Enterovirus 71 virus-like particles, methods of making and uses thereof

Abstract

The application discloses an enterovirus 71 virus-like particle and a preparation method and application thereof, and belongs to the technical field of genetic engineering and biological medicine. The enterovirus 71 virus-like particle is obtained by expressing and self-assembling in insect cells after the insect cells are infected by a recombinant baculovirus of a coding gene of a recombinant enterovirus 71 capsid protein P1 and a coding gene of a protease 3CD. The application further discloses a preparation and purification method of the enterovirus 71 virus-like particle and application thereof. Compared with a wild-type enterovirus 71 virus particle, the enterovirus 71 virus-like particle has a completely similar appearance structure and size, does not contain virus nucleic acid, has no replication and infection capacity, has a high in-vitro assembly efficiency, has the function of exciting a host adaptive immune response, can be prepared into an enterovirus vaccine in cooperation with an adjuvant, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and biomedicine, and relates to an enterovirus 71 (EV71) virus-like particle, its preparation method and application. Background Technology

[0002] Hand, foot, and mouth disease (HFMD) is a common infectious disease in children, caused by various enteroviruses. It is most prevalent in preschool children, with infants under five years old being generally susceptible. EV71 is one of the important pathogens causing HFMD. Besides EV71, Coxsackievirus A16 and echoviruses can also cause HFMD. Most patients experience mild symptoms, but some cases progress rapidly, leading to central nervous system complications and even death, with cases caused by EV71 infection being particularly severe. In recent years, HFMD caused by EV71, especially severe and fatal cases, has become a serious public health problem. Due to the widespread susceptibility of children and the lack of effective treatments, safe and effective targeted vaccination has become the most economical and effective means of controlling the disease.

[0003] Currently, the hand-foot-and-mouth disease (HFMD) vaccines available in China include the Beijing Sinovac HFMD (EV71) vaccine, the Wuhan Biological Products HFMD (EV71) vaccine using Vero cell technology, and the Chinese Academy of Medical Sciences Biological Products HFMD (EV71) vaccine using human diploid cell technology; all of these are inactivated vaccines. However, developing an inactivated vaccine requires rigorous and complex processes such as strain screening, strain domestication, and virus culture. This not only results in a long development cycle and high costs but also poses biosafety risks during the production process, failing to meet the needs of emergency prevention and control of emerging infectious diseases.

[0004] EV71 virus is a single-stranded positive-sense RNA virus belonging to the family Picornaviridae, genus Enterovirus, and type A enterovirus. Based on significant differences in gene sequence, genome, and biological properties, human enterovirus serotypes include four types: Enterovirus A, B, C, and D. EV71 virus particles are non-enveloped, icosahedral, and 20-30 nm in diameter. The viral capsid contains a single-stranded positive-sense RNA nucleotide (SRNA). The RNA is approximately 7.4 kb long. The EV71 viral RNA coding region is roughly divided into three subregions: P1, P2, and P3, encoding the P1, P2, and P3 proteins, respectively. The P1 protein is the precursor protein of four viral structural proteins: VP1, VP2, VP3, and VP4. The P2 and P3 proteins are precursor proteins of seven non-structural proteins, including 2A-2C and 3A-3D, which constitute RNA-dependent RNA polymerase and the protease used in the cleavage of the P1 protein. VP1, VP2, and VP3 are exposed on the surface of the viral capsid, allowing them to access the external environment. Therefore, these structural proteins can be easily recognized by the host's immune system. In particular, the VP1 protein contains major neutralizing epitopes, possesses antigenicity, and plays an important role in viral function. VP4 is located inside the capsid proteins.

[0005] 3CD is a protease essential for cleaving the P1 protein into VP1, VP0, and VP3 proteins. VP0 can be further cleaved into VP2 and VP4. Simultaneous expression of P1 and 3CD in cells yields structural proteins such as VP1. The four viral structural proteins VP1 to VP4 form a protomer. Five protomers assemble to form a pentamer, and twelve pentamers form a single EV71 virus-like particle (VLP). This VLP is similar in size and morphology to the wild-type EV71 virus, contains no viral nucleic acid, lacks replication and infectivity, and maintains the native conformation of the wild-type EV71 antigenic proteins, thus possessing the ability to elicit both innate and adaptive immune responses in the host.

[0006] Currently, three VLP-based human vaccines have been officially approved for the prevention of hepatitis B virus, human papillomavirus, and hepatitis E virus. There are currently no enterovirus-related VLP vaccines on the market. Summary of the Invention

[0007] In order to overcome the shortcomings of the existing technology, the purpose of this invention is to provide an EV71 virus-like particle, its preparation method and application.

[0008] Based on the efficient multi-gene co-expression characteristics of the baculovirus-insect cell system, this invention achieves efficient assembly and high-purity preparation of EV71 virus-like particles (VLPs) by synergistically expressing EV71 structural proteins and proteases and optimizing the multiplicity of infection, harvest time, and purification process.

[0009] This invention provides an EV71 P1 protein, the amino acid sequence of which is shown in SEQ ID NO.3.

[0010] The present invention also provides an EV71 3CD enzyme, the amino acid sequence of which is shown in SEQ ID NO.4.

[0011] The present invention also provides a gene encoding the EV71 P1 protein as described above, the nucleotide sequence of which is shown in SEQ ID NO.1;

[0012] The present invention also provides a gene encoding the EV71 3CD enzyme as described above, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0013] The present invention also provides a recombinant plasmid comprising the encoding gene of EV71 P1 protein as described above and the encoding gene of EV71 3CD enzyme as described above.

[0014] The present invention also provides a recombinant rod granule, which includes the encoding gene of EV71 P1 protein as described above and the encoding gene of EV71 3CD enzyme as described above.

[0015] The present invention also provides a recombinant baculovirus, which includes the recombinant baculovirus as described above.

[0016] The present invention also provides a host cell comprising one or more of the recombinant plasmids or recombinant rods as described above.

[0017] The present invention also provides an EV71 virus-like particle, which is assembled from the expression of the coding gene of the EV71 P1 protein as described above.

[0018] The present invention also provides a method for preparing EV71 virus-like particles as described above, comprising the following steps:

[0019] (1) Synthesize the gene encoding the EV71 P1 protein as described above and the gene encoding the EV71 3CD enzyme as described above, etc.;

[0020] (2) Construct the recombinant plasmid as described above;

[0021] (3) Construct the recombinant rod particles as described above;

[0022] (4) Transfect cells with the recombinant baculovirus described in step (3) to obtain the recombinant baculovirus as described above;

[0023] (5) Infect insect cells with the recombinant baculovirus described in step (4) to express EV71 virus-like particles as described above.

[0024] (6) The insect cells infected with recombinant baculovirus in step (5) are lysed, and the resulting cell lysate is subjected to two-step column chromatography and concentration to obtain the purified EV71 virus-like particles as described above.

[0025] In step (1), the nucleotide sequences of the gene encoding the EV71 P1 protein and the gene encoding the EV71 3CD enzyme are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively, and the amino acid sequences of the corresponding proteins encoded by the nucleotide sequences of the gene encoding are shown in SEQ ID NO.3-4.

[0026] In step (2), the specific steps for constructing the recombinant plasmid are as follows: the coding gene of EV71 P1 protein and the coding gene of EV71 3CD enzyme in step (1) are inserted into two different promoters of the vector plasmid, and the integrity and accuracy of the recombinant plasmid are verified by sequencing.

[0027] In step (2), the vector plasmid used to construct the recombinant plasmid can be selected from at least one of pFastBac Dual, pFastBac l, pFastBac HTA, pFastBac HTB, pFastBac HTC, or other similar baculovirus homologous recombination or transposon vectors. The pFastBac Dual baculovirus vector is preferred.

[0028] In a preferred embodiment, the constructed recombinant plasmid is pFastBac Dual-P1-3CD, which contains the nucleotide sequences of the gene encoding the EV71 P1 protein and the gene encoding the EV713CD enzyme.

[0029] In step (3), the specific steps for constructing the recombinant rod-like granules are as follows: The recombinant plasmid constructed in step (2) is transformed into competent cells containing rod-like granules. Blue-white screening is performed on LB plates containing 30-50 μg / mL kanamycin, 5-10 μg / mL gentamicin, 5-15 μg / mL tetracycline, 340-400 μg / mL Bluo-Gal, and 10-40 μM IPTG. P1-3CD cells that have successfully transposonized from the recombinant plasmid to the rod-like granules will appear as white spots on the plate. White spots are selected and cultured in liquid LB medium containing 30-50 μg / mL kanamycin, 5-10 μg / mL gentamicin, and 5-15 μg / mL tetracycline for expansion. The bacterial cells are lysed, and the recombinant rod-like granules are extracted.

[0030] In a preferred embodiment, the recombinant plasmid pFastBac Dual-P1-3CD constructed in step (2) was transformed into DH10Bac competent cells containing rod granules. Blue-white screening was performed on LB agar plates containing 0.05 mg / mL kanamycin, 0.007 mg / mL gentamicin, 0.01 mg / mL tetracycline, 0.4 mg / mL Bluo-Gal, and 10 μM IPTG. Successful transposition of P1-3CD from the recombinant plasmid to the rod granules resulted in white spots on the agar plates. Selected white spots were then cultured in liquid LB medium containing 0.05 mg / mL kanamycin, 0.007 mg / mL gentamicin, and 0.01 mg / mL tetracycline for expansion. The bacterial cells were lysed using a rod granule extraction kit to extract the recombinant rod granules.

[0031] In step (4), the cells refer to susceptible cells of baculoviruses, including but not limited to one or more cell lines such as SF9 and High Five. SF9 cell line is preferred.

[0032] In step (4), the specific steps of transfection are as follows: the recombinant baculovirus rBV-P1-3CD constructed in step (3) is transfected into insect cells, and the P0 generation recombinant baculovirus rBV-P1-3CD in the supernatant is harvested after 72-96 h. The P0 generation recombinant baculovirus rBV-P1-3CD infects insect cells with a multiplicity of infection (MOI) of less than 0.1, and the P1 generation recombinant baculovirus rBV-P1-3CD in the supernatant is harvested after 48-72 h, and so on, to amplify to obtain the P2 generation or a titer greater than or equal to 1×10 8 Recombinant baculovirus rBV-P1-3CD at pfu / mL.

[0033] In a preferred embodiment, the recombinant baculovirus rBV-P1-3CD constructed in step (3) was transfected into SF9 insect cells, and the P0 generation recombinant baculovirus rBV-P1-3CD in the supernatant was harvested after 72 h. The P0 generation recombinant baculovirus rBV-P1-3CD was used to infect SF9 insect cells with a multiplicity of infection (MOI) of less than 0.1, and the P1 generation recombinant baculovirus rBV-P1-3CD in the supernatant was harvested after 48 h. This process was repeated to amplify the P2 generation or cells with a titer of 1×10⁻⁶. 8 Recombinant baculovirus rBV-P1-3CD at pfu / mL was used. SF9 insect cells were cultured in ESF 921 medium (Expression Systems, Cat. 9600101) at 27°C under static conditions.

[0034] In step (5), the cells refer to susceptible cells of baculoviruses, including but not limited to one or more cell lines such as SF9 and High Five. The High Five cell line is preferred.

[0035] In step (5), the specific steps of the infection are as follows: the recombinant baculovirus rBV-P1-3CD amplified in step (4) is used to infect insect cells with an MOI of 1-5 to express EV71 VLP, and the cell pellet is harvested on the 3rd-4th day after infection.

[0036] In a preferred embodiment, the recombinant baculovirus rBV-P1-3CD amplified in step (4) was used to infect SF9 insect cells with an MOI of 1 to express EV71 VLP (EV71 virus-like particles), and the cell pellet was harvested on day 4 post-infection. The SF9 insect cells were cultured in ESF 921 medium (Expression Systems, Cat. 9600101) at 27°C with shaking at 110 rpm.

[0037] In step (6), the specific steps of lysis include: lysing cells under sterile conditions using mechanical lysis methods (including ultrasonic disruption, repeated freeze-thaw cycles, etc.) or chemical detergent lysis methods (including non-ionic detergents, zwitterionic detergents, etc.), centrifuging to remove cell debris, and obtaining cell lysate. Nuclease and Mg2+ are added to the cell lysate. 2+ Incubate at 10-20 mM for 0.5-12 h at 4-42 ℃. Centrifuge to remove precipitate, and filter the cell lysate supernatant through a 0.22 μm or 0.45 μm filter membrane.

[0038] In a preferred embodiment, an EDTA-free protease inhibitor (Shanghai Yisheng Biotechnology Co., Ltd., Cat. 20123ES50) was added to 1% NP-40 (in PBS) to prepare a lysis buffer, which was then sterilized by 0.22 μm filtration. Cells were lysed under aseptic conditions using this lysis buffer, and cell debris was removed by centrifugation to obtain cell lysate. Nuclease (Shanghai Beyotime Biotechnology Co., Ltd., Cat. D7121-100KU) was added to the cell lysate to a final concentration of 2 kU / 100 mL, and MgCl2 was added to a final concentration of 20 mM. The reaction was carried out at 37°C for 1 h. The precipitate was removed by centrifugation, and the cell lysate supernatant was filtered through a 0.22 μm filter.

[0039] In step (6), the specific steps for purifying the lysate include: concentrating the cell lysate through a tangential flow filter and replacing the liquid phase with PBS, followed by two-step column chromatography to purify the cell lysate. After each column chromatography, the flow-through component containing EV71 virus-like particles is collected and concentrated by ultrafiltration to obtain purified EV71 virus-like particles.

[0040] In step (6), before each column chromatography, particulate matter in the cell lysate is removed by centrifugation or 0.22 μm filtration. The column is equilibrated with at least one column volume of PBS until the UV absorption baseline and conductivity stabilize. The column chromatography consists of two steps, using a Capto Core 400 composite chromatography column and a Sephacryl S-500 High Resolution size exclusion chromatography column, respectively. When purifying with the Capto Core 400 composite chromatography column at room temperature, the flow rate should not exceed 3.9 mL / min; when purifying with the Sephacryl S-500 High Resolution size exclusion chromatography column at room temperature, the flow rate should not exceed 1 mL / min. When purifying at 4 °C, the flow rate should be halved accordingly.

[0041] The present invention also provides an immunogen, which includes the EV71 P1 protein as described above.

[0042] The present invention also provides a drug / drug composition comprising, as described above, EV71 P1 protein, etc.

[0043] In this invention, the drug / drug composition may further include pharmaceutically acceptable adjuvants and / or excipients, etc.

[0044] Excipients refer to the excipients and additives used in the production of pharmaceutical compositions and formulations. They have important functions such as shaping, protecting active ingredients, improving stability, solubilizing, aiding in solubility, and providing sustained or controlled release, so as to enable the pharmaceutical composition to achieve a certain shelf life and bioavailability, thereby improving the safety and efficacy of the pharmaceutical composition. Excipients that can be used in conjunction with the drugs / pharmaceutical compositions of the present invention include, but are not limited to, sugars, proteins, amino acids, and polymers.

[0045] Adjuvants are substances added to vaccines or other immunizing agents to enhance the immunogenicity of antigens and strengthen, prolong, or regulate the body's immune response to antigens. They include aluminum salt adjuvants, water-in-oil emulsions, microbial derivatives, and saponin adjuvants.

[0046] Specifically, the drug / drug composition includes liquid dosage forms, gaseous dosage forms, solid dosage forms, and semi-solid dosage forms.

[0047] Preferably, the dosage form of the drug / drug composition includes injections, sterile powders for injection, tablets, pills, capsules, lozenges, liniments, powders, granules, syrups, solutions, tinctures, aerosols, powder inhalers, or suppositories, etc. All of the above dosage forms of the drug / drug composition can be prepared according to conventional methods in the pharmaceutical field.

[0048] Specifically, the drug / drug composition is administered orally, by injection, nasal administration, transdermal administration, or mucosal administration.

[0049] Preferably, the route of administration of the drug / drug composition is parenteral administration, injection, or oral administration. Injection administration preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection, or subcutaneous injection. The drug composition is in various dosage forms conventional in the art, preferably in solid, semi-solid, gaseous, or liquid form, i.e., aqueous solution, non-aqueous solution, or suspension; more preferably, tablets, capsules, granules, injections, or infusions. More preferably, it is administered intravascularly, subcutaneously, intraperitoneally, or intramuscularly. Preferably, the drug composition can also be administered as an aerosol or coarse spray, i.e., nasal administration; or intrathecal, intramedullary, or intraventricular administration. More preferably, the drug / drug composition can also be administered transdermally, percutaneously, topically, intraenterically, intravaginally, sublingually, or rectally. The drug / drug composition of the present invention can be formulated into various dosage forms as needed, and the dosage beneficial to the patient can be determined by a physician based on factors such as patient type, age, weight, general disease condition, and administration method. Administration methods may include injection or other treatment methods.

[0050] The dosage level of the drug / pharmaceutical composition of the present invention can be adjusted according to the amount of composition required to achieve the desired diagnostic or therapeutic outcome. The administration regimen can also be a single injection or multiple injections, or adjustments can be made. The selected dosage level and regimen depend on various factors, including the activity and stability (i.e., half-life) of the drug / pharmaceutical composition, the formulation, the route of administration, combination with other drugs or treatments, the disease or condition to be detected and / or treated, and the health status and prior medical history of the subject to be treated, and require reasonable adjustment.

[0051] The therapeutically effective dose of the drug / pharmaceutical composition of the present invention can initially be estimated in cell culture experiments or animal models such as rodents, rabbits, dogs, pigs, and / or primates. Animal models can also be used to determine suitable concentration ranges and routes of administration. These can then be used to determine the useful dose and route of administration in humans. Generally, the determination and adjustment of the effective amount or dose, and the assessment of when and how to make such adjustments, are known to those skilled in the art.

[0052] For further guidance on formulations, dosages, administration regimens, and measurable treatment outcomes, see Berkow et al. (2000) The Merck Manual of Medical Information and Merck & Co. Inc., Whitehouse Station, New Jersey; Ebadi (1998) CRC Desk Reference of Clinical Pharmacology.

[0053] Furthermore, the drug / drug composition can be used alone and / or in combination with other drugs.

[0054] The other drugs mentioned include one or more of the following: other immunogens, other vaccines, nucleic acid drugs, nanomedicines, traditional Chinese medicine or natural drugs.

[0055] The present invention also provides an EV71 vaccine, which is prepared by adding adjuvants to EV71 virus-like particles as described above.

[0056] The adjuvant includes one or more of aluminum adjuvant, MF59, AS01, AS04, CpG 1018, Matrix-M, etc.

[0057] The present invention also provides the use of the P1 protein as described above, or the EV71 3CD enzyme as described above, or the encoding gene as described above, or the recombinant plasmid as described above, or the recombinant baculosome as described above, or the recombinant baculovirus as described above, or the host cell as described above, or the recombinant baculovirus as described above, or the EV71 virus-like particle as described above, or the preparation method as described above, or the immunogen as described above, or the drug / drug composition as described above, or the EV71 vaccine as described above, in the preparation of EV71 vaccines and / or drugs, multivalent vaccines and / or drugs containing EV71, etc.

[0058] In one specific embodiment of the present invention, the nucleotide sequence described above further includes a nucleotide sequence having at least 85% sequence identity with the nucleotide sequence; or a nucleic acid sequence having the same function as the nucleotide sequence formed by substitution, deletion or addition of one or more nucleic acid bases; or a nucleotide sequence that hybridizes with the nucleotide sequence or its full-length complement under stringent conditions; or a nucleotide sequence that is distinct from the nucleotide sequence due to the degeneracy of the genetic codon.

[0059] In one specific embodiment of the present invention, the amino acid sequence described above further includes: an amino acid sequence having at least 85% sequence identity with the amino acid sequence; or an amino acid sequence having the same function but with one or more amino acid residues substituted, deleted, or added; or an amino acid fragment expressed having the same function after substitution, deletion, or addition of one or more nucleic acid bases based on the coding nucleotide sequence of the amino acid sequence; or an amino acid sequence having the same function that can hybridize with the coding nucleotide sequence of the amino acid sequence under moderately stringent conditions and encode the same function.

[0060] The beneficial effects of the enterovirus 71 (EV71) virus-like particles of the present invention are as follows: Compared with wild-type enterovirus 71 virus-like particles, the enterovirus 71 virus-like particles of the present invention have a completely similar appearance, structure, and size, do not contain viral nucleic acid, have no replication or infectivity, possess the function of stimulating the host's innate and adaptive immune responses, and have a high in vitro assembly efficiency. The enterovirus 71 virus-like particles of the present invention retain the natural conformation of the antigenic protein of wild-type enterovirus 71 virus particles, and retain the immunogenicity and immunoprotective properties against enterovirus 71. After immunizing mice with the enterovirus 71 virus-like particles of the present invention, the production of binding antibodies and neutralizing antibodies against EV71 can be induced, with a seroconversion rate of 100%; through maternally transmitted antibodies, newborn mice can be protected from lethal EV71 challenge, with a protection efficiency of 100%. Attached Figure Description

[0061] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0062] Figure 1 A schematic diagram of the process for expressing EV71 VLP via a baculovirus-insect cell expression system;

[0063] Figure 2 The results of Coomassie Brilliant Blue staining of EV71 VLP gel after SDS-PAGE;

[0064] Figure 3 The results of immunoblotting of EV71 VLP protein onto an NC membrane after SDS-PAGE using three antibodies against EV71 structural proteins (Anti-EV71 VP1, Anti-EV71 VP2, and Anti-EV71 VP3).

[0065] Figure 4For transmission electron microscopy observation of the morphology of EV71 VLP (red arrow), (A) scale bar = 200 nm; (B) local magnification, scale bar = 100 nm;

[0066] Figure 5 The total IgG content of Anti-EV71 in the serum of mice after three doses of immunization;

[0067] Figure 6 The titer of Anti-EV71 neutralizing antibodies in the serum of mice after three doses of immunization; the numbers above the box plot represent the geometric mean titer. Detailed Implementation

[0068] The present invention will be further described in detail below through specific implementation examples and accompanying drawings. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art fall within the scope of the appended claims.

[0069] The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the invention or its application or use. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention.

[0070] In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values.

[0071] This invention discloses an enterovirus 71 virus-like particle, its preparation method, and its applications, belonging to the fields of genetic engineering and biomedicine. The enterovirus 71 virus-like particle is obtained by expressing and self-assembling insect cells after infection with a recombinant baculovirus encoding the P1 gene of the enterovirus 71 capsid protein and the 3CD protein of the enterovirus 71. This invention also discloses the preparation, purification method, and applications of the above-mentioned enterovirus 71 virus-like particle. Compared with wild-type enterovirus 71 virus-like particles, the enterovirus 71 virus-like particle of this invention has a completely similar appearance, structure, and size, does not contain viral nucleic acid, has no replication or infectivity, has high in vitro assembly efficiency, and possesses the function of stimulating an adaptive immune response in the host. It can be used in combination with adjuvants to prepare enterovirus vaccines.

[0072] Unless otherwise specified, the culture media, reagents and solutions used in the following examples are commercially available products or can be prepared by methods known in the art.

[0073] Example 1: A method for preparing enterovirus 71 virus-like particles, comprising the following steps:

[0074] 1. The nucleotide sequences encoding the EV71 P1 protein and the EV71 3CD enzyme (as shown in SEQ ID NO.1 and SEQ ID NO.2, respectively) were cloned into the polyhedral promoter (PH) and p10 promoter of the pFastBac Dual plasmid vector, respectively, to obtain the recombinant plasmid pFastBac Dual-P1-3CD. pFastBac Dual is a commercially available expression vector (Gibco, Cat. 10712024); the recombinant plasmid pFastBac Dual-P1-3CD was constructed by Nanjing Genscript Biotech Co., Ltd.

[0075] 2. The recombinant plasmid pFastBac Dual-P1-3CD was transformed into DH10Bac competent cells (Beijing Qingke Biotechnology Co., Ltd., Cat.TSC-C15) containing rod granules. Blue-white screening was performed on LB agar plates containing 0.05 mg / mL kanamycin, 0.007 mg / mL gentamicin, 0.01 mg / mL tetracycline, 0.4 mg / mL Bluo-Gal, and 10 μM IPTG (formulation: 1 g tryptone, 0.5 g yeast extract, 1 g sodium chloride (NaCl), 1.5 g agar powder, and ddH2O to a final volume of 100 mL, autoclaved). Successful transposition of P1-3CD from the recombinant plasmid to the rod granule resulted in white spots on the agar plate. White spots were selected and cultured in liquid LB medium (formulation: 1 g tryptone, 0.5 g yeast extract, 1 g sodium chloride (NaCl), adjusted to 100 mL with ddH2O, autoclaved) containing 0.05 mg / mL kanamycin, 0.007 mg / mL gentamicin, and 0.01 mg / mL tetracycline, for expansion. The bacterial cells were lysed using a bacterial cell extraction kit (Shanghai Shangbao Biotechnology Co., Ltd., Cat. 10123) to extract recombinant bacterial cells (…). Figure 1 ).

[0076] 3. The recombinant baculovirus rBV-P1-3CD was transfected into SF9 insect cells using Cellfectin II reagent (Gibco, Cat. 10362100). The P0 generation recombinant baculovirus rBV-P1-3CD was harvested from the supernatant after 72 h. SF9 insect cells were then infected with the P0 generation recombinant baculovirus rBV-P1-3CD at a multiplicity of infection (MOI) below 0.1. The P1 generation recombinant baculovirus rBV-P1-3CD was harvested from the supernatant after 48 h, and this process was repeated to amplify the P2 generation or cells with a titer of 1 × 10⁻⁶. 8Recombinant baculovirus rBV-P1-3CD at pfu / mL. SF9 insect cells were cultured in ESF 921 medium (Expression Systems, Cat. 9600101) at 27°C under static incubation conditions. Figure 1 ).

[0077] 4. The amplified recombinant baculovirus rBV-P1-3CD was used to infect SF9 insect cells with an MOI of 1-5 to express EV71 VLP. Cell pellet was harvested on day 4 post-infection. SF9 insect cells were cultured in ESF 921 medium (Expression Systems, Cat. 9600101) at 27°C with shaking at 110 rpm. Figure 1 ).

[0078] 5. Prepare a lysis buffer by adding an EDTA-free protease inhibitor (Shanghai Yisheng Biotechnology Co., Ltd., Cat. 20123ES50) and 1% ethylphenyl polyethylene glycol (NP-40, Shanghai Yisheng Biotechnology Co., Ltd., Cat. 20103ES60) to sterile PBS. Lyse the collected cell pellet, centrifuge at 10,000 rpm for 10 min at 4°C to remove cell debris. Add sterile MgCl2 to a final concentration of 20 mM and nuclease (Shanghai Beyotime Biotechnology Co., Ltd., Cat. D7121) to the cell lysis buffer, and react at 37°C for 1 h. Centrifuge at 10,000 × g for 10 min at 4°C, collect the supernatant, and filter through a 0.22 μm filter membrane. Concentrate the cell lysis buffer using a tangential flow filtration device (Merck, Cat. Z615382), and replace the liquid phase with PBS. "Lysis buffer" refers to 1% NP-40 (in PBS, containing protease inhibitors), which does not contain components from the cells. "Cell lysate" refers to the lysate in which components from the cells are released after cells have been lysed using the above-mentioned "lysis buffer," resulting in a mixture of components from the cells.

[0079] 6. The concentrated cell lysate was purified in two steps using a Capto Core 400 column and a Sephacryl S-500 High Resolution column. After each column chromatography, the flow-through was collected and immunoblot detected with Anti-EV71 antibodies (Anti-EV71 VP1, Abnova, Cat.MAB1255-M05; Anti-EV71 VP2, GeneTex, Cat.GTX132340; Anti-EV71 VP3, GeneTex, Cat.GTX132341). The flow-through fractions containing EV71 VLP were combined and concentrated using an ultrafiltration tube (Millipore, Cat.UFC9100).

[0080] Example 2: Characterization of the enterovirus 71 virus-like particles prepared in Example 1 of this invention, including the following steps and results:

[0081] 1. The EV71 VLP prepared according to the above steps (Example 1 of the present invention) was subjected to SDS-PAGE, and the gel was stained with Coomassie Brilliant Blue to show protein purity. Figure 2 The proteins were transferred onto an NC membrane and immunoblotting was performed using three antibodies against EV71 structural proteins (Anti-EV71 VP1, Abnova, Cat.MAB1255-M05; Anti-EV71 VP2, GeneTex, Cat.GTX132340; Anti-EV71 VP3, GeneTex, Cat.GTX132341). Figure 3 As a precursor protein of VP2, VP0 was also detected by the Anti-EV71 VP2 antibody. The results showed that the prepared EV71 VLP had high purity and intact protein composition.

[0082] 2. Dilute the EV71 VLP prepared above (Example 1 of this invention) and drop it onto a copper grid. Allow it to adsorb for 2-3 minutes, then remove excess liquid. Add 5 μL of 1% uranium acetate, quickly remove, then add another 5 μL of 1% uranium acetate, stain for 30 seconds, and remove excess liquid. Observe the morphology of the EV71 VLP on the copper grid using a transmission electron microscope. Figure 4 The results showed that the EV71 VLP particles prepared above had uniform size and a morphology similar to wild-type EV71 virus particles.

[0083] Example 3: The vaccine formulation of enterovirus 71 virus-like particles prepared in Example 1 of this invention and its application, including the following steps and results:

[0084] 1. The EV71 VLP prepared above (Example 1 of this invention) was supplemented with aluminum adjuvant (OZ Biosciences, Cat.AH0050) to prepare a vaccine, with each dose being 30 μg. Inactivated EV71 (BrCr strain, from China Center for Type Culture Collection, accession number: VR784, heat inactivated at 56 ℃ for 30 min) and PBS were used as control groups. The above preparation was administered intramuscularly to SPF-grade Balb / c female mice once each at weeks 0, 2, and 4. Blood was collected from the immunized mice, serum was separated, and the serum was inactivated at 56 ℃ for 30 min.

[0085] 2. The total Anti-EV71 IgG content in the serum of the mice in the above (step 1) was detected by ELISA.

[0086] Step (1) Coating: Dilute the purified EV71VLP in Example 1 of this invention with coating buffer (Beijing Solarbio Science & Technology Co., Ltd., Cat.C1055), add 8 ng to each well of the microplate, and coat overnight at 4°C;

[0087] Step (2) Washing: Wash three times with 1×PBST the next day. 400 μL per well, 5 min each time;

[0088] Step (3) Blocking: Block with 1% BSA (in PBST) blocking solution, 200 μL per well, at 37°C for 2 h;

[0089] Step (4) Primary antibody incubation: Dilute the serum to be tested with blocking solution (1% BSA) at a ratio of 1:1000, add 100 μL to each of the above-coated reaction wells, and incubate at 37°C for 2 hours;

[0090] Step (5) Washing: Wash 3 times with 1×PBST, 400 μL per well, 5 min each time;

[0091] Step (6) Secondary antibody incubation: Dilute Anti-Mouse IgG and HRP (CellSignaling Technology, Cat.7076) at a ratio of 1:5000 with blocking buffer (1% BSA), add 100 μL to each well, and incubate at 37°C for 1 h;

[0092] Step (7) Washing: Wash 3 times with 1×PBST, 400 μL per well, 5 min each time;

[0093] Step (8) Color development: Add 100 μL of TMB substrate (Suzhou Xinsaimei Biotechnology Co., Ltd., Cat.M30500) that has been equilibrated at room temperature for 30 min to each reaction well, and develop color in the dark for 10-20 min;

[0094] Step (9) Termination: Add 50 μL of 1 mol / L hydrochloric acid to each reaction well to terminate the reaction, and use an enzyme-linked immunosorbent assay (ELISA) reader to detect the OD value at a wavelength of 450 nm.

[0095] The results showed that the EV71 VLP described in this invention could induce a strong humoral immune response in mice, with a significant difference compared to the control group. Figure 5 ).

[0096] 3. The titer of Anti-EV71 neutralizing antibody in the serum of mice in the above (step 1) was detected by micro-neutralization assay.

[0097] Step (1) Cell seeding: One day before the experiment, human rhabdomyosarcoma (RD) cells were seeded into 96-well plates (1.5 × 10⁶ cells per well, except for the edge wells). 4 Add PBS buffer to each cell in the edge wells. Incubate the cell culture plate overnight at 37°C in a 5% CO2 incubator.

[0098] Step (2) Virus dilution: Based on the TCID of EV71 virus (BrCr strain, from China Center for Type Culture Collection, accession number: VR784) 50 Dilute the virus to 50 TCID per 60 μL. 50 The working concentration.

[0099] Step (3) Serum dilution and neutralization reaction: Set up negative control wells, serum gradient dilution wells and positive control wells in another 96-well plate; start the serum to be tested from 1:50 and perform 2-fold serial dilution, with the dilutions ranging from 1:50 to 1:6400; add an equal volume of EV71 virus solution diluted in step (2) to each dilution serum well, mix gently, and incubate at 37°C for 2 hours.

[0100] Step (4) Neutralization reaction detection: Discard the original culture medium in the cell culture plate from step (1), and inoculate the neutralization reaction system from step (3) into the corresponding wells containing RD cells. Continue culturing at 37°C and 5% CO2 for 72 hours. The neutralizing antibody titer is determined by the reciprocal of the highest serum dilution that can inhibit 50% cytopathic effect (CPE). The experimental conditions are: good cell growth in the negative control wells and complete CPE in the positive control wells.

[0101] The results showed that the EV71 VLP prepared in this invention could induce mice to produce high levels of neutralizing antibodies, which were significantly different from the control group. Figure 6 ).

[0102] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0103] As used in this invention, the terms "comprising" and "including" are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0104] As used in this invention, the term "and / or" includes any one or more of the related listed items and all combinations thereof.

[0105] The scope of protection of this invention is not limited to the above embodiments. Any variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of this invention are included in this invention and are protected by the appended claims.

Claims

1. An EV71 P1 protein, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

3.

2. An EV71 3CD enzyme, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

4.

3. A gene encoding the EV71 P1 protein as described in claim 1, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

1.

4. A gene encoding the EV71 3CD enzyme as described in claim 2, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

2.

5. A recombinant plasmid, characterized in that, It includes the gene encoding the EV71 P1 protein as described in claim 3 and the gene encoding the EV71 3CD enzyme as described in claim 4.

6. A recombinant rod-like particle, characterized in that, It includes the gene encoding the EV71 P1 protein as described in claim 3 and the gene encoding the EV71 3CD enzyme as described in claim 4.

7. A recombinant baculovirus, characterized in that, It contains the recombinant rod particles as described in claim 6.

8. A host cell, characterized in that, It comprises one or both of the recombinant plasmid as described in claim 5 or the recombinant rod-like particle as described in claim 6.

9. An EV71 virus-like particle, characterized in that, It is assembled from the gene encoding the EV71 P1 protein as described in claim 3 after expression.

10. A method for preparing EV71 virus-like particles as described in claim 9, characterized in that, Includes the following steps: (1) Synthesize the gene encoding the EV71 P1 protein as described in claim 3 and the gene encoding the EV71 3CD enzyme as described in claim 4; (2) Construct the recombinant plasmid as described in claim 5; (3) Construct the recombinant rod particles as described in claim 6; (4) Transfect cells with the recombinant baculovirus described in step (3) to obtain the recombinant baculovirus as described in claim 7; (5) Infect insect cells with the recombinant baculovirus described in step (4) to express the EV71 virus-like particles; (6) The insect cells infected with recombinant baculovirus in step (5) are lysed, and the resulting cell lysate is subjected to two-step column chromatography and concentration to obtain the purified EV71 virus-like particles.

11. The preparation method according to claim 10, characterized in that, In step (1), the nucleotide sequences of the gene encoding the EV71 P1 protein and the gene encoding the EV71 3CD enzyme are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively, and the amino acid sequences of the corresponding proteins encoded by the nucleotide sequences of the gene encoding are shown in SEQ ID NO.3-4. And / or, In step (2), the specific steps for constructing the recombinant plasmid are as follows: the coding gene of the EV71 P1 protein and the coding gene of the EV71 3CD enzyme in step (1) are inserted after two different promoters of the vector plasmid, and the integrity and accuracy of the recombinant plasmid are verified by sequencing; and / or, the vector plasmid used to construct the recombinant plasmid is selected from at least one of pFastBac Dual, pFastBac l, pFastBac HTA, pFastBac HTB, pFastBac HTC or other similar baculovirus homologous recombination or transposon vectors; and / or, In step (3), the specific steps for constructing the recombinant rod-like granules are as follows: the recombinant plasmid constructed in step (2) is transformed into competent cells containing rod-like granules; blue-white screening is performed on LB agar containing 30-50 μg / mL kanamycin, 5-10 μg / mL gentamicin, 5-15 μg / mL tetracycline, 340-400 μg / mL Bluo-Gal, and 10-40 μM IPTG; P1-3CD will appear as white spots on the agar after successfully transposition from the recombinant plasmid to the rod-like granules; white spots are selected and expanded in liquid LB agar containing 30-50 μg / mL kanamycin, 5-10 μg / mL gentamicin, and 5-15 μg / mL tetracycline; the bacterial cells are lysed and the recombinant rod-like granules are extracted; and / or, In step (4), the cells refer to susceptible cells of baculovirus, including one or more of the SF9 cell line and the High Five cell line; and / or, the specific steps of the transfection are as follows: transfect the recombinant baculovirus rBV-P1-3CD constructed in step (3) into insect cells, and harvest the P0 generation recombinant baculovirus rBV-P1-3CD from the supernatant after 72-96 h; infect insect cells with the P0 generation recombinant baculovirus rBV-P1-3CD at a multiplicity of infection (MOI) of less than 0.1, and harvest the P1 generation recombinant baculovirus rBV-P1-3CD from the supernatant after 48-72 h, and so on, to amplify to obtain the P2 generation or a titer greater than or equal to 1×10 8 Recombinant baculovirus rBV-P1-3CD at pfu / mL; and / or, In step (5), the cells refer to susceptible cells of baculovirus, including one or more of the SF9 cell line and the High Five cell line; and / or, the specific steps of the infection are: infecting insect cells with the recombinant baculovirus rBV-P1-3CD amplified in step (4) at a multiplicity of infection (MOI) of 1-5 to express EV71 VLP, and harvesting the cell pellet on day 3-4 post-infection; and / or, In step (6), cells are lysed under aseptic conditions using mechanical lysis or chemical detergent lysis, and cell debris is removed by centrifugation to obtain cell lysate; nuclease and Mg are added to the cell lysate. 2+ Incubate at 10-20 mM for 0.5-12 h at 4-42 ℃; remove precipitate by centrifugation, and filter the cell lysate supernatant through a 0.22 μm or 0.45 μm filter membrane.

12. An immunogen, characterized in that, It includes the EV71 P1 protein as described in claim 1.

13. A drug / drug composition, characterized in that, It includes the EV71 P1 protein as described in claim 1.

14. The pharmaceutical / pharmaceutical composition according to claim 13, characterized in that, The drug / drug composition may be used alone and / or in combination with other drugs.

15. The pharmaceutical / pharmaceutical composition as claimed in claim 14, characterized in that, The other drugs include one or more of the following: other immunogens, other vaccines, nucleic acid drugs, nanomedicines, traditional Chinese medicine, or natural drugs.

16. An EV71 vaccine, characterized in that, It is prepared by adding an adjuvant to the EV71 virus-like particles as described in claim 9.

17. The EV71 vaccine as described in claim 16, characterized in that, The adjuvant includes one or more of aluminum adjuvant, MF59, AS01, AS04, CpG 1018, and Matrix-M.

18. The use of the EV71 P1 protein as claimed in claim 1, or the EV71 3CD enzyme as claimed in claim 2, or the encoding gene as claimed in claim 3 or 4, or the recombinant plasmid as claimed in claim 5, or the recombinant baculovirus as claimed in claim 6, or the recombinant baculovirus as claimed in claim 7, or the host cell as claimed in claim 8, or the EV71 virus-like particle as claimed in claim 9, or the preparation method as claimed in claim 10 or 11, or the immunogen as claimed in claim 12, or the drug / drug composition as claimed in any one of claims 13-15, or the EV71 vaccine as claimed in claim 16 or 17 in the preparation of EV71 vaccines and / or drugs, multivalent vaccines and / or drugs containing EV71.

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