Construction and preparation method of novel HPV (human papillomavirus) fusion protein antigen and application of novel HPV fusion protein antigen in preparation of polyclonal egg yolk antibody

Abstract

The invention provides a construction and preparation method of a novel HPV fusion protein antigen and an application of the novel HPV fusion protein antigen in preparation of a polyclonal egg yolk antibody. Core antigen peptides of human papilloma virus 16 subtype and HPV 18 subtype capsid protein L1 are respectively connected in series with an Fc of immunoglobulin IgY and a tail peptide of immunoglobulin IgM for the first time; the recombinant fusion protein antigen is prepared through escherichia coli expression and an inclusion body renaturation technology, the method is beneficial for improving the stability and presentation capacity of antigen peptide, laying hens are directly immunized with the purified novel HPV fusion protein antigen, after an antibody is generated, the antibody is purified through the steps of salting out, dialysis and the like, and the antibody is obtained. Verification shows that the obtained egg yolk antibody can be combined with L1 proteins of HPV16 and HPV18, and has the capability of preventing or treating infection of HPV16 and HPV18.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a construction and preparation method of a novel HPV fusion protein antigen and its application in preparing polyclonal egg yolk antibodies. Background Art

[0002] Human papillomavirus (HPV) is the primary cause of cervical cancer, anogenital cancer, oropharyngeal cancer, and other diseases. Approximately 5.3% of cancers worldwide are linked to infection with this virus. Over 200 HPV subtypes have been identified, categorized by their carcinogenicity into high-risk and low-risk types. HPV 16 / 18 are the core oncogenic types, responsible for over 70% of cervical cancers worldwide. Low-risk types primarily cause benign lesions and, while not carcinogenic, may foster infection with high-risk types.

[0003] The HPV capsid is composed of the major capsid protein L1 and the minor capsid protein L2, both of which play key roles in viral assembly, infection, and the immune response. The antigenic properties of the L1 protein are primarily determined by the structure of its surface loop regions (loops). Studies have shown that the five loop regions located on the pentamer surface—BC, DE, EF, FG, and HI—are the primary antigenic epitope regions, containing a large number of type-specific neutralizing epitopes. The L1 protein can self-assemble in vitro to form virus-like particles (VLPs). These VLPs are morphologically highly similar to native virus particles, approximately 50-60 nm in diameter, but do not contain viral DNA and, therefore, pose no infectious risk. This property makes L1 VLPs an ideal antigenic form for HPV preventive vaccines, effectively inducing high titers of type-specific neutralizing antibodies to block the initial binding of the virus to host cells. However, conventional L1 VLP production technologies face several challenges: high expression costs and long production cycles in eukaryotic systems; and the VLP assembly process requires disassembly and reassembly, resulting in low yields.

[0004] Fc fusion proteins are a new type of recombinant protein formed by fusing the Fc domain of an immunoglobulin (such as IgG, IgM, etc.) with the target protein sequence. Fc fusion proteins have significant advantages as antigens in antibody development, mainly due to their structural characteristics and biological functions:

[0005] (1) Enhanced immunogenicity:

[0006] 1) Fcγ receptor-mediated antigen presentation: The Fc region of the Fc fusion protein can be recognized and bound by Fcγ receptors on the surface of antigen-presenting cells (APCs), such as dendritic cells, macrophages, and B cells. This binding promotes efficient internalization and processing of the antigen by APCs, and presentation of target antigen peptides to T cells, thereby significantly enhancing the adaptive immune response (T and B cell activation) against the target antigen.

[0007] 2) Multimerization Effect: The Fc region of immunoglobulins tends to form dimers under physiological conditions. Therefore, Fc fusion proteins are typically bivalent or multivalent molecules. This multimerization can more effectively cross-link B cell receptors, providing stronger activation signals, promoting B cell activation, proliferation, and differentiation into plasma cells, and producing more high-affinity antibodies.

[0008] 3) Activation of the complement pathway: The Fc region can bind to complement components (such as C1q), activating the complement cascade. Complement activation products (such as C3d) are potent B cell helper signaling molecules that enhance stability and solubility, further amplifying the immune response.

[0009] (2) Improved stability and solubility: The Fc domain is usually very stable and has high solubility. Fusion of the Fc domain to target proteins that are originally unstable, prone to aggregation, or have poor solubility can significantly improve the physicochemical properties of the entire fusion protein, making it less susceptible to degradation or precipitation during storage and immunological operations, and maintaining the native conformation of the antigen epitope.

[0010] (3) Prolonging the half-life in vivo: The Fc segment can significantly prolong the half-life of the protein in the blood circulation by binding to the receptor. For experimental animals that require multiple immunizations, prolonging the antigen exposure time may help maintain stronger immune stimulation and continuously produce high-titer antibodies.

[0011] Not only that, by modifying Fc through genetic engineering to form a higher aggregation state, it is beneficial to further enhance its stability and improve its antigen presentation ability.

[0012] Yolk antibodies (Immunoglobulin of yolk, IgY) are antigen-stimulated avian B lymphocytes that differentiate into plasma cells, which then secrete specific antibodies into the bloodstream. As blood flows through the ovaries, they gradually accumulate in the oocytes, forming IgY. These antibodies are considered analogs of mammalian IgG, with their Fc domains possessing similar functions and properties to those of IgG. Furthermore, IgY offers advantages such as high stability, strong specificity, low production cost, and oral safety. They hold great promise for the diagnosis, prevention, and treatment of various human and animal diseases. Summary of the Invention

[0013] Traditional production techniques for VLP antigens using HPV capsid protein L1 face multiple challenges: high eukaryotic expression costs and long production cycles; and the VLP assembly process requires disassembly and reassembly, resulting in low yields. To address these technical challenges, the present invention provides novel HPV fusion protein antigens and their application in egg yolk antibodies. This study concatenated the core antigen epitopes of HPV16 and HPV18 capsid protein L1 with the Fc domain of IgY and the tail peptide sequence of IgM, respectively. Gene sequences encoding these fusion proteins were obtained through codon optimization. Furthermore, inclusion bodies containing the HPV fusion protein antigens were generated through recombinant expression in Escherichia coli, and highly pure, water-soluble HPV fusion protein antigens were prepared through protein renaturation. Egg-yolk antibodies were generated by direct immunization of laying hens with this antigen, and the antibodies were purified through salting out and dialysis. Validation demonstrated that these antibodies recognize HPV16 or HPV18 capsid protein L1 and have the potential to prevent or treat HPV16 and HPV18 infections.

[0014] Specifically, the present invention first provides a method for constructing a novel HPV fusion protein antigen, which is obtained by sequentially connecting an antigenic peptide, the Fc region of immunoglobulin IgY, and the tail peptide of immunoglobulin IgM through a polypeptide linker, wherein the antigenic peptide is the core antigen sequence of HPV16 or HPV18 capsid protein L1.

[0015] Preferably, the antigen peptide is fused to the Fc region of immunoglobulin IgY through a polypeptide linker (GSSG)n, where n is a natural number between 1 and 3, that is, the core antigen epitope of HPV16 or HPV18 capsid protein L1 and the Fc region of IgY are connected in series through the polypeptide linker (GSSG)n.

[0016] Preferably, the Fc region of immunoglobulin IgY is linked to the tail peptide of immunoglobulin IgM via a polypeptide linker TGS.

[0017] Preferably, the amino acid sequence of the HPV fusion protein antigen is shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0018] On the other hand, the present invention also provides a method for preparing a novel HPV fusion protein antigen, comprising the following steps:

[0019] Step 1: codon optimization of the novel HPV fusion protein antigen to obtain matching nucleotide sequences, as shown in SEQ ID NO: 3 and SEQ ID NO: 4;

[0020] Step 2: The nucleotide sequence in step 1 is constructed into a prokaryotic expression vector by homologous recombination to obtain a recombinant expression vector, wherein the expression vector includes an expression vector including pET21b, pET28 or pET30a;

[0021] Step 3: The recombinant expression vector of step 2 is introduced into Escherichia coli for recombinant expression, wherein the strain is selected from BL21 (DE3), HMS174, Rosetta or Origami B, etc., and the recombinant E. coli is selected and cultured in a constant temperature shaking incubator for recombinant expression. The culture conditions are: 37°C, 220 rpm;

[0022] Step 4: Collect the Escherichia coli in step 3, and use the protein inclusion body renaturation technology to separate and purify the HPV fusion protein to obtain the HPV fusion protein antigen. The specific operation is: use an ultrasonic instrument to crush the bacterial suspension, collect the precipitate by centrifugation, repeatedly wash the precipitate to obtain inclusion bodies containing the HPV fusion protein antigen, then dissolve the inclusion bodies with a denaturing solution, and dialyze the dissolved inclusion bodies in a renaturing solution to obtain water-soluble HPV fusion protein antigen, wherein the denaturing solution contains urea or guanidine hydrochloride; the renaturing solution contains arginine, oxidized glutathione (GSSG), reduced glutathione (GSH) and glycerol.

[0023] On the other hand, the present invention also provides a use of a novel HPV fusion protein antigen in the preparation of polyclonal egg yolk antibodies, wherein polyclonal egg yolk antibodies for preventing or treating HPV virus infection are prepared from immunized laying hens carrying HPV fusion protein antigens.

[0024] Preferably, the method for preparing polyclonal egg yolk antibodies comprises the following steps:

[0025] Step 1: Immunization procedure for laying hens: The primary immunogen is a novel HPV fusion protein antigen with an antigen content of 200 μg, which is thoroughly mixed with an equal volume of Freund's complete adjuvant to form an oil-in-water emulsion. Six points on the chest of the laying hens are disinfected with conventional alcohol, and 0.8 mL of the oil-in-water emulsion is distributed intramuscularly for immunization. The subsequent booster immunization is an antigen with an antigen content of 200 μg of the novel HPV fusion protein antigen, which is thoroughly mixed with Freund's incomplete adjuvant. The booster immunization is repeated twice at an interval of 15 days. Seven days after the first booster immunization, the eggs are collected and stored at 4°C for later use.

[0026] Step 2: Isolation and purification of polyclonal egg yolk antibodies: Egg yolks are separated and collected, diluted with 10 volumes of PBS buffer, and centrifuged at 10,000 g for 10 minutes. The precipitate is discarded and the supernatant is filtered through cotton wool. A saturated ammonium sulfate solution is then added until the supernatant reaches 40%-50% ammonium sulfate saturation. The mixture is allowed to stand at 4°C for 10 hours and then centrifuged. 30 minutes after centrifugation, the precipitate is collected and 10 mL of PBS buffer is added per mL of precipitate. The mixture is dissolved at 4°C for 2 hours and then centrifuged at 15,000 g for 20 minutes. The supernatant is placed in a dialysis bag and dialyzed against PBS buffer for 12 hours. The dialysate is concentrated by ultrafiltration and sterilized by filtration to obtain polyclonal egg yolk antibodies based on the novel HPV fusion protein antigen.

[0027] Among them, the above-mentioned polyclonal yolk antibodies based on the novel HPV fusion protein antigen are used in the preparation of preparations for preventing or treating HPV virus infection diseases, including but not limited to ointments, creams, suspensions, solutions, gels, sprays or transdermal patches, etc.

[0028] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0029] The present invention is the first to fuse the core antigen epitopes of HPV16 and HPV18 capsid protein L1 with the Fc region of IgY and the tail piece of IgM in series to obtain HPV fusion protein antigens. The fusion protein antigens are expressed and purified using the E. coli system and inclusion body renaturation technology, and are then applied to the preparation of egg yolk antibodies. This has the following advantages:

[0030] (1) Improve antigen stability: Since the Fc region of IgY is highly similar to the Fc region of IgG, it can significantly improve the stability of the antigen peptide;

[0031] (2) Improve immunogenicity: The Fc region can bind to the IgY receptor, improve sensitization efficiency, and prolong the half-life of the fusion protein antigen in vivo;

[0032] (3) The IgM tail peptide can trigger Fc fusion protein oligomerization, further improving the stability and antigen presentation ability of the fusion protein;

[0033] (4) Improve the yield of effective antibodies: The fusion protein contains only core antigen peptides, which can increase the yield of effective antibodies;

[0034] (5) Purified HPV fusion protein antigens were used to directly immunize laying hens. After the antibodies were produced, polyclonal egg yolk antibodies were obtained through salting out, dialysis, and concentration. The antibodies were verified to bind to the capsid protein L1 of HPV16 or HPV18 and can effectively prevent or treat human papillomavirus (HPV) infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0036] Figure 1 This is a process flow chart for the construction, preparation and application of a novel HPV fusion protein antigen of the present invention.

[0037] Figure 2 The present invention is a binding analysis of the polyclonal egg yolk antibody and HPV16 capsid protein L1.

[0038] Figure 3 This is a binding analysis of the polyclonal egg yolk antibody of the present invention and HPV18 capsid protein L1. DETAILED DESCRIPTION

[0039] In order to more clearly understand the above-mentioned objects, features and advantages of the present invention, Figure 1-3 It should be noted that, in the absence of conflict, the technical solutions and features in the technical solutions of this application can be combined with each other.

[0040] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Therefore, the scope of protection of the present invention is not limited to the specific technical solutions disclosed below.

[0041] The technical solutions of the present invention will be described below in conjunction with specific embodiments. The embodiments described are only part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the present invention without making any creative work shall fall within the scope of protection of the present invention.

[0042] Example 1

[0043] A method for constructing a novel HPV fusion protein antigen. The fusion protein is obtained by connecting the core antigen epitope of HPV16 capsid protein L1 to the Fc region of IgY via a polypeptide linker GSSG, and the Fc region of IgY to the tail peptide of IgM via a polypeptide linker TGS. The amino acid sequence of the fusion protein is shown in SEQ ID NO: 1.

[0044] Example 2

[0045] A method for constructing a novel HPV fusion protein antigen. The fusion protein is obtained by connecting the core antigen epitope of HPV18 capsid protein L1 to the Fc region of IgY via a polypeptide linker GSSG, and the Fc region of IgY to the tail peptide of IgM via a polypeptide linker TGS. The amino acid sequence of the fusion protein is shown in SEQ ID NO: 2.

[0046] Example 3

[0047] A method for preparing an HPV16 fusion protein antigen, comprising the following steps:

[0048] Step 1: Based on the amino acid sequence of the fusion protein antigen described in Example 1, codon optimization was performed to obtain a matching nucleotide sequence; the nucleotide sequence is shown in SEQ ID NO: 3;

[0049] Step 2: The nucleotide sequence from step 1 was constructed into the expression vector pET28a by homologous recombination;

[0050] Step 3: The recombinant expression vector from step 2 was introduced into Escherichia coli strain BL21(DE3) by heat shock for recombinant expression. The recombinant E. coli was selected and cultured in a shake flask at 37°C and 220 rpm. When the E. coli concentration reached OD450 = 0.6, IPTG was added to a final concentration of 0.6 mM and the culture was continued for 5 hours.

[0051] Step 4: Collect the Escherichia coli in step 3, use an ultrasonic instrument to break up the bacterial suspension, and then collect the precipitate by centrifugation; at 25°C, wash the precipitate with washing solution 1, washing solution 2 and washing solution 3 in sequence to remove impurities from the inclusion body to obtain a higher purity inclusion body protein; add 10 times the volume of denaturing solution 1 to the inclusion body protein, mix thoroughly for 1 hour, and collect the supernatant by high-speed centrifugation; transfer the supernatant to a dialysis bag, dialyze in refolding solution 1 at 4°C for 36 hours, replace the refolding solution once every 12 hours, and then transfer the dialysis bag to PBS solution and continue dialysis for 12 hours to obtain the water-soluble HPV16 fusion protein antigen as described in Example 1.

[0052] The main components of the solution are as follows:

[0053] Wash solution 1: 25 mM Tris HCl, 150 mM NaCl;

[0054] Wash solution 2: 25 mM Tris HCl, 150 mM NaCl, 1% Triton X-100;

[0055] Wash solution 3: 25 mM Tris HCl, 150 mM NaCl, 1 M urea;

[0056] Denaturation solution 1: 25 mM Tris HCl, 300 mM NaCl, 6 M guanidine hydrochloride;

[0057] Refolding Solution 1: 25 mM Tris HCl, 150 mM NaCl, 100 mM arginine, 5% glycerol, 15 mM oxidized glutathione, 5 mM reduced glutathione.

[0058] Example 4

[0059] A method for preparing an HPV18 fusion protein antigen comprises the following steps:

[0060] Step 1: Based on the amino acid sequence of the fusion protein antigen described in Example 2, codon optimization was performed to obtain a matching nucleotide sequence; the nucleotide sequence is shown in SEQ ID NO: 4;

[0061] Step 2: The nucleotide sequence from step 1 was constructed into the expression vector pET28b by homologous recombination;

[0062] Step 3: Introduce the recombinant expression vector from step 2 into Escherichia coli for recombinant expression; the Escherichia coli is Rosseta; select the recombinant E. coli and culture it in a shake flask; the shake flask culture conditions are 37°C, 220 rpm; when the E. coli concentration reaches OD450 = 0.4, IPTG is added to a final concentration of 0.2 mM and the culture is continued for 6 hours;

[0063] Step 4: Collect the Escherichia coli in step 3, use an ultrasonic instrument to break up the bacterial suspension, and then collect the precipitate by centrifugation; at 25°C, wash the precipitate with washing solution 4, washing solution 5 and washing solution 6 in sequence to remove impurities from the inclusion body to obtain a higher purity inclusion body protein; add 10 times the volume of denaturing solution 2 to the inclusion body protein, mix thoroughly for 1 hour, and collect the supernatant by high-speed centrifugation; transfer the supernatant to a dialysis bag, dialyze in refolding solution 2 at 4°C for 36 hours, replace the refolding solution once every 12 hours, and then transfer the dialysis bag to PBS solution and continue dialysis for 12 hours to obtain the water-soluble HPV18 fusion protein antigen as described in Example 2.

[0064] The main components of the solution are as follows:

[0065] Wash solution 4: 25 mM Tris HCl, 100 mM NaCl, 50 mM KCl;

[0066] Wash solution 5: 25 mM Tris HCl, 100 mM NaCl, 50 mM KCl, 1% Tween 20;

[0067] Wash solution 6: 25 mM Tris HCl, 100 mM NaCl, 50 mM KCl, 1 M urea;

[0068] Denaturing solution 2: 25 mM HEPES, 200 mM NaCl, 100 mM KCl, 6 M guanidine hydrochloride;

[0069] Refolding solution 2: 25 mM HEPES, 100 mM NaCl, 50 mM KCl, 150 mM arginine, 5% glycerol, 20 mM oxidized glutathione, 5 mM reduced glutathione.

[0070] Example 5

[0071] The present invention separates and purifies for the first time an egg yolk antibody based on a novel HPV fusion protein antigen. The egg yolk antibody is prepared by immunizing laying hens with the novel HPV fusion protein antigen of Example 1 or Example 2.

[0072] Furthermore, the preparation method of egg yolk antibody comprises the following steps:

[0073] (1) Obtain the novel HPV fusion protein antigen described in Example 1 or Example 2 according to the preparation method of the novel HPV fusion protein antigen described in Example 3 or Example 4;

[0074] (2) The primary immunogen was an HPV fusion protein antigen solution (HPV fusion protein antigen content was 200 μg) mixed with an equal volume of Freund's complete adjuvant to form an oil-in-water emulsion. Six points on the breast of laying hens were disinfected with conventional alcohol and 0.8 mL of the emulsion was distributed intramuscularly injected for immunization. The subsequent booster immunization antigen was an HPV fusion protein antigen solution (HPV fusion protein antigen content was 200 μg) mixed with Freund's incomplete adjuvant to be boosted twice at an interval of 15 days. Seven days after the first booster immunization, the eggs were collected and stored at 4°C for later use.

[0075] (3) Isolation and purification of egg yolk antibodies: Separate and collect egg yolks and dilute them with 10 times the volume of PBS buffer. Centrifuge the egg yolk dilution at 10,000 g for 10 minutes, discard the precipitate, filter the supernatant with cotton wool, and then add a saturated ammonium sulfate solution to make the supernatant ammonium sulfate saturation reach 40%-50%. Let it stand at 4°C for 10 hours. After centrifugation at 10,000 g for 30 minutes, collect the precipitate, add 10 mL of PBS buffer per milliliter of precipitate, dissolve it at 4°C for 2 hours, and then centrifuge it at 15,000 g for 20 minutes. Put the supernatant into a dialysis bag and dialyze it with PBS buffer for 12 hours. After the dialysate is concentrated by ultrafiltration and sterilized by filtration, egg yolk antibodies based on the novel HPV fusion protein antigen are obtained.

[0076] Example 6

[0077] like Figure 2-3 As shown, the binding titers of egg yolk antibodies based on the novel HPV fusion protein antigen against HPV16 and HPV18 subtypes were determined using an ELISA. 50 μg / mL of HPV16 and HPV18 L1 proteins, respectively, were used as detection antigens and coated onto 96-well plates. The purified egg yolk antibodies were then added at varying dilutions and incubated at 37°C for 1 hour. After washing, a 1:5000 dilution of horseradish peroxidase-labeled rabbit anti-chicken IgY antibody was added and incubated at 37°C for 1 hour. After washing, the substrate was added for color development. Absorbance at 450 nm was measured using a microplate reader. Egg yolk antibodies extracted from non-immunized chicken egg yolk fluid served as a control.

[0078] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for constructing a novel HPV fusion protein antigen, characterized by: The HPV fusion protein antigen is obtained by sequentially connecting an antigen peptide, the Fc region of immunoglobulin IgY and the tail peptide of immunoglobulin IgM through a polypeptide linker, wherein the antigen peptide is the core antigen sequence of HPV16 or HPV18 capsid protein L1.

2. The method for constructing a novel HPV fusion protein antigen according to claim 1, characterized in that: The antigen peptide is fused to the Fc region of immunoglobulin IgY via a polypeptide linker (GSSG)n, wherein n is a natural number between 1-3.

3. The method for constructing a novel HPV fusion protein antigen according to claim 1, characterized in that: The Fc region of immunoglobulin IgY is linked to the tail peptide of immunoglobulin IgM via a polypeptide linker TGS.

4. The method for constructing a novel HPV fusion protein antigen according to claim 1, characterized in that: The amino acid sequences of the HPV fusion protein antigen are shown in SEQ ID NO: 1 and SEQ ID NO:

2.

5. A method for preparing a novel HPV fusion protein antigen, characterized by: The following steps are involved: Step 1: codon-optimizing the novel HPV fusion protein antigen according to any one of claims 1 to 4 to obtain a matching nucleotide sequence; Step 2: The nucleotide sequence in step 1 is constructed into a prokaryotic expression vector by homologous recombination to obtain a recombinant expression vector; Step 3: Introduce the recombinant expression vector from step 2 into Escherichia coli for recombinant expression; Step 4: Collect the E. coli from step 3 and separate and purify the HPV fusion protein using protein inclusion body renaturation technology.

6. The method for preparing a novel HPV fusion protein antigen according to claim 5, characterized in that: In step 1, the nucleotide sequences of the HPV fusion protein antigen after codon optimization are shown in SEQ ID NO: 3 and SEQ ID NO:

4.

7. The method for preparing a novel HPV fusion protein antigen according to claim 5, characterized in that: The protein refolding solution used in the protein inclusion body refolding technique in step 4 contains arginine, oxidized glutathione, reduced glutathione and glycerol.

8. Use of a novel HPV fusion protein antigen in the preparation of polyclonal egg yolk antibodies, characterized in that: Polyclonal egg yolk antibodies for preventing or treating HPV virus infection are prepared from immunized laying hens carrying the HPV fusion protein antigen according to claim 5.

9. Use of a novel HPV fusion protein antigen in the preparation of polyclonal egg yolk antibodies according to claim 8, characterized in that: The method for preparing polyclonal egg yolk antibodies comprises the following steps: Step 1: Immunization procedure for laying hens: The primary immunogen is 200 µg of the novel HPV fusion protein antigen mixed with an equal volume of Freund's complete adjuvant to form an oil-in-water emulsion. Six points on the chest of the laying hens are disinfected with conventional alcohol and 0.8 mL of the oil-in-water emulsion is distributed intramuscularly for immunization. The subsequent booster immunization is the novel HPV fusion protein antigen mixed with Freund's incomplete adjuvant. Two booster immunizations are performed at intervals of 15 days. Seven days after the first booster immunization, the eggs are collected and stored at 4°C for later use. Step 2: Isolation and purification of polyclonal egg yolk antibodies: Egg yolks were separated and collected, and diluted with 10-fold volume of PBS buffer. The egg yolk dilution was centrifuged at 10,000 g for 10 minutes, the precipitate was discarded, and the supernatant was filtered through absorbent cotton. A saturated ammonium sulfate solution was then added until the ammonium sulfate saturation of the supernatant reached 40%-50%. The mixture was allowed to stand at 4°C for 10 hours and centrifuged. After 30 minutes of centrifugation, the precipitate was collected and 10 mL of PBS buffer was added per mL of precipitate. The mixture was dissolved at 4°C for 2 hours, and then centrifuged at 15,000 g for 20 minutes. The supernatant was placed in a dialysis bag and dialyzed with PBS buffer for 12 hours. The dialysate was concentrated by ultrafiltration and sterilized by filtration to obtain polyclonal egg yolk antibodies based on the novel HPV fusion protein antigen.

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