Broad-spectrum cervical cancer vaccine based on human papillomavirus L2 protein and preparation method thereof

Abstract

The present application relates to the field of biopharmaceutical technology, and in particular to a broad-spectrum vaccine for cervical cancer based on human papillomavirus L2 protein and a preparation method thereof; the broad-spectrum vaccine comprises a nano-vaccine formed by encapsulating L2 protein polypeptide with nano-materials, the nano-vaccine being a core-shell structure, wherein the core is L2 protein polypeptide and the shell is nano-materials; the method comprises: epitope analysis and screening of HPV L2 protein antigen to obtain L2 protein polypeptide; encapsulating and preparing the L2 protein polypeptide and the nano-material to obtain a nano-vaccine; encapsulating and preparing the adjuvant and the nano-material to obtain a nano-adjuvant; collecting the nano-vaccine and the nano-adjuvant to obtain a broad-spectrum vaccine; by encapsulating the L2 protein polypeptide with nano-materials to form a nano-vaccine, the L2 protein polypeptide can be prevented from being destroyed, and the L2 protein polypeptide can be slowly released in the body, thereby comprehensively improving the stability of L2 protein polypeptide vaccines.

Description

Technical Field

[0001] The present application relates to the field of biopharmaceutical technology, and in particular to a broad-spectrum cervical cancer vaccine based on human papillomavirus L2 protein and a preparation method thereof. Background Art

[0002] High-risk human papillomavirus (HPV) infection is the main cause of cervical cancer. There are many HPV subtypes, and more than 220 types have been discovered so far. According to the degree of their pathogenicity to humans, they can be roughly divided into two categories: high-risk and low-risk. Among them, high-risk types mainly include HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58 and HPV59, which are related to the occurrence of human cervical cancer and high-grade cervical intraepithelial lesions (CINII / III); low-risk types are mainly represented by HPV6 and HPV11, which mainly cause benign proliferative lesions such as genital warts.

[0003] Virus preventive vaccines are biological products that use viral antigens as starting materials and are prepared using biotechnology. They stimulate the body's immune system to produce specific immune responses, thereby preventing corresponding viral infections. The representative virus preventive vaccine is the HPV vaccine. HPV vaccines generally use the virus's major capsid protein L1 and minor capsid protein L2 as target antigens to induce the body to produce high-titer neutralizing antibodies and effective local immune responses to prevent long-term infection and repeated infection of HPV.

[0004] At present, most HPV vaccines are composed of virus-like particles formed by the recombination of the main capsid protein L1 gene of the contained virus type. They are type-specific and can only prevent infection against the contained type, but cannot completely protect the body from infection with other HPV types. The L2 protein is highly conserved among various HPV types, and there are many polypeptide epitopes that can cross-neutralize with other types of HPV. Most of them are linear epitopes, and their polypeptides have a small molecular weight and can be produced in a prokaryotic system. They have the advantages of simple process, low cost, and few side effects. However, compared with the L1 protein, the antibody titer induced by it is lower. This is because traditional protein polypeptide vaccines will be destroyed by enzymes and other factors in the body, which makes the L2 protein polypeptide poorly stable in the body and leads to a shorter half-life of the L2 protein polypeptide in the body. Therefore, how to improve the stability of L2 protein polypeptide vaccines in the body to increase the half-life of the L2 protein polypeptide in the body is a technical problem that urgently needs to be solved. Summary of the invention

[0005] The present application provides a broad-spectrum cervical cancer vaccine based on human papillomavirus L2 protein and a preparation method thereof, in order to solve the technical problem of poor in vivo stability of L2 protein polypeptide vaccines in the prior art.

[0006] In a first aspect, the present application provides a broad-spectrum vaccine for cervical cancer based on human papillomavirus L2 protein, wherein the broad-spectrum vaccine comprises a nanovaccine formed by encapsulating L2 protein polypeptide with nanomaterials, wherein the nanovaccine is a core-shell structure, wherein the core is the L2 protein polypeptide and the shell is the nanomaterial.

[0007] Optionally, the sequence of the L2 protein polypeptide is as shown in SEQ ID NO.1.

[0008] Optionally, the broad-spectrum vaccine further includes a nano-adjuvant formed by encapsulating an adjuvant with a nano-material, and the nano-adjuvant is a core-shell structure, wherein the core is the adjuvant and the shell is the nano-material.

[0009] Optionally, the adjuvant comprises imiquimod (ie, R837).

[0010] Optionally, the nanomaterial includes: TPGS-b-(PCL-ran-PGA) copolymer nanomaterial.

[0011] In a second aspect, the present application provides a method for preparing the broad-spectrum vaccine according to the first aspect, the method comprising:

[0012] Performing epitope analysis and screening on HPV L2 protein antigens to obtain an L2 protein polypeptide as shown in SEQ ID NO.1;

[0013] Encapsulating the L2 protein polypeptide and the nanomaterial to obtain a nano vaccine;

[0014] Encapsulating the adjuvant and the nanomaterial to obtain a nano adjuvant;

[0015] The nano vaccine and the nano adjuvant are collected to obtain a broad-spectrum vaccine.

[0016] Optionally, the epitope analysis and screening of the HPV L2 protein antigen to obtain the L2 protein polypeptide as shown in SEQ ID NO.1 specifically includes:

[0017] The gene fragment of HPV L2 protein antigen is compared with the gene sequence in the DNA sequence database, and then analyzed to obtain the amino acid sequence of L2 protein;

[0018] Predicting the secondary structure, hydrophilicity, plasticity, antigenic index and surface accessibility of the amino acid sequence, and then investigating and screening it under target conditions to obtain multiple L2 protein polypeptide sequences;

[0019] A plurality of L2 protein polypeptide sequences were screened to obtain the L2 protein polypeptide as shown in SEQ ID NO.1.

[0020] Optionally, the target conditions include the hydrophilicity of the polypeptide, the spatial structure of the polypeptide, the sequence conservation of the polypeptide and the immunogenicity of multiple sequences.

[0021] Optionally, the screening includes screening based on a preset sequence length of amino acids and whether there is a secondary structure, and the preset sequence length is 8 to 20 amino acids.

[0022] Optionally, the particle sizes of the nano vaccine and the nano adjuvant are both 100 nm to 600 nm.

[0023] The above technical solution provided by the embodiment of the present application has the following advantages compared with the prior art:

[0024] The embodiment of the present application provides a broad-spectrum vaccine for cervical cancer based on human papillomavirus L2 protein, which forms a nano vaccine by encapsulating L2 protein polypeptide with nano materials. Since the L2 protein polypeptide has a small molecular weight, the encapsulation of the nano materials can to a certain extent prevent the enzymes and other factors in the body from damaging the L2 protein polypeptide. At the same time, the L2 protein polypeptide is slowly released in the body through the support of the nano materials, exerting the effects of slow-controlled release and long circulation, thereby comprehensively improving the stability of the L2 protein polypeptide vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0027] Figure 1 A schematic diagram of a process for a method provided in an embodiment of the present application;

[0028] Figure 2 A detailed flowchart of the method provided in the embodiment of the present application;

[0029] Figure 3 A schematic diagram of the immunogenicity analysis of the amino acid sequence of the L2 protein polypeptide provided in the examples of the present application;

[0030] Figure 4 A schematic diagram of the L2 protein polypeptide sequence structure analysis provided in the examples of the present application;

[0031] Figure 5 A schematic diagram of the process of the double emulsion solvent volatilization method provided in the embodiment of the present application;

[0032] Figure 6 This is a scanning electron microscope image of the nanovaccine TPP-L2pep nanoparticles provided in the examples of the present application;

[0033] Figure 7 The particle size distribution diagram of the nanovaccine TPP-L2pep nanoparticles provided in the examples of the present application;

[0034] Figure 8 The particle size distribution diagram of the nanoadjuvant TPP-R837 nanoparticles provided in the examples of the present application;

[0035] Fig. 9 Schematic diagram of the in vitro release curves of the nanoadjuvant and nanovaccine provided in the examples of the present application;

[0036] Fig.10 A schematic diagram of the uptake of blank nanoparticles by cells provided in the examples of the present application;

[0037] Fig.11 A schematic diagram of the uptake of fluorescently labeled nanoparticles TPP-FITC-BSA by cells provided in the examples of the present application;

[0038] Fig.12 This is a graph comparing the serum antibody levels of the TPP-L2pep group and the TPP-L2pep and TPP-R837 mixed group at a dilution of 1:200 provided in the examples of the present application. DETAILED DESCRIPTION

[0039] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are for illustrating the present invention, rather than limiting the present invention.

[0040] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0041] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0042] The technical solution provided by the embodiment of the present invention is to solve the above technical problems, and the overall idea is as follows:

[0043] In one embodiment of the present application, a broad-spectrum vaccine for cervical cancer based on human papillomavirus L2 protein is provided, wherein the broad-spectrum vaccine comprises a nanovaccine formed by encapsulating L2 protein polypeptide with nanomaterials, wherein the nanovaccine is a core-shell structure, wherein the core is the L2 protein polypeptide and the shell is the nanomaterial.

[0044] In some optional embodiments, the sequence of the L2 protein polypeptide is as shown in SEQ ID NO.1.

[0045] In the embodiment of the present application, the positive effect of controlling the L2 protein polypeptide sequence to be CTSSTPIPGSRPT (Cys-Thr-Ser-Ser-Thr-Pro-Ile-Pro-Gly-Ser-Arg-Pro-Thr) is due to the fact that this sequence is a polypeptide sequence with amino acid positions 211-222 in the HPV52 L2 protein, and the sequence has homologous sequences to HPV16 / 33 / 35 / 58 types, so it has good broad spectrum, and the overall polypeptide sequence has good immunogenicity and hydrophilicity, and has no secondary structure, so it can be used as an immunogen for the preparation of nanovaccines.

[0046] In some optional embodiments, the broad-spectrum vaccine further includes a nano-adjuvant formed by encapsulating an adjuvant with a nano-material, and the nano-adjuvant is a core-shell structure, wherein the core is the adjuvant and the shell is the nano-material.

[0047] In the embodiments of the present application, the broad-spectrum vaccine also includes an adjuvant, which can assist the immune response of the nanovaccine and improve the preventive effect of the nanovaccine.

[0048] In some alternative embodiments, the adjuvant comprises imiquimod (R837).

[0049] In the embodiments of the present application, the control adjuvant includes imiquimod. Since Toll-like receptors (TLRs) are an important class of protein molecules involved in nonspecific immunity, their ligands or agonists can be used as important immune adjuvants to upregulate the specific immune response induced by preventive vaccines. TLR7 is a type I transmembrane protein that can induce the production of inflammatory factors, chemokines and IFN-I to exert biological effects, and artificially synthesized imiquimod (R837) can activate TLR7, and can exert immunomodulatory effects by inducing the production of inflammatory cytokines such as IFN-α and IL-12, thereby activating natural and acquired immunity. Therefore, the TLR7 agonist R837 can be used to assist the nanovaccine to improve the preventive effect of the nanovaccine.

[0050] In some optional embodiments, the nanomaterial includes: TPGS-b-(PCL-ran-PGA) copolymer nanomaterial.

[0051] In the embodiments of the present application, the positive effect of controlling the nanomaterial to include TPGS-b-(PCL-ran-PGA) copolymer nanomaterial is due to the fact that the nanomaterial TPGS-b-(PCL-ran-PGA) combines the excellent drug permeability of PCL, the rapid degradation of PGA and the excellent emulsification and solubility of TPGS. As a drug carrier, it can improve the stability and effect of the encapsulated drug and has good biocompatibility. It has been studied in the treatment of breast cancer, cervical cancer, and the application of oral chemotherapy drugs for lung cancer and bladder cancer. However, it has not yet been used in the field of preventive vaccines. Therefore, the use of copolymer nanomaterials to encapsulate L2 protein polypeptides to form preventive nanovaccines can greatly improve the stability of the nanovaccines.

[0052] In one embodiment of the present application, Figure 1 As shown, a method for preparing a broad-spectrum cervical cancer vaccine based on human papillomavirus L2 protein is provided, the method comprising:

[0053] S1. Perform epitope analysis and screening on HPV L2 protein antigen to obtain L2 protein polypeptide as shown in SEQ ID NO.1;

[0054] S2. Encapsulating the L2 protein polypeptide and the nanomaterial to obtain a nanovaccine;

[0055] S3. Encapsulating the adjuvant and the nanomaterial to obtain a nano adjuvant;

[0056] S4. collecting the nanovaccine and the nanoadjuvant to obtain a broad-spectrum vaccine;

[0057] Wherein, the encapsulation preparation includes any one of the preparation by double emulsion solvent evaporation method and the preparation by solvent evaporation method.

[0058] In some optional embodiments, such as Figure 2 As shown, the epitope analysis and screening of the HPV L2 protein antigen to obtain the L2 protein polypeptide as shown in SEQ ID NO.1 specifically includes:

[0059] S101. Comparing the gene fragment of HPV L2 protein antigen with the gene sequence in the DNA sequence database, and then analyzing to obtain the amino acid sequence of L2 protein;

[0060] S102. predicting the secondary structure, hydrophilicity, plasticity, antigenic index and surface accessibility of the amino acid sequence, and then investigating and screening according to target conditions to obtain multiple L2 protein polypeptide sequences;

[0061] S103. Screening the plurality of L2 protein polypeptide sequences to obtain the L2 protein polypeptide as shown in SEQ ID NO.1, wherein the DNA sequence database may be Genbank.

[0062] In the examples of the present application, the gene fragments of the HPV L2 protein antigen are first compared, and then the amino acid sequence of the L2 protein translated therefrom is analyzed, and then a plurality of L2 protein polypeptide sequences are screened out through the structural characteristics and performance characteristics of the amino acid sequence, and finally the L2 protein polypeptide sequences are further screened according to the characteristics of actual needs to obtain L2 polypeptide protein sequences with homologous sequences to HPV16 / 33 / 35 / 58 types, so as to make them have a broad spectrum.

[0063] In some optional embodiments, the target conditions include the hydrophilicity of the polypeptide, the spatial structure of the polypeptide, the sequence conservation of the polypeptide and the immunogenicity of the polypeptide.

[0064] In the examples of the present application, the types of target conditions are controlled so as to effectively screen out multiple L2 protein polypeptide sequences that meet the conditions, thus paving the way for subsequently obtaining the final single sequence L2 protein polypeptide.

[0065] In some optional embodiments, the screening includes screening based on a preset sequence length, hydrophilicity, and presence of a secondary structure of amino acids, wherein the preset sequence length is 8 to 20 amino acids.

[0066] In the embodiments of the present application, the positive effect of the preset sequence length of 8 to 20 is that within the preset sequence length range, it is ensured that the generated antibodies have affinity and specificity and no secondary structure, and it is easy to obtain high-purity polypeptide products, facilitate their preparation, and reduce the preparation cost of preventive vaccines.

[0067] In some optional embodiments, the particle size of the nanovaccine and the nanoadjuvant are both 100 nm to 600 nm.

[0068] In the examples of the present application, controlling the particle size of the nanovaccine and the nanoadjuvant is important to ensure the quality stability and effect of the nanovaccine.

[0069] Example 1

[0070] S1. Epitope analysis and screening of HPV L2 protein antigens to obtain an L2 protein polypeptide as shown in SEQ ID NO.1, specifically comprising:

[0071] 1. Analysis of HPV L2 protein antigen epitopes and screening of antigenic peptides:

[0072] 1. Perform PCR amplification on the gene fragment of HPV L2 protein antigen and compare it with the gene sequence submitted in Genbank, such as Figure 3 As shown, the amino acid sequence of L2 protein was analyzed;

[0073] 2. If Figure 4 As shown, the Gamier-Robson and Chou-Fasman methods were used to predict the secondary structure of the amino acid sequence, including the α, β regions, corners, curls and other regions of the protein formed by the amino acid sequence, the Kyte-Doolittle and Eisenberg methods were used to predict the hydrophilicity of the protein, the Karplus-Schulz method was used to predict its plasticity, the Jameson-Wolf method was used to predict the antigenic index, and the Emini method was used to predict the surface accessibility. Under the premise of taking the hydrophilicity, spatial structure, sequence conservation and immunogenicity of the polypeptide as the inspection indicators, multiple L2 protein polypeptide sequences were obtained;

[0074] 3. Select peptide sequences with a sequence length of 8-20 amino acid residues, no secondary structure, hydrophilicity and located on the protein surface as much as possible. After comparative analysis, the L2 protein polypeptide sequence CTSSTPIPGSRP at amino acid position 211-222 in HPV52 L2 protein was selected.

[0075] Prediction results of antigenic epitopes of L2 protein

[0076]

[0077]

[0078] Example 2

[0079] Comparing Example 2 with Example 1, the difference between Example 2 and Example 1 is:

[0080] S2. The L2 protein polypeptide and the nanomaterial are prepared by a double emulsion solvent evaporation method to obtain a nano vaccine, specifically comprising:

[0081] 2. Preparation of Nano-Vaccines:

[0082] 1. 100 mg of L2 protein polypeptide with a synthetic purity of more than 95% was used as an immunogen to prepare a nano vaccine. The nano material TPGS-b-(PCL-ran-PGA) was used to encapsulate the L2 polypeptide fragment and the adjuvant R837, respectively, to prepare the nano vaccine TPP-L2pep and the nano adjuvant TPP-R837.

[0083] 2. Prepare nanoparticles TPP-L2pep containing nano vaccine according to the double emulsion solvent evaporation method, such as Figure 5As shown, using this method, the immiscible water phase and oil phase can be emulsified by ultrasound and mechanical stirring to obtain an emulsion, evaporate to remove the internal phase solvent, precipitate spherical materials, and solidify into microspheres, which is suitable for small-batch preparation of nanoparticles. The specific method includes:

[0084] (1) Weigh 100 mg of TPGS-b-(PCL-ran-PGA) and dissolve it in 2 mL of dichloromethane (DCM). Dissolve 10 mg of the peptide in 200 μL of PBS and add it to the DCM solution. Ultrasonicate at 40% power on ice for 30 s to obtain an emulsion.

[0085] (2) Slowly add 6 mL of 7% (W / V) PVA aqueous solution to the emulsion and sonicate on ice at 40% power for 25 s.

[0086] (3) Using a 1 mL syringe, the obtained emulsion was added dropwise into 50 mL of a 1% (W / V) PVA aqueous solution containing 2% isopropanol on a magnetic stirrer, with magnetic stirring. After the addition was completed, magnetic stirring was carried out at 600 rpm overnight to obtain a nano solution.

[0087] (4) The nanoparticle solution was poured into a centrifuge tube and centrifuged at 20,000 rpm for 20 min at 4°C. The supernatant was collected and an appropriate amount of sterile water was added to the nanoparticle precipitate. The nanoparticles were centrifuged at 20,000 rpm for 20 min at 4°C and the supernatant was removed.

[0088] (5) Repeat the centrifugation washing three times, then add sterile water to resuspend and freeze in a -80°C refrigerator.

[0089] (6) Take out the centrifuge tube with the nanoparticles frozen and agglomerated from the refrigerator, remove the tube cap, seal the tube mouth with a sealing film, poke several small holes in the film, and immediately put it into a freeze dryer for freeze drying for 2 days. When the nanoparticles become dry powder or cotton wool, take them out and store them at 4°C for future use to obtain the nano vaccine.

[0090] S3. The adjuvant and the nanomaterial are prepared by solvent evaporation to obtain a nano adjuvant, specifically comprising:

[0091] (1) Weigh 5 mg of R837 and dissolve it in DMSO, heat and dissolve it, then weigh 100 mg of TPGS-b-(PCL-ran-PGA) and dissolve it in 2 mL of DCM, add R837 to the DCM solution and mix well to obtain a mixed solution.

[0092] (2) Slowly add 6 mL of 5% (W / V) PVA aqueous solution to the mixed solution, and sonicate for 30 seconds at 40% power in an ice bath to obtain an emulsion.

[0093] (3) Using a 1 mL syringe, the obtained emulsion was added dropwise into 50 mL of a 1% (W / V) PVA aqueous solution containing 2% isopropanol on a magnetic stirrer, with magnetic stirring. After the addition was completed, magnetic stirring was carried out at 600 rpm overnight to obtain a nano solution.

[0094] (4) The nanoparticle solution was poured into a centrifuge tube and centrifuged at 20,000 rpm for 20 min at 4°C. The supernatant was collected and an appropriate amount of sterile water was added to the nanoparticle precipitate. The nanoparticles were centrifuged at 20,000 rpm for 20 min at 4°C and the supernatant was removed.

[0095] (5) Repeat the centrifugation washing three times, then resuspend in sterile water and freeze in a -80°C refrigerator.

[0096] (6) Take out the centrifuge tube after the nanoparticles are frozen and agglomerated from the refrigerator, remove the tube cap, seal the tube mouth with a sealing film, poke several small holes in the film, and immediately put it into a freeze dryer for freeze drying for 2 days. When the nanoparticles become dry powder or cotton wool, take them out and store them at 4°C for future use to obtain the nano adjuvant.

[0097] S4. Collect nano vaccines and nano adjuvants to obtain a broad-spectrum vaccine.

[0098] Example 3

[0099] Comparing Example 3 with Example 2, the difference between Example 3 and Example 2 is:

[0100] Testing of the obtained nano-vaccine and nano-adjuvant:

[0101] 3. Broad-spectrum vaccine characteristics detection:

[0102] Broad-spectrum vaccine property testing includes nanoparticle characterization, nanoparticle encapsulation efficiency and drug loading testing, in vitro sustained release of nanoparticles, and identification of cell uptake of nanoparticles. The process is as follows:

[0103] 1. Characterization of the physical and chemical properties of nanoparticles:

[0104] The surface morphology of the nanoparticles was observed using a scanning electron microscope. A small amount of freeze-dried powder of the nanoparticles including the nanovaccine and nanoadjuvant was taken and the poured powder was pasted onto the sample copper base with a conductive adhesive. The surface morphology of the nanoparticles was observed and photographed under a scanning electron microscope. The particle size, zeta potential and polydispersity index of the nanoparticles were measured using a dynamic light scattering instrument and a laser particle size analyzer. Each sample was measured three times and the average value was calculated.

[0105] like Figure 6 As shown, the nanoparticles of the nanovaccine TPP-L2pep prepared in this application have smooth and round surfaces and are spherical. Figure 7As shown, the average particle size is between 100-600 nm, and the particle size distribution diagram shows that the particle size distribution peak of the nanoparticles is relatively concentrated and the size is uniform.

[0106] At the same time, if Figure 8 As shown, the average particle size of the prepared nanoadjuvant TPP-R837 nanoparticles is between 100-600 nm, and the particle size distribution diagram shows that the particle size distribution peak of the nanoparticles is relatively concentrated and the size is uniform.

[0107] 2. Detection of encapsulation efficiency and drug loading of nanoparticles:

[0108] The standard curve method is used to detect the encapsulation efficiency and drug loading of nanoparticles. In this method, the standard component is first prepared into solutions of different known concentrations, the absorbance value is measured, and a standard curve is drawn. In the preparation process of nanoparticles, the supernatant after the nanoparticles are centrifuged is collected, and the content of the unencapsulated component is obtained by measuring the absorbance value of the supernatant of the nanoparticles centrifugation, thereby calculating the encapsulation efficiency of the nanoparticles, and the encapsulation efficiency and drug loading of the nanoparticles are calculated according to the following formula:

[0109] Encapsulation efficiency (%) = (total amount of L2pep or R837 added in the preparation of nanoparticles - content of free L2pep or R837 in the supernatant) / (total amount of L2pep or R837 added in the preparation of nanoparticles) × 100%;

[0110] Drug loading = (total amount of L2pep or R837 added in the preparation of nanoparticles - content of free L2pep or R837 in the supernatant) / total weight of nanoparticles.

[0111] The specific implementation methods are as follows:

[0112] The L2 protein polypeptide of the standard was prepared into known solutions of different concentrations, and the absorbance values ​​of the different concentrations at 214 nm were measured, and then a standard curve was drawn.

[0113] The absorbance value at 214 nm in the nanocentrifugation supernatant was measured and recorded as the A214 value. The content of the unencapsulated L2 protein polypeptide was calculated based on the standard curve, thereby calculating the encapsulation efficiency and drug loading of the nanovaccine encapsulated with the L2 protein polypeptide. The nanovaccine was prepared three times and its average value was measured and calculated.

[0114] The adjuvant R837 was dissolved in dimethyl sulfoxide (DMSO) and diluted in multiple ratios to different concentration solutions (0, 0.05, 0.1, 0.2, 0.4 mg / mL). The absorbance at 325 nm was measured, and a standard curve was drawn to calculate the content of unencapsulated adjuvant R837 in the supernatant after centrifugation of the nanoadjuvant. The encapsulation rate and drug loading of the nanoparticles including the nanoadjuvant were obtained, and the average value was taken after multiple measurements.

[0115] According to the standard curve, the encapsulation efficiency of the nanovaccine TPP-L2pep was about 51.24%, and the drug loading was about 73.20μ / mg; the encapsulation efficiency of the nanoadjuvant TPP (R837) was about 27.25%, and the drug loading was about 20.96μg / mg.

[0116] 3. In vitro sustained release of nanoparticles:

[0117] Weigh the nanoparticles including the nanovaccine or nanoadjuvant, and divide them into several tubes, 5 mg in each tube, add 1 mL of PBS to 5 mg of the nanoparticles, mix well, and incubate in a constant temperature water bath oscillator at 37°C and 150 rpm. Take out samples at different time points (4 h, 8 h, 12 h, 24 h, 48 h, 72 h, 96 h, 7 d, 14 d, 21 d, 28 d), collect the supernatant by centrifugation at 15000 rpm for 15 min, detect the absorbance value, and then calculate the release amount of L2 protein polypeptide and adjuvant R837 in the supernatant with reference to the standard curve.

[0118] like Fig. 9 As shown, the in vitro release curves of the nanovaccine and nanoadjuvant showed that both nanoparticles were released rapidly in the initial stage, and the L2 protein polypeptide and adjuvant R837 released after 24 hours were 8.4% and 6.5%, respectively, and continued to release to reach a peak, after which the speed gradually slowed down. At the end of the experiment 28 days later, the contents of L2 polypeptide and R837 were 44.2% and 34.8%, respectively.

[0119] 4. Identification of Nanoparticle Uptake by Cells:

[0120] According to the double emulsion solvent evaporation method, bovine serum albumin (BSA) labeled with fluorescein isothiocyanate (FITC) was encapsulated by nanomaterials to prepare fluorescent labeled nanoparticles TPP-FITC-BSA and blank control nanoparticles TPP-blank, and the encapsulation efficiency was detected.

[0121] Hela cells were prepared, inoculated into 12-well plates, cultured at 37°C overnight, washed twice with PBS the next day, 1 mL of serum-free culture medium was added to each well, incubated at 37°C for 1 h, and then nanoparticles TPP-FITC-BSA and control TPP-blank (the final concentration of nanoparticles was 0.2 mg / ml) were added, and incubated with Hela cells at 37°C for 4 h.

[0122] The incubated cells were washed three times with 1 mL of pre-cooled PBS to remove free nanoparticles, and complete culture medium was added to continue the culture. After 24 hours, the fluorescence intensity of the cells was observed under a fluorescence microscope to study the uptake of the nanoparticles.

[0123] In order to observe the uptake of nanoparticles by cells, fluorescently labeled nanoparticles TPP-FITC-BSA were prepared, and blank nanoparticles TPP-blank were used as a control, such as Fig.10 and Fig.11 As shown in the figure, after incubation with HeLa cells, it was found that after TPP-FITC-BSA interacted with cells, obvious green fluorescence was visible under the microscope, indicating that the protein FITC-BSA was successfully encapsulated into the nanoparticles and could be taken up by cells. Based on this, it is speculated that after the L2 polypeptide is encapsulated into TPP, it can also be taken up by cells and release the L2 polypeptide antigen, inducing the occurrence of an immune response.

[0124] Example 4

[0125] Comparing Example 4 with Example 3, the difference between Example 4 and Example 3 is:

[0126] 4. Evaluation of Nanovaccine Effects:

[0127] In order to detect whether the prepared nanovaccine TPP-L2pep and nanoadjuvant TPP-R837 can effectively induce humoral immune response in vivo, the ELISA method was used to detect the serum of immune mice:

[0128] Female BALB / C mice aged 6 to 8 weeks were selected and randomly divided into different experimental groups, including PBS group, TPP-blank group, L2pep group, TPP-L2pep group, R837 group, TPP-R837 group, TPP-L2pep and TPP-R837 mixed group, and L2pep and R837 mixed group. After each nanoparticle dry powder was dissolved with sterile PBS, each group was injected subcutaneously into the abdominal cavity to immunize the mice. One week later, the immunization was boosted once. After 14 days, the mice's eyeballs were removed and blood was collected. After the blood was collected, it was placed at room temperature for 15 minutes, and then stood at 4°C for 3 hours. After the blood coagulated, it was centrifuged at 3000rpm for 10 minutes to separate the serum, and the HPV antibody level in the serum was detected.

[0129] Among them, the specific operation steps of ELISA are as follows:

[0130] (1) Antigen coating: Dilute the antigen appropriately with coating solution to a coating concentration of 2 μg / mL. Add 100 μL to each well, incubate at 37°C for 2 h, and place in a refrigerator at 4°C for 16 to 18 h to complete the coating.

[0131] (2) Washing: After coating, pour out all the liquid in the wells, fill up with PBST buffer for washing, let it stand for 5 min, discard the wash solution, refill, and repeat the washing 3 to 5 times. Finally, turn the reaction plate upside down on absorbent paper to allow the wash solution in the wells to drain out.

[0132] (3) Blocking: Add 200 μL of blocking solution to each well of the ELISA plate and incubate at 37°C for 2 h.

[0133] (4) Washing: Wash the blocked ELISA plate using the same method as (2).

[0134] (5) Adding the test serum: Use diluent to dilute the serum in a five-fold series from a ratio of 1:40 to 1:40, 1:200, 1:1000, 1:5000, 1:25000, 1:125000, and 1:625000. Add 100 μL of serum diluent to each well. Set up blank and negative controls and incubate at 37°C for 2 h.

[0135] (6) Washing: Same as (2).

[0136] (7) Add secondary antibody: Add HRP-labeled secondary antibody, dilute with diluent, refer to the instructions for dilution, dilute the secondary antibody 1:5000, take 2 μL of antibody and add it to 10 mL of diluent. Add the diluted secondary antibody to each well, 100 μL per well, and react at 37°C for 1 h.

[0137] (8) Washing: Same as (2).

[0138] (9) Color development: The color development solution must be prepared freshly, 100 μL per well. The specific method is as follows:

[0139] When (8) is halfway through, prepare the color developing solution. First, prepare 2 mg / mL tetramethylbenzidine (TMB) working solution (weigh 10 mg of TMB powder and dissolve it in 5 mL of anhydrous ethanol until it is completely dissolved). Take 0.5 mL of TMB working solution and add it to 9.5 mL of substrate buffer. After (8) is completed, take 10 uL of 30% hydrogen peroxide solution and add it to the previously prepared 10 mL solution to mix well. Immediately add this color developing solution to each well of ELISA, and then react the ELISA plate at 37°C for about 10 min to 20 min.

[0140] (10) Termination: Take out the ELISA plate and add 50 μL of stop solution (2 mol / L sulfuric acid solution) to each well.

[0141] (11) Observation results: Immediately after termination, read the result on a microplate reader at 450 nm. After adjusting the value to zero with the blank control well, measure the OD value of each well. If the value is greater than 2.1 times the OD value of the negative control, it is considered positive.

[0142] like Fig.12As shown, the data of the present application show that at a dilution of 1:200, the TPP-L2pep group, the TPP-L2pep and TPP-R837 mixed group all showed higher serum antibody levels, and compared with the free L2pep group, the antibody level in the mouse serum was increased after being encapsulated with nanomaterials, which indicates that the L2 polypeptide encapsulated with nanomaterials can protect the antigen and prolong the retention time of the antigen in the body, thereby more effectively stimulating the production of antibodies.

[0143] One or more technical solutions in the embodiments of the present application also have at least the following technical effects or advantages:

[0144] (1) The broad-spectrum vaccine provided in the embodiments of the present application is formed by encapsulating the L2 protein polypeptide with nanomaterials. Since the L2 protein polypeptide has a small molecular weight, the encapsulation with nanomaterials can prevent the enzymes and other factors in the body from damaging the L2 protein polypeptide. At the same time, the L2 protein polypeptide can be slowly released in the body, exerting the effects of slow-controlled release and long circulation, thereby comprehensively improving the stability and action time of the L2 protein polypeptide vaccine.

[0145] (2) The broad-spectrum vaccine provided in the embodiments of the present application can be synthesized in vitro or produced in a prokaryotic system due to the small molecular weight of the L2 protein polypeptide, with a simple process and low cost, which can effectively reduce the preparation cost of the HPV vaccine.

[0146] (3) The broad-spectrum vaccine provided in the examples of the present application uses the nanomaterial TPGS-b-(PCL-ran-PGA) in a preventive vaccine, and utilizes it to encapsulate the L2 protein polypeptide, which can effectively improve the stability of the L2 protein polypeptide vaccine.

[0147] (4) The broad-spectrum vaccine provided in the embodiments of the present application can effectively assist the function of the nano-vaccine and improve the preventive effect of the preventive vaccine by encapsulating the TLR7 agonist R837 with nano-materials.

[0148] (5) The method provided in the examples of the present application has a simple overall process and is suitable for small-scale preparation of nanovaccines and nanoadjuvants.

[0149] It should be noted that, in this article, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "include..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0150] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0151] The foregoing is merely a specific embodiment of the present invention, which enables those skilled in the art to understand or implement the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features claimed herein. Sequence Listing <110> Jianghan University <120> Broad-spectrum cervical cancer vaccine based on human papillomavirus L2 protein and preparation method thereof <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 13 <212> PRT <213> Artificial Sequence <400> 1 Cys Thr Ser Ser Thr Pro Ile Pro Gly Ser Arg Pro Thr 1 5 10

Claims

1. A broad-spectrum cervical cancer vaccine based on human papillomavirus L2 protein, characterized in that: The broad-spectrum vaccine includes a nano-vaccine formed by nano-materials encapsulating L2 protein polypeptides, wherein the nano-vaccine is a core-shell structure, wherein the core is the L2 protein polypeptide and the shell is the nano-material; The sequence of the L2 protein polypeptide is as shown in SEQ ID NO.1; The nano material is a TPGS-b-(PCL-ran-PGA) copolymer nano material.

2. The broad-spectrum vaccine according to claim 1, characterized in that The broad-spectrum vaccine also includes a nano-adjuvant formed by encapsulating an adjuvant with a nano-material, and the nano-adjuvant is a core-shell structure, wherein the core is the adjuvant and the shell is the nano-material.

3. The broad-spectrum vaccine according to claim 2, characterized in that The adjuvant includes imiquimod.

4. A method for preparing the broad-spectrum vaccine according to any one of claims 1 to 3, characterized in that: The method comprises: Performing epitope analysis and screening on HPV L2 protein antigens to obtain an L2 protein polypeptide as shown in SEQ ID NO.1; Encapsulating the L2 protein polypeptide and the nanomaterial to obtain a nano vaccine; Encapsulating the adjuvant and the nanomaterial to obtain a nano adjuvant; The nano vaccine and the nano adjuvant are collected to obtain a broad-spectrum vaccine.

5. The method according to claim 4, characterized in that The particle sizes of the nano vaccine and the nano adjuvant are respectively 100nm to 600nm.

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