Influenza b virus specific antigenic short peptides and methods for their production

Abstract

This invention discloses a short peptide specific to influenza B virus and its preparation method. This invention relates to the field of biotechnology and provides a polypeptide combination composed of polypeptide B and polypeptide P; polypeptide B is a polypeptide with the amino acid sequence SEQ ID No. 1; and polypeptide P is a polypeptide with the amino acid sequence SEQ ID No. 2. The two polypeptides provided by this invention can be conjugated with a carrier protein to immunize animals to obtain high-titer, high-specificity polyclonal antibodies, which can be used to identify influenza B viruses of the Victoria and Yamagata lineages, respectively. They not only show no cross-reactivity with each other but also with influenza A virus fluid. This characteristic fundamentally solves the problem of false positives and misjudgments caused by the similarity of antigenic epitopes between lineages in traditional detection, ensuring the absolute accuracy of virus lineage identification results and providing core technical support for the accurate typing of influenza viruses.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to short peptides of influenza B virus-specific antigens and their preparation methods. Background Technology

[0002] Influenza B virus is an important pathogen causing seasonal influenza epidemics. Based on differences in hemagglutinin (HA) antigenicity, it can be divided into two main lineages: B / Yamagata (BY) and B / Victoria (BV). The strains of the two lineages have significant differences in antigenicity, and both can spread in the human population and cause infection. Therefore, accurate typing and identification of the BY and BV lineages is a key link in influenza virus surveillance, epidemiological investigation, and clinical diagnosis and treatment.

[0003] HA protein is a key glycoprotein on the surface of influenza B virus. It consists of an HA1 receptor-binding domain and an HA2 membrane fusion domain linked by disulfide bonds. The HA1 globular head can bind to sialic acid receptors on the surface of host cells, mediating viral adsorption and invasion of host cells. It is the main target of neutralizing antibodies. Both the antigenic site on its head and the conserved region of its stem can be recognized by antibodies.

[0004] It is noteworthy that the BY lineage HA protein primarily binds to α-2,6-linked sialic acid receptors on the surface of human respiratory epithelial cells, while the BV lineage HA protein can bind to both α-2,3 and α-2,6-linked sialic acid receptors simultaneously, thus exhibiting a broader host range. However, the amino acid sequences of the BY and BV lineage HA proteins are highly homologous, posing a significant technical challenge to the differentiation and identification of the two lineages.

[0005] Currently, the standard HA antibodies used for influenza B virus detection on the market are all universal antibodies, which can only achieve overall recognition of influenza B virus and cannot specifically distinguish between the BY and BV lineages. While the genotyping method that relies on gene sequencing is accurate, it has drawbacks such as cumbersome operation procedures, long detection cycles, high requirements for instruments and equipment, and difficulty in meeting the needs of primary healthcare institutions and rapid on-site testing.

[0006] Against this backdrop, the preparation of lineage-specific antibodies based on short peptide fragments from the hypervariable region of the HA protein has become a potential technical pathway for achieving precise typing—these short peptides possess natural lineage specificity and, theoretically, can serve as ideal immunogens for preparing lineage-specific antibodies. However, those skilled in the art generally believe that short peptides, due to their small molecular weight and single antigenic epitope, have extremely weak immunogenicity and are difficult to induce the body to produce high-titer antibodies when used alone as immunogens.

[0007] To improve the immunogenicity of short peptides, existing techniques typically employ carrier protein conjugation. However, related research has focused on short peptides with a length of 10 amino acids or more (usually 10-20 amino acids). For ultrashort peptides (less than 10 amino acids), there is a common technical bias among those skilled in the art, who generally believe that even with carrier protein conjugation, it is difficult to overcome the core bottleneck of weak immunogenicity. This is mainly because ultrashort peptides (less than 10 amino acids) usually contain only a single epitope, and it is speculated that during conjugation, the epitope may be masked due to steric hindrance from the carrier protein, thus failing to be effectively recognized by the immune system. Based on this speculation, the antibody titer induced by this method will be very low, and may even be insufficient for basic detection requirements.

[0008] In summary, if a BY / BV lineage differentiation antibody based on an ultrashort peptide of less than 10 aa that combines high titer and high specificity can be developed, it will overcome the technical challenges that urgently need to be addressed in this field. Summary of the Invention

[0009] The purpose of this invention is to provide a short peptide specific to influenza B virus and its preparation method.

[0010] In a first aspect, the present invention claims protection for a polypeptide combination.

[0011] The polypeptide combination claimed in this invention consists of polypeptide B and polypeptide P; The polypeptide B is an 8a polypeptide with the amino acid sequence SEQ ID No. 1; The polypeptide P is an 8aa polypeptide with the amino acid sequence SEQ ID No. 2.

[0012] The cysteine ​​residues in SEQ ID NO:1 and SEQ ID NO:2 are used to provide thiol groups to enable coupling with the carrier protein.

[0013] Secondly, the present invention claims protection for a coupling combination.

[0014] The coupling combination claimed in this invention consists of coupling B and coupling P; The conjugate B is a complete antigen obtained by conjugating the polypeptide B described in the first aspect above with a carrier protein. The conjugate P is a complete antigen obtained by conjugating the polypeptide P described in the first aspect above with a carrier protein.

[0015] The carrier protein may be keyhole hemocyanin, bovine serum albumin, human serum albumin, ovalbumin, mouse serum albumin, thyroglobulin, or rabbit serum albumin, etc.

[0016] In some embodiments of the present invention, the carrier protein is keyhole hemocyanin (KLH). In some embodiments of the present invention, the carrier protein is fetal bovine serum albumin (BSA).

[0017] In some embodiments of the present invention, the polypeptide B / polypeptide P is coupled to the carrier protein via a cysteine ​​residue carried by the polypeptide itself.

[0018] Thirdly, the present invention claims the use of the polypeptide combination described in the first aspect above or the conjugate combination described in the second aspect above as an immunogen in the preparation of antibody combinations for identifying and distinguishing between Victoria lineage and Yamagata lineage influenza B viruses.

[0019] Furthermore, the antibody combination consists of a polyclonal antibody specific to the Victoria lineage influenza B virus (denoted as antibody B) and a polyclonal antibody specific to the Yamagata lineage influenza B virus (denoted as antibody P).

[0020] Furthermore, antibody B is prepared using polypeptide B described in the first aspect above or conjugate B described in the second aspect above as an immunogen; antibody P is prepared using polypeptide P described in the first aspect above or conjugate P described in the second aspect above as an immunogen.

[0021] Furthermore, the antibody combination can not only distinguish between Victoria and Yamagata lineage influenza B viruses, but also shows no cross-reaction with influenza A viruses (such as influenza A H1N1).

[0022] Fourthly, the present invention claims a method for preparing an antibody combination for identifying and distinguishing between Victoria lineage and Yamagata lineage influenza B viruses.

[0023] The method for preparing an antibody combination for identifying and distinguishing between Victoria and Yamagata lineage influenza B viruses, as claimed in this invention, may include the following steps: (A1) Using the polypeptide B described in the first aspect above or the conjugate B described in the second aspect above as an immunogen, a polyclonal antibody specific to the Victoria lineage influenza B virus was prepared, denoted as antibody B. (A2) Using the polypeptide P described in the first aspect above or the conjugate P described in the second aspect above as an immunogen, a polyclonal antibody specific to the Yamagata lineage influenza B virus was prepared, denoted as antibody P; The antibody B and the antibody P constitute the antibody combination.

[0024] Furthermore, the antibody combination can not only distinguish between Victoria and Yamagata lineage influenza B viruses, but also shows no cross-reaction with influenza A viruses (such as influenza A H1N1).

[0025] The application of the antibody combination in differentiating between Victoria and Yamagata lineage influenza B viruses also falls within the scope of protection of this invention. Furthermore, the application is not for disease diagnosis or treatment.

[0026] Fifthly, the present invention claims protection for an antibody combination.

[0027] The antibody combination claimed in this invention consists of antibody B and antibody P described in the fourth aspect above.

[0028] Regarding the aforementioned aspects, in this invention, antibody B does not cross-react with Yamagata lineage influenza B virus, and antibody P does not cross-react with Victoria lineage influenza B virus.

[0029] Sixthly, the present invention claims protection for a polypeptide.

[0030] The polypeptide claimed in this invention is polypeptide B as described in the first aspect above.

[0031] Seventhly, the present invention claims protection for a coupling.

[0032] The coupling claimed in this invention is coupling B as described in the second aspect above.

[0033] Eighthly, the present invention claims the use of the polypeptide described in the sixth aspect or the conjugate described in the seventh aspect as an immunogen in the preparation of antibodies for identifying Victoria lineage influenza B virus.

[0034] Furthermore, the antibody is a polyclonal antibody (denoted as antibody B) specific to the Victoria lineage influenza B virus.

[0035] Furthermore, the antibody B is prepared using the polypeptide B described in the sixth aspect above or the conjugate B described in the seventh aspect above as an immunogen.

[0036] Furthermore, the antibody can not only identify Victoria lineage influenza B virus, but also shows no cross-reaction with Yamagata lineage influenza B virus and influenza A virus (such as influenza A H1N1).

[0037] Ninthly, the present invention claims a method for preparing antibodies for identifying Victoria lineage influenza B virus.

[0038] The method for preparing antibodies for identifying Victoria lineage influenza B virus claimed in this invention may include the following steps: using the polypeptide described in the sixth aspect above or the conjugate described in the seventh aspect above as an immunogen to prepare a polyclonal antibody specific to Victoria lineage influenza B virus, which is the target antibody.

[0039] Furthermore, the antibody can not only identify Victoria lineage influenza B virus, but also shows no cross-reaction with Yamagata lineage influenza B virus and influenza A virus (such as influenza A H1N1).

[0040] The application of the antibody in identifying Victoria lineage influenza B virus also falls within the scope of protection of this invention. Furthermore, the application is not for disease diagnosis or treatment.

[0041] In a tenth aspect, the present invention claims protection for antibodies prepared using the method described in the ninth aspect above.

[0042] Regarding the aforementioned aspects, in this invention, when preparing antibodies, various healthy and qualified mammals can be selected as immunizing animals to prepare target polyclonal antibodies, including but not limited to rats, New Zealand white rabbits, Balb / c mice, guinea pigs, and goats. In some embodiments of this invention, rats are specifically used.

[0043] In an eleventh aspect, the present invention also claims protection for any of the following biological materials: (B1) A set of nucleic acid molecules, consisting of nucleic acid molecule B and nucleic acid molecule P; wherein nucleic acid molecule B is a nucleic acid molecule capable of encoding polypeptide B described in the first aspect above; wherein nucleic acid molecule P is a nucleic acid molecule capable of encoding polypeptide P described in the first aspect above; (B2) A complete expression cassette, consisting of expression cassette B and expression cassette P; wherein expression cassette B is an expression cassette containing the nucleic acid molecule B described in (B1); and expression cassette P is an expression cassette containing the nucleic acid molecule P described in (B1). (B3) A set of recombinant vectors, consisting of recombinant vector B and recombinant vector P; wherein recombinant vector B is a recombinant vector containing the nucleic acid molecule B described in (B1); and recombinant vector P is a recombinant vector containing the nucleic acid molecule P described in (B1). (B4) A set of recombinant bacteria, consisting of recombinant bacteria B and recombinant bacteria P; wherein recombinant bacteria B is a recombinant bacteria containing the nucleic acid molecule B described in (B1); and recombinant bacteria P is a recombinant bacteria containing the nucleic acid molecule P described in (B1). (B5) A complete set of transgenic cell lines, consisting of transgenic cell line B and transgenic cell line P; wherein transgenic cell line B is a transgenic cell line containing the nucleic acid molecule B described in (B1); wherein transgenic cell line P is a transgenic cell line containing the nucleic acid molecule P described in (B1); (B6) Nucleic acid molecule, which is nucleic acid molecule B described in (B1); (B7) Expression box, which is the expression box B described in (B2); (B8) The recombinant vector is the recombinant vector B described in (B3); (B9) Recombinant bacteria, specifically recombinant bacteria B described in (B4); (B10) Transgenic cell line, which is the transgenic cell line B described in (B5).

[0044] In a twelfth aspect, the present invention also claims the use of the biomaterials described in the eleventh aspect above in the preparation of the aforementioned polypeptide combinations or polypeptide or conjugate combinations or conjugates.

[0045] In a thirteenth aspect, the present invention also claims a kit containing the aforementioned combination of polypeptides or combination of polypeptides or conjugates or conjugates.

[0046] The kit can be used to detect the antibody combination or the antibody described above. Further, the kit can be an enzyme-linked immunosorbent assay (ELISA) kit. Even further, in the ELISA kit, the polypeptide or conjugate described above is used as the coating antigen. In some embodiments of the present invention, the ELISA kit also contains horseradish peroxidase-labeled secondary antibody, coating buffer, washing solution, chromogenic solution, and / or stop solution.

[0047] In a fourteenth aspect, the present invention also claims a kit containing the antibody combination described in the fifth aspect above or the antibody described in the tenth aspect above.

[0048] When the kit contains the antibody combination described in the fifth aspect above, it can be used to distinguish between Victoria lineage and Yamagata lineage influenza B viruses. When the kit contains the antibody described in the tenth aspect above, it can be used to identify Victoria lineage influenza B viruses. In some embodiments of the present invention, the kit is a Western blot kit. Further, the Western blot kit may also include electrophoresis buffer, transfer buffer, blocking buffer, washing buffer, protein marker, chromogenic / luminescent substrate, etc.

[0049] In some embodiments of the present invention, the Victoria lineage influenza B virus is B / Brisbane / 60 / 2008; and the Yamagata lineage influenza B virus is B / Phuket / 3073 / 2013.

[0050] In some embodiments of the present invention, the influenza B virus mentioned above is a Victoria lineage influenza B virus (e.g., B / Brisbane / 60 / 2008), a Yamagata lineage influenza B virus (e.g., B / Phuket / 3073 / 2013), or influenza A H1N1 (e.g., A / Puerto Rico / 8 / 34).

[0051] This invention identifies two ultrashort antigenic peptides, each 8 aa in length. When conjugated with a carrier protein, both peptides induce high-titer, high-specificity polyclonal antibodies in immunized animals. These antibodies can be used to identify influenza B viruses of the Victoria and Yamagata lineages, respectively, showing no cross-reactivity with each other or with influenza A virus. This characteristic fundamentally solves the problem of false positives and misjudgments caused by the similarity of antigenic epitopes between lineages in traditional detection methods, ensuring the absolute accuracy of virus lineage identification results and providing core technical support for the precise typing of influenza viruses. Attached Figure Description

[0052] Figure 1 Serum titer was measured in four rats immunized with BSA-peptide B.

[0053] Figure 2 Serum titer was determined in four rats immunized with BSA-peptide P.

[0054] Figure 3 This study aimed to identify the specific binding of polyclonal antibodies to the HA1 protein of influenza B virus. In the figure, A represents SDS-PAGE detection of the B / Brisbane / 60 / 2008 and B / Phuket / 3073 / 2013 HA1 proteins; B represents Western blot detection using polyclonal antibody B as the primary antibody; and C represents Western blot detection using polyclonal antibody P as the primary antibody. In the figure, lane 1 represents the B / Brisbane / 60 / 2008 HA1 protein, and lane 2 represents the B / Phuket / 3073 / 2013 HA1 protein.

[0055] Figure 4This study aimed to identify the specific binding of polyclonal antibodies to influenza A and B virus strains. In the figure, A represents SDS-PAGE detection of A / PuertoRico / 8 / 34 virus, B / Brisbane / 60 / 2008 virus, and B / Phuket / 3073 / 2013 virus; B represents Western blot detection using polyclonal antibody B as the primary antibody; and C represents Western blot detection using polyclonal antibody P as the primary antibody. In the figure, lane 1 represents A / Puerto Rico / 8 / 34 virus; lane 2 represents B / Brisbane / 60 / 2008 virus; and lane 3 represents B / Phuket / 3073 / 2013 virus. Detailed Implementation

[0056] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0057] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0058] Example 1: Preparation of specific polyclonal antibodies against Victoria and Yamagata lineage influenza B viruses I. Obtaining the sequence of the antigenic peptide (1) The full amino acid sequences of HA of Victoria lineage standard strain B / Brisbane / 60 / 2008 and Yamagata lineage standard strain B / Phuket / 3073 / 2013 were obtained from The Global Initiative on Sharing All Influenza Data (GISAID) database.

[0059] (2) The amino acid sequences of the two strains were compared using the Megalign software to screen for positions with continuous amino acid sequence differences and then extracted.

[0060] (3) After comparison, only three positions of the full-length HA protein from the two lineages showed differences of four consecutive amino acids. This invention ultimately determined that the amino acids at positions P70-P77 (which had not been reported in any literature prior to this application as antigenic epitopes for the Victoria / Yamagata lineage HA protein), sequence PKCTGKIP (SEQ ID NO:1), would serve as the hapten for obtaining Victoria lineage HA-specific polyclonal antibodies. Sequence PMCVGTTP (SEQ ID NO:2) would serve as the hapten for obtaining Yamagata lineage HA-specific polyclonal antibodies.

[0061] II. Synthesis of Immunogens 1. Solid-phase peptide synthesis: The polypeptide sequence PKCTGKIP (SEQ ID NO:1, corresponding to the Victoria lineage) is denoted as polypeptide B.

[0062] 2. Solid-phase peptide synthesis: The polypeptide sequence PMCVGTTP (SEQ ID NO:2, corresponding to the Yamagata lineage) is denoted as polypeptide P.

[0063] 3. The complete antigen is synthesized by coupling the thiol group on the cysteine ​​side chain of the polypeptide with keyhole hemocyanin (KLH) or fetal bovine serum albumin (BSA). The specific operation is as follows: (1) Prepare reagents Anhydrous DMSO; PBS buffer (pH 7.2); 0.5M EDTA buffer (pH 8.1); lysine; 3-maleimide propionic acid hydroxysuccinimide ester (CAS: 55750-62-4; molecular weight: 266.21); keyhole limpethemocyanin (KLH); fetal bovine serum albumin (BSA).

[0064] (2) Solution preparation Coupling buffer: Take 50 mL of PBS buffer (pH 7.2) and add 500 μL of EDTA buffer (pH 8.1).

[0065] 100mM crosslinking reagent stock solution: Weigh 26.6mg of 3-maleimide propionic acid hydroxysuccinimide ester into a 2mL centrifuge tube, add 1mL of anhydrous DMSO, and dissolve and mix thoroughly.

[0066] Using pre-prepared coupling buffer, KLH and BSA were dissolved and diluted to prepare two protein solutions with a concentration of 5 mg / mL.

[0067] (3) Synthesis of complete antigen Step 1: Add 10 μL of crosslinking reagent stock solution to 1 mL of the KLH solution prepared above, invert and mix well, and react at room temperature for 30 min.

[0068] Step 2: Add 10 μL of crosslinking reagent stock solution to 1 mL of the BSA solution prepared above, mix by inverting, and react at room temperature for 30 min.

[0069] Step 3: After the reaction, add 10 μL of lysine to the KLH solution from step 1 and the BSA solution from step 2 respectively to block the excess succinimide groups.

[0070] Step 4: Take 1 mg of peptide (set up two parallel steps: peptide B and peptide P), add 100 μL of coupling buffer, and disperse thoroughly to obtain a peptide solution.

[0071] Step 5: Add 50 μL of the peptide solution prepared above to the KLH solution obtained in Step 3; add 30 μL of the peptide solution prepared above to the BSA solution obtained in Step 3.

[0072] Step 6: Incubate at room temperature for 30 minutes.

[0073] Step 7: Add the above-conjugated protein solution into the ultrafiltration tube.

[0074] Step 8: Add 10 ml of PBS buffer (pH 7.2) to the ultrafiltration tube, then centrifuge to replace the protein solution at 3000 rpm for 30 min at room temperature.

[0075] Step 9: After performing three replacements as described in Step 8, dilute the replaced protein solution to 2 mL with PBS buffer (pH 7.2) and quantify the BCA protein in the resulting protein solution.

[0076] III. Preparation and Potency Detection of Rat Antiserum 1. Immunity in rats (1) Select four healthy male SD rats, 6 weeks old (220-260g).

[0077] (2) The complete antigen synthesized in step two (two parallels: KLH-conjugated polypeptide B and KLH-conjugated polypeptide P) was diluted with PBS and mixed with Freund's complete adjuvant at a 1:1 ratio and thoroughly emulsified. Rats were immunized by subcutaneous injection at multiple points on the back, 50 μl / point, with a total injection volume of 400 μl / rat, and each rat received 100 μg of total immunized protein.

[0078] (3) Two weeks after the initial immunization, Freund's incomplete adjuvant was mixed with the complete antigen synthesized in step two (two parallel mixtures: KLH-conjugated peptide B and KLH-conjugated peptide P) in a 1:1 ratio, emulsified, and booster immunization was performed in the same manner (the booster immunization dose for each mouse was the same as the initial immunization dose). A second booster immunization was performed four weeks later. The dose for each immunization was 100 μg per rat (calculated based on peptide content).

[0079] 2. Determination of antiserum titer in rats by ELISA method (1) Material preparation: ELISA plate, 0.05 mol / L carbonate buffer (pH 9.6), BSA-conjugated antigen (two parallels: BSA-conjugated peptide B and BSA-conjugated peptide P prepared in step 2); PBST buffer (pH 7.2), BSA, HRP-labeled goat anti-rat IgG (H+L), 3,3',5,5'-tetramethylbenzidine (TMB single-component chromogenic solution), 1M sulfuric acid solution, 5% skim milk.

[0080] (2) One week after the third immunization, the tails of the rats were cut off and blood was collected. The blood volume was about 300 μl per rat. (3) After collecting the blood, let it stand at room temperature for 2 hours, centrifuge at 5000 rpm for 10 minutes, carefully aspirate the supernatant, aliquot it and store it at -20℃ for later use.

[0081] (4) Dilute the BSA synthetic antigen (5 mg / mL) with carbonate buffer to a coating solution of 2 μg / mL, add 100 μl of coating solution to each well of the microplate and incubate at 4°C overnight; (5) Discard the liquid in the well plate, add 300 μL of PBST to each well and wash 3 times, then block with 300 μL / well of 5% skim milk powder and incubate at 37°C for 2 hours; (6) Dilute the serum sample to be tested 100 times, and then perform continuous 3-fold dilutions to obtain dilutions of 1:100, 1:300, 1:900, ..., 1:590-4900, for later use; (7) After blocking, wash once with 300 μl PBST in each well, dry, and transfer the diluted serum to the corresponding enzyme label strips, 50 μl per well, and incubate at 37°C for 1 h. (8) After washing each well three times with 300 μl PBST, HRP-labeled goat anti-rat IgG (H+L) antibody was diluted 1:1000, 50 μl per well, and incubated at 37°C for 1 h; (9) After washing three times with 300 μl / well PBST, use TMB for color development, 50 μl / well, incubate at 37°C for 10 min in the dark; (10) Add 1M concentrated sulfuric acid to terminate the reaction, 50 μl / well, protected from light; (11) Detect the OD450nm reading. If the result is greater than 2.1 times the OD450 value of the negative serum (using unimmunized rat serum as a control) as a substitute for the test serum, it is considered positive.

[0082] The results show: A 96-well ELISA plate was coated with BSA-conjugated peptide B (corresponding to the Victoria lineage) at a concentration of 2 μg / mL. Antisera from four rats (B-1, B-2, B-3, and B-4) one week after their third immunization were serially diluted 3-fold, and the OD450 values ​​of the antisera were measured using an indirect ELISA method. Table 1 and... Figure 1 The table shows the OD450 readings of serum from four rats. Based on the OD450 values, the antiserum titers of rats B-1, B-2, B-3, and B-4 were determined to be 1:218700, 1:656100, 1:656100, and 1:218700, respectively. The results indicate that the antiserum titers from all four rats were generally very high.

[0083] A 96-well ELISA plate was coated with a BSA-conjugated polypeptide P (corresponding to the Yamagata lineage) at a concentration of 2 μg / mL. Antisera from four rats (P-1, P-2, P-3, P-4) one week after their third immunization were serially diluted 3-fold, and the OD450 values ​​of the antisera were measured using an indirect ELISA method. Table 2 and... Figure 2 The table shows the OD450 readings of serum from four rats. Based on the OD450 values, the titers of the antiserum from rats P-1, P-2, P-3, and P-4 were determined to be 1:218700, 1:656100, 1:24300, and 1:72900, respectively. The results indicate that the antiserum from rats P-1 and P-2 had very high titers.

[0084]

[0085]

[0086] IV. Purification of total IgG from rats (1) Antiserum treatment: The antiserum (rat serum 1 week after the third immunization) was slowly thawed in a 4°C freezer to avoid protein aggregation and precipitation. After thawing, the antiserum was centrifuged at 10,000 rpm for 15 min in a low-temperature centrifuge at 4°C. The supernatant was transferred and filtered through a 0.22 μm filter membrane to remove impurities such as fat and precipitated fibrin. The antiserum was diluted with PBS at a ratio of 1:5, and sodium azide was added to a final concentration of 0.05%, and then filtered again through a 0.22 μm filter membrane.

[0087] (2) Pack the Protein A+G pre-packed column, diluted antiserum and other buffers to room temperature.

[0088] (3) Open the cap of the pre-packed column and add 6 mL of PBS. Open the cap at the outlet of the chromatography column to allow the liquid to flow out naturally. Add another 6 mL of PBS to replace the buffer in the column.

[0089] (4) Place the chromatography column above the sterile collection tube. Add the diluted antiserum to the column tube, allowing the liquid to flow out slowly until the liquid level drops to about 3 mm above the sedimentation layer. Collect the antiserum eluent using a sterile collection tube. After all the antiserum to be purified has been loaded and collected through the column, add the eluent back into the column tube, allowing it to flow out naturally and collect it again. Avoid running the liquid dry during the operation.

[0090] (5) Fill the tube with PBS and allow the liquid to flow out naturally until the liquid level drops to 5 mm above the sedimentation layer. Repeat the operation three times to wash away unbound protein components in the sample solution. Detect the absorption wavelength at A280 of the outflow.

[0091] (6) Antibody elution: Add 300 μL of neutralization buffer (formulation: 1M Tris·HCl pH 8.8) to a sterile collection tube beforehand. After placing the column above the collection tube, add 2.7 mL of 0.2 M glycine solution (pH 2.5-3.0) to the column tube. After allowing the liquid to flow out naturally, drop it directly into the collection tube containing the neutralization buffer and mix thoroughly immediately.

[0092] (7) Wash the column with ultrapure water. Fill the column with ultrapure water and let it flow out naturally. Repeat three times. Then fill the column with PBS and let the liquid flow out naturally. Next, fill the column with PBS containing 0.03% NaN3 to wash the column. When the liquid level drops to about 1 cm above the quartz filter at the top, cover the column outlet and inlet in sequence and store in a refrigerator at 4°C.

[0093] (8) Transfer the collected antibody eluent to the inner tube of an ultrafiltration concentration tube and centrifuge at 12,000 rpm for 5 min. Discard the eluent, add PBS to the inner tube of the ultrafiltration tube, and centrifuge at 12,000 rpm for 5 min to replace the buffer in the ultrafiltration tube. Discard the eluent, and repeat the replacement with PBS once more. Invert the inner tube of the ultrafiltration tube into a sterile centrifuge tube and centrifuge at 12,000 rpm for 30 s. The resulting liquid is the purified antibody.

[0094] (9) Add an equal volume of glycerol to the collected antibodies and store at -20°C for later use.

[0095] The two polyclonal antibodies used in the following experiments were prepared from rat B-2 (corresponding to polyclonal antibody B below) and rat P-2 (corresponding to polyclonal antibody P below), respectively.

[0096] V. Detection of the specificity of rat polyclonal antibodies 1. Material preparation B / Brisbane / 60 / 2008 HA1 protein and B / Phuket / 3073 / 2013 HA1 protein are products of Sinopharm Biotechnology Co., Ltd.

[0097] The influenza virus strains A / Puerto Rico / 8 / 34 (influenza A H1N1), B / Brisbane / 60 / 2008 (Victoria lineage influenza B virus), and B / Phuket / 3073 / 2013 (Yamagata lineage influenza B virus) were prepared by the laboratory.

[0098] 1×SDS-PAGE electrophoresis buffer, Coomassie Brilliant Blue staining solution G250 (prepared in the laboratory).

[0099] The 10% SDS-PAGE preform, Western blocking solution, Western primary antibody diluent, and Western secondary antibody diluent were products of Beyotime Biotechnology Co., Ltd.

[0100] 0.45 μM PVDF membrane; Western chemiluminescence HRP substrate was a Merck Millipore product.

[0101] For the semi-dry transfer, use 7.5*10cm thickened filter paper, and the semi-dry transfer buffer is a product of Bio-Rad Laboratories.

[0102] 2. Western blot method for determining the antigen-binding ability and specificity of polyclonal antibodies. (1) SDS-PAGE Take the HA1 protein of two different lineages of influenza virus (Victoria lineage and Yamagata lineage of influenza B virus), as well as the inactivation solutions of three influenza viruses A / Puerto Rico / 8 / 34 (influenza A H1N1), B / Brisbane / 60 / 2008 (influenza B Victoria lineage), and B / Phuket / 3073 / 2013 (influenza B Yamagata lineage), add 5×SDS-PAGE loading buffer to each, boil for 10 min and prepare for loading.

[0103] Place the pre-made gel onto the gel casting stand and remove the comb teeth from the gel. Pour SDS-PAGE electrophoresis buffer into the cathode electrophoresis tank until it is full; then pour electrophoresis buffer into the anode tank until it reaches the center of the glass plate.

[0104] Add the samples into the comb wells and connect the power supply. Electrophoresis is performed at a constant voltage of 120V for 1 hour, after which the electrophoresis is completed. The gel is then stained or subjected to Western blot.

[0105] (2) Coomassie brilliant blue staining After cutting the protein gel, place it in an appropriate amount of Coomassie Brilliant Blue staining solution and gently shake it on a horizontal shaker at room temperature for 30-60 minutes. After recovering the staining solution, add distilled water and place it in a microwave oven for destaining on medium heat for 5 minutes, changing the solution 3 times during this period. Observe the bands by imaging the gel.

[0106] (3) Western blot Activate the 0.45 μM PVDF membrane with methanol and place it in transfer buffer for later use. After electrophoresis, remove the gel and use a Bio-Rad semi-dry transfer apparatus, setting the transfer current to 2.5 A and the transfer time to 6 minutes. After transfer, immediately place the PVDF membrane in PBST and rinse for 1-2 minutes to remove the transfer buffer. Place the PVDF membrane in a Western blot antibody incubation box, add an appropriate amount of Western blocking buffer, and gently shake on a shaker at room temperature for 60 minutes. Rinse the PVDF membrane with PBST once, 10 minutes each time. Dilute the prepared and purified antibody 1:1000 with Western primary antibody dilution buffer (set up two replicates: polyclonal antibody B and polyclonal antibody P). After rinsing the PVDF membrane, place it in the diluted primary antibody solution at 4°C overnight. Rinse the PVDF membrane with PBST 5-6 times, 10 minutes each time. HRP-labeled goat anti-rat IgG (H+L) antibody was diluted 1:3000 with Western blotting secondary antibody dilution buffer. After rinsing, the PVDF membrane was placed in the diluted enzyme-labeled secondary antibody and incubated at 37°C for 1 hour. After incubation, the PVDF membrane was washed 5-6 times with PBST for 10 minutes each time. Protein bands were detected by chemiluminescence immunoassay.

[0107] The results show: The results of the identification of the specific binding of polyclonal antibodies B and P to the HA1 protein of influenza B virus are as follows: Figure 3 .like Figure 3As shown in SDS-PAGE A, a distinct band (approximately 70 kDa) was detected in both lanes 1 and 2. Its molecular weight is consistent with the expected bands of the influenza B virus B / Brisbane / 60 / 2008 HA1 protein and B / Phuket / 3073 / 2013 HA1 protein. Figure 3 As shown in the Western blot of lane B (using polyclonal antibody B as the primary antibody), only one distinct band (approximately 70 kDa) was detected in lane 1, indicating that polyclonal antibody B can only specifically bind to the B / Brisbane / 60 / 2008 HA1 protein and cannot bind to the B / Phuket / 3073 / 2013 HA1 protein. Figure 3 As shown in the Western blot of lane C (using polyclonal antibody P as the primary antibody), only one distinct band (approximately 70 kDa) was detected in lane 2, indicating that polyclonal antibody P can only specifically bind to the B / Phuket / 3073 / 2013 HA1 protein and cannot bind to the B / Brisbane / 60 / 2008 HA1 protein.

[0108] The results of the specific binding identification of polyclonal antibodies to influenza A and B virus strains are as follows: Figure 4 .like Figure 4 As shown in SDS-PAGE A, a distinct band between 40-55 kDa was detected in lanes 1, 2, and 3, with a molecular weight consistent with the expected band of HA1 in influenza virus particles A / Puerto Rico / 8 / 34, B / Brisbane / 60 / 2008, and B / Phuket / 3073 / 2013. Figure 4 As shown in the Western blot of influenza B (using polyclonal antibody B as the primary antibody), only one distinct band (40-55 kDa) was detected in lane 2, indicating that polyclonal antibody B specifically binds only to influenza virus strain B / Brisbane / 60 / 2008, and not to influenza virus strains A / Puerto Rico / 8 / 34 and B / Phuket / 3073 / 2013. Figure 4 As shown in the Western blot of lane C (using polyclonal antibody P as the primary antibody), only one distinct band (40-55 kDa) was detected in lane 3, indicating that polyclonal antibody P can only specifically bind to influenza virus strain B / Phuket / 3073 / 2013, and cannot bind to influenza virus strains A / Puerto Rico / 8 / 34 and B / Brisbane / 60 / 2008.

[0109] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A polypeptide combination, characterized in that: The polypeptide combination consists of polypeptide B and polypeptide P; The polypeptide B is a polypeptide with the amino acid sequence SEQ ID No. 1; The polypeptide P is a polypeptide with the amino acid sequence SEQ ID No.

2.

2. A combination of couplings, characterized in that: The coupling assembly consists of coupling B and coupling P; The conjugate B is a complete antigen obtained by conjugating the polypeptide B and the carrier protein as described in claim 1. The conjugate P is a complete antigen obtained by conjugating the polypeptide P described in claim 1 and a carrier protein.

3. The use of the polypeptide combination of claim 1 or the conjugate combination of claim 2 as an immunogen in the preparation of antibody combinations for identifying and distinguishing between Victoria lineage and Yamagata lineage influenza B viruses.

4. A method for preparing an antibody combination for identifying and distinguishing between Victoria lineage and Yamagata lineage influenza B viruses, characterized in that: The method includes the following steps: (A1) Using the polypeptide B described in claim 1 or the conjugate B described in claim 2 as an immunogen, a polyclonal antibody specific to the Victoria lineage influenza B virus is prepared, denoted as antibody B; (A2) Using the polypeptide P described in claim 1 or the conjugate P described in claim 2 as an immunogen, a polyclonal antibody specific to the Yamagata lineage influenza B virus is prepared, denoted as antibody P; The antibody B and the antibody P constitute the antibody combination.

5. An antibody combination, characterized in that: The antibody combination consists of antibody B and antibody P as described in claim 4.

6. A polypeptide, characterized in that: The polypeptide is polypeptide B as described in claim 1.

7. A coupling agent, characterized in that: The coupling agent is coupling agent B as described in claim 2.

8. The use of the polypeptide of claim 6 or the conjugate of claim 7 as an immunogen in the preparation of antibodies for identifying Victoria lineage influenza B virus.

9. A method for preparing antibodies for identifying Victoria lineage influenza B virus, characterized in that: The method includes the following steps: using the polypeptide of claim 6 or the conjugate of claim 7 as an immunogen to prepare a polyclonal antibody specific to the Victoria lineage influenza B virus, which is the target antibody.

10. The antibody prepared using the method of claim 9.

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