CN103740656A - Method for purifying recombined hansenula polymorpha hepatitis E virus-like particles and application - Google Patents
Method for purifying recombined hansenula polymorpha hepatitis E virus-like particles and application Download PDFInfo
- Publication number
- CN103740656A CN103740656A CN201410029495.0A CN201410029495A CN103740656A CN 103740656 A CN103740656 A CN 103740656A CN 201410029495 A CN201410029495 A CN 201410029495A CN 103740656 A CN103740656 A CN 103740656A
- Authority
- CN
- China
- Prior art keywords
- hepatitis
- culture
- concentration
- volume
- virus
- Prior art date
- Legal status (The legal status 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 status listed.)
- Pending
Links
Images
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a method for purifying recombined hansenula polymorpha hepatitis E virus-like particles and application. The method comprises the following steps: cultivating and fermenting working seed lot recombined hepatitis E vaccine engineering strains, and collecting a fermented culture; sequentially performing cell disruption, clarification, ultra-filtration, silica gel adsorption, ultra-filtration and concentration liquid change, chromatographic purification, desalination, disinfection and filtration on the culture to obtain the purified recombined hansenula polymorpha hepatitis E virus-like particles. The size of the purified recombined hansenula polymorpha hepatitis E virus-like particles is in accordance with the theoretical size of natural hepatitis E virus particles and is 27-34nm, a gene sequence is in accordance with a theoretical gene sequence, the expressed foreign protein molecular weight is in accordance with the expected target protein size, and the recombined hansenula polymorpha hepatitis E virus-like particles are good in immunogenicity. The invention also discloses the application of the recombined hansenula polymorpha hepatitis E virus-like particles in preparation of vaccine. The recombined hansenula polymorpha hepatitis E virus-like particles are mixed with an aluminum adjuvant to form the vaccine. An animal immunogenicity detection result shows that the vaccine prepared from the recombined hansenula polymorpha hepatitis E virus-like particles is good in immunogenicity.
Description
Technical field
The invention belongs to medical bioengineering technical field, be specifically related to a kind of purification process of the debaryomyces hansenii hepatitis E viruslike particle of recombinating and prepare the application of vaccine.
Background technology
Hepatitis E (HepatitisE, HE) is the popular intestinal tract diseases of a kind of acute region, mainly by fecal oral route, is propagated, and hepatitis E mostly is acute onset, generally can not develop into chronic hepatitis.Viral hepatitis type E can account for 50% of acute viral hepatitis as a kind of infectious intestinal disease in some developing countries.Hepatitis E clinical symptom is substantially identical with hepatitis A, and patient's main manifestations is jaundice, apocleisis, hepatomegaly, abdominal pain, feels sick, the symptoms such as vomiting and fever, and mortality ratio is about 1%, higher than hepatitis A.
The Identification of etiology for the first time of hepatitis E is that nineteen eighty-three Balayan etc. obtains by a volunteer's research, be the scholars such as Balayan utilize immunoelectronmicroscopy patient's acute phase ight soil and convalescence serum in find the nonenveloped virus particle of a diameter 27nm (scope of 27-34nm).But the cell cultures of hepatitis E virus and tissue culture are all unsuccessful, do not find yet the means that obtain a large amount of viruses of enforcement at present.Along with people are in recent years to the deepening continuously with perfect of hepatitis E virus research, also more and more clear for hepatitis E virus genomic expression, the recombinant vaccine of hepatitis E virus becomes the emphasis of bio-science field research.In recombinant vaccine, viruslike particle (Virus Like Particle, VLP) is than soluble antigen vaccine, because of immunogenicity and security higher, the first candidate of Chang Zuowei vaccine.
The expression system of succeeding in developing at present the recombined hepatitis E hepatitis vaccine employing of listing in worldwide is protokaryon escherichia expression system, the hepatitis E virus antigen of expressing could form hepatitis E viroid sample particle by external suitable assembling condition, the shortcoming of prokaryotic expression system be express recombinant protein cannot process modification, affect its biological activity.
Summary of the invention
The present invention is directed to above-mentioned the deficiencies in the prior art, the purifying of a kind of restructuring debaryomyces hansenii hepatitis E viruslike particle (HEV VLPs) and the preparation method of vaccine are provided, the viruslike particle that this expressed by Hansenula yeast system can directly be expressed, formation is consistent with natural hepatitis E virus granular size, by a series of purifying process, obtain hepatitis E viruslike particles, and be prepared into and there is fine immunogenic vaccine.
Technical scheme of the present invention is as follows:
The recombinate purification process of debaryomyces hansenii hepatitis E viruslike particle, comprises the following steps:
(1) work seed lot recombined hepatitis E hepatitis vaccine engineering strain is carried out to cultivation and fermentation, collect the culture after fermentation;
(2) to the culture after the fermentation of collecting in step (1) successively carry out cytoclasis, clarification and ultrafiltration, silica gel adsorption, ultrafiltration and concentration changes liquid, chromatogram purification and desalination, Sterile Filtration, makes the restructuring debaryomyces hansenii hepatitis E viruslike particle of purifying.
The target protein that described work seed lot recombined hepatitis E hepatitis vaccine engineering strain is expressed is HEV gene type-IV infection structural area ORF2 proteins encoded 112-6O7 amino acid fragment.
The size of described restructuring debaryomyces hansenii hepatitis E viruslike particle is 27-34nm.
The concrete grammar of the cultivation and fermentation described in step (1) is: work seed lot recombined hepatitis E hepatitis vaccine engineering strain is inoculated in the yeast nitrogen substratum of 200ml by 1%, carry out one-level cultivation, when detect one-level culture absorbance A with ultraviolet visible spectrophotometry
600nmduring for 10-20, by culture culture transferring to the yeast nitrogen substratum of 2L, when detect secondary culture absorbance A with ultraviolet visible spectrophotometry
600nmduring for 10-18, culture culture transferring is carried out to cultivation and fermentation, methanol induction expression to glycerin medium, collect the culture after fermentation;
Preferably, the temperature that described one-level is cultivated is 30-33 ℃, and incubation time is 20h;
Preferably, the temperature that described secondary is cultivated is 30-33 ℃, and incubation time is 20h;
Preferably, the culture temperature in glycerin medium is 30 ℃, and incubation time is 13-15h, and volume of culture is 15-25L, then with the speed stream of final concentration 1%, adds methyl alcohol and carries out inducing culture 75-80h.
Described cytoclastic concrete grammar is: the culture after the fermentation of collection is carried out to centrifugal 20min with the rotating speed of 4500 turn/min, in yeast cell after centrifugation, add PB damping fluid, making cell concn is 50%, after fully mixing, with similarity condition centrifuging and taking, precipitate again, in centrifugation, add cytoclasis liquid, making cell concn is 30%, add again 1% long-pending Tween-80 of cytoclasis liquid, the NaCl of 0.1M/L, with the fragmentation of granulated glass sphere polishing, the PMSF solution that the concentration that adds its volume 4% in broken liquid is 1%, it is 8.0 that the NaOH of 1M/L regulates pH value, with the centrifugal 40-50min of rotating speed of 7000 turn/min, remove and precipitate to obtain supernatant liquor, the preferred 2-8 ℃ of centrifuging temperature.
Described clarification and the concrete grammar of ultrafiltration are: supernatant liquor is carried out to clarifying treatment cell debris by 0.65 μ m hollow fiber column, with 9-10 times of supernatant liquor volume of PB damping fluid flushing, under collection membrane, see through solution, to by 0.2 μ m hollow fiber column, carry out ultrafiltration and concentration through liquid, with PB damping fluid, rinse 5-6 doubly long-pending through liquid, on collection membrane, liquid is preserved; Described PB ph value of buffer solution preferably 8.0.
The concrete grammar of described silica gel adsorption is: on film, in liquid, add silica gel solution, and whip attachment, then remove supernatant liquor with the centrifugal 20min of rotating speed of 4000 turn/min; In precipitation, add the silica gel elutriant of former supernatant liquor volume 44%, in 57 ℃ of stirring 90min, then with the centrifugal 15min of rotating speed of 4000 turn/min, remove precipitation, in supernatant liquor, add sodium-chlor to mix, standing, with the centrifugal 10min of 7000 turn/min rotating speed, remove precipitation, collect supernatant liquor;
Preferably, the concentration of described silica gel solution is 7.5%, and the volume that adds of silica gel solution is the 40-50% of supernatant liquor volume;
Preferably, described whip attachment temperature is 2-8 ℃, and the whip attachment time is 10-16h, and described centrifuging temperature is 2-8 ℃;
Preferably, the volume of described sodium-chlor is 1/11 silica gel effluent volume, and the concentration of sodium-chlor is 4M/L;
Preferably, described dwell temperature is 2-8 ℃, and time of repose is 30min.
Described silica gel elutriant is comprised of the component that comprises following mass body volume concentrations:
Sodium tetraborate 3.814g/L,
EDTA 0.742g/L，
Sodium desoxycholate 2.5g/L.
The concrete grammar that described ultrafiltration and concentration changes liquid is: the supernatant liquor obtaining after silica gel adsorption operation is crossed to 750K hollow fiber column, 5-6 times of supernatant liquor volume of PB buffer solution for cleaning, on film, retain solution and be concentrated into protein concentration 2mg/ml-3mg/ml, collect and retain concentrated solution.
The concrete grammar of described chromatogram purification is: to retaining concentrated solution employing chromatographic media, be that Sepharose4FF carries out purifying, and loading, flow velocity is 20ml/min, and elutriant is PBS, and monitoring wavelength 280nm, collects A
280nmthe 1st elution peak; Adopting chromatographic media is DEAE Sepharose; Loading, flow velocity is 2-3ml/min, and elutriant is the mixed solution of PB and sodium-chlor, and monitoring wavelength 280nm, collects absorbance A
280nmbe greater than 0.5 elution peak;
Preferably, the volume of described loading is 2%-3% column volume, and the concentration of described PBS is 0.003mol/L, and the pH value of PBS is 8.0;
Preferably, the concentration of described PB is 50mmol/L, and described sodium chloride concentration is 0.5mol/L; Further preferred, described PB and the volume ratio of sodium-chlor are 1:(0.8-2.5).
The concrete grammar of described desalination is: adopt column chromatography desalination, chromatographic media is Sepharose4FF, loading, and flow velocity is 20ml/min, and elutriant is PBS, and monitoring wavelength 280nm, collects A
280nmfrom 0.5 to 0.3 elution peak;
Preferably, the volume of described loading is 4%-8% column volume, and the concentration of described PBS is 0.003mol/L, and the PH of PBS is 7.0.
Above-mentioned restructuring debaryomyces hansenii hepatitis E viruslike particle is prepared an application for vaccine, comprises the following steps:
In the restructuring debaryomyces hansenii hepatitis E viruslike particle of purifying, add the aluminium hydroxide solution through degerming, shake up absorption, make hepatitis E virus vaccine work in-process; The preferred 1.0mg/ml of concentration of described aluminium hydroxide solution, aluminum ions concentration 0.5mg/ml in described hepatitis E virus vaccine work in-process.
Compared with prior art, beneficial effect of the present invention has:
(1) restructuring debaryomyces hansenii hepatitis E virus (HEV) viruslike particle of purifying in the present invention is carried out to gene order order-checking, sequencing result is with theoretical consistent; The vaccine making in the present invention is carried out to amino acid sequencing, and sequencing result is with theoretical consistent.
(2) SDS-PAGE detected result shows, the foreign protein molecular weight that the restructuring debaryomyces hansenii hepatitis E viruslike particle of purifying is expressed is 55kD, the target protein of molecular weight and expection is in the same size, the copy number of hepatitis E virus gene is higher, the expression amount of target protein is higher, more than the foreign gene of the high expression level bacterial strain that screening obtains is two copies.
(3) Western blot detected result shows, the target protein of specific reaction band molecular size range and expection is in the same size, without nonspecific reaction band.
(4) by phospho-wolframic acid negative staining electron microscopic observation, find, the copy number of hepatitis E virus gene affects hepatitis E virus sample and forms size, it is consistent with natural hepatitis E virus granular size that bacterial strain single, two copies hepatitis E virus gene is expressed the viruslike particle forming, and the above virus-like particle size of 4 copy is obviously greater than natural hepatitis E virus sample particle.
(5) the active detected result of hepatitis E viruslike particle ELISA purifying being obtained, 10,000 times of dilutions of cytoclasis liquid, OD value more than 1.0, illustrates that the hepatitis E viruslike particle that purifying obtains has good immunoreactivity.
(6) hepatitis E viruslike particle and aluminium adjuvant are configured to animal immune originality detected result, mouse immune ED50(median effective dose) be 0.3 μ g, show to have good immunogenicity.
Accompanying drawing explanation
Fig. 1 is the expressed hepatitis E viroid sample particle electron microscopic observation picture of recombined hepatitis E hepatitis vaccine engineering strain in embodiment;
Fig. 2 is recombined hepatitis E hepatitis vaccine engineering strain expression product SDS-PAGE analytical results figure in embodiment;
Fig. 3 is for being SDS-PAGE analytical results figure after recombined hepatitis E hepatitis vaccine engineering strain expression product purifying in embodiment;
Fig. 4 is Western blot analytical results figure after recombined hepatitis E hepatitis vaccine engineering strain expression product purifying;
Fig. 5 be in embodiment after recombined hepatitis E hepatitis vaccine engineering strain expression product purifying HPLC analyze collection of illustrative plates;
Wherein, in Fig. 2,1 is before HEV debaryomyces hansenii engineering strain is induced, 2 is HEV debaryomyces hansenii engineering strain 30L tank fermentation thalline, and 3 is HEV prokaryotic expression positive control, and 4 is prokaryotic expression negative control, 5-is the little shaking flask abduction delivering of HEV debaryomyces hansenii engineering strain 20ml thalline, 6 is HEV debaryomyces hansenii engineering strain 30L tank fermentation bacterial cell disruption liquid, and 7 is debaryomyces hansenii negative control, and 8 is albumen Marker;
In Fig. 3,1 is Marker, and 2,3 is that Isosorbide-5-Nitrae-7,4FF target protein peak are DEAE wash-out object peak;
In Fig. 4,1 is Marker, and 2 is the HEVVLPs after purifying, 3 negative contrasts.
Embodiment
Below in conjunction with embodiment, the present invention will be further described.Work seed lot recombined hepatitis E hepatitis vaccine engineering strain in embodiment can build according to disclosed method in < < biotechnology journal > > the 23rd the 1st phase of volume 73-78 page paper January in 2007 " expression of hepatitis E virus IV type ORF2 albumen in debaryomyces hansenii ".
The recombinate purification process of debaryomyces hansenii hepatitis E viruslike particle, comprises the following steps:
(1) cultivation and fermentation: work seed lot recombined hepatitis E hepatitis vaccine engineering strain is inoculated in the yeast nitrogen substratum of 200ml by 1%, carries out one-level and cultivate 20h under 30 ℃ of conditions, with ultraviolet visible spectrophotometry detection one-level culture absorbance A
600nmbe 10 o'clock, culture culture transferring is carried out to secondary cultivation 20h to the yeast nitrogen substratum of 2L under 30 ℃ of conditions, with ultraviolet visible spectrophotometry detection secondary culture absorbance A
600nmbe 10 o'clock, by culture culture transferring to glycerin medium, 30 ℃ of culture temperature, incubation time 15h, volume of culture is 15L, then with the speed stream of final concentration 1%, add methyl alcohol and carry out inducing culture 80h, collect fermentation after culture;
(2) to the culture after the fermentation of collecting in step (1) successively carry out cytoclasis, clarification and ultrafiltration, silica gel adsorption, ultrafiltration and concentration changes liquid, chromatogram purification and desalination, Sterile Filtration, makes the restructuring debaryomyces hansenii hepatitis E viruslike particle of purifying.
Cytoclasis: the culture after the fermentation of collection is carried out to centrifugal 20min with the rotating speed of 4500 turn/min at 2 ℃, in yeast cell after centrifugation, add PB damping fluid, making cell concn is 50%, after fully mixing, with similarity condition centrifuging and taking, precipitate again, in centrifugation, add cytoclasis liquid, making cell concn is 30%, add again 1% long-pending Tween-80 of cytoclasis liquid, the NaCl of 0.1M/L, with the fragmentation of granulated glass sphere polishing, the PMSF solution that the concentration that adds its volume 4% in broken liquid is 1%, it is 8.0 that the NaOH of 1M/L regulates pH value, with the centrifugal 50min of rotating speed of 7000 turn/min, remove and precipitate to obtain supernatant liquor,
Clarification and ultrafiltration: supernatant liquor is carried out to clarifying treatment cell debris by 0.65 μ m hollow fiber column, the PB damping fluid that is 8.0 with PH rinses 9 times of supernatant liquor volumes, under collection membrane, see through solution, to by 0.2 μ m hollow fiber column, carry out ultrafiltration and concentration through liquid, the PB damping fluid that is 8.0 with PH rinses 5 times and amasss through liquid, and on collection membrane, liquid is preserved;
Silica gel adsorption: the concentration that adds the 40-50% of supernatant liquor volume on film in liquid is 7.5% silica gel solution, whip attachment 10h at 2 ℃, then remove supernatant liquor at 2 ℃ of centrifugal 20min of the rotating speed with 4000 turn/min; In precipitation, add the silica gel elutriant of former supernatant liquor volume 44%, 1L contains containing sodium tetraborate 3.814g, EDTA0.742g, Sodium desoxycholate 2.5g, in 57 ℃, stir 90min, then with the centrifugal 15min of rotating speed of 4000 turn/min, remove precipitation, in supernatant liquor, add the NaCl that 1/11 silica gel eluting liquid volume concentrations is 4M/L to mix, standing 30min at 2 ℃, with the centrifugal 10min of 7000 turn/min rotating speed, remove precipitation, collect supernatant liquor;
Ultrafiltration and concentration changes liquid: the supernatant liquor obtaining after silica gel adsorption operation is crossed to 750K hollow fiber column, and 5 times of supernatant liquor volumes of PB buffer solution for cleaning, retain solution and be concentrated into protein concentration 2mg/ml on film, collects and retains concentrated solution;
Chromatogram purification: be that Sepharose4FF carries out purifying to retaining concentrated solution employing chromatographic media, with 2% column volume loading, flow velocity is 20ml/min, and elutriant concentration is that 0.003mol/LPH value is 8.0 PBS, and monitoring wavelength 280nm, collects A
280nmthe 1st elution peak; Adopting chromatographic media is DEAE Sepharose; With 3% column volume loading, flow velocity is 3ml/min, and elutriant is that volume ratio is the PB of 1:0.8 and the mixed solution of sodium-chlor, and the concentration of PB is 50mmol/L, and the concentration of sodium-chlor is 0.5mol/L, and monitoring wavelength 280nm, collects absorbance A
280nmbe greater than 0.5 elution peak;
Desalination: adopt column chromatography desalination, chromatographic media is Sepharose4FF, and with 4% column volume loading, flow velocity is 20ml/min, and elutriant is that concentration is that 0.003mol/LPH is 7.0 PBS, monitoring wavelength 280nm, collects A
280nmfrom 0.5 to 0.3 elution peak;
The detection of the restructuring debaryomyces hansenii hepatitis E viruslike particle of purifying:
Electron microscopic observation: the former drop of restructuring debaryomyces hansenii hepatitis E viruslike particle that takes a morsel is in copper mesh, and 2% phospho-wolframic acid negative staining 3min, is dried 4min under room temperature, and sample is placed under transmission electron microscope (H-7000FA) and observes.
ELISA detects: with double antibody sandwich ELISA, detect, concrete steps are as follows: 4 ℃ of coated E type hepatitis virus monoclonal antibodies spend the night; 0.5% 37 ℃ of bovine serum albumins sealing lh; Add 37 ℃ of the cytoclasis supernatant liquors (establishing the positive, feminine gender, blank) of gradient doubling dilution to hatch lh; Add again 37 ℃ of anti-hepatitis E virus ORF2 sheep polyclonal antibodies to hatch lh; Then add the anti-sheep IgG of rabbit of HRP mark, hatch lh for 37 ℃; Add 1%TMB nitrite ion colour developing 10min, 2mol/L sulfuric acid termination reaction is measured ELISA and is reacted each hole OD on enzyme micro-plate reader
450nmvalue, P/N>=2.1, are judged as the positive, and A
450nm>=0.1.
SDS-PAGE detects (SDS-polyacrylamide gel electrophoresis): preparation 12% separation gel, 6% concentrated glue, sample is processed through NaOH, SDS Buffer, 4uL loading, point sample positive control and negative control simultaneously, under constant current, 12mA20min, 24mA1.5h, electrophoresis is complete, take off glue, coomassie brilliant blue staining, the expression amount of gel imaging system scanning analysis target protein.
Westernblot identifies: SDS-PAGE glue electrotransfer is to pvdf membrane, with the constant current transferase 12 h of 160mA; Sealing: the pvdf membrane after shifting is put to 37 ℃ of sealings in confining liquid PBST and spend the night; In conjunction with: add goat-anti HEV polyclonal antibody (1:500) as first antibody, in conjunction with 2h, then add the anti-sheep IgG of rabbit (1:1000) second antibody of HRP mark with 37 ℃ of pvdf membranes, 37 ℃ of effect lh; OPD develops the color until there is clear band, deionized water termination reaction.
Above-mentioned restructuring debaryomyces hansenii hepatitis E viruslike particle is prepared an application for vaccine, comprises the following steps:
In the restructuring debaryomyces hansenii hepatitis E viruslike particle of purifying, add the aluminium hydroxide solution through degerming, shake up absorption, make hepatitis E virus vaccine work in-process; The preferred 1.0mg/ml of concentration of described aluminium hydroxide solution, aluminum ions concentration 0.5mg/ml in described hepatitis E virus vaccine work in-process.
The recombinate purification process of debaryomyces hansenii hepatitis E viruslike particle, comprises the following steps:
(1) cultivation and fermentation: work seed lot recombined hepatitis E hepatitis vaccine engineering strain is inoculated in the yeast nitrogen substratum of 200ml by 1%, carries out one-level and cultivate 20h under 33 ℃ of conditions, with ultraviolet visible spectrophotometry detection one-level culture absorbance A
600nmbe 20 o'clock, culture culture transferring is carried out to secondary cultivation 20h to the yeast nitrogen substratum of 2L under 33 ℃ of conditions, with ultraviolet visible spectrophotometry detection secondary culture absorbance A
600nmbe 18 o'clock, by culture culture transferring to glycerin medium, 30 ℃ of culture temperature, incubation time 15h, volume of culture is 25L, then with the speed stream of final concentration 1%, add methyl alcohol and carry out inducing culture 75h, collect fermentation after culture;
(2) to the culture after the fermentation of collecting in step (1) successively carry out cytoclasis, clarification and ultrafiltration, silica gel adsorption, ultrafiltration and concentration changes liquid, chromatogram purification and desalination, Sterile Filtration, makes the restructuring debaryomyces hansenii hepatitis E viruslike particle of purifying.
Cytoclasis: the culture after the fermentation of collection is carried out to centrifugal 20min with the rotating speed of 4500 turn/min at 8 ℃, in yeast cell after centrifugation, add PB damping fluid, making cell concn is 50%, after fully mixing, with similarity condition centrifuging and taking, precipitate again, in centrifugation, add cytoclasis liquid, making cell concn is 30%, add again 1% long-pending Tween-80 of cytoclasis liquid, the NaCl of 0.1M/L, with the fragmentation of granulated glass sphere polishing, the PMSF solution that the concentration that adds its volume 4% in broken liquid is 1%, it is 8.0 that the NaOH of 1M/L regulates pH value, with the centrifugal 50min of rotating speed of 7000 turn/min, remove and precipitate to obtain supernatant liquor,
Clarification and ultrafiltration: supernatant liquor is carried out to clarifying treatment cell debris by 0.65 μ m hollow fiber column, the PB damping fluid that is 8.0 with PH rinses 10 times of supernatant liquor volumes, under collection membrane, see through solution, to by 0.2 μ m hollow fiber column, carry out ultrafiltration and concentration through liquid, the PB damping fluid that is 8.0 with PH rinses 6 times and amasss through liquid, and on collection membrane, liquid is preserved;
Silica gel adsorption: the concentration that adds the 40-50% of supernatant liquor volume on film in liquid is 7.5% silica gel solution, whip attachment 16h at 8 ℃, then remove supernatant liquor at 8 ℃ of centrifugal 20min of the rotating speed with 4000 turn/min; In precipitation, add the silica gel elutriant of former supernatant liquor volume 44%, 1L contains containing sodium tetraborate 3.814g, EDTA0.742g, Sodium desoxycholate 2.5g, in 57 ℃, stir 90min, then with the centrifugal 15min of rotating speed of 4000 turn/min, remove precipitation, in supernatant liquor, add the NaCl that 1/11 silica gel eluting liquid volume concentrations is 4M/L to mix, standing 30min at 8 ℃, with the centrifugal 10min of 7000 turn/min rotating speed, remove precipitation, collect supernatant liquor;
Ultrafiltration and concentration changes liquid: the supernatant liquor obtaining after silica gel adsorption operation is crossed to 750K hollow fiber column, and 6 times of supernatant liquor volumes of PB buffer solution for cleaning, retain solution and be concentrated into protein concentration 3mg/ml on film, collects and retains concentrated solution.
Chromatogram purification: be that Sepharose4FF carries out purifying to retaining concentrated solution employing chromatographic media, with 3% column volume loading, flow velocity is 20ml/min, and elutriant is that concentration is that 0.003mol/LPH value is 8.0 PBS, and monitoring wavelength 280nm, collects A
280nmthe 1st elution peak; Adopting chromatographic media is DEAE Sepharose; With 3% column volume loading, flow velocity is 3ml/min, and elutriant is that volume ratio is 1:2.5, PB and the mixed solution of sodium-chlor, the concentration of PB is 50mmol/L, the concentration of sodium-chlor is 0.5mol/L, monitoring wavelength 280nm, collects absorbance A
280nmbe greater than 0.5 elution peak;
Desalination: adopt column chromatography desalination, chromatographic media is Sepharose4FF, and with 8% column volume loading, flow velocity is 20ml/min, and elutriant is that concentration is that 0.003mol/LPH is 7.0 PBS, monitoring wavelength 280nm, collects A
280nmfrom 0.5 to 0.3 elution peak.
The detection of the restructuring debaryomyces hansenii hepatitis E viruslike particle of purifying:
Electron microscopic observation: the former drop of restructuring debaryomyces hansenii hepatitis E viruslike particle that takes a morsel is in copper mesh, and 2% phospho-wolframic acid negative staining 3min, is dried 4min under room temperature, and sample is placed under transmission electron microscope (H-7000FA) and observes.
ELISA detects: with double antibody sandwich ELISA, detect, concrete steps are as follows: 4 ℃ of coated E type hepatitis virus monoclonal antibodies spend the night; 0.5% 37 ℃ of bovine serum albumins sealing lh; Add 37 ℃ of the cytoclasis supernatant liquors (establishing the positive, feminine gender, blank) of gradient doubling dilution to hatch lh; Add again 37 ℃ of anti-hepatitis E virus ORF2 sheep polyclonal antibodies to hatch lh; Then add the anti-sheep IgG of rabbit of HRP mark, hatch lh for 37 ℃; Add 1%TMB nitrite ion colour developing 10min, 2mol/L sulfuric acid termination reaction is measured ELISA and is reacted each hole OD on enzyme micro-plate reader
450nmvalue, P/N>=2.1, are judged as the positive, and A
450nm>=0.1.
SDS-PAGE detects (SDS-polyacrylamide gel electrophoresis): preparation 12% separation gel, 6% concentrated glue, sample is processed through NaOH, SDS Buffer, 4uL loading, point sample positive control and negative control simultaneously, under constant current, 12mA20min, 24mA1.5h, electrophoresis is complete, take off glue, coomassie brilliant blue staining, the expression amount of gel imaging system scanning analysis target protein.
Western blot identifies: SDS-PAGE glue electrotransfer is to pvdf membrane, with the constant current transferase 12 h of 160mA; Sealing: the pvdf membrane after shifting is put to 37 ℃ of sealings in confining liquid PBST and spend the night; In conjunction with: add goat-anti HEV polyclonal antibody (1:500) as first antibody, in conjunction with 2h, then add the anti-sheep IgG of rabbit (1:1000) second antibody of HRP mark with 37 ℃ of pvdf membranes, 37 ℃ of effect lh; OPD develops the color until there is clear band, deionized water termination reaction.
Above-mentioned restructuring debaryomyces hansenii hepatitis E viruslike particle is prepared an application for vaccine, comprises the following steps:
In the restructuring debaryomyces hansenii hepatitis E viruslike particle of purifying, add the aluminium hydroxide solution through degerming, shake up absorption, make hepatitis E virus vaccine work in-process; The preferred 1.0mg/ml of concentration of described aluminium hydroxide solution, aluminum ions concentration 0.5mg/ml in described hepatitis E virus vaccine work in-process.
The recombinate purification process of debaryomyces hansenii hepatitis E viruslike particle, comprises the following steps:
(1) cultivation and fermentation: be inoculated in the yeast nitrogen substratum of 200ml by 1% all work seed lot recombined hepatitis E hepatitis vaccine engineering bacteria, under 31.5 ℃ of conditions, carry out one-level and cultivate 20h, with ultraviolet visible spectrophotometry detection one-level culture absorbance A
600nmbe 15 o'clock, culture culture transferring is carried out to secondary cultivation 20h to the yeast nitrogen substratum of 2L under 31.5 ℃ of conditions, with ultraviolet visible spectrophotometry detection secondary culture absorbance A
600nmbe 15 o'clock, by culture culture transferring to glycerin medium, 30 ℃ of culture temperature, incubation time 15h, volume of culture is 20L, then with the speed stream of final concentration 1%, add methyl alcohol and carry out inducing culture 78h, collect fermentation after culture;
(2) to the culture after the fermentation of collecting in step (1) successively carry out cytoclasis, clarification and ultrafiltration, silica gel adsorption, ultrafiltration and concentration changes liquid, chromatogram purification and desalination, Sterile Filtration, makes the restructuring debaryomyces hansenii hepatitis E viruslike particle of purifying.
Cytoclasis: the culture after the fermentation of collection is carried out to centrifugal 20min with the rotating speed of 4500 turn/min at 5 ℃, in yeast cell after centrifugation, add PB damping fluid, making cell concn is 50%, after fully mixing, with similarity condition centrifuging and taking, precipitate again, in centrifugation, add cytoclasis liquid, making cell concn is 30%, add again 1% long-pending Tween-80 of cytoclasis liquid, the NaCl of 0.1M/L, with the fragmentation of granulated glass sphere polishing, the PMSF solution that the concentration that adds its volume 4% in broken liquid is 1%, it is 8.0 that the NaOH of 1M/L regulates pH value, with the centrifugal 50min of rotating speed of 7000 turn/min, remove and precipitate to obtain supernatant liquor,
Clarification and ultrafiltration: supernatant liquor is carried out to clarifying treatment cell debris by 0.65 μ m hollow fiber column, the PB damping fluid that is 8.0 with PH rinses 9.5 times of supernatant liquor volumes, under collection membrane, see through solution, to by 0.2 μ m hollow fiber column, carry out ultrafiltration and concentration through liquid, the PB damping fluid that is 8.0 with PH rinses 5.5 times and amasss through liquid, and on collection membrane, liquid is preserved;
Silica gel adsorption: the concentration that adds the 40-50% of supernatant liquor volume on film in liquid is 7.5% silica gel solution, whip attachment 13h at 5 ℃, then remove supernatant liquor at 5 ℃ of centrifugal 20min of the rotating speed with 4000 turn/min; In precipitation, add the silica gel elutriant of former supernatant liquor volume 44%, 1L contains containing sodium tetraborate 3.814g, EDTA0.742g, Sodium desoxycholate 2.5g, in 57 ℃, stir 90min, then with the centrifugal 15min of rotating speed of 4000 turn/min, remove precipitation, in supernatant liquor, add the NaCl that 1/11 silica gel eluting liquid volume concentrations is 4M/L to mix, standing 30min at 5 ℃, with the centrifugal 10min of 7000 turn/min rotating speed, remove precipitation, collect supernatant liquor; Ultrafiltration and concentration changes liquid: the supernatant liquor obtaining after silica gel adsorption operation is crossed to 750K hollow fiber column, and 6 times of supernatant liquor volumes of PB buffer solution for cleaning, retain solution and be concentrated into protein concentration 2.5mg/ml on film, collects and retains concentrated solution;
Chromatogram purification: be that Sepharose4FF carries out purifying to retaining concentrated solution employing chromatographic media, with 3% column volume loading, flow velocity is 20ml/min, and elutriant is that concentration is that 0.003mol/LPH value is 8.0 PBS, and monitoring wavelength 280nm, collects A
280nmthe 1st elution peak; Adopting chromatographic media is DEAE Sepharose; With 3% column volume loading, flow velocity is 3ml/min, and elutriant is that volume ratio is the PB of 1:1.5 and the mixed solution of sodium-chlor, and the concentration of PB is 50mmol/L, and the concentration of sodium-chlor is 0.5mol/L, and monitoring wavelength 280nm, collects absorbance A
280nmbe greater than 0.5 elution peak;
Desalination: adopt column chromatography desalination, chromatographic media is Sepharose4FF, and with 5% column volume loading, flow velocity is 20ml/min, and elutriant is that concentration is that 0.003mol/LPH is 7.0 PBS, monitoring wavelength 280nm, collects A
280nmfrom 0.5 to 0.3 elution peak;
The detection of the restructuring debaryomyces hansenii hepatitis E viruslike particle of purifying:
Electron microscopic observation: the former drop of restructuring debaryomyces hansenii hepatitis E viruslike particle that takes a morsel is in copper mesh, and 2% phospho-wolframic acid negative staining 3min, is dried 4min under room temperature, and sample is placed under transmission electron microscope (H-7000FA) and observes.
ELISA detects: with double antibody sandwich ELISA, detect, concrete steps are as follows: 4 ℃ of coated E type hepatitis virus monoclonal antibodies spend the night; 0.5% 37 ℃ of bovine serum albumins sealing lh; Add 37 ℃ of the cytoclasis supernatant liquors (establishing the positive, feminine gender, blank) of gradient doubling dilution to hatch lh; Add again 37 ℃ of anti-hepatitis E virus ORF2 sheep polyclonal antibodies to hatch lh; Then add the anti-sheep IgG of rabbit of HRP mark, hatch lh for 37 ℃; Add 1%TMB nitrite ion colour developing 10min, 2mol/L sulfuric acid termination reaction is measured ELISA and is reacted each hole OD on enzyme micro-plate reader
450nmvalue, P/N>=2.1, are judged as the positive, and A
450nm>=0.1.
SDS-PAGE detects (SDS-polyacrylamide gel electrophoresis): preparation 12% separation gel, 6% concentrated glue, sample is processed through NaOH, SDS Buffer, 4uL loading, point sample positive control and negative control simultaneously, under constant current, 12mA20min, 24mA1.5h, electrophoresis is complete, take off glue, coomassie brilliant blue staining, the expression amount of gel imaging system scanning analysis target protein.
Westernblot identifies: SDS-PAGE glue electrotransfer is to pvdf membrane, with the constant current transferase 12 h of 160mA; Sealing: the pvdf membrane after shifting is put to 37 ℃ of sealings in confining liquid PBST and spend the night; In conjunction with: add goat-anti HEV polyclonal antibody (1:500) as first antibody, in conjunction with 2h, then add the anti-sheep IgG of rabbit (1:1000) second antibody of HRP mark with 37 ℃ of pvdf membranes, 37 ℃ of effect lh; OPD develops the color until there is clear band, deionized water termination reaction.
Above-mentioned restructuring debaryomyces hansenii hepatitis E viruslike particle is prepared an application for vaccine, comprises the following steps:
In the restructuring debaryomyces hansenii hepatitis E viruslike particle of purifying, add the aluminium hydroxide solution through degerming, shake up absorption, make hepatitis E virus vaccine work in-process; The preferred 1.0mg/ml of concentration of described aluminium hydroxide solution, aluminum ions concentration 0.5mg/ml in described hepatitis E virus vaccine work in-process.
Restructuring debaryomyces hansenii hepatitis E viruslike particle to the purifying in embodiment 1 detects, and detected result is as follows:
Electron microscopic observation: the former drop of restructuring debaryomyces hansenii hepatitis E viruslike particle that takes a morsel is in copper mesh, and 2% phospho-wolframic acid negative staining 3min, is dried 4min under room temperature, and sample is placed under transmission electron microscope (H-7000FA) and observes, and observations is shown in accompanying drawing 1.
ELISA detects: with double antibody sandwich ELISA, detect, concrete steps are as follows: 4 ℃ of coated E type hepatitis virus monoclonal antibodies spend the night; 0.5% 37 ℃ of bovine serum albumins sealing lh; Add 37 ℃ of cytoclasis supernatant liquors (establishing the positive, feminine gender, blank) that gradient doubly dilutes to hatch lh; Add again 37 ℃ of anti-hepatitis E virus ORF2 sheep polyclonal antibodies to hatch lh; Then add the anti-sheep IgG of rabbit of HRP mark, hatch lh for 37 ℃; Add 1%TMB nitrite ion colour developing 10min, 2mol/L sulfuric acid termination reaction is measured ELISA and is reacted each hole OD on enzyme micro-plate reader
450nmvalue, P/N>=2.1, are judged as the positive, and A
450nm>=0.1, what the high dilution of antigen was target protein tires, and detected result is in Table 1.
SDS-PAGE detects (SDS-polyacrylamide gel electrophoresis): preparation 12% separation gel, 6% concentrated glue, sample is processed through NaOH, SDS Buffer, 4uL loading, point sample positive control and negative control simultaneously, under constant current, 12mA20min, 24mA1.5h, electrophoresis is complete, takes off glue, coomassie brilliant blue staining, the expression amount of gel imaging system scanning analysis target protein, the molecular weight of albumen of the expression of restructuring debaryomyces hansenii hepatitis E viruslike particle is about 55kD, and SDS-PAGE detected result is shown in accompanying drawing 2 and accompanying drawing 3.
Western blot identifies: SDS-PAGE glue electrotransfer is to pvdf membrane, with the constant current transferase 12 h of 160mA; Sealing: the pvdf membrane after shifting is put to 37 ℃ of sealings in confining liquid PBST and spend the night; In conjunction with: add goat-anti HEV polyclonal antibody (1:500) as first antibody, in conjunction with 2h, then add the anti-sheep IgG of rabbit (1:1000) second antibody of HRP mark with 37 ℃ of pvdf membranes, 37 ℃ of effect lh; Until there is clear band in OPD colour developing, deionized water termination reaction, and Western blot identifies and sees accompanying drawing 4.
In embodiment, after recombined hepatitis E hepatitis vaccine engineering strain expression product purifying, HPLC analytical results is shown in accompanying drawing 5 and table 2, and table 2 is the explanations to ratio in accompanying drawing 5.
Table 1
Table 2
The above embodiment is preferred embodiments of the present invention, is not to limit the scope of the present invention, and the equivalence of doing according to structure, feature and principle described in the present patent application the scope of the claims therefore all changes or modifies, and all should comprise in patent claim of the present invention.
Claims (10)
1. the recombinate purification process of debaryomyces hansenii hepatitis E viruslike particle, is characterized in that, comprises the following steps:
(1) work seed lot recombined hepatitis E hepatitis vaccine engineering strain is carried out to cultivation and fermentation, collect the culture after fermentation;
(2) to the culture after the fermentation of collecting in step (1) successively carry out cytoclasis, clarification and ultrafiltration, silica gel adsorption, ultrafiltration and concentration changes liquid, chromatogram purification and desalination, Sterile Filtration, makes the restructuring debaryomyces hansenii hepatitis E viruslike particle of purifying.
2. the purification process of restructuring debaryomyces hansenii hepatitis E viruslike particle according to claim 1, it is characterized in that, the concrete grammar of the cultivation and fermentation described in step (1) is: work seed lot recombined hepatitis E hepatitis vaccine engineering strain is inoculated in the yeast nitrogen substratum of 200ml by 1%, carry out one-level cultivation, when detect one-level culture absorbance A with ultraviolet visible spectrophotometry
600nmduring for 10-20, by culture culture transferring to the yeast nitrogen substratum of 2L, when detect secondary culture absorbance A with ultraviolet visible spectrophotometry
600nmduring for 10-18, culture culture transferring is carried out to cultivation and fermentation to glycerin medium, methanol induction is expressed, and collects the culture after fermentation;
Preferably, the temperature that described one-level is cultivated is 30-33 ℃, and incubation time is 20h;
Preferably, the temperature that described secondary is cultivated is 30-33 ℃, and incubation time is 20h;
Preferably, the culture temperature in glycerin medium is 30 ℃, and incubation time is 13-15h, and volume of culture is 15-25L, then with the speed stream of final concentration 1%, adds methyl alcohol and carries out inducing culture 75-80h.
3. the purification process of restructuring debaryomyces hansenii hepatitis E viruslike particle according to claim 1, it is characterized in that, described cytoclastic concrete grammar is: the culture after the fermentation of collection is carried out to centrifugal 20min with the rotating speed of 4500 turn/min, in yeast cell after centrifugation, add PB damping fluid, making cell concn is 50%, after fully mixing, with similarity condition centrifuging and taking, precipitate again, in centrifugation, add cytoclasis liquid, making cell concn is 30%, add again 1% long-pending Tween-80 of cytoclasis liquid, the NaCl of 0.1M/L, with the fragmentation of granulated glass sphere polishing, the PMSF solution that the concentration that adds its volume 4% in broken liquid is 1%, it is 8.0 that the NaOH of 1M/L regulates pH value, with the centrifugal 40-50min of rotating speed of 7000 turn/min, remove and precipitate to obtain supernatant liquor, the preferred 2-8 ℃ of centrifuging temperature.
4. the purification process of restructuring debaryomyces hansenii hepatitis E viruslike particle according to claim 1, it is characterized in that, described clarification and the concrete grammar of ultrafiltration are: supernatant liquor is carried out to clarifying treatment cell debris by 0.65 μ m hollow fiber column, with 9-10 times of supernatant liquor volume of PB damping fluid flushing, under collection membrane, see through solution, to by 0.2 μ m hollow fiber column, carry out ultrafiltration and concentration through liquid, and rinse 5-6 doubly long-pending through liquid with PB damping fluid, on collection membrane, liquid is preserved; Described PB ph value of buffer solution preferably 8.0.
5. the purification process of restructuring debaryomyces hansenii hepatitis E viruslike particle according to claim 1, it is characterized in that, the concrete grammar of described silica gel adsorption is: on film, in liquid, add silica gel solution, and whip attachment, then remove supernatant liquor with the centrifugal 20min of rotating speed of 4000 turn/min; In precipitation, add the silica gel elutriant of former supernatant liquor volume 44%, in 57 ℃ of stirring 90min, then with the centrifugal 15min of rotating speed of 4000 turn/min, remove precipitation, in supernatant liquor, add sodium-chlor to mix, standing, with the centrifugal 10min of 7000 turn/min rotating speed, remove precipitation, collect supernatant liquor;
Preferably, the concentration of described silica gel solution is 7.5%, and the volume that adds of silica gel solution is the 40-50% of supernatant liquor volume;
Preferably, described whip attachment temperature is 2-8 ℃, and the whip attachment time is 10-16h, and described centrifuging temperature is 2-8 ℃;
Preferably, the volume of described sodium-chlor is 1/11 silica gel effluent volume, and the concentration of sodium-chlor is 4M/L;
Preferably, described dwell temperature is 2-8 ℃, and time of repose is 30min.
6. the purification process of restructuring debaryomyces hansenii hepatitis E viruslike particle according to claim 5, is characterized in that, described silica gel elutriant is comprised of the component that comprises following mass body volume concentrations:
Sodium tetraborate 3.814g/L,
EDTA 0.742g/L，
Sodium desoxycholate 2.5g/L.
7. the purification process of restructuring debaryomyces hansenii hepatitis E viruslike particle according to claim 1, it is characterized in that, the concrete grammar that described ultrafiltration and concentration changes liquid is: the supernatant liquor obtaining after silica gel adsorption operation is crossed to 750K hollow fiber column, 5-6 times of supernatant liquor volume of PB buffer solution for cleaning, on film, retain solution and be concentrated into protein concentration 2mg/ml-3mg/ml, collect and retain concentrated solution.
8. the purification process of restructuring debaryomyces hansenii hepatitis E viruslike particle according to claim 1, it is characterized in that, the concrete grammar of described chromatogram purification is: to retaining concentrated solution employing chromatographic media, be that Sepharose4FF carries out purifying, loading, flow velocity is 20ml/min, elutriant is PBS, and monitoring wavelength 280nm, collects A
280nmthe 1st elution peak; Adopting chromatographic media is DEAE Sepharose; Loading, flow velocity is 2-3ml/min, and elutriant is the mixed solution of PB and sodium-chlor, and monitoring wavelength 280nm, collects absorbance A
280nmbe greater than 0.5 elution peak;
Preferably, the volume of described loading is 2%-3% column volume, and the concentration of described PBS is 0.003mol/L, and the pH value of PBS is 8.0;
Preferably, the concentration of described PB is 50mmol/L, and described sodium chloride concentration is 0.5mol/L; Further preferred, described PB and the volume ratio of sodium-chlor are 1:(0.8-2.5).
9. the purification process of restructuring debaryomyces hansenii hepatitis E viruslike particle according to claim 1, the concrete grammar of described desalination is: adopt column chromatography desalination, chromatographic media is Sepharose4FF, loading, flow velocity is 20ml/min, elutriant is PBS, monitoring wavelength 280nm, collects A
280nmfrom 0.5 to 0.3 elution peak;
Preferably, the volume of described loading is 4%-8% column volume, and the concentration of described PBS is 0.003mol/L, and the PH of PBS is 7.0.
10. the application of preparing vaccine of the restructuring debaryomyces hansenii hepatitis E viruslike particle of an employing as described in as arbitrary in claim 1-9, it is characterized in that, comprise the following steps: in the restructuring debaryomyces hansenii hepatitis E viruslike particle of purifying, add the aluminium hydroxide solution through degerming, shake up absorption, make hepatitis E virus vaccine work in-process; The preferred 1.0mg/ml of concentration of described aluminium hydroxide solution, aluminum ions concentration 0.5mg/ml in described hepatitis E virus vaccine work in-process.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410029495.0A CN103740656A (en) | 2014-01-22 | 2014-01-22 | Method for purifying recombined hansenula polymorpha hepatitis E virus-like particles and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410029495.0A CN103740656A (en) | 2014-01-22 | 2014-01-22 | Method for purifying recombined hansenula polymorpha hepatitis E virus-like particles and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103740656A true CN103740656A (en) | 2014-04-23 |
Family
ID=50497723
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410029495.0A Pending CN103740656A (en) | 2014-01-22 | 2014-01-22 | Method for purifying recombined hansenula polymorpha hepatitis E virus-like particles and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103740656A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104845944A (en) * | 2015-04-18 | 2015-08-19 | 湖北创瑞生物科技有限公司 | Hollow fiber purification method of recombinant herpes simplex virus |
| CN105907729A (en) * | 2015-05-05 | 2016-08-31 | 广东东阳光药业有限公司 | Method for preparing human papilloma virus (HPV) L1 protein virus-like particles (VLP) |
| CN109652384A (en) * | 2019-02-21 | 2019-04-19 | 昆明理工大学 | A kind of method of in vitro culture Hepatitis E virus |
| CN109689862A (en) * | 2016-12-28 | 2019-04-26 | 北京民海生物科技有限公司 | Recombinate purifying and its vaccine preparation method of EV71 virus-like particle |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009011413A1 (en) * | 2007-07-13 | 2009-01-22 | Japan As Represented By Director General Of National Institute Of Infectious Diseases | Production and use of epitope-tagged hepatitis c virus particle |
| CN101875921A (en) * | 2009-12-22 | 2010-11-03 | 中国人民解放军第三军医大学 | Method for separating and purifying hepatitis B virus particles |
-
2014
- 2014-01-22 CN CN201410029495.0A patent/CN103740656A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009011413A1 (en) * | 2007-07-13 | 2009-01-22 | Japan As Represented By Director General Of National Institute Of Infectious Diseases | Production and use of epitope-tagged hepatitis c virus particle |
| CN101875921A (en) * | 2009-12-22 | 2010-11-03 | 中国人民解放军第三军医大学 | Method for separating and purifying hepatitis B virus particles |
Non-Patent Citations (2)
| Title |
|---|
| 李津等: "《生物制药设备和分离纯化技术》", 30 April 2003, article "重组酵母乙型肝炎疫苗", pages: 349 * |
| 苏彩霞等: "戊型肝炎病毒Ⅳ型ORF2 蛋白在汉逊酵母中的表达", 《生物工程学报》, 31 January 2007 (2007-01-31) * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104845944A (en) * | 2015-04-18 | 2015-08-19 | 湖北创瑞生物科技有限公司 | Hollow fiber purification method of recombinant herpes simplex virus |
| CN105907729A (en) * | 2015-05-05 | 2016-08-31 | 广东东阳光药业有限公司 | Method for preparing human papilloma virus (HPV) L1 protein virus-like particles (VLP) |
| CN105907729B (en) * | 2015-05-05 | 2019-08-16 | 广东东阳光药业有限公司 | A method of preparing human mammilla tumor virus L 1 protide virus-like particle |
| CN109689862A (en) * | 2016-12-28 | 2019-04-26 | 北京民海生物科技有限公司 | Recombinate purifying and its vaccine preparation method of EV71 virus-like particle |
| CN109689862B (en) * | 2016-12-28 | 2022-09-30 | 北京民海生物科技有限公司 | Purification of recombinant EV71 virus-like particle and vaccine preparation method thereof |
| CN109652384A (en) * | 2019-02-21 | 2019-04-19 | 昆明理工大学 | A kind of method of in vitro culture Hepatitis E virus |
| CN109652384B (en) * | 2019-02-21 | 2021-10-26 | 昆明理工大学 | Method for culturing hepatitis E virus in vitro |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103740656A (en) | Method for purifying recombined hansenula polymorpha hepatitis E virus-like particles and application | |
| CN102154325B (en) | Vaccine against human papillomavirus (HPV) as well as preparation method and application thereof | |
| CN103255163A (en) | EV71virus-like particles as well as preparation method and application thereof | |
| CN103760365A (en) | BAS-ELISA kit for detecting hog cholera virus Erns and E2 antigen | |
| CN101602805A (en) | Be used for agarose compatible medium of purifying hand foot mouth disease immunoglobulin (Ig) and preparation method thereof | |
| CN113512096A (en) | Weever rhabdovirus recombinant G2 protein and application thereof | |
| CN101864447A (en) | Construction of prokaryotic expression vector of grass carp reovirus outer capsid protein and preparation method of polyclonal antibody | |
| CN105126096A (en) | Foot and mouth disease virus-like particle vaccine and preparation method thereof | |
| CN116396959A (en) | Anti-porcine astrovirus five-type PAstV5 monoclonal antibody, primer, hybridoma cell strain, preparation and application | |
| CN104211784A (en) | Protein for preparation of hepatitis E virus-like particles and method | |
| CN105018435A (en) | Purifying method for virus-like particle | |
| CN111378030A (en) | Method for separating and purifying nano antibody | |
| KR20230129506A (en) | Stable formulation of human papillomavirus-like particle vaccine | |
| CN103539839A (en) | Neutralizing epitope peptide of enterovirus 71-type VP2 antigen and application thereof | |
| CN105801691A (en) | HPV16E7 monoclonal antibody and preparation method and application thereof | |
| CN113150126A (en) | Rabbit-derived porcine parvovirus 6-type VP2 protein antibody and preparation method thereof | |
| CN102242083B (en) | 3D protein monoclonal antibody for resisting novel duck hepatitis virus serotype I | |
| CN103983782A (en) | ELISA kit for detecting hog cholera virus Erns IgM antibody | |
| CN106754981A (en) | A kind of method that utilization E. coli system prepares Goose Parvovirus sample particle | |
| CN110627910A (en) | Schistosoma mansoni recombinant fusion protein SmSAP and application thereof in schistosomiasis immune diagnosis | |
| CN103204942B (en) | Pig gyrate virus II type genetic engineering subunit vaccine and its preparation method and application | |
| CN105907729A (en) | Method for preparing human papilloma virus (HPV) L1 protein virus-like particles (VLP) | |
| CN105424928B (en) | Immunochromatography strip detecting porcine reproductive and respiratory syndrome virus antibodies and preparation method and application thereof | |
| CN116284431A (en) | Preparation and application of novel coronavirus polyclonal antibody | |
| CN102757493A (en) | Method for preparing chicken pox virus antibody |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20140423 |