A novel HPV fusion protein antigen, preparation method and application in preparation of polyclonal yolk antibody

Abstract

The application provides a novel HPV fusion protein antigen, a preparation method and application in preparation of polyclonal yolk antibody, which first connects core antigen peptides of human papilloma virus 16 subtype and HPV 18 subtype capsid protein L1 with Fc of immunoglobulin IgY and tail peptides of immunoglobulin IgM in series, and the above-mentioned recombinant fusion protein antigen is prepared by means of E. coli expression and inclusion body renaturation technology, which is beneficial to improving antigen peptide stability and presentation capacity, and the novel HPV fusion protein antigen is directly used for immunizing laying hens, after antibody production, the antibody is purified through steps such as salting-out and dialysis, and verification shows that the obtained yolk antibody can combine with L1 proteins of HPV16 and HPV18, and has the ability of preventing or treating HPV16 and HPV18 infection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical technology, in particular to a novel HPV fusion protein antigen, a preparation method and application in preparing polyclonal yolk antibody. BACKGROUND

[0002] Human papillomavirus (HPV) is the main pathogen causing cervical cancer, anogenital cancer and oropharyngeal cancer, etc. About 5.3% of cancers worldwide are associated with HPV infection. More than 200 subtypes of HPV have been found, which are divided into high-risk and low-risk types according to the carcinogenic risk. Among them, HPV 16 / 18 is the core carcinogenic type, which causes more than 70% of cervical cancer worldwide. Low-risk types mainly cause benign lesions, although they are not carcinogenic, but they can create conditions for high-risk type infection. The HPV capsid is composed of major capsid protein L1 and minor capsid protein L2, which play a key role in virus assembly, infection and immune response. The antigenic properties of L1 protein mainly depend on the structure of its surface loop. Studies have shown that the BC, DE, EF, FG and HI five loop regions on the surface of the pentamer are the main antigen epitope regions, which contain a large number of type-specific neutralizing epitopes. L1 protein can self-assemble into virus-like particles (VLPs) in vitro. These VLPs are highly similar in morphology to natural virus particles, with a diameter of about 50-60 nm, but do not contain viral DNA, so there is no risk of infection. This characteristic makes L1 VLP an ideal antigen form for HPV preventive vaccines, which can effectively induce the body to produce high titers of type-specific neutralizing antibodies to block the initial binding of the virus to host cells. However, the traditional L1 VLP production technology faces several challenges: high cost and long production cycle in eukaryotic systems; VLP assembly process requires disassembly and reassembly, resulting in low yield. Fc fusion protein is a new type of recombinant protein formed by fusion of the Fc domain of immunoglobulin (such as IgG, IgM, etc.) with the target protein sequence. Fc fusion protein as an antigen has significant advantages in antibody development, mainly due to its structural characteristics and biological functions:

[0003] (1) Enhanced immunogenicity:

[0004] 1) Fcγ receptor-mediated antigen presentation: The Fc region in the Fc fusion protein can be recognized and bound by the Fcγ receptor on the surface of antigen-presenting cells (such as dendritic cells, macrophages, B cells). This binding promotes efficient endocytosis, processing of the antigen by APC, and presentation of the target antigen peptide to T cells, thereby significantly enhancing the adaptive immune response (activation of T and B cells) against the target antigen.

[0005] 2) Multimerization effect: The Fc region of immunoglobulin tends to form dimers under physiological conditions. Therefore, Fc fusion proteins are usually bivalent or multivalent molecules. This multimerization can more effectively cross-link B cell receptors, providing a stronger activation signal, promoting B cell activation, proliferation, and differentiation into plasma cells, and producing more high-affinity antibodies.

[0006] 3) Activation of the complement alternative pathway: The Fc region can bind to complement components (such as C1q) to activate the complement cascade. Complement activation products (such as C3d) are potent B cell helper signal molecules that enhance stability and solubility, further amplifying the immune response.

[0007] (2) Improved stability and solubility:

[0008] Improved stability and solubility: Fc domains are usually very stable and have high solubility. Fusing them to some target proteins that are unstable, prone to aggregation, or have poor solubility can significantly improve the physicochemical properties of the entire fusion protein, making it less likely to degrade or precipitate during storage and immunization operations, and maintaining the native conformation of the antigen epitope.

[0009] (3) Prolonged half-life in vivo: The Fc segment can significantly prolong the half-life of the protein in the blood circulation by binding to receptors, which may help maintain stronger immune stimulation and continuous production of high-titer antibodies for experimental animals that require multiple immunizations. Not only that, but by genetically engineering the Fc to form higher aggregation states, it is beneficial to further enhance its stability and improve antigen presentation capacity. Immunoglobulin of yolk (IgY) is an antigen-stimulated avian B lymphocyte that differentiates into a plasma cell and secretes specific antibodies into the blood circulation, which gradually accumulates in the oocyte when the blood flows through the ovary, forming yolk antibodies. This antibody is considered a mammalian IgG analog, and its Fc domain has similar functions and characteristics to IgG Fc. In addition, yolk antibodies have the advantages of high stability, strong specificity, low preparation cost, oral safety, and good application value in the diagnosis, prevention, and treatment of various diseases in humans and animals. SUMMARY

[0010] The production technology of VLP antigens prepared by using HPV capsid protein L1 in the prior art faces multiple challenges: high cost and long production cycle of eukaryotic system expression; and low yield caused by the disassembly and reassembly process of VLP assembly. In view of the technical problems existing in the prior art, the application provides a novel HPV fusion protein antigen and application of yolk antibodies thereof. In the research, the core antigen epitope of HPV16 and HPV18 capsid protein L1 is connected in sequence with the Fc domain of IgY and the tail peptide sequence of IgM, respectively, and the gene sequence encoding the fusion protein is obtained by codon optimization. Further, the inclusion body of the HPV fusion protein antigen is obtained by using Escherichia coli recombinant expression, and the high-purity and water-soluble HPV fusion protein antigen is prepared by the method of protein renaturation. The yolk antibody is directly immunized by using the antigen to produce an egg-laying chicken, and the antibody is purified through steps such as salting-out and dialysis. Verification shows that the antibody can recognize HPV16 or HPV18 capsid protein L1, and has the ability to prevent or treat HPV16 or HPV18 infection.

[0011] Specifically, the application first provides a novel HPV fusion protein antigen, which is obtained by connecting in sequence an antigen peptide, an Fc region of immunoglobulin IgY and a tail peptide of immunoglobulin IgM through a polypeptide linker, wherein the antigen peptide is a core antigen sequence of HPV16 or HPV18 capsid protein L1, and the amino acid sequence of the HPV fusion protein antigen is shown as SEQ ID NO: 1 or SEQ ID NO: 2.

[0012] Preferably, the antigen peptide and the Fc region of immunoglobulin IgY are fused through a polypeptide linker (GSSG)n, wherein 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 sequence through the polypeptide linker (GSSG)n.

[0013] Preferably, the Fc region of immunoglobulin IgY and the tail peptide of immunoglobulin IgM are connected through a polypeptide linker TGS. On the other hand, the application also provides a preparation method of the novel HPV fusion protein antigen, comprising the following steps:

[0014] Step 1: codon optimization is performed on the novel HPV fusion protein antigen to obtain a matched nucleotide sequence, as shown in SEQ ID NO: 3 or SEQ ID NO: 4;

[0015] 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 pET21b, pET28 or pET30a, etc.

[0016] Step 3: The recombinant expression vector in step 2 is introduced into E. 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, cultured and recombinantly expressed in a constant temperature shaker incubator under the following conditions: 37°C, 220 rpm.

[0017] Step 4: The E. coli in step 3 is collected, and the HPV fusion protein is separated and purified by protein inclusion body renaturation technology, i.e. the HPV fusion protein antigen is obtained, and the specific operation is as follows: the bacterial suspension is broken by using an ultrasonic instrument, the precipitate is collected by centrifugation, and the precipitate is repeatedly washed to obtain inclusion bodies containing the HPV fusion protein antigen, then the inclusion bodies are dissolved with a denaturing solution containing urea or guanidine hydrochloride, and the dissolved inclusion bodies are dialyzed in a refolding solution, i.e. the water-soluble HPV fusion protein antigen is obtained.

[0018] On the other hand, the application also provides a use of the novel HPV fusion protein antigen in the preparation of polyclonal yolk antibodies, wherein the polyclonal yolk antibodies for preventing or treating HPV virus infection are prepared from immune laying hens carrying the HPV fusion protein antigen.

[0019] Preferably, the preparation method of the polyclonal yolk antibodies comprises the following steps:

[0020] Step 1: Laying hen immunization program: the primary immunogen is the novel HPV fusion protein antigen with an antigen content of 200µg mixed with an equal volume of Freund's complete adjuvant to form a water-in-oil emulsion, the 6 points on the chest of the laying hen are conventionally sterilized with alcohol, and the laying hen is immunized by intramuscular injection of 0.8mL of the water-in-oil emulsion, then the antigen for the booster immunization is the novel HPV fusion protein antigen with an antigen content of 200µg mixed with Freund's incomplete adjuvant, and the booster immunization is performed twice at an interval of 15 days, and the chicken eggs are collected 7 days after the first booster immunization and stored at 4°C for standby use.

[0021] Step 2: Isolation and purification of polyclonal yolk antibody: the egg yolk of the collected chicken egg is diluted with 10 times volume of PBS buffer, the diluted egg yolk is centrifuged at 10000g for 10 minutes, the precipitate is discarded, the supernatant is filtered with defatted cotton, then saturated ammonium sulfate solution is added to the supernatant to reach 40%-50% saturation of ammonium sulfate. 4℃ for 10 hours, centrifugation, 30 minutes after centrifugation, the precipitate is collected, 10mL PBS buffer is added to each milliliter of the precipitate, and the solution is dissolved at 4℃ for 2 hours, then 15000g centrifugation is performed for 20 minutes, the supernatant is loaded into a dialysis bag and dialyzed with PBS buffer for 12 hours, and the dialysate is concentrated and filtered to remove bacteria to obtain the polyclonal yolk antibody based on the novel HPV fusion protein antigen.

[0022] The polyclonal yolk antibody based on the novel HPV fusion protein antigen is used in the preparation of a preparation for preventing or treating HPV virus infection diseases, and the preparation includes but is not limited to ointments, creams, suspensions, solutions, gels, sprays or transdermal patches and the like.

[0023] Compared with the prior art, the polyclonal yolk antibody based on the novel HPV fusion protein antigen has the advantages and beneficial effects that:

[0024] The polyclonal yolk antibody based on the novel HPV fusion protein antigen has the following advantages:

[0025] (1) Improve the stability of the antigen: the Fc region of IgY has high similarity with the Fc of IgG, which can significantly improve the stability of the antigen peptide;

[0026] (2) Improve immunogenicity: the Fc region can bind to the receptor of IgY, improve the sensitization efficiency, and prolong the half-life of the fusion protein antigen in the body;

[0027] (3) The tailpiece of IgM can induce Fc fusion protein oligomerization, further improve the stability of the fusion protein and the antigen presentation ability;

[0028] (4) Improve the yield of effective antibodies: the fusion protein only contains a core antigen peptide segment, which can increase the yield of effective antibodies;

[0029] (5) Directly immunize the egg-laying chicken with the purified HPV fusion protein antigen, and after the antibody is produced, the polyclonal yolk antibody is obtained through the processes of salting-out, dialysis and concentration. It is verified that the antibody can bind to the capsid protein L1 of HPV16 or HPV18, and can effectively prevent or treat human papilloma virus (HPV) infection. BRIEF DESCRIPTION OF DRAWINGS

[0030] The above and / or additional aspects and advantages of the present application will become apparent and be readily understood from the following description, taken in conjunction with the accompanying drawings, in which:

[0031] Fig. 1 is a process flow chart of a novel HPV fusion protein antigen, preparation and application of the present application.

[0032] Fig. 2 is a binding analysis of the polyclonal yolk antibody of the present application and the HPV16 capsid protein L1.

[0033] Fig. 3 is a binding analysis of the polyclonal yolk antibody of the present application and the HPV18 capsid protein L1. DETAILED DESCRIPTION

[0034] In order to more clearly understand the above-mentioned purposes, features and advantages of the present application, the present application will be further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be noted that the technical solutions and features in the technical solutions of the present application can be combined with each other without conflict. Figures 1-3 and the specific embodiments. It should be noted that the technical solutions and features in the technical solutions of the present application can be combined with each other without conflict.

[0035] In the following description, a large number of specific details are set forth in order to facilitate a thorough understanding of the present application, however, the present application can also be implemented in other ways different from those described herein, and therefore, the protection scope of the present application is not limited by the specific technical solutions disclosed below.

[0036] The technical solutions of the present application will be described below in conjunction with specific embodiments. The described embodiments are only a part of the embodiments of the present application, and not all the embodiments. All other embodiments obtained by those skilled in the art without creative labor based on the present application are within the scope of protection of the present application. EMBODIMENT

[0037] A novel HPV fusion protein antigen, the fusion protein is obtained by fusing the core antigen epitope of HPV16 capsid protein L1 with the Fc region of IgY through a polypeptide linker GSSG, and the tail peptide of IgM with the Fc region of IgY through a polypeptide linker TGS, the amino acid sequence is shown as SEQ ID NO: 1. EMBODIMENT

[0038] A novel HPV fusion protein antigen, which is obtained by fusing the core antigen epitope of HPV18 coat protein L1 with the Fc region of IgY through a polypeptide linker GSSG and the tail peptide of IgM with the Fc region of IgY through a polypeptide linker TGS, and the amino acid sequence is shown as SEQ ID NO: 2. Embodiment

[0039] A preparation method of an HPV16 fusion protein antigen, the method comprising the following steps:

[0040] Step 1: Based on the amino acid sequence of the fusion protein antigen described in Embodiment 1, a matching nucleotide sequence is obtained through codon optimization; the nucleotide sequence is shown as SEQ ID NO: 3;

[0041] Step 2: The nucleotide sequence of step 1 is constructed into an expression vector pET28a by homologous recombination;

[0042] Step 3: The recombinant expression vector of step 2 is introduced into E. coli strain BL21(DE3) by heat shock for recombinant expression; the recombinant E. coli is selected and subjected to shake flask culture; the shake flask culture conditions are 37℃ and 220rpm; when the concentration of E. coli reaches OD450=0.6, IPTG is added to a final concentration of 0.6mM, and the culture is continued for 5 hours;

[0043] Step 4: The E. coli in step 3 is collected, the bacterial suspension is subjected to disruption treatment using an ultrasonic instrument, and the precipitate is collected by centrifugation; the precipitate is sequentially washed with wash solution 1, wash solution 2 and wash solution 3 at 25℃ to remove impurities, and the inclusion body protein with high purity is obtained; 10 times the volume of denaturing solution 1 is added to the inclusion body protein, mixed thoroughly for 1 hour, and the supernatant is collected by high-speed centrifugation; the supernatant is transferred to a dialysis bag and dialyzed in refolding solution 1 at 4℃ for 36 hours, and the refolding solution is replaced every 12 hours; then the dialysis bag is transferred to PBS solution for continuous dialysis for 12 hours, and the water-soluble HPV16 fusion protein antigen as described in Embodiment 1 is obtained.

[0044] The main components of the solution are as follows:

[0045] Wash solution 1: 25mM Tris HCl, 150mM NaCl;

[0046] Wash solution 2: 25mM Tris HCl, 150mM NaCl, 1% Triton X-100;

[0047] Washing solution 3: 25 mM Tris HC1, 150 mM NaCl, 1M urea;

[0048] Denaturing solution 1: 25 mM Tris HC1, 300 mM NaCl, 6M guanidine HC1;

[0049] Renaturing solution 1: 25 mM Tris HC1, 150 mM NaCl, 100 mM arginine, 5% glycerol, 15 mM oxidized glutathione, 5 mM reduced glutathione. Example

[0050] A method for preparing an HPV18 fusion protein antigen, the method comprising the following steps:

[0051] Step 1: based on the amino acid sequence of the fusion protein antigen described in Example 2, a matching nucleotide sequence is obtained by codon optimization; the nucleotide sequence is shown as SEQ ID NO: 4;

[0052] Step 2: the nucleotide sequence of step 1 is constructed into an expression vector pET28b by homologous recombination;

[0053] Step 3: the recombinant expression vector of step 2 is introduced into Escherichia coli for recombinant expression; the Escherichia coli is Rossetta; the recombinant Escherichia coli is selected and subjected to flask culture; the flask culture conditions are 37°C, 220rpm; when the concentration of the Escherichia coli reaches OD450=0.4, then IPTG is added to a final concentration of 0.2mM, and the culture is continued for 6 hours;

[0054] Step 4: the Escherichia coli in step 3 is collected, the bacterial suspension is subjected to crushing treatment using an ultrasonic instrument, and the precipitate is collected by centrifugation; the precipitate is sequentially washed with washing solution 4, washing solution 5 and washing solution 6 at 25°C to remove impurities from the inclusion body, so that a high-purity inclusion body protein is obtained; 10 times the volume of denaturing solution 2 is added to the inclusion body protein, mixed thoroughly for 1 hour, and then the supernatant is collected by high-speed centrifugation; the supernatant is transferred to a dialysis bag, dialyzed in renaturing solution 2 at 4°C for 36 hours, the renaturing solution is replaced every 12 hours, then the dialysis bag is transferred to PBS solution for continued dialysis for 12 hours, and thus the water-soluble HPV18 fusion protein antigen described in Example 2 is obtained.

[0055] The main components of the solution are as follows:

[0056] Washing solution 4: 25 mM Tris HC1, 100 mM NaCl, 50 mM KCl;

[0057] Washing solution 5: 25 mM Tris HC1, 100 mM NaCl, 50 mM KCl, 1% Tween 20;

[0058] Washing solution 6: 25 mM Tris HC1, 100 mM NaCl, 50 mM KCl, 1M urea;

[0059] Denaturing solution 2: 25 mM HEPES, 200 mM NaCl, 100 mM KCl, 6M guanidine HC1;

[0060] Renaturing solution 2: 25 mM HEPES, 100 mM NaCl, 50 mM KCl, 150 mM arginine, 5% glycerol, 20 mM oxidized glutathione, 5 mM reduced glutathione. Example

[0061] The present application first isolates and purifies a yolk antibody based on a novel HPV fusion protein antigen, which is immunized by the novel HPV fusion protein antigen of Example 1 or Example 2

[0062] The yolk antibody is prepared from laying hens.

[0063] Further, the preparation method of the yolk antibody comprises the following steps:

[0064] (1) The novel HPV fusion protein antigen of Example 1 or Example 2 is obtained according to the preparation method of the novel HPV fusion protein antigen described in Example 3 or Example 4;

[0065] (2) The primary immunogen is the HPV fusion protein antigen solution (the HPV fusion protein antigen content is 200 µg) mixed with an equal volume of Freund's complete adjuvant to form a water-in-oil emulsion, the 6 points on the chest of the laying hen are sterilized with conventional alcohol, and the distributed intramuscular injection of 0.8 mL emulsion is performed for immunization, and then the antigen for the subsequent booster immunization is the HPV fusion protein antigen solution (the HPV fusion protein antigen content is 200 µg) mixed with Freund's incomplete adjuvant, and the booster immunization is performed twice at an interval of 15 days. Seven days after the first booster immunization, the chicken eggs are collected and stored at 4°C for standby;

[0066] (3) Isolation and purification of egg yolk antibody: collect egg yolk and add 10 times volume of PBS buffer solution for dilution, centrifuge the egg yolk dilution at 10000g for 10 minutes, discard the precipitate, filter the supernatant with absorbent cotton, then add saturated ammonium sulfate solution to make the saturation of ammonium sulfate in the supernatant reach 40%-50%, stand at 4°C for 10 hours, centrifuge at 10000g for 30 minutes, collect the precipitate, add 10ml of PBS buffer solution per milliliter of precipitate, dissolve at 4°C for 2 hours, then centrifuge at 15000g for 20 minutes, load the supernatant into a dialysis bag and dialyze with PBS buffer solution for 12 hours, concentrate and filter sterilize the dialysate, and the egg yolk antibody based on the novel HPV fusion protein antigen is obtained. Embodiment

[0067] As shown in Figures 2-3, the binding titer of the egg yolk antibody based on the novel HPV fusion protein antigen to HPV16 and 18 subtypes was determined by ELISA. 50μg / mL of L1 protein of HPV16 and HPV18 was selected as the detection antigen, respectively, to coat the 96-well plate, and finally different dilution multiples of the above purified egg yolk antibody were added, incubated at 37°C for 1 hour, washed the plate, then added 1:5000 diluted rabbit anti-chicken IgY antibody labeled with horseradish peroxidase, incubated at 37°C for 1 hour, washed the plate, then added substrate for color development. The absorbance value at 450nm wavelength was determined by an enzyme marker, and the egg yolk antibody extracted from the yolk of non-immunized chicken was used as a control.

[0068] The above is a preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A novel HPV fusion protein antigen, characterized in that: The HPV fusion protein antigen is obtained by sequentially linking the antigenic peptide, the Fc region of immunoglobulin IgY, and the tail peptide of immunoglobulin IgM through a polypeptide linker. The antigenic peptide is the core antigen sequence of HPV16 or HPV18 capsid protein L1. The amino acid sequence of the HPV fusion protein antigen is shown in SEQ ID NO: 1 or SEQ ID NO:

2.

2. A method for preparing a novel HPV fusion protein antigen, characterized in that: Includes the following steps: Step 1: Codon optimization is performed on the novel HPV fusion protein antigen described in claim 1 to obtain a matching nucleotide sequence; Step 2: The nucleotide sequence from Step 1 is constructed into a prokaryotic expression vector via homologous recombination to obtain a recombinant expression vector; Step 3: The recombinant expression vector from Step 2 is introduced into E. coli for recombinant expression; Step 4: Collect the E. coli from Step 3, and use protein inclusion body refolding technology to separate and purify the HPV fusion protein.

3. The method for preparing a novel HPV fusion protein antigen according to claim 2, characterized in that: In step 1, the codon-optimized nucleotide sequence of the HPV fusion protein antigen is shown in SEQ. As shown in ID NO: 3 or SEQ ID NO:

4.

4. The method for preparing a novel HPV fusion protein antigen according to claim 2, characterized in that: The protein refolding solution used in step 4 for the protein inclusion body refolding technique contains arginine, oxidized glutathione, reduced glutathione, and glycerol.

Images