Recombinant Coxsackievirus A10 VLP and its use

Abstract

The present invention relates to the field of medicine, and in particular to recombinant Coxsackievirus A10 VLP and its use. The recombinant Coxsackievirus A10 VLP is produced by a cell line in which VP4, VP2, VP3 and VP1 capsid proteins encoding Coxsackievirus A10 are integrated into the genome. The present invention also provides the use of the recombinant Coxsackievirus A10 VLP in the preparation of a product for preventing hand, foot and mouth disease. The product for preventing hand, foot and mouth disease is a pharmaceutical composition, such as a vaccine composition. In the immunogenicity study, it was found that the Coxsackievirus A10 VLP of the present invention can avoid the degradation problem of VP0 and can induce a good immune response in mice, indicating that the VLP can be used as a candidate vaccine for Coxsackievirus A10.

Description

[0001] This application is a divisional application of application number 2022104944929. The filing date of the original application is May 7, 2022. The name of the invention is "Recombinant Coxsackievirus A10 VLP and its use". Technical Field

[0002] The present invention relates to the field of medicine, and in particular to recombinant Coxsackievirus A10 VLP and uses thereof. Background Art

[0003] Hand, foot and mouth disease is a common infant infectious disease in children under 5 years old. The main clinical manifestations are small blisters or ulcers on the hands, feet and mouth. A small number of children may develop pulmonary edema, aseptic meningoencephalitis, myocarditis and other complications, and even death. Hand, foot and mouth disease is caused by infection with coxsackievirus A group 16, 4, 5, 7, 9, 10, group B 2, 5, enterovirus 71 and echovirus 30 of the human enterovirus genus. Enterovirus 71 and coxsackievirus A16 are the main pathogens causing outbreaks of hand, foot and mouth disease. In recent years, reports of infection by coxsackievirus A10 have become more and more common. There is currently no specific drug for coxsackievirus A10, so vaccines have become the most effective way to prevent coxsackievirus A10 infection. Coxsackievirus A10 belongs to the genus Enterovirus of the Picornaviridae family. It is a non-enveloped, 20-sided, symmetrical spherical particle. Its genome is a single-stranded positive-strand RNA with conserved non-coding regions at both ends and an open reading frame coding region in the middle. This coding region encodes the structural protein P1 and non-structural proteins P2 and P3. P1 can be cleaved by protease 3CD into capsid proteins VP0, VP3 and VP1, and assembled into the capsid of the virus. These capsid proteins and viral RNA can be further assembled into true viruses, accompanied by cleavage of VP0 into VP4 and VP2. Virus-like particles (VLPs) are similar to viruses in morphology and composition, can induce a good immune response, and provide a solution for the development of Coxsackievirus A10 vaccines. Studies have shown that after co-expression of Coxsackievirus A10 structural proteins P1 and 3CD, they can be cleaved into VP0, VP3 and VP1 capsid proteins, and further assembled into Coxsackievirus A10 VLPs, and the VLPs can protect mice from lethal attacks by Coxsackievirus A10. However, VP0 of the prepared VLPs will be degraded to varying degrees to produce VP4 and VP2, thus affecting the uniformity of VLPs. Summary of the invention

[0004] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a recombinant Coxsackievirus A10 VLP and uses thereof, so as to solve the problems in the prior art.

[0005] To achieve the above objectives and other related objectives, the present invention provides a polynucleotide, which is any of the following:

[0006] 1) A first polynucleotide comprising nucleotides encoding VP2, VP3 and VP1 capsid proteins of Coxsackievirus A10, excluding RBS sequences and nucleotides encoding other capsid proteins of Coxsackievirus A10;

[0007] 2) The second polynucleotide comprises nucleotides encoding the VP4, VP2, VP3 and VP1 capsid proteins of Coxsackievirus A10, but does not include the RBS sequence and nucleotides encoding other capsid proteins of Coxsackievirus A10.

[0008] The present invention also provides a nucleic acid construct, which comprises any one of the polynucleotides.

[0009] The present invention also provides a cell line, which comprises the nucleic acid construct, or has any of the polynucleotides integrated into its genome.

[0010] The present invention also provides a recombinant Coxsackievirus A10 virus-like particle, wherein the recombinant Coxsackievirus A10 virus-like particle is selected from any one of the following:

[0011] 1) Includes VP2, VP3 and VP1 capsid proteins, excluding other capsid proteins of Coxsackievirus A10;

[0012] Includes VP4, VP2, VP3 and VP1 capsid proteins, but excludes other capsid proteins of Coxsackievirus A10.

[0013] The present invention also provides a method for preparing the recombinant Coxsackievirus A10 virus-like particles, which comprises the following steps:

[0014] 1) culturing the cell line to express recombinant Coxsackievirus A10 virus-like particles;

[0015] 2) Isolating the recombinant Coxsackievirus A10 virus-like particles expressed by the cell line.

[0016] The present invention also provides use of the recombinant Coxsackievirus A10 virus-like particles in preparing a product for preventing hand, foot and mouth disease.

[0017] The present invention also provides a pharmaceutical composition for preventing hand, foot and mouth disease, which comprises the recombinant Coxsackievirus A10 virus-like particles and a pharmaceutically acceptable carrier.

[0018] As described above, the recombinant Coxsackievirus A10 VLP and its use of the present invention have the following beneficial effects: -231 and VLP-4231 The problem of VP0 degradation was solved, and further immunogenicity studies found that Coxsackievirus A10 VLP -231 and VLP -4231 It can induce good specific antibody levels in mice, providing new ideas for the development of Coxsackievirus A10 vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Shown as pPink / HC-A10 VP of the present invention -231 Schematic diagram of the expression vector, PAOX1 is the AOX1 promoter, CYC1 TT is the CYC1 terminator, VP2, VP3 and VP1 are the Coxsackievirus A10 capsid protein sequences.

[0020] Figure 2 A10 high expression strain A10 VLP of the present invention -231 The A10 high expression strain VLP obtained by screening was -231 Induce expression, collect samples 24h and 48h after induction to determine the target protein content (VLP) and soluble protein content (TSP), the ordinate is the ratio of target protein to soluble protein, ie, VLP / TSP.

[0021] Figure 3 Coxsackievirus A10 VLPs of the present invention are shown -231 Particle size detection.

[0022] Figure 4 Coxsackievirus A10 VLPs of the present invention are shown -231 Immune serum antibody titer. For the convenience of statistics, the reciprocal of the highest dilution factor with an OD450nm absorbance value greater than 0.15 is used as the specific antibody titer of the sample, and the horizontal line shows the geometric mean.

[0023] Figure 5 Shown as pPink / HC-A10 VP of the present invention -4231 Schematic diagram of expression vector, P AOX1 is the AOX1 promoter, CYC1TT is the CYC1 terminator, and VP4, VP2, VP3 and VP1 are the Coxsackievirus A10 capsid protein sequences.

[0024] Figure 6 A10 high expression strain A10 VLP of the present invention -4231 The A10 high expression strain A10 VLP was screened and -4231Induce expression, collect samples 24h and 48h after induction to determine the target protein content (VLP) and soluble protein content (TSP), the ordinate is the ratio of target protein to soluble protein, ie, VLP / TSP.

[0025] Figure 7 Coxsackievirus A10 VLPs of the present invention are shown -4231 Particle size detection.

[0026] Figure 8 Coxsackievirus A10 VLPs of the present invention are shown -4231 For the convenience of statistics, the specific antibody titer of immune serum was determined by taking the reciprocal of the highest dilution multiple with an OD450nm absorbance value greater than 0.15 as the specific antibody titer of the sample, and the horizontal line showed the geometric mean. DETAILED DESCRIPTION

[0027] The present invention provides a polynucleotide, which is any of the following:

[0028] 1) A first polynucleotide comprising nucleotides encoding VP2, VP3 and VP1 capsid proteins of Coxsackievirus A10, excluding RBS sequences and nucleotides encoding other capsid proteins of Coxsackievirus A10;

[0029] 2) The second polynucleotide comprises nucleotides encoding the VP4, VP2, VP3 and VP1 capsid proteins of Coxsackievirus A10, but does not include the RBS sequence and nucleotides encoding other capsid proteins of Coxsackievirus A10.

[0030] like Figure 1 As shown, the first polynucleotide includes nucleotides encoding the VP2, VP3 and VP1 capsid proteins of Coxsackievirus A10, and the arrangement order is: VP2-VP3-VP1.

[0031] like Figure 5 As shown, the second polynucleotide includes nucleotides encoding the VP2, VP3 and VP1 capsid proteins of Coxsackievirus A10, and the arrangement order is: VP4-VP2-VP3-VP1.

[0032] The expression cassettes for expressing each capsid protein in the polynucleotide are connected in series in the polynucleotide.

[0033] In such Figure 1In the illustrated embodiment, the specific tandem mode is promoter-VP2-terminator-promoter-VP3-terminator-promoter-VP1-terminator. Of course, in other embodiments, the tandem mode of the three expression frames can also be any one of the following: promoter-VP2-terminator-promoter-VP1-terminator-promoter-VP3-terminator, promoter-VP3-terminator-promoter-VP2-terminator-promoter-VP1-terminator, promoter-VP3-terminator-promoter-VP1-terminator-promoter-VP2-terminator, promoter-VP1-terminator-promoter-VP3-terminator-promoter-VP2-terminator, promoter-VP1-terminator-promoter-VP2-terminator, promoter-VP1-terminator-promoter-VP2-terminator-promoter-VP3-terminator. In one embodiment, the promoter is AOX1 promoter, and the terminator is CYC1 terminator. Since each protein is an independent open reading frame, different tandem modes can achieve the same effect as in the embodiment.

[0034] In such Figure 5 In the illustrated embodiment, the specific tandem connection is promoter-VP4-terminator-promoter-VP2-terminator-promoter-VP3-terminator-promoter-VP1-terminator. Of course, in other embodiments, the tandem connection of the four expression frames can also be any permutation and combination, for example: promoter-VP4-terminator-promoter-VP2-terminator-promoter-VP1-terminator-promoter-VP3-terminator, promoter-VP3-terminator-promoter-VP2-terminator-promoter-VP1-terminator-promoter-VP4-terminator, promoter-VP4-terminator-promoter-VP3-terminator-promoter-VP1-terminator-promoter-VP2-terminator, promoter-VP1-terminator-promoter-VP3-terminator-promoter-VP2-terminator-promoter-VP4-terminator, promoter-VP1-terminator-promoter-VP3-terminator-promoter-VP2-terminator-promoter-VP4-terminator. In one embodiment, the promoter is AOX1 promoter, and the terminator is CYC1 terminator. Since each protein is an independent open reading frame, different tandem methods can achieve the same effect as in the embodiment.

[0035] The nucleotides encoding the VP4 capsid protein of Coxsackievirus A10 are the full-length nucleotide sequence of VP4 or truncated nucleotides, the nucleotides encoding the VP2 capsid protein of Coxsackievirus A10 are the full-length nucleotide sequence of VP2 or truncated nucleotides, the nucleotides encoding the VP1 capsid protein of Coxsackievirus are the full-length nucleotide sequence of VP1 or truncated nucleotides, and the nucleotides encoding the VP3 capsid protein of Coxsackievirus are the full-length nucleotide sequence of the VP3 capsid protein of Coxsackievirus A10 or truncated nucleotide sequence.

[0036] In one embodiment, the nucleotide sequence encoding the VP4 capsid protein of Coxsackievirus A10 is shown in SEQ ID NO: 7, and the nucleotide sequence encoding the VP2 capsid protein of Coxsackievirus A10 is shown in SEQ ID NO: 8. The nucleotide sequence encoding the VP3 capsid protein of Coxsackievirus A10 is shown in SEQ ID NO: 9. The nucleotide sequence encoding the VP1 capsid protein of Coxsackievirus A10 is shown in SEQ ID NO: 10.

[0037] In one embodiment, a sequence having a homology of more than 95% with the sequence shown in SEQ ID NO. 7 to 10, or a sequence complementary to the sequence shown in SEQ ID NO. 7 to 10 can also achieve the same effect as the embodiment of the present invention.

[0038] The polynucleotide does not include nucleotides encoding P1 and 3CD proteins.

[0039] The sequence of the polynucleotide is a sequence obtained after codon optimization.

[0040] In one embodiment, the nucleotide encoding the VP4 capsid protein of Coxsackievirus A10 encodes an amino acid sequence such as the VP4 capsid protein shown in SEQ ID NO:3; the nucleotide encoding the VP2 capsid protein of Coxsackievirus A10 encodes an amino acid sequence such as the VP2 capsid protein shown in SEQ ID NO:4; the nucleotide encoding the VP3 capsid protein of Coxsackievirus A10 encodes an amino acid sequence such as the VP3 capsid protein shown in SEQ ID NO:5; the nucleotide encoding the VP1 capsid protein of Coxsackievirus A10 encodes an amino acid sequence such as the VP1 capsid protein shown in SEQ ID NO:6.

[0041] The present invention also provides a nucleic acid construct, which comprises any one of the polynucleotides.

[0042] The term "nucleic acid construct" refers to an artificially constructed nucleic acid segment that can be introduced into target cells or tissues. The nucleic acid construct includes a vector backbone, namely an expression vector and an expression cassette. The nucleic acid construct can be a plasmid.

[0043] The VP4, VP2, VP3 and VP1 expression cassettes in the nucleic acid construct may be independent single copies or multiple copies. Preferably, the VP4, VP2, VP3 and VP1 expression cassettes in the nucleic acid construct are all single copies.

[0044] The nucleic acid construct includes not only the capsid protein nucleotides of Coxsackievirus A10 contained in the polynucleotide, but also does not include nucleotides encoding other capsid proteins of Coxsackievirus A10.

[0045] In one embodiment, the nucleic acid construct further comprises an expression vector. The expression vector may be any expression vector suitable for expressing Coxsackie virus in the prior art, such as a yeast expression vector. Preferably, it is a Pichia pastoris expression vector pPink-HC (manufacturer: Invitrogen).

[0046] In such Figure 1 In the illustrated embodiment, the nucleotide sequence of the nucleic acid construct is shown in SEQ ID NO:11.

[0047] In such Figure 5 In the illustrated embodiment, the nucleotide sequence of the nucleic acid construct is shown in SEQ ID NO:18.

[0048] The present invention also provides a cell line, which comprises the nucleic acid construct, or has any of the polynucleotides integrated into its genome.

[0049] 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.

[0050] The present invention also provides a recombinant Coxsackievirus A10 virus-like particle, wherein the recombinant Coxsackievirus A10 virus-like particle is selected from any one of the following:

[0051] 1) Includes VP2, VP3 and VP1 capsid proteins, excluding other capsid proteins of Coxsackievirus A10;

[0052] 2) Includes VP4, VP2, VP3 and VP1 capsid proteins, excluding other capsid proteins of Coxsackievirus A10.

[0053] The recombinant Coxsackievirus A10 virus-like particles are produced by the cell line.

[0054] The hydrated diameter of the recombinant Coxsackievirus A10 virus-like particles is 35nm to 45nm. The recombinant Coxsackievirus A10 virus-like particles have uniform sizes.

[0055] The present invention also provides a method for preparing the recombinant Coxsackievirus A10 virus-like particles, which comprises the following steps:

[0056] 1) culturing the cell line to express recombinant Coxsackievirus A10 virus-like particles;

[0057] 2) Isolating the recombinant Coxsackievirus A10 virus-like particles expressed by the cell line.

[0058] In one embodiment, the cell line is cultured at 28°C-30°C and 250-300 rpm.

[0059] In one embodiment, the cell line is obtained by transducing the nucleic acid construct into a host cell. In one embodiment, the host cell is a Pichia pastoris cell.

[0060] In one embodiment, the method for preparing the nucleic acid construct comprises the following steps:

[0061] 1) Cloning the codon-optimized nucleotides expressing the Coxsackievirus A10 capsid protein into different expression vectors to obtain intermediate constructs;

[0062] 2) Recombining the intermediate construct obtained in step 1) to obtain the nucleic acid construct.

[0063] The present invention also provides use of the recombinant Coxsackievirus A10 virus-like particles in preparing a product for preventing hand, foot and mouth disease.

[0064] In one embodiment, the hand, foot and mouth disease is hand, foot and mouth disease caused by Coxsackievirus A10 infection.

[0065] The product for preventing hand, foot and mouth disease is a pharmaceutical composition, which is, for example, a vaccine composition.

[0066] The present invention also provides a pharmaceutical composition for preventing hand, foot and mouth disease, which comprises the recombinant Coxsackievirus A10 virus-like particles and a pharmaceutically acceptable carrier.

[0067] The pharmaceutical composition can be monovalent (containing only one type of virus-like particle) or multivalent (containing multiple types of virus-like particles).

[0068] The pharmaceutical composition can be prepared into various conventional dosage forms, such as injections, granules, tablets, pills, suppositories, capsules, suspensions, sprays, etc.

[0069] The pharmaceutical composition comprises a preventive or therapeutically effective amount of the virus-like particle or polynucleotide of the present invention.

[0070] The term "prophylactically or therapeutically effective amount" refers to the amount of a pharmaceutical composition that treats, alleviates or prevents a target disease or condition, or an amount that exhibits a detectable therapeutic or preventive effect. That is, the amount of virus-like particles is sufficient to induce an immune response in the selected route of administration, and can effectively promote the protection of 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 exact effective amount for a subject depends on the subject's size and health status, the nature and extent of the disease, and the therapeutic agent and / or combination of therapeutic agents selected for administration. Therefore, it is useless to specify an exact effective amount in advance. However, for a given condition, the effective amount can be determined by routine experiments.

[0071] In one embodiment, for the purposes of the present invention, an effective dose is about 0.001 mg / kg to 1000 mg / kg, preferably about 0.01 mg / kg to 100 mg / kg of virus-like particles administered to an individual.

[0072] The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier used for the administration of a pharmaceutical composition (e.g., a recombinant virus-like particle of the present invention). The term refers to pharmaceutical carriers that do not induce the production of antibodies harmful to the individual receiving the composition and are not excessively toxic after administration. Suitable carriers may be large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, and the like. 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. In addition, auxiliary substances such as wetting agents or emulsifiers, pH buffering substances, and the like may also be present in these carriers. Typically, 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 being formulated into a solution or suspension, or a liquid excipient before injection. Liposomes are also included in the definition of a pharmaceutically acceptable carrier.

[0073] Once formulated, the composition of the present invention can be administered directly to a subject. The subject to be treated can be a mammal, especially a human.

[0074] The pharmaceutical composition is, for example, a vaccine composition. The vaccine composition can be directly administered to an individual using known methods to administer the virus-like particles of the present invention. These vaccines are usually administered using the same administration routes as conventional vaccines and / or by simulating pathogen infection routes.

[0075] The routes of administering the pharmaceutical composition of the present invention include: intramuscular, subcutaneous, intradermal, intrapulmonary, intravenous, nasal, oral or other parenteral routes of administration. If necessary, the routes of administration 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 the administration of booster doses to induce and / or maintain immunity.

[0076] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0077] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0078] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints 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 those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0079] Example 1 Coxsackievirus A10 expression plasmid pPink / HC-A10 VPN -231 Construction

[0080] In order to achieve optimal expression, the amino acid sequence of the structural protein P1 of Coxsackievirus A10 was optimized and synthesized according to the codon preference of Pichia pastoris. The amino acid sequence of the structural protein P1 of Coxsackievirus A10 is shown in SEQ ID NO:1, wherein 1-69 is the VP4 amino acid sequence (SEQ ID NO:3), 70-324 is the VP2 amino acid sequence (SEQ ID NO:4), 325-564 is the VP3 amino acid sequence (SEQ ID NO:5), and 565-862 is the VP1 amino acid sequence (SEQ ID NO:6). The optimized nucleotide sequence is shown in SEQ ID NO:2. The nucleotide sequence of VP4 is shown in SEQ ID NO:7, i.e., 1-207 in SEQ ID NO:2. The nucleotide sequence of VP2 is shown in SEQ ID NO:8, i.e., 208-972 in SEQ ID NO:2. The nucleotide sequence of VP3 is shown in SEQ ID NO:9, i.e., 973-1692 in SEQ ID NO:2. The nucleotide sequence of VP1 is shown in SEQ ID NO:10, i.e., 1693-2586 in SEQ ID NO:2.

[0081] Preparation of expression plasmids: According to the optimized nucleotide sequences of VP2, VP3, and VP1 and the sequence at the multiple cloning site of the expression vector pPink-HC (purchased from Invitrogen), synthetic recombination primers were designed (Table 1). Using a homologous recombination kit (purchased from Novozymes), VP2, VP3, and VP1 were ligated into the vector pPinK-HC by recombination using homologous recombination enzymes to obtain intermediate plasmids pPink / HC-A10 VP2, pPink / HC-A10 VP3, and pPink / HC-A10 VP1, respectively. Using the homologous tail enzymes Bgl Ⅱ and BamH Ⅰ, the A10 VP3 expression cassette and the A10 VP2 expression cassette in the intermediate plasmid were successively ligated into pPink / HC-A10 VP1 by enzyme cutting and ligation to obtain the final plasmids pPink / HC-A10 VPN -231 (The nucleotide sequence is shown in SEQ ID NO: 11), and the schematic diagram is shown in Figure 1 shown.

[0082] Table 1 Homologous recombination primer sequences

[0083] Primer name Serial Number Primer sequences A10VP2-F SEQ ID NO:12 5'-caactaattattcgaaacggaattcaccatgtctccttctgttgaggcttg-3' A10VP2-R SEQ ID NO:13 5'-ctgtatttaaatggccggccggtacctcattattgagaaacagcttgtctca-3' A10VP3-F SEQ ID NO:14 5'-caactaattattcgaaacggaattcaccatgggtattccagctgagttg-3' A10VP3-R SEQ ID NO:15 5'-ctgtatttaaatggccggccggtacctcattattgcaaaacagcttgttgag-3' A10VP1-F SEQ ID NO:16 5'-caactaattattcgaaacggaattcaccatgggcgaccgggtggccg-3' A10VP1-R SEQ ID NO:17 5'-ctgtatttaaatggccggccggtacctcattacagtgtggtgatggcggtc-3'

[0084] Example 2 Screening, expression and purification of coxsackievirus A10 high expression strain

[0085] Screening of high expression strains

[0086] The final plasmid pPink / HC-A10 VPN prepared in Example 1-231 The linearization was performed using endonuclease AflⅡ, and the purification and recovery were performed using ethanol precipitation. The linearized plasmids were introduced into different Pichia yeasts for gene recombination using electroporation, and the PAD plates were coated and cultured at 30°C. After 3 days, large white colonies were picked and placed in 24-well deep-well plates using BMMY medium for methanol-induced expression (28°C, 250rpm). After 48 hours of induction, the induced bacteria were collected and expression was detected using nutritional screening and sandwich ELISA methods. The strain with the highest expression level was used as the high-expression strain A10 VLP -23l The target gene sequence of the high-expression strain was consistent with the theoretical nucleotide sequence after sequencing analysis.

[0087] The rabbit anti-coxsackievirus A10 polyantiserum and coxsackievirus A10-specific mouse monoclonal antibody used in the sandwich ELISA method were both produced by our company. The preparation steps are as follows: (1) Rabbit anti-coxsackievirus A10 polyantiserum: the purified coxsackievirus A10 VLP (500 μg / rabbit) was mixed with Freund's adjuvant in a ratio of 1:1 and then emulsified and injected subcutaneously into adult New Zealand white rabbits (1 ml / rabbit). The interval between the two immunizations was 4 weeks, and the rabbit serum was collected 2 weeks after the 4th immunization for future use; (2) Coxsackievirus A10-specific mouse monoclonal antibody: the purified coxsackievirus A10 VLP was mixed with Freund's adjuvant in a ratio of 1:1 and then emulsified and injected subcutaneously into adult New Zealand white rabbits (1 ml / rabbit). The interval between the two immunizations was 4 weeks, and the rabbit serum was collected 2 weeks after the 4th immunization for future use; VLP (5 μg / mouse) was fully mixed with aluminum adjuvant (500 μg / mouse) and injected intraperitoneally into mice. The mice were immunized 4 times with an interval of 2 weeks between the two immunizations. Spleen cells were collected 2 weeks after the 4th immunization and fused with myeloma cells to obtain a Coxsackievirus A10-specific mouse monoclonal antibody cell line. The cell line was injected intraperitoneally into mice to obtain ascites, which was purified with protein G filler to obtain A10-specific mouse monoclonal antibody for use.

[0088] The operation steps of the sandwich ELISA method are as follows: the rabbit anti-Coxsackie virus A10 polyclonal antibody is diluted 1:2000 and coated in a 96-well ELISA plate, 50 μl / well, and blocked with 5% skim milk powder after overnight coating at 4°C; the bacteria are resuspended in PBS, broken at 70HZ for 120s after adding an equal volume of glass beads, and the supernatant is taken for use after centrifugation; the supernatant of broken bacteria and the homemade standard of Coxsackie virus A10VLP are appropriately diluted with 2% skim milk powder and added to the blocked ELISA plate, and incubated at 37°C; after 2 hours, coxsackie virus A10-specific mouse monoclonal antibody is added and incubated at 37°C; after 2 hours, HRP-labeled goat anti-mouse secondary antibody is added at a dilution of 1:5000, incubated at 37°C for 1 hour, color development is performed, and the absorbance value at 450nm is read, and the VLP content is calculated based on the standard curve.

[0089] Expression and purification

[0090] The high expression strain A10 VLP was screened -231Inoculate in BMGY medium, culture for 24h (30℃, 250rpm), then change to BMMY medium for induced expression (28℃, 250rpm), harvest the cells by centrifugation after 24h and 48h induction. Resuspend the cells in PBS and use a high-pressure homogenizer at 1200bar to break the cells, collect the supernatant after centrifugation for PEG precipitation, and perform sucrose gradient centrifugation on the reconstituted supernatant to finally obtain the target VLPs, named A10 VLP -231 . Determine the target protein content (VLP) and soluble protein content (TSP), the results are as follows Figure 2 As shown, the production can reach 21μg / mg in 48h.

[0091] Example 3 Study on the particle size of Coxsackievirus A10 VLP

[0092] Purified Coxsackievirus A10 VLPs -231 Dilute with PBS to 50-200 ng / μl, take 1 ml and add it to the sample pool, avoid bubbles, put the sample pool into the Zetasizer device, use dynamic light scattering method to measure its hydration diameter and perform data analysis. A10 VLP -231 The hydrated diameter is about 40nm. Figure 3 As shown, A10 VLP -231 Well assembled.

[0093] Example 4 Immunogenicity of Coxsackievirus A10 VLPs

[0094] To identify Coxsackievirus A10 VLP -231 The immunogenicity of A10 VLP --231 Immunize mice. The specific steps are as follows: 6-8 week old female Balb / C mice were divided into 2 groups, 5 mice in each group. -231 (5μg / mouse) and aluminum adjuvant (500μg / mouse) were adsorbed at room temperature for 1-2h, and then intramuscularly injected (100μL / mouse). The immunization was performed twice, with an interval of 4 weeks between the two immunizations. The mouse serum was collected 2 weeks after the second immunization to determine the specific antibody titer.

[0095] Determination of Coxsackievirus A10 VLP-specific antibody titer

[0096] Rabbit anti-Coxsackie virus A10 VLP was coated in a 96-well ELISA plate at 20 ng / well, and then blocked with 5% skim milk powder after overnight coating at 4°C; serum samples were diluted with 2% skim milk powder in multiples and added to the blocked ELISA plate, and incubated at 37°C; after 2 hours, HRP-labeled goat anti-mouse secondary antibody was added at a dilution of 1:5000, and color was developed after incubation at 37°C for 1 hour, and the absorbance value at 450 nm was read. Figure 4 As shown, VLP-231 Can induce good specific antibodies.

[0097] The above example is Coxsackie virus A10 VLP -231 The following examples are Coxsackie virus A10 VLP -4231 Embodiment of the invention.

[0098] Example 5 Coxsackievirus A10 expression plasmid pPink / HC-A10 VPN -4231 Preparation and immunogenicity analysis of

[0099] In order to achieve optimal expression, the amino acid sequence of the structural protein P1 of Coxsackievirus A10 was optimized and synthesized according to the codon preference of Pichia pastoris. The amino acid sequence of the structural protein P1 of Coxsackievirus A10 is shown in SEQ ID NO:1. The optimized nucleotide sequence is shown in SEQ ID NO:2. The nucleotide sequence of VP4 is shown in SEQ ID NO:7, i.e., 1-207 in SEQ ID NO:2. The nucleotide sequence of VP2 is shown in SEQ ID NO:8, i.e., 208-972 in SEQ ID NO:2. The nucleotide sequence of VP3 is shown in SEQ ID NO:9, i.e., 973-1692 in SEQ ID NO:2. The nucleotide sequence of VP1 is shown in SEQ ID NO:10, i.e., 1693-2586 in SEQ ID NO:2.

[0100] Synthetic recombination primers were designed based on the optimized nucleotide sequences of VP4, VP2, VP3, and VP1 and the sequence at the multiple cloning site of the expression vector pPink-HC (purchased from Invitrogen) (Table 2). A homologous recombination kit (purchased from Novozymes) was used to connect VP4, VP2, VP3, and VP1 to the vector pPinK-HC by recombination using homologous recombination enzymes to obtain intermediate plasmids pPink / HC-A10 VP4, pPink / HC-A10 VP2, pPink / HC-A10 VP3, and pPink / HC-A10VP1, respectively. The A10 VP4 expression cassette, A10VP3 expression cassette, and A10 VP2 expression cassette in the intermediate plasmid were connected to pPink / HC-A10 VP1 in turn by enzyme cutting and ligation using the homologous tail enzymes BglⅡ and BamHI to obtain the final plasmids pPink / HC-A10VPN -4231 (The nucleotide sequence is shown in SEQ ID NO: 18), and the schematic diagram is shown in Figure 5 shown.

[0101] Table 2 Homologous recombination primer sequences

[0102] Primer name Serial Number Primer sequences A10 VP4-F SEQ ID NO:19 5'-caactaattattcgaaacggaattcaccatgggtgctcaagtttctac-3' A10VP4-R SEQ ID NO:20 5'-tttaaatggccggccggtacctcattagttcaatggagcagacaattc-3' A10VP2-F SEQ ID NO:21 5'-caactaattattcgaaacggaattcaccatgtctccttctgttgaggcttg-3' A10VP2-R SEQ ID NO:22 5'-ctgtatttaaatggccggccggtacctcattattgagaaacagcttgtctca-3' A10VP3-F SEQ ID NO:23 5'-caactaattattcgaaacggaattcaccatgggtattccagctgagttg-3' A10VP3-R SEQ ID NO:24 5'-ctgtatttaaatggccggccggtacctcattattgcaaaacagcttgttgag-3' A10VP1-F SEQ ID NO:25 5’-caactaattattcgaaacggaattcaccatgggcgaccgggtggccg-3’ A10VP1-R SEQ ID NO:26 5’-ctgtatttaaatggccggccggtacctcattacagtgtggtgatggcggtc-3’

[0103] The expression plasmid pPink / HC-A10 VP -4231 After linearization, the strain was introduced into Pichia pastoris for gene recombination, and then screened by nutritional screening and sandwich ELISA to obtain the high-expression strain A10 VLP. -4231 The specific experimental method is the same as in Example 2. The results are as follows Figure 6 As shown, the yield of this high expression strain can reach 5μg / mg.

[0104] The A10 VLP of the high expression strain of Coxsackievirus A10 was obtained -4231 Fermentation and purification were performed, and sucrose density gradient centrifugation was used to purify the samples, and finally Coxsackievirus A10 VLP was obtained. -4231 The hydrated diameter is about 40 nm ( Figure 7 ).

[0105] To determine the expression of recombinant coxsackievirus A10 VLP -4231 The immunogenicity of the purified Coxsackievirus A10 VLP was -4231 Mice were immunized by intramuscular injection, and mouse serum was collected 2 weeks after the second immunization to determine the antibody titer. The experimental steps were the same as in Example 5. The results are shown in Figure 8 As shown, Coxsackievirus A10 VLP -4231 Can induce a good immune response.

[0106] In this example, Pichia pastoris was used to express the VP4, VP2, VP3 and VP1 capsid proteins of Coxsackievirus A10 in tandem, and the Coxsackievirus A10 VLPs were successfully assembled into particles. -4231 , solving the problem of uneven bands caused by VP0 degradation, and providing a new method for the preparation of enterovirus VLP. Further immunogenicity studies found that this Coxsackievirus A10 VLP -4231 Good antibody levels can be induced in mice, suggesting that this VLP can be used as a candidate vaccine for Coxsackievirus A10.

[0107] The above examples are for the purpose of illustrating the embodiments disclosed by the present invention and are not to be construed as limiting the present invention. In addition, the various modifications listed herein and the variations of the methods in 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, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of the present invention.

Claims

1. A polynucleotide, characterized in that The polynucleotide is a polynucleotide of a coxsackievirus A10 virus-like particle, and the polynucleotide is a nucleotide encoding the VP4, VP2, VP3 and VP1 capsid proteins of the coxsackievirus A10, excluding the RBS sequence and the nucleotide encoding other capsid proteins of the coxsackievirus A10. The nucleotides encoding the VP4, VP2, VP3 and VP1 capsid proteins of the coxsackievirus A10 are arranged in the order of: VP4-VP2-VP3-VP1. The nucleotide encoding the VP2 capsid protein of the coxsackievirus A10 encodes the VP2 capsid protein with an amino acid sequence such as the VP2 capsid protein shown in SEQ ID NO:4; the nucleotide encoding the VP3 capsid protein of the coxsackievirus A10 encodes the VP3 capsid protein with an amino acid sequence such as the VP3 capsid protein shown in SEQ ID NO:5; the nucleotide encoding the VP1 capsid protein of the coxsackievirus A10 encodes the VP1 capsid protein with an amino acid sequence such as the VP1 capsid protein shown in SEQ ID NO:6; the nucleotide encoding the VP4 capsid protein of the coxsackievirus A10 encodes the amino acid sequence such as the SEQ ID NO: The VP4 capsid protein shown in NO:3, the arrangement order of the nucleotides in the polynucleotide is: promoter-VP4-terminator-promoter-VP2-terminator-promoter-VP3-terminator-promoter-VP1-terminator.

2. The polynucleotide according to claim 1, wherein Also includes any one or more of the following: 1) The nucleotide encoding the VP2 capsid protein of Coxsackievirus A10 is the full-length VP2 nucleotide sequence; 2) The nucleotide encoding the VP1 capsid protein of Coxsackievirus A10 is the full-length VP1 nucleotide sequence; 3) The nucleotide encoding the VP3 capsid protein of Coxsackievirus A10 is the full-length nucleotide sequence of the VP3 capsid protein; 4) The nucleotide encoding the VP4 capsid protein of Coxsackievirus A10 is the full-length nucleotide sequence of the VP4 capsid protein.

3. The polynucleotide according to claim 1, wherein Also includes one or more of the following: 1) The nucleotide sequence encoding the VP2 capsid protein of Coxsackievirus A10 is shown in SEQ ID NO: 8; 2) The nucleotide sequence encoding the VP3 capsid protein of Coxsackievirus A10 is shown in SEQ ID NO:9; 3) The nucleotide sequence encoding the VP1 capsid protein of Coxsackievirus A10 is shown in SEQ ID NO: 10; 4) The nucleotide sequence encoding the VP4 capsid protein of Coxsackievirus A10 is shown in SEQ ID NO:

7.

4. A nucleic acid construct, characterized in that The nucleic acid construct comprises the polynucleotide according to any one of claims 1 to 3.

5. The nucleic acid construct according to claim 4, characterized in that The expression vector of the nucleic acid construct is a yeast expression vector.

6. The nucleic acid construct according to claim 4, characterized in that The nucleotide sequence of the nucleic acid construct is shown in SEQ ID NO:

18.

7. A cell line, characterized in that The cell line comprises the nucleic acid construct of claim 6, or the polynucleotide of any one of claims 1 to 3 is integrated into its genome.

8. The cell line according to claim 7, characterized in that The cell line is a Pichia pastoris cell line.

9. A recombinant coxsackievirus A10 virus-like particle, characterized in that: The recombinant Coxsackievirus A10 virus-like particles include VP4, VP2, VP3 and VP1 capsid proteins, but do not include other capsid proteins of Coxsackievirus A10. The recombinant Coxsackievirus A10 virus-like particles are produced by the cell line described in claim 7 or 8.

10. Use of the recombinant Coxsackievirus A10 virus-like particles according to claim 9 in the preparation of a product for preventing hand, foot and mouth disease.

11. A pharmaceutical composition for preventing hand, foot and mouth disease, characterized in that: The pharmaceutical composition comprises the recombinant Coxsackievirus A10 virus-like particle according to claim 9 and a pharmaceutically acceptable carrier.

12. The pharmaceutical composition according to claim 11, which is a vaccine composition.

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