Preparation of poliovirus type 1 thermostable virus-like particles and immunogenicity research of poliovirus type 1 thermostable virus-like particles

Abstract

The invention relates to the field of medicine, in particular to preparation of poliovirus type 1 thermostable virus-like particles and immunogenicity research of the poliovirus type 1 thermostable virus-like particles. The invention provides a poliovirus type 1 virus-like particle, which 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; the capsid protein of the virus-like particle does not contain or contains a mutant capsid protein fragment sVP4; 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 PV1 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 1 and the study of their immunogenicity. Background Art

[0002] Poliovirus (PV) is a non-enveloped small RNA virus, whose genome is single-stranded positive-sense RNA. 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 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 strain-related poliomyelitis epidemics (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 true viruses, but do not contain viral nucleic acids, and are good candidate vaccines.

[0003] PV belongs to the Enterovirus genus and has an icosahedral structure without an envelope. Its genome is single-stranded positive-sense RNA, encoding an open reading frame that contains the structural protein P1 and the non-structural proteins P2 and P3. The structural protein P1 can be cleaved by the protease 3CD into the capsid proteins VP0, VP3, and VP1, and these capsid proteins can assemble with viral RNA into mature virus particles and are accompanied by 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 non-infectious empty particles with poor thermal stability and immunogenicity (referred to as C antigen). The wild-type VLP of PV1 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 above-mentioned disadvantages of the prior art, the applicant's research found that: amino acid mutations in the PV structural protein can improve the stability of its capsid protein. The non-structural protein 2C helps to maintain the morphology of the viral capsid protein, and the modification of myristoylation at the N-terminus of the VP4 protein also contributes to the stability of the viral 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 viral particles. However, in the resolved VLP structure, VP4 is very dynamic and difficult to resolve, indicating that VP4 plays an important role in maintaining the morphology of the viral 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, this application introduced 7 amino acid mutations into the wild-type PV1 structural protein, and optimized the sequence of a single subunit based on literature reports and structural information. VLPs were successfully prepared in Pichia pastoris by using the method of tandem expression frames of VP0, VP3, and VP1, etc. The purified VLPs were named the sVLP series. The thermal stability results showed that the thermal stability of the sVLP series was much better than that of the wild-type VLP (wtVLP) and could tolerate higher temperatures. In addition, animal immunization tests showed that the immunogenicity of the sVLP series was also better, and the neutralization titer of its immune serum was much higher than that of the wtVLP immune serum. In summary, the sVLP series is a good candidate strategy for PV1 vaccines.

[0006] The purpose of this application is to provide the preparation of poliovirus type 1 heat-stable virus-like particles and their immunogenicity research to solve the problems in the prior art.

[0007] To achieve the above object and other related objects, the first aspect of this application provides a virus-like particle of poliovirus type 1, selected from any one of the following:

[0008] a. The capsid protein of the virus-like particle contains mutant capsid protein fragments sVP2, mutant capsid protein fragments sVP3, and mutant capsid protein fragments sVP1; and the capsid protein of the virus-like particle does not contain or contains mutant capsid protein fragments sVP4;

[0009] b. The capsid protein of the virus-like particle contains mutant capsid protein fragments sVP0 or its variants, mutant capsid protein fragments sVP3, and mutant capsid protein fragments sVP1 or its variants.

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

[0011] The third aspect of the present application provides a nucleic acid construct, which 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 the virus-like particles of poliovirus type 1;

[0015] 2) Isolating virus-like particles with improved thermal stability and immunogenicity compared to the wild-type poliovirus PV1 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, which includes 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 separated and connected in series by operable elements, and through sequence optimization, the sVLP series is obtained. The thermal stability of the sVLP series is much better than that of the wild-type VLP (wtVLP) and can tolerate higher temperatures.

[0021] 2. The immunogenicity of the sVLP series of 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 PV1 vaccine. Description of the Drawings

[0022] Figure 1Schematic diagram of PV1 VLP expression vector.

[0023] Figure 2 Observation of PV1 VLP morphology. Among them, Figure 2 A is wtVLP 031 , Figure 2 B is sVLP 031 , Figure 2 C is sVLP 231 , Figure 2 D is sVLP 4231 , Figure 2 E is sVLP 031M , Figure 2 F is sVLP 031MC , Figure 2 G is sVLP 031t1 , Figure 2 H is sVLP 031t2 . Specific implementation mode

[0024] The first aspect of this application provides a virus-like particle of poliovirus type 1, selected from any of the following:

[0025] a. The capsid protein of the virus-like particle contains mutant capsid protein fragment sVP2, mutant capsid protein fragment sVP3, and mutant capsid protein fragment sVP1; and the capsid protein of the virus-like particle does not contain or contains mutant capsid protein fragment sVP4;

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

[0027] In the virus-like particles provided by the present application, the capsid protein of the poliovirus PV1 mutant is obtained by optimization, specifically: seven amino acids of the wild-type poliovirus PV1 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 PV1 structural protein P1 is derived from Gene Bank: KU866422.1 and is named wtP1. The specific mutations are as follows: the arginine at the 18th position of the wild-type poliovirus PV1 capsid protein is mutated to glycine, the threonine at the 94th position is mutated to alanine, the aspartic acid at the 126th position is mutated to glutamic acid, the leucine at the 460th position is mutated to methionine, the glutamine at the 519th position is mutated to leucine, the valine at the 775th position is mutated to leucine, and the histidine at the 827th position is mutated to proline. The poliovirus PV1 capsid protein formed after mutation is named sP1. 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 particles provided by the present application, the amino acid sequence of the mutant capsid protein fragment sVP2 is shown in SEQ ID No:21. The amino acid sequence of the mutant capsid protein fragment sVP3 is shown in SEQ ID No:23. The amino acid sequence of the mutant capsid protein fragment sVP4 is shown in SEQ ID No:19. The amino acid sequence of the mutant capsid protein fragment sVP1 is shown in SEQ ID No:25. The amino acid sequence of the mutant capsid protein fragment sVP0 is shown in SEQ ID No:11.

[0029] In the virus-like particles 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 are from the 29 amino acids at the N-terminus of the mutant capsid protein fragment sVP0. Specifically, the amino acid sequence of sVP1 P29 is shown in SEQ ID No:27.

[0030] In the virus-like particles provided by the present application, the variant of the mutant capsid protein fragment sVP0 is the myristoylated variant sVP0 M , 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 . Myristoylation modification refers to replacing the amino acid sequence GAQVSSQK (SEQ ID NO: 39) at the N-terminus with GNAAAARR (SEQ ID NO: 40), and this modification helps protein stability. Specifically, the amino acid sequence of sVP0 M is as shown in SEQ ID No: 13, the amino acid sequence of the said sVP0 N2 is as shown in SEQ IDNo: 15, and the amino acid sequence of the said sVP0 N29 is as shown in SEQ ID No: 17.

[0031] In the virus-like particles provided by the present application, it is selected from any one of the following:

[0032] [[ID=~18]]1) The capsid protein of the said virus-like particles contains sVP0 with the amino acid sequence as shown in SEQ ID No: 11, sVP3 with the amino acid sequence as shown in SEQ ID No: 23, and sVP1 with the amino acid sequence as shown in SEQ ID No: 25;

[0033] 2) The capsid protein of the said virus-like particles contains sVP2 with the amino acid sequence as shown in SEQ ID No: 21, sVP3 with the amino acid sequence as shown in SEQ ID No: 23, and sVP1 with the amino acid sequence as shown in SEQ ID No: 25;

[0034] 3) The capsid protein of the said virus-like particles contains sVP4 with the amino acid sequence as shown in SEQ ID No: 19, sVP2 with the amino acid sequence as shown in SEQ ID No: 21, sVP3 with the amino acid sequence as shown in SEQ ID No: 23, and sVP1 with the amino acid sequence as shown in SEQ ID No: 25;

[0035] 4) The capsid protein of the said virus-like particles contains sVP0 with the amino acid sequence as shown in SEQ ID No: 13 M, sVP3 with an amino acid sequence as shown in SEQ ID No: 23, and sVP1 with an amino acid sequence as shown in SEQ ID No: 25;

[0036] 5) The capsid protein of the virus-like particle contains sVP0 with an amino acid sequence as shown in SEQ ID No: 15 N2 , sVP3 with an amino acid sequence as shown in SEQ ID No: 23, and sVP1 with an amino acid sequence as shown in SEQ ID No: 25;

[0037] 6) The capsid protein of the virus-like particle contains sVP0 with an amino acid sequence as shown in SEQ ID No: 17 N29 , sVP3 with an amino acid sequence as shown in SEQ ID No: 23, and sVP1 with an amino acid sequence as shown in SEQ ID No: 27 P29 .

[0038] 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.

[0039] 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.

[0040] In the polynucleotide provided by the present application, the capsid protein of the virus-like particle contains 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 as shown in SEQ ID NO: 6; the nucleotide sequence encoding wtVP3 is as shown in SEQ ID NO: 8, and the nucleotide sequence encoding wtVP1 is as shown in SEQ ID NO: 10.

[0041] In the polynucleotide provided by the present application, the virus-like particle further includes protease 2C. Specifically, the nucleotide sequence encoding protease 2C is as shown in SEQ ID NO: 30.

[0042] In the polynucleotide provided by the present application, the nucleotide sequence encoding sVP0 is as shown in SEQ ID No: 12, the nucleotide sequence encoding sVP0 M is as shown in SEQ ID No: 14, the nucleotide sequence encoding sVP0 N2 is as shown in SEQ ID No: 16, the nucleotide sequence encoding sVP0 N29 is as shown in SEQ ID No: 18.

[0043] Among the polynucleotides provided by the present application, the nucleotide sequence encoding sVP3 is as shown in SEQ ID NO:24.

[0044] Among the polynucleotides provided by the present application, the nucleotide sequence encoding sVP1 is as shown in SEQ ID NO:26, and the nucleotide sequence encoding sVP1 P29 is as shown in SEQ ID NO:28.

[0045] Among the polynucleotides provided by the present application, the nucleotide sequence encoding the sVP2 capsid protein is as shown in SEQ ID NO:22.

[0046] Among the polynucleotides provided by the present application, the nucleotide sequence encoding the sVP4 capsid protein is as shown in SEQ ID NO:20.

[0047] Among the polynucleotides provided by the present application, the fragments in the polynucleotides are concatenated in any of the following ways:

[0048] 1) Promoter - wtVP0 - Terminator - Promoter - wtVP3 - Terminator - Promoter - wtVP1 - Terminator;

[0049] 2) Promoter - sVP0 - Terminator - Promoter - sVP3 - Terminator - Promoter - sVP1 - Terminator;

[0050] 3) Promoter - sVP2 - Terminator - Promoter - sVP3 - Terminator - Promoter - sVP1 - Terminator;

[0051] 4) Promoter - sVP4 - Terminator - Promoter - sVP2 - Terminator - Promoter - sVP3 - Terminator - Promoter - sVP1 - Terminator;

[0052] 5) Promoter - sVP0 M - Terminator - Promoter - sVP3 - Terminator - Promoter - sVP1 - Terminator;

[0053] 6) Promoter - 2C - Terminator - Promoter - sVP0 M - Terminator - Promoter - sVP3 - Terminator - Promoter - sVP1 - Terminator;

[0054] 7) Promoter - sVP0 N2 - Terminator - Promoter - sVP3 - Terminator - Promoter - sVP1 - Terminator;

[0055] 8) Promoter - sVP0 N29 - Terminator - Promoter - sVP3 - Terminator - Promoter - sVP1 P29 - Terminator.

[0056] In the polynucleotide provided by the present application, the expression cassettes for expressing each capsid protein in the polynucleotide are in tandem in the polynucleotide. The specific tandem manner is as Figure 1 shown.

[0057] The third aspect of the present application provides a nucleic acid construct, and the nucleic acid construct includes the aforementioned polynucleotide.

[0058] 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.

[0059] The VP0, VP4, VP2, VP3, and VP1 expression cassettes in the nucleic acid construct can be single copies or multiple copies that are independent of each other. Preferably, the VP4, VP2, VP3, and VP1 expression cassettes in the nucleic acid construct are all single copies.

[0060] In addition to including the nucleotide sequences of the capsid proteins of poliovirus PV1 contained in the polynucleotide, the nucleic acid construct does not include the nucleotide sequences encoding other capsid proteins of poliovirus PV1.

[0061] 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, such as a yeast expression vector. Preferably, it is the Pichia pastoris expression vector pPink-HC (manufacturer: Invitrogen).

[0062] In the embodiment as Figure 1 shown, the nucleotide sequence of the nucleic acid construct is as shown in SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, or SEQ ID NO:38.

[0063] In one embodiment, the method for preparing the nucleic acid construct includes the following steps:

[0064] 1) Cloning the nucleotides encoding the capsid proteins of poliovirus PV1 into different expression vectors respectively to obtain an intermediate construct;

[0065] 2) Recombining the intermediate construct obtained in step 1) to obtain the nucleic acid construct.

[0066] 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.

[0067] 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 transfecting the nucleic acid construct into Pichia pastoris cells.

[0068] The fifth aspect of the present application provides a method for preparing the aforementioned virus-like particles, comprising the following steps:

[0069] 1) Culturing the aforementioned cell line to express poliovirus PV1 virus-like particles;

[0070] 2) Isolating virus-like particles with improved thermal stability and immunogenicity compared to wild-type poliovirus PV1 virus-like particles.

[0071] In one embodiment, the conditions for culturing the cell line are 28-30 °C and 250-300 rpm. In the poliovirus PV1 virus-like particles provided by the present application, the diameter of the poliovirus PV1 virus-like particles is about 30 nm. The sizes of the poliovirus PV1 virus-like particles are uniform.

[0072] Any suitable technique can be used to measure the stability of VLPs. Standard techniques are known in the art, including but 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% upon 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%).

[0073] The method for detecting the thermal stability temperature is as follows: After incubating PV1 VLPs at different incubation temperatures for 10 min, the detection of D antigen is performed by sandwich ELISA. 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 non-linear regression (curve fitting).

[0074] Any suitable technique can be used to measure the immunogenicity of VLPs. 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 the neutralization titer.

[0075] The method for detecting the neutralization titer is as follows: Dilute the test sample two-fold with DMEM containing 2% FBS, take 50 μl of the diluted serum and add it to OPV1 (NIBSC, 100 TCID 50(50 μL) was placed in a 5% CO₂ incubator and incubated at 36 °C for 2 h, followed by incubation at 4 °C overnight; after the virus and serum were incubated overnight, the hep-2 cells were digested into single cells with trypsin, counted, the supernatant was discarded after centrifugation, and the cells were resuspended with 2% DMEM (containing 2% double antibiotics). The resuspended cells were added dropwise to the incubated 96-well plate, 1.5×10 4 cells / 100 μL / well, and cultured in a 5% CO₂ incubator at 36 °C for 7 days. Observation of cytopathic effects was started on Day 7, and the results were determined according to the cytopathic effect (CPE). The maximum dilution that could neutralize more than 50% of the CPE was used as the neutralization titer.

[0076] The sixth aspect of the present application provides the use of the foregoing virus-like particles, the foregoing polynucleotides, the foregoing nucleic acid constructs, and the foregoing cell lines in the preparation of products for preventing poliovirus. In some embodiments, the product for preventing polio is a polio vaccine.

[0077] The seventh aspect of the present application provides a pharmaceutical composition for preventing poliovirus, which comprises the foregoing virus-like particles, the foregoing polynucleotides, the foregoing nucleic acid constructs, the foregoing cell lines, and a pharmaceutically acceptable carrier.

[0078] 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.

[0079] The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for administering a pharmaceutical composition (such as the virus-like particles of the present invention). This term refers to such pharmaceutical carriers that do not themselves induce the production of antibodies harmful to the individual receiving the composition and have no excessive toxicity 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 suitable for formulation into a solution or suspension, a liquid excipient before injection. Liposomes are also included in the definition of pharmaceutically acceptable carriers.

[0080] The vaccine can be directly administered to an individual with the virus-like particles of the present invention by known methods. These vaccines are usually administered by the same route of administration as conventional vaccines and / or by mimicking the pathogen infection pathway.

[0081] Routes for administering the pharmaceutical composition or vaccine of the present invention include: intramuscular, subcutaneous, intradermal, intralung, intravenous, nasal, oral or other parenteral administration routes. If needed, 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 can include administering booster doses to initiate and / or maintain immunity.

[0082] 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, which is dissolved or suspended in a suitable carrier before use.

[0083] 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.

[0084] 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 the target disease or condition, or is an amount that exhibits a detectable therapeutic or preventive effect. That is, the amount of virus-like particles is sufficient to initiate an immune response in the selected administration route and can effectively protect the host against related diseases. This effect can be detected by, for example, antigen levels. The therapeutic effect also includes a reduction in physiological symptoms. The precise effective amount for a particular subject depends on the size and health 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.

[0085] In one embodiment, for the purpose of the present invention, the effective dose is about 0.001 mg / kg to 1000 mg / kg, preferably about 0.01 mg / kg to 100 mg / kg body weight of virus-like particles administered to an individual.

[0086] The poliovirus-like particles (VLP) of the present invention can be used on a small scale, such as as an antigen in serological assays (e.g., as part of seroprevalence studies or other monitoring activities) or on a large scale in vaccine production. Since the VLP of the present invention is non-infectious (lacking the poliovirus genome) and recombinant expression does not require live virus, containment is not required before and after the eradication of poliovirus. In some embodiments, the sVLP series of the present application can exhibit good immunogenicity without an adjuvant.

[0087] In summary, the thermal stability of the sVLP series of the present application is far superior to that of wild-type VLP (wtVLP), 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 PV1 vaccines.

[0088] The following specific examples illustrate the implementation manners of the present invention. Those skilled in the art can easily understand 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 implementation manners. 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.

[0089] Before further describing the specific implementation manners of the present invention, it should be understood that the protection scope of the present invention is not limited to the specific implementation manners described below; it should also be understood that the terms used in the embodiments of the present invention are for describing specific implementation manners, rather than 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.

[0090] When the embodiments give a numerical range, it should be understood that unless otherwise specified in the present invention, any value at both ends of each numerical range and any value between the two ends 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 similar to or equivalent to those described in the embodiments of the present invention can also be used to implement the present invention.

[0091] Example 1 Construction of expression plasmid

[0092] The amino acid sequence of the PV1 structural protein P1 (wtP1, Gene Bank: KU866422.1) and the amino acid sequence of P1 containing 7 mutations (sP1) were optimized and synthesized by GenScript. The amino acid sequence of the PV1 structural protein P1 obtained by introducing 7 mutations (R18G, T94A, D126E, L460M, Q519L, V775L, and H827P) into wtP1 was named sP1. wtP1 and sP1 were respectively optimized and synthesized according to the codon preference of Pichia pastoris.

[0093] The amino acid sequence of wtP1 is as follows, where 1-341 is the amino acid sequence of VP0 (where 1-69 is the amino acid sequence of VP4, and 70-341 is the amino acid sequence of VP2), 342-579 is the amino acid sequence of VP3, and 580-881 is the amino acid sequence of VP1:

[0094]

[0095] The optimized nucleotide sequence of wtP1 is as follows, where 1-1023 is the nucleotide sequence of VP0 (where 1-207 is the nucleotide sequence of VP4, and 208-1023 is the nucleotide sequence of VP2), 1024-1737 is the nucleotide sequence of VP3, and 1738-2643 is the nucleotide sequence of VP1:

[0096]

[0097]

[0098] The amino acid sequence of sP1 is as follows, where 1-341 is the amino acid sequence of VP0 (where 1-69 is the amino acid sequence of VP4, and 70-341 is the amino acid sequence of VP2), 342-579 is the amino acid sequence of VP3, and 580-881 is the amino acid sequence of VP1:

[0099]

[0100]

[0101] The optimized nucleotide sequence of sP1 is as follows, where 1-1023 is the nucleotide sequence of VP0 (where 1-207 is the nucleotide sequence of VP4, and 208-1023 is the nucleotide sequence of VP2), 1024-1737 is the nucleotide sequence of VP3, and 1738-2643 is the nucleotide sequence of VP1:

[0102]

[0103]

[0104] The amino acid sequence of wtVP0 is as follows:

[0105]

[0106] The nucleotide sequence of wtVP0 is as follows:

[0107]

[0108]

[0109] The amino acid sequence of wtVP3 is as follows:

[0110]

[0111] The nucleotide sequence of wtVP3 is as follows:

[0112]

[0113]

[0114] The amino acid sequence of wtVP1 is as follows:

[0115]

[0116] The nucleotide sequence of wtVP1 is as follows:

[0117]

[0118] The amino acid sequence of sVP0 is as follows:

[0119]

[0120] The nucleotide sequence of sVP0 is as follows:

[0121]

[0122] sVP0 M (Replacing the amino acid sequence GAQVSSQK (SEQ ID NO: 39) at the N-terminus of sVP0 with GNAAAARR (SEQ ID NO: 40) helps protein stabilization) The amino acid sequence is as follows:

[0123]

[0124]

[0125] sVP0 M The nucleotide sequence is as follows:

[0126]

[0127] sVP0 N2 (Truncating the first two amino acids at the N-terminus of VP0, which is also the N-terminus of VP4, is expected to help protein expression) The amino acid sequence is as follows:

[0128]

[0129]

[0130] sVP0 N2 The nucleotide sequence is as follows:

[0131]

[0132] sVP0 N29 (Delete the first two β - sheets at the N - terminus of VP4, which are the first 29 amino acids of VP0, and then connect them to the N - terminus of VP1, so that the VLP structurally mimics the structure of virus particles, which helps to stabilize) The amino acid sequence is as follows:

[0133]

[0134] sVP0 N29 The nucleotide sequence is as follows:

[0135]

[0136] The amino acid sequence of sVP4 is as follows:

[0137]

[0138] The nucleotide sequence of sVP4 is as follows:

[0139]

[0140] The amino acid sequence of sVP2 is as follows:

[0141]

[0142] The nucleotide sequence of sVP2 is as follows:

[0143]

[0144] The amino acid sequence of sVP3 is as follows:

[0145]

[0146] The nucleotide sequence of sVP3 is as follows:

[0147]

[0148]

[0149] The amino acid sequence of sVP1 is as follows:

[0150]

[0151] The nucleotide sequence of sVP1 is as follows:

[0152]

[0153] sVP1 P29(The first 29 amino acids of VP0, i.e., the first two β-sheets at the N-terminus of VP4, are linked to the N-terminus of VP1, enabling the VLP to structurally mimic the structure of the virus particle, which helps with stability.) The amino acid sequence is as follows:

[0154]

[0155] sVP1 P29 The nucleotide sequence is as follows:

[0156]

[0157] The amino acid sequence of 2C is as follows:

[0158]

[0159]

[0160] The nucleotide sequence of 2C is as follows:

[0161]

[0162] The 2C nucleotide sequence (Gene Bank ID: V01149.1) in the replicon was also synthesized by GenScript. According to the 2C nucleotide sequence, the wtP1 nucleotide sequence, the sP1 nucleotide sequence, 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-PV1 wtVP0, pPink / HC-PV1 sVP0, pPink / HC-PV1 sVP0 M , pPink / HC-PV1 sVP0 N2 , pPink / HC-PV1 sVP0 N29 , Pink / HC-PV1 sVP4, Pink / HC-PV1 sVP2, pPink / HC-PV1wtVP3, Pink / HC-PV1 sVP3, pPink / HC-PV1 wtVP1, Pink / HC-PV1 sVP1, pPink / HC-PV1sVP1 P29 and pPink / HC-PV1 2C were obtained by recombination respectively; then, using the isocaudomers BglⅡ and BamHⅠ, the expression cassettes in the corresponding intermediate plasmids were ligated in sequence to pPink / HC-PV1 wtVP1, Pink / HC-PV1 sVP1 or pPink / HC-PV1 sVP1 Figure 1 as shown in the order, and P29 , were obtained respectively Figure 1The shown expression final plasmids pPink / HC-PV1 wtVP 031 and pPink / HC-PV1 sVP series.

[0163] The complete plasmid sequences are as follows:

[0164] pPink / HC-PV1 wtVP 031 :

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187] pPink / HC-PV1 sVP 4231 :

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204] pPink / HC-PV1 sVP 031MC :

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215] pPink / HC-PV1 sVP 031t1

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224] pPink / HC-PV1 sVP 031t2 :

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233] 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), and intermediate plasmids pPink / HC-PV1 wtVP0, pPink / HC-PV1 sVP0, pPink / HC-PV1 sVP0 M , pPink / HC-PV1 sVP0 N2 , pPink / HC-PV1 sVP0 N29 , Pink / HC-PV1 sVP4, Pink / HC-PV1 sVP2, pPink / HC-PV1 wtVP3, Pink / HC-PV1sVP3, pPink / HC-PV1 wtVP1, Pink / HC-PV1 sVP1, pPink / HC-PV1 sVP1 P29 and pPink / HC-PV12C were obtained by recombination respectively.

[0234] In the order of Figure 1 , using the isocaudamer enzymes BglⅡ and BamHⅠ, the expression cassettes of the corresponding intermediate plasmids were successively ligated into pPink / HC-PV1 wtVP1, Pink / HC-PV1 sVP1 or pPink / HC-PV1 sVP1 P29 to obtain the final expression plasmids pPink / HC-PV1 wtVP 031 and pPink / HC-PV1 sVP 031 , pPink / HC-PV1 sVP 231 , pPink / HC-PV1 sVP 4231 , pPink / HC-PV1 sVP 031M , pPink / HC-PV1 sVP 031MC , pPink / HC-PV1 sVP 031t1 , pPink / HC-PV1 sVP 031t2 .

[0235] Table 1 Homologous recombination primer sequences

[0236]

[0237]

[0238]

[0239] Example 2 Screening, expression and purification of high-expression strains

[0240] 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 coated on PAD plates and cultured at 30 °C (nutritional screening); after 3 days, large white colonies were picked and placed in 24-well deep-well plates 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 taken 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.

[0241] 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 of the D antigen-specific antibody MAb234 (NIBSC) (diluted 1:1000) 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 (diluted 1:5000) was added and incubated at 37 °C for 1 h; the plate was washed 5 times with PBST, and TMB substrate was added for color development and the OD450nm absorbance value was read.

[0242] Sandwich ELISA

[0243] The PV1-specific monoclonal antibody 5C6 used in the sandwich ELISA method was prepared in-house by our company, and the preparation steps are as follows: The purified PV1 sVLP 031 (5 μg / animal) was fully mixed with aluminum adjuvant (60 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 5C6 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 5C6 monoclonal antibody was HRP-labeled using an HRP conjugation kit (Thermo).

[0244] The operating steps of the sandwich ELISA method are as follows: Dilute the antibody mAb234 with PBS at a ratio of 1:500, coat a 96-well ELISA plate, 50 μl per well, and incubate overnight at 4°C; Wash the plate 3 times with PBST, add 200 μl per well of 5% skim milk powder, and incubate at room temperature for 1 hr; Wash the plate 3 times with PBST, add the diluted test sample and / or standard (PV1 sVLP 031 ), and incubate at room temperature for 2 hr; Wash the plate 3 times with PBST, add the HRP-labeled monoclonal antibody 5C6 (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 fitting and calculate the expression level per unit wet weight (μg / mg) of each high-expression strain.

[0245] Expression and purification

[0246] 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 cells by centrifugation after 48 h of induction. Resuspend the cells with PBS and lyse the cells 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. The expression level per unit wet weight of each high-expression strain is relatively high, up to 2.55 μg / mg at most, which can meet the industrialization requirements.

[0247] Table 2 Yield of high-expression strains

[0248]

[0249] We carried out large-scale fermentation of the high-expression strains, resuspended the collected cells and lysed them by high pressure and PEG precipitation, and performed ultracentrifugation on the reconstituted supernatant to finally obtain the corresponding VLPs: wtVLP 031 , sVLP 031 , sVLP 231 , sVLP 4231 , sVLP 031M , sVLP 031MC , sVLP 031t1 and sVLP 031t2 .

[0250] Example 3 Morphological study of PV1 VLP

[0251] 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, and then observe it using a transmission electron microscope at 120 kV. The results are as Figure 2 shown, wtVLP 031 , sVLP031 、sVLP 231 、sVLP 4231 、sVLP 031M 、sVLP 031MC 、sVLP 031t1 and sVLP 031t2 all showed regular spherical structures with a diameter of approximately 30 nm. Therefore, tandem expression of the PV1 capsid protein in Pichia pastoris can spontaneously assemble into VLPs.

[0252] Example 4 Thermal Stability and Immunogenicity of PV1 VLPs

[0253] After incubating PV1 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 defined as the thermal stability temperature (Tm) using the method of non-linear regression (curve fitting).

[0254] The results are shown in Table 3. The thermal stability of the sVLP series was much better than that of wtVLP. Among them, sVLP 031t1 was the best, with a Tm value reaching 44.75 °C.

[0255] Table 3 Thermal Stability of VLPs

[0256]

[0257]

[0258] To determine the immunogenicity of poliovirus PV1 VLPs, the specific steps are as follows:

[0259] wtVLP 031 and sVLP 031 Comparison: Adsorb 5 μg VLP per mouse with 60 μg Al per mouse by shaking at room temperature for 1 - 2 h, then immunize female Balb / C mice aged 6 - 8 weeks intraperitoneally (10 mice per group), immunize 3 times in total, with a 2 - week interval between two immunizations. Two weeks after the third immunization, collect mouse sera for neutralization titer determination.

[0260] sVLP 031 and sVLP 231 、sVLP 4231 Comparison: Adsorb 5 μg VLP per mouse with 60 μg Al per mouse by shaking at room temperature for 1 - 2 h, then inject female Balb / C mice aged 6 - 8 weeks intraperitoneally (10 mice per group), immunize 3 times in total, with a 2 - week interval between two immunizations. Two weeks after the third immunization, collect mouse sera for neutralization titer determination.

[0261] sVLP 031and sVLP 031M 、sVLP 031MC Comparison: 5 μg VLP per mouse was adsorbed with 60 μg Al per mouse at room temperature with shaking for 1 - 2 h, and then intraperitoneally injected into female Balb / C mice aged 6 - 8 weeks (5 mice / group). Immunization was carried out 3 times in total, with a 3 - week interval between two immunizations. Mouse sera were collected 2 weeks after the third immunization for neutralization titer determination.

[0262] sVLP 031 and sVLP 031t1 、sVLP 031t2 Comparison: 1 μg VLP per mouse was intraperitoneally immunized into female Balb / C mice aged 6 - 8 weeks (10 mice / group). Immunization was carried out 3 times in total, with a 2 - week interval between two immunizations. Mouse sera were collected 2 weeks after the third immunization for neutralization titer determination.

[0263] Neutralization titer determination: The test sample was serially diluted 2 - fold with DMEM containing 2% FBS. 50 μl of the diluted serum was taken and mixed with OPV1 (NIBSC, 100 TCID 50 / 50 μL) and placed in a 5% CO2 incubator at 36 °C for 2 h, and then incubated overnight at 4 °C; after the virus and serum were incubated overnight, the hep - 2 cells were digested into single cells with trypsin, counted, the supernatant was discarded after centrifugation, and the cells were resuspended with 2% DMEM (containing 2% double antibiotics). The resuspended cells were added drop - wise to the 96 - well plates after incubation, 1.5×10 4 cells / 100 μL / well, and cultured in a 5% CO2 incubator at 36 °C for 7 days. CPE was observed starting from Day 7, and based on the CPE determination result, the maximum dilution that could neutralize more than 50% of the CPE was taken as the neutralization titer.

[0264] As shown in Table 4, in the case of using aluminum adjuvant, sVLP 031 、sVLP 231 、sVLP 4231 、sVLP 031M and sVLP 031MC could induce relatively high neutralizing antibody levels, which were much higher than those of wtVLP 031 ; to determine whether sVLP could achieve the immunization effect of commercially available IPV, we tested the immunogenicity of sVLP 031 、sVLP 031t1 and sVLP 031t2 without adding aluminum adjuvant. The results showed that the neutralizing antibody levels of sVLP 031 、sVLP 031t1 were comparable to those of IPV, while the neutralizing antibody titer of sVLP 031t2 was slightly inferior to that of the IPV vaccine.

[0265] Table 4 Serum neutralization titers

[0266]

[0267]

[0268] Without introducing protease 3CD, VLPs were successfully prepared by tandemly expressing the capsid proteins VP0, VP3, and VP1 of PV1 in Pichia pastoris. These VLPs avoid the risk of 3CD protease residue. The PV1 wild-type VLP is wtVLP 031 It has extremely poor thermal stability and induces a relatively low level of neutralizing antibodies, and thus cannot yet be used as a candidate vaccine for PV1. According to the report by Fox et al., introducing mutations at the interface and hydrophobic pockets, etc. can increase the stability of empty particles. However, empty particles are naturally formed during the virus replication process, and the assembly of the virus is related to proteases such as 2C. Therefore, the assembly or stability of empty particles may also be affected by the virus's own protease. We introduced 7 amino acid mutations into the PV1 capsid protein and tandemly expressed it in Pichia pastoris, successfully preparing sVLP 031 , and the thermal stability results showed that its Tm value was 43.02 °C and it could induce a relatively high antibody level. In addition, since VP0 can be cleaved into VP4 and VP2, in order to improve the homogeneity of VLP, we truncated VP4 or tandemly expressed VP4 and VP2 and successfully obtained sVLP 231 and sVLP 4231 ; in order to further promote assembly and improve thermal stability, we introduced a yeast myristoylation modification sequence at the N-terminus of VP0, co-expressed 2C, truncated the N-terminus of VP0 or grafted the N-terminus of VP4 into the N-terminus of VP1 sequence and successfully prepared sVLP 031M , sVLP 031MC , sVLP 031t1 and sVLP 031t2 ; the thermal stability and immunogenicity of these sVLPs are superior to those of wtVLP 031 , among which sVLP 031t1 has the best thermal stability and can induce an antibody level equivalent to that of the IPV vaccine. In summary, the sVLP series can be used as candidate vaccines for PV1.

[0269] 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 1, characterized in that, Selected from any of the following: a. The capsid protein of the virus-like particle contains mutant capsid protein fragment sVP2, mutant capsid protein fragment sVP3, and mutant capsid protein fragment sVP1; and the capsid protein of the virus-like particle does not contain or contains mutant capsid protein fragment sVP4; b. The capsid protein of the virus-like particle contains mutant capsid protein fragment sVP0 or its variant, mutant capsid protein fragment sVP3, mutant capsid protein fragment sVP1 or its variant.

2. The virus-like particle according to claim 1, wherein The amino acid sequence of the mutant capsid protein fragment sVP2 is as shown in SEQ ID No:21; and / or, the amino acid sequence of the mutant capsid protein fragment sVP3 is as shown in SEQ ID No:23; and / or, the amino acid sequence of the mutant capsid protein fragment sVP4 is as shown in SEQ ID No:19; and / or, the amino acid sequence of the mutant capsid protein fragment sVP1 is as shown in SEQ ID No:25; and / or, the amino acid sequence of the mutant capsid protein fragment sVP0 is as 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 - 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 myristoylated variant sVP0 M 、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.

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, characterized in that, The sVP1 P29 has the amino acid sequence shown in SEQ ID No: 27; and / or, the sVP0 M has an amino acid sequence as shown in SEQ ID No: 13, the sVP0 N2 has an amino acid sequence as shown in SEQ ID No: 15, the sVP0 N29 has an amino acid sequence as shown in SEQ ID No:

17.

6. A polynucleotide encoding the virus-like particle according to any one of claims 1 to 5, characterized in that, The capsid proteins of the virus-like particle are separated by operable elements.

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

8. The polynucleotide according to claim 6, wherein The virus-like particle also includes protease 2C.

9. The polynucleotide according to claim 8, wherein, The nucleotide sequence encoding protease 2C is as shown in SEQ ID NO:

30.

10. The polynucleotide according to claim 6, wherein Also includes one or several of the following: 1) The nucleotide sequence encoding sVP0 is shown in SEQ ID NO:12, and the nucleotide sequence encoding sVP0 M is shown in SEQ ID NO:14, and the nucleotide sequence encoding sVP0 N2 is shown in SEQ ID NO:16, and the nucleotide sequence encoding sVP0 N29 is shown in SEQ ID NO:18; 2) The nucleotide sequence encoding sVP3 is as shown in SEQ ID No:24; 3) The nucleotide sequence encoding sVP1 is as shown in SEQ ID NO:26, and the nucleotide sequence encoding sVP1 P29 is as shown in SEQ ID NO:28; 4) The nucleotide sequence encoding the sVP2 capsid protein is as shown in SEQ ID No:22; 5) The nucleotide sequence encoding the sVP4 capsid protein is as shown in SEQ ID No:

20.

11. The polynucleotide according to any one of claims 6 to 10, wherein The fragments in the polynucleotide are concatenated 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 - sVP4 - Terminator - Promoter - sVP2 - Terminator - Promoter - sVP3 - Terminator - Promoter - sVP1 - Terminator; 4) Promoter - sVP0 M - Terminator - Promoter - sVP3 - Terminator - Promoter - sVP1 - Terminator; 5) Promoter - 2C - Terminator - Promoter - sVP0 M - Terminator - Promoter - sVP3 - Terminator - Promoter - sVP1 - Terminator; 6) Promoter - sVP0 N2 - Terminator - Promoter - sVP3 - Terminator - Promoter - sVP1 - Terminator; 7) Promoter - sVP0 N29 - Terminator - Promoter - sVP3 - Terminator - Promoter - sVP1 P29 - Terminator.

12. A nucleic acid construct, characterized in that, The nucleic acid construct includes the polynucleotide according to any one of claims 6 - 11.

13. The nucleic acid construct according to claim 12, characterized in that, The expression vector of the nucleic acid construct is a yeast expression vector.

14. The nucleic acid construct according to claim 13, wherein, The nucleotide sequence of the nucleic acid construct is as shown in SEQ ID No:31, SEQ ID No:32, SEQ ID No:33, SEQ ID No:34, SEQ ID No:35, SEQ ID No:36, SEQ ID No:37 or SEQ ID No:

38.

15. A cell line, characterized in that, The cell line includes the nucleic acid construct according to any one of claims 12 to 14, or the polynucleotide according to any one of claims 6 to 11 is integrated into the genome.

16. The cell line according to claim 15, wherein, The cell line is a Pichia pastoris cell line.

17. The method for preparing virus-like particles according to any one of claims 1 to 5, the preparation method comprising the following steps: 1) Culturing the cell line according to claim 15 or 16 to express virus-like particles of poliovirus type 1; 2) Isolating virus-like particles with improved thermal stability and immunogenicity compared to wild-type poliovirus PV1 virus-like particles.

18. Use of the virus-like particles according to any one of claims 1 to 5, the polynucleotide according to any one of claims 6 to 11, the nucleic acid construct according to any one of claims 12 to 14, the cell line according to claim 15 or 16 in the preparation of a product for preventing poliomyelitis.

19. The use according to claim 18, wherein, The product for preventing poliomyelitis is a poliomyelitis vaccine.

20. A pharmaceutical composition for preventing poliovirus, characterized in that, The pharmaceutical composition includes the virus-like particles according to any one of claims 1 to 5, the polynucleotide according to any one of claims 6 to 11, the nucleic acid construct according to any one of claims 12 to 14, the cell line according to claim 15 or 16, and a pharmaceutically acceptable carrier.

21. A method for preventing poliovirus, comprising administering to a subject an effective amount of the virus-like particles according to any one of claims 1 to 5, the polynucleotide according to any one of claims 6 to 11, the nucleic acid construct according to any one of claims 12 to 14, the cell line according to claim 15 or 16, the poliomyelitis vaccine in the use according to any one of claims 18 or 19, or the pharmaceutical composition according to claim 20.

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