Preparation method of influenza C virus HEF protein polyclonal antibody

Abstract

The invention discloses an influenza C virus HEF protein polyclonal antibody preparation method, which comprises: carrying out signal peptide prediction, antigen epitope screening and hydrophobicity analysis through bioinformatics, constructing a recombinant prokaryotic expression vector pET32a-HEF by using a seamless cloning technology, and efficiently expressing the HEF protein in Escherichia coli to obtain the influenza C virus HEF protein polyclonal antibody. The inclusion body protein yield is improved by optimizing conditions; and purifying by nickel column affinity chromatography to obtain high-purity recombinant protein, emulsifying the high-purity recombinant protein and a Freund's adjuvant, immunizing a Japanese white rabbit, immunizing for three times, and collecting high-titer serum. The method breaks through detection limitation caused by antigen tag concealment in a eukaryotic expression system, the obtained polyclonal antibody is high in specificity, the eukaryotic expression HEF protein can be accurately recognized, and a key tool is provided for ICV virus-like particle identification, vaccine research and development and diagnosis technologies. Compared with a traditional method, the scheme is simple and convenient to operate, low in cost and high in antibody titer, and has remarkable application value.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing polyclonal antibodies against influenza C virus HEF protein. Background Art

[0002] Influenza C virus (ICV) belongs to the genus Influenzavirus in the family Orthomyxoviridae. Unlike influenza A and B viruses, it has a narrower host range, primarily infecting humans and causing mild upper respiratory tract infections. However, research suggests that ICV can also infect pigs and other mammals. The ICV genome consists of seven single-stranded RNA segments and is transmitted via respiratory droplets. Infections occur at low levels year-round, and no large-scale outbreaks have been documented. However, due to the virus's ability to mutate antigenically, it still poses a potential public health risk. The HEF protein, the only surface glycoprotein of ICV, has multiple biological functions. It not only recognizes and binds to receptors but also plays a crucial role in the fusion of the viral envelope with the cell membrane, making it a core target for diagnostic and vaccine development.

[0003] Polyclonal antibodies have advantages in detection sensitivity due to their ability to recognize multiple sites on the HEF protein. However, traditional polyclonal antibody preparation methods often suffer from issues such as low antigen purity and poor antibody specificity, which impacts antibody quality and effectiveness. The complexity of ICV and the specificity of the HEF protein make the preparation of polyclonal antibodies targeting the HEF protein particularly challenging. Therefore, an efficient and reliable antibody preparation method is urgently needed to provide a reliable tool for accurate diagnosis of ICV and vaccine development.

[0004] The present invention aims to provide a new method for preparing polyclonal antibodies against influenza C virus HEF protein. Summary of the Invention

[0005] The object of the present invention is to provide a method for preparing polyclonal antibodies to the HEF protein of influenza C virus, so as to solve the problems raised in the above background technology.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A method for preparing polyclonal antibodies to influenza C virus HEF protein comprises the following steps:

[0008] S1. Construction of recombinant plasmid pET32a-HEF

[0009] S11. Bioinformatics Analysis

[0010] (1) Signal peptide prediction: Analyze the HEF protein sequence using SignalP 6.0 based on GenBank C / China / Taian106 / 2020 (OQ733382.1) to determine the signal peptide cleavage site, and select the mature peptide coding region for subsequent design.

[0011] (2) Antigenic epitope screening: Predict B-cell linear epitopes through the IEDB database, and select epitope regions with an antigenicity score > 0.7.

[0012] (3) Hydrophobicity analysis: Analyze the hydrophobicity of the HEF protein using the ProtScale tool (Kyte-Doolittle algorithm), and avoid highly hydrophobic regions to optimize soluble expression.

[0013] S12, Primer design and target gene amplification: Based on the bioinformatics analysis in S11 and combined with the sequence of the expression vector pET32a, design specific primers containing BamHI and XhoI restriction sites. Using the cDNA of the sample stored in the laboratory as a template, use the specific primers pET32a+HEF-F and pET32a+HEF-R to amplify the uniseamless-pET32a-HEF fragment for the prokaryotic expression of the HEF protein;

[0014] S13, Plasmid construction and verification: Perform double digestion on the target gene uniseamless-pET32a-HEF and the expression vector pET32a respectively, and recover the linearized vector and the HEF fragment; after purifying the digestion products, use seamless cloning technology for ligation to construct the recombinant plasmid pET32a-HEF, transform the reaction products into E. coli DH5ɑ competent cells, screen positive clones and perform plasmid extraction and sequencing to verify the correctness of pET32a-HEF;

[0015] S2, Prokaryotic expression and purification of HEF protein

[0016] S21, Small-scale induction expression: Transform the pET32a-HEF plasmid into Transetta(DE3) competent cells, and after optimizing the expression conditions, identify the products by SDS-PAGE;

[0017] S22, Large-scale induction expression: Perform large-scale induction according to the optimized conditions, collect the bacteria and lyse them to obtain the lysate containing the target protein;

[0018] S23, Protein purification and identification: Purify the HEF protein using nickel column affinity chromatography. Through the steps of filtration sterilization, nickel column equilibration, nickel column binding to the protein, and protein elution, obtain high-purity HEF protein, and verify the solubility and purity of the purified HEF protein;

[0019] S3. Preparation and Identification of Polyclonal Antibodies

[0020] S31. Animal Immunization Protocol: The purified pET32a-HEF protein emulsified with Freund's adjuvant was used as an antigen to immunize 2-month-old Japanese white rabbits. Complete Freund's adjuvant was used for the first immunization, and an equal amount of antigen was injected with Freund's incomplete adjuvant at 2-week and 4-week intervals for two booster immunizations;

[0021] S32. Serum Collection and Processing: Serum was collected 7 days after the last immunization to obtain polyclonal antibodies against the HEF protein and stored at -80 °C;

[0022] S33. Antibody Titer Detection: The antibody titer was determined by the indirect ELISA detection method;

[0023] S34. Antibody Specificity Identification: The antibody specificity was determined by Western blot identification.

[0024] S35. Antibody Sensitivity Identification: The lowest detection limit (LOD) of the antibody for a series of diluted antigens was determined by the indirect ELISA method.

[0025] Preferably, the primer sequences are as follows:

[0026] The nucleotide sequence of pET32a+HEF-F is:

[0027] 5’-GGCTGATATCGGATCCATGCAATGTGACATGATCCAGCT-3’;

[0028] The nucleotide sequence of the primer pET32a+HEF-R is:

[0029] 5’-GGTGGTGGTGCTCGAGGATTCCAAAAATCCTGCTT-3’;

[0030] The nucleotide sequence of the primer T7-F is: 5’-TAATACGACTCACTATAGGG-3’;

[0031] The nucleotide sequence of the primer T7-R is: 5’-GCTAGTTATTGCTCAGCGG-3’.

[0032] Preferably, in a method for preparing a polyclonal antibody against the HEF protein of influenza C virus according to S12, the specific method for amplifying the HEF gene is as follows: Take 1.5 μL of the cDNA of the ICV sample stored in the laboratory as a template, take 1 μL each of the specific primers pET32a+HEF-F and pET32a+HEF-R, take 12.5 μL of 2×HIFI Mix, and make up the remaining volume to 25 μL with ddH2O. Take a PCR tube after autoclaving, 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-denature at 95°C for 5 min; 30 cycles of amplification steps, each cycle including denaturation at 95°C for 30 s, annealing at 62°C for 30 s, and extension at 72°C for 1 s; finally, perform a final extension at 72°C for 5 min; after the amplification product is separated by 1% agarose gel electrophoresis, it is purified by gel recovery technology to obtain the uniseamless-pET32a-HEF fragment.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0034] The present invention provides an efficient method for extracting and purifying the HEF protein from influenza C virus (ICV).

[0035] By constructing the uniseamless-pET32a-HEF recombinant plasmid and achieving efficient expression and purification in Escherichia coli, high-purity HEF protein is obtained, which serves as an ideal antigen for preparing polyclonal antibodies. The optimized antibody preparation process: Using the purified HEF protein as an antigen, polyclonal antibodies are prepared by immunizing Japanese white rabbits. The process is simple, controllable, and has a high antibody yield. This method effectively solves the problems of low antigen purity and poor antibody specificity in the traditional antibody preparation process.

[0036] The prepared polyclonal antibodies have a high titer, good activity, high specificity for the HEF protein, and low cross-reactivity with other related proteins, ensuring their quality and application feasibility.

[0037] At the same time, the present invention has a wide application prospect. The method for preparing HEF polyclonal antibodies of the present invention not only provides a powerful tool for the in-depth study of ICV, but also may play an important role in the development of diagnostic reagents and vaccines for ICV. In addition, this method also has potential generality and can be applied to the preparation of polyclonal antibodies against other viruses or proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is the signal peptide prediction result in the embodiment of the present invention

[0039] Figure 2 It is the antigenic epitope screening result in the embodiment of the present invention

[0040] Figure 3 Hydrophobicity analysis results in the embodiments of the present invention

[0041] Figure 4 Amplification results of pET32a-HEF gene in the embodiments of the present invention

[0042] Figure 5 Identification results of colony PCR in the embodiments of the present invention

[0043] Figure 6 SDS-PAGE identification of HEF protein purification in the embodiments of the present invention

[0044] Figure 7 SDS-PAGE solubility analysis of HEF protein purification in the embodiments of the present invention

[0045] Figure 8 Western blot identification of purified HEF protein in the embodiments of the present invention

[0046] Figure 9 Western blot identification of HEF polyclonal antibody in the embodiments of the present invention

[0047] Figure 10 Sensitivity identification of HEF polyclonal antibody in the embodiments of the present invention Specific implementation manners

[0048] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0049] Example 1:

[0050] (1) Construction of pET32a-HEF plasmid

[0051] 1. Primer design: Antigenic epitopes were screened through bioinformatics analysis based on the nucleic acid sequence of C / China / Taian106 / 2020 (OQ733382.1), and primers containing BamHI and XhoI restriction enzyme sites were designed in combination with the sequence of the expression vector pET32a. The detailed primer sequences are shown in Table 1 and were sent to Jilin Kumei Biotechnology Co., Ltd. for synthesis.

[0052] Table 1 Primer information

[0053]

[0054] 2. Amplification of target gene: Using the cDNA of ICV stored in the laboratory as a template, specific primers pET32a+HEF-F and pET32a+HEF-R were used to amplify the uniseamless-pET32a-HEF fragment for prokaryotic expression of HEF protein according to the reaction system in Table 2 and the reaction procedure in Table 3.

[0055] Table 2 PCR reaction system

[0056]

[0057] Table 3 PCR reaction procedure

[0058]

[0059] The amplified product was separated and verified by agarose gel electrophoresis, and the uniseamless-pET32a-HEF fragment was recovered and purified by gel extraction. The purified sample was entrusted to Jilin Kumei Biotechnology for bidirectional sequencing analysis.

[0060] 3. Double digestion of target fragment and expression vector

[0061] The pET32a(+) 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 enzyme digestion system is shown in Table 4:

[0062] Table 4 Double digestion reaction system table

[0063]

[0064] 4. Ligation of target fragment and expression vector

[0065] The enzyme digestion products were separated by agarose gel electrophoresis, and the target fragment was recovered using a kit. The pET32a(+) linearized vector and the uniseamless-pET32a-HEF fragment were cloned seamlessly using Assembly Mix. The seamless cloning reaction system is shown in Table 5:

[0066] Table 5 Seamless cloning reaction system

[0067]

[0068]

[0069] 5. Transformation of recombinant vector

[0070] It was placed in a PCR instrument and reacted at 50 °C for 15 minutes, then placed on ice for a few seconds waiting for transformation. The reaction product was transformed into E.coli DH5ɑ competent cells. The specific operation is as follows:

[0071] (1) Add the reaction product to 50 μL of competent cells and place it on ice-water mixture for 30 min.

[0072] (2) Heat shock in a 42 °C water bath for 1 min, then quickly take it out and place it on ice bath for 3 min.

[0073] (3) Add 0.5 mL of antibiotic-free LB in a laminar flow hood and incubate it on a shaker at 37 °C for 1 h.

[0074] Take 200 μL and spread it evenly on an LB plate with kanamycin resistance. Incubate it in a 37 °C incubator, first upright for 1 h and then inverted overnight.

[0075] 6. Screen positive clone colonies

[0076] Observe the colony growth the next day. In the laminar flow hood, pick a single colony with a sterile pipette tip and transfer it to a liquid LB medium with Kna resistance, and incubate it on a shaker at 37 °C for 4 h. Identify the bacterial liquid by PCR using T7 universal primer and specific primers pET32a+HEF-F / R. The reaction system and reaction program are shown in Table 6 and Table 7:

[0077] Table 6 Bacterial liquid PCR reaction system

[0078]

[0079] Table 7 Bacterial liquid PCR reaction program

[0080]

[0081] 7. Plasmid extraction and sequencing

[0082] Extract the plasmid from the positive bacterial liquid and send it to Jilin Kumei Biotechnology Co., Ltd. for sequencing. The plasmid with correct sequencing is named pET32a-HEF.

[0083] (2) Prokaryotic expression and purification of HEF protein

[0084] 1. Small-scale induction expression and condition optimization: Transform the pET32a-HEF plasmid into Transetta(DE3) competent cells, inoculate a single colony into 5 mL of LB medium (containing 50 μg / mL kanamycin), and incubate it at 37 °C with shaking until OD600≈0.6. Identify the product by SDS-PAGE;

[0085] 2. Large-scale induced expression: Select the optimal expression conditions for large-scale induction. Centrifuge the bacterial solution at 8000 g for 10 min, discard the supernatant, collect the bacterial cells, resuspend and wash the bacterial cells with PBS, and then centrifuge at 8000 g for 10 min to discard the supernatant. Wash the bacterial cells twice. Resuspend the bacterial cells in the lysis buffer (20 mM Tris-HCl, 500 mM NaCl, 10 mM imidazole, pH 8.0), and ultrasonically disrupt under ice bath conditions with a power of 200 w, a pulse of 5 s and an interval of 5 s, for a total of 30 min. Centrifuge the ultrasonic lysate at 8000 g for 30 min, and collect the supernatant for subsequent protein purification.

[0086] 3. Protein purification: Purify the HEF protein by nickel column affinity chromatography.

[0087] (1) Filter sterilization: Filter the sample, buffer, eluent and double-distilled water with a 0.22 μm filter respectively.

[0088] (2) Nickel column equilibration: Equilibrate the Ni-NTA column with 5 column volumes of the binding buffer Binding Buffer.

[0089] (3) Binding of nickel column and protein: Load the sample and the equilibrated Ni-NTA Resin packing material into the column, load the sample at a flow rate of 1 mL / min, and bind on a shaker at 50 rpm in a chromatography cabinet at 4 °C for 2 h.

[0090] (4) Elution of protein: Let the supernatant in the column flow out, use the Binding Buffer with a low imidazole concentration of 5 column volumes to elute the miscellaneous proteins, and then use the Elution Buffer with a high imidazole concentration of 3 column volumes to elute the target protein. Label the liquids flowing out successively as: loading solution, washing solution and elution solution.

[0091] 4. Identification: Prepare protein samples from the purified sample and the uninduced sample respectively, and identify them by SDS-PAGE and Western blot.

[0092] (1) The specific operation of SDS-PAGE is as follows:

[0093] Sample treatment: Take 40 μL of the sample to be tested and mix it with the 5× protein loading buffer at a volume ratio of 4:1, boil in a boiling water bath for 10 min to fully denature the protein, and then centrifuge at 8000 g for 5 min. Take the supernatant for use.

[0094] Gel preparation: Prepare a 10% SDS-PAGE separating gel using the Yaenzyme protein gel preparation kit, and let it stand for polymerization for 30 minutes.

[0095] Loading and Electrophoresis: Precisely load 10 μL of the treated sample into each well. Initially, set the constant voltage electrophoresis at 80 V. After the bromophenol blue indicator migrates to the interface of the separating gel, adjust it to a constant voltage of 120 V and continue electrophoresis until the indicator reaches the bottom of the gel.

[0096] Staining and Decolorization: After electrophoresis, immerse the separating gel in Coomassie Brilliant Blue staining solution and shake for staining for 2 hours. Transfer it to the decolorizing solution (10% acetic acid + 20% methanol) and shake for decolorization for 8 - 12 h until the background is transparent. Finally, use a gel imaging system to collect and analyze.

[0097] (2) The specific operation of Western blot is as follows:

[0098] Transfer Membrane Operation: After completing the gel preparation and electrophoresis according to the SDS - PAGE standard procedure, precisely cut the gel area containing the target protein and protein Marker; Immerse the gel block, filter paper, and PVDF membrane activated with methanol for 30 s in the transfer buffer for equilibration for 10 min; Stack the transfer sandwich in the order of "filter paper - PVDF membrane - gel - filter paper", completely remove air bubbles, and transfer the membrane at a constant voltage of 20 V for 30 min.

[0099] Blocking and Antibody Incubation: After membrane transfer, immerse the PVDF membrane in 5% non - fat milk powder (dissolved in PBST) and block at room temperature for 1 h; Transfer the membrane to the diluted primary antibody solution (diluted 1:2000 in 5% non - fat milk powder) and incubate with shaking at 4°C for 12 - 16 h; Wash with PBST with shaking at room temperature (5 min × 3 times) to remove unbound antibodies.

[0100] Secondary Antibody Reaction and Development: Immerse the membrane in the HRP - labeled secondary antibody solution (diluted 1:5000 in 5% non - fat milk powder) and incubate with shaking at room temperature for 40 min; After washing three times with PBST, mix the ECL chemiluminescent solution A / B in a ratio of 1:1 and evenly cover the surface of the membrane; Expose in a darkroom on a chemiluminescent imager for 0.2 - 5 min to capture specific signals.

[0101] (3) Preparation and Identification of Polyclonal Antibodies

[0102] 1. Animal Immunization Protocol

[0103] (1) Antigen Emulsification: Mix the purified pET32a - HEF protein with Freund's complete adjuvant (for primary immunization) or Freund's incomplete adjuvant (for booster immunization) in a volume ratio of 1:1, and vortex - emulsify until a stable water - in - oil emulsion is formed.

[0104] (2) Immunization Program: Subcutaneously inject the antigen containing complete Freund's adjuvant (dose 500 μg / rabbit) at multiple points on the back of 2 - month - old Japanese white rabbits; At intervals of 2 weeks and 4 weeks, inject the same amount of antigen containing Freund's incomplete adjuvant respectively.

[0105] 2. Serum Collection and Processing

[0106] On the 7th day after the last immunization, 5 mL of blood was collected from the marginal ear vein. After standing at 4 °C for 2 h, it was centrifuged at 3000 g for 15 min to separate the serum, which was then aliquoted and stored at -80 °C for a long time.

[0107] 3. Antibody titer detection (indirect ELISA)

[0108] (1) Coating antigen: Dilute the HEF protein with CBS buffer (pH 9.6) to 1 μg / mL, and coat a 96-well ELISA plate with 100 μL per well. Incubate at 4 °C for 12 - 16 h.

[0109] (2) Blocking and washing: Discard the coating solution, wash 3 times with 200 μL per well of PBST (0.05% Tween - 20), block with 100 μL per well of 5% skim milk powder, and incubate at 37 °C for 1 h.

[0110] (3) Serum incubation: Dilute the serum in a 2-fold gradient (800 - 1,638,400 times), add 100 μL per well, incubate at 37 °C for 1 h, and wash 3 times with PBST.

[0111] (4) Secondary antibody reaction: Dilute the goat anti-rabbit HRP secondary antibody (1:5000) with 5% skim milk powder, add 100 μL per well, incubate at 37 °C in the dark for 1 h, and wash 3 times with PBST.

[0112] (5) Color development and detection: Add 100 μL per well of TMB substrate, develop color in the dark for 15 min, terminate with 50 μL per well of 2 M sulfuric acid, measure the OD450 value with an ELISA reader, and determine the titer with a P / N value ≥ 2.1.

[0113] 4. Antibody specificity verification (Western blot)

[0114] (1) Sample treatment: Take the eukaryotic-expressed HEF protein sample and separate it according to the optimized SDS-PAGE procedure;

[0115] (2) Membrane transfer and blocking: After transferring to the PVDF membrane, block with 5% skim milk powder for 1 h;

[0116] (3) Antibody incubation:

[0117] Primary antibody: Dilute the HEF polyclonal antibody at 1:2000 and incubate with shaking at 4 °C for 12 h;

[0118] Secondary antibody: Incubate the goat anti-rabbit HRP (1:5000) at room temperature for 40 min;

[0119] Signal detection: Develop color with ECL chemiluminescence solution and capture specific bands with an imager.

[0120] 5. Antibody sensitivity identification

[0121] (1) Antigen dilution: Dilute the HEF protein in gradients (from 1 ng / mL to 0.01 ng / mL) and coat a 96-well plate;

[0122] (2) Antibody incubation: Incubate with polyclonal antibodies at a fixed dilution (such as 1:10,000);

[0123] (3) Color development and analysis: After TMB color development, measure the OD450 value, and determine the effective detection limit when the signal-to-noise ratio (S / N) ≥ 3.

Claims

1. A method for preparing a polyclonal antibody against the HEF protein of influenza C virus, characterized in that, It includes the following steps: S1. Construction of recombinant plasmid pET32a-HEF S11. Bioinformatics analysis (1) Signal peptide prediction: According to GenBank C / China / Taian106 / 2020 (OQ733382.1), the HEF protein sequence was analyzed using SignalP6.0 to determine the signal peptide cleavage site, and the mature peptide coding region was selected for subsequent design; (2) Antigenic epitope screening: B cell linear epitopes were predicted through the IEDB database, and epitope regions with an antigenicity score > 0.7 were selected; (3) Hydrophobicity analysis: The hydrophobicity of the HEF protein was analyzed using the ProtScale tool (Kyte-Doolittle algorithm), and high hydrophobic regions were avoided to optimize soluble expression; S12. Primer design and target gene amplification: Based on the bioinformatics analysis in S11 and combined with the sequence of the expression vector pET32a, specific primers containing BamHI and XhoI restriction sites were designed. Using the cDNA sample stored in the laboratory as a template, the uniseamless-pET32a-HEF fragment was amplified using specific primers pET32a+HEF-F and pET32a+HEF-R for the prokaryotic expression of the HEF protein; S13. The target gene uniseamless-pET32a-HEF and the expression vector pET32a were respectively double digested, and the linearized vector and the HEF fragment were recovered; after purifying the digested products, seamless cloning technology was used for ligation to construct the recombinant plasmid pET32a-HEF. The reaction products were transformed into E.coli DH5ɑ competent cells, positive clones were screened, and plasmid extraction and sequencing were performed to verify the correctness of pET32a-HEF; S2. Prokaryotic expression and purification of HEF protein S21. Small-scale induction expression and condition optimization: The pET32a-HEF plasmid was transformed into Transetta(DE3) competent cells, and the expression conditions were optimized from the induction temperature, IPTG concentration, and induction time. The products were identified by SDS-PAGE; S22. Large-scale induction expression: Large-scale induction was carried out according to the optimized conditions, the bacteria were collected and lysed to obtain a lysate containing the target protein; S23. Protein purification: The HEF protein was purified by affinity chromatography. Through the steps of filtration sterilization, nickel column equilibration, nickel column binding to the protein, and protein elution, high-purity HEF protein was obtained, and the solubility and purity of the purified HEF protein were verified; S3. Preparation and identification of polyclonal antibodies S31. Animal immunization protocol: The purified pET32a-HEF protein was emulsified with Freund's adjuvant and used as an antigen to immunize 2-month-old Japanese white rabbits. Complete Freund's adjuvant was used for the first immunization, and equal amounts of antigen were injected with Freund's incomplete adjuvant at intervals of 2 weeks and 4 weeks for two booster immunizations; S32. Serum collection and treatment: Serum was collected 7 days after the last immunization to obtain polyclonal antibodies against the HEF protein and stored at -80°C; S33. Antibody titer detection: The antibody titer was determined by the indirect ELISA detection method; S34. Antibody specificity identification: Determine the antibody specificity by Western blot identification.

2. The method for preparing a polyclonal antibody against the HEF protein of influenza C virus according to claim 1, characterized in that, The specific primers in S11 are as follows: The nucleotide sequence of primer pET32a+HEF-F is: 5’-GGCTGATATCGGATCCATGCAATGTGACATGATCCAGCT-3’; The nucleotide sequence of primer pET32a+HEF-R is: 5’-GGTGGTGGTGCTCGAGGATTCCAAAAATCCTGCTT-3’; The nucleotide sequence of primer T7-F is: 5’-TAATACGACTCACTATAGGG-3’; The nucleotide sequence of primer T7-R is: 5’-GCTAGTTATTGCTCAGCGG-3’.

3. A method for preparing a polyclonal antibody against the HEF protein of influenza C virus according to claim 1, characterized in that, The specific method for amplifying the HEF gene is as follows: Take 1.5 μL of the cDNA of the ICV sample stored in the laboratory as the template, take 1 μL each of the specific primers pET32a+HEF-F and pET32a+HEF-R, take 12.5 μL of 2×HIFI Mix, and make up the remaining volume to 25 μL with ddH2O. Take a PCR tube sterilized by autoclaving, prepare the system on ice, mix well and place it in a PCR instrument, and perform a thermal cycling program including the following steps for amplifying the target gene: Pre-denature at 95 °C for 5 min; 30 cycles of amplification steps, each cycle including denaturation at 95 °C for 30 s, annealing at 62 °C for 30 s, and extension at 72 °C for 1 s; finally, perform a final extension at 72 °C for 5 min; After the amplification product is separated by 1% agarose gel electrophoresis, the uniseamless-pET32a-HEF fragment is purified by gel recovery technology.

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