In the step 4, the specific method is: Vero cells are cultured with DMEM complete medium containing 10% fetal bovine serum and 1% double antibody, and the cells are inoculated into a 12-well plate to a cell density of 80%, and then the DMEM complete medium is discarded, and the cells are washed 3 times with PBS; DMEM medium containing 2% FBS is added, and 0.5 MOI of PEDV infection fluid is inoculated into Vero cells, and TGEV and PDCoV infection fluid are inoculated into ST cells respectively; 272 h after infection, the cells were digested with 1x trypsin, centrifuged at 3500 r / min for 5 min, and the cell pellets were collected.

6. The method for preparing and identifying a PEDV RBD protein polypeptide antibody according to claim 1, characterized in that: In the step 5, the protein sample was mixed with 5x SDS Loading Buffer at a ratio of 4:1 and then boiled for 10 minutes, subjected to SDS-PAGE and then transferred to the NC membrane; 5% skim milk powder was used for blocking at room temperature for 2 hours; the PEDVRBD protein expressed by the transfected Expi293F cells and the PEDV RBD protein expressed by the SF9 cells were diluted respectively and Strep tag and mouse gradient dilution serum were used as primary antibodies, with concentrations of 1:500, 1:1000, and 1:2000, respectively; for the PEDV S protein expressed by the PEDV virus-infected Vero cells, 1:1000 diluted mouse serum was used as the primary antibody for Western Blot detection, and after overnight incubation at 4°C, the membrane was washed 3 times with TBST; HRP-labeled goat anti-mouse IgG was used as the secondary antibody at a dilution of 1:5000, incubated at room temperature for 40 min, and finally developed with ECL luminescent solution for observation.

7. The method for preparing and identifying a PEDV RBD protein polypeptide antibody according to claim 1, characterized in that: In step 6, the specific method is: inoculate Vero cells in good condition into a 12-well plate, discard the culture medium after the cells grow to 90%, wash the cells twice with PBS, and then inoculate 0.5 MOI of PEDV infection solution into the Vero cells in each well, discard the virus infection solution after culturing for 48 hours, and wash the cells twice with PBS.

8. The method for preparing and identifying a PEDV RBD protein polypeptide antibody according to claim 1, characterized in that: In step 6, fix with 4% paraformaldehyde for 20 minutes, wash with PBS three times; permeabilize with TrionX-100 for 10 minutes, wash with PBS three times, block with 5% skim milk for 1 hour, and wash with PBST three times.

9. The method for preparing and identifying a PEDV RBD protein polypeptide antibody according to claim 1, characterized in that: In step 6, the PEDV polypeptide antibody was diluted 1:500 and incubated at room temperature for 2 hours, and then washed 3 times with PBST; the FITC-labeled goat anti-mouse IgG was diluted 1:1000 and incubated at room temperature in the dark for 30 minutes; the PBST was washed 3 times; the DAPI staining solution was incubated at room temperature in the dark for 20 minutes; and the cells were washed 3 times with PBST and observed under a fluorescence microscope.

10. The method for preparing and identifying a PEDV RBD protein polypeptide antibody according to claim 1, characterized in that: In the step 7, the specific method is: 5 μg / mL PEDV RBD protein is coated on a 96-well plate, overnight at 4°C, and blocked with 5% skim milk and PBST at 37°C for 1 hour; the mouse serum polyclonal antibody is diluted by 1:400, 1:800, 1:1600, 1:3200, 1:6400, and 1:12800; 100 μL of the diluted antibody is added to each well and incubated at 37°C for 1 hour; 100 μL of HRP-labeled goat anti-mouse IgG is added to each well at a dilution of 1:5000, and incubated at 37°C for 30 minutes; 100 μL of TMB color development solution is added for color development for 5 minutes, 100 μL of stop solution is added to terminate the reaction, and the 0D450nm value is measured.

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