CN103160477B - Method for improving Japanese encephalitis virus titer - Google Patents
Method for improving Japanese encephalitis virus titer Download PDFInfo
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- CN103160477B CN103160477B CN201310058501.0A CN201310058501A CN103160477B CN 103160477 B CN103160477 B CN 103160477B CN 201310058501 A CN201310058501 A CN 201310058501A CN 103160477 B CN103160477 B CN 103160477B
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract
The invention provides a method for proliferation of a Japanese encephalitis virus. The method for proliferation of the Japanese encephalitis virus comprises the steps of culturing cells, inoculating Japanese encephalitis viruses to the cells which are provided with mono-layers in an overgrowing mode, and directly obtaining a virus solution until a rate of a cytopathic effect reaches to 75%-95% after inoculation. The invention further provides a preparation method of Japanese encephalitis vaccines. The method for proliferation of the Japanese encephalitis virus and the preparation method of the Japanese encephalitis vaccines can remarkably improve a Japanese encephalitis virus titer, and meanwhile shorten production periods, and can prepare the Japanese encephalitis vaccines which are better, stable, and good in immunogenicity.
Description
Technical field
The present invention relates to the method for a kind of encephalitis b virus propagation, and prepare vaccine for virus of encephalitis B.In particular to a kind of method that can significantly improve encephalitis b virus and tire.
Background technology
Epidemic encephalitis type B (epidemic encephalitis B, referred to as " encephalitis ") also known as Japanese encephalitis (Japanese encephalitis, JE), by encephalitis b virus (Japaneseencephalitis virus, JEV, referred to as " encephalitis b virus ") cause, through the acute infectious disease of killing propagation.This disease can constitute a serious infringement and infect central nervous system, causes clinical onset and presents nervous symptoms.Meanwhile, this disease is a kind of disease of natural focus, not only can infect people, can also infect other animals such as horse, pig.In recent years, the scope of breaking out of this disease spreads to other areas such as Australia by Asia.Mosquito is the primary vehicle of encephalitis b virus, and when there is no specific medicament and the method for the treatment of encephalitis B disease at present, controlling killing propagation and carrying out vaccine immunization is significant defense means present stage controlling encephalitis B disease.
At present, Vaccinum Encephalitis B mainly comprises three major types: cultivate the inactivated vaccine of preparation through murine brain, inactivated vaccine prepared by suslik kidney primary cell or Vero cell cultures and attenuated live vaccine prepared by suslik kidney primary cell culture.Wherein, Inactivated Japanese Encephalitis Vaccine prepared by suslik kidney primary cell is applied and attenuated live vaccine serves vital role in the prevention of encephalitis B disease.But then, because suslik kidney primary cell receives all many-sided impacts such as trysinization condition, culture condition and viral biological nature own in preparation process, encephalitis b virus tiring in breeding is caused not to be also fine, comparison in difference between every batch is large, production cycle is long, therefore affect immunogenicity, even can also cause clinical immunization failure.
Summary of the invention
In order to overcome the above problems, the present inventor is by optimizing the condition of encephalitis b virus propagation, improvement of production process and sum up method and the preparation method of vaccine thereof of encephalitis b virus propagation.Tiring of encephalitis b virus can be significantly improved by the method, shorten the production cycle simultaneously, and can prepare thus there is higher, more stable, the better Vaccinum Encephalitis B of immunogenicity.
The method that encephalitis b virus according to the present invention is bred, after the method comprises culturing cell, to the cell inoculation encephalitis B kind poison covering with individual layer, wherein, after inoculation, directly gathers in the crops virus liquid when cytopathy reaches 75-95%.
According to the present invention, the encephalitis B kind poison inoculated is the 0.5-1 volume % of cell maintenance medium amount.
According to the present invention, described cell is hamster kidney cell, and the suslik nephridial tissue that the method for described culturing cell comprises collecting carries out trysinization, and isolated cell carries out rotation and cultivates after moving into nutrient solution; After described trysinization comprises add pancreatin in the suslik nephridial tissue collected, at being placed in 6-12 DEG C, digest 9-12 hour.
According to the present invention, the volume ratio of the pancreatin added and suslik nephridial tissue is 2-3.5:1, and the temperature of carrying out trysinization is 8-10 DEG C, and the time is 9.5-10.5 hour.
According to the present invention, before carrying out trysinization, with 7-8 quality %NaHCO
3the PH of pancreatin is adjusted to 7.2-7.4.
According to the present invention, the temperature of carrying out cultivating is 35.5-38.5 DEG C, and carrying out cultured cells concentration is 1.8-2.5 × 10
8the cell quantity of individual/L.
According to the present invention, described nutrient solution is the dual anti-newborn Chinese liquid containing 10% calf serum and 100 units/ml.
According to the present invention, described nutrient solution is also containing RPMI1640, and the volume ratio of newborn Chinese liquid and RPMI1640 is 1:0.8-1.2.
Present invention also offers a kind of method preparing Vaccinum Encephalitis B, the method comprises carries out joining seedling with encephalitis b virus, and wherein, this encephalitis b virus obtains the method that encephalitis b virus is bred by according to of the present invention.
According to method of the present invention, described Vaccinum Encephalitis B is inactivated vaccine and/or attenuated live vaccine.
Embodiment
The invention provides the method for a kind of encephalitis b virus propagation, the method comprises the cell inoculation encephalitis B kind poison to covering with individual layer, wherein, after inoculation, when cytopathy reaches 75-95%, directly gathers in the crops virus liquid.
In conventional process of breeding encephalitis b virus, after the cytopathy being vaccinated with encephalitis B kind poison acquires a certain degree, also need to gather in the crops virus liquid again after freeze thawing operation at least one times.This is because general theory is all thought just can must make cytoclasis discharge viral ion by freeze thawing repeatedly, and virus titer obtains and improves, and can prepare the vaccine of high-quality thus.And the present inventor finds, method according to the present invention is directly gathered in the crops without freeze thawing the virus liquid obtained and is had higher tiring on the contrary, and can prepare homogeneous, stable, that immunogenicity is good Vaccinum Encephalitis B.
According to the present invention, described cell is preferably hamster kidney cell, and the suslik nephridial tissue that the method for described culturing cell comprises collecting carries out trysinization, and isolated cell carries out rotation and cultivates after moving into nutrient solution.
Generally speaking, pancreatin refers to trypsin Trypsin), commercially available from GIBCO, the brands such as Sigma (Sigma).Preferably the pH value of pancreatin is adjusted to neutral before carrying out trysinization according to method of the present invention.Such as, can 7-8 quality %(such as 7.5 quality % be used) NaHCO
3the PH of pancreatin is adjusted to 7.2-7.4.The pancreatin added and the volume ratio of tissue are 2-3.5:1, and the volume ratio of such as pancreatin and suslik nephridial tissue is 2:1,2.2:1,2.4:1,2.6:1,2.8:1,3:1,3.1:1,3.2:1,3.3:1,3.5:1 etc.Carrying out the temperature that digests for being 6-12 DEG C, such as, can be 6 DEG C, 7 DEG C, 8 DEG C, 9 DEG C, 10 DEG C, 11 DEG C, 12 DEG C etc., be preferably 8-10 DEG C, such as, can be 8.3 DEG C, 8.6 DEG C, 8.9 DEG C, 9.2 DEG C, 9.6 DEG C, 9.8 DEG C, 10 DEG C etc.
Digestion nutrient solution can be jiggled in the process of carrying out trysinization, contact more fully with pancreatin to make suslik kidney tissue block.The time of carrying out digesting can be 9-12 hour, is preferably 9.5-10.5 hour.Treat that suslik kidney tissue block becomes bulk, and when batt layer appears in upper strata, namely pourable pancreatin stops digestion.Subsequently, enzyme operation can be taken off at least one times to tissue further, preferably carry out 2-3 de-enzyme operation with newborn Chinese liquid.
Described newborn Chinese liquid for adding the Chinese krebs solution of 5 ‰ milk-proteins (with Chinese krebs solution mass ratio), wherein, milk-protein and all commercially available GIBCO brand of Chinese krebs solution.Nutrient solution with newborn Chinese liquid for solvent, containing 10% calf serum (commercially available from Jing Niu bio tech ltd, Jinan) with 100 units/ml's is dual anti-.Also GIBCO brand is purchased containing RPMI1640(in preferred nutrient solution), there is no particular limitation for the volume ratio of newborn Chinese liquid and RPMI1640, is preferably 1:0.8-1.2, is more preferably 1:0.9-1.1.Dual anti-ly refer to penicillin and Streptomycin sulphate, to dual anti-ly having no particular limits of using in the present invention, be limited so that method of the present invention can be realized, can be commercially available from companies such as such as GIBCO.There is no particular limitation to carry out the temperature of cultivating and cell concn, is limited to perform the methods of the present invention.As a kind of embodiment, the temperature of cultivation is 35.5-38.5 DEG C, and carrying out cultured cells concentration is 1.8-2.5 × 10
8the cell quantity of individual/L.
Detect by an unaided eye cell growth status in the process of culturing cell, when cell covers with individual layer (adherent), nutrient solution can be poured out, add the cell maintenance medium of the calf serum of 2%, there is no particular limitation for described cell maintenance medium, can be the dual anti-of 2% calf serum that adds in newborn Chinese liquid and 100 units/ml.To the amount of maintenance medium, there is no particular limitation, and general consumption did not have cell.Subsequently, according to the 0.5-1% volume inoculation encephalitis b virus of cell maintenance medium amount.
Described encephalitis b virus is had no particular limits, as long as object of the present invention can be met.As a kind of exemplary embodiment, encephalitis b virus of the present invention can be encephalitis b virus SA
14-14-2strain, P
3any strain in other strain isolateds of strain, Nakayama strain (commercially available from Wuhan Biological Products Inst.) and encephalitis b virus.
After inoculation encephalitis b virus, by the degree of microscope observing cell pathology, determine according to the needs produced the cell degree can gathering in the crops virus liquid.Usually, according to the standard in industry, treat that cytopathy variability reaches 75-100% and can gather in the crops virus liquid.
Described suslik nephridial tissue preferably gathers the suslik from 10-14 age in days under aseptic technique, and suslik is commercially available from Beijing laboratory animal company limited of dimension tonneau China etc.Subsequently, with pancreatin, trysinization is carried out to collected suslik nephridial tissue, after the method comprises add pancreatin in the suslik nephridial tissue collected, at being placed in 6-12 DEG C, digest 9-12 hour.
Present invention also offers a kind of method preparing Vaccinum Encephalitis B, encephalitis b virus carries out joining seedling, it is characterized in that, this encephalitis b virus obtains the method that encephalitis b virus is bred by according to of the present invention.There is no particular limitation for described method of joining seedling; method well known in the art can be adopted; refer to by encephalitis b virus and lyophilized vaccine according to a certain percentage (such as 6-8:1) mix; can contain 8 quality % gelatin and 40 quality % sucrose in described gelatin protective material, gelatin and sucrose are all purchased from China Medicine (Group) Shanghai Chemical Reagent Co..
Vaccinum Encephalitis B method is prepared according to of the present invention, owing to eliminating freeze thawing operation in the process of breeding encephalitis b virus, tiring of the virus liquid that results are obtained is improved significantly, therefore, can to tire height, the vaccine for virus of encephalitis B that stable, immunogenicity is good by joining seedling preparation with it.As a kind of embodiment, described Vaccinum Encephalitis B is inactivated vaccine and/or attenuated live vaccine.
To sum up, method of the present invention, by saving the freeze thawing operation in ordinary method, is directly gathered in the crops virus liquid without freeze thawing, and is joined seedling to it and then prepare Vaccinum Encephalitis B after the lesion degree that cell reaches required.Such one side can carry out higher, the more stable encephalitis b virus of obtained quality by significantly improving virus titer.On the other hand, the method can shorten process cycle significantly, reduces production cost, improves production efficiency.
Embodiment
Below in conjunction with embodiment, with encephalitis b virus (SA
14-14-2) set forth method of the present invention in detail for example.It is to be appreciated that the present embodiment is only for exemplarily explaining and content of the present invention, the present invention is not limited thereto.
Preparation embodiment 1:
The process of suslik kidney: the suslik kidney (being purchased from Beijing laboratory animal company limited of dimension tonneau China) of the 10-14 age in days gathered is cleaned twice with the Chinese krebs solution (being purchased GIBCO brand) containing 400 units/ml dual anti-(being purchased GIBCO brand) of 500ml.Be advisable by nephridial tissue ten thousand ml cells bottles of 20 susliks.With scissors, tissue is shredded, cut even.Then repeatedly rinse beaker inner tissue with containing the dual anti-Chinese krebs solution of 400 units/ml, in beaker supernatant liquor limpid till, last fully precipitation, removes supernatant liquor.
Trysinization: regulate the PH of pancreatin to be 7.2-7.4 with the NaHCO3 of 7.5 quality %, is pouring into the tissue precipitated above in digestion bottle (aseptic technique).By pancreatin: suslik nephridial tissue be 2:1 volume ratio add 0.25 quality % pancreatin (being purchased GIBCO brand), put into 6 DEG C of refrigerators carry out 11 hours digestion.Centre can jog several times.Tissue block is fully contacted with pancreatin, treats that tissue block becomes bulk, and occur batt layer on upper strata, terminate digestion.
Cell dispersal and cultivation: in cell harvesting bottle by cultivate liquid measure add be purchased from GIBCO containing the dual anti-newborn Chinese liquid of 10% calf serum (being purchased from Jing Niu bio tech ltd, Jinan), 100 units/ml and RPMI1640() nutrient solution, wherein the volume ratio of newborn Chinese liquid and RPMI1640 is 1:0.8.Gone gently by the pancreatin of digestion bottle, carry out de-enzyme with the newborn Chinese liquid of about 500ml, during each de-enzyme, jolting dispersion bottle, after shaking up precipitation, pours in receiving flask by upper strata cell dispersion, in triplicate.Carrying out third time de-enzyme, first firmly jolting digestion bottle, then liquid feeding shakes up precipitation, pours receiving flask into, so carries out repeatedly, until supernatant liquor becomes clear, in digestion bottle, only discard residue till surplus a small amount of flocculated sludge.Make the cell in receiving flask to discard supernatant liquor after the centrifugal 10min of 1000rpm.With containing 10% calf serum and 100 units/ml dual anti-nutrient solution re-suspended cell and count, with 1.8 × 10
8the cell quantity of individual/L is inoculated in rolling bottle, put 37 DEG C of greenhouses, revolution be 7-8 turn/hour Rotary Machine on cultivate.
Virus inoculation and results: cover with after individual layer until cell, pour out nutrient solution, add the cell maintenance medium containing 2% calf serum, there be not cell.Then press the 0.5 volume % maintaining liquid measure and inoculate encephalitis kind poison, cultivate at inoculation is placed on 35.5 DEG C, when cytopathy reaches 95%, gather in the crops virus liquid.
The virus liquid gathered in the crops, without freeze thawing, is applied directly to join in seedling and goes.
Preparation embodiment 2:
Be prepared according to the method identical with preparing embodiment 1, unlike:
In trysinization step, pancreatin: suslik nephridial tissue volume ratio is 2.5:1, is placed in the digestion that 7 DEG C are carried out 12 hours.
In cell dispersal culturing step, the volume ratio of newborn Chinese liquid and RPMI1640 is that to carry out cultured cells concentration be 2 × 10 to 1:0.9
8the cell quantity of individual/L.
Virus inoculation, with in results, after inoculating encephalitis kind poison, is cultivated, when cytopathy reaches 100%, gather in the crops virus liquid by the 0.8 volume % maintaining liquid measure at 37 DEG C.
Preparation embodiment 3:
Be prepared according to the method identical with preparing embodiment 1, unlike:
In trysinization step, pancreatin: suslik nephridial tissue volume ratio is 3.5:1, is placed in the digestion that 12 DEG C are carried out 9 hours.
In cell dispersal culturing step, the volume ratio of newborn Chinese liquid and RPMI1640 is that to carry out cultured cells concentration be 2.5 × 10 to 1:1.2
8the cell quantity of individual/L.
Virus inoculation, with in results, after inoculating encephalitis kind poison, is cultivated, when cytopathy reaches 75%, gather in the crops virus liquid by the 1 volume % maintaining liquid measure at 38.5 DEG C.
Simultaneous test 1: trysinization condition optimizing
The suslik of 200 10-14 ages in days is divided into 5 groups at random, often organizes 40.The nephridial tissue of aseptic collection 5 groups of susliks, and being shredded by tissue in sterile beaker, cuts even, then repeatedly rinses beaker inner tissue with the Chinese krebs solution dual anti-containing 400 units/ml, in beaker supernatant liquor limpid till.
After fully precipitating, remove supernatant liquor, by pancreatin: suslik nephridial tissue is the pancreatin that the volume ratio of 3:1 adds 0.25 quality %.5 groups of nephridial tissues are placed in respectively in 37 DEG C, 8 DEG C, 9 DEG C, 10 DEG C and 4 DEG C of refrigerators and digest.Treat that tissue block becomes bulk, and occur batt layer on upper strata, terminate digestion, record the digestion time of each group.
Take out digestion bottle, remove pancreatin gently, and take off enzyme with the newborn Chinese liquid of about 500ml, discard supernatant, then add newborn Chinese liquid, after shaking up precipitation, upper strata cell dispersion is poured in receiving flask, repeat three times.Enchylema the most at last in receiving flask, after the centrifugal 10min of 1000rpm, abandons supernatant liquor.With containing 10% calf serum and 100 units/ml dual anti-nutrient solution re-suspended cell and count, check cell viability with Trypan Blue.
According to above grouping and digestion method, this test is repeated twice again, the test-results obtained is listed in the table below in 1.
Table 1 hamster kidney cell digestion method compares
Single performance in no matter testing for three times from table 1, or the performance of three test-results mean values, all be not difficult to find: after suslik nephridial tissue is digested at 8-10 DEG C, the survival rate of cell all exceedes the cell survival rate and total cellular score that to carry out digesting at 37 DEG C and 4 DEG C; The time used from trysinization is analyzed, digest under selecting 8-10 DEG C of condition, can cell survival rate be reached the highest in the digestion time of 10 hours, compare with the situation of carrying out digesting at 37 DEG C with 4 DEG C, obviously shorten trysinization time and virus production cycle.
Simultaneous test 2: liquid medium-selection
By the method for simultaneous test 1 10 DEG C of digestion suslik nephridial tissues, with three kinds of different nutrient solution cultivation hamster kidney cells, often kind of cell culture fluid cultivates two bottles of cells, and every bottle of cell dilution number is 2 × 10
8individual/L, the Rotary Machine being placed in 37 DEG C of greenhouses is cultivated.
According to above clustering method, this test is repeated twice again, record cell grows up to time of individual layer, and the cell growth state by being observed visually, and the test-results obtained is listed in the table below in 2.
The different nutrient solution composition of table 2-1 tri-kinds
Table 2-2 hamster kidney cell growing state in different nutrient solution
As can be seen from result shown in table 2, when cell count is identical with serum-concentration, with containing 10% calf serum volume ratio be 1:1 breast Chinese liquid and RPMI1640 as cell culture fluid time, only need within about 4 days, to make cell grow up to individual layer.Only compare as the situation of nutrient solution with 10% calf serum of RPMI1640 containing newborn Chinese liquid with other two groups, not only highly shortened the time that cell grows up to individual layer, and the cell count of cell surface apoptosis is few, cell refractivity might as well.
Simultaneous test 3: virus liquid number of freezing and thawing is tested
The encephalitis b virus liquid 201201001 batches of being bred by hamster kidney cell is after 0,1,2,3 freeze thawing, and carry out doubling dilution to virus, choosing extent of dilution is 10
-5, 10
-6, 10
-7with 10
-8virus liquid is inoculated into and covers with in 96 hole hamster kidney cell culture plates of individual layer, and each extent of dilution inoculates 6 porocytes, every hole inoculation 0.1ml virus liquid, then adds 0.1ml not containing the newborn Chinese liquid+RPMI1640 cell maintenance medium of serum, puts 37 DEG C of CO
27 days result of determination are cultivated in incubator.Virus titer is calculated according to Read-Munch method.
Detect tiring of encephalitis b virus liquid 201202002 batches and 201203002 batches according to above method, the test-results obtained is listed in the table below in 3.
The viral freeze thawing of table 3 different number of times bioactivity result
The encephalitis b virus liquid bioactivity result of three batches as can be seen from table 3: it tires the highest encephalitis b virus liquid before without freeze thawing, and along with number of freezing and thawing increases, it is tired and reduces gradually.Can reach a conclusion thus, encephalitis b virus aborning without tiring of freeze thawing be the highest.
Simultaneous test 4: before and after condition optimizing, encephalitis b virus potency ratio comparatively
This experiment is divided into two groups to carry out.
1st group is not carried out encephalitis b virus Optimization of proliferation conditions: when namely preparing hamster kidney cell under 37 DEG C of conditions with 0.25 quality % trysinization 25min, with 2 × 10
8the cell count of individual/L joins in rolling bottle cultivates, and nutrient solution is containing the dual anti-newborn Chinese liquid of 10% calf serum, 100 units/ml, is designated as nutrient solution 4.1;
2nd group is carried out encephalitis b virus Optimization of proliferation conditions: when namely preparing hamster kidney cell under 10 DEG C of conditions 0.25 quality % trysinization 10h, with 2 × 10
8the cell count of individual/L joins in rolling bottle cultivates, and nutrient solution is that both volume ratios are 1:1, are designated as nutrient solution 4.2 containing the dual anti-newborn Chinese liquid of 10% calf serum, 100 units/ml and RPMI1640 mixed solution.
After two groups of cells cover with individual layer, discard cell culture fluid, inoculating tiring of 0.5 volume % after adding cell maintenance medium is 10
8.25tCID
50the encephalitis b virus liquid of/ml, be placed in 37 DEG C of greenhouses, revolution be 7-8 turn/hour Rotary Machine on cultivate.Results virus liquid.
1st papova gathers in the crops virus liquid when cytopathy (CPE) reaches 95% after freeze thawing 1 time.2nd papova directly gathers in the crops virus liquid without freeze thawing when cytopathy (CPE) reaches 95%.By the method in simultaneous test 3, two papovas are tired into detection.
In triplicate, the test-results obtained is listed in the table below in 4.
Encephalitis b virus bioactivity result before and after table 4 condition optimizing
As can be seen from the above results, the result that the 2nd group of virus titer recorded for 3 times all records for 3 times far above the 1st group, and the virus titer mean value of the virus titer mean value of the 2nd group than the 1st group at least improves 11 times, after this illustrates and is optimized encephalitis b virus proliferation conditions by method of the present invention, virus titer obtains remarkable lifting.
Claims (5)
1. a method for encephalitis b virus propagation, after the method comprises culturing cell, to the cell inoculation encephalitis B kind poison covering with individual layer, it is characterized in that, after inoculation, directly gather in the crops virus liquid when cytopathy reaches 75-95% after, be applied directly to join in seedling without freeze thawing and go; Described cell is hamster kidney cell, and the suslik nephridial tissue that the method for described culturing cell comprises collecting carries out trysinization, and isolated cell carries out rotation and cultivates after moving into nutrient solution; After described trysinization comprises add pancreatin in the suslik nephridial tissue collected, 9-12 hour is digested at being placed in 6-12 DEG C, the volume ratio of the pancreatin added and suslik nephridial tissue is 2-3.5:1, described nutrient solution is the dual anti-newborn Chinese liquid containing 10% calf serum and 100 units/ml, described nutrient solution is also containing RPMI 1640, and the volume ratio of newborn Chinese liquid and RPMI 1640 is 1:0.8-1.2.
2. method according to claim 1, wherein, the encephalitis B kind poison inoculated is the 0.5-1 volume percent of cell maintenance medium amount.
3. method according to claim 1, wherein, the temperature of carrying out trysinization is 8-10 DEG C, and the time is 9.5-10.5 hour.
4. method according to claim 1, wherein, before carrying out trysinization, with 7-8 mass percent NaHCO
3the PH of pancreatin is adjusted to 7.2-7.4.
5. method according to claim 1, wherein, the temperature of carrying out cultivating is 35.5-38.5 DEG C, and carrying out cultured cells concentration is 1.8-2.5 × 10
8the cell quantity of individual/L.
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| CN1109782A (en) * | 1994-09-21 | 1995-10-11 | 卫生部长春生物制品研究所 | Production of inactivated vaccine for epidemic hemorrhagic fever using primary cells of kidney of golden yellow hamster |
| CN102038943A (en) * | 2010-09-15 | 2011-05-04 | 武汉中博生物股份有限公司 | Method for industrially producing porcine Japanese encephalitis (JE) vaccines by utilizing bioreactor |
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|---|---|---|---|---|
| CN1109782A (en) * | 1994-09-21 | 1995-10-11 | 卫生部长春生物制品研究所 | Production of inactivated vaccine for epidemic hemorrhagic fever using primary cells of kidney of golden yellow hamster |
| CN102038943A (en) * | 2010-09-15 | 2011-05-04 | 武汉中博生物股份有限公司 | Method for industrially producing porcine Japanese encephalitis (JE) vaccines by utilizing bioreactor |
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