HANSENULA ENGINEERING FUNGI EFFICIENTLY EXPRESSING CA10 VIRUS-LIKE PARTICLES AND USE THEREOF

Abstract

The present invention provides a Hansenula engineering fungus that efficiently expresses CA10 virus-like particles and use thereof. The engineering fungus comprises a recombinant vector carrying P1 and 3CD genes of the CA10 virus, and the starting strain of the engineering fungus is uracil auxotroph. The Hansenula AU-0501, P1 and 3CD genes are optimized according to preferred codons of Hansenula. The present invention also provides a preparation method of CA10 virus-like particles, comprising: culturing engineering fungi, expressing CA10 virus-like particles, and separating and purifying virus-like particles by ultrafiltration and three-step chromatography. The CA10 virus-like particles provided by the present invention and the vaccine prepared therefrom have good immunogenicity, safety and biological activity, the process is simple, the chromatography method is adopted for purification, and large-scale preparation and purification can be realized to obtain a VLP protein stock solution with high purity (greater than 99%), which can be used to prepare a vaccine to prevent CA10 infection, with good economic value and application prospects.

Claims

1. A Hansenula engineering fungus that efficiently expresses CA10 virus-like particles, wherein, the engineering fungus comprises a recombinant vector carrying P1 and 3CD genes of the CA10 virus; wherein, starting vector of the recombinant vector is PMV-05; starting strain of the engineering fungus is uracil auxotroph Hansenula AU-0501; the P1 and 3CD genes of the CA10 virus are sequence-optimized genes according to preferred codons of Hansenula, and nucleotide sequences of the P1 and 3CD genes of the CA10 virus are represented by SEQ ID NOs: 1 and 2, respectively.

2. The engineering fungus according to claim 1, wherein, the engineering fungus is obtained by subjecting the P1 and 3CD genes of the CA10 virus to double enzyme digestion with EcoRI and BamHI, then respectively constructing with a vector PMV-05 having been double-enzyme digested by EcoRI and BamHI to obtain a PMV-05-P1 recombinant expression vector and a PMV-05-3CD recombinant expression vector, then respectively subjecting the PMV-05-P1 recombinant expression vector and the PMV-05-3CD recombinant expression vector to enzyme digestion with SacI and XhoI, SacI and SalI, and constructing to obtain a PMV-05-P1-3CD co-expression vector, and finally constructing by introducing the co-expression vector into Hansenula AU-0501; preferably, the engineering fungus is Hansenula polymorpha CA10-W, with an accession number of CGMCC No. 19850.

3. (canceled)

4. A method for preparing CA10 virus-like particles, comprising the following steps: 1) culturing the engineering fungi of claim 1, so as to express CA10 virus P1 protein and 3CD protein in the engineering fungal cells, and self-assemble into immunogenic virus-like particles; and 2) separating and purifying the virus-like particles.

5. The method according to claim 4, wherein ultrafiltration and three-step chromatography are adopted in the step 2) to separate and purify the virus-like particles, and specific steps comprises: A. centrifuging to collect the fungi, breaking the fungi, performing ultrafiltration after the target product is clarified, and collecting the ultrafiltrate; B. ion exchange chromatography; C. hydroxyapatite chromatography; and D. molecular sieve chromatography.

6. The method according to claim 4, wherein the method for breaking the fungi in step A comprises: resuspending the engineering fungi using a cell lysis buffer, and breaking the cells for 2 to 4 times under a pressure of 1100 to 1400 bar, preferably, breaking the engineering fungi twice under a pressure of 1300 bar; the cell lysis buffer comprises 20 to 100 mM of Tris, 1 to 5 mM of EDTA-Na2, 200 to 1000 mM of NaCl, 1 to 5 mM of PMSF, and 0.01% to 1.0% of Tween-80, with a pH of 7.5 to 8.5; the method for clarifying the target product comprises: filtering the cell-broken liquid through a depth filter at a filtration flow rate of 900 to 1850 ml/min/m.sup.2, washing the filter membrane package with a washing solution, and collecting the clarified liquid; the washing solution comprises 20 to 100 mM of Tris, 1 to 5 mM of EDTA-Na2, 200 to 1000 mM of NaCl, and 0.01% to 1.0% of Tween-80, with a pH of 7.5 to 8.5; or centrifuging the cell-broken liquid at 6000 to 8000 rpm for 40 to 60 min, and collecting the clarified liquid; or subjecting the cell-broken liquid to microfiltration through a 0.65 μm membrane package, and collecting the clarified liquid; the ultrafiltration method comprises: subjecting the collected clarified liquid to ultrafiltration with a 100 to 500 KD membrane package using a buffer of pH 7.0 to 8.5, and collecting the ultrafiltrate; the buffer is an aqueous solution comprising 20 to 100 mM of tris(hydroxymethyl)aminomethane, 150 to 300 mM of NaCl and 0 to 10% by weight of glycerol; preferably, the buffer has a pH of 8.0, and comprises 50 mM of tris(hydroxymethyl)aminomethane and 250 mM of NaCl, and the buffer also comprises 5% by mass fraction of glycerol.

7. The method according to claim 6, wherein the step B comprises: performing equilibrium using a buffer with 5 to 10 column volumes, then loading the sample, collecting the eluate having an ultraviolet absorption peak at UV 280 nm, which is a first-step chromatography protein solution; the buffer is an aqueous solution comprising 20 to 100 mM of tris(hydroxymethyl)aminomethane, 150 to 300 mM of NaCl and 0 to 10% by weight of glycerol, and has a pH of 7.5 to 8.5; preferably, an aqueous solution comprising 50 mM of tris(hydroxymethyl)aminomethane, 250 mM of NaCl and 5% by weight of glycerol is used to form a buffer with a pH of 8.0 for equilibrium elution; an anion exchange chromatography column is used for ion exchange chromatography, with a medium of Capto Q.

8. The method according to claim 6, wherein the step C comprises: adding 500 mM of PB solution to the first-step chromatography protein solution to have a final concentration of 10 to 120 mM of PB; using an buffer with a pH of 6.8 to 8.5 formed of an aqueous solution comprising 10 to 120 mM of PBS and 0 to 10% by weight of glycerol to perform equilibrium with 5 to 10 column volumes, then loading the sample, then using an buffer with a pH of 6.8 to 8.5 formed of an aqueous solution comprising 150 to 400 mM of PBS and 0 to 10% by weight of glycerol for elution, and collecting the eluate having an ultraviolet absorption peak at UV 280 nm, which is a second-step chromatography protein solution; preferably, a buffer with a pH of 8.0 comprising 60 mM of PBS and 5% by weight of glycerol is used for equilibrium; preferably, a buffer with a pH of 8.0 comprising 200 mM of PBS and 5% by weight of glycerol is used for elution.

9. The method according to claim 7, wherein the step D comprises: loading the second-step chromatography protein solution onto a molecular sieve chromatography column, eluting using a buffer with a pH of 6.8 to 7.4 containing 100 to 300 mM of NaCl 0.05%0 to 3%0 (w/v) of Tween-80, and 10 to 50 mM of PBS, and collecting the eluate having an ultraviolet absorption peak at UV 280 nm, which is the target protein solution; preferably, 20 mM of PBS, 150 mM of NaCl and 0.1%0 (w/v) of Tween-80 are used to prepare a buffer with a pH of 7.2 for elution; and the medium used for molecular sieve chromatography is Sephacryl S-300HR.

10. A vaccine for hand-foot-and-mouth disease, wherein the vaccine is prepared by subjecting the CA10 virus-like particles prepared according to the method of claim 4 to adsorption with an aluminum hydroxide adjuvant; wherein, the content of CA10 virus-like particles is 5 to 40 μg/ml.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0061] FIG. 1 shows the Western blot detection result of inducibly expressed protein of some strains screened from the recombinant Hansenula engineering strain in Example 1 of the present invention.

[0062] FIG. 2 shows the Western blot detection result of protein in samples of the engineering fungi CA10-W at different time (0, 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, and 44 h) after induction in Example 2 of the present invention. M is a low molecular weight protein standard (Beijing Quanshijin Biotechnology Co., Ltd.).

[0063] FIG. 3 shows the HPLC purity detection result in Example 3 of the present invention.

[0064] FIG. 4 shows a dynamic light scattering spectrum of the purified CA10 virus-like particles in Example 3 of the present invention.

[0065] FIG. 5 shows a transmission electron micrograph of the purified CA10 virus-like particles in Example 3 of the present invention (magnification: 120000).

[0066] FIG. 6 shows the protection effect of immune serum of the CA10 virus-like particle vaccine in Example 7 of the present invention.

SPECIFIC MODES FOR CARRYING OUT THE EMBODIMENTS

[0067] The present invention provides a method for preparing and purifying CA10 virus-like particles, and use thereof in the field of vaccines. The recombinantly expressed Hansenula engineering fungi were cultured by high-density fermentation and induced by methanol to express CA10 virus-like particle proteins. The fungi were collected by centrifugation and subjected to breaking by high-pressure homogenization. The supernatant was subjected to purification such as ultrafiltration, ion exchange chromatography, hydroxyapatite chromatography and molecular sieve chromatography to obtain the CA10 virus-like particles. The CA10 virus-like particles provided by the present invention and the vaccine prepared therefrom have good immunogenicity, safety, immune characteristics and biological activity, and simple process. The purification adopts chromatography methods, which are more favorable to linear amplification compared with density gradient centrifugation, and a large-scale preparation and purification can be performed to obtain a high-purity (greater than 99%) VLP protein stock solution, which can be used to prepare a vaccine for preventing CA10 infection, with good economic value and application prospects.

[0068] The present invention first provides a high-efficiency strain expressing A10 virus-like particles obtained from Hansenula expression system, comprising the following steps:

[0069] (1) Gene sequence optimization and synthesis: The sequence of the yeast secretion signal peptide or the sequence of the transcription termination signal recognized by yeast is deleted from the nucleotide sequence encoding the P1 and 3CD proteins of CA10 virus. The preferred codon of Hansenula was used for sequence optimization and synthesis. The optimized nucleotide sequences of the P1 and 3CD genes of the CA10 virus are represented by SEQ ID NOs: 1 and 2, respectively.

[0070] (2) Construction of recombinant expression vector: The optimized synthesized P1 and 3CD genes were subjected to double enzyme digestion with EcoRI and BamHI, and respectively constructing with a vector PMV-05 (refer to ZL201210592813.5 for the construction method of the vector PMV-05) having been double-enzyme digested by EcoRI/BamHI to obtain a PMV-05-P1 recombinant expression vector and a PMV-05-3CD recombinant expression vector, and then the PMV-05-P1 recombinant expression vector and the PMV-05-3CD recombinant expression vector were subjected to enzyme digestion with SacI/XhoI and SacI/SalI, respectively, and constructing to obtain a PMV-05-P1-3CD co-expression vector.

[0071] (3) Obtaining a CA10 recombinant Hansenula engineering strain: The recombinant co-expression vector PMV-05-P1-3CD was transformed into Hansenula ATCC26012 uracil auxotroph host cell AU-0501 by electroporation (see ZL201210592813.5 for the strain, with an accession number of CGMCC No. 7013), and a CA10 recombinant Hansenula engineering strain was obtained by stabilizing culturing and screening.

[0072] The present invention also provides a purification method of CA10 virus-like particles expressed from the Hansenula expression system, which is more favorable to linear amplification, can realize large-scale preparation and purification, and can obtain a high-purity (greater than 99%) VLP protein (a virus-like particle vaccine) stock solution, which can be used to prepare a vaccine for preventing CA10 infection, with good economic value and application prospects.

[0073] The method for preparing the CA10 virus-like particles comprises the following steps:

[0074] (1) subjecting the recombinant Hansenula engineering fungi containing CA10 coat protein P1 gene and 3CD protease gene to fermentation;

[0075] (2) breaking the engineering fungi, clarifying the target product and performing ultrafiltration;

[0076] (3) ion exchange chromatography;

[0077] (4) hydroxyapatite chromatography; and

[0078] (5) molecular sieve chromatography.

[0079] The above method can be used to obtain a high-purity (greater than 99%) VLP stock solution for preparing a human vaccine. The present invention adopts three-step chromatography, the process is simple, the antigen recovery rate is up to 40% or more, the production process is highly controllable, and can realize large-scale production, with high social value and economic value.

[0080] The following examples are used to illustrate the present invention, but not to limit the scope of the present invention. Unless otherwise specified, the examples are in accordance with conventional experimental conditions, such as conditions suggested by Sambrook J & Russell DW (Molecular Cloning: a Laboratory Manual, 2001), or conditions suggested by the manufacturer's instructions.

[0081] The percentage “%” involved in the present invention refers to mass percentage unless otherwise specified; however, the percentage of a solution, unless otherwise specified, refers to the number of grams of solute contained in 100 mL of the solution. “% o” refers to the number of grams of solute contained in 1000 mL of the solution.

Example 1: Obtaining of CA10 Recombinant Hansenula Engineering Fungi

[0082] According to the nucleotide sequences of the P1 and 3CD proteins of the recently prevalent Coxsackie virus A10 strain, the Vector software was used to optimize the design of the P1 and 3CD gene sequences according to preferred codons of Hansenula to increase the expression level thereof.

[0083] In the optimization process, the sequence of the yeast secretion signal peptide or the sequence of the transcription termination signal recognized by the yeast was deleted. In order to prevent an excessively high GC content of the translated mRNA and the secondary structure of the mRNA from affecting the translation efficiency, and considering the sites of enzyme digestion, the gene sequence at certain positions was adjusted appropriately while ensuring that the amino acid remained unchanged. The optimized nucleotide sequences of the P1 and 3CD genes of the CA10 virus are represented by SEQ ID NOs: 1 and 2, respectively.

[0084] The present invention uses the expression vector PMV-05 to construct the CA10 recombinant expression vector. Specifically, the optimized synthesized P1 and 3CD genes were subjected to double enzyme digestion with EcoRI and BamHI, and respectively constructing with the expression vector PMV-05 having been double-enzyme digested by EcoRI/BamHI to obtain a PMV-05-P1 recombinant expression vector and a PMV-05-3CD recombinant expression vector, and then the PMV-05-P1 recombinant expression vector and the PMV-05-3CD recombinant expression vector were subjected to enzyme digestion with SacI/XhoI and SacI/SalI, respectively, and constructing to obtain a PMV-05-P1-3CD co-expression vector.

[0085] The present invention uses the patented Hansenula uracil auxotroph host strain AU-0501 to screen the CA10 recombinant Hansenula engineering strain, specifically the recombinant co-expression vector PMV-05-P1-3CD was transformed into Hansenula ATCC26012 uracil auxotroph host cell AU-0501 by electroporation, and a CA10 recombinant Hansenula engineering strain was obtained by stabilizing culturing and screening by methods such as PCR, SDS-PAGE and WB.

[0086] The Western blot detection result of induced expression of some strains screened from the CA10 recombinant Hansenula engineering strain is shown in FIG. 1. In FIG. 1, 1: negative control; 2-5: PMV-05-P1 transformed strain; 6-14: PMV-05-P1-3CD transformed strain. It should be noted that 3CD is a protease that can cleave P1 protein to assemble into VLP, and the 3CD protease is removed in subsequent purification. Not the P1 protein in all transformants can be efficiently cleaved by 3CD protease and assembled into VLP.

[0087] The Western blot result shows that: the expression product of the PMV-05-P1-3CD transformed strain can specifically bind to the monoclonal antibody, and there is a relatively obvious reaction band at 33 KD, indicating that a CA10 recombinant Hansenula expression strain (a Hansenula engineering strain that efficiently expresses CA10 virus-like particles) is obtained by screening.

[0088] An engineering strain—Hansenula polymorpha CA10-W that can stably express the target protein at the highest protein expression level was selected therefrom, and sent to the China General Microbiological Culture Collection Center for depositing, the accession number is CGMCC No. 19850, and the depositing date is May 20, 2020.

Example 2: Cultivation of the Recombinant CA10 Yeast Expression Strain by Fermentation in a 30 L Fermenter

[0089] The strain CA10-W was inoculated into 100 ml of a primary seed culture medium (0.67% yeast nitrogen-source medium, 0.5% ammonium sulfate, and 2% glucose), and cultured in a shaker at 33° C. and 200 rpm for 18 to 22 h. The primary seed culture solution was taken and inoculated into 1000 ml of a secondary seed culture medium, and cultivation was performed in a shaker at 33° C. and 200 rpm for 21 to 23 h. The secondary seed culture solution was inoculated into a 30 L fermenter, the pH value of the fermentation broth was adjusted by ammonia water to maintain at 5.0±0.5, the fermentation temperature was 30±1° C., the rotating speed was controlled at 350 to 750 rpm, and the air flow rate was 0.5 to 1.0 m.sup.3/h. High-density fermentation requires supplementation of pure oxygen. Dissolved oxygen was controlled at 20% to 60%. The carbon source in the fermentation medium was exhausted at 16 h to 18 h. A total of glycerol added was 2.0 L, and the total growth period of fungi was about 26 h to 28 h. The weight of wet fungi can reach about 0.3 to 0.4 g/ml. Derepression stage: the stirring speed was set to 750 rpm, the air flow rate was 1.0 m.sup.3/h, the dissolved oxygen was controlled at 20% to 60%, and 1 L of a derepressor solution (the derepressor solution was a mixture of methanol and glycerol, and the volume ratio of glycerol to methanol was 1:4) was added for derepression culturing at 36 h to 41 h (totally 11 h to 13 h) during fermentation. Induction stage: methanol induction was performed at 41 h to 86 h (40 h to 44 h) during fermentation, dissolved oxygen was maintained at about 20% to 40%. The end of fermentation: at 82 h to 86 h, when methanol was consumed completely, the dissolved oxygen rose to 80% or more, the fermentation was finished to discharge after the temperature was lowered to 2° C. to 8° C., and the weight of wet fungi was maintained at 0.26 to 0.30 g/ml.

[0090] Identification of CA10 virus-like particles expressed by Hansenula: The samples were taken at different time (0 h, 4 h, 8 h, 12 h, 16 h, 20 h, 24 h, 28 h, 32 h, 36 h, 40 h, 44 h) after the above induction for protein western blot detection by using rabbit anti-CA10-VP1 polyclonal antibody (the preparation method of the polyclonal antibody was as follows: after purification of CA10-VP1 protein expressed by E. coli, a Freund's adjuvant was added, a rabbit was immunized with the above CA10-VP1 protein for four times, the serum was separated, and purification was performed using Protein A to obtain the polyclonal antibody) as the primary antibody, HRP-goat anti-rabbit-IgG (Sigma company) as the secondary antibody, and DAB color development. The results were shown in FIG. 2.

[0091] Western blot results show that: the expression product can specifically bind to the monoclonal antibody, and there is a relatively obvious reaction band at 33 KD, indicating that the foreign protein can be expressed well in yeast cells.

[0092] Determination of the expression level of recombinant CA10 virus-like particles by fermentation: The rabbit polyclonal antibody was 1000-fold diluted, and 100 μl was added to a 96-well ELISA plate, to be coated overnight at 4° C. The coating solution was removed completely, and a washing solution PBST was filled to wash the ELISA plate. A blocking solution (1% BSA in PBST solution) was filled in the ELISA plate to incubate at 37° C. for 2 h. The supernatant collected by centrifugation after breaking and the reference sample were diluted in gradient series. After the blocking solution was completely removed, 100 μl of the sample to be tested and the reference sample (the initial concentration of the reference sample was 100 ng/ml) were added to each well to incubate at 37° C. for 1 h. A washing solution PBST was filled to wash the ELISA plate for 3 times. 100 μl of a HRP-labeled mouse monoclonal antibody (1:5000 diluted) was added to each well to incubate at 37° C. for 1 h. The enzyme-labeled solution was completely removed, and a washing solution PBST was filled to wash the ELISA plate for 3 times. 100 μl of a TMB color developing solution was added to each well to keep in the dark at 37° C. for 15 min. 50 μl of 2 mol/L H.sub.2SO.sub.4 was added to each well to terminate. The OD.sub.450 nm value was measured with a microplate reader, and the antigen content was calculated by the double parallel line method. The measurement results were shown in Table 1.

TABLE-US-00002 TABLE 1 Test results of antigen content of cell-broken fermentation broth (ELISA method) Dilution folds of the reference sample Standard 1 Standard 2 Standard 3 Standard 4 Standard 5 20 40 80 160 320 OD.sub.450 nm average value 1.756 0.999 0.561 0.284 0.116 Sample dilution 1500 3000 6000 12000 24000 Sample OD.sub.450 nm average value 1.187 0.672 0.422 0.218 0.117 Antigen content of cell-broken fermentation 225 broth (μg/ml) ELISA measurement results show that the antigen content of CA10 virus-like particles in the cell-broken fermentation broth is 225 μg/ml. The CA10 virus-like particles expressed by Hansenula cells have a relatively high expression level.

Example 3: Separation and Purification of CA10 Virus-Like Particles

[0093] Cell collection: The fermentation broth of strain CA10-W was collected, centrifugation was performed at 6500 rpm to collect precipitates, and a cell washing buffer was used to wash the cells twice.

[0094] Breaking: The collected Hansenula cells were resuspended in a cell lysis buffer (50 mM Tris, 2 mM EDTA-Na2, 500 mM NaCl, 2 mM PMSF, 0.05% Tween-80, pH 8.0), and a high-pressure homogenizer was used to break the cells twice under the pressure of 1300 bar, and the cell breakage rate was up to 85% or more.

[0095] Clarification: The cell-broken liquid was subjected to depth filtration, the filter membrane package was washed with a washing solution (50 mM Tris, 2 mM EDTA-Na2, 500 mM NaCl, 0.05% Tween-80, pH 8.0), the filtration flow rate was 1200 ml/min/m.sup.2, and the clarified liquid was collected.

[0096] Ultrafiltration: The collected clarified liquid was subjected to ultrafiltration by a 300 KD membrane package using 50 mM Tris+250 mM NaCl+5% glycerol (pH 8.0) to remove small molecular substances, and the ultrafiltrate was collected to obtain a crude pure product.

[0097] Ion exchange chromatography: with the Capto Q chromatography medium as an example, a buffer of 5 to 10 column volumes comprising 50 mM Tris+250 mM NaCl+5% glycerol (pH 8.0) was used to perform equilibrium, then the sample was loaded, the eluate (i.e., a first-step chromatography protein solution) having an ultraviolet absorption peak at UV 280 nm was collected, and 1 mol/L NaOH was used to regenerate the chromatography medium.

[0098] Hydroxyapatite chromatography: a 500 mM PB solution was added to the first-step chromatography protein solution to have a final concentration of 60 mmol/L PB, a buffer of 5 column volumes comprising 60 mM PBS+5% glycerol solution (pH 8.0) was used to perform equilibrium, then the sample was loaded, equilibrium was performed with another 2 column volumes, 200 mM PBS+5% glycerol solution (pH 8.0) were used for eluting, and the eluate (i.e., a second-step chromatography protein solution) having an ultraviolet absorption peak at UV 280 nm was collected.

[0099] Molecular sieve chromatography: a Sephacryl S-300HR packing material was used, 20 mM PB (pH6.8) and 150 mM NaCl solution and 0.1%0 Tween-80 buffer were used to purify the second-step chromatography protein solution, and the target protein having an ultraviolet absorption peak at UV 280 nm was collected. The purity of the protein solution was determined to be 100% by HPLC. The detection results were shown in FIG. 3. The VLP has a peak corresponding to the retention time of 7.242 min, and the peaks corresponding to the retention time of 9.621 min and 10.183 min are solvent peaks.

[0100] Detection of the target protein concentration (Lowry method): 0 ml, 0.2 ml, 0.4 ml, 0.6 ml, 0.8 ml, and 1.0 ml of a standard protein bovine serum albumin solution (200 μg/ml) were accurately measured, and added in test tubes, respectively, distilled water was added to supplement to 1 ml, and meanwhile, 1 ml of the 2-fold diluted purified protein solution was measured to add in a test tube, 5 ml of an alkaline copper solution and 0.5 ml of a phenol reagent were added, respectively, and in a cuvette, the absorbance value at the wavelength of 650 nm was measured. A standard curve was drawn with the protein content of the standard protein as the abscissa and the absorbance value as the ordinate, and the concentration of the protein solution to be tested was calculated. The test results were shown in Table 2.

TABLE-US-00003 TABLE 2 Test results of purified protein concentration (Lowry method) Standard concentration μg/ml Standard 1 Standard 2 Standard 3 Standard 4 Standard 5 Standard 6 0 40 80 120 160 200 OD650 nm 0.000 0.140 0.270 0.391 0.506 0.622 Sample OD650 nm 0.253 0.247 OD650 nm average value 0.250 Calculated 78.8 Dilution 4 Final 315.2 concentration μg/ml folds concentration of purified protein solution μg/ml

[0101] The test results of Lowry method show that the final concentration of the purified protein is 315.2 μg/ml.

[0102] Dynamic light scattering analysis of the target protein solution: an appropriate amount of the purified CA10 protein solution was taken and added into a sample pool, the temperature was set to 25° C., the equilibrating time was 90 s, the number of automatic cycles was set, the measurement was started, and the results were analyzed. The results show that the recombinant CA10 virus-like particles are intact, with a particle diameter of more than 99% in the distribution of 24 nm to 30 nm, the PDI was 0.08, and the dynamic light scattering spectrum is shown in FIG. 4.

[0103] Electron microscopy analysis of the target protein solution: an appropriate amount of purified CA10 protein solution was taken, dropped on a copper mesh, and stored in the dark for 5 min, excessive liquid was removed, 1% phosphotungstic acid was used for staining for 2 min, and the CA10 VLPs were analyzed by a transmission electron microscope (TEM). The results show that the CA10 protein is in form of a virus-like particle, with the icosahedral structure of a natural virus, the diameter of the virus particle is about 30 nm, and the particles are intact and regular (FIG. 5).

[0104] Determination of the antigen content of the target protein solution (double antibody sandwich ELISA): the rabbit polyclonal antibody was 1000-fold diluted, 100 μl of the sample was taken and added into a 96-well ELISA plate, to be coated overnight at 4° C. The coating solution was removed completely, and a washing solution PBST was filled to wash the ELISA plate. A blocking solution (1% BSA in PBST solution) was filled in the ELISA plate to incubate at 37° C. for 2 h. The blocking solution was removed completely, 100 μl of the sample to be tested and the reference sample (the initial concentration was 100 ng/ml) were added to each well to incubate at 37° C. for 1 h. A washing solution PBST was filled to wash the ELISA plate for 3 times. 100 μl of a HRP-labeled mouse monoclonal antibody (1:5000 diluted) was added to each well to incubate at 37° C. for 1 h. The enzyme-labeled solution was completely removed, and a washing solution PBST was filled to wash the ELISA plate for 3 times. 100 μl of a TMB color developing solution was added to each well to keep in the dark at 37° C. for 15 min. 50 μl of 2 mol/L H.sub.2SO.sub.4 was added to each well to terminate. The OD450 nm value was measured with a microplate reader, and the antigen content was calculated by the double parallel line method. The test results of double antibody sandwich ELISA were shown in Table 3.

TABLE-US-00004 TABLE 3 Test results of double antibody sandwich ELISA Dilution folds of the reference sample Standard 1 Standard 2 Standard 3 Standard 4 Standard 5 20 40 80 160 320 OD.sub.450 nm average value 1.186 0.672 0.422 0.218 0.117 Dilution folds of the sample 500 1000 2000 4000 8000 OD.sub.450 nm average value of 1.137 0.860 0.471 0.234 0.121 the sample Antigen content of the purified protein 310.4 solution (μg/ml)

Example 4: Preparation of a Recombinant CA10 Virus-Like Particle Vaccine

[0105] Main index control: the content of antigen (CA10 virus-like particles) was 20 μg/ml; the content of aluminum was controlled at 0.40 to 0.60 mg/ml; and the pH value was controlled at 6.6 to 7.4.

[0106] Preparation method: The homemade aluminum hydroxide adjuvant was diluted to 0.50 mg/ml with a sterile 0.9% sodium chloride solution. The purified CA10 stock solution was slowly added dropwise to the adjuvant to make it fully adsorbed, and the vaccine preparation was completed. The test standards and test results of the prepared vaccine were shown in Table 4.

TABLE-US-00005 TABLE 4 Test results of the recombinant CA10 virus-like particle vaccine Test items Quality standard Test results Antigen content Not less 20.1 μg/ml than 16.0 μg/ml Aluminum content 0.40 to 0.60 mg/ml 0.49 mg/ml pH value 6.0 to 8.0 6.8 Adsorption rate >95.0% 99.4% Osmolality 300 ± 65 mOsmol/kg 293 mOsmol/kg Fungal endotoxin Less than 5 EU/ml In compliance with the regulation

[0107] The results in Table 4 show that the test indicators of the recombinant CA10 virus-like particle vaccine all meet the test standards.

Example 5: Immunogenicity (ED.SUB.50.) Test of the Recombinant CA10 Virus-Like Particle Vaccine

[0108] Test vaccine: The recombinant CA10 virus particle vaccine containing aluminum hydroxide adjuvant prepared in Example 3.

[0109] Experimental animals: 50 SPF-grade NIH mice of 18 to 22 g were selected and purchased from China Food and Drug Research Institute.

[0110] Animal immunization: 10 mice were injected with proportionally diluted vaccines of 2 μg/0.5 ml, 0.5 μg/0.5 ml, 0.125 μg/0.5 ml, and 0.03125 μg/0.5 ml and aluminum hydroxide adjuvant, respectively, each with 0.5 ml intraperitoneal injection, and 28 days after immunization, eyeballs were taken and blood was collected. The collected blood was kept at 37° C. for 1 h and 4° C. for 3 to 4 h, centrifugation was performed at 4000 rpm for 10 min, and the supernatant was pipetted for detection.

[0111] Neutralizing antibody detection: The serum sample was diluted with MEM culture solution of 2% newborn calf serum at a ratio of 1:8, and inactivation was performed in a water bath at 56° C. for 30 min. A 96-well cell culture plate was used, and 50 μl of a diluting solution was added to each well. The corresponding sample of 50 μl was added to each well, after being mixed well, a multichannel pipette was used to suck 50 μl from row A to row B to mix well, mixing and diluting were performed in sequence till row D, and 50 μl was discarded. The CA10 virus attack strain was serially diluted to 100 CCID.sub.50/0.05 ml, 50 μl was taken and dropped vertically into each well, the cell culture plate was tapped gently to mix well, and neutralizing was performed at 37° C. for 2 h. The RD cells (the cells were resuscitated and expanded in advance) were digested with a digestive solution to prepare a cell suspension with a concentration of 2×10.sup.5 cells/ml, 0.1 ml of the cell suspension was added to each well (including the virus back titration well) to mix well, and incubation and culture was performed in a CO.sub.2 incubator at 35° C. An inverted microscope was used to observe the CPE every day, and the virus titration results were recorded, with the reciprocal of the highest dilution of serum to inhibit 50% cytopathy as the endpoint titer. The final result was determined within 6 to 7 days. According to the test results, the antibody positive conversion rate was shown in Table 5.

TABLE-US-00006 TABLE 5 Calculation results of the antibody positive conversion rate Cumulative total Positive Positive Mouse antibody conversion conversion Dilution Protein content Positive Negative rate Positive Negative rate 1:5      2 μg/0.5 ml 10 0 100%  19 0 100%  1:20   0.5 μg/0.5 ml 7 3 50% 9 3 75% 1:80  0.125 μg/0.5 ml 2 8 10% 2 11 15%  1:320 0.03125 μg/0.5 ml 0 10  0% 0 21  0% Calculation according to Reed-Muench method: ED.sub.50 = 0.28 (μg)

[0112] According to the results of the mouse ED.sub.50 experiment, the CA10 virus-like particle vaccine prepared by the present invention can make the antibody positive conversion rate reach 50% after immunizing the mice with only 0.28 μg. Therefore, the CA10 virus-like particle of the present invention has strong immunogenicity.

Example 6: Abnormal Toxicity Test of the Recombinant CA10 Virus-Like Particle Vaccine

[0113] Vaccine sample: the recombinant CA10 virus-like particle vaccine prepared in Example 3.

[0114] Experimental animals: 10 SPF-grade KM mice of 18 to 22 g, and 4 SPF-grade Hartley guinea pigs of 250 to 350 g, all purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.

[0115] Experimental method: The weight of each experimental animal was weighed before injection, the mouse was 18 to 22 g, and the guinea pig was 250 to 350 g. The vaccine was injected into 5 mice and 2 guinea pigs, the mice were intraperitoneally injected with 0.5 ml/mouse, the guinea pigs were intraperitoneally injected with 5.0 ml/guinea pig, and observation was performed for 7 days. At the same time, a blank control of the same batch of animals was set up. Qualification criteria: During the observation period, the animals in the blank control and the experimental groups survived without abnormal reaction, and each animal gained weight in due time. The animal test results were shown in Table 6.

TABLE-US-00007 TABLE 6 Animal experiment results Initial weight Final weight Animal species Number (g) (g) Mouse experimental group 1 20.2 29.8 2 19.3 28.7 3 20.0 31.0 4 21.0 30.2 5 21.2 29.9 Mouse control group 1 19.5 31.3 2 21.4 32.2 3 21.0 29.6 4 20.5 28.7 5 21.5 30.4 Guinea pig experimental 1 300.2 412.3 group 2 322.2 412.9 Guinea pig control group 1 286.5 389.6 2 310.2 401.3

[0116] The results show that the animals in the blank control and experimental groups survived during the observation period without abnormal reaction, and the weight of each animal increased on the 8th day. It is proved that the recombinant CA10 virus-like particle vaccine has no abnormal toxicity and good safety to the experimental animals.

Example 7: Study on the Immune Serum Protective Effect of the CA10 Virus-Like Particle Vaccine

[0117] Test serum: immune serum obtained from the mice immunized with the CA10 virus-like particle vaccine prepared in Example 3.

[0118] Experimental animals: 5 litters of 1-day-old Blab/c suckling mice, SPF grade, purchased from China Institute for Food and Drug Control.

[0119] Experimental method: 50 μl of virus (CA10 virus, strain source: FZ-2014 KY012321, virus titer: 8.5 lgCCID.sub.50/ml) was used for intraperitoneally attacking, respectively, and within 1 hour each litter of suckling mice were simultaneously immunized intraperitoneally with 50 μl immune serum having neutralizing antibody titers of 341.33U, 56.89U, 9.48U, and 1.58U obtained from mice immunized with the CA10 virus-like particle vaccine and negative control (virus culture solution), respectively. The morbidity and mortality of the suckling mice were recorded every day for 21 consecutive days, and the morbidity and mortality of the suckling mice in each group were counted at the end of the observation period.

[0120] The test results were shown in FIG. 6. The suckling mice in the control group all died and the test was successful. The vaccine groups all show different protective effects. According to the immune serum titers of 341.33U, 56.89U, 9.48U, and 1.58U, the protection rates of the recombinant CA10 virus-like particle vaccine were 100%, 100%, 100%, and 50%, respectively. According to the Reed-Muench method, the average half animal protection neutralizing antibody titer (ED.sub.50) was calculated to be less than 1.58U. The recombinant CA10 virus-like particle vaccine has a good immune protection effect in 1-day-old suckling mice.

[0121] Although the present invention has been described in detail above with general descriptions and specific embodiments, some modifications or improvements can be made on the basis of the present invention, which is obvious to a person skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention belong to the scope of the present invention.

INDUSTRIAL APPLICABILITY

[0122] The present invention provides a Hansenula engineering fungus that efficiently expresses CA10 virus-like particles and use thereof. The engineering fungus comprises a recombinant vector carrying P1 and 3CD genes of the CA10 virus, and the starting strain of the engineering fungus is uracil auxotroph Hansenula AU-0501. P1 and 3CD genes are optimized according to preferred codons of Hansenula. The present invention also provides a preparation method of CA10 virus-like particles, comprising: culturing engineering fungi, expressing CA10 virus-like particles, and separating and purifying virus-like particles by ultrafiltration and three-step chromatography. The CA10 virus-like particles provided by the present invention and the vaccine prepared therefrom have good immunogenicity, safety and biological activity, the process is simple, the chromatography method is adopted for purification, and large-scale preparation and purification can be realized to obtain a VLP protein stock solution with high purity (greater than 99%), which can be used to prepare a vaccine to prevent CA10 infection, with good economic value and application prospects.