EV71 virus-like particles and preparation method and application thereof
09782471 · 2017-10-10
Assignee
Inventors
- Meirong Gu (Beijing, CN)
- Wenjin Wei (Beijing, CN)
- Jiankai Liu (Beijing, CN)
- Linlin Song (Beijing, CN)
- Shanshan Xu (Beijing, CN)
- Guoshun Li (Beijing, CN)
- Lin Guo (Beijing, CN)
Cpc classification
C12N2770/32351
CHEMISTRY; METALLURGY
C12N7/00
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention provides EV71 virus-like particles and a preparation method and application thereof. The method comprises: connecting a P1 protein gene and a 3CD protease gene of an EV71 virus with a PMV plasmid to construct a PMV-P1-3CD recombinant expression plasmid; then transforming a Hansenula polymorpha AU-0501 expression strain with the PMV-P1-3CD recombinant expression plasmid to obtain an AU-PMV-P1-3CD recombinant expression strain; fermenting and culturing the recombinant expression strain, and inducing the recombinant expression strain to express the EV71 virus-like particle protein with methanol; centrifuging and collecting mycelia for homogeneous breakage at a high pressure; and purifying the supernatant through ion-exchange chromatography, hydrophobic chromatography, and molecular sieve chromatography, so as to obtain EV71 virus-like particles.
Claims
1. A recombinant expression vector, comprising P1 gene and 3CD gene of EV71 virus, wherein the expression vector is PMV-P1-3CD, and wherein the P1 gene of the EV71 virus has a nucleotide sequence as shown in SEQ ID No. 1 or the 3CD gene of the EV71 virus has a nucleotide sequence as shown in SEQ ID No. 3.
2. A host cell comprising the recombinant expression vector according to claim 1.
3. The recombinant expression vector according to claim 1, wherein the P1 gene of the EV71 virus has a nucleotide sequence as shown in SEQ ID No. 1.
4. A host cell comprising the recombinant expression vector according to claim 3.
5. The recombinant expression vector according to claim 1, wherein the 3CD gene of the EV71 virus has a nucleotide sequence as shown in SEQ ID No. 3.
6. A host cell comprising the recombinant expression vector according to claim 5.
7. The recombinant expression vector according to claim 1, comprising a methanol oxidase promoter or a formaldehyde dehydrogenase promoter.
8. A host cell comprising the recombinant expression vector according to claim 7.
9. A method of preparing a compostion, comprising: transforming an expression strain with the recombinant expression vector according to claim 1 to obtain a recombinant expression strain; fermenting and culturing the recombinant expression strain; isolating and purifying EV71 virus-like particles; and absorbing the EV71 virus-like particles with an aluminum phosphate adjuvant to prepare the EV71 viral vaccine.
10. The method of claim 9, wherein the recombinant expression strain is AU-PMV-P1-3CD.
11. The method of claim 9, wherein the expression strain is Hansenula polymorpha AU-0501.
12. EV71 virus-like particles, wherein the EV71 virus-like particles are prepared by the steps of: (1) transforming a Hansenula polymorpha AU-0501 expression strain with the PMV-P1-3CD recombinant expression vector to obtain an AU-PMV-P1-3CD recombinant expression strain; (2) fermenting and culturing the recombinant expression strain; (3) isolating and purifying the EV71 virus-like particles, further comprising centrifuging and collecting mycelia for homogeneous breakage, clarifying, ultrafiltering, and precipitating the virus particles, ultracentrifuging, and collecting the supernatant, purifying the supernatant through ion-exchange chromatography, hydrophobic chromatography or molecular sieve chromatography, wherein the PMV-P1-3CD recombinant expression vector comprises a P1 gene of the EV71 virus having a nucleotide sequence as shown in SEQ ID No. 1 or a 3CD gene of the EV71 virus having a nucleotide sequence as shown in SEQ ID No. 3.
13. A hand, foot and mouth disease vaccine, comprising virus-like particles prepared from the process of claim 12.
14. A method for preparing the vaccine according to claim 13, further comprising absorbing the EV71 virus-like particles with an aluminum phosphate adjuvant to prepare a vaccine comprising 20 μg/ml virus-like particles.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
(14) The following Examples are used for illustrating the present invention, but not for limiting the scope of the present invention. Unless otherwise indicated, all Examples are carried out according to the conventional experiment conditions, such as Sambrook J & Russell D W, Molecular cloning: a laboratory manual, 2001, or according to the conditions recommended by the manufacturer's instructions.
Example 1
Optimization of Gene Sequences Encoding P1 and 3CD Proteins of EV71
(15) According to the nucleotide sequences of P1 and 3CD proteins of C4a subtype of EV71 epidemic in China, the gene sequences of P1 and 3CD proteins were optimized with the most biased codons of Hansenula polymorpha using vector software to increase the expression level of the P1 and 3CD genes in Hansenula polymorpha cells. The nucleotide sequences of genes encoding P1 and 3CD proteins provided in the present invention were deleted the sequences encoding the secretion signal peptide and the transcription termination signal recognized by the yeast. Codons of genes encoding the P1 and 3CD proteins used in the present invention are the most biased codons of Hansenula polymorpha. For the codon usage frequency of Hansenula polymorpha (Pichia angusta), please refer to kazusa.or.jp/codon/. In order to avoid the translated mRNA having higher content of GC and the secondary structure of mRNA affecting the efficiency of translation, secondary biased codons are used for some amino acids in the present invention, provided that the usage frequency of the secondary biased codon is very close to that of the most biased codon, and the order of the original amino acid sequence remained unchanged. In certain very special cases, the order of the sequences at certain positions may be appropriately adjust in order to decrease or increase the enzyme digested sites. Therefore, the present invention optimally designed a P1 gene, the sequence of P1 gene is shown in SEQ ID NO. 1 and the gene P1 sequence is synthesized by Shanghai Generay Biotech Co., Ltd and cloned onto the Dev-C vector (purchased from Shanghai Generay Biotech Co., Ltd), and the amino acid sequence of the P1 protein is shown in SEQ ID NO. 2; 3CD gene was optimally designed in the present invention, the sequence of 3CD gene is shown in SEQ ID NO. 3 and the gene 3CD sequence is synthesized by Shanghai Generay Biotech Co., Ltd, and cloned onto the Dev-C vector (purchased from Shanghai Generay Biotech Co., Ltd). The amino acid sequence of the 3CD protein is shown in SEQ ID NO. 4.
Example 2
Construction of the Recombinant Expression Vector PMV-P1-3CD
(16) 1. Construction of Expression Vector PMV-05:
(17) The expression vector PMV-05 of the present invention consists of 6 parts: promoter (MOX-P), terminator (MOX-T), replicon HARS, ura3 gene, ColE1 replicon, and Amp resistance gene.
(18) In order to obtain the genes MOXP, MOXT, HARS and Ura3, PCR amplification was performed using a yeast genomic DNA as a template and using MOXP-F (sequence shown in SEQ ID NO. 5), MOXP-R (shown in SEQ ID NO. 6); MOXT-F (shown in SEQ ID NO. 7), MOXT-R (shown in SEQ ID NO. 8); HARS-F (shown in SEQ ID NO. 9), HARS-R (shown in SEQ ID NO. 10); Ura3-F (shown in SEQ ID NO. 11) and Ura3-R (shown in SEQ ID NO. 12) as primers respectively. In order to obtain the gene Amp+ColE1, PCR amplification was performed using a PBR-SK plasmid (purchased from Takara Biotechnology (Dalian) Co., Ltd., Article No.: D3050) as a template and using primers Amp+ColE1-F (as shown in SEQ ID NO. 13), Amp+ColE1-R (as shown in SEQ ID NO. 14). PCR reaction system was as follows: 10 μl of PCR buffer, 10 μl of dNTP, 1 μl of primer F, 1 μl of primer R, 1 μl of Taq DNA polymerase, 157 μl of distilled water, 200 μl of total volume. Reaction conditions were as follows: 95° C. for 2 min; 95° C. for 30 s, 55° C. for 30 s, 72° C. for 2 min; performing 30 cycles. The PCR amplified DNA fragments were detected by 1% agarose gel electrophoresis, the sizes of PCR products were as follows: MOXP: around 1500 bp, MOXT: around 300 bp, HARS: around 500 bp, Ura3: around 1100 bp, Amp+ColE1: around 2200 bp respectively. The above 5 gene fragments were purified with DNA fragment purification kit. PCR amplification was performed using 1 μl of each of the purified MOXP, MOXT gene fragments respectively as templates, and using MOXP-F and MOXT-R as primers to obtain MOXP+MOXT gene fragments, and the reaction system and reaction conditions were the same as above. PCR amplification was performed using 1 μl of each of the purified HARS, Ura3, Amp+ColE1 gene fragments respectively as templates and using HARS-R and Amp+ColE1-R as primers to obtain HARS+Ura3+Amp+ColE1 gene fragments, PCR reaction system was as follows: 10 μl of PCR buffer, 10 μl of dNTP, 1 μl of primer F, 1 μl of primer R, 1 μl of Taq DNA polymerase, 155 μl of distilled water, 200 μl of total volume. Reaction conditions were as follows: 95° C. for 2 min; 95° C. for 30 s, 55° C. for 30 s, 72° C. for 4 min; performing 30 cycles. The gene fragments MOXP+MOXT, HARS+Ura3+Amp+ColE1 were purified with a DNA fragment purification kit. The purified gene fragments MOXP+MOXT and HARS+Ura3+Amp+ColE1 were digested with double enzymes (SacI, SalI) respectively, and the digestion reaction was as follows: 60 μl of gene fragment, 3 μl of each of SacI and SalI, 10 μl of 10×Basal butter, 10 μl of 10×BSA, adding water to 100 μl. The enzyme digestion was performed at 37° C. overnight. The product obtained by the enzyme digestion was separated by agarose gel electrophoresis, and then gene fragments produced by the enzyme digestion were recovered by excising the gel, and ligated using solution I ligation kit (purchased from Takara Biotechnology (Dalian) Co., Ltd.). The ligation reaction system was as follows: 1 μl (SacI, SalI) double enzyme digested and purified products of HARS+Ura3+Amp+ColE1, 4 μl (SacI, SalI) double enzymes digested and purified products of MOXP+MOXT, 5 μl of solution I, a total 10 μl of reaction system, and the ligation reaction was performed for 1 hour at 16° C. The ligated recombinant expression vectors were transformed into a 100 μl of E. coli (DH5α) competent cells, plated on LB+Amp (100 μg/ml) solid medium, and cultured in an incubator at 37° C. overnight.
(19) Screen of transformed clones: in order to identify transformed clones, PCR was performed by picking a monoclonal colony on a LB+Amp solid medium as a template and using MOXP-F, MOXP-R, Ura3-F, Ura3-R as primers respectively, the PCR reaction system was as follows: 2 μl of PCR buffer, 2 μl of dNTP, 0.1 μl of primer F, 0.1 μl of primer R, 0.1 μl of Taq DNA polymerase, 15.7 μl of distilled water, and a total volume of 20 μl. Reaction conditions were follows: 95° C. for 2 min; 95° C. for 30 s, 55° C. for 30 s, 72° C. for 1 min; and performing 30 cycles. The PCR amplified DNA fragments were detected by 1% agarose gel electrophoresis, and the size of the PCR product should be around 1500 bp for MOXP-F and MOXP-R as primers, and around 1000 bp for Ura3-F and Ura3-R as primers. The correct transformed clone 5 identified by PCR was named as recombinant expression plasmid PMV-05, inoculated in 10 ml LB+Amp liquid medium, and cultured at 37° C. in a shaker at 200 rpm overnight. The recombinant expression plasmid PMV-05 was extracted according to the instructions of the plasmid extraction kit (from Beijing TransGen Biotech Co., Ltd.), and the extracted recombinant expression vector was detected by 1% agarose electrophoresis. The obtained expression vector was identified by enzyme digestion. The enzyme digestion reaction volume was as follows: 5 μl of plasmid, 1 μl of each of SacI and SalI, 2 μl of 10×Basal buffer and 2 μl of 10×BSA buffer and adding water to 20 μl. The enzyme digestion was performed for 4 hours at 37° C. The results show that the recombinant expression vector PMV-05 was constructed successfully.
(20) 2. Construction of Recombinant Expression Vectors PMV-P1 and PMV-3CD
(21) The genes P1 and 3CD which were synthesized optimally in Example 1 were cloned into a Dev-C vector. EcoRI and BamHI digestion sites were added to both ends of the gene sequence while optimally synthesizing the genes P1 and 3CD, so that the genes can be cloned into an expression vector.
(22) Dev-C vector and the expression vector (PMV-05) into which P1 and 3CD genes have been cloned were subjected to double enzyme digestion with EcoRI and BamHI (both were purchased from Takara Biotechnology (Dalian) Co., Ltd.). The enzyme digestion reaction system was as follows: 30 μl of plasmid, 3 μl of each of EcoRI and BamHI, 10 μl of 10×K buffer buffer and adding water to 100 μl. The enzyme digestion was performed at 37° C. overnight. After isolation by agarose gel electrophoresis, the product obtained by the enzyme digestion, the gene fragment and linear expression vector were recovered by excising the gel, and ligated via Solution I ligation kit (purchased from Takara Biotechnology (Dalian) Co., Ltd.). The ligation reaction system was as follows: 1 μl of double enzyme digested and purified product of the vector PMV-05 (EcoRI/BamHI), 5 μl of double enzyme digested and purified products of each of P1 and 3CD genes (EcoRI/BamHI), 6 μl of Solution I and a total 12 μl of reaction system. The ligation reaction was performed for 1 hour at 16° C. The ligated recombinant expression vectors were transformed into 100 μl of E. coli (DH5α) competent cells, plated on LB+Amp (100 μg/ml) solid medium and cultured in an incubator at 37° C. overnight.
(23) Screen of transformed clones: the transformed clones were identified by PCR by picking a monoclonal colony on a LB+Amp solid medium as a template. The primer sequences of P1 gene were as follows: primer SEQ ID NO. 15, and primer SEQ ID NO. 16; the primer sequences of 3CD gene were as follows: primer SEQ ID NO. 17, and primer SEQ ID NO. 18 PCR reaction system was as follows: 2 μl of PCR buffer, 2 μl of dNTP, 0.1 μl of primer F, 0.1 μl of primer R, 0.1 μl of Taq DNA polymerase, 15.7 μl of distilled water, and a total volume of 20 μl. Reaction conditions were as follows: 95° C. for 2 min; 95° C. for 30s, 55° C. for 30s, 72° C. for 1 min; performing 30 cycles. The PCR amplified DNA fragments were detected by 1% agarose gel electrophoresis, and the size of the PCR product of P1 gene was about 1000 bp, and the size of the PCR product of 3CD gene was about 630 bp. Results of PCR electrophoresis are shown in
(24) 3. Construction of Recombinant Expression Vector PMV-P1-3CD
(25) The recombinant expression vector PMV-P1 was digested with double enzymes SacI and XhoI (both are purchased from Takara Biotechnology (Dalian) Co., Ltd.) and the recombinant expression vector PMV-3CD was digested with double enzymes SacI and SalI (both are purchased from Takara Biotechnology (Dalian) Co., Ltd.). The enzyme digestion reaction volume was as follows: 50 μl of plasmid, 5 μl of each of SalI/XhoI and SacI, 10 μl of 10×K buffer and adding water to 100 μl. The enzyme digestion was performed at 37° C. overnight. After isolation by agarose gel electrophoresis, the products obtained by the enzyme digestion, 3CD gene fragment and PMV-P1 linear vector fragment were recovered by excising the gel, and ligated via Solution I ligation kit (purchased from Takara Biotechnology (Dalian) Co., Ltd.). The ligation reaction system was as follows: 1 μl of double enzyme digested and purified product of vector PMV-P1(SacI/XhoI), 5 μl of double enzyme digested and purified product of 3CD gene (SacI/SalI), 6 μl of Solution I and a total 12 μl of reaction system, and the ligation was performed for 1 hour at 16° C. The ligated recombinant expression vectors were transformed into 100 μl of E. coli (DH5α) competent cells, plated on LB+Amp (100 μg/ml) solid medium and cultured in an incubator at 37° C. overnight.
(26) Screen of transformed clones: the transformed clones were identified by PCR by picking monoclonal colonies on a LB+Amp solid medium as a template. The primer sequences of P1 gene were as follows: primer F SEQ ID NO. 15, and primer SEQ ID NO. 16; the primer sequences of 3CD gene were as follows: primer SEQ ID NO. 17, and primer SEQ ID NO. 18. PCR reaction system was as follows: 2 μl of PCR buffer, 2 μl of dNTP, 0.1 μl of primer F, 0.1 μl of primer R, 0.1 μl of Taq DNA polymerase, 15.7 μl of distilled water, and a total volume of 20 μl. Reaction conditions were as follows: 95° C. for 2 min; 95° C. for 30s, 55° C. for 30s, 72° C. for 1 min; performing 30 cycles. The PCR amplified DNA fragments were detected by 1% agarose gel electrophoresis, and the size of the PCR product of P1 gene was about 1000 bp, and the size of the PCR product of 3CD gene was about 630 bp. Results of PCR electrophoresis were shown in
Example 3
Inducible Expression and Detection of EV71 Strain Expressed by Hansenula polymorpha
(27) A Hansenula polymorpha ATCC26012 uracil defective host cell AU-0501 (wildtype host stain was from ATCC26012, and the ATCC26012 uracil defective host cell was obtained by method of gene knockout), with an Accession No. of CGMCC No. 7013, was transformed with the recombinant expression vector PMV-P1-3CD by electroporation.
(28) The transformed clone was screened and identified by selective medium and method of PCR, and the transformed clones containing both P1 and 3CD genes which had been identified were subcultured in the selective medium to obtain a strain AU-PMV-P1-3CD co-expressing P1 protein and 3CD protein. The strain was inoculated in the selective medium MDL (0.67% yeast medium with nitrogen source, purchased from SIGMA-ALDRICH CO. LLC., specification: Y1251-KG, batch no. 030M1754), 0.5% ammonium sulfate, 2% glucose), cultured for 20 hours in a shaker at 33° C. and centrifuged at 5000 rpm for 6 min. The precipitate was collected, added into a induction medium MM (0.67% yeast medium with nitrogen source, 0.5% ammonium sulfate, 1% methanol) with supplementing 1% methanol every day, for inducing expression for 3 days.
(29) SDS-PAGE gel electrophoresis analysis: 100 μl of induced samples were taken and centrifuged, the precipitate was collected, washed with sterile water, treated with NaOH for 3 min and centrifuged. The supernatant was discarded, the precipitate was resuspended in 100 μl SDS sample butter, boiled for 10 min, and centrifuged. 10 μl of supernatant was taken and loaded for electrophoresis. After electrophoresis, the gel was taken down for silver staining and scanned with a gel imaging system software for analysis of the expression level of the target protein.
(30) The results detected by SDS-PAGE show that (see
(31) Detection by Western-blot: an anti-EV71-VP1 antibody (AbMax Biotechnology (Beijing) Co., LTD, 22A12) was used as a primary antibody, and an HRP-goat anti-mouse-IgG (Beijing Bioss Biotechnology Co., LTD) was used as a secondary antibody, DAB was used for development.
(32) The results of Western-blot show that (see
Example 4
Fermentation and Culture of the Recombinant EV71 Yeast Expression Strain in a 30 L Fermentation Tank
(33) The recombinant Hansenula polymorpha strain was inoculated in a 150 ml first-grade seed medium (0.67% yeast medium with nitrogen source (purchased from SIGMA-ALDRICH CO. LLC., specification Y1251-KG, batch No.: 030M1754), 0.5% ammonium sulfate, 2% glucose), and cultured with shaking at 200 rpm for 20 hours in a shaker at 33° C. Then all the culture was inoculated in a 1500 ml second-grade seed medium (0.67% yeast medium with nitrogen source, 0.5% ammonium sulfate, 2% glycerol), and cultured with shaking at 200 rpm for 20 hours in a shaker at 33° C. (OD.sub.600nm up to 8-10). Then all the culture was inoculated in a 30 L fermentation tank which contained 15 L fermentation medium (glycerol, ammonium dihydrogen phosphate, potassium chloride, calcium chloride, sodium chloride, magnesium sulfate, sodium ethylene diamine tetracetate, with a mass ratio of 140:70:20:15:2:1). The pH of the fermentation fluid was adjusted by supplementing ammonia water to maintain at 5.0. The fermentation temperature was 30° C., the rotate speed was controlled at 350-750 rpm, the air velocity was 0.5-1.0 m.sup.3/h, high density fermentation needs to supplement with pure oxygen, and the dissolved oxygen was controlled at 20-60%. At 20 h, the carbon source in the fermentation medium was depleted and a total 2.0 L of glycerol was added in 5 times with 0.40 L per time. Whenever the carbon source was depleted and the dissolved oxygen increased, glycerol was added. The mycelium grows for a total of about 36 h, and the weight of the wet mycelia was up to about 0.3-0.4 g/ml. At the stage of derepression: rotate speed was 750 rpm, air velocity was 1.0 m.sup.3/h, and the dissolved oxygen was controlled at 20-60%, 1 L of mixed solution of glycerol and methanol (400 ml of glycerol, 600 ml of methanol) was added to perform derepression culture at 36-54 h (a total of 16-18 h). At the stage of induction: the methanol induction was carried out at 54-94 h (36-40 h), and the dissolved oxygen was controlled at about 40%. At the end of fermentation: at 92-94 h, when the methanol was consumed completely, the dissolved oxygen was increased to more than 80% and the temperature was cooled to 20° C., the fermentation tank was removed to finish the fermentation, and the weight of the wet mycelia was in the range of 0.3-0.4 g/ml.
(34) Identification of the EV71 VLPs expressed in Hansenula polymorpha: the above samples at different fermentation time (44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96 hours) were taken and used for detection by Western blot using the EV71-VP1 monoclonal antibody (AbMax Biotechnology (Beijing) Co., LTD, 22A12) as a primary antibody and using a HRP-goat anti-mouse-IgG (Bioss Biotechnology Co., LTD) as a secondary antibody, with development with DAB, and the results are shown as
(35) The results of the Western-blot show that the expression product can specifically bind to the monoclonal antibody, and had a more clear reaction band at 32.7 KD, indicating that the expression product has good immune reactivity.
(36) Determination of the expression level of fermentation of the recombinant EV71 VLPs:
(37) The supernatant collected by centrifugation after cell breakage was diluted in a series of 10 times of dilution, and the expression level of the recombinant EV71 VLP was determined via a EV71 antigen ELISA detection kit (AbMax Biotechnology (Beijing) Co., LTD, this kit mainly detects the EV71-VP1 protein, Lot#100408) using the purified product of EV71-VP1 expressed in a prokaryotic system as standard, and the result was shown in Table 1.
(38) TABLE-US-00001 TABLE 1 Detection result of the content of antigen in the fermented and broken liquid (ELISA method) Concentration of standard ng/ml Standard 1 Standard 2 Standard 3 Standard 4 Standard 5 200 100 50 25 12.5 OD.sub.450 nm 2.491 1.295 0.713 0.314 0.234 OD.sub.450 nm of 0.509 0.502 average of OD.sub.450 nm 0.5055 sample Calculated 36.02 Dilution 10.sup.4 Concen- 360 concen- factor tration tration of sample ng/ml μg/ml
(39) The results detected by ELISA show that: the expression level of the EV71 VLPs in the fermented and broken cell liquid was 360 mg/L. EV71 expressed by Hansenula polymorpha cell had high expression level.
Example 5
Isolation and Purification of EV71 VLPs
(40) Cell breakage: the Hansenula polymorpha cells obtained by fermentation in Example 4 were resuspended in the cell lysis buffer (20 mmol/L NaH.sub.2PO.sub.4, 2 mmol/L EDTA-Na.sub.z, 0.4 mol/L NaCl, pH 7.5) for washing for 2 times, and then suspended in a cell lysis buffer containing 2 mmol/L PMSF, 1% Tween-20, 3% PEG 6000 in a ratio of 1:4 (w/v), and broken twice at a pressure of 1100 bar with a high pressure homogenizer, so as to make the cell broken rate reach more than 95%.
(41) Clarification: the broken cell lysate was poured into a centrifuge bowl, centrifuged for 20 min at 8000 rpm, and the supernatant was collected and microfiltered through a 0.45 μm membrane to remove the macromolecular substances.
(42) Ultrafiltration: the clarified protein solution was ultrafiltered through a 50 KD membrane to remove the small molecule substances.
(43) Precipitation of the virus particles: the ultrafiltered protein solution was adjusted with 0.5M NaOH to pH 6.5, 5M NaCl solution was added into the solution, and 0.5 g/ml PEG 6000 solution was added to the solution in drop-wise under stirring to a final concentration of 0.12 g/ml. The resultant solution stood for 2 h at 4° C., was centrifuged at 12000 rpm for 30 min at 4° C., the supernatant was discarded, and the precipitate was dissolved with proper volume of 20 mmol/L PB and re-centrifuged at 5000 rpm for 30 min at 4° C., and the supernatant obtained by the second centrifugation was collected to obtain the crude pure protein solution.
(44) Ultra-centrifugation: solid potassium bromide is added into the crude pure protein solution to adjust the density to 1.25 g/ml. The order for loading samples for ultracentrifugation was: 150 ml of NaCl-EDTA; 300 ml of 1.25 KBr sample solution; 700 ml of 1.30 KBr gradient solution; 540 ml of 1.35 KBr gradient solution. Ultracentrifugation was performed at 25000 rpm for 4 h at 8° C. 1.35 KBr solution was used as the upper solution to collect the solutions having ultraviolet absorption peaks at UV 280 nm in individual tubes, and the solution in each tube was sampled for detection by SDS-PAGE, and the detection results were shown as
(45) Molecular sieve chromatography: taking Sephacryl S-500HR as an example, elution was performed with PBS (pH7.0), the eluent having ultraviolet absorption peaks at UV 280 nm were collected to obtain the purified protein solution.
(46) Detection of the concentration of purified proteins (Lowry method): 0 ml, 0.2 ml, 0.4 ml, 0.6 ml, 0.8 ml, 1.0 ml of the standard protein, viz., bovine serum albumin solution (100 μg/ml) were accurately weighted and placed in test tubes respectively, distilled water was added to the tests to 1 ml, meanwhile 1 ml of 4-fold diluted purified protein solution was added into the test tubes, and 5 ml alkaline copper solution and 0.5 ml phenol reagent were added into the test tubes respectively, for determining the absorbance value at 650 nm of wave length in cuvettes. The standard curve was plotted using the protein content of the standard protein as abscissa and the absorbance value as coordinate, and the concentration of the purified protein to be detected was calculated. The detection results were shown as Table 2.
(47) TABLE-US-00002 TABLE 2 Detection results of the concentration of the purified protein (Lowry method) Concentration of standard μg/ml Standard 1 Standard 2 Standard 3 Standard 4 Standard 5 20 40 60 80 100 OD.sub.650 nm 0.052 0.118 0.176 0.237 0.285 OD.sub.650 nm of 0.150 0.147 average of OD.sub.650 nm 0.1485 sample Calculated 51.86 final concentration of 207 concen- the purified protein tration solution μg/ml μg/ml
(48) The final concentration of the purified protein obtained according to the detection results of Lowry method was 207 μg/ml.
(49) Electronic microscope analysis for the purified protein: 10 ul of purified EV71 protein solution was added drop-wise onto the copper grid, kept away from light for 5 min, the extra liquid was removed completely. Then the purified EV71 protein solution was stained with 1% phosphotungstic acid for 2 min and the EV71 VLPs were analyzed through a transmission electron microscope (TEM). The results indicate that the EV71 protein appeared as a VLP, had a icosahedron structure of the naturally occurring virus with a particle size of around 30 nm, and the particle was integral and regular (as shown in
Example 6
Preparation of EV71 VLPs Vaccine by Aluminium Phosphate Adsorption In Situ Method
(50) Control of the main indexes: the content of the antigen (EV71 VLP) was 20 μg/ml; the content of aluminium was controlled at 0.45-0.60 mg/ml; and the pH value was controlled at 5.8-6.0.
(51) Preparation method: the sterilized and filtered sodium chloride solution, AlCl.sub.3 solution, and EV71 VLPs were added in a sterile- and pyrogen-free reactor successively, stirred with a magnetic stirrer, and after stirring well the mixed solution of NaOH and phosphate was added in a proper constant speed flow to initiate the adsorption reaction in situ while adjusting the pH value. The acceleration of flow should not too fast, and was controlled at 2-5 ml/min to enable the adsorption reaction to take place at a relatively constant pH value. When pH was 5.8-6.0, the addition of the mixed solution of NaOH and phosphate was stopped. Stirring was performed for 30 min, sampling was performed for testing pH value, the adsorption reaction was ended and the preparation of the vaccine was completed. The test standard and test results of the prepared vaccine were shown in Table 3.
(52) TABLE-US-00003 TABLE 3 Test results of EV71 VLP vaccine prepared by aluminium phosphate adsorption in situ method Test items Content of aluminium pH value Adsorption rate Test criteria Chinese
Chinese refer to
Chinese Pharmacopoeia
Volume III, Pharmacopoeia
Volume Pharmacopoeia
Appendix VII F Testing III, Volume III, Appendix method of aluminium Appendix V A Testing X A hydroxide method of pH value Test standard 0.45-0.60 mg/ml 5.8-6.0 >95% Test results 0.55 mg/ml 5.83 99.3%
(53) The results in Table 3 show that: all the test indexes of the EV71 VLP vaccine prepared by aluminium phosphate adsorption in situ method meet with the test standard.
Example 7
Immunogenicity (ED50) Test of the Vaccine Prepared from EV71 VLPs
(54) Vaccine sample: EV71 VLP vaccine containing an aluminium phosphate adjuvant prepared in the Example 6.
(55) Experimental animals: 60 SPF grade BALB/c mice with a weight 18-22 g per mouse were chosen as experimental animals, which were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.
(56) Immune procedure: the prepared vaccine was diluted to 0.5, 0.25, 0.125, 0.0625, 0.03125 μg/ml in a series of doubling dilution, each dilution was injected intraperitoneally into 10 mice with each mouse receiving 1 ml. 30 days after immunization, blood was collected from the eyeballs of mice. The collected blood was placed at 37° C. for 2 h, centrifuged at 4000 g for 10 min, and the supernatant was sucked out as antiserum to be detected.
(57) Detection of the antiserum by indirect ELISA method: each of the purified antigen solutions in Example 5 was diluted with coating buffer to a concentration of 1 μg/ml and the dilution was added into the shrinkage pool of a 96 well microplate reader and incubated at 4° C. overnight. After removing the coating buffer completely, the microplate reader was washed by filling with the cleaning solution PBST. The microplate reader was filled with blocking buffer (1% BSA in PBST) and incubated for 1 hour at 37° C. After removing the blocking buffer completely, 100 μl of antiserum to be detected was added into each well, and incubated for 1 hour at 37° C. After removing the serum liquid completely, the microplate reader was washed for 3 times by filing with cleaning solution PBST. 100 μl of HRP labeled goat-anti-mouse IgG (in 1:4000 dilution) was added into each well and incubated for 1 hour at 37° C. After removing the liquid from microplate completely, the microplate reader was washed for 3 times by filing with cleaning solution PBST. 100 μl TMB developing solution was added into each well, and protected from light for 15 min at 37° C. To each well, 50 μl of 2M H.sub.2SO.sub.4 was added for completion. OD.sub.450nm value was determined by ELISA instrument.
(58) The detection results of the antiserum by indirect ELISA were shown in Table 4.
(59) TABLE-US-00004 TABLE 4 Detection result of the antiserum by indirect ELISA method Conc. 0.5 0.25 0.125 0.0625 0.03125 Negative Positive No. μg/ml μg/ml μg/ml μg/ml μg/ml serum control 1 0.810 3.003 1.094 0.195 0.083 0.051 3.009 2 1.742 1.678 2.349 0.096 0.055 0.049 3.013 3 2.144 0.783 0.107 0.729 0.092 — — 4 2.902 2.192 1.448 1.051 0.101 — — 5 2.650 0.372 0.205 0.103 0.822 — — 6 1.815 1.545 1.338 0.301 1.038 — — 7 1.633 2.232 2.051 2.009 0.058 — — 8 2.589 1.351 0.793 1.473 0.078 — — 9 1.988 1.628 0.098 0.098 0.109 — — 10 1.902 2.749 0.229 0.672 0.991 — — Positive 100% 100% 80% 70% 30% — — rate
(60) According the detection results, the positive conversion rate of an antibody was shown as Table 5.
(61) TABLE-US-00005 TABLE 5 Calculated result of the positive conversion rate of an antibody Sum up Content of Antibody of positive positive antigen mouse conversion conversion (μg/ml) positive negative rate positive negative rate 0.5 10 0 100% 38 0 100% 0.25 10 0 100% 28 0 100% 0.125 8 2 80% 18 2 90.0% 0.0625 7 3 70% 10 5 66.67% 0.03125 3 7 30% 3 12 20.0%
(62) Calculated in accordance with Reed-Muench method: ED.sub.50=0.049 (μg)
(63) It can be seen from the experimental results obtained by mice ED.sub.50 that: when only 0.049 μg of EV71 VLP vaccine prepared according to the present invention was used to immune the mice, the positive conversion rate of the antibody can reach 50%. Therefore, the EV71 VLPs of the present invention had strong immunogenicity.
Example 8
Abnormal Toxicity Test of the Vaccine Prepared from EV71 VLPs
(64) Vaccine sample: EV71 VLP vaccine containing an aluminium phosphate adjuvant prepared in Example 6.
(65) Experimental animals: 5 SPF grade BALB/c mice with a weight of 18-22 g per mouse, and 2 SPF grade Hartley guinea pig with a weight of 250-350 g per guinea pig were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.
(66) Experimental method: every experimental animal was weighted before injection with the mouse being 18-22 g and the guinea pig being 250-350 g. 5 mice were intraperitoneally injected with vaccines in the amount of 0.5 ml/mouse, 2 guinea pigs were intraperitoneally injected with vaccines in the amount of 5.0 ml/guinea pig, and then the experimental animals were observed for 7 days. Meanwhile, the same batch of experimental animals were used as blank control. Criterion of acceptability: the blank control and the experimental animals were healthy and had no abnormal reactions during the observation period, and every experimental animal had a weight gain at end. The details for animal experiments were shown in Table 6.
(67) TABLE-US-00006 TABLE 6 Details for animal experiments Labeled Initial Final Species of animals No. position weight (g) weight (g) Experimental group of mice 1 Head 18.5 19.6 2 Spine 19.0 20.3 3 Tail 18.6 19.5 4 Leg 19.6 21.5 5 None 19.2 20.9 Experimental group of guinea 1 Head 338.7 360.8 pig 2 Spine 357.0 403.6 Control group of mice 1 Head 19.4 20.8 2 Spine 19.7 21.3 3 Tail 19.0 19.6 4 Leg 18.5 20.7 5 None 18.9 19.9 Control group of guinea pig 1 Head 336.2 376.3 2 Spine 340.3 399.6
(68) Conclusion: the blank control and the experimental animals were healthy and had no abnormal reactions during the observation period, and every experimental animal had a weight gain on day 8. It was demonstrated that vaccines prepared from EV71 VLPs had no abnormal toxicity and the experimental animals had good safety.
(69) While the present invention has been described in detail through general description and specific embodiments above, it is obvious to a person skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, all these modifications or improvements made without departing from the spirit of the present invention belong to the scope claimed in the present invention.
INDUSTRIAL APPLICABILITY
(70) The present invention provides EV71 VLPs and a preparation method and application thereof. The method comprises: connecting a P1 protein gene and a 3CD protease gene of an EV71 virus with a PMV plasmid to construct a PMV-P1-3CD recombinant expression plasmid; then transforming a Hansenula polymorpha AU-0501 expression strain with the PMV-P1-3CD recombinant expression plasmid to obtain an AU-PMV-P1-3CD recombinant expression strain; fermenting and culturing the recombinant expression strain, and inducing the strain to express the EV71 VLP protein with methanol; centrifuging and collecting cells for homogeneous breakage at a high pressure; and purifying the supernatant through ion-exchange chromatography, hydrophobic chromatography, and molecular sieve chromatography and the like to obtain EV71 VLPs.
(71) The EV71 VLP vaccine provided in the present invention has excellent immunogenicity, safety, immunological property and biological activity, and it can be prepared and purified in a large scale and used for preparing vaccines for preventing EV71 infection, and thus has good economic value and application prospect.