CN107174658A - People is with varicella virus inactivated vaccine and preparation method thereof - Google Patents
People is with varicella virus inactivated vaccine and preparation method thereof Download PDFInfo
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- CN107174658A CN107174658A CN201710297864.8A CN201710297864A CN107174658A CN 107174658 A CN107174658 A CN 107174658A CN 201710297864 A CN201710297864 A CN 201710297864A CN 107174658 A CN107174658 A CN 107174658A
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Classifications
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- 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
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
- A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
- A61L2/0082—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts using chemical substances
- A61L2/0088—Liquid substances
-
- 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
- A61K2039/5252—Virus inactivated (killed)
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- 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
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2202/00—Aspects relating to methods or apparatus for disinfecting or sterilising materials or objects
- A61L2202/20—Targets to be treated
- A61L2202/21—Pharmaceuticals, e.g. medicaments, artificial body parts
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- 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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
- C12N2710/16734—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
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- Engineering & Computer Science (AREA)
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to biological technical field, a kind of people is specifically provided with varicella virus inactivated vaccine and preparation method thereof, is by β propiolactone and varicella virus concentrate by volume 1：2000~1：8000 mixing, inactivate 12~36h under the conditions of 3~5 DEG C, and 1.5~3h is then hydrolyzed under the conditions of 34~37 DEG C, inactivation of viruses liquid is obtained；Inactivation of viruses liquid is purified, packing, it is lyophilized after be prepared into inactivated vaccine.Safely, effectively, reaction of inoculation is slight for varicella virus inactivated vaccine prepared by this method, more applicable to not applying to the crowd of immunologic hypofunction of attenuated live vaccine.
Description
Technical field
The present invention relates to biological technical field, and in particular to people is with varicella virus inactivated vaccine and preparation method thereof.
Background technology
Varicella and the pathogen of herpes zoster be varicella virus (Varicella-zoster virus,
VZV).VZV belongs to herpetoviridae, is double-stranded DNA virus.
VZV is a kind of herpesviral with highly infectious, can both trigger varicella, can also trigger herpes zoster
(herpes zoster, HZ)；Childhood that the former is commonly found in and young adult, the latter are then latent VZV reactivations and replicated
Cause.After primary infection VZV, virus can keep resting state in sensory ganglion, and such VZV reactivations are with being immunized work(
Energy decline is relevant, and mostly occur (Weinberg et al., the Journal of in the elderly or the crowd of immunologic hypofunction
Infectious Diseases(2009)200：1068-77).Some patients pain related to HZ after the recovery from illness of HZ fash is also
Can last very long.
Accepted in the world using the effect of vaccine prevention varicella.Based on live attenuated virus (VZV Oka
Strain) chicken pox vaccine develop and carry out clinical test the seventies in last century and the eighties.1984, the vaccine was first in Germany
Listing license is obtained with Sweden.Presently commercially available has several freeze-dried live attenuated varicella vaccines, can refrigerator cold-storage or freezing.These epidemic diseases
Seedling have for univalent vaccine (only containing VZV viral composition), what is had is then made connection with MMR vaccine (measles (MMR)
Close vaccine.
Research shows that monovalent varicella attenuation live vaccine tolerance is good, is most often reported after healthy children inoculation chicken pox vaccine
Adverse events are relatively slight, including injection site mild tenderness, rubescent and fash.However, attenuated vaccine living may be uncomfortable
Patient for immunologic hypofunction.Include pneumonia, hepatitis, herpes zoster meninx by Oka plants of caused rare complications of VZV
Inflammation, recurrent herpes zoster, serious rash and Secondary cases VZV are propagated, this takes place mostly in immune function depression person or inoculation
The patient of other diagnosed serious conditions of Shi Youwei.The individual of immunologic hypofunction, including the trouble with hematologic malignancies
Person, the patient for undergoing immunosuppressive therapy, the patient for receiving HSCT (HCT) or solid organ transplantation (SOT),
The patient of HIV and the patient with autoimmune disease, the morbidity with the development HZ higher relative to general groups
Rate.In addition, these PATIENT POPULATIONs are in the increased risk of serious and life-threatening the complication of development.At present, to varicella
The no specific drug for the treatment of, to put prevention first, can only by be inoculated with chicken pox vaccine method prevent infection VZV virus, so peace
Complete effective chicken pox vaccine people at highest risk is immunized significant.
The vaccine is used to adapt to above-mentioned immunocompromised person, current researcher has researched and developed inactivated vaccine, disease
Malicious sample particle vaccines, subunit vaccine, DNA vaccination.By heat inactivation VZV or by using subunit vaccine can develop for
Safer vaccine (Cohen, the J.I., (2008) J.Infect.Dis.197 (Suppl 2) of immunologic hypofunction subject:
S237-S241).Redman et al. (J.Infectious Diseases (1997) 176:578-85) describe by 50 DEG C
Heating and inactivate and cause<1.2pfu/0.5mL infectious virus content inactivation VZV vaccine.But for immunologic function
Low patient, even if the infectivity of the level is also with risk.U.S. Patent No. 6214354 and 5997880 is mentioned
Using inactivation VZV vaccine potential and disclose the example of the vaccine for producing and testing heat treatment；The patent of MSD Corp.
γ radiological inactivation VZV viruses have been used to manufacture varicella inactivated vaccine in (CN 103167880A).If developing inactivation VZV
Method, safety can be made in the patient of immunologic hypofunction and effectively (i.e. without infectious but to retain it immune in methods described
Originality and antigenicity) VZV samples, this will meet great needs of medical treatment.
At present it is known that for chicken pox vaccine inactivate method have heat inactivation and gamma-rays inactivate.Heat inactivation earliest by
Smith etc. proposes that this method is simple and easy to apply when developing hog cholera inactivated vaccine.But the method that microorganism is killed in heating is thicker
It is rough, protein denaturation is easily caused, and then immunogenicity is significantly affected, and also to there may be inactivation not thorough for this method
Possibility, the viral level of residual is still unsafe for the patient of hypoimmunity；In addition, there are some researches show60Coγ
Singlet oxygen and free radical produced by x ray irradiation x have larger damaging action to protein, are mainly shown as Protein primary
Peptide bond fracture, amino acid composition in structure change and reduced with β-pleated sheet content in space conformation, and close structure tends to pine
Dissipate, random coil substantially increases.The virus lived in cell system has maximum radiation resistance, and double-stranded viruses is without single
Deactivation of the chain virus to ionising radiation is sensitive.The uniformity control of irradiation technique middle dosage is bad also to influence vaccine matter
Amount.
Beta-propiolactone (beta-propiolacto ne, BPL) also known as hydracrylic acid β lactones, are a kind of heterocycle compounds
(C3H4O2), there is very strong deactivation to virus, be a kind of good virus inactivator.BPL Inactivations are to act on disease
Pathogen DNA or RNA, by the purine bases (mainly guanine) with nucleic acid, reaction destroys the structure of nucleic acid, but does not make directly
For protein, therefore good immunogenicity can be kept.BPL is easily hydrolyzed, and is disappeared after 37 DEG C of water-bath hydrolysis 2h, it is hydrolyzed
For avirulent body fat metabolite ethylene lactic acid.Further, since its can in vaccine liquid complete hydrolysis, it is not necessary to examine
Consider the residual in finished product vaccine, reaction of inoculation is also slight.Meanwhile, the beta-propiolactone inactivation cause of disease time is short, so as to significantly contract
In the short production of vaccine cycle, improve economic benefit.
The content of the invention
Present invention aim to address above-mentioned the deficiencies in the prior art there is provided people with varicella virus inactivated vaccine and its preparation
Method.
The present invention is achieved by the following technical solutions：
People is comprised the following steps with the preparation method of varicella virus inactivated vaccine：
(1) Human embryo was no more than for 34 generations into fiber (MRC-5) passage；
(2) when degrees of fusion more than 90% is arrived in cell amplification, varicella virus is inoculated into cell and infected；
(3) cell for having infected varicella virus is added into minimum necessary (MEM) containing 2%~5% hyclone to cultivate
In base, 34~37 DEG C of cell culture incubator cultures are placed in；
(4) cytopathy degree is observed, when induced cytopathic effect (CPE) reaches more than 50%, culture medium is outwelled, used
Phosphate buffer solution (PBS) washs cell；
(5) add 0.02~0.05% ethylenediamine tetra-acetic acid (EDTA) solution elution infection cell, by cell suspension from
Heart processing, removes supernatant, adds stabilizer and merges, mixes, Sterility testing is done in single bottle sampling, and -65~-80 DEG C freeze；
(6) it will detect that sterile sample carries out ultrasonication, supernatant, i.e. virus stock solution used are collected in centrifugation；
(7) virus stock solution used is concentrated by ultrafiltration using milipore filter bag；
(8) virus liquid protein content and virus titer after detection is concentrated by ultrafiltration；
(9) it is the virus of 35~95mg/mL, virus titer not less than 4.0lgPFU/mL by beta-propiolactone and protein content
Concentrate by volume 1：2000~1：8000 mixing, inactivate 12~36h, then in 34~37 DEG C of conditions under the conditions of 3~5 DEG C
1.5~3h of lower hydrolysis, obtains inactivation of viruses liquid；
(10) inactivation of viruses liquid is purified using column chromatography, obtains inactivation of viruses refined solution；
(11) by inactivation of viruses refined solution in Human embryo into fiber (MRC-5) cell at least generation of blind passage 3, detection virus drop
Degree, as a result all should be without plaque test.
(12) dispense, lyophilized inactivation of viruses refined solution produces varicella virus inactivated vaccine.
Preferably, step (2) infection multiplicity (M.O.I.) for being infected virus inoculation into cell is 0.01
~0.1.
Preferably, the centrifugal condition described in step (5) is centrifuged 10~15 minutes for 3000~4000rpm at 3~5 DEG C.
Preferably, the centrifugal condition described in step (6) is centrifuged 10~15 minutes for 7000~8000rpm at 3~5 DEG C.
Preferably, it is using retention molecule that the use milipore filter bag described in step (7), which carries out ultrafiltration concentration to virus stock solution used,
Measure and 30~60 times are concentrated by ultrafiltration to virus stock solution used for 100KD or 300KD milipore filter bag.
Preferably, the protein content described in step (8) is detected using Lowry methods.
Preferably, the inactivation condition described in step (9) is beta-propiolactone and viral concentration liquid by volume 1：8000 mixing,
24h is inactivated under the conditions of 4 DEG C, then 2h is hydrolyzed under the conditions of 37 DEG C.
Preferably, the way of purification described in step (10) is to be divided inactivation of virus liquid using Sepharose 4FF chromatographic columns
Loading is criticized, varicella virus protein peak, as inactivation of viruses refined solution are collected under 280nm ultraviolet detections.
Preferably, step (8) and (11) described virus titer are detected using plaque method.
The present invention people that also protection any of the above-described preparation method is prepared uses varicella virus inactivated vaccine.
The beneficial effects of the present invention are：
Beta-propiolactone directly acts on pathogen DNA or RNA, anti-by the purine bases (mainly guanine) with nucleic acid
The structure of nucleic acid should be destroyed, but is not directly placed on protein, compared with heat inactivation, gamma-rays inactivation, formalin-inactivated, with more
Good immunogenicity, inactivating efficacy is thorough.Further, since beta-propiolactone is easily hydrolyzed, can in vaccine liquid complete hydrolysis, water
Solve as avirulent body fat metabolite ethylene lactic acid, therefore the residual in finished product vaccine need not be considered, inoculation is anti-
Should be also slight.Meanwhile, the beta-propiolactone inactivation cause of disease time is short, so as to significantly reduce the production of vaccine cycle, improves economy
Benefit.Safely, effectively, reaction of inoculation is slight for varicella virus inactivated vaccine prepared by this method, to not applying to attenuated live vaccine
The crowd of immunologic hypofunction is more applicable.
Embodiment
To be best understood from the present invention, with reference to embodiment, the invention will be further described, and following examples are only pair
The present invention illustrates rather than it is limited.
The people of embodiment 1 is with varicella virus inactivated vaccine preparation method
(1) MRC-5 cells were passaged to for 28 generations in Tissue Culture Flask and cell factory；
(2) when degrees of fusion more than 90% is arrived in cell amplification, virus inoculation is infected into cell, M.O.I. is
0.01-0.1；
(3) the MEM culture mediums containing 2%~5% hyclone are added, 34~37 DEG C of cell culture incubator cultures are placed in；
(4) cytopathy degree is observed daily, when cell CPE reaches more than 50%, is outwelled culture medium, is washed with PBS
Cell；
(5) the EDTA solution for adding 0.03% elutes infection cell, cell suspension is collected in centrifuge tube, at 4 DEG C
4000rpm is centrifuged 10 minutes, removes supernatant, is added stabilizer and is merged, mixes, Sterility testing is done in single bottle sampling, and -80 DEG C freeze；
(6) it will detect that sterile sample is placed in ultrasonic bottle and carry out ultrasonication, 8000rpm is centrifuged 10 minutes at 4 DEG C,
Collect supernatant, i.e. virus stock solution used；
(7) molecular cut off is used to carry out 50 times of concentrations for 100KD milipore filter bag；
(8) Lowry methods detection protein content is 52mg/mL, and plaque method detection virus titer is 4.9lgPFU/mL；
(9) by beta-propiolactone and viral concentration liquid by volume 1：6000 mixing, 3~5 DEG C of 12~36h of inactivation, 35 DEG C of water
Solve 2h；
(10) column chromatography purifying inactivation of viruses liquid, collects viral peak according to ultraviolet detection value, obtains inactivation of viruses refined solution；
(11) inactivation of viruses refined solution is in the generation of MRC-5 cells blind passage 3, plaque method detection virus titer；
(12) dispense, lyophilized inactivation of viruses refined solution produces varicella inactivated vaccine；
The selection of the viral concentration liquid protein concentration of embodiment 2
(1) 5 batches of inactivation of viruses liquid are prepared with varicella virus inactivated vaccine preparation method step (1)-(10) by people；
(2) measure viral concentration liquid concentration and inactivation after it is infectious as shown in the table：
According to upper table result, we select to inactivate protein content for 35~95mg/ml viral concentration liquid.
The plaque method of embodiment 3 determines the infectivity of varicella virus inactivated vaccine
MRC-5 cells are spread into 6 orifice plates, 3*10 is inoculated with per hole5Individual cell, 5%CO234~37 DEG C of incubator is incubated 3~4 days
Afterwards, cell, which grows up to, can determine varicella virus titre after individual layer.Culture medium is abandoned in suction, and varicella virus inactivated vaccine is diluted to suitably
Concentration infection cell, in 5%CO234~37 DEG C of incubator is adsorbed 1~2 hour, then adds 3mL virus-culturing fluids per hole,
5%CO234~37 DEG C of cultures of incubator.Culture is dyed after 7~10 days.Counting is averaged, and is multiplied by the coefficient of dilution and is connect
Volume correction factor is planted, varicella virus inactivated vaccine titre is drawn, i.e., is represented with lg PFU/mL, the chickenpox through inactivating
Malicious inactivated vaccine titre results are without plaque test.
The varicella virus of embodiment 4 inactivates and inactivation checking
(1) viral concentration liquid is obtained with varicella virus inactivated vaccine preparation method step (1)-(7) by people；
(2) the viral concentration liquid for obtaining (1) step detects its protein content and virus titer, by protein content be 35~
The viral concentration liquid of 95mg/mL and virus titer not less than 4.0lgPFU/ml adds inactivator beta-propiolactone (BPL) by table 1~4
Inactivated and hydrolysis：
Table 1
Table 2
Table 3
Table 4
(3) determine that protein content should be 35~95mg/mL according to (2) step results, virus titer is not less than 4lgPFU/mL
When, inactivator additional proportion is 1：2000~1：8000, inactivation temperature is 3~5 DEG C, and inactivation time is 12~36h, hydrolysis temperature
It is 1.5h~3h for 34~37 DEG C of hydrolysis times, the virus liquid access inactivated with this condition grows up in 24 orifice plates of cell monolayer,
Supplement virus-culturing fluid 1mL after 34~37 DEG C of 1~2h of absorption, during 34~37 DEG C of 70~96h of culture, collects virus liquid access and grows up to
In 24 orifice plates of cell monolayer, in the so continuous generation of blind passage 3, collect virus liquid and survey virus titer with plaque method；
(4) it is 0 the virus titer after inactivation to be measured according to the method in embodiment 2, that is, illustrates that inactivated vaccine does not infect
Property.
The people of embodiment 5 is with varicella virus inactivated vaccine purification process
By people inactivation of viruses liquid is obtained with varicella virus inactivated vaccine preparation method (1)-(9).Use Sepharose4FF
Chromatographic column carries out the purifying of inactivation restrovirus liquid, purple in 280nm according to the applied sample amount of chromatographic column by inactivation of virus liquid loading in batches
Varicella virus protein peak, as inactivation of viruses refined solution are collected under outer detection.
The Lyophilized Human of embodiment 6 varicella virus inactivated vaccine preparation method
Protective agent is added after obtaining inactivation of viruses refined solution with varicella virus inactivated vaccine preparation method (1)-(10) by people,
Packing is lyophilized into white loose body per dosage 0.5mL, is clear liquid after redissolution.
Embodiment 7
(1) viral concentration liquid is obtained with varicella virus inactivated vaccine preparation method (1)-(7) by people；
(2) appropriate formaldehyde is added in viral concentration liquid, is inactivated, in different time points sampling detection virus infection
Property, the generation of blind passage 3, as a result such as following table：
| Inactivation time (h) | It is infectious |
| 24 | There is plaque test |
| 48 | There is plaque test |
| 72 | Without plaque test |
| 96 | Without plaque test |
(3) take isometric viral concentration liquid to be separately added into formaldehyde and beta-propiolactone inactivation, BALB/c mouse, 4 weeks is immunized
Venous blood sampling, the VZV IgG levels such as following table in serum is detected with ELISA method and FAMA methods afterwards：
| VZV IgG Conversion rates | Formaldehyde | Beta-propiolactone |
| ELISA method (%) | 62 | 89 |
| FAMA methods (%) | 100% | 100% |
(4) two kinds of inactivators are compared as follows table：
Embodiment described above is only that the preferred embodiment of the present invention is described, not to the model of the present invention
Enclose and be defined, on the premise of design spirit of the present invention is not departed from, technical side of the those of ordinary skill in the art to the present invention
In various modifications and improvement that case is made, the protection domain that claims of the present invention determination all should be fallen into.
Claims (10)
1. people is with the preparation method of varicella virus inactivated vaccine, it is characterised in that comprise the following steps：
(1) human embryonic fibroblast's passage was no more than for 34 generations；
(2) when degrees of fusion more than 90% is arrived in cell amplification, varicella virus is inoculated into cell and infected；
(3) cell for having infected varicella virus is added in the minimum necessary culture medium containing 2%~5% hyclone, be placed in
34~37 DEG C of cell culture incubator cultures；
(4) cytopathy degree is observed, when induced cytopathic effect reaches more than 50%, culture medium is outwelled, it is slow with phosphate
Rush solution washing cell；
(5) 0.02%-0.05% EDTA solution elution infection cell is added, by cell suspension centrifugal treating, removes supernatant, closes
And, mix, single bottle sampling do Sterility testing, -65~-80 DEG C freeze；
(6) it will detect that sterile sample carries out ultrasonication, supernatant, i.e. virus stock solution used are collected in centrifugation；
(7) virus stock solution used is concentrated by ultrafiltration using milipore filter bag；
(8) virus liquid protein content and virus titer after detection is concentrated by ultrafiltration；
(9) it is the viral concentration of 35~95mg/mL, virus titer not less than 4.0lgPFU/mL by beta-propiolactone and protein content
Liquid by volume 1：2000~1：8000 mixing, inactivate 12~36h, then the water under the conditions of 34~37 DEG C under the conditions of 3~5 DEG C
1.5~3h is solved, inactivation of viruses liquid is obtained；
(10) inactivation of viruses liquid is purified using column chromatography, obtains inactivation of viruses refined solution；
(11) by inactivation of viruses refined solution in human embryonic fibroblast's at least generation of blind passage 3, virus titer is detected, as a result all should nothing
Plaque test.
(12) dispense, lyophilized inactivation of viruses refined solution produces varicella virus inactivated vaccine.
2. people according to claim 1 is with the preparation method of varicella virus inactivated vaccine, it is characterised in that：Step (2) institute
It is 0.01~0.1 to state the infection multiplicity for being infected virus inoculation into cell.
3. people according to claim 1 is with the preparation method of varicella virus inactivated vaccine, it is characterised in that：Step (5) institute
The centrifugal condition stated is centrifuged 10-15 minutes for 3000~4000rpm at 3~5 DEG C.
4. people according to claim 1 is with the preparation method of varicella virus inactivated vaccine, it is characterised in that：Step (6) institute
The centrifugal condition stated is centrifuged 10-15 minutes for 7000~8000rpm at 3~5 DEG C.
5. people according to claim 1 is with the preparation method of varicella virus inactivated vaccine, it is characterised in that：Step (7) institute
That states uses milipore filter bag to be the ultrafiltration for using molecular cut off for 100KD or 300KD to virus stock solution used progress ultrafiltration concentration
Film bag is concentrated by ultrafiltration 30~60 times to virus stock solution used.
6. people according to claim 1 is with the preparation method of varicella virus inactivated vaccine, it is characterised in that：Step (8) institute
The protein content stated is detected using Lowry methods.
7. people according to claim 1 is with the preparation method of varicella virus inactivated vaccine, it is characterised in that：Step (9) institute
The inactivation condition stated is beta-propiolactone and viral concentration liquid by volume 1：8000 mixing, inactivate 24h, then under the conditions of 4 DEG C
2h is hydrolyzed under the conditions of 37 DEG C.
8. people according to claim 1 is with the preparation method of varicella virus inactivated vaccine, it is characterised in that：Step (10) institute
The way of purification stated is, by inactivation of virus liquid loading in batches, to be received under 280nm ultraviolet detections using Sepharose 4FF chromatographic columns
Water intaking poxvirus protein peak, as inactivation of viruses refined solution.
9. people according to claim 1 is with the preparation method of varicella virus inactivated vaccine, it is characterised in that：Step (8) and
(11) virus titer is detected using plaque method.
10. people prepared by a kind of any one of claim 1~9 uses varicella virus inactivated vaccine.
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| CN110743007A (en) * | 2019-09-10 | 2020-02-04 | 安徽智飞龙科马生物制药有限公司 | Combined vaccine and preparation method and application thereof |
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