CN102657857A - 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 the gerbil jird nephrocyte to prepare enterovirus vaccine.
(2) background technology
Hand-foot-mouth disease can cause 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.
Enterovirus 71 types (EV71) are to belong to the people enterovirus to belong to, and Picornaviridae is one of main pathogen of hand-foot-mouth disease.Cause hand-foot-mouth disease and herpangina behind the infant infection, complication such as aseptic meningitis, BBE, neurogenic pulmonary edema and acute flaccid paralysis can take place in minority, serious nervous system, respiratory system and blood circulation symptom occur.First since California, USA is isolated virus, the whole world experienced repeatedly the outbreak of epidemic of EV71 from 1969.Since 1997, the number that infects EV71 significantly increases, and especially in the Asian-Pacific area, Malaysian EV71 infected about 6000 people in 1997, more than 30 death of child, 1.5 years old mean age.Taiwan in 1998 reports that altogether 129106 routine hand-foot-mouth disease infect, 405 serious central nervous system infections by name, and 78 examples are dead.Spring in 2008, China breaks out the 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 is listed this disease in statutory report infection disease notification sick kind from May, 2008.In recent years, hand-foot-mouth disease infected more and more widely popular, and effective medicine of Shang Weiyou and vaccine can be used for the treatment and the prevention of this disease.
The existing how tame at present research EV71 of research unit vaccine, but some unsafty places are still arranged.The stromal cell that is used to produce the EV71 vaccine at present can be divided into two types: one type is diploid cell, mainly is the human embryonic lung cell; One type is the Vero passage cell.The former, EV71 virus breeds in this diploid cell that titre is low, and output is restricted, and enlarges to produce relatively difficulty; For the latter, because the Vero cell is passage cell, contained passage cell dna content need be below 30ng in the vaccine, but removes the DNA difficulty again in the viral liquid, causes that production cost is higher, efficient is lower.Therefore, the improvement of still needing of the stromal cell of producing the EV71 vaccine.
(3) summary of the invention
The object of the invention provides the method for preparing of enterovirus vaccine such as EV71, CoxA16.
The technical scheme that the present invention adopts is:
A kind of method for preparing of enterovirus vaccine, said 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 that contains the 100U/ml kanamycin, centrifugally goes most washing liquid;
What (2) nephridial tissue added 10 times of quality contains the tryptic Digestive system of 0.25% (w/v), and 4 ℃ of digestion are spent the night; W/v representes the mass and size percent concentration, and this constituent content is 1g in certain concentration of component 1% expression 100mL solution;
(3) stop discarding Digestive system after the digestion, wash with the 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 NBCS to its final concentration is 3% (v/v), is seeded to the cell bottle, cultivates in 36 ℃ of incubators;
(4) reach 50%～60% when merging when being cultured to cell growth, change the MEM culture fluid that contains 3% (v/v) NBCS and continue to cultivate,, be used for virus inoculation when cell reaches 95% when merging;
(5) get the 95% kidney cell monolayer culture bottle that merges, abandon culture fluid, the virus inoculation titre is 1 * 10
3.0～5 * 10
3.0TCID
50The viral liquid that contains enterovirus; Put and make virus absorption 2h in 35 ℃ of incubators; Abandon viral liquid; Add the MEM culture fluid that contains 1% (v/v) NBCS, 100U/ml kanamycin, pathological changes (show as cell shrinkage, become circle and come off), results supernatant (this is the 2nd a generation virus) appear in 35 ℃ of cultured cells;
(6) supernatant of results is seeded to the freshly prepd 95% kidney cell monolayer that merges as viral liquid, and (5) repetitive operation is 6～7 times set by step, and virus titer is greater than 1 * 10 in the supernatant of results
6.0TCID
50(after 7～8 generations of continuous passage, the time that cytopathy occurs can be foreshortened to about 3 days by original about 10 days EV71 in monolayer gerbil jird nephrocyte; 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) NBCS, adds the skim milk of 2 times of volumes of viral liquid again, is viral seed culture of viruses, and after the packing ,-80 ℃ of preservations are subsequent use;
(7) get the viral seed culture of viruses of step (6); Be seeded to the kidney cell monolayer culture bottle of freshly prepd 95% fusion, put and make virus absorption 2h in 35 ℃ of incubators, abandon viral liquid; Wash 3 times with the MEM culture fluid; The MEM culture fluid that adds the no Ox blood serum that contains the 100U/ml kanamycin was cultivated 2～3 days, and was gathered in the crops viral supernatant for 35 ℃;
(8) after the deactivation of gained virus supernatant, the ultrafilter membrane through 10 sub-very much metering-orifice footpaths concentrates, and after Sepharose or Sephacryl gel filtration chromatography, the results first peak is vaccinogen liquid; It is about 1: 160 that vaccinogen liquid makes the antigen amount through the PBS dilution, adds the human albumin in 0.5% (w/v) ratio, gets the vaccine semi-finished product, and the vaccine semi-finished product through check, packing, are the vaccine finished product again; Conventional β-third lactone or formalin-inactivated are adopted in deactivation; β-third lactone the volumetric usage that is used for inactivation of virus is one of one of 3,000 to 5,000 of the viral volume of results; As with formalin-inactivated virus, volumetric usage is one of one of 2,000 to 4,000 of the viral volume of results.
Said enterovirus is enterovirus EV 71 or CoxA16, can from hand, mouth and foot diseases patient BF, separate acquisition.
Said vaccine also can be EV71 and CoxA16 bivalent vaccine, with enterovirus EV 71 and CoxA16 respectively set by step (1)～(8) method make the vaccine semi-finished product, mix by 1: 1 volume ratio again, through checking, after the packing, obtaining EV71 and CoxA16 bivalent vaccine finished product.
Beneficial effect of the present invention is mainly reflected in: the present invention selects the gerbil jird nephrocyte as the stromal cell of cultivating EV71 or CoxA16 virus, and EV71 or the CoxA16 virus titre height of on this cell, grow can repeatedly be received liquid.Secondly it is a primary cell, does not exist passage cell DNA to be difficult to problems such as processing, and preparation production of vaccine technology is simple relatively, convenient purification, and its prepared vaccine is inoculated infant, and is safer, reliable.
(4) specific embodiment
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) preparation cell monolayer
Get gerbil jird in 10～15 day age, cut off the carotid artery sacrificed by exsanguination, put in 0.2% (v/v) benzalkonium bromide disinfectant solution and to soak 30 minutes, the aseptic kidney of getting shreds, and the MEM culture fluid that contains the 100U/ml kanamycin is given a baby a bath on the third day after its birth time, centrifugally goes most washing liquid; Add 0.25% (w/v) tryptic digestive juice that kidney weighs 10 times of quality, put 4 ℃ of digestion; Abandon Digestive system after 16 hours, once with the washing of MEM culture fluid.Add the MEM liquid that kidney weighs 10 times of quality and blow and beat cell dispersion repeatedly, adding NBCS to ultimate density is 3% (v/v), and the inoculating cell bottle was cultivated 2～3 days for 36 ℃.When the cell growth reached 50%～60% fusion, the MEM that changes 3% (v/v) NBCS cultivated and continues to cultivate.When cell reaches 95% fusion, be used for virus inoculation;
(2) virus inoculation and going down to posterity
Original EV71 Strain is existing known strain (CGMCC No.3650), and the liquid of keeping of preserving virus is the pH7.2 that contains 10% (v/v) calf serum, 100U/ml kanamycin, the 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 and keep liquid; Make virus absorption 2h in 35 ℃ of incubators, abandon virus and keep liquid, add 35 ℃ of MEM culture fluid and be cultured to cell and pathological changes (show as cell shrinkage, become circle and come off) occurs; Gather in the crops viral liquid, freeze thawing once discharges the virus in the cell; The centrifugal cell debris that goes is collected supernatant;
Virus goes down to posterity: the supernatant of results is added in the kidney cell monolayer culture bottle of 95% new fusion as seed culture of viruses, makes virus absorption 2h in 35 ℃ of incubators, abandons supernatant; Adding the MEM culture fluid is cultured to cell for 35 ℃ and pathological changes occurs; Gather in the crops viral liquid, freeze thawing once, the centrifugal cell debris that goes; Collect supernatant and repeat aforesaid operations until the supernatant that obtains the 7th generation virus, its virus titer is about 1 * 10
6.75TCID
50, behind assay approval, use seed culture of viruses as production of vaccine;
(3) virus multiplication
Get above viral seed culture of viruses, the kidney cell monolayer culture bottle that inoculation 95% is merged is put and is made virus absorption 2h in 35 ℃ of incubators; Abandon viral liquid, wash 3 times, add the MEM culture fluid of the no Ox blood serum that contains the 100U/ml kanamycin with the MEM culture fluid; Cultivated 2～3 days the results supernatant for 35 ℃;
(4) inactivation of virus, concentrated and purification
The viral liquid of results is added β-third lactone of 1/4000 volume, inactivation of viruses 2h.The virus of deactivation, the ultrafilter membrane through 10 sub-very much metering-orifice footpaths concentrates suitable multiple, and after Sepharose or Sephacryl gel filtration chromatography, the 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.Equal eligible is tested and measured to each item, gets into next process.
(5) add the human albumin
It is about 1: 160 that vaccinogen liquid is made the antigen amount through the PBS dilution, adds the human albumin by 0.5% (w/v) again, is the vaccine semi-finished product.Qualified through sterility test, carry out packing, 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 of the vaccine system inspection rules that all meet country, be the EV71 inactivated vaccine.
Embodiment 2: preparation CoxA16 vaccine
In embodiment 1, virus inoculation is replaced by CoxA16 Strain (Sixth Man people hospital provides by Hangzhou), other method for preparing is identical, can prepare CoxA16 vaccine semi-finished product and vaccine finished product.
Embodiment 3: preparation EV71 and CoxA16 bivalent vaccine
At the EV71 vaccine semi-finished product of instance 1 preparation and the CoxA16 vaccine semi-finished product of experimental example 2 preparations, mix by 1: 1 volume ratio, carry out packing, be two valency enterovirus vaccine.
Claims (3)
1. method of utilizing the gerbil jird nephrocyte to prepare enterovirus vaccine, said 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 that contains the 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 ℃ of digestion are spent the night;
(3) stop discarding Digestive system after the digestion, wash with the 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 NBCS to its final concentration is 3%, is seeded to the cell bottle, cultivates in 36 ℃ of incubators;
(4) reach 50%～60% when merging when being cultured to cell growth, change the MEM culture fluid that contains 3% NBCS and continue to cultivate,, be used for virus inoculation when cell reaches 95% when merging;
(5) get the 95% kidney cell monolayer culture bottle that merges, abandon culture fluid, the virus inoculation titre is 1 * 10
3.0～5 * 10
3.0TCID
50The viral liquid that contains enterovirus, put and make virus absorption 2h in 35 ℃ of incubators, abandon viral liquid, add the MEM culture fluid that contains 1% NBCS, 100U/ml kanamycin, 35 ℃ are cultured to cell and pathological changes occur, the results supernatant;
(6) supernatant of results is seeded to the freshly prepd 95% kidney cell monolayer that merges as viral liquid, and (5) repetitive operation is 6～7 times set by step, and virus titer is greater than 1 * 10 in the supernatant of results
6.0TCID
50, centrifugal removal cell debris adds 10% NBCS, adds the skim milk of 2 times of volumes of viral liquid again, and mixing is viral seed culture of viruses, and after the packing ,-80 ℃ of preservations are subsequent use;
(7) get the viral seed culture of viruses of step (6); Be seeded to the kidney cell monolayer culture bottle of freshly prepd 95% fusion, put and make virus absorption 2h in 35 ℃ of incubators, abandon viral liquid; Wash 3 times with the MEM culture fluid; The MEM culture fluid that adds the no Ox blood serum that contains the 100U/ml kanamycin was cultivated 2～3 days, and was gathered in the crops viral supernatant for 35 ℃;
(8) after the deactivation of gained virus supernatant, the ultrafilter membrane through 10 sub-very much metering-orifice footpaths concentrates, and after Sepharose or Sephacryl gel filtration chromatography, the results first peak is vaccinogen liquid; It is 1: 160 that vaccinogen liquid makes the antigen amount through the PBS dilution, adds the human albumin in 0.5% ratio, gets the vaccine semi-finished product, and the vaccine semi-finished product through check, packing, are the vaccine finished product again.
2. the method for claim 1 is characterized in that said enterovirus is enterovirus EV 71 or CoxA16.
3. the method for claim 1; It is characterized in that said vaccine is EV71 and CoxA16 bivalent vaccine; With enterovirus EV 71 and CoxA16 respectively set by step (1)～(8) method make the vaccine semi-finished product; Proportional mixing after check, packing, obtains EV71 and CoxA16 bivalent vaccine finished product again.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104490937A (en) * | 2014-12-09 | 2015-04-08 | 浙江省疾病预防控制中心 | Animal model of CA16 virus infected gerbil and application thereof |
| CN106318914A (en) * | 2016-09-29 | 2017-01-11 | 浙江省疾病预防控制中心 | Human enterovirus strain and application thereof |
Citations (3)
| 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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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104490937A (en) * | 2014-12-09 | 2015-04-08 | 浙江省疾病预防控制中心 | Animal model of CA16 virus infected gerbil and application thereof |
| CN104490937B (en) * | 2014-12-09 | 2018-08-21 | 浙江省疾病预防控制中心 | A kind of CA16 viruses infect animal model and the application of gerbil jird |
| CN106318914A (en) * | 2016-09-29 | 2017-01-11 | 浙江省疾病预防控制中心 | Human enterovirus strain and application thereof |
| 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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