CN102657858B - Method for preparing enterovirus vaccines - Google Patents
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- CN102657858B CN102657858B CN201210127077.6A CN201210127077A CN102657858B CN 102657858 B CN102657858 B CN 102657858B CN 201210127077 A CN201210127077 A CN 201210127077A CN 102657858 B CN102657858 B CN 102657858B
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Abstract
The invention provides a method for preparing enterovirus vaccines, which uses gerbil muscle cells as stroma cells of virus culture. The method uses the gerbil muscle cells as the stroma cells for culturing EV71 or CoxA16 viruses, growth titer of the EV71 or CoxA16 viruses on the cells is high, and liquid collection can be conducted repeatedly. The cells can be performed with passage repeatedly, muscle cell strains can be built for production of vaccines, and consumption of gerbil is saved. The cells are primary cells so that the problems that passage cell deoxyribonucleic acids (DNA) are difficult to process and the like do not exist. The vaccine production process is relatively easy, purification is convenient, and the prepared vaccines are safe and reliable when used for vaccination of infants.
Description
(1) technical field
The present invention relates to a kind of preparation method of enterovirus vaccine.
(2) background technology
Hand-foot-mouth disease can be caused by multiple enterovirus, comprising CoxA5, and A10, A16, A19, EV71, and part echovirus and change of coxsackie b virus, the most common with CoxA16 and EV71.
Enteric virus71 type (EV71) is to belong to people enterovirus to belong to, and Picornaviridae, is one of main pathogen of hand-foot-mouth disease.After infant infection, cause hand-foot-mouth disease and herpangina, can there is the complication such as aseptic meningitis, brain stem encephalitis, neurogenic pulmonary edema and acute flaccid paralysis in minority, occur serious nervous system, respiratory system and blood circulation symptom.From 1969, first since California, USA is isolated virus, the whole world experienced repeatedly the outbreak of epidemic of EV71.Since 1997, the number that infects EV71 significantly increases, and especially in the Asian-Pacific area, within 1997, Malaysian EV71 infects approximately 6000 people, more than 30 death of child, 1.5 years old mean age.Taiwan in 1998 reports that 129106 routine hand-foot-mouth disease infect altogether, and 405 severe central nervous systems by name infect, and 78 examples are dead.Spring in 2008, China breaks out Fuyang hand-foot-mouth disease epidemic situation, 78 death of child, and nearly 10000 people infect, and have caused the common concern of various circles of society.China's Ministry of Public Health, from May, 2008, is listed this disease in notifiable infectious diseases report sick kind.In recent years, hand-foot-mouth disease infected popular more and more extensive, not yet had effective medicine and vaccine to can be used for treatment and the prevention of this disease.
The at present existing Duo Jia research EV71 of research unit vaccine, but still have some unsafty places.At present can be divided into two classes for the production of the stromal cell of EV71 vaccine: a class is diploid cell, is mainly human embryonic lung cell; One class is Vero passage cell.The former, EV71 virus breeds in this diploid cell that titre is low, and output is restricted, and expanding production is more difficult; For the latter, because Vero cell is passage cell, in vaccine, contained passage cell DNA content need to be below 30ng, but it is more difficult again to remove DNA in virus liquid, causes that production cost is higher, efficiency is lower.Therefore the improvement of still needing of the stromal cell of, producing EV71 vaccine.
(3) summary of the invention
The object of the invention is to provide the preparation method of the enterovirus vaccine such as EV71, CoxA16.
The technical solution used in the present invention is:
A preparation method for enterovirus vaccine, described method comprises:
(1) get the gerbil jird in 10～15 day age, put to death, the aseptic muscular tissue (being mainly skeletal muscle) of taking off, shreds, and washes 2 times with the cell culture fluid containing 100U/ml kanamycin, goes most washing liquid;
(2) muscular tissue add 10 times of quality containing 0.2% (w/v) collagenase II, 0.25% (w/v) tryptic Digestive system, 4 DEG C of digestion are spent the night; W/v represents quality concentration of volume percent, and certain concentration of component 1% represents that in 100mL solution, this constituent content is 1g;
(3) stop discarding Digestive system after digestion, the cell culture fluid that adds quality and be 10 times of muscular tissue quality is blown and beaten repeatedly, cell dispersion, crosses 100,200 and 400 object stainless steel filtering nets successively, collects filtrate, centrifugal, abandon supernatant, sedimentation cell cell culture fluid Eddy diffusion, adding new-born calf serum to its final concentration is 10% (v/v), be seeded to cell bottle, in 36 DEG C of incubators, cultivate;
(4) after differential velocity adherent 2h, draw cell conditioned medium liquid, inoculate new culture bottle, 36 DEG C be cultured to Growth of Cells reach 70%～80% merge time, change containing the cell culture fluid of 5% (v/v) new-born calf serum and continue to cultivate, in the time that cell reaches 95% fusion, for virus inoculation;
(5) get the muscle monolayer cell culture bottle of 95% fusion, abandon culture fluid, virus inoculation titre is 1 × 10
3.0～5 × 10
3.0tCID
50containing the virus liquid of enterovirus, put in 35 DEG C of incubators and make viruses adsorption 2h, abandon virus liquid, add the cell culture fluid containing 1% (v/v) new-born calf serum, 100U/ml kanamycin, there are pathological changes (show as cell shrinkage, become circle and come off), results supernatant (this is 2nd generation virus) in 35 DEG C of cultured cells;
(6) supernatant of results is seeded to the freshly prepd 95% muscle cell monolayer merging as virus liquid, by step (5) repetitive operation 4～5 times, until virus titer is greater than 1 × 10 in the supernatant of results
6.0tCID
50(EV71 is after 5～6 generations of continuous passage in monolayer gerbil jird muscle cell, and the time that cytopathy occurs can be foreshortened to about 3 days by original about 10 days; Virus titer can be by original 1～5 × 10
3.0tCID
50be increased to 1 × 10
7.0tCID
50above), centrifugal removal cell debris, adds 10% (v/v) new-born calf serum, then adds the skim milk of 2 times of volumes of virus liquid, is viral seed culture of viruses, and after subpackage ,-80 DEG C save backup;
(7) get the viral seed culture of viruses of step (6), be seeded to the freshly prepd 95% muscle monolayer cell culture bottle merging, put in 35 DEG C of incubators and make viruses adsorption 2h, abandon virus liquid, wash 3 times with cell culture fluid, add the cell culture fluid without Ox blood serum containing 100U/ml kanamycin, cultivate 2～3 days, gather in the crops viral supernatant for 35 DEG C;
(8) after the deactivation of gained virus supernatant, concentrated through the ultrafilter membrane in 10 sub-very much metering-orifice footpaths, after Sepharose or Sephacryl gel filtration chromatography, results first peak, is vaccinogen liquid; It is 1: 160 left and right that vaccinogen liquid makes antigen amount through PBS dilution, adds human albumin in 0.5% (w/v) ratio, obtains vaccine semi-finished product, and vaccine semi-finished product through inspection, subpackage, are vaccine finished product again; Deactivation adopts conventional β-the third lactone or formalin-inactivated, be one of one of 3,000 to 5,000 of results virion accumulated amount for the β-the third lactone volumetric usage of inactivation of virus, as to use formalin-inactivated virus, volumetric usage be one of one of 2,000 to 4,000 of results virion accumulated amount.
In step (1)～(7), described cell culture fluid is one of following: MEM culture fluid, F10 culture fluid or F10/DMEM culture fluid.
Described enterovirus is enterovirus EV 71 or CoxA16, can from hand, mouth and foot diseases patient blister fluid, separate and obtain.
Described vaccine also can be EV71 and CoxA16 bivalent vaccine, enterovirus EV 71 and CoxA16 are made to vaccine semi-finished product by step (1)～(8) method respectively, mix by 1: 1 volume ratio again, after inspection, subpackage, obtain EV71 and CoxA16 bivalent vaccine finished product.
Beneficial effect of the present invention is mainly reflected in: the present invention selects gerbil jird muscle cell as the stromal cell of cultivating EV71 or CoxA16 virus, and EV71 or the CoxA16 virus titre of growing on this cell is high, can repeatedly receive liquid; This cell can repeatedly go down to posterity, and can be created as muscle cell line, for the production of vaccine, saves the consumption of gerbil jird; It is primary cell, does not exist passage cell DNA to be difficult to the problems such as processing, and preparation production of vaccine technique is relatively simple, and purification is convenient, and its prepared vaccine is inoculated infant, safer, reliable.
(4) detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
Embodiment 1: preparation EV71 univalent vaccine
(1) prepare cell monolayer
Get gerbil jird in 10～15 day age, cut off carotid artery sacrificed by exsanguination, set to 0 in .2% (v/v) benzalkonium bromide disinfectant solution and soak 30 minutes, the aseptic back leg muscle cell of getting, shreds, and washes secondary containing the MEM culture fluid of 100U/ml kanamycin, goes most washing liquid; Add 0.2% (w/v) collagenase II, 0.25% (w/v) tryptic digestive juice that muscular tissue weighs 10 times of quality, put 4 DEG C of digestion; After 16～20 hours, abandon Digestive system, add the MEM liquid that muscular tissue weighs 10 times of quality and repeatedly blow and beat cell dispersion, cross successively 100,200 and 400 object stainless steel filtering nets, collect filtrate, centrifugal, abandon supernatant, sedimentation cell MEM Eddy diffusion, adding new-born calf serum to ultimate density is 10% (v/v), and inoculating cell bottle is cultivated 2～3 days for 36 DEG C.In the time that Growth of Cells reaches 70%～80% fusion, the MEM that changes 5% (v/v) new-born calf serum cultivates and continues to cultivate.When cell reaches 95% fusion, for virus inoculation;
(2) virus inoculation and going down to posterity
Original EV71 Strain is existing known strain (CGMCC No.3650), preserves viral maintenance medium for the pH7.2MEM culture fluid containing 10% (v/v) calf serum, 100U/ml kanamycin, and wherein virus titer is about 1 × 10
3.0tCID
50;
Get the well-grown cell bottle of above-mentioned steps, abandon culture fluid, add EV71 virus, in 35 DEG C of incubators, make viruses adsorption 2h, abandon virus liquid, add 35 DEG C of MEM culture fluid be cultured to cell occur pathological changes (show as cell shrinkage, become circle and come off), results virus liquid, freeze thawing once, discharges the virus in cell, the centrifugal cell debris that goes, collects supernatant;
Virus goes down to posterity: the supernatant of results is added in the 95% new muscle monolayer cell culture bottle merging as virus liquid, in 35 DEG C of incubators, make viruses adsorption 2h, abandon supernatant, adding 35 DEG C of MEM culture fluid is cultured to cell and occurs pathological changes, results virus liquid, freeze thawing once, the centrifugal cell debris that goes, collect supernatant repeat aforesaid operations until obtain the 5th generation virus supernatant, its virus titer is about 1 × 10
7.0tCID
50, add 10% (v/v) new-born calf serum, then add the skim milk of 2 times of volumes of virus liquid, after assay approval, as production of vaccine seed culture of viruses;
(3) virus multiplication
Get above viral seed culture of viruses, the muscle monolayer cell culture bottle that inoculation 95% is merged, puts in 35 DEG C of incubators and makes viruses adsorption 2h, abandon virus liquid, wash 3 times with MEM culture fluid, add the MEM culture fluid without Ox blood serum containing 100U/ml kanamycin, cultivate 2～3 days results supernatant for 35 DEG C;
(4) inactivation of virus, concentrated and purification
The virus liquid of results is added to β-the third lactone of 1/4000 volume, inactivation of viruses 2h.The virus of deactivation, the concentrated suitable multiple of ultrafilter membrane through 10 sub-very much metering-orifice footpaths, then through Sepharose or Sephacryl gel filtration chromatography, results first peak, is vaccinogen liquid.Virus stock solution used need carry out sterility test, antigen quantitative determination, remaining Ox blood serum mensuration and safety test.Every test and measure equal eligible, enters next process.
(5) add human albumin
It is 1: 160 left and right that vaccinogen liquid is made to antigen amount through PBS dilution, then adds human albumin by 0.5% (w/v), is vaccine semi-finished product.Qualified through sterility test, carry out subpackage, every 1.1ml, is finished product.
Finished product vaccine liquid need carry out visual examination, sterility test, pH value mensuration, efficacy determinations, antiseptic test, toxicity test.More than detect the requirement that all meets national vaccine system inspection code, be EV71 inactivated vaccine.
Embodiment 2: preparation CoxA16 vaccine
Virus inoculation is replaced by CoxA16 Strain (Sixth Man people hospital provides by Hangzhou) in embodiment 1, other preparation method is identical, can prepare CoxA16 vaccine semi-finished product and vaccine finished product.
Embodiment 3: preparation EV71 and CoxA16 bivalent vaccine
CoxA16 vaccine semi-finished product prepared by the EV71 vaccine semi-finished product of preparing at example 1 and experimental example 2, mix by 1: 1 volume ratio, carry out subpackage, are bivalent enterovirus vaccine.
Claims (2)
1. a preparation method for enterovirus vaccine, described method comprises:
(1) get the gerbil jird in 10～15 day age, put to death, the aseptic muscular tissue of taking off, shreds, and washes 2 times with the cell culture fluid containing 100U/ml kanamycin, goes most washing liquid;
(2) add 10 times of quality of muscular tissue containing 0.2% collagenase II, 0.25% tryptic Digestive system, 4 DEG C of digestion are spent the night;
(3) stop abandoning Digestive system after digestion, the cell culture fluid that adds quality and be 10 times of muscular tissue quality is blown and beaten repeatedly, cell dispersion, crosses 100,200 and 400 object stainless steel filtering nets successively, collects filtrate, centrifugal, abandon supernatant, sedimentation cell cell culture fluid Eddy diffusion, adding new-born calf serum to its final concentration is 10%, be seeded to Tissue Culture Flask, put in 36 DEG C of incubators and cultivate;
(4) after differential velocity adherent 2h, supernatant suspension cell is transferred to new Tissue Culture Flask, and 36 DEG C are cultured to Growth of Cells and reach 70%～80% while merging, change containing the cell culture fluid of 5% new-born calf serum and continue to cultivate, in the time that cell reaches 95% fusion, for virus inoculation;
(5) get the muscle monolayer cell culture bottle of 95% fusion, abandon culture fluid, virus inoculation titre is 1 × 10
3.0～5 × 10
3.0tCID
50containing the virus liquid of enterovirus, put in 35 DEG C of incubators and make viruses adsorption 2h, abandon virus liquid, add the cell culture fluid containing 1% new-born calf serum, 100U/ml kanamycin, 35 DEG C are cultured to cell and occur pathological changes, results supernatant; Described enterovirus is enterovirus EV 71 or CoxA16;
(6) supernatant of results is seeded to the freshly prepd 95% muscle cell monolayer merging as virus liquid, by step (5) repetitive operation 4～5 times, until virus titer is greater than 1 × 10 in the supernatant of results
6.0tCID
50, centrifugal removal cell debris, adds 10% new-born calf serum, then adds the skim milk of 2 times of volumes of virus liquid, mixes, and is viral seed culture of viruses, and after subpackage ,-80 DEG C save backup;
(7) get the viral seed culture of viruses of step (6), be seeded to the freshly prepd 95% muscle monolayer cell culture bottle merging, put in 35 DEG C of incubators and make viruses adsorption 2h, abandon virus liquid, wash 3 times with cell culture fluid, add the cell culture fluid without Ox blood serum containing 100U/ml kanamycin, cultivate 2～3 days, gather in the crops viral supernatant for 35 DEG C;
(8) after the deactivation of gained virus supernatant, concentrated through the ultrafilter membrane in 10 sub-very much metering-orifice footpaths, after Sepharose or Sephacryl gel filtration chromatography, results first peak, is vaccinogen liquid; Vaccinogen liquid dilutes through PBS, and dilution ratio is 1:160, adds human albumin in 0.5% ratio, obtains vaccine semi-finished product, and vaccine semi-finished product through inspection, subpackage, are vaccine finished product again; Described deactivation adopts β-the third lactone or formaldehyde, is one of one of 3,000 to 5,000 of results virion accumulated amount for the β-the third lactone volumetric usage of inactivation of virus, and formaldehyde volumetric usage is one of one of 2,000 to 4,000 of results virion accumulated amount;
In step (1)～(7), described cell culture fluid is MEM culture fluid.
2. the method for claim 1, it is characterized in that described vaccine is EV71 and CoxA16 bivalent vaccine, enterovirus EV 71 and CoxA16 are made to vaccine semi-finished product by step (1)～(8) method respectively, be mixed in proportion again, after inspection, subpackage, obtain EV71 and CoxA16 bivalent vaccine finished product.
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| CN101402944A (en) * | 2008-11-17 | 2009-04-08 | 中国医学科学院医学生物学研究所 | EV-71 virus seed, inactivated vaccine for human and method of producing the same |
| CN101695570A (en) * | 2009-11-03 | 2010-04-21 | 中国人民解放军军事医学科学院微生物流行病研究所 | Univalent and bivalent inactivated vaccine for hand-foot-and-mouth disease and preparation method thereof |
| CN101897963A (en) * | 2010-03-18 | 2010-12-01 | 北京绿竹生物技术有限责任公司 | Vaccine for hand-foot-and-mouth disease viruses |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101402944A (en) * | 2008-11-17 | 2009-04-08 | 中国医学科学院医学生物学研究所 | EV-71 virus seed, inactivated vaccine for human and method of producing the same |
| CN101695570A (en) * | 2009-11-03 | 2010-04-21 | 中国人民解放军军事医学科学院微生物流行病研究所 | Univalent and bivalent inactivated vaccine for hand-foot-and-mouth disease and preparation method thereof |
| CN101897963A (en) * | 2010-03-18 | 2010-12-01 | 北京绿竹生物技术有限责任公司 | Vaccine for hand-foot-and-mouth disease viruses |
Non-Patent Citations (4)
| Title |
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| A mouse muscle-adapted enterovirus 71 strain with increased virulence in mice;Wei Wang et al.;《Microbes and Infection》;20110512;第13卷(第10期);862-863 * |
| C4亚型人肠道病毒71型的小鼠肌肉适应株对小鼠具有致死毒力;马春梅 等;《中国比较医学杂志》;20120530;第22卷(第5期);31-36 * |
| Wei Wang et al..A mouse muscle-adapted enterovirus 71 strain with increased virulence in mice.《Microbes and Infection》.2011,第13卷(第10期),862-870. |
| 马春梅 等.C4亚型人肠道病毒71型的小鼠肌肉适应株对小鼠具有致死毒力.《中国比较医学杂志》.2012,第22卷(第5期),31-36. |
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