Preparation of poliovirus type 3 thermostable virus-like particles and immunogenicity research of poliovirus type 3 thermostable virus-like particles

Abstract

The invention relates to the field of medicine, in particular to preparation of poliovirus type 3 thermostable virus-like particles and immunogenicity research of the poliovirus type 3 thermostable virus-like particles. The invention provides a poliovirus type 3 virus-like particle. The poliovirus type 3 virus-like particle is selected from any one of the following: a, a capsid protein of the virus-like particle comprises a mutant capsid protein fragment sVP2, a mutant capsid protein fragment sVP3 and a mutant capsid protein fragment sVP1; and b, the capsid protein of the virus-like particle comprises a mutant capsid protein fragment sVP0 or a variant thereof, a mutant capsid protein fragment sVP3 and a mutant capsid protein fragment sVP1 or a variant thereof. According to the application, capsid proteins are connected in series at intervals through an operable element, the VLP is successfully prepared in pichia pastoris through sequence optimization, and the purified VLP is named as an sVLP series. The thermal stability result shows that the thermal stability of the sVLP series is far better than that of the wild type VLP (wtVLP), and the sVLP series can tolerate higher temperature; in addition, animal immune tests show that the immunogenicity of the sVLP series is better, and the neutralizing titer of immune serum of the sVLP series is far higher than that of wtVLP immune serum. In conclusion, the sVLP series is a good candidate strategy for PV3 vaccines.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and particularly to the preparation of heat-stable virus-like particles of poliovirus type 3 and the study of their immunogenicity. Background Art

[0002] Poliovirus (PV) is a non-enveloped small RNA virus with a single-stranded positive-sense RNA genome. It mainly infects children under 5 years old through the fecal-oral route, and can cause symptoms such as fever, fatigue, headache, vomiting, neck stiffness, and limb pain. In severe cases, it can lead to paralysis or even death due to respiratory paralysis. Poliovirus (PV) is the pathogen of poliomyelitis, a disabling disease, and can be divided into three different serotypes, namely PV1, PV2, and PV3. The live-attenuated oral PV vaccine (OPV) and the inactivated PV vaccine (IPV) have been used in large-scale global immunization programs and have finally formed a feasible strategic ultimate plan for eradicating poliomyelitis. It is reported that OPV can regain virulence through reverse mutation or recombination, thus causing vaccine-associated paralytic poliomyelitis (VAPP) or vaccine-derived poliovirus epidemic (cVDPV). Chronic carriers of cVDPV can also act as sources of infection and cause further transmission. The WHO announced in 1999 and 2020 respectively that wild-type PV2 and PV3 have been eradicated globally, but in recent years, there have still been reports of PV1 infections, which are wild-type PV1 or cVDPV. Although the inactivated poliovirus vaccine IPV can avoid this problem, IPV involves the cultivation of a large number of viruses and there is a risk of accidental release. Therefore, it is necessary to find new alternative vaccines to achieve the global poliomyelitis eradication plan. Virus-like particles (VLPs) are composed of viral capsid proteins, are structurally similar to real viruses, but do not contain viral nucleic acids, and are good candidate vaccines.

[0003] PV belongs to the Enterovirus genus and has a non-enveloped icosahedral structure. Its genome is a single-stranded positive-sense RNA, encoding an open reading frame that contains structural protein P1 and non-structural proteins P2 and P3. Structural protein P1 can be cleaved by protease 3CD into capsid proteins VP0, VP3, and VP1, and these capsid proteins can assemble with viral RNA into mature virus particles and accompany the cleavage of VP0 into VP4 and VP2. For PV, there are two types of particles in its natural culture, namely mature virus particles with infectivity and good immunogenicity (referred to as D antigen) and empty particles without infectivity and with poor thermal stability and immunogenicity (referred to as C antigen). The wild-type VLP of PV3 has poor thermal stability and immunogenicity and needs to be optimized and modified to become a candidate vaccine. Summary of the Invention

[0004] In view of the disadvantages of the above-mentioned prior art, the applicant's research found that: amino acid mutations in the PV structural protein can improve the stability of its capsid protein. At the structural level, in PV virions, VP4 is located on the inner surface of the capsid protein, and the N-terminal amino acids of VP4 and VP1 form a stable sheet structure that contributes to the stability of the virus particles. However, VP4 is very dynamic and difficult to resolve in the analyzed VLP structure, indicating that VP4 plays an important role in maintaining the morphology of the virus particles. The structure of VP1 contains a hydrophobic cavity that can accommodate a pocket factor (usually sphingosine), which is very important for the assembly and conformational stability of the PV capsid protein. In summary, these basic studies provide a theoretical basis for the modification of PV VLPs.

[0005] Based on the above research and analysis, the present application introduced 8 amino acid mutations into the wild-type PV3 structural protein, and optimized the sequence of a single subunit based on literature reports and structural information. The sVLP series was successfully prepared in Pichia pastoris by tandemly expressing related expression cassettes such as VP0, VP3, and VP1. The results of thermal stability and animal immunization tests showed that the sVLP series had better thermal stability and immunogenicity, and was an excellent candidate for PV3 vaccines.

[0006] The purpose of the present application is to provide the preparation of a thermostable virus-like particle of poliovirus type 3 and its immunogenicity study, aiming to solve the problems in the prior art.

[0007] To achieve the above and other related purposes, the first aspect of the present application provides a virus-like particle of poliovirus type 3, selected from any one of the following:

[0008] a. The capsid protein of the virus-like particle comprises a mutant capsid protein fragment sVP2, a mutant capsid protein fragment sVP3, and a mutant capsid protein fragment sVP1;

[0009] b. The capsid protein of the virus-like particle comprises a mutant capsid protein fragment sVP0 or its variant, a mutant capsid protein fragment sVP3, and a mutant capsid protein fragment sVP1 or its variant.

[0010] The second aspect of the present application provides a polynucleotide that encodes the aforementioned virus-like particle, and the capsid proteins of the virus-like particle are separated by operable elements.

[0011] The third aspect of the present application provides a nucleic acid construct that includes the aforementioned polynucleotide.

[0012] The fourth aspect of the present application provides a cell line, which includes the aforementioned nucleic acid construct, or the aforementioned polynucleotide is integrated into the genome.

[0013] The fifth aspect of the present application provides a method for preparing the aforementioned virus-like particles, and the preparation method includes the following steps:

[0014] 1) Culturing the aforementioned cell line to express virus-like particles of poliovirus type 3;

[0015] 2) Isolating virus-like particles with improved thermal stability and immunogenicity compared to wild-type poliovirus PV3 virus-like particles.

[0016] The sixth aspect of the present application provides the use of the aforementioned virus-like particles, the aforementioned polynucleotide, the aforementioned nucleic acid construct, and the aforementioned cell line in the preparation of anti-poliovirus products.

[0017] The seventh aspect of the present application provides a pharmaceutical composition for preventing poliovirus, and the pharmaceutical composition includes the aforementioned virus-like particles, the aforementioned polynucleotide, the aforementioned nucleic acid construct, the aforementioned cell line, and a pharmaceutically acceptable carrier.

[0018] The eighth aspect of the present application provides a method for preventing poliovirus, including administering an effective amount of the aforementioned virus-like particles, the aforementioned polynucleotide, the aforementioned nucleic acid construct, the aforementioned cell line, the poliovirus vaccine in the aforementioned use, or the aforementioned pharmaceutical composition to a subject.

[0019] Compared with the prior art, the beneficial effects of the present application are as follows:

[0020] 1. In the present application, the capsid proteins are tandemly spaced through operable elements, and through sequence optimization, the sVLP series is obtained. The thermal stability of the sVLP series is far superior to that of wild-type VLP (wtVLP) and can tolerate higher temperatures.

[0021] 2. The immunogenicity of the sVLP series in the present application is also better, and the neutralization titer of its immune serum is much higher than that of the wtVLP immune serum. Therefore, the sVLP series is a good candidate strategy for the PV3 vaccine. Description of the Drawings

[0022] Figure 1 It is a schematic diagram of the PV3 VLP expression vector.

[0023] Figure 2 It is the morphological observation of PV3 VLP. Among them, Figure 2 A is wtVLP 031 , Figure 2 B is sVLP 031 , Figure 2C is sVLP 231 , Figure 2 D is sVLP 031t1 , Figure 2 E is sVLP 031t2 . Detailed implementation manners

[0024] The first aspect of the present application provides a virus-like particle of poliovirus type 3, selected from any of the following:

[0025] a. The capsid protein of the virus-like particle comprises a mutant capsid protein fragment sVP2, a mutant capsid protein fragment sVP3, and a mutant capsid protein fragment sVP1;

[0026] b. The capsid protein of the virus-like particle comprises a mutant capsid protein fragment sVP0 or its variant, a mutant capsid protein fragment sVP3, and a mutant capsid protein fragment sVP1 or its variant.

[0027] In the virus-like particle provided by the present application, the mutant capsid protein of poliovirus PV3 is obtained by optimization, specifically: 8 amino acids of the wild-type poliovirus PV3 capsid protein are mutated and codon-optimized. Specifically, the codon optimization is based on the host cell preference, and the host is, for example, Pichia pastoris. In some embodiments, the amino acid sequence of the PV3 structural protein P1 is derived from Gene Bank: KP247597.1, named wtP1. wtP1 and sP1 are respectively optimized and synthesized according to the codon preference of Pichia pastoris. The amino acid sequence of wtP1 is shown in SEQ ID NO:1, the nucleotide sequence of wtP1 is shown in SEQ ID NO:2, the amino acid sequence of sP1 is shown in SEQ ID NO:3, and the nucleotide sequence of sP1 is shown in SEQ ID NO:4.

[0028] In the virus-like particle provided by the present application, the amino acid sequence of the mutant capsid protein fragment sVP2 is shown in SEQ ID NO:17. The amino acid sequence of the mutant capsid protein fragment sVP3 is shown in SEQ ID NO:19. The amino acid sequence of the mutant capsid protein fragment sVP1 is shown in SEQ ID NO:21. The amino acid sequence of the mutant capsid protein fragment sVP0 is shown in SEQ ID NO:11.

[0029] In the virus-like particle provided by the present application, the variant of the mutant capsid protein fragment sVP1 is a polypeptide formed by adding 15 - 45 amino acids to the N-terminus of the mutant capsid protein fragment VP1. Preferably, the variant of the mutant capsid protein fragment sVP1 is a polypeptide sVP1 formed by adding 29 amino acids to the N-terminus of the mutant capsid protein fragment sVP1 P29. Preferably, sVP1 P29 The 29 amino acids added in P29 are the 29 amino acids at the N-terminus of the mutant capsid protein fragment sVP0. Specifically, sVP1 P29 has the amino acid sequence shown in SEQ ID No:23.

[0030] In the virus-like particles provided by the present application, the variant of the mutant capsid protein fragment sVP0 is a polypeptide formed by truncating 1 to 5 amino acids at the N-terminus of the mutant capsid protein fragment sVP0 or a polypeptide formed by truncating 15 to 45 amino acids at the N-terminus of the mutant capsid protein fragment sVP0. Preferably, the variant of the mutant capsid protein fragment sVP0 is the polypeptide sVP0 formed by truncating 2 amino acids at the N-terminus of the mutant capsid protein fragment sVP0 N2 or the polypeptide sVP0 formed by truncating 29 amino acids at the N-terminus of the mutant capsid protein fragment sVP0 N29 . Specifically, the sVP0 N2 has the amino acid sequence shown in SEQ ID No:13, and the sVP0 N29 has the amino acid sequence shown in SEQ ID No:15.

[0031] The virus-like particles provided by the present application are selected from any of the following:

[0032] 1) The capsid protein of the virus-like particle contains sVP0 with the amino acid sequence shown in SEQ ID No:11, sVP3 with the amino acid sequence shown in SEQ ID No:19, and sVP1 with the amino acid sequence shown in SEQ ID No:21;

[0033] 2) The capsid protein of the virus-like particle contains sVP2 with the amino acid sequence shown in SEQ ID No:17, sVP3 with the amino acid sequence shown in SEQ ID No:19, and sVP1 with the amino acid sequence shown in SEQ ID No:21;

[0034] 3) The capsid protein of the virus-like particle contains sVP0 with the amino acid sequence shown in SEQ ID No:13 N2 , sVP3 with the amino acid sequence shown in SEQ ID No:19, and sVP1 with the amino acid sequence shown in SEQ ID No:21;

[0035] 4) The capsid protein of the virus-like particle contains sVP0 with the amino acid sequence shown in SEQ ID No:15 N29 , sVP3 with the amino acid sequence shown in SEQ ID No:19, and sVP1 with the amino acid sequence shown in SEQ ID No:23 P29 .

[0036] In a second aspect of the present application, a polynucleotide is provided, which encodes the aforementioned virus-like particles, and the capsid proteins of the virus-like particles are separated by operable elements.

[0037] In the polynucleotide provided by the present application, the operable element includes a promoter, a terminator, a transposon or an enhancer. In some embodiments, the promoter is the AOX1 promoter; the terminator is the CYC1 terminator.

[0038] In the polynucleotide provided by the present application, the capsid proteins of the virus-like particles include a wild-type capsid protein fragment wtVP0, a wild-type capsid protein fragment wtVP3, and a wild-type capsid protein fragment wtVP1. Specifically, the nucleotide sequence encoding wtVP0 is shown in SEQ ID NO: 6; the nucleotide sequence encoding wtVP3 is shown in SEQ ID NO: 8, and the nucleotide sequence encoding wtVP1 is shown in SEQ ID NO: 10.

[0039] In the polynucleotide provided by the present application, the nucleotide sequence encoding sVP0 is shown in SEQ ID NO: 12, the nucleotide sequence encoding sVP0 N2 is shown in SEQ ID NO: 14, and the nucleotide sequence encoding sVP0 N29 is shown in SEQ ID NO: 16.

[0040] In the polynucleotide provided by the present application, the nucleotide sequence encoding sVP3 is shown in SEQ ID NO: 20.

[0041] In the polynucleotide provided by the present application, the nucleotide sequence encoding sVP1 is shown in SEQ ID NO: 22, and the nucleotide sequence encoding sVP1 P29 is shown in SEQ ID NO: 24.

[0042] In the polynucleotide provided by the present application, the nucleotide sequence encoding the sVP2 capsid protein is shown in SEQ ID NO: 18.

[0043] In the polynucleotide provided by the present application, the fragments in the polynucleotide are concatenated in any of the following ways:

[0044] 1) Promoter - wtVP0 - Terminator - Promoter - wtVP3 - Terminator - Promoter - wtVP1 - Terminator;

[0045] 2) Promoter - sVP0 - Terminator - Promoter - sVP3 - Terminator - Promoter - sVP1 - Terminator;

[0046] 3) Promoter - sVP2 - Terminator - Promoter - sVP3 - Terminator - Promoter - sVP1 - Terminator;

[0047] 4) Promoter - sVP0 N2 - Terminator - Promoter - sVP3 - Terminator - Promoter - sVP1 - Terminator;

[0048] 5) Promoter - sVP0 N29 - Terminator - Promoter - sVP3 - Terminator - Promoter - sVP1 P29 - Terminator.

[0049] In the polynucleotide provided by the present application, the expression cassettes for expressing each capsid protein in the polynucleotide are tandemly arranged in the polynucleotide. The specific tandem arrangement is as Figure 1 shown.

[0050] The third aspect of the present application provides a nucleic acid construct, and the nucleic acid construct includes the aforementioned polynucleotide.

[0051] The term "nucleic acid construct" refers to an artificially constructed nucleic acid segment that can be introduced into a target cell or tissue. The nucleic acid construct includes a vector backbone, i.e., an expression vector and an expression cassette, and the nucleic acid construct can be a plasmid.

[0052] In the nucleic acid construct, the VP0, VP2, VP3, and VP1 expression cassettes can be independent single - copies or multiple - copies respectively. Preferably, the VP2, VP3, and VP1 expression cassettes in the nucleic acid construct are all single - copies.

[0053] In addition to the nucleotide of the capsid protein of poliovirus PV3 contained in the polynucleotide in the nucleic acid construct, the nucleic acid construct does not include the nucleotide encoding other capsid proteins of poliovirus PV3.

[0054] In one embodiment, the nucleic acid construct further includes an expression vector. The expression vector can be any expression vector suitable for expressing poliovirus in the prior art, for example, a yeast expression vector. Preferably, it is the Pichia pastoris expression vector pPink - HC (manufacturer: Invitrogen).

[0055] In the embodiment as Figure 1 shown, the nucleotide sequence of the nucleic acid construct is as shown in SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:29.

[0056] In one embodiment, the preparation method of the nucleic acid construct includes the following steps:

[0057] 1) Clone the nucleotides expressing the capsid protein of poliovirus PV3 into different expression vectors respectively to obtain intermediate constructs;

[0058] 2) Recombine the intermediate constructs obtained in step 1) to obtain the nucleic acid construct.

[0059] The fourth aspect of the present application provides a cell line, which includes the aforementioned nucleic acid construct, or the aforementioned polynucleotide is integrated into the genome.

[0060] In the cell line provided by the present application, the cell line is a eukaryotic cell. In one embodiment, the cell line is obtained by transducing the nucleic acid construct into Pichia pastoris cells.

[0061] The fifth aspect of the present application provides a method for preparing the aforementioned virus-like particles, including the following steps:

[0062] 1) Cultivate the aforementioned cell line to express poliovirus PV3 virus-like particles;

[0063] 2) Isolate virus-like particles with improved thermal stability and immunogenicity compared to wild-type poliovirus PV3 virus-like particles.

[0064] In one embodiment, the conditions for culturing the cell line are 28 - 30 °C and 250 - 300 rpm. In the poliovirus PV3 virus-like particles provided by the present application, the diameter of the poliovirus PV3 virus-like particles is about 30 nm. The sizes of the poliovirus PV3 virus-like particles are uniform.

[0065] Any suitable technique can be used to measure the stability of VLPs. Standard techniques known in the art include, but are not limited to, the retention of the native D antigen structure upon heating (represented as the temperature at which the ELISA reactivity with a D antigen-specific monoclonal antibody (MAb) decreases by 50% after a 10-minute incubation) or the retention of the native D antigen structure upon long-term incubation at 37 °C (represented as the length of time (in days) at which the ELISA reactivity with a D antigen-specific MAb decreases by 50%).

[0066] The detection method for the thermal stability temperature is as follows: After incubating PV3 VLPs at different incubation temperatures for 10 min, the detection of D antigen is carried out by the sandwich ELISA method. According to the OD450nm readings of samples at different temperatures, the temperature at which the D antigen content decreases by 50% is determined as the thermal stability temperature (Tm) by the method of non-linear regression (curve fitting).

[0067] Any suitable technique can be used to measure the immunogenicity of the VLP. Standard techniques are known in the art, including but not limited to C / D antigen ELISA and in vivo potency testing in rats (a relative potency ≥ 1.0 indicates that the immunogenicity is at least as good as that of the international reference preparation). In some embodiments, the immunogenicity is expressed as a neutralization titer.

[0068] The method for detecting the neutralization titer is as follows: Dilute the sample to be tested two-fold with DMEM containing 2% FBS. Take 50 μl of the diluted serum and OPV1 (NIBSC, 100 TCID 50 / 50 μL) and place them in a 5% CO2 incubator at 36 °C for 2 h, and then incubate overnight at 4 °C; After the virus and serum have been incubated overnight, digest the hep-2 cells with trypsin into single cells, count them, centrifuge and discard the supernatant, resuspend them with 2% DMEM (containing 2% double antibiotics), and add the resuspended cells dropwise to the 96-well plate after incubation, 1.5×10 4 cells / 100 μL / well, and culture in a 5% CO2 incubator at 36 °C for 7 days. Observe the cytopathic effect starting from Day 7, and determine the result according to the cytopathic effect (CPE). The maximum dilution that can neutralize more than 50% of the CPE is used as the neutralization titer.

[0069] The sixth aspect of the present application provides the use of the aforementioned virus-like particles, the aforementioned polynucleotides, the aforementioned nucleic acid constructs, and the aforementioned cell lines in the preparation of products for preventing poliovirus. In some embodiments, the product for preventing polio is a polio vaccine.

[0070] The seventh aspect of the present application provides a pharmaceutical composition for preventing poliovirus, which comprises the aforementioned virus-like particles, the aforementioned polynucleotides, the aforementioned nucleic acid constructs, the aforementioned cell lines, and a pharmaceutically acceptable carrier.

[0071] The pharmaceutical composition can be monovalent (containing only one type of virus-like particle) or multivalent (containing multiple types of virus-like particles). The pharmaceutical composition can be prepared into various conventional dosage forms, such as: injections, granules, tablets, pills, suppositories, capsules, suspensions, sprays, etc.

[0072] The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for the administration of a pharmaceutical composition (such as the virus-like particles of the present invention). This term refers to those pharmaceutical carriers that do not themselves induce the production of antibodies harmful to the individual receiving the composition and are not unduly toxic after administration. Suitable carriers can be large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, etc. These carriers are well-known to those of ordinary skill in the art. Pharmaceutically acceptable carriers may include liquids, such as water, saline, glycerol, and ethanol. Additionally, auxiliary substances, such as wetting agents or emulsifiers, pH buffering substances, etc., may also be present in these carriers. Generally, the composition can be made into an injectable, such as a liquid solution or suspension; it can also be made into a solid form that is suitable for formulation into a solution or suspension, a liquid excipient before injection. Liposomes are also included in the definition of pharmaceutically acceptable carriers.

[0073] The vaccines of the present invention can be directly administered to an individual by known methods using the virus-like particles of the present invention. Generally, the same administration routes as those of conventional vaccines and / or the routes that mimic the pathogen infection pathway are used to administer these vaccines.

[0074] The routes of administration of the pharmaceutical composition or vaccine of the present invention include: intramuscular, subcutaneous, intradermal, intranasal, intravenous, oral, or other parenteral administration routes. If necessary, the administration routes can be combined or adjusted according to the disease condition. The vaccine composition can be administered in a single dose or multiple doses, and booster doses can be included to initiate and / or maintain immunity.

[0075] The pharmaceutical composition or vaccine suitable for injection can be in the form of a solution, suspension, or emulsion, or in the form of a dry powder that is dissolved or suspended in a suitable carrier before use.

[0076] The eighth aspect of the present application provides a method for preventing poliovirus, including administering an effective amount of the aforementioned virus-like particles, the aforementioned polynucleotides, the aforementioned nucleic acid constructs, the aforementioned cell lines, the aforementioned vaccines, or the aforementioned pharmaceutical compositions to a subject.

[0077] In the method provided by the present application, the subject can be a mammal, especially a human. The term "effective amount" refers to the amount that treats, alleviates or prevents a target disease or condition, or the amount that exhibits a detectable therapeutic or preventive effect. That is, the amount of virus-like particles is sufficient to elicit an immune response in the selected administration route and can effectively prompt the protection of the host against related diseases. This effect can be detected, for example, by the antigen level. The therapeutic effect also includes the reduction of physiological symptoms. The precise effective amount for a particular subject depends on the size and health status of the subject, the nature and degree of the disease condition, and the therapeutic agent and / or combination of therapeutic agents selected for administration. Therefore, it is not useful to pre-specify an accurate effective amount. However, for a given condition, the effective amount can be determined by routine experiments.

[0078] In one embodiment, for the purposes of the present invention, an effective dose is from about 0.001 mg / kg to 1000 mg / kg, preferably from about 0.01 mg / kg to 100 mg / kg body weight of virus-like particles administered to an individual.

[0079] The poliovirus-like particles (VLPs) of the present invention can be used on a small scale, for example, as an antigen in serological assays (such as as part of a seroprevalence study or other surveillance activities) or on a large scale in vaccine production. Since the VLPs of the present invention are non-infectious (lacking the poliovirus genome) and recombinant expression does not require live virus, containment is not required before and after the eradication of the poliovirus. In some embodiments, the sVLP series of the present application can exhibit good immunogenicity without an adjuvant.

[0080] In summary, the thermal stability of the sVLP series of the present application is far superior to that of wild-type VLPs (wtVLPs), and the immunogenicity of the sVLP series is also better. The neutralization titer of its immune serum is much higher than that of the wtVLP immune serum. Therefore, the sVLP series is a good candidate strategy for PV3 vaccines.

[0081] The following specific examples illustrate the embodiments of the present invention. Those skilled in the art can easily understand the other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0082] Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are for describing the specific embodiments and not for limiting the protection scope of the present invention; in the specification and claims of the present invention, unless otherwise clearly indicated in the text, the singular forms "a", "an" and "the" include the plural forms.

[0083] When the embodiments give a numerical range, it should be understood that, unless otherwise specified in the present invention, either endpoint of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art of this technology. In addition to the specific methods, devices and materials used in the embodiments, according to the knowledge of those skilled in the art of this technology and the description of the present invention, any methods, devices and materials of the prior art similar or equivalent to those described in the embodiments of the present invention can also be used to implement the present invention.

[0084] Example 1 Construction of expression plasmid

[0085] The amino acid sequences of the PV3 structural protein P1 (wtP1, Gene Bank: KP247597.1) and the P1 amino acid sequence with 8 mutations (sP1) were optimized and synthesized by GenScript. The amino acid sequence of the PV3 structural protein P1 obtained by introducing 8 mutations (T67A, L87I, L284M, D310E, H359Y, L425F, T683M and F710L) into wtP1 was named sP1. wtP1 and sP1 were respectively optimized and synthesized according to the codon preference of Pichia pastoris.

[0086] The amino acid sequence of wtP1 is as follows, where 1-340 is the amino acid sequence of VP0 (where 1-69 is the amino acid sequence of VP4, 70-340 is the amino acid sequence of VP2), 341-578 is the amino acid sequence of VP3, and 579-878 is the amino acid sequence of VP1:

[0087]

[0088]

[0089] The optimized nucleotide sequence of wtP1 is as follows, where 1-1020 is the nucleotide sequence of VP0 (where 1-207 is the nucleotide sequence of VP4, 208-1020 is the nucleotide sequence of VP2), 1021-1734 is the nucleotide sequence of VP3, and 1735-2634 is the nucleotide sequence of VP1:

[0090]

[0091]

[0092] The amino acid sequence of sP1 is as follows, where 1 - 340 is the amino acid sequence of VP0 (where 1 - 69 is the amino acid sequence of VP4, and 70 - 340 is the amino acid sequence of VP2), 341 - 578 is the amino acid sequence of VP3, and 579 - 878 is the amino acid sequence of VP1:

[0093]

[0094] The optimized nucleotide sequence of sP1 is as follows, where 1 - 1020 is the nucleotide sequence of VP0 (where 1 - 207 is the nucleotide sequence of VP4, and 208 - 1020 is the nucleotide sequence of VP2), 1021 - 1734 is the nucleotide sequence of VP3, and 1735 - 2634 is the nucleotide sequence of VP1:

[0095]

[0096]

[0097]

[0098] The amino acid sequence of wtVP0 is as follows:

[0099]

[0100] The nucleotide sequence of wtVP0 is as follows:

[0101]

[0102] The amino acid sequence of wtVP3 is as follows:

[0103]

[0104]

[0105] The nucleotide sequence of wtVP3 is as follows:

[0106]

[0107] The amino acid sequence of wtVP1 is as follows:

[0108]

[0109] The nucleotide sequence of wtVP1 is as follows:

[0110]

[0111]

[0112] The amino acid sequence of sVP0 is as follows:

[0113]

[0114] The nucleotide sequence of sVP0 is as follows:

[0115]

[0116]

[0117] sVP0 N2 (Delete the first two amino acids at the N-terminus of VP0, which are also the first two amino acids at the N-terminus of VP4) The amino acid sequence is as follows:

[0118]

[0119] sVP0 N2 The nucleotide sequence is as follows:

[0120]

[0121]

[0122] sVP0 N29 (Delete the first 29 amino acids of VP0, that is, the first two β-sheets at the N-terminus of VP4, and then connect them to the N-terminus of VP1 to make the VLP structurally mimic the structure of the virus particle, which helps to stabilize) The amino acid sequence is as follows:

[0123]

[0124] sVP0 N29 The nucleotide acid sequence is as follows:

[0125]

[0126] The amino acid sequence of sVP2 is as follows:

[0127]

[0128] The nucleotide sequence of sVP2 is as follows:

[0129]

[0130] The amino acid sequence of sVP3 is as follows:

[0131]

[0132] The nucleotide sequence of sVP3 is as follows:

[0133]

[0134]

[0135] The amino acid sequence of sVP1 is as follows:

[0136]

[0137] The nucleotide sequence of sVP1 is as follows:

[0138]

[0139]

[0140] sVP1 P29 (The first 29 amino acids of VP0, i.e., the first two β-sheets at the N-terminus of VP4, are truncated and linked to the N-terminus of VP1, enabling the VLP to structurally mimic the structure of virus particles, which helps with stability) The amino acid sequence is as follows:

[0141]

[0142] sVP1 P29 The nucleotide sequence is as follows:

[0143] NO:24)

[0144] Based on the nucleotide sequences of wtP1, sP1, and the sequence at the multiple cloning site of the expression vector pPink-HC (purchased from Invitrogen), recombinant primers were designed (Table 1). Using a homologous recombination kit (purchased from Novoprotein), intermediate plasmids pPink / HC-PV3 wtVP0, pPink / HC-PV3 sVP0, pPink / HC-PV3sVP0 N2 , pPink / HC-PV3 sVP0 N29 , Pink / HC-PV3 sVP2, pPink / HC-PV3 wtVP3, Pink / HC-PV3sVP3, pPink / HC-PV3 wtVP1, Pink / HC-PV3 sVP1, pPink / HC-PV3 sVP1 P29 ; were obtained by recombination respectively. Then, using the isocaudomers BglⅡ and BamHⅠ, the expression cassettes in the corresponding intermediate plasmids were successively ligated in the order shown by Figure 1 to pPink / HC-PV3wtVP1, Pink / HC-PV3 sVP1, or pPink / HC-PV3 sVP1 P29 , and the final expression plasmids pPink / HC-PV3 wtVP Figure 1 and pPink / HC-PV3 sVP series were obtained respectively. 031 ​

[0145] The complete plasmid sequences are as follows:

[0146] pPink / HC-PV3 wtVP 031 :

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155] pPink / HC-PV3 sVP 031 :

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164] pPink / HC-PV3 sVP 231 :

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173] pPink / HC-PV3 sVP 031t1

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182] pPink / HC-PV3 sVP 031t2 :

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191] The construction process is as follows: First, primers were designed according to the nucleotide sequences of optimized wtP1 and sP1 and the nucleotide sequence at the multiple cloning site of the expression vector pPink-HC (Table 1). By means of recombination, intermediate plasmids pPink / HC-PV3 wtVP0, pPink / HC-PV3 sVP0, pPink / HC-PV3 sVP0 N2 , pPink / HC-PV3 sVP0 N29 , Pink / HC-PV3 sVP2, pPink / HC-PV3 wtVP3, Pink / HC-PV3 sVP3, pPink / HC-PV3 wtVP1, Pink / HC-PV3sVP1 and pPink / HC-PV3 sVP1 P29 .

[0192] In the order of Figure 1 , using the isocaudomers BglⅡ and BamHⅠ, the expression frames of the corresponding intermediate plasmids were successively ligated into pPink / HC-PV3 wtVP1, Pink / HC-PV3 sVP1 or pPink / HC-PV3 sVP1 P29 to obtain the final expression plasmids pPink / HC-PV3 wtVP 031 and pPink / HC-PV3 sVP 031 , pPink / HC-PV3 sVP 231 , pPink / HC-PV3 sVP 031t1 , pPink / HC-PV3 sVP 031t2 .

[0193] Table 1 Homologous recombination primer sequences

[0194]

[0195]

[0196] Example 2 Screening, expression and purification of high-expression strains

[0197] The expression final plasmid in Example 1 was linearized using the restriction enzyme AflⅡ and purified and recovered by the ethanol precipitation method; the linearized plasmid was introduced into Pichia pastoris by electroporation, and the PAD plate was coated and cultured at 30 °C (nutritional screening); after 3 days, large white colonies were picked and placed in a 24-well deep-well plate for methanol-induced expression (28 °C, 250 rpm), and the cells were collected after 48 h of induced expression; after the induced cells were resuspended in PBS, an equal volume of glass beads was added and broken at 70 Hz for 120 s, and the supernatant was collected by centrifugation; the expression of the target protein in the cell lysate supernatant was determined by the indirect ELISA method, and the strain with a high OD450nm was used as the high-expression strain; the cell lysate supernatant of the high-expression strain was detected by the sandwich ELISA method to determine its expression level.

[0198] Indirect ELISA: The total protein concentration of the cell lysate supernatant was determined according to the instructions of the Bradford kit (Pierce); the cell lysate supernatant was coated on a 96-well ELISA plate (25 - 50 μg / well) and incubated at 37 °C for 2 h; the plate was washed 3 times with PBST, 200 μl / well of 5% skim milk powder was added, and blocked at 37 °C for 1 h; the plate was washed 3 times with PBST, 50 μl / well of the antibody dilution solution (1:1000 dilution) of the D antigen-specific antibody mAb520 (NIBSC) was added to the 96-well ELISA plate, and incubated at 37 °C for 2 h; the plate was washed 3 times with PBST, 50 μl / well of the HRP-labeled goat anti-mouse secondary antibody (1:5000 dilution) was added, and incubated at 37 °C for 1 h; the plate was washed 5 times with PBST, TMB substrate was added for color development and the OD450nm absorbance value was read.

[0199] Sandwich ELISA

[0200] The PV3-specific monoclonal antibody 2E4 used in the sandwich ELISA method was self-prepared by the company, and the preparation steps were as follows: The purified PV3 sVLP 031 (5 μg / animal) was fully mixed with aluminum adjuvant (500 μg / animal) and intraperitoneally injected into female Balb / c mice (6 - 8 weeks old), and immunized 3 times in total, with an interval of 2 weeks between two immunizations. Two weeks after the third immunization, spleen cells were taken and fused with myeloma cells, and the 2E4 murine monoclonal antibody cell line was screened. The cell line was intraperitoneally injected into mice to obtain ascites, and purified using protein G packing material. The purified 2E4 monoclonal antibody was HRP-labeled using an HRP conjugation kit (Thermo).

[0201] The operation steps of the sandwich ELISA method are as follows: Dilute the antibody mAb520 with PBS at a ratio of 1:500, coat a 96-well ELISA plate, 50 μl / well, and incubate overnight at 4°C; Wash the plate 3 times with PBST, add 200 μl / well of 5% skim milk powder, and incubate at room temperature for 1 hr; Wash the plate 3 times with PBST, add the diluted sample to be tested and / or standard (PV3 sVLP 031 ), and incubate at room temperature for 2 hr; Wash the plate 3 times with PBST, add the HRP-labeled monoclonal antibody 2E4 (1:2000), and incubate at room temperature for 2 hr; Wash the plate 5 times with PBST, add the TMB substrate for color development and read the absorbance value at OD450nm. Fit the standard with four-parameter curve, and calculate the expression level per unit wet weight (μg / mg) of each high-expression strain.

[0202] Expression and purification

[0203] Inoculate the screened high-expression strains into BMGY medium respectively, culture for 24 h (30°C, 250 rpm), then change to BMMY medium for induction expression (28°C, 250 rpm), and harvest the bacteria by centrifugation after 48 h of induction. Resuspend the bacteria with PBS and disrupt the bacteria using a high-pressure homogenizer at 1200 bar, collect the supernatant after centrifugation for PEG precipitation, and perform sucrose density gradient centrifugation on the reconstituted supernatant to finally obtain the target VLP. The results are shown in Table 2, and the corresponding high-expression strains PPS1_wtVP 031 、PPS1_sVP 031 、PPS1_sVP 231 、PPS1_sVP 031t1 and PPS1_sVP 031t2 were finally obtained. The expression level per unit wet weight of each high-expression strain is relatively high, up to 1.21 μg / mg at most, which can meet the industrialization requirements.

[0204] Table 2 Yield of high-expression strains

[0205]

[0206] Example 3 Morphological study of PV3 VLP

[0207] Dilute the sample to be analyzed with PBS to 100 - 200 ng / μl, load it onto a copper grid and stain it with uranyl acetate, then observe it using a transmission electron microscope at 120 kV. The results are as Figure 2 shown, wtVLP 031 、sVLP 031 、sVLP 231 、sVLP 031t1 and sVLP 031t2Both showed regular spherical structures with a diameter of approximately 30 nm. Therefore, tandem expression of the PV3 capsid protein in Pichia pastoris can spontaneously assemble into VLPs.

[0208] Example 4 Thermal Stability and Immunogenicity of PV3 VLPs

[0209] After incubating PV3 VLPs at different incubation temperatures for 10 min, the detection of D antigen was performed using the above sandwich ELISA method. According to the OD450nm readings of samples at different temperatures, the temperature at which the D antigen content decreased by 50% was determined as the thermal stability temperature (Tm) using the method of non-linear regression (curve fitting).

[0210] The results are shown in Table 3. The thermal stability of the sVLP series is much better than that of the wtVLP. The sVLP 031t2 is the best, and the Tm value can reach

[0211] 58.25 °C. Table 3 Thermal Stability of VLPs

[0212]

[0213] To determine the immunogenicity of poliovirus PV3 VLPs, the specific steps are as follows:

[0214] wtVLP 031 and sVLP 031 Comparison: Adsorb 5 μg VLP / animal with 60 μg Al / animal by shaking at room temperature for 1 - 2 h, then immunize female Balb / C mice (5 animals / group) at 6 - 8 weeks old intraperitoneally, immunize 3 times in total, with a 2-week interval between two immunizations. Collect mouse sera 2 weeks after the third immunization for neutralization titer determination.

[0215] sVLP 031 and sVLP 231 Comparison: Adsorb 5 μg VLP / animal with 60 μg Al / animal by shaking at room temperature for 1 - 2 h, then inject female Balb / C mice (10 animals / group) at 6 - 8 weeks old intraperitoneally, immunize 3 times in total, with a 2-week interval between two immunizations. Collect mouse sera 2 weeks after the third immunization for neutralization titer determination.

[0216] sVLP 031 and sVLP 031t1 、sVLP 031t2 Comparison: Immunize female Balb / C mice (10 animals / group) at 6 - 8 weeks old intraperitoneally with 0.7 μg VLP / animal, immunize 3 times in total, with a 2-week interval between two immunizations. Collect mouse sera 2 weeks after the third immunization for neutralization titer determination.

[0217] Pseudovirus packaging: Replace the P1 sequence in the PV1 Mahoney virus strain (Gene Bank ID: V01149.1) with GFP and add a T7 promoter sequence in front of the 5’UTR. After synthesis, it is ligated into pUC57simple to obtain the replicon plasmid pUC57-PV1M-eGFP (GenScript); the P1 sequence of the Saukett virus strain (Gene Bank ID: KP247597.1) and the dsRed gene sequence are synthesized by GenScript. After ligating the Saukett P1 gene into pcDNA3.3 (Thermo), dsRed is then ligated to the N-terminus of P1 to obtain pcDNA3.3-dsRed-SK3 P1; after synthesizing T7 polymerase (Gene Bank ID: M38308), it is ligated into pcDNA3.3 to obtain pcDNA3.3-T7. Mix 2 μg of pcDNA3.3-dsRed-SK3 P1 with lipo 3000 (Thermo) and add it to a 6-well plate containing 50 - 60% 293T cells; after 1 day, add a mixture of 1 μg of pcDNA3.3-T7 and 1.5 μg of pUC57-PV1M-eGFP with lipo3000 (Thermo); after 4 days, harvest the transfected cells, freeze-thaw them 3 times at -80 °C, and centrifuge to take the supernatant. Infect 293T cells with the packaged virus, count the number of GFP after 19 h, and calculate the titer of the pseudovirus.

[0218] Neutralization titer determination: Serially dilute the test sample 2-fold with DMEM containing 2% FBS. Take 50 μl of the diluted serum and OPV1 (NIBSC, 100 TCID 50 / 50 μL) and place them in a 5% CO2 incubator at 36 °C for 2 h, then incubate overnight at 4 °C; after the virus and serum have been incubated overnight, digest the hep-2 cells with trypsin into single cells, count them, centrifuge and discard the supernatant, resuspend with 2% DMEM (containing 2% double antibiotics), and add the resuspended cells dropwise to the incubated 96-well plate, 1.5×10 4 cells / 100 μL / well, and culture in a 5% CO2 incubator at 36 °C for 7 days. Start observing cytopathic effect on Day 7, and based on the CPE to judge the result. The maximum dilution that can neutralize more than 50% of the CPE is taken as the neutralization titer.

[0219] As shown in Table 4, when using aluminum adjuvant, sVLP 031 and sVLP 231 can induce a relatively high level of neutralizing antibodies, and are much higher than those of wtVLP 031 ; to determine whether sVLP can achieve the immunization effect of commercially available IPV, we tested sVLP 031 and sVLP 031t1 and sVLP031t2 Immunogenicity without the addition of aluminum adjuvant. The results showed that except for sVLP 031 and sVLP 031t2 , the neutralizing antibody level was not weaker than that of IPV, and the antibody titer of sVLP 031t1 was slightly lower than that of the IPV vaccine.

[0220] Table 4 Serum neutralizing titers

[0221] Immunogen Immunogen dose (per animal) Aluminum adjuvant dose (μg per animal) Neutralizing titer (GMT) <![CDATA[wtVLP 031 > 5 μg 60 16 <![CDATA[sVLP 031 > 5 μg 60 512 <![CDATA[sVLP 231 > 5 μg 60 2352.5 <![CDATA[wtVLP 031 > 2.8 μg 0 11.31 IPV 0.5 dose 0 256 <![CDATA[sVLP 031 > 2.8 μg 0 388 <![CDATA[sVLP 031t1 > 2.8 μg 0 157.6 <![CDATA[sVLP 031t2 > 2.8 μg 0 362

[0222] In this application, without introducing protease 3CD, VLPs were successfully prepared by tandemly expressing the capsid proteins VP0, VP3, and VP1 of PV3 in Pichia pastoris. These VLPs avoided the risk of 3CD protease residue. The PV3 wild-type VLP, namely wtVLP 031 had extremely poor thermal stability and induced a relatively low level of neutralizing antibodies, and thus could not yet be used as a candidate vaccine for PV3. According to the report by Fox et al., introducing mutations at the interface and hydrophobic pockets, etc. could increase the stability of empty particles. We introduced 8 amino acid mutations into the PV3 capsid protein and tandemly expressed it in Pichia pastoris, and successfully prepared sVLP 031 . The thermal stability results showed that its Tm value was 56.69 °C and it could induce a relatively high antibody level. In addition, since VP0 could be cleaved into VP4 and VP2, in order to improve the homogeneity of VLP, we tandemly expressed VP2 and successfully obtained sVLP 231 ; in order to further promote assembly and improve thermal stability, we truncated the N-terminus of VP0 or grafted the N-terminal sequence of VP4 into the N-terminus of VP1 and successfully prepared sVLP 031t1 and sVLP 031t2 ; the thermal stability and immunogenicity of these sVLPs were both superior to those of wtVLP 031 , among which sVLP 031t2 had the best thermal stability and could induce an antibody level slightly higher than that of the IPV vaccine. In summary, the sVLP series could be used as candidate vaccines for PV3.

[0223] The above embodiments are for illustrating the implementation schemes disclosed in the present invention and should not be construed as limitations on the present invention. In addition, various modifications listed herein and changes in the methods of the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, all obvious modifications to those skilled in the art as described above to obtain the invention should be included within the scope of the present invention.

Claims

1. A virus-like particle of poliovirus type 3, characterized in that, Select any of the following: a. The capsid protein of the virus-like particle comprises a mutant capsid protein fragment sVP2, a mutant capsid protein fragment sVP3, and a mutant capsid protein fragment sVP1; b. The capsid protein of the virus-like particle comprises a mutant capsid protein fragment sVP0 or a variant thereof, a mutant capsid protein fragment sVP3, a mutant capsid protein fragment sVP1 or a variant thereof.

2. The virus-like particle according to claim 1, wherein The amino acid sequence of the mutant capsid protein fragment sVP2 is shown in SEQ ID No: 17; And / or, the amino acid sequence of the mutant capsid protein fragment sVP3 is shown in SEQ ID No: 19; And / or, the amino acid sequence of the mutant capsid protein fragment sVP1 is shown in SEQ ID No: 21; And / or, the amino acid sequence of the mutant capsid protein fragment sVP0 is shown in SEQ ID No:

11.

3. The virus-like particle according to claim 1, wherein The variant of the mutant capsid protein fragment sVP1 is a polypeptide formed by adding 15 to 45 amino acids to the N-terminus of the mutant capsid protein fragment VP1; And / or, the variant of the mutant capsid protein fragment sVP0 is a polypeptide formed by truncating 1 to 5 amino acids from the N-terminus of the mutant capsid protein fragment sVP0 or a polypeptide formed by truncating 15 to 45 amino acids from the N-terminus of the mutant capsid protein fragment sVP0.

4. The virus-like particle according to claim 3, wherein The variant of the mutant capsid protein fragment sVP1 is a polypeptide sVP1 formed by adding 29 amino acids to the N-terminus of the mutant capsid protein fragment sVP1 P29 ; Preferably, the 29 amino acids added to the sVP1 P29 are from the 29 amino acids at the N-terminus of the mutant capsid protein fragment sVP0; and / or, the variant of the mutant capsid protein fragment sVP0 is a polypeptide sVP0 formed by truncating 2 amino acids at the N-terminus of the mutant capsid protein fragment sVP0 N2 or a polypeptide sVP0 formed by truncating 29 amino acids at the N-terminus of the mutant capsid protein fragment sVP0 N29 .

5. The virus-like particle according to claim 4, wherein The sVP1 P29 has the amino acid sequence shown in SEQ ID No: 23; and / or, said sVP0 N2 has the amino acid sequence shown in SEQ ID No: 13, and said sVP0 N29 has the amino acid sequence shown in SEQ ID No:

15.

6. A polynucleotide encoding a virus-like particle as described in any one of claims 1 to 5, characterized in that, Each capsid protein of the virus-like particle is separated by an operable element.

7. The polynucleotide according to claim 6, wherein The operable element comprises a promoter, a terminator, a transposon or an enhancer; Preferably, the promoter is AOX1 promoter; and / or the terminator is CYC1 terminator.

8. The polynucleotide of claim 6, wherein Also includes one or more of the following: 1) The nucleotide sequence encoding sVP0 is shown in SEQ ID NO: 12, encoding sVP0 N2 The nucleotide sequence is shown in SEQ ID NO: 14, encoding sVP0 N29 The nucleotide sequence is shown in SEQ ID NO: 16; 2) The nucleotide sequence encoding sVP3 is shown in SEQ ID NO: 20; 3) The nucleotide sequence encoding sVP1 is as shown in SEQ ID NO: 22, and the nucleotide sequence encoding sVP1 P29 is as shown in SEQ ID NO: 24; 4) The nucleotide sequence encoding the sVP2 capsid protein is shown in SEQ ID NO:

18.

9. The polynucleotide according to any one of claims 6 to 8, characterized in that Each fragment in the polynucleotide is connected in series in any of the following ways: 1) Promoter-sVP0-terminator-promoter-sVP3-terminator-promoter-sVP1-terminator; 2) Promoter-sVP2-terminator-promoter-sVP3-terminator-promoter-sVP1-terminator; 3) Promoter - sVP0 N2 - Terminator - Promoter - sVP3 - Terminator - Promoter - sVP1 - Terminator; 4) Promoter-sVP0 N29 -Terminator-Promoter-sVP3-Terminator-Promoter-sVP1 P29 -Terminator.

10. A nucleic acid construct, characterized in that, The nucleic acid construct comprises the polynucleotide according to any one of claims 6 to 9.

11. The nucleic acid construct according to claim 10, characterized in that, The expression vector of the nucleic acid construct is a yeast expression vector.

12. The nucleic acid construct according to claim 11, characterized in that The nucleotide sequence of the nucleic acid construct is shown in SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO:

29.

13. A cell line, characterized in that, The cell line comprises the nucleic acid construct according to any one of claims 10 to 12, or the polynucleotide according to any one of claims 6 to 9 is integrated into its genome.

14. The cell line according to claim 13, characterized in that, The cell line is a Pichia pastoris cell line.

15. The method for preparing virus-like particles according to any one of claims 1 to 5, comprising the following steps: 1) Cultivating the cell line according to claim 13 or 14 to express virus-like particles of poliovirus type 3; 2) Viral-like particles with improved thermal stability and immunogenicity compared to wild-type poliovirus PV3 are isolated.

16. Use of the viral-like particles according to any one of claims 1 to 5, the polynucleotide according to any one of claims 6 to 9, the nucleic acid construct according to any one of claims 10 to 12, and the cell line according to claim 13 or 14 in the preparation of a product for preventing poliomyelitis.

17. The use according to claim 16, characterized in that, The product for preventing poliomyelitis is a poliomyelitis vaccine.

18. A pharmaceutical composition for preventing poliovirus, characterized in that, The pharmaceutical composition comprises the viral-like particles according to any one of claims 1 to 5, the polynucleotide according to any one of claims 6 to 9, the nucleic acid construct according to any one of claims 10 to 12, the cell line according to claim 13 or 14, and a pharmaceutically acceptable carrier.

19. A method for preventing poliovirus, comprising administering to a subject an effective amount of the viral-like particles according to any one of claims 1 to 5, the polynucleotide according to any one of claims 6 to 9, the nucleic acid construct according to any one of claims 10 to 12, the cell line according to claim 13 or 14, the poliomyelitis vaccine in the use according to any one of claims 16 or 17, or the pharmaceutical composition according to claim 18.

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