WO2018119746A1 - 重组ev71病毒样颗粒的纯化及其疫苗制备方法 - Google Patents

重组ev71病毒样颗粒的纯化及其疫苗制备方法 Download PDF

Info

Publication number
WO2018119746A1
WO2018119746A1 PCT/CN2016/112636 CN2016112636W WO2018119746A1 WO 2018119746 A1 WO2018119746 A1 WO 2018119746A1 CN 2016112636 W CN2016112636 W CN 2016112636W WO 2018119746 A1 WO2018119746 A1 WO 2018119746A1
Authority
WO
WIPO (PCT)
Prior art keywords
virus
chromatography
collected
purification method
vaccine
Prior art date
Application number
PCT/CN2016/112636
Other languages
English (en)
French (fr)
Inventor
李国顺
顾美荣
刘建凯
郭林
陈磊
张改梅
徐颖之
李进
肖海峰
Original Assignee
北京民海生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 北京民海生物科技有限公司 filed Critical 北京民海生物科技有限公司
Priority to CN201680088253.6A priority Critical patent/CN109689862B/zh
Priority to US16/333,290 priority patent/US10987416B2/en
Priority to PCT/CN2016/112636 priority patent/WO2018119746A1/zh
Publication of WO2018119746A1 publication Critical patent/WO2018119746A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/125—Picornaviridae, e.g. calicivirus
    • A61K39/135—Foot- and mouth-disease virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/125—Picornaviridae, e.g. calicivirus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C12N7/04—Inactivation or attenuation; Producing viral sub-units
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32311—Enterovirus
    • C12N2770/32334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/027—Liquid chromatography

Definitions

  • the present invention relates to the field of biological products, and in particular to a method for purifying recombinant EV71 virus-like particles and a method for preparing a vaccine using the purified EV71 virus-like particles.
  • Hand, foot and mouth disease is a global infectious disease and is reported in most parts of the world.
  • Hand, foot and mouth disease is a common and pediatric acute infectious disease caused by enterovirus, which can cause fever and mouth, hand, cavity and other parts to form rash, herpes and other major symptoms.
  • enterovirus a common and pediatric acute infectious disease caused by enterovirus, which can cause fever and mouth, hand, cavity and other parts to form rash, herpes and other major symptoms.
  • a small number of patients can cause aseptic meningitis, neurogenic pulmonary edema, brainstem encephalitis, myocarditis and so on.
  • the pathogens of hand, foot and mouth disease are mainly enteric viruses, including Coxsackie virus, EV71 and Echovirus, of which EV71 and Cox16 are the most common.
  • the international research on the vaccine against foot and mouth disease mainly includes inactivated vaccine, virus-like particle vaccine, subunit vaccine, DNA vaccine and live attenuated vaccine.
  • Subunit vaccines, DNA vaccines, and live attenuated vaccines have not achieved satisfactory animal protection despite reports of research progress.
  • Certain live attenuated vaccines can also cause mild neurological symptoms, and there are uncertainties in the safety of the vaccine.
  • the research progress is faster inactivated vaccine.
  • the EV71 inactivated vaccine developed by Kunming Institute and Beijing Kexing Biological Products Co., Ltd. has been listed and approved, but some of the inactivated vaccines have been inactivated and some antigen targets have been inactivated.
  • VLP Virus Like Particle
  • a virus-like particle is a hollow particle containing one or more structural proteins of a certain virus.
  • the nucleic acid (DNA/RNA) without a virus cannot be autonomously replicated, and is morphologically identical or similar to a true virion, and can be infected by a virus.
  • the same route is presented to immune cells, effectively inducing the body's immune system to produce an immune protective response.
  • the capsid proteins of the virus generally have a natural self-assembly ability.
  • VLPs vaccines are not infectious, have good stability, are not easy to inactivate, and have broad development prospects.
  • the enterovirus belongs to the picornavirus family.
  • the virus particles are icosahedral symmetrical spherical structures, stable, not easily mutated, without envelopes and protrusions, and have a diameter of 24-30 nm.
  • the nucleic acid is a single strand of positive strand RNA.
  • the capsid of the virion consists of 60 subunits, each of which is assembled from four capsid proteins (VPl-VP4) into a pentameric structure. Studies have shown that the four structural proteins of EV71 virus can be assembled into a virus-like particle structure (VLP) in the cell, and have a spatial structure similar to that of the natural virus.
  • VLP virus-like particle structure
  • the expression systems commonly used for virus-like particles mainly include prokaryotic expression systems and eukaryotic expression systems.
  • Most of the proteins expressed by prokaryotic expression systems lose their natural conformation and cannot produce protective antibodies. Or the expression products are mostly inclusion bodies, and the inclusion body denaturation and renaturation steps are complicated, especially the expression of VLP, and the purification process is more complicated.
  • Eukaryotic expression system has mammalian cell expression system, insect rod disease Toxic expression system, yeast expression system. In the eukaryotic expression system, proteins can form VLP spontaneously, which provides great convenience for the purification process.
  • the use of insect cells to prepare VLPs requires higher culture conditions and complicated purification processes, which limits the large-scale production requirements.
  • the baculovirus-insect cell expression system produces baculovirus particles and other pollutants that affect the vaccine effect. Baculovirus particles are difficult to separate from the prepared VLP and require inactivation treatment and the like, and thus have a great influence on the quality of the vaccine.
  • the Hansenula polymorpha expression system has the characteristics of stable genetic property, simple operation, high-density fermentation culture, high yield of target product, low production cost, suitable for industrialized large-scale production, and foreign protein not found in prokaryotic expression system.
  • the advantages of post-translational processing are a more advanced VLP vaccine expression system superior to E. coli and other eukaryotic expression systems.
  • Another object of the invention is to provide a recombinant EV71 vaccine.
  • the present invention provides a method for purifying recombinant EV71 virus-like particles, comprising the steps of:
  • the high-density formula medium is used for the fermentation of the engineering bacteria, and the rapid growth of the cells is carried out by intermittent addition of glycerol, and the inducer methanol is continuously added to express the virus-like particles.
  • the engineered bacteria are at pH 5. Induction under conditions of 36 to 42 h.
  • the crushing of the engineering bacteria is specifically: using the engineering bacteria in a cell lysis buffer (20 mM NaH 2 PO 4 , 2 mM EDTA-Na 2 , 0.2-1.0 M NaCl, 2 mM PMSF, 0.1% to 1.0% Tween-20, pH 6). .8 ⁇ 7.4), using a high-pressure homogenizer to break the cells 2-4 times under the pressure of 1100-1400 bar, the cell breakage rate is 80%-95%.
  • the high pressure homogenizer breaks the cells 2 times at a pressure of 1200 bar.
  • the target product is precipitated by specifically pouring the crushed cell liquid into a centrifuge tube, performing centrifugation at 6000 to 8000 rpm for 40 to 60 minutes, and collecting the supernatant.
  • the collected supernatant was added with ammonium sulfate to a final concentration of ammonium sulfate of 20% to 28% and allowed to stand overnight.
  • the final concentration to ammonium sulphate is 22%.
  • the reconstitution is specifically: the ammonium sulfate precipitation product is centrifuged at 8000 to 10000 rpm for 40 to 60 minutes, the precipitate is collected, and a reconstitution buffer (20 mM NaH 2 PO 4 , 0.1 to 1.0 M NaCl, pH 6.8 to 7.4) is added. Stir for 30 to 60 minutes, centrifuge at 8000 to 10000 rpm for 40 to 60 minutes, and collect the reconstituted supernatant.
  • a reconstitution buffer (20 mM NaH 2 PO 4 , 0.1 to 1.0 M NaCl, pH 6.8 to 7.4
  • the ultrafiltration is specifically: the collected reconstituted supernatant is ultrafiltered with 20-50 mM Tris (pH 7.5-8.5) in a membrane package of 100-500 KD to remove small molecular substances, and the ultrafiltrate is collected. It is a crude protein solution.
  • the ion exchange chromatography is specifically: taking Capto Q chromatography medium as an example, using a 50-50 mM Tris (pH 7.5 to 8.5) to equilibrate 5 to 10 column volumes, and then loading 20-50 mM Tris and 150 to 300 mM.
  • the NaCl eluate was eluted, and the UV absorption peak of UV 280 nm of the eluate was collected, which was a one-step chromatography protein solution.
  • elution is carried out using an eluate of 20-50 mM Tris and 230 mM N aCl.
  • the molecular sieve chromatography is specifically as follows: Sephacryl S-300HR is taken as an example, and the purification step is carried out by using 10-50 mM PBS (pH 6.8-7.4) and 0.1-0.3 M NaCl solution and 0.01%-0.1% Tween-80. The protein solution was collected, and the UV absorption peak of UV280 nm was collected as the target protein solution.
  • the hydroxyapatite chromatography is specifically: one-step chromatography protein solution is ultrafiltered with 10 mM PBS (pH 6.8-7.4) in a 300 KD membrane package to remove small molecular substances, and the ultrafiltrate is collected. After 5 to 10 column volumes were equilibrated in 10 to 50 mM PBS (pH 6.8 to 7.4), the sample was applied, and eluted with an eluent (30 to 200 mM) in PBS to collect an ultraviolet absorption peak of UV 280 nm of the eluate, which was the target protein solution. .
  • each dose of the human vaccine comprises:
  • Recombinant EV71 virus particles (calculated by protein) 2 ⁇ 10 ⁇ g
  • Aluminum adjuvant (calculated as Al ion) 0.10 ⁇ 0.30mg
  • the invention provides a method for extracting and purifying a recombinant protein, which has the following advantages:
  • the invention adopts high-density formula medium for fermentation of engineering bacteria, and intermittently supplements glycerin and continuously supplements the inducer methanol, so that the wet weight of the bacteria after the fermentation of the engineering bacteria can reach 300-400 g/L, and the antigen expression amount Up to 35 ⁇ 10 4 ⁇ 45 ⁇ 10 4 U / ml.
  • the invention adopts ammonium sulfate precipitation, PBS reconstitution and two-step chromatography, has simple operation and high recovery rate, and is favorable for large-scale industrial production.
  • the novel vaccine of the present invention is a highly safe recombinant vaccine that does not cause adverse effects of cancer and potential RNA carcinogenesis.
  • the vaccine of the present invention uses an aluminum adjuvant, and the recombinant EV71 virus particle vaccine containing no aluminum adjuvant is significantly less immunogenic than the vaccine containing the aluminum adjuvant.
  • Figure 1 shows Western blotting of samples of EV71 strains expressed by engineered bacteria at different fermentation times (44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96 hours).
  • M is a low molecular weight protein standard (Beijing Quanjin Company).
  • Figure 2 is an ion exchange chromatography purification map.
  • Figure 3 is a molecular sieve chromatogram purification map.
  • Figure 4 is a dynamic light scattering spectrum of purified EV71 virus particles.
  • Figure 5 is a transmission electron micrograph of a purified EV71 virus particle (magnification: 135000).
  • Example 1 Recombinant EV71 Yeast Expression Strains Fermented in a 30 L Fermentor
  • the recombinant Hansenula strain (preparation method see Chinese patent ZL201310179673.3) was inoculated into 150 ml of primary seed culture medium (0.67% yeast nitrogen source medium, purchased from SIGMA, 0.5% ammonium sulfate, 2% glucose). Incubate at 33 ° C, shaking at 200 rpm for 20 to 24 hours. Then, the whole amount was inoculated into 1500 ml of secondary seed culture medium (0.67% yeast nitrogen source medium, 0.5% ammonium sulfate, 2% glycerol), and cultured at 33 ° C, shaking at 200 rpm for 20-24 hours (OD 600nm up to 8-10). ).
  • primary seed culture medium 0.67% yeast nitrogen source medium, purchased from SIGMA, 0.5% ammonium sulfate, 2% glucose. Incubate at 33 ° C, shaking at 200 rpm for 20 to 24 hours. Then, the whole amount was inoculated into 1500 ml of secondary seed culture medium (0.67% yeast
  • the rabbit polyclonal antibody was diluted 1000-fold, 100 ⁇ l was added to the well of a 96-well microtiter plate, and coated at 4 ° C overnight. In addition to the net coating solution, top up the washing solution with PBST wash ELISA plate. The plate was incubated with blocking solution (1% BSA in PBST) for 2 hours at 37 °C. The supernatant collected by centrifugation and the national antigen standard were serially diluted, and 100 ⁇ l of the sample to be tested and the standard were added to each well, and incubated at 37 ° C for 1 hour. The washing solution was topped up with PBST washing enzyme plate.
  • the results of ELISA showed that the antigen content of EV71 virus-like particles in the fermentation cell disrupted solution was 43 ⁇ 10 4 U/ml. EV71 expressed by Hansenula cells has a high expression level.
  • Cell collection The fermentation broth was collected, and the precipitate was collected by centrifugation at 6000 rpm, and washed twice with cell washing buffer (20 mmol/L NaH 2 PO 4 , 2 mmol/L EDTA-Na 2 , 0.4 mol/L NaCl, pH 7.4) to wash the cells. Times.
  • Clarification pour the broken cell liquid into a centrifuge tube, centrifuge at 6000 to 8000 rpm for 40 to 60 minutes, and collect the supernatant.
  • Precipitating virus-like particles adding the above supernatant to ammonium sulfate to ammonium sulfate The degree is about 22% to 28% and is left overnight.
  • Reconstitution The above precipitated liquid is decanted, and the precipitate is centrifuged at 10,000 rpm for 40 to 60 minutes, and the precipitate is collected and added to a reconstitution buffer (20 mmol/L NaH 2 PO 4 , 0.01 to 1 mol/L NaCl, pH 7.4). Stir for 30 to 60 minutes, centrifuge at 10,000 rpm for 40 to 60 minutes, and collect the reconstituted supernatant.
  • a reconstitution buffer (20 mmol/L NaH 2 PO 4 , 0.01 to 1 mol/L NaCl, pH 7.4
  • Ion exchange chromatography Take Capto Q chromatography medium as an example, equilibrate 5 to 10 column volumes with 50 mmol/L Tris (pH 8.0), and then use an eluent (50 mmol/L Tris-NaCl (150-300 mmol). /L)) Elution, collection of UV absorbance UV280nm UV absorption peak, which is a one-step chromatography protein solution, using 1mol / L NaOH regeneration of the chromatographic medium, the purification map shown in Figure 2.
  • Molecular sieve chromatography Sephaacryl S-300HR packing was used to purify one-step chromatography protein solution with 10-50 mm PB (pH 6.8-7.2) and 0.1-0.3 M NaCl solution and Tween-80 (0.01%-0.1%).
  • the UV absorption peak of UV280nm is the target protein solution, and the purity of the protein solution is 100% by HPLC.
  • the purification map is shown in Fig. 3.
  • Determination of target protein concentration (Lowry method): accurately measure standard protein bovine serum albumin solution (200 ⁇ g/ml) 0ml, 0.2ml, 0.4ml, 0.6ml, 0.8ml, 1.0ml, respectively, placed in a test tube, add distilled water to make up To 1 ml, simultaneously take 1 ml of the diluted protein solution 1 ml in a test tube, add 5 ml of alkaline copper solution, 0.5 ml of phenol reagent, and measure the absorbance value in a cuvette with a wavelength of 650 nm. The protein content of the standard protein is taken as the abscissa, the absorbance value is plotted on the ordinate, and the concentration of the protein to be detected is calculated. The test results are shown in Table 2.
  • the final low concentration of purified protein was 948 ⁇ g/ml by Lowry test.
  • Dynamic light scattering analysis of the target protein solution The purified EV71 protein solution was added to the sample cell in an appropriate amount, set the temperature at 25 ° C, the equilibrium time was 90 s, set the number of automatic cycles, start measurement, and analyze the results. The results showed that the recombinant EV71 virus particles were intact, and the particle diameter was more than 99% in the range of 24 to 30 nm.
  • the dynamic light scattering spectrum is shown in Fig. 4.
  • the objective protein was analyzed by electron microscopy.
  • the purified EV71 protein solution was added to the copper mesh and stored in the dark for 5 min. The excess liquid was removed and stained with 1% phosphotungstic acid for 2 min.
  • the EV71 VLPs were analyzed by transmission electron microscopy (TEM). The results showed that the EV71 protein showed virus-like particles and had the icosahedral structure of the natural virus. The diameter of the virus particles was about 30 nm, and the particles were completely regular (as shown in Fig. 5, magnification: 135000).
  • target protein liquid antigen content double-antibody sandwich ELISA: The rabbit polyclonal antibody was diluted 5000 times, 100 ⁇ l was added to the well of a 96-well microtiter plate, and coated at 4 ° C overnight. In addition to the net coating solution, top up the washing solution with PBST wash ELISA plate. Incubate the plate with blocking solution (1% BSA in PBST) for 2 hours at 37 °C. 100 ⁇ l of the test sample was added to each well except the net blocking solution, and incubated at 37 ° C for 1 hour. The washing solution was topped up with PBST washing enzyme plate.
  • blocking solution 1% BSA in PBST
  • the preparation method comprises the following steps: injecting a sterile PBS solution into a tank, using a mechanical stirring speed of 500-700 rpm, and maintaining a pressure of 0.05-0.1 MPa in the tank, and slowly adding the potassium aluminum sulfate solution after sterilization removal to the tank. Then, the sodium hydroxide solution was added dropwise to the pH at 6.6-7.0 to stop. After standing, the preparation of the aluminum hydroxide adjuvant was completed after using a sterile 0.9% sodium chloride solution and replacing the supernatant 4-5 times. The stock solution of the purified EV71 was slowly added dropwise to the adjuvant to make it fully adsorbed, and the preparation of the vaccine was completed.
  • Table 4 The preparation standards for vaccine preparation and the test results are shown in Table 4.
  • Test vaccine A recombinant EV71 virus particle vaccine containing an aluminum hydroxide adjuvant prepared in Example 3.
  • Test animals 50-22 g of SPF grade BALB/c mice were selected and purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.
  • Animal immunization 0.5, 0.125, 0.03125, 0.0078125 ⁇ g/0.5ml vaccine and aluminum hydroxide adjuvant were injected into 10 mice, 0.5ml per intraperitoneal injection, and 28 days after immunization, the eyeball was taken for blood. .
  • the collected blood was placed at 37 ° C for 1 h, allowed to stand at 4 ° C for 3-4 hours, centrifuged at 4000 rpm for 10 min, and the supernatant was aspirated for examination.
  • Neutralizing antibody detection serum samples were diluted 1:8 in MEM medium supplemented with 2% newborn bovine serum, and extinguished in a 56 ° C water bath for 30 minutes. A 96-well cell culture plate was used, and 50 ⁇ l of the dilution was added to each well. Add 50 ⁇ l of the corresponding sample to each well, mix the row of guns and mix 50 ⁇ l to B line, mix and dilute to D line and discard 50 ⁇ l (diluted EV71 virus attack poison series to 100CCID 50 /0.05ml, take 50 ⁇ l Drip vertically into each well, gently mix the cell culture plate, and neutralize for two hours at 37 ° C.
  • the EV71 virus-like particle vaccine prepared by the invention can achieve the antibody positive conversion rate of 50% after immunizing the mouse with only 0.011 ⁇ g, so the EV71 virus-like particle of the invention has a strong immunogen. Sex.
  • Vaccine sample Recombinant EV71 virus-like particle vaccine prepared in Example 3.
  • mice 18-22 g of SPF grade KM mice, 250-350 g of SPF grade Hartley guinea pigs, both purchased from Beijing Vital Lihua Experimental Animal Technology Co., Ltd.
  • mice The body weight of each experimental animal was weighed before injection, and the mice were 18-22 g, and the guinea pigs were 250-350 g. Five mice and two guinea pigs were vaccinated. The mice were intraperitoneally injected with 0.5 ml/mouse, and the guinea pigs were intraperitoneally injected with 5.0 ml/only for 7 days. At the same time, the same batch of animal blanks were set. Eligibility Criteria: During the observation period, the blank control and the experimental group of animals survived, no abnormal reaction, and the weight of each animal increased. The animal test conditions are shown in Table 6.
  • the invention provides a method for purifying EV71 virus-like particles and a method for preparing a vaccine by using the purified EV71 virus-like particles.
  • High-density fermentation culture of recombinantly expressed engineering bacteria and induction of expression of EV71 virus granule protein by methanol the cells were collected by centrifugation for high-pressure homogenization, and the supernatant was precipitated by ammonium sulfate, reconstituted, ion-exchanged, and molecular sieved. It is obtained after purification by hydroxyapatite chromatography or the like.
  • the EV71 virus particle vaccine provided by the invention has good immunogenicity, safety, immune characteristics and biological activity, has simple process, does not use a large equipment of an ultracentrifuge, and is more convenient to be prepared and purified on a large scale, and can be used for preparing the EV71 for prevention. Infected vaccines have good economic value and application prospects.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Virology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Organic Chemistry (AREA)
  • Mycology (AREA)
  • Engineering & Computer Science (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Zoology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Epidemiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Oncology (AREA)
  • Communicable Diseases (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

本发明提供一种EV71病毒样颗粒的纯化方法及其疫苗制备方法。通过将重组表达的工程菌高密度发酵培养和甲醇诱导表达EV71类病毒颗粒蛋白，离心收集菌体进行高压均浆破碎，上清液经硫酸铵沉淀，复溶、超滤、离子交换层析、分子筛层析和羟基磷灰石层析等纯化后获得。

Description

重组EV71病毒样颗粒的纯化及其疫苗制备方法 技术领域
本发明涉及生物制品领域，具体地，涉及一种重组EV71病毒样颗粒的纯化方法以及利用纯化后的EV71病毒样颗粒制备疫苗的方法。
背景技术
手足口病是全球性传染病，世界大部分地区均有此病流行的报道。手足口病是由肠道病毒引起的，可引发发热和口、手、空腔等部位形成皮疹、疱疹等主要症状的一种常见与小儿的急性传染病。少数患者可引发无菌性脑膜炎、神经性肺水肿、脑干脑炎、心肌炎等。手足口病的病原体主要为肠道病毒，包括柯萨奇病毒、EV71和埃可病毒，其中以EV71、Cox16最多见。
1957年，首次在新西兰爆发。1969年EV 71型在美国被首次确认。20世纪70年代中期，保加利亚、匈牙利相继爆发以中枢神经系统为主要临床特征的EV71流行，仅保加利亚报告750例发病，149人致瘫，44人死亡。英国1994年4季度爆发了一起遍布英格兰威尔士由CoxAl6引起的手足口病流行，监测哨点共观察到952个病例，为该国有记录以来的最大一次。患者大多1-4岁。1997年马来西亚发生了由EV 71型引起的手足口病流行，4月一8月发病2628例，死亡29例。2000年新加坡发生HFMD暴发，发病3790例，死亡5例。2000年9月至10月，新加坡报告手足口病3790例，5例死亡，EV71是主要的病原体。
我国1981年上海报告了手足口病，病原体为EV 71型。1983年厦门发生HFMD流行，从患儿标本分离出Cox A16型病毒。同年天津发生了由CoxA16引发的手足口病。5～10月发病7000余例，1986年再次暴发。1995年在武汉、1998年在深圳均从手足口病患儿分离到EV 71型病毒。1998年在台湾发生了EV 71型引起的手足口病和疱疹性咽峡炎。病例达13万人。2000年5～8月山东招远发病近1700例。2008年后，手足口病呈蔓延趋势。2008年3月至5月，安徽阜阳累计报告病例3736例，其中 22例死亡。在我国大部分省区都有此病报导。
目前，国际上对手足口病疫苗的研究主要有灭活疫苗、病毒样颗粒疫苗、亚单位疫苗、DNA疫苗和减毒活疫苗等。亚单位疫苗、DNA疫苗和减毒活疫苗尽管有研究进展方面的报道，但是都没有达到满意的动物保护效果。某些减毒活疫苗还能导致轻度的神经性临床症状，疫苗的安全性还存在不确定因素。研究进展较快的为灭活疫苗，现已有昆明所和北京科兴生物制品有限公司研发的EV71灭活疫苗经过批签发陆续上市，但此类灭活疫苗经过灭活后部分抗原靶位被破坏，免疫原性受此影响，疫苗的抗原纯度低，临床副反应相对较大。因此，研制更加安全、有效的针对EV71的预防性疫苗对控制婴幼儿手足口病的流行具有十分重大的意义。
病毒样颗粒(Virus Like Particle，VLP)疫苗的出现为研发新型安全有效的疫苗提供了一个新的契机。病毒样颗粒是含有某种病毒的一个或多个结构蛋白的空心颗粒，没有病毒的核酸(DNA/RNA)，不能自主复制，其在形态上与真正病毒粒子相同或相似，可通过和病毒感染一样的途径呈递给免疫细胞，有效地诱导机体的免疫系统产生免疫保护反应。病毒的衣壳蛋白一般具有天然的自我装配能力。VLPs疫苗没有感染性，稳定性好，不易失活，具有广阔的发展前景。
肠道病毒属于微小核糖核酸病毒科(picornavirus)。病毒颗粒为二十面体对称的球状结构，稳定、不易变异、无包膜和突起、直径为24—30nm。核酸为单股正链RNA。病毒粒子的衣壳由60个亚单位组成，每个亚单位均由4种衣壳蛋白(VPl-VP4)拼装成有五聚体样结构。研究表明，EV71病毒的四种结构蛋白在细胞内可自行组装成病毒样颗粒结构(Virus Like Particle，VLP)，具有与天然病毒相似的空间结构。
目前常用于病毒样颗粒的表达体系主要有原核表达系统和真核表达系统。原核表达系统表达的蛋白大多失去天然构象，不能产生保护性抗体。或者表达产物多为包涵体，包涵体变性、复性步骤复杂，尤其VLP的表达，纯化工艺更为复杂。真核表达系统有哺乳动物细胞表达系统、昆虫杆状病 毒表达系统、酵母表达系统。在真核表达系统中蛋白能自发的形成VLP，为纯化工艺提供极大的便利。但是，采用昆虫细胞制备VLP，培养条件要求较高，纯化过程复杂，限制了大规模的生产需求；另外，杆状病毒-昆虫细胞表达系统产生杆状病毒颗粒及其它影响疫苗效果的污染物，杆状病毒颗粒很难与制备的VLP分离，且需要进行灭活处理等措施，因此对疫苗质量有很大的影响。多形汉逊酵母表达系统具遗传性质稳定、操作简单、高密度发酵培养、目的产物产量高、生产成本低、适合于工业化大生产等特点，还具有原核生物表达系统所不具有的外源蛋白翻译后加工等优势，是一种优于大肠杆菌和其它真核表达系统的较为先进的VLP疫苗表达系统。
发明内容
本发明的目的是提供一种从汉逊酵母表达系统中表达的EV71类病毒颗粒的纯化方法。
本发明的另一种目在于提供一种重组EV71疫苗。
为实现上述目的，本发明提供了一种重组EV71病毒样颗粒的纯化方法，包括以下步骤：
(1)将含EV71外壳蛋白P1基因和3CD蛋白酶基因的重组汉逊酵母工程菌发酵；
(2)破碎工程菌，目的产物沉淀、复溶、超滤；
(3)离子交换层析；
(4)分子筛层析或羟基磷灰石层析。
本发明的含EV71外壳蛋白P1基因和3CD蛋白酶基因的重组汉逊酵母工程菌的制备方法参见中国专利ZL201310179673.3。
步骤(1)中，采用高密度配方培养基进行工程菌发酵，通过间歇性补加甘油使菌体快速生长、连续性补加诱导剂甲醇进行类病毒颗粒表达，发酵后期，工程菌在pH5.5条件下诱导36～42h。
所述工程菌的破碎具体为：将工程菌采用细胞裂解缓冲液中(20mM NaH2PO4，2mM EDTA-Na2，0.2～1.0M NaCl，2mM PMSF，0.1％～1.0％ Tween-20，pH6.8～7.4)，使用高压匀浆机在压力1100～1400bar的条件下破碎细胞2-4次，细胞破碎率达80％-95％。
优选地，高压匀浆机在压力1200bar，破碎细胞2次。
所述目的产物沉淀具体为：将破碎后的细胞液倒入离心筒中，进行6000～8000rpm离心40～60min，收集上清液。将收集的上清液中加入硫酸铵，至硫酸铵终浓度为20％～28％，放置过夜。优选地，至硫酸铵终浓度为22％。
所述复溶具体为：将上述硫酸铵沉淀产物采用8000～10000rpm离心40～60min，收集沉淀，并加入复溶缓冲液(20mM NaH2PO4，0.1～1.0M NaCl，pH6.8～7.4)，搅拌30～60min，离心8000～10000rpm离心40～60min，收集复溶上清液。
所述超滤具体为：将收集的复溶上清液以100～500KD的膜包采用20-50mM Tris(pH7.5～8.5)进行超滤以除掉小分子物质，收集超滤液，即为粗纯蛋白液。
所述离子交换层析具体为：以Capto Q层析介质为例，采用20-50mM Tris(pH7.5～8.5)平衡5～10个柱体积后上样，采用20-50mM Tris和150～300mM NaCl洗脱液进行洗脱，收集洗脱液UV280nm紫外吸收峰，即为一步层析蛋白液。优选地，采用20-50mM Tris和230mM N aCl洗脱液进行洗脱。
所述分子筛层析具体为：以Sephacryl S-300HR为例，以10-50mM PBS(pH6.8～7.4)和0.1-0.3M NaCl溶液和0.01％-0.1％的吐温-80进行纯化一步层析蛋白液，收集UV280nm紫外吸收峰即为目的蛋白液。
所述羟基磷灰石层析具体为：一步层析蛋白液以300KD的膜包采用10mM PBS(pH6.8～7.4)进行超滤以除掉小分子物质，收集超滤液。以10～50mM PBS(pH6.8～7.4)平衡5～10个柱体积后上样，采用洗脱液(30～200mM)PBS洗脱，收集洗脱液UV280nm紫外吸收峰，即为目的蛋白液。
本发明还提供了一种疫苗配方，每剂人用疫苗中含有：
重组EV71类病毒颗粒(按蛋白质计算)        2～10μg
铝佐剂(按Al离子计算)                    0.10～0.30mg
本发明提供了一种重组蛋白提取纯化方法，具有以下优点：
1、本发明采用高密度配方培养基进行工程菌发酵，并通过间歇性补加甘油和连续性补加诱导剂甲醇，使得工程菌发酵结束菌湿重可达300～400g/L，抗原表达量可达35×104～45×104U/ml。
2、本发明采用硫酸铵沉淀，PBS复溶和两步层析，操作简单，回收率高，有利于大规模工业化生产。
3、本发明的新疫苗属于安全性极高的重组疫苗，不会引起逆毒和潜在的RNA致癌风险。本发明的疫苗使用铝佐剂，不含铝佐剂的重组EV71类病毒颗粒疫苗其免疫原性明显低于含铝佐剂的疫苗。
附图说明
图1为工程菌表达的EV71菌株在不同发酵时间(44、48、52、56、60、64、68、72、76、80、84、88、92、96小时)样品的蛋白质免疫印迹(Western blot)检测结果，M为低分子量蛋白标准(北京全式金公司)。
图2为离子交换层析纯化图谱。
图3为分子筛层析纯化图谱。
图4为纯化的EV71类病毒颗粒动态光散射图谱。
图5为纯化的EV71类病毒颗粒透射电镜照片(放大倍数：135000)。
具体实施方式
在以下的实施例中提供了本发明的示例性的实施方案。以下的实施例仅通过示例的方式给出，并用于帮助普通技术人员使用本发明。所述实施例并不能以任何方式来限制本发明的范围。
实施例1重组EV71酵母表达菌株在30L发酵罐中发酵培养
将重组的汉逊酵母菌株(制备方法参见中国专利ZL201310179673.3)接种到150ml一级种子培养基(0.67％酵母氮源培养基，购自SIGMA公 司，0.5％硫酸铵，2％葡萄糖)中，在33℃，200rpm摇床振荡培养20～24小时。然后全量接种于1500ml二级种子培养基(0.67％酵母氮源培养基，0.5％硫酸铵，2％甘油)中，在33℃，200rpm摇床振荡培养20～24小时(OD600nm达8-10)。之后全量接种至30L发酵罐中，其中装有15L发酵培养基(甘油、磷酸二氢铵、氯化钾、氯化钙、氯化钠、硫酸镁、乙二胺四乙酸钠，其质量比为140:70:20:15:2:1)，氨水调节发酵液pH值维持在5.0，发酵温度为30℃，转速控制在350-750rpm，空气流速0.5-1.0m3/h，高密度发酵需要纯氧补充，溶氧控制在20-60％，20～24h时发酵培养基中碳源耗尽，补加甘油共计2.0L，分5次补加0.40L/次，每次碳源耗尽，溶氧上升时补加甘油，菌体生长共计36～39h左右，湿菌重最高可达0.3-0.4g/ml左右；去阻遏阶段：转速750rpm，空气流速1.0m3/h，溶氧控制在20-60％，加入1L甘油和甲醇混合液(甘油200ml，甲醇800ml)进行去阻遏培养，36-54h(共计15-18h)之间；诱导阶段：54-96h(36-42h)之间进行甲醇诱导，溶氧维持在20～40％左右。发酵结束：92-96h时，待甲醇消耗完全，溶氧上升至80％以上，降温至20℃开始下罐发酵结束，湿菌重维持在0.3-0.4g/ml。
汉逊酵母表达的EV71病毒样颗粒的鉴定：取上述不同发酵时间(44、48、52、56、60、64、68、72、76、80、84、88、92、96小时)的样品进行蛋白质免疫印迹(Western blot)检测，用抗EV71-VP1单克隆抗体(京天成生物技术(北京)有限公司，)作为一抗，使用HRP-羊抗鼠-IgG(北京博奥森公司)作为二抗，DAB显色，结果如图1所示。
Western-blot结果显示：表达产物能与单克隆抗体特异性结合，并在32.7KD处有较为明显的反应条带，说明该表达产物具有良好的免疫反应性。
重组EV71病毒样颗粒发酵表达量测定：将兔多抗稀释1000倍，取100μl加样于96孔酶标板孔内，4℃包被过夜。除净包被液，加满洗涤液PBST洗涤酶标板。用封闭液(1％BSA的PBST液)加满酶标板37℃孵 育2小时。将破碎后离心收集的上清液和国家抗原标准品进行梯度系列稀释，除净封闭液每孔加入100μl待检样品和标准品，37℃孵育1小时。加满洗涤液PBST洗涤酶标板3次。每孔加入100μl HRP标记的鼠单抗(1:1000稀释)37℃孵育1小时。除净酶标液，加满洗涤液PBST洗涤酶标板3次。每孔加入100μl TMB显色液，37℃避光作用15分钟。每孔加入50μl 2mol/L H2SO4终止。用酶标仪测定OD450nm值，并采用双平行线法计算抗原含量。测定结果如表1所示。
表1 发酵破碎液抗原含量(ELISA法)检测结果
Figure PCTCN2016112636-appb-000001
ELISA测定结果显示：发酵细胞破碎液中EV71病毒样颗粒的抗原含量43×104U/ml。汉逊酵母细胞表达的EV71具有较高的表达量。
实施例2EV71类病毒颗粒的分离纯化
细胞收集：收集发酵液，6000rpm离心收集沉淀，采用细胞洗涤缓冲液(20mmol/L NaH2PO4，2mmol/L EDTA-Na2，0.4mol/L NaCl，pH 7.4)洗2次，洗涤细胞两次。
破碎：将收集得到的汉逊酵母细胞，重悬于细胞裂解缓冲液中(20mmol/L NaH2PO4，2mmol/L EDTA-Na2，400m mol/L NaCl，2mmol/L PMSF，0.1％～1％Tween-20，pH 7.4)，使用高压匀浆机在压力1200bar的条件下破碎细胞2次，细胞破碎率达80％以上。
澄清：将破碎后的细胞液倒入离心筒中，进行6000～8000rpm离心40～60min，收集上清液。
沉淀病毒样颗粒：将上述收集的上清液中加入硫酸铵，至硫酸铵浓 度为22％～28％左右，放置过夜。
复溶：将上述沉淀液倾去上清，沉淀液采用10000rpm离心40～60min，收集沉淀加入复溶缓冲液(20mmol/L NaH2PO4，0.01～1mol/L NaCl,pH7.4))，搅拌30～60min，离心10000rpm离心40～60min，收集复溶上清液。
超滤：将收集的复溶上清液以300KD的膜包采用50mmol/L Tris(pH8.0)进行超滤以除掉小分子物质，收集超滤液，即为粗纯蛋白液。
离子交换层析：以Capto Q层析介质为例，采用50mmol/L Tris(pH8.0)平衡5～10个柱体积后上样，采用洗脱液(50mmol/L Tris-NaCl(150～300mmol/L))洗脱，收集洗脱液UV280nm紫外吸收峰，即为一步层析蛋白液，采用1mol/L NaOH再生层析介质，纯化图谱如图2所示。
分子筛层析：采用Sephacryl S-300HR填料，以10-50mmPB(pH6.8-7.2)和0.1-0.3M NaCl溶液和吐温-80(0.01％-0.1％)进行纯化一步层析蛋白液，收集UV280nm紫外吸收峰即为目的蛋白液，采用HPLC检测蛋白液纯度为100％，纯化图谱如图3所示。采用含吐温-80洗脱液洗脱蛋白得到的目的蛋白液稳定性更好，4℃存放12个月未有沉淀产生。
羟基磷灰石层析：一步层析蛋白液以300KD的膜包采用10mmol/L PBS(pH6.8)进行超滤以除掉小分子物质，收集超滤液。以10mmol/L PBS(pH6.8)平衡5～10个柱体积后上样，采用洗脱液(30～100mmol/L)PBS洗脱，收集洗脱液UV280nm紫外吸收峰，即为目的蛋白液。
目的蛋白浓度检测(Lowry法)：精确量取标准蛋白牛血清白蛋白溶液(200μg/ml)0ml、0.2ml、0.4ml、0.6ml、0.8ml、1.0ml，分别置于试管中，加蒸馏水补至1ml，同时量取5倍稀释纯化蛋白液1ml于试管中，分别加5ml碱性铜液，0.5ml酚试剂，于比色杯中用650nm波长测定吸光度值。以标准蛋白的蛋白含量做为横坐标，吸光度值为纵坐标绘制标准曲线，计算待检测蛋白液浓度。检测结果如表2所示。
表2 纯化蛋白浓度(Lowry法)检测结果
Figure PCTCN2016112636-appb-000002
Lowry法检测结果获得纯化蛋白终浓度为948μg/ml。
目的蛋白液动态光散射分析：取经纯化的EV71蛋白液适量加入样品池中，设置温度25℃，平衡时间90s，设置为自动循环次数，开始测量，对结果分析。结果表明，重组EV71类病毒颗粒完整，颗粒直径在24～30nm分布大于99％，动态光散射图谱如图4所示。
目的蛋白液电镜分析：取经纯化的EV71蛋白液适量滴加于铜网上，避光保存5min，除净多余液体，用1％磷钨酸染色2min，通过透射电镜(TEM)对EV71VLPs进行分析。结果表明EV71蛋白呈现病毒样颗粒，具有天然病毒的正二十面体结构，病毒颗粒直径在30nm左右，颗粒完整规则(如图5所示，放大倍数：135000)。
目的蛋白液抗原含量测定(双抗夹心ELISA)：将兔多抗稀释5000倍，取100μl加样于96孔酶标板孔内，4℃包被过夜。除净包被液，加满洗涤液PBST洗涤酶标板。用封闭液(1％BSA的PBST液)加满酶标板37℃孵育2小时。除净封闭液每孔加入100μl待检样品，37℃孵育1小时。加满洗涤液PBST洗涤酶标板3次。每孔加入100μl HRP标记的鼠单抗(1:1000稀释)37℃孵育1小时。除净酶标液，加满洗涤液PBST洗涤酶标板3次。每孔加入100μl TMB显色液，37℃避光作用15分钟。每孔加入50μl 2mol/L H2SO4终止。用酶标仪测定OD450nm值，并采用双平行线法计算抗原含量。双抗夹心ELISA检测结果如表3所示。
表3 双抗夹心ELISA检测结果
Figure PCTCN2016112636-appb-000003
实施例3重组EV71病毒样颗粒疫苗的制备
主要指标控制：抗原(EV71病毒样颗粒)含量为10μg/ml；铝含量控制在0.40-0.60mg/ml；pH值控制在6.6-7.0。
制备方法：将无菌的PBS溶液注入罐中，采用机械搅拌转速为500～700转/分，向罐中通空气保持压力0.05～0.1MPa，除菌过滤后的硫酸铝钾溶液缓慢的加入罐中，再将氢氧化钠溶液滴加到pH在6.6-7.0时停止。静置后，采用无菌的0.9％氯化钠溶液，置换上清液4-5次后，氢氧化铝佐剂制备完成。向佐剂中加入缓慢滴加纯化EV71原液，使之充分吸附，疫苗制备完成。制备疫苗检测标准及检测结果如表4所示。
表4 重组EV71病毒样颗粒疫苗的检测结果
Figure PCTCN2016112636-appb-000004
表4结果显示：重组EV71病毒样颗粒疫苗检测指标均符合检测标准。
实施例4重组EV71病毒样颗粒疫苗的免疫原性(ED50)试验
试验疫苗：实施例3制备的含有氢氧化铝佐剂的重组EV71类病毒颗粒疫苗。
试验动物：挑选18-22g的SPF级BALB/c小鼠50只，购自北京维通利华实验动物技术有限公司。
动物免疫：将倍比稀释好的0.5、0.125、0.03125、0.0078125μg/0.5ml的疫苗、氢氧化铝佐剂，分别注射10只小鼠，每只腹腔注射0.5ml，免疫28天后摘眼球取血。将采集的血液置37℃放置1h，4℃静置3-4小时，4000rpm离心10min，吸取上清待检。
中和抗体检测：将血清样本采用2％新生牛血清的MEM培养液以1：8比例稀释，56℃水浴锅灭能30分钟。采用96孔细胞培养板，每孔加入50μl稀释液。每孔加入相应样品50μl，排枪由A行混匀后吸50μl至B行混匀，依次混匀稀释至D行弃取50μl(将EV71病毒攻击毒种系列稀释至100CCID50/0.05ml，取50μl垂直悬滴入每孔，将细胞培养板轻拍混匀，至37℃中和两小时。用消化液消化RD细胞(细胞提前复苏扩增)，制备浓度为2×105个/ml的细胞悬液，每孔(包括病毒回滴孔)分别加入0.1ml细胞悬液，混匀，放入35℃CO2孵箱中孵育培养。使用倒置显微镜每天观察CPE，并记录病毒滴定结果，以抑制50％细胞病变的血清最高稀释度的倒数为终点效价。6～7天判定最终结果。根据检测结果，抗体阳转率如表5所示。
表5 抗体阳转率计算结果
Figure PCTCN2016112636-appb-000005
根据Reed-Muench法计算：ED50＝0.011(μg)
由小鼠ED50实验结果可见：本发明制备的EV71病毒样颗粒疫苗仅0.011μg免疫小鼠后就能使抗体阳转率达到50％，因此本发明的EV71病毒样颗粒具有较强的免疫原性。
实施例5重组EV71病毒样颗粒疫苗异常毒性试验
疫苗样品：实施例3制备的重组EV71病毒样颗粒疫苗。
实验动物：18-22g的SPF级KM小鼠5只，250-350g的SPF级Hartley豚鼠2只，均购自北京维通利华实验动物技术有限公司。
实验方法：注射前称量每只实验动物体重，小鼠为18-22g，豚鼠为250-350g。疫苗注射5只小鼠和2只豚鼠，小鼠腹腔注射0.5ml/只，豚鼠腹腔注射5.0ml/只，观察7天。同时设同批动物空白对照。合格标准：观察期内空白对照和实验组动物健存，无异常反应，到期每只动物体重增加。动物试验情况如表6所示。
表6 动物实验情况
Figure PCTCN2016112636-appb-000006
Figure PCTCN2016112636-appb-000007
结论：观察期内空白对照和实验组动物健存，无异常反应，第8天每只动物体重均增加。证明重组EV71病毒样颗粒疫苗无异常毒性，试验动物安全性较好。
工业实用性
本发明提供一种EV71病毒样颗粒的纯化方法及利用纯化后的EV71病毒样颗粒制备疫苗的方法。通过将重组表达的工程菌高密度发酵培养和甲醇诱导表达EV71类病毒颗粒蛋白，离心收集菌体进行高压均浆破碎，上清液经硫酸铵沉淀，复溶、离子交换层析、分子筛层析和羟基磷灰石层析等纯化后获得。本发明提供的EV71类病毒颗粒疫苗具有良好的免疫原性、安全性、免疫特性和生物学活性，工艺简单，不使用超速离心机大型设备，更易于大规模制备和纯化，可用于制备预防EV71感染的疫苗，具有较好的经济价值和应用前景。

Claims (10)

  1. 重组EV71病毒样颗粒的纯化方法，其特征在于，包括以下步骤：
    (1)将含EV71外壳蛋白P1基因和3CD蛋白酶基因的重组汉逊酵母工程菌发酵；
    (2)破碎工程菌，目的产物沉淀、复溶、超滤；
    (3)离子交换层析；
    (4)分子筛层析或羟基磷灰石层析。
  2. 如权利要求1所述的纯化方法，其特征在于，步骤(2)破碎工程菌的方法为将工程菌采用细胞裂解缓冲液重悬，使用高压匀浆机在压力1100～1400bar的条件下破碎细胞2-4次；所述细胞裂解缓冲液为20mM NaH2PO4，2mM EDTA-Na2，0.2-1.0M NaCl，2mM PMSF，0.1％～1％Tween-80，pH6.8～7.4。
  3. 如权利要求1所述的纯化方法，其特征在于，步骤(2)目的产物沉淀方法为：将破碎后的细胞液倒入离心筒中，进行6000～8000rpm离心40～60min，收集上清液，将收集的上清液中加入硫酸铵浓度至终浓度为20～28％。
  4. 如权利要求3所述的纯化方法，其特征在于，步骤(2)的复溶为：将硫酸铵沉淀产物采用8000～10000rpm离心40～60min，收集沉淀，并加入复溶缓冲液，搅拌30～60min，8000～10000rpm离心45～60min，收集上清液；所述复溶缓冲液为20～50mM NaH2PO4，0.1～1M NaCl，pH6.8～7.4。
  5. 如权利要求1所述的纯化方法，其特征在于，步骤(2)的超滤为：复溶后收集的上清液以100～500KD的膜包采用pH7.5～8.5、20～50mM Tris进行超滤以除掉小分子物质，收集超滤液，得到粗纯蛋白液。
  6. 如权利要求1-5任一所述的纯化方法，其特征在于，步骤(3)的离子交换层析为：采用pH7.5～8.5、20～50mM Tris平衡5～10个柱体积后上样，采用20～50mM Tris和150～300mM NaCl洗脱液洗脱，收集洗脱液UV280nm紫外吸收峰，得到一步层析蛋白液。
  7. 如权利要求6所述的纯化方法，其特征在于，步骤(4)的分子筛层析方法为，将步骤(3)获得的一步层析蛋白液，以pH6.8-7.2的10-50mmPB和0.1-0.3M NaCl溶液和0.01％-0.1％的吐温-80进行纯化一步层析蛋白液，收集UV280nm紫外吸收峰即为目的蛋白液。
  8. 如权利要求6所述的纯化方法，其特征在于，步骤(4)的羟基磷灰石层析其方法为：将步骤(3)获得的一步层析蛋白液以100～500KD的膜包采用pH6.8～7.4、10～50mM PBS进行超滤以除掉小分子物质，收集超滤液；以pH6.8～7.4、10～50mM PBS平衡5～10个柱体积后上样，采用30～200mM PBS洗脱，即得目的蛋白液。
  9. 权利要求1-8任一所述的纯化方法在制备手足口疫苗中的应用。
  10. 一种手足口病疫苗，其特征在于，按照权利要求1-8任一所述的纯化方法纯化重组EV71类病毒颗粒后，经氢氧化铝佐剂吸附制成疫苗，EV71蛋白含量为4～20μg/ml。
PCT/CN2016/112636 2016-12-28 2016-12-28 重组ev71病毒样颗粒的纯化及其疫苗制备方法 WO2018119746A1 (zh)

Priority Applications (3)

Application Number Priority Date Filing Date Title
CN201680088253.6A CN109689862B (zh) 2016-12-28 2016-12-28 重组ev71病毒样颗粒的纯化及其疫苗制备方法
US16/333,290 US10987416B2 (en) 2016-12-28 2016-12-28 Purification of recombinant EV71 virus-like particle and method for preparing vaccine thereof
PCT/CN2016/112636 WO2018119746A1 (zh) 2016-12-28 2016-12-28 重组ev71病毒样颗粒的纯化及其疫苗制备方法

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2016/112636 WO2018119746A1 (zh) 2016-12-28 2016-12-28 重组ev71病毒样颗粒的纯化及其疫苗制备方法

Publications (1)

Publication Number Publication Date
WO2018119746A1 true WO2018119746A1 (zh) 2018-07-05

Family

ID=62706979

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2016/112636 WO2018119746A1 (zh) 2016-12-28 2016-12-28 重组ev71病毒样颗粒的纯化及其疫苗制备方法

Country Status (3)

Country Link
US (1) US10987416B2 (zh)
CN (1) CN109689862B (zh)
WO (1) WO2018119746A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111187720A (zh) * 2019-12-27 2020-05-22 深圳康泰生物制品股份有限公司 表达肠道病毒71型抗原的重组汉逊酵母的细胞破碎方法及其在制备手足口病疫苗中的应用
CN111534492A (zh) * 2020-04-28 2020-08-14 江苏金迪克生物技术股份有限公司 一种人用狂犬病疫苗中宿主dna去除方法

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111197109A (zh) * 2019-12-27 2020-05-26 深圳康泰生物制品股份有限公司 重组肠道病毒71型病毒样颗粒粒径检测方法及其在制备手足口病疫苗中的应用
CN111778168B (zh) * 2020-06-19 2022-06-03 北京民海生物科技有限公司 高效表达ca10病毒样颗粒的汉逊酵母工程菌及其应用
CN113670914B (zh) * 2021-08-19 2024-03-08 青岛易邦生物工程有限公司 一种油乳剂灭活疫苗的细菌内毒素检测方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103255163A (zh) * 2013-05-15 2013-08-21 北京民海生物科技有限公司 一种ev71病毒样颗粒及其制备方法与应用
CN104673760A (zh) * 2013-11-29 2015-06-03 江苏先声药业有限公司 一种原核细胞表达类病毒颗粒的纯化方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103740656A (zh) * 2014-01-22 2014-04-23 大连汉信生物制药有限公司 重组汉逊酵母戊型肝炎类病毒颗粒的纯化方法及应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103255163A (zh) * 2013-05-15 2013-08-21 北京民海生物科技有限公司 一种ev71病毒样颗粒及其制备方法与应用
CN104673760A (zh) * 2013-11-29 2015-06-03 江苏先声药业有限公司 一种原核细胞表达类病毒颗粒的纯化方法

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111187720A (zh) * 2019-12-27 2020-05-22 深圳康泰生物制品股份有限公司 表达肠道病毒71型抗原的重组汉逊酵母的细胞破碎方法及其在制备手足口病疫苗中的应用
CN111534492A (zh) * 2020-04-28 2020-08-14 江苏金迪克生物技术股份有限公司 一种人用狂犬病疫苗中宿主dna去除方法

Also Published As

Publication number Publication date
CN109689862B (zh) 2022-09-30
US10987416B2 (en) 2021-04-27
US20190240314A1 (en) 2019-08-08
CN109689862A (zh) 2019-04-26

Similar Documents

Publication Publication Date Title
WO2018119746A1 (zh) 重组ev71病毒样颗粒的纯化及其疫苗制备方法
CN109680026B (zh) 重组ca16病毒样颗粒的纯化、在疫苗中的应用及疫苗
US9782471B2 (en) EV71 virus-like particles and preparation method and application thereof
WO2021253645A1 (zh) 高效表达ca10病毒样颗粒的汉逊酵母工程菌及其应用
CN109750054B (zh) 一种牛支原体蛋白基因MbovGdpP及其应用
CN104099301B (zh) 柯萨奇病毒a16型病毒株、用途、疫苗及其制备方法
AU9004798A (en) An attenuated japanese encephalitis virus adapted to vero cell and a japanese encephalitis vaccine
CN111420044B (zh) 一种四价流感病毒亚单位疫苗及其制备方法
CN111789941B (zh) 一种山羊支原体肺炎、鹦鹉热衣原体病的二联灭活疫苗及其制备方法
CN107973841B (zh) Cho细胞表达的重组牛病毒性腹泻病毒e2蛋白及亚单位疫苗的制备方法和应用
WO2023246639A1 (zh) 柯萨奇病毒a6型毒株及其免疫原性组合物和应用
WO2023246621A1 (zh) 柯萨奇病毒a10型毒株及其应用
KR101617464B1 (ko) Ｉｐｖ―ｄｐｔ 백신
CN106167518A (zh) 截短的轮状病毒vp4蛋白及其用途
WO2024125100A1 (zh) 一种ev71-ca16二价灭活疫苗及其制备方法和应用
US9862933B2 (en) Method for in-vitro preparation of double-layered virus-like particles of rotavirus
WO2000020565A1 (fr) Immunogene ameliore pour un vaccin inactive contre une infection a virus de l'encephalite japonaise et procede de production associe
CN111789942B (zh) 一种绵羊支原体肺炎、鹦鹉热衣原体病的二联灭活疫苗及其制备方法
CN109602899B (zh) 一种增强病毒抗原免疫原性的皮内注射技术及其在手足口病疫苗研究中的应用
CN108715607B (zh) 重组霍乱毒素b亚基蛋白、pedv灭活疫苗及制备与应用
KR101942626B1 (ko) 노다바이러스 캡시드 단백질의 정제 수율 증가 방법
CN1112934C (zh) 流行性出血热传代细胞多价纯化疫苗
CN118267463A (zh) 手足口病疫苗及其制备方法和应用
CN116350773A (zh) 手足口病疫苗及其制备方法和应用
CN118272316A (zh) 手足口病疫苗及其制备方法和应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16925278

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 16925278

Country of ref document: EP

Kind code of ref document: A1