CN102657857B - Method for preparing intestinal virus vaccine by using gerbil kidney cell - Google Patents
Method for preparing intestinal virus vaccine by using gerbil kidney cell Download PDFInfo
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Abstract
The invention provides a method for preparing an intestinal virus vaccine by using a gerbil kidney cell as a matrix cell for virus culture. According to the method, the gerbil kidney cell is used as the matrix cell for culturing EV71 or CoxA16 virus, wherein the growth titer of the EV71 or CoxA16 virus on the cell is high and liquid can be collected for many times. Secondly, the cell is a primary cell, so that the problem of difficulty in treatment of DNA (Deoxyribonucleic Acid) of a passage cell is solved; a production process for preparing the vaccine is relatively simple; the purification is convenient; and the prepared vaccine is safer and more reliable for inoculation of infants and young children.
Description
(1) technical field
The present invention relates to a kind of method of utilizing Cultured in M. unguiculatus Kidney Cell cell to prepare 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 nephridial tissue of taking off, shreds, and washes 2 times with the MEM culture fluid containing 100U/ml kanamycin, centrifugally goes most washing liquid;
(2) nephridial tissue add 10 times of quality containing 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, with the washing of MEM culture fluid, the MEM culture fluid that adds quality and be 10 times of nephridial tissue quality is blown and beaten repeatedly, cell dispersion, adding new-born calf serum to its final concentration is 3% (v/v), is seeded to cell bottle, in 36 DEG C of incubators, cultivates;
(4) reach 50%～60% while merging when being cultured to Growth of Cells, change containing the MEM culture fluid of 3% (v/v) new-born calf serum and continue to cultivate, in the time that cell reaches 95% fusion, for virus inoculation;
(5) get the kidney 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 MEM 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% kidney cell monolayer merging as virus liquid, by step (5) repetitive operation 6～7 times, until virus titer is greater than 1 × 10 in the supernatant of results
6.0tCID
50(EV71 is after 7～8 generations of continuous passage in monolayer Cultured in M. unguiculatus Kidney Cell 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
6.5～1 × 10
7.5tCID
50), 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% kidney monolayer cell culture bottle merging, put in 35 DEG C of incubators and make 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, 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.
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 Cultured in M. unguiculatus Kidney Cell 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.Secondly 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 kidney of getting, shreds, and washes three times containing the MEM culture fluid of 100U/ml kanamycin, centrifugally goes most washing liquid; Add 0.25% (w/v) tryptic digestive juice that kidney weighs 10 times of quality, put 4 DEG C of digestion; After 16 hours, abandon Digestive system, use MEM culture fluid to wash once.Add the MEM liquid that kidney weighs 10 times of quality and repeatedly blow and beat cell dispersion, adding new-born calf serum to ultimate density is 3% (v/v), and inoculating cell bottle is cultivated 2～3 days for 36 DEG C.In the time that Growth of Cells reaches 50%～60% fusion, the MEM that changes 3% (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 pH7.2 containing 10% (v/v) calf serum, 100U/ml kanamycin, MEM culture fluid, 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 maintenance medium, in 35 DEG C of incubators, make viruses adsorption 2h, abandon viral maintenance medium, 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 kidney monolayer cell culture bottle merging as seed culture of viruses, 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 7th generation virus supernatant, its virus titer is about 1 × 10
6.75tCID
50, after assay approval, as production of vaccine seed culture of viruses;
(3) virus multiplication
Get above viral seed culture of viruses, the kidney 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, after 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. utilize Cultured in M. unguiculatus Kidney Cell cell to prepare a method for enterovirus vaccine, described enterovirus is enterovirus EV 71 or CoxA16, and described method comprises:
(1) get the gerbil jird in 10～15 day age, put to death, the aseptic nephridial tissue of taking off, shreds, and washes 2 times with the MEM culture fluid containing 100U/ml kanamycin, centrifugally goes most washing liquid;
(2) nephridial tissue adds the 0.25% tryptic Digestive system that contains of 10 times of quality, and 4 DEG C of digestion are spent the night;
(3) stop discarding Digestive system after digestion, with the washing of MEM culture fluid, the MEM culture fluid that adds quality and be 10 times of nephridial tissue quality is blown and beaten repeatedly, cell dispersion, and adding new-born calf serum to its final concentration is 3%, is seeded to cell bottle, in 36 DEG C of incubators, cultivates;
(4) reach 50%～60% while merging when being cultured to Growth of Cells, change containing the MEM culture fluid of 3% new-born calf serum and continue to cultivate, in the time that cell reaches 95% fusion, for virus inoculation;
(5) get the kidney 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 MEM culture fluid containing 1% new-born calf serum, 100U/ml kanamycin, 35 DEG C are cultured to cell and occur pathological changes, results supernatant;
(6) supernatant of results is seeded to the freshly prepd 95% kidney cell monolayer merging as virus liquid, by step (5) repetitive operation 6～7 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% kidney monolayer cell culture bottle merging, put in 35 DEG C of incubators and make 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, 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 that vaccinogen liquid makes antigen amount through PBS dilution, 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.
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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| CN106318914B (en) * | 2016-09-29 | 2019-10-29 | 浙江省疾病预防控制中心 | A kind of human enterovirus strain and its application |
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| CN101575593A (en) * | 2009-06-03 | 2009-11-11 | 唐山怡安生物工程有限公司 | Vaccine for hand-foot-mouth disease and preparation method and application thereof |
| 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 |
| CN101695569A (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 gene engineered subunit vaccine for hand-foot-and-mouth disease and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101575593A (en) * | 2009-06-03 | 2009-11-11 | 唐山怡安生物工程有限公司 | Vaccine for hand-foot-mouth disease and preparation method and application thereof |
| 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 |
| CN101695569A (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 gene engineered subunit vaccine for hand-foot-and-mouth disease and preparation method thereof |
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