Preparation method of influenza D virus HEF protein polyclonal antibody
By constructing the pET28a-HEF recombinant plasmid in E. coli and purify the HEF protein, the immune process is optimized, and the high-titer and specificity polyclonal antibodies are prepared, which solves the problems of poor antigen purity and antibody specificity in traditional methods, and the preparation of high-quality antibodies is achieved.
Patent Information
- Application Number
- CN202510498964.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-21
- Publication Date
- 2025-07-08
AI Technical Summary
The antigen purity and poor antibody specificity in traditional polyclonal antibody preparation methods affect the research and application effect of HEF protein of influenza D virus.
By constructing a pET28a-HEF recombinant plasmid, HEF protein was efficiently expressed and purified in E. coli, the immune process was optimized, polyclonal antibodies were prepared, and the purified HEF protein was used as an antigen to immunize Japanese white rabbits, and the antibody preparation process was optimized.
Obtaining polyclonal antibodies with high titer and strong specificity solves the problems of antigen purity and antibody specificity, improves the quality and application feasibility of the antibodies, and has wide application prospects.
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Figure CN120271700A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of influenza D virus HEF protein technology, specifically relating to a method for preparing polyclonal antibodies against influenza D virus HEF protein. Background Technology
[0002] In biological and medical research, polyclonal antibodies are widely used because they are relatively easy to prepare, inexpensive, and can recognize multiple antigenic epitopes.
[0003] HEF protein is a key factor in the replication and pathogenesis of influenza D virus (IDV), and the preparation of its polyclonal antibody is of great significance for the research, diagnosis and vaccine development of IDV.
[0004] However, traditional polyclonal antibody preparation methods often suffer from problems such as low antigen purity and poor antibody specificity, affecting antibody quality and application efficacy. In particular, the preparation of polyclonal antibodies against the HEF protein, due to the complexity of IDV and the specific characteristics of the HEF protein, urgently requires an efficient and reliable antibody preparation method.
[0005] The present invention aims to provide a novel method for preparing polyclonal antibodies against the HEF protein of influenza D virus. Summary of the Invention
[0006] The purpose of this invention is to provide a method for preparing a polyclonal antibody against the HEF protein of influenza D virus, so as to solve the problems mentioned in the background art.
[0007] To achieve the above objectives, the present invention provides the following technical solution:
[0008] A method for preparing polyclonal antibodies against the HEF protein of influenza D virus includes the following steps:
[0009] Construction of S1 and pET28a-HEF plasmids
[0010] S11. Primer design: Based on the nucleic acid sequence of GenBank InfluenzaD virus HEF (NC_036620.1), antigenic epitopes were screened through bioinformatics analysis, and primers containing BamHI and XhoI restriction sites were designed in conjunction with the sequence of the expression vector pET28a.
[0011] S12. Target gene amplification: Using the cDNA of D / swine / Oklahoma / 1334 / 2011 preserved in the laboratory as a template, the uniseamless-pET28a-HEF fragment was amplified using specific primers pET28a+HEF-F and pET28a+HEF-R for prokaryotic expression of HEF protein.
[0012] S13. Plasmid construction: The target gene uniseamless-pET28a-HEF and the expression vector pET28a were double-digested with enzymes, the digestion products were purified and then ligated using seamless cloning technology to construct the recombinant plasmid pET28a-HEF. The reaction product was transformed into E. coli DH5α competent cells, positive clones were screened and plasmids were extracted and sequenced for verification.
[0013] Expression and purification of S2 and HEF proteins
[0014] S21. Small-scale induction expression: pET28a-HEF plasmid was transformed into Transetta(DE3) competent cells, and the expression conditions were optimized by induction temperature, IPTG concentration and induction time. The product was identified by SDS-PAGE.
[0015] S22. Large-scale induction of expression: Select the optimal expression conditions for large-scale induction, collect the bacterial cells and lyse them to obtain a lysate containing the target protein;
[0016] S23. Purification: HEF protein is purified using affinity chromatography. The purification process involves steps such as filtration sterilization, nickel column equilibration, nickel column binding to protein, and protein elution to obtain high-purity HEF protein.
[0017] S3. Preparation and Identification of Polyclonal Antibodies
[0018] S31. Immunization of animals: Two-month-old Japanese rabbits were immunized with purified pET28a-HEF protein emulsified with Freund's adjuvant as antigen. The first immunization used complete Freund's adjuvant, and two booster immunizations were performed at 2 and 4 weeks apart using incomplete Freund's adjuvant.
[0019] S32. Collect serum: Collect serum 7 days after the last immunization to obtain polyclonal antibodies against HEF protein and store them at -80℃.
[0020] S33. Antibody titer detection: Antibody titer is determined by indirect ELISA detection method;
[0021] S34. Antibody specificity identification: Antibody specificity is determined by Western blot identification.
[0022] S35. Indirect immunofluorescence detection: This method identifies that the prepared HEF polyclonal antibody can specifically bind to eukaryotic expressed proteins.
[0023] Preferably, the primer sequence in S11 is:
[0024] The pET28a+HEF-F nucleotide sequence is as follows:
[0025] 5'-AATGGGTCGCGGATCCATGGCACAAGAACAACTACTTGCT-3';
[0026] The nucleotide sequence of primer pET28a+HEF-R is as follows:
[0027] 5'-GGTGGTGGTGCTCGAGCTTCCAGTCTCTTTTTAGGGCAAGAT-3';
[0028] The nucleotide sequence of primer T7-F is: 5'-TAATACGACTCACTATAGGG-3';
[0029] The nucleotide sequence of primer T7-R is: 5'-GCTAGTTATTGCTCAGCGG-3'.
[0030] Preferably, according to the method for preparing a polyclonal antibody against influenza D virus HEF protein as described in S12, the specific method for amplifying the HEF gene is as follows: using 1.5 μL of cDNA from D / swine / Oklahoma / 1334 / 2011 stored in the laboratory as a template, taking 1 μL of specific primers pET28a+HEF-F and pET28a+HEF-R, taking 12.5 μL of 2×HIFIMix, and using ddH2O to make up the remaining 25 μL of the system. Take the autoclaved PCR tubes, prepare the system on ice, mix well, and place them in a PCR instrument to perform a thermal cycling program containing the following steps for target gene amplification: 95℃ pre-denaturation for 5 min; 30 cycles of amplification steps, each cycle including 95℃ denaturation for 30 s, 62℃ annealing for 30 s, and 72℃ extension for 1 s; and a final extension at 72℃ for 5 min; after separation by agarose gel electrophoresis, the amplified product is purified by gel recovery technology to obtain the uniseamless-pET28a-HEF fragment.
[0031] Compared with the prior art, the beneficial effects of the present invention are:
[0032] This invention provides an efficient method for extracting and purifying HEF protein from influenza D virus (IDV).
[0033] By constructing the uniseamless-pET28a-HEF recombinant plasmid and achieving efficient expression and purification in *E. coli*, high-purity HEF protein was obtained, serving as an ideal antigen for the preparation of polyclonal antibodies. The optimized antibody preparation process involves using the purified HEF protein as the antigen to prepare polyclonal antibodies by immunizing Japanese white rabbits. This process is simple, controllable, and yields high antibody production. This method effectively solves the problems of low antigen purity and poor antibody specificity in traditional antibody preparation processes.
[0034] The prepared polyclonal antibody has high titer and good activity, exhibits high specificity for HEF protein, and has low cross-reactivity with other related proteins, ensuring its quality and application feasibility.
[0035] This invention also has broad application prospects. The HEF polyclonal antibody preparation method of this invention not only provides a powerful tool for in-depth research on IDV, but may also play an important role in the development of diagnostic reagents and vaccines for IDV. Furthermore, this method has potential versatility and can be applied to the preparation of polyclonal antibodies against other viruses or proteins. Attached Figure Description
[0036] Figure 1 The pET28a-HEF gene amplification results in the embodiments of the present invention.
[0037] Figure 2 The results of bacterial culture PCR identification in the embodiments of the present invention.
[0038] Figure 3 Optimization of induced temperature conditions in embodiments of the present invention
[0039] Figure 4 Optimization of IPTG induction conditions in embodiments of the present invention
[0040] Figure 5 Optimization of induction time conditions in embodiments of the present invention
[0041] Figure 6 SDS-PAGE identification of HEF protein in the embodiments of the present invention
[0042] Figure 7 Western blot identification of purified HEF protein in this embodiment of the invention.
[0043] Figure 8 Western blot identification of HEF polyclonal antibodies in this embodiment of the invention.
[0044] Figure 9 This is for the indirect immunofluorescence identification of HEF polyclonal antibodies in the embodiments of the present invention. Detailed Implementation
[0045] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0046] Example 1:
[0047] (I) Construction of pET28a-HEF plasmid
[0048] 1. Primer Design: Based on the nucleic acid sequence of GenBank Influenza D virus HEF (NC_036618.1), antigenic epitopes were screened through bioinformatics analysis. Primers containing BamHI and XhoI restriction sites were designed in conjunction with the sequence of the expression vector pET28a. Detailed primer sequences are shown in Table 1 and were synthesized by Jilin Kumei Biotechnology Co., Ltd.
[0049] Table 1 Primer Information
[0050]
[0051] 2. Target gene amplification: Using the cDNA of D / swine / Oklahoma / 1334 / 2011 preserved in the laboratory as a template, the uniseamless-pET28a-HEF fragment was amplified for prokaryotic expression of HEF protein using specific primers pET28a+HEF-F and pET28a+HEF-R, following the reaction system in Table 2 and the reaction procedure in Table 3.
[0052] Table 2 PCR reaction system
[0053]
[0054] Table 3 PCR reaction procedure
[0055]
[0056] The amplified products were separated and verified by agarose gel electrophoresis. The uniseamless-pET28a-HEF fragment was purified by gel recovery, and the purified sample was sent to Jilin Kumei Biotechnology for bidirectional sequencing analysis.
[0057] 3. Double digestion of the target fragment and expression vector
[0058] The pET28a(+) empty vector was double-digested with restriction endonucleases BamHI and XhoI in a water bath at 37°C for 30 min to obtain a linearized vector. The digestion system is shown in Table 4.
[0059] Table 4. Double Enzyme Digestion Reaction System
[0060]
[0061] 4. Connection between target fragment and expression carrier
[0062] The enzyme digestion products were subjected to agarose gel electrophoresis, and the target fragment was recovered using a kit. The pET28a(+) linearized vector and the uniseamless-pET28a-HEF fragment were seamlessly cloned using Assembly Mix. The seamless cloning reaction system is shown in Table 5.
[0063] Table 5 Seamless Cloning Reaction System
[0064]
[0065] 5. Recombinant vector transformation
[0066] After reacting at 50°C for 15 minutes in a PCR instrument, place the cells on ice for a few seconds to allow for transformation. Transform the reaction product into E. coli DH5α competent cells, as follows:
[0067] (1) After adding the reaction product to 50 μL of competent cells, let it stand in an ice-water mixture for 30 min.
[0068] (2) Place it in a 42℃ water bath for 1 minute for heat shock, and then quickly remove it and place it in an ice bath for 3 minutes.
[0069] (3) Add 0.5 mL of non-resistant LB to the clean bench and shake at 37°C for 1 h.
[0070] Spread 200 μL evenly onto kanamycin-resistant LB agar plates. Incubate upright at 37°C for 1 hour, then invert and incubate overnight.
[0071] 6. Screening for positive clones
[0072] The following day, colony growth was observed. In a clean bench, single colonies were picked using a sterile pipette tip and placed in Kna-resistant liquid LB medium, then incubated at 37°C for 4 hours using a shaker. Bacterial PCR was performed using the universal T7 primers and the specific primers pET28a+HEF-F / R. The reaction system and procedure are shown in Tables 6 and 7.
[0073] Table 6. Bacterial PCR Reaction System
[0074]
[0075] Table 7. PCR reaction procedure for bacterial culture
[0076]
[0077] 7. Plasmid extraction and sequencing
[0078] Plasmids were extracted from positive bacterial cultures and sent to Jilin Kumei Biotechnology Co., Ltd. for sequencing. The correctly sequenced plasmid was named pET28a-HEF.
[0079] (II) Expression and purification of HEF protein
[0080] 1. Small-scale induction expression: pET28a-HEF plasmid was transformed into Transetta(DE3) competent cells, and the expression conditions were optimized by induction temperature, IPTG concentration and induction time. The product was identified by SDS-PAGE.
[0081] (1) Induction temperature: Add an inducer with a final concentration of 0.1 mM IPTG to the bacterial culture, and then incubate at 16℃, 28℃, and 37℃ for 8 h at 180 rpm. After ultrasonic disruption, the supernatant and precipitate suspension are identified by SDS-PAGE.
[0082] (2) IPTG concentration: IPTG inducer with final concentrations of 0.05mM, 0.1mM, 0.3mM, 0.5mM, 0.7mM and 1.0mM was added to the bacterial culture. pET28a-HEF was cultured at 37℃ and 180rpm for 8h. After the culture was disrupted by sonication with Binding Buffer containing 6M urea, the supernatant was identified by SDS-PAGE.
[0083] (3) Induction time: IPTG inducer with a final concentration of 0.05 mM was added to pET28a-HEF bacterial culture and induced at 37℃ for 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h, respectively. The bacterial cells were sonicated with binding buffer containing 6 M urea, and the supernatant after disruption was identified by SDS-PAGE.
[0084] 2. Large-scale induction of expression: Select optimal expression conditions for large-scale induction. Centrifuge the bacterial culture at 8000g for 10 min, discard the supernatant and collect the bacterial cells. Resuspend the cells in PBS and wash them, then centrifuge at 8000g for 10 min and discard the supernatant. Wash the cells twice. Add binding buffer containing 6M urea and sonicate under ice bath conditions at 200W power, with a 5-second pulse interval of 5 seconds, for a total of 30 min. Centrifuge the sonicated lysis buffer at 8000g for 30 min and collect the supernatant for subsequent purification.
[0085] 3. Purification: The HEF protein was purified using affinity chromatography.
[0086] (1) Filtration sterilization: The sample, buffer solution, eluent and double-distilled water were filtered through a 0.22μm filter.
[0087] (2) Nickel column balancing: The nickel columns were balanced using Binding Buffer, balancing the volumes of 5 columns.
[0088] (3) Nickel column binding to protein: The sample and the equilibrated Ni-NTAResin packing material were packed into the column and bound in a chromatography cabinet at 4°C with a shaker at 50 rpm for 2 h.
[0089] (4) Protein elution: Elute the column supernatant and elute contaminating proteins with Binding Buffer (5 column volume, low imidazole concentration), followed by elution with Elution Buffer (3 column volume, high imidazole concentration). Label the eluents sequentially as: loading buffer, washing buffer, and elution buffer.
[0090] 4. Identification: Protein samples were prepared from the purified sample and the uninduced sample, and identified by SDS-PAGE and Western blot.
[0091] (1) The specific operation of SDS-PAGE is as follows:
[0092] Sample preparation: Take 40 μL of the sample to be tested and mix it with 5× protein loading buffer at a volume ratio of 4:1. Boil in a water bath for 10 min to fully denature the protein. Then centrifuge at 8000g for 5 min and collect the supernatant for later use.
[0093] Gel preparation: A 10% SDS-PAGE separating gel was prepared using the YARN protein gel preparation kit and allowed to stand for polymerization for 30 minutes.
[0094] Sample loading and electrophoresis: Accurately load 10 μL of the processed sample into each well. Initially set the electrophoresis voltage to 80 V. After the bromophenol blue indicator migrates to the separating gel interface, adjust the voltage to 120 V and continue electrophoresis until the indicator reaches the bottom of the colloid.
[0095] Staining and destaining: After electrophoresis, the separating gel was immersed in Coomassie Brilliant Blue staining solution and stained with shaking for 2 hours. Then it was transferred to destaining solution (10% acetic acid + 20% methanol) and stained with shaking for 8-12 hours until the background was transparent. Finally, the gel was collected and analyzed using a gel imaging system.
[0096] (2) The specific operation of Western blot is as follows:
[0097] Transfer procedure: After preparing the gel and performing electrophoresis according to the standard SDS-PAGE procedure, accurately cut out the gel region containing the target protein and protein marker; immerse the gel block, filter paper, and PVDF membrane activated with methanol for 30 seconds into the transfer buffer and equilibrate for 10 minutes; stack the transfer layers in the order of "filter paper-PVDF membrane-gel-filter paper", thoroughly remove air bubbles, and transfer at a constant voltage of 20V for 30 minutes.
[0098] Blocking and antibody incubation: After the transfer was completed, the PVDF membrane was immersed in 5% skim milk powder (dissolved in PBST) and blocked at room temperature for 1 h; the membrane was then transferred to a diluted primary antibody solution (1:2000 diluted in 5% skim milk powder) and incubated at 4°C with shaking for 12-16 h; the membrane was washed with PBST with shaking at room temperature (5 min × 3 times) to remove unbound antibodies.
[0099] Secondary antibody reaction and development: Immerse the membrane in HRP-labeled secondary antibody solution (1:5000 diluted in 5% skim milk powder) and incubate at room temperature with shaking for 40 min; after washing three times with PBST, mix ECL chemiluminescence solution A / B at a 1:1 ratio and evenly cover the membrane surface; expose in a dark room with a chemiluminescence imager for 0.2-5 min to capture specific signals.
[0100] (III) Preparation and Identification of Polyclonal Antibodies
[0101] 1. Animal immunization program
[0102] (1) Antigen preparation: The purified pET28a-HEF protein was emulsified with Freund's adjuvant at a volume ratio of 1:1 and vortexed to form a stable emulsion.
[0103] (2) Immunization program: 2-month-old Japanese rabbits were injected subcutaneously at multiple points on their backs with antigen containing complete Freund's adjuvant (dose 500 μg / rabbit); at 2 and 4 weeks later, the same amount of antigen containing incomplete Freund's adjuvant was injected.
[0104] 2. Serum collection and preservation
[0105] Seven days after the last immunization, blood was collected from the marginal ear vein, incubated at 4°C for 2 hours, and then centrifuged at 3000g for 15 minutes to separate the serum. The serum was then aliquoted and stored at -80°C for long-term preservation.
[0106] 3. Antibody titer detection (indirect ELISA)
[0107] (1) Antigen coating: Dilute HEF protein to 1 μg / mL with CBS buffer, coat 96-well microplates with 100 μL / well, and incubate at 4℃ for 12-16 h.
[0108] (2) Blocking and washing: Discard the coating solution, wash 3 times with 200 μL / well of PBST (0.05% Tween-20), block with 100 μL / well of 5% skim milk powder, and incubate at 37°C for 1 h.
[0109] (3) Serum incubation: Dilute the serum in a 2-fold gradient (800-1,638,400 times), add 100 μL / well, incubate at 37°C for 1 h, and wash 3 times with PBST.
[0110] (4) Secondary antibody reaction: Dilute goat anti-rabbit HRP secondary antibody (1:5000) with 5% skim milk powder, add 100 μL / well, incubate at 37℃ in the dark for 1 h, and wash 3 times with PBST.
[0111] (5) Color development and detection: Add 100 μL of TMB substrate per well, develop color in the dark for 15 min, stop with 50 μL of 2M sulfuric acid per well, measure the OD450 value with an ELISA reader, and determine the potency with a P / N value ≥ 2.1.
[0112] 4. Antibody specificity verification (Western blot)
[0113] (1) Sample processing: Take eukaryotic HEF protein samples and separate them according to the optimized SDS-PAGE procedure;
[0114] (2) Transfer and sealing: After transferring to PVDF film, it is sealed with 5% skim milk powder for 1 hour;
[0115] (3) Antibody incubation:
[0116] Primary antibody: HEF polyclonal antibody diluted 1:2000, incubated at 4°C with shaking for 12 hours;
[0117] Secondary antibody: Goat anti-rabbit HRP (1:5000) incubated at room temperature for 40 min;
[0118] Signal detection: ECL chemiluminescent liquid is used for development, and an imager captures specific bands.
[0119] 5. Rescuing recombinant baculovirus expressing influenza D virus HEF protein using an insect baculovirus expression system, infecting sf9 cells, and performing IFA identification using the prepared HEF protein polyclonal antibody as the primary antibody, the steps are as follows:
[0120] (1) Cell infection and culture: Sf9 cells in the logarithmic growth phase were seeded in 24-well plates. When the cell density reached 70%, the experimental group was added with recombinant baculovirus rBV-HEF, while the control group was not added with virus. The cells were incubated in a 27°C incubator for 48 hours.
[0121] (2) Cell fixation: Discard the culture medium, add 200 μL of 4% paraformaldehyde solution to each well, and fix at room temperature for 10 minutes;
[0122] (3) Washing treatment: After removing the fixative, add 200 μL of PBST buffer (containing 0.1% Tween-20, pH 7.4) to each well, shake and wash for 5 min, repeat 3 times;
[0123] (4) Cell membrane permeability: Add 200 μL of 0.4% Triton X-100 permeation solution to each well and incubate at room temperature for 10 min;
[0124] (5) Repeat the PBST washing process in step (3);
[0125] (6) Blocking non-specific binding sites: Add 5% skim milk powder blocking solution to each well and incubate at 37°C for 1 hour;
[0126] (7) Repeat the PBST washing process in step (3);
[0127] (8) Primary antibody incubation: Dilute HEF polyclonal antibody at 1:500 in PBS buffer, add 500 μL of dilution buffer to each well, and incubate at 4°C for 16 h;
[0128] (9) Repeat the PBST washing process of step (3) to remove unbound primary antibody;
[0129] (10) Secondary antibody labeling: Dilute FITC-labeled secondary antibody at a ratio of 1:1000 in PBS buffer, add 200 μL of dilution buffer to each well, and incubate at room temperature in the dark for 1 h;
[0130] (11) Repeat the PBST washing process of step (3) to remove unbound secondary antibody;
[0131] (12) Cell nuclear staining: Add 200 μL DAI staining solution to each well and stain at room temperature for 10 min in the dark;
[0132] (13) Final wash: Add 200 μL of PBST buffer to each well, shake and wash 3 times, 5 min each time;
[0133] (14) Fluorescence detection: The 24-well plate was placed under an inverted fluorescence microscope, and the protein expression and nuclear localization of the experimental group and the control group were observed through the FITC channel (excitation wavelength 490nm / emission wavelength 525nm) and the DAPI channel (excitation wavelength 358nm / emission wavelength 461nm).
Claims
1. A method for preparing polyclonal antibodies to influenza D virus HEF protein, characterized in that: The following steps are involved: S1. Construction of pET28a-HEF plasmid S11. Primer design: Based on the nucleic acid sequence of Influenza D virus HEF (NC_036620.1) in GenBank, the antigenic epitope was screened by bioinformatics analysis. In combination with the sequence of the expression vector pET28a, primers containing BamHI and XhoI restriction sites were designed. S12. Target gene amplification: Using the laboratory-stored cDNA of D / swine / Oklahoma / 1334 / 2011 as a template, specific primers pET28a+HEF-F and pET28a+HEF-R were used to amplify the uniseamless-pET28a-HEF fragment for prokaryotic expression of HEF protein; S13. Plasmid construction: The target gene, uniseamless-pET28a-HEF, and the expression vector, pET28a, were double-digested separately. The digestion products were purified and ligated using seamless cloning technology to construct the recombinant plasmid pET28a-HEF. The reaction products were transformed into E. coli DH5ɑ competent cells, and positive clones were screened and verified by plasmid extraction and sequencing. Expression and purification of S2 and HEF proteins S21. Small-scale induced expression: The pET28a-HEF plasmid was transformed into Transetta (DE3) competent cells. The expression conditions were optimized in terms of induction temperature, IPTG concentration, and induction time. The product was identified by SDS-PAGE. S22. Large-scale induction expression: Select the optimal expression conditions for large-scale induction, collect the bacteria and lyse them to obtain a lysate containing the target protein; S23. Purification: The HEF protein is purified by affinity chromatography, and high-purity HEF protein is obtained through the steps of filtration sterilization, nickel column equilibration, nickel column-protein binding, and protein elution; S3. Preparation and identification of polyclonal antibodies S31. Immunization of Animals: Purified pET28a-HEF protein emulsified with Freund's adjuvant was used as an antigen to immunize 2-month-old Japanese rabbits. Complete Freund's adjuvant was used for the first immunization, followed by two booster immunizations with incomplete Freund's adjuvant at intervals of 2 and 4 weeks, respectively. S32. Serum collection: Serum was collected 7 days after the last immunization to obtain polyclonal antibodies against HEF protein and stored at -80°C; S33. Antibody titer detection: Antibody titer was determined by indirect ELISA method; S34. Antibody specificity identification: Determine antibody specificity by Western blot identification; S35. Indirect immunofluorescence assay: Identify that the prepared HEF polyclonal antibody can specifically bind to the eukaryotic expressed protein.
2. The method for preparing a polyclonal antibody against influenza D virus HEF protein according to claim 1, characterized in that: The specific primers in the S11 are as follows: The nucleotide sequence of primer pET28a+HEF-F is: 5'-AATGGGTCGCGGATCCATGGCACAAGAACAACTACTTGCT-3'; The nucleotide sequence of primer pET28a+HEF-R is: 5'-GGTGGTGGTGCTCGAGCTTCCAGTCTCTTTTTAGGGCAAGAT-3'; The nucleotide sequence of primer T7-F is: 5′-TAATACGACTCACTATAGGG-3′; The nucleotide sequence of primer T7-R is: 5'-GCTAGTTATTGCTCAGCGG-3'.
3. The method for preparing a polyclonal antibody against influenza D virus HEF protein according to claim 1, characterized in that: The specific method for amplifying the HEF gene is to take 1.5 μL of laboratory-preserved D / swine / Oklahoma / 1334 / 2011 cDNA as a template, take 1 μL of specific primers pET28a+HEF-F and pET28a+HEF-R, take 12.5 μL of 2×HIFI Mix, and fill the remaining 25 μL system with ddH2O. Take a high-pressure sterilized PCR tube, prepare the system on ice, mix well, and place it in a PCR instrument, and perform a thermal cycling program including the following steps to amplify the target gene: pre-denaturation at 95°C for 5 minutes; 30 cycles of amplification steps, each cycle including denaturation at 95°C for 30 seconds, annealing at 62°C for 30 seconds, and extension at 72°C for 1 second; and finally, final extension at 72°C for 5 minutes; after the amplified product is separated by agarose gel electrophoresis, it is purified by gel recovery technology to obtain an uniseamless-pET28a-HEF fragment.