CN1456355A - Forest cerebritis purifying vaccinum - Google Patents
Forest cerebritis purifying vaccinum Download PDFInfo
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Abstract
A purified vaccine for forest encephalitis contains deactivated forest encephalitic virus and vaccine adjuvant, and is prepared through inoculating the "Zhang" strain P9-3 virus to cells, purifying and deactivating.
Description
Technical field
The invention belongs to field of biological pharmacy, relate to deactivation forest encephalitis purified vaccine and preparation method thereof, particularly the method for preparation of industrialization deactivation forest encephalitis purified vaccine.The invention still further relates to a kind of fores encephalitis virus, gloomy " opening " strain P
9-3
Background technology
Forest encephalitis is caused by fores encephalitis virus, is the main source of infection with the Ticks class, and to invade central nervous system's the impatient sexually transmitted disease in natural epidemic disease source, case fatality rate is a kind of viral infectious of serious harm people ' s health at 20-30%.Forest encephalitis mainly is distributed in Changbai Mountain, the Xiaoxinanlin Mountains and the Daxinganling District in northeast in China, in Yunnan and Xinjiang also have the forest encephalitis natural epidemic foci to exist.In recent years along with the rise of forest zone expanding economy and tourist industry, the crowd who enters the forest zone increases year by year, therefore, to the epidemic-stricken area high-risk group with will enter epidemic-stricken area work or the people of tourism carry out the specific immunity inoculation, be prevention infection, reduce sickness rate, effectively control the fundamental way of forest encephalitis eruption and prevalence.
After the Beiye political affairs of nineteen forty-four Japan ground force time successfully were separated to a kind of fores encephalitis virus first, the scientific research personnel began to carry out the separation of fores encephalitis virus and the development work of vaccine both at home and abroad.The fifties, China began to develop polytype Teck-borne Encephalitis Vaccine, was used to prevent the vaccine of forest encephalitis.In early stage former generation,, vaccine was as the Mus brain Inactivated Tick-born Encephalitis Vaccine of nineteen fifty-two development, owing to contain and to cause allergic composition, the allergic encephalomyelitis incidence rate height that causes after the vaccination, the Embryo Gallus domesticus Inactivated Tick-born Encephalitis Vaccine of development in 1954, the inoculation side reaction is lower than Mus brain vaccine, but effect is not certainly.In order to reduce side reaction, using cellular matrix instead is the method for tissue culture virus of proliferation of chick-embryo cell, hamster kidney cell, prepares vaccine behind the results virus-culturing fluid.But the antigenic content of these vaccines is lower, and foreign protein is many, has sensitizer, and it is serious and immune effect is relatively poor to cause inoculating side reaction.
First external Teck-borne Encephalitis Vaccine is the 1939-1940 The former Russian scholar with the preparation of Mus brain, the Teck-borne Encephalitis Vaccine that adopted cell culture (chick embryo fibroblast) technological development in 1961, and be used for people's immunity.Developed a kind of partially purified chick-embryo cell inactivated vaccine (with the hydroxyapatite is adsorbent, chromatography) in 1973, but major defect be inoculate first that the back finds that headache is arranged, heating, side reaction such as uncomfortable, the child particularly more to be seen.
In sum, the Teck-borne Encephalitis Vaccine of development all needs further to improve and improve both at home and abroad at present, and, employed fores encephalitis virus in all unexposed both at home and abroad so far Teck-borne Encephalitis Vaccine.
Detailed Description Of The Invention
Main purpose of the present invention is:
1. provide a kind of be adapted to the white mice brain, gloomy " opening " strain that virulence is stable is a seed culture of viruses, but the method for preparation of industrialization forest encephalitis purified vaccine, especially with the cellular matrix of golden hamster kidney cells as infective virus, through continuous flow centrifugation, step such as ultrafiltration and concentration and column chromatography is carried out purification, prepares the method for deactivation forest encephalitis purified vaccine.
2. provide a kind of new selection-breeding isolating fores encephalitis virus strain.
3. provide a kind of antigenic content height, immunogenicity good, be suitable for that use in each epidemic-stricken area, the forest encephalitis of human deactivation safely and effectively purified vaccine.
Other purposes of the present invention are below to embodying in the specific descriptions of the present invention.
Description of drawings
Accompanying drawing 1 is the schematic flow sheet that the present invention preferably prepares deactivation forest encephalitis purified vaccine.
Accompanying drawing 2 is the Sepharose 4FF column chromatography collection of illustrative plates of forest encephalitis purified vaccine of the present invention.The I peak is the fores encephalitis virus peak, and the II peak is the foreign protein peak.
Gloomy " opening " strain is to separate the russian spring-summer encephalitis virus strain that obtains June nineteen fifty-two from certain patient's brain tissue, proves russian spring-summer encephalitis virus through the test of small white mouse cross-protection and discrimination test. In order to obtain the antigenicity wide spectrum, the russian spring-summer encephalitis virus vaccine strain that immunogenicity is good, the applicant has carried out for a long time and widely research. Go down to posterity in the mouse brain after 15 times with gloomy " opening " strain seed culture of viruses, the applicant has obtained a kind of russian spring-summer encephalitis virus strain, gloomy " opening " strain P9-3, belong to the Flavivirus Togaviridae, Flaviviridae-Togavivirus. This Strain has been deposited in Chinese Typical Representative culture collection center, and the address is the Wuhan University of Wuhan, China, and its preserving number is CCTCC V202004, and preservation date is on September 9th, 2002. The virus infections titre of this Strain reaches and is stabilized in 9.001gLD50More than/the ml, viral biology is activity stabilized, and antigenicity wide spectrum and immunogenicity are good, through comprehensively calibrating, proves no exogenous factor in the seed culture of viruses.
According to the present invention, a kind of deactivation russian spring-summer encephalitis virus purified vaccine comprises deactivation russian spring-summer encephalitis virus and the adjuvant of effective dose. This deactivation russian spring-summer encephalitis virus is from gloomy " opening " of the present invention strain P9-3, CCTCC V202004. This adjuvant is adjuvant, for example Al (OH) commonly used3 This purified vaccine also contains viral stabilizing agent. This virus stabilizing agent is viral stabilizing agent, for example human albumin commonly used.
The invention still further relates to a kind of preparation method of deactivation russian spring-summer encephalitis virus purified vaccine, particularly the method for suitability for industrialized production deactivation russian spring-summer encephalitis virus purified vaccine.
According to the present invention, a kind of preparation method of deactivation russian spring-summer encephalitis virus purified vaccine comprises by cell preparing virus liquid with russian spring-summer encephalitis virus, and with adjuvant virus liquid is mixed with purified vaccine. The preparation of this virus liquid comprises
(1) in virus-culturing fluid, uses russian spring-summer encephalitis virus, gloomy " opening " strain P9-3, CCTCC V202004, infection cell,
(2) virus liquid of propagation and/or propagative viruses suspensions in infection cell,
(3) the viral liquid that obtains in results (2) step,
(4) remove residue culture and/or foreign protein in this virus liquid.
Preparation method of the present invention also comprises the step of virus in the inactivation of viruses liquid.This inactivation step can be carried out before or after residue culture in removing this virus liquid and/or foreign protein step.
There are various kinds of cell and tissue to be applicable to infection step of the present invention, for example, hamster kidney cell, chick-embryo cell, murine brain, Embryo Gallus domesticus tissue, preferred golden hamster kidney cells.If desired, infecting the used cell of step can be obtained by the cell culture fluid cultivation, preferably cell culture is become cell monolayer.Used cell culture fluid can be the cell culture fluid of using always, for example, and the Earle ' s liquid of the calf serum lactalbumin hydrolysate of 4-8%, 199 liquid of suitable concn, MEM liquid or the like.
With gloomy " opening " strain P
9-3During infection cell, used infective dose is equivalent to infection multiplicity (M.O.I.) less than 0.5, and is preferred 0.02～0.5, obtains good productive rate for virus multiplication and breeding.The viral infection of cell is to carry out in the presence of virus-culturing fluid.Used virus-culturing fluid can be the Earle ' s liquid that contains 0.1～0.2% human albumin, 0.005～0.01% cysteine; 0.5～2.0% calf serum, 0.005～0.01% cysteine Earle ' s liquid; 199 liquid that contain 0.1～0.2% human albumin or 0.5～2.0% calf serum.
According to the present invention, the collection of viral liquid can repeatedly be carried out.After collecting viral liquid for the first time, by adding virus-culturing fluid, cell and virus are continuously bred, so that continue to collect viral liquid.By repeatedly adding virus-culturing fluid, can realize the repeatedly collection of viral liquid.Adopt Strain of the present invention and cultural method of the present invention, the collection of viral liquid can 8 times or more than.
Method of repeatedly collecting viral liquid of the present invention is particularly conducive to suitability for industrialized production deactivation fores encephalitis virus of the present invention purified vaccine.
According to the present invention, the inactivation step in the preparation method uses chemical reagent as inactivator, for example, adopts formaldehyde as inactivator.Therefore, when using the toxicity strain to be used for virus multiplication and/or breeding, still can produce inactivated vaccine.Adopt the inactivator of debita spissitudo, under suitable temperature and time, make virus lose appeal, but keep the antigenicity and the immunogenicity of virus.For example, during deactivation, the concentration of formaldehyde is 1/2000～1/6000; The deactivation condition is at 37 ℃, 2～3 days; 20～25 ℃, 7～14 days; 4～8 ℃, 14～21 days, but equal inactivation of viruses, the concentration of the preferred formaldehyde of the present invention is 1/4000, the deactivation temperature and time is 20～25 ℃, 16～24 hours; 4～8 ℃, 14 days.
According to the present invention, deactivation together after the viral liquid of repeatedly collecting can being merged.
Purified vaccine of the present invention also contains for example thimerosal of antiseptic commonly used.
Can adopt several different methods to remove residual culture and the foreign protein in the viral liquid after the deactivation, to obtain purified vaccine of the present invention.For example: high velocity band centrifugation method, the zinc acetate sedimentation method, column chromatography etc.Viral liquid before or after the deactivation can come purification through ultrafiltration and concentration and chromatography.Chromatography can be an ion-exchange chromatography, adsorption chromatography, gel filtration or molecular sieve, before ultrafiltration and concentration, viral liquid can be handled through centrifugal or clarification film earlier, with impurity such as place to go cell debris and albumen, 8000～12000 rev/mins of the conditions of the preferred continuous flow centrifugation of the present invention, 4～8 ℃; The ultrafilter membrane of the preferred 100Kd of ultrafiltration or clarify preferred 0.4um clarification film; The elution speed of gel filtration or molecular sieve is 10～200ml/ minute, and temperature is 4～25 ℃.
The preferred suitability for industrialized production deactivation of the present invention fores encephalitis virus purified vaccine is characterised in that, the viral liquid of deactivation is used the continuous flow centrifuge high speed centrifugation, through ultrafiltration and concentration, pass through Sepharose 4FF column chromatography again, the viral liquid that obtains is used for purified vaccine of the present invention.
Viral liquid behind deactivation and the purification adds suitable adjuvant and/or carrier, can be made into purified vaccine of the present invention.Commonly used adjuvant and/or carrier be, for example, and Al (OH)
3Can also add an amount of viral stabilizing agent, viral stabilizing agent commonly used is, for example, and human albumin.
Deactivation fores encephalitis virus purified vaccine of the present invention can be used for preventing the infection of fores encephalitis virus.Deactivation fores encephalitis virus purified vaccine of the present invention can adopt the mode administration of intramuscular injection, also can adopt other suitable manner administrations.
The invention still further relates to fores encephalitis virus of the present invention is used to prepare deactivation fores encephalitis virus purified vaccine of the present invention.
With reference to the accompanying drawings 1, a kind of preferred embodiment to suitability for industrialized production deactivation fores encephalitis virus purified vaccine of the present invention is further described.Related herein raw material and equipment if no special instructions, all are commercially available.
1. preparation hamster kidney cell
With two the week age health Golden Hamster, take out kidney digestion through the sterile working, shredding the back, to add pH7.6～8.0 concentration be 0.1～0.5% pancreatin, put 4-8 ℃ 18-20 hour or 33～37 ℃, after the digestion in 0.5～1.0 hour, discard trypsin solution, Earle ' s liquid with the calf serum that contains 6-8%, 0.1～0.2% lactalbumin hydrolysate is the cell growth medium cell dispersion, is prepared into cell suspension, is sub-packed in 10～15 liter Tissue Culture Flasks, the 1000ml-1500ml/ bottle, 15～25 pairs of kidney/bottles.Place 37 ℃ of thermostatic chamber internal rotation to cultivate 2-3 day Tissue Culture Flask, on the bottle wall of cell bottle, can form even, fine and close cell monolayer.
2. viral infection, viral liquid results, deactivation
Method one: select even, the fine and close cell monolayer bottle of cell growth, discard cell growth medium, with Earle ' s liquid or normal saline flushing 2-3 minute postoperative infection, the final concentration of the seed culture of viruses of inoculation is 10 of a seed culture of viruses
-4-10
-6Doubly, infection multiplicity is 0.02～0.5, the seed culture of viruses diluent is for containing 0.1～0.5% human albumin, 0.005 the Earle ' s liquid of～0.01% cysteine, cell bottle behind the infective virus is put 32-34 ℃ of thermostatic chamber internal rotation to be cultivated 16-20 hour, discard viral liquid, add and contain 0.1～0.2% human albumin, 0.005 the Earle ' s liquid of～0.01% cysteine, continue to put 32-34 ℃ of thermostatic chamber internal rotation and be cultured to 60-72 hour, visual cell pathological changes situation is gathered in the crops viral liquid, sterility test and titration of virus are made in sampling, the viral liquid of results is added 1/2000～1/6000 formalin solution (inactivator) and 1/20000 thimerosal, put 37 ℃, after 16～24 hours again in 4-8 ℃ of deactivation 14 days; Or in 4-8 ℃ of deactivation 21 days
Method two: select even, the fine and close cell monolayer bottle of cell growth to wash postoperative infection with Earle ' s liquid, the final concentration of the seed culture of viruses of inoculation is 10 of a seed culture of viruses
-4-10
-6Doubly, infection multiplicity is 0.02～0.5, the seed culture of viruses diluent is for containing 0.5～1.5% calf serum, 0.1 the Earle ' s liquid of～0.5% lactalbumin hydrolysate, the cell bottle of infective virus is put 32-34 ℃ of thermostatic chamber internal rotation to be cultivated 60-72 hour, visual cell pathological changes situation is gathered in the crops viral liquid, add again in the time of results and contain 0.5～1.5% calf serum, 0.005 the Earle ' s liquid of～0.01% cysteine, putting 32-34 ℃ of thermostatic chamber internal rotation cultivated 24-48 hour, look the pathological changes situation and gather in the crops viral liquid once more, gather in the crops virus-culturing fluid so continuously, till cell detachment, each time results liquid is all sampled and is made sterility test and titration of virus, and presses the method deactivation of method one.The viral liquid that obtains after the deactivation is the semi-finished product (vaccinogen liquid) of vaccine.
3. the centrifugal merging of vaccinogen liquid
With the semi-finished product of vaccine behind sterility test, inactivation test assay approval, remove through continuous flow centrifugation or clarification film be incorporated in the airtight bucket of rustless steel behind the impurity such as cell debris to be purified.
4. the purification of vaccine
The method is suitable for the purification of forest encephalitis purified vaccine.The vaccine semi-finished product of impurity such as cell debris will have been removed through continuous flow centrifugation.With the ultrafilter membrane ultrafiltration and concentration of molecular cut off 100kd, and, concentrate 50-100 doubly, collect concentrated solution with pH7.4～7.7,0.01MPBS buffer balance.Select Sepharose 4FF medium then, after pH7.4～7.7,0.01MPBS buffer balance, carry out column chromatography, with ultraviolet monitoring instrument (A
280Nm) detect eluent, the first peak (I) of collecting eluting is fores encephalitis virus antigen (referring to an accompanying drawing 2), and second peak (II) is the foreign protein peak.Sampling Detection albumen or antigenic content carry out aseptic filtration after being diluted to finite concentration according to albumen or antigenic content, and to add final concentration be the AL (OH) of 0.1-0.2% human albumin and 0.45-0.55mg/ml
3Adjuvant.Make sterility test, pyrogen testing then, carry out packing behind the assay approval, be finished product.The finished product calibrating comprises: visual examination, sterility test, potency test, toxicity test, content of formaldehyde mensuration, thimerosal assay, AL (OH)
3Assay, pH value mensuration etc.
1. forest encephalitis purified vaccine sensitization test of the present invention
Three batches of purified vaccine sensitization tests are all qualified, and the positive and negative contrast is all set up.Negative control group: 6 Cavia porcelluss are attacked the no any symptom in back; 6 Cavia porcelluss of positive controls allergic symptom all occurs after attacking, and agitation is arranged, cough, grab nose, occur extremity then and feel like jelly, lie on one's side, are slow in action, attack back 30 minutes after, dead one, 5 sxs in addition; Three batches of purified vaccines all do not have any symptom after 6 Cavia porcelluss of each batch are attacked, all strong depositing in 3 days.The results are shown in Table 1.
Table 1 forest encephalitis purified vaccine of the present invention sensitivity test result
Cavy number of elements vaccine lot number sum 0-I level II level III level IV level was good for and is deposited negative control 6 6---6 positive control 6-1 32 520,010,701 6 6---620,010,702 6 6---620,010,703 6 6---6 in 3 days afterwards
2. forest encephalitis purified vaccine of the present invention is renderd a service and heat stability
Every batch of vaccine, by 0,7 day program, in peritoneal immunity 2 times, immunizing dose was every each 0.3ml, to 14 days with gloomy " opening " strain P
9-30.3ml/ is only attacked in the viral suspension abdominal cavity, 21 days result of determination, and vaccine immunity protection index is all greater than 1.0 * 10
5Standard, and exceed more than 10 times than working standard.Vaccine is in 37 ℃ of 1 weeks of placement, 2 weeks, and the potency test testing result is fine, and the protection index is still greater than 1.0 * 10
5Standard, show that vaccine stability is good, the results are shown in Table 2.(attack poison is gloomy " opening " strain P to table 2 forest encephalitis purified vaccine of the present invention stability test result
9-3) vaccine placement condition matched group (LD
50/ ml) test group (LD
50/ ml) 20,010,701 4 ℃ of former surveys 10 of protection index
-8.4010
-2.002.51 * 10
6
37 ℃ of 1 week 10
-8.4010
-2.468.71 * 10
5
37 ℃ of 2 week 10
-8.4010
-3.002.51 * 10
520,010,702 4 ℃ of former surveys 10
-8.4010
-1.883.31 * 10
6
37 ℃ of 1 week 10
-8.4010
-2.311.23 * 10
6
37 ℃ of 2 week 10
-8.4010
-2.803.98 * 10
520,010,703 4 ℃ of former surveys 10
-8.4010
-2.002.51 * 10
6
37 ℃ of 1 week 10
-8.4010
-2.371.07 * 10
6
37 ℃ of 2 week 10
-8.4010
-2.655.62 * 10
5
3. the comprehensive verification result of forest encephalitis purified vaccine of the present invention
Table 3 forest encephalitis purified vaccine of the present invention verification result
| Project/lot number | Quality standard | ????20010701????20010702????20010703 |
| Inactivation of virus test protein content (Lowry ' s) pyrogen test sensitization test cow's serum residual quantity abnormal toxicity test potency test heat stabilization test PE chemical assay aluminium hydroxide (mg/ml) formaldehyde % (g/ml) thimerosal % (g/ml) acid-base value (pH value) | Small white mouse should be good for and deposit≤3 rabbit of 120 (ug/ml) heat up be lower than all that 0.60 ℃ of summation cavy should not have allergic symptom and in 3 days strong depositing≤50 (ng/ml) small white mouse, cavy should be good for and deposit and body weight increases by 5.0 * 1055.0×10 5Milky suspendible liquid is as good as thing≤0.70≤0.01≤0.01 7.2-8.0 | Qualified qualified 52 50 54 qualified qualified＜10＜10＜10 qualified qualified 2.51 * 106???3.31×10 6??2.51×10 6??8.71×10 5???1.23×10 6??1.07×10 6Qualified qualified 0.65 0.60 0.60＜0.01＜0.01＜0.01＜0.01＜0.01＜0.01 7.48 7.49 7.45 |
The deactivation forest encephalitis purified vaccine of the present invention's preparation, shortcoming such as can solve that existing vaccine antigen content is low, foreign protein is many, injected dose is big, inoculation back side reaction weighs and human immunity weak effect, serological conversion rate be low, this vaccine is suitable for each forest encephalitis epidemic-stricken area use.
Below by embodiment, content of the present invention is done further to describe.
Embodiment 1
Get two the week age health Golden Hamster, water is put to death the back and is sterilized 5-6 time with 0.3～0.5% bromo geramine, kidney is got by the sterile working, shred, clean 0.1～0.5% the pancreatin that adds pH7.6～8.0 behind the blood, put 4-8 ℃ of cold digestion 18-20 hour, discard trypsin solution, wash with Earle ' s liquid, jog cell dispersion 5-6 time is collected each time cell suspension, through two-layer filtered through gauze, make it to be suspended from the calf serum that contains 6-8%, in the cell growth medium of the Earle ' s liquid of 0.2% lactalbumin hydrolysate, be prepared into cell suspension, be sub-packed in 10 liter culture bottles, the 1000ml-1500ml/ bottle after shaking all.Tissue Culture Flask was placed 37 ℃ of thermostatic chamber rotating and culturing 3, and cell grows up to even, fine and close monolayer.
Select even, the fine and close cell monolayer bottle of cell growth, discard cell growth medium, with 2-3 minute postoperative infection of Earle ' s liquid flushing, with seed culture of viruses with contain 0.1～0.2% human albumin, 0.005～0.01% cysteine Earle ' s liquid dilutes 10
5Doubly, add in the cell bottle to be infected, the 2000-2500ml/ bottle, putting 32-34 ℃ of thermostatic chamber internal rotation cultivated 18 hours, discard viral liquid, add the Earle ' s liquid that contains 0.1～0.2% human albumin, 0.005～0.01% cysteine, continue to put 32-34 ℃ of thermostatic chamber internal rotation and be cultured to 68 hours, gather in the crops viral liquid, sterility test and titration of virus are made in sampling, with results viral liquid add 1/4000 formalin solution (inactivator) and 1/20000 thimerosal, put 37 ℃, after 16～24 hours again in 4-8 ℃ of deactivation 14 days; Or in 4-8 ℃ of deactivation 21 days, the viral liquid that obtains after the deactivation was the semi-finished product of vaccine.The vaccine semi-finished product are behind sterility test, inactivation test assay approval, after handling impurity such as removing cell debris, continuous flow centrifugation or clarification film be incorporated in the airtight bucket of 100 liter rustless steels, use the ultrafilter membrane ultrafiltration and concentration of molecular cut off 100kd then, and with pH7.4～7.7,0.01MPBS buffer balance, concentrate 100 times, collect concentrated solution.Use Sepharose 4FF medium then, after pH7.4～7.7,0.01MPBS buffer balance, carry out column chromatography, with ultraviolet monitoring instrument (A
280Nm) detect eluent, the first peak of collecting eluting is a fores encephalitis virus antigen, and the sampling Detection antigenic content carries out aseptic filtration after the dilution, and the adding final concentration is 0.1～0.2% human albumin and 0.45～0.55mg/ml AL (OH)
3Adjuvant.Make sterility test, pyrogen testing then, packing behind the assay approval is purified vaccine finished product of the present invention.The finished product calibrating comprises: visual examination, sterility test, potency test, toxicity test, content of formaldehyde mensuration, thimerosal assay, AL (OH)
3Assay, pH value mensuration etc.
Embodiment 2
Get two the week age health Golden Hamster, water is put to death the back and is sterilized 5-6 time with 0.3～0.5% bromo geramine, kidney is got by the sterile working, shred, 0.1～0.5% of adding pH 7.6～8.0 pancreatin behind the clean blood, put 4-8 ℃ of cold digestion 18-20 hour, discard trypsin solution, wash with Earle ' s liquid, jog cell dispersion 5-6 time is collected time cell suspension fully, through two-layer filtered through gauze, make it to be suspended from the calf serum that contains 6-8%, in the cell growth medium of the Earle ' s liquid of 0.2% lactalbumin hydrolysate, be prepared into cell suspension, be sub-packed in 10 liter Tissue Culture Flasks, the 1000ml-1500ml/ bottle after shaking all.18 pairs of kidney/bottles placed 37 ℃ of thermostatic chamber rotating and culturing 3 with Tissue Culture Flask, and cell grows up to even, fine and close monolayer.
Select even, the fine and close cell monolayer bottle of cell growth, with Earle ' s liquid flushing postoperative infection, the seed culture of viruses Earle ' s liquid dilution 10 that contains 0.5～2.0% calf serum, 0.005～0.01% cysteine
5Doubly, add in the cell bottle to be infected, the 2000-3000ml/ bottle, cell bottle behind the infective virus is put 32-34 ℃ of thermostatic chamber internal rotation to be cultivated 68～72 hours, gather in the crops viral liquid, add the Earle ' s liquid that contains 0.5～2.0% calf serum, 0.005～0.01% cysteine in the time of results again, 2000～3000ml/ bottle, putting 32-34 ℃ of thermostatic chamber internal rotation cultivated 24-48 hour, gather in the crops viral liquid once more, gather in the crops virus-culturing fluid so continuously, the viral liquid of each time results is all sampled and is made sterility test and titration of virus, and presses the method deactivation of method one.Viral liquid after the deactivation is the vaccine semi-finished product.
The vaccine semi-finished product are behind sterility test, inactivation test assay approval, be incorporated in the airtight bucket of 100 liter rustless steels remove impurity such as cell debris through continuous flow centrifugation after, use the ultrafilter membrane ultrafiltration and concentration of molecular cut off 100kd then, and with pH7.4～7.7,0.01MPBS balance concentrated solution, concentrate 40～100 times, collect concentrated solution.Use Sepharose 4FF medium then, after pH7.4～7.7,0.01MPBS balance, carry out column chromatography, with ultraviolet monitoring instrument (A
280Nm) detect eluent, the first peak of collecting eluting is a fores encephalitis virus antigen, and sampling Detection albumen or antigenic content carry out aseptic filtration after the dilution, and to add final concentration be 0.1～0.2% human albumin and the AL (OH) of 0.45～0.55mg/ml
3Adjuvant.Make sterility test, pyrogen testing then, carry out packing behind the assay approval, be purified vaccine finished product of the present invention.The finished product calibrating comprises: visual examination, sterility test, potency test, toxicity test, content of formaldehyde mensuration, thimerosal assay, AL (OH)
3Assay, pH value mensuration etc.
Claims (12)
1. deactivation forest encephalitis purified vaccine comprises the deactivation fores encephalitis virus and the vaccine adjuvant of effective dose, and wherein this deactivation fores encephalitis virus is from gloomy " opening " strain P
9-3, CCTCC V202004.
2. the deactivation fores encephalitis virus purified vaccine of claim 1, wherein this adjuvant is Al (OH)
3
3. the deactivation forest encephalitis purified vaccine of claim 1 wherein also contains viral stabilizing agent, is preferably the human serum albumin.
4. isolating fores encephalitis virus strain, gloomy " opening " strain P
9-3, CCTCC V202004.
5. the preparation method of a deactivation forest encephalitis purified vaccine comprises the following step:
(1) in virus-culturing fluid, uses fores encephalitis virus, gloomy " opening " strain P
9-3, CCTCC V202004, infection cell,
(2) in infection cell propagation and/or breeding with the viral liquid of viral suspension form,
(3) the viral liquid that obtains in results (2) step,
(4) remove residue culture and/or foreign protein in this virus liquid,
(5) the viral liquid that deactivation obtains before or after (4) step,
(6) the viral liquid that obtains in (5) step is mixed with purified vaccine with adjuvant.
6. the method for claim 5, wherein this cell is a Golden Hamster kidney cell monolayer.
7. the method for claim 5, wherein the virus inoculation dosage during infection cell is equivalent to infection multiplicity (M.O.I.) less than 0.5.
8. the method for claim 5 after wherein this (3) step is gathered in the crops viral liquid, is added culture fluid and is repeated (2) and (3) step, can repeatedly gather in the crops viral liquid.
9. the method for claim 8, wherein the viral liquid of each time that will collect merges back reuse chemical reagent and carries out deactivation.
10. the method for claim 5 should (4) step be to be undertaken by ultrafiltration and concentration and/or column chromatography wherein.
11. the method for claim 10, wherein this column chromatography is a Sepharose 4FF column chromatography.
12. the method for claim 10, wherein carry out this ultrafiltration and concentration before, the viral liquid of these results carried out continuous flow centrifugation separates and/or the clarification membrane filtration is collected viral liquid, to remove cell debris and foreign protein.
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| CN 02159942 CN1228085C (en) | 2002-12-30 | 2002-12-30 | Forest cerebritis purifying vaccinum |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102335198A (en) * | 2011-10-13 | 2012-02-01 | 长春生物制品研究所有限责任公司 | Production method for tick-borne encephalitis virus horse antiserum |
| CN109432413A (en) * | 2018-12-29 | 2019-03-08 | 长春生物制品研究所有限责任公司 | A kind of russian spring-summer encephalitis virus inactivated vaccine and preparation method thereof |
| CN110669738A (en) * | 2019-09-27 | 2020-01-10 | 长春生物制品研究所有限责任公司 | Preparation method of forest encephalitis virus based on cell factory |
-
2002
- 2002-12-30 CN CN 02159942 patent/CN1228085C/en not_active Expired - Lifetime
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102335198A (en) * | 2011-10-13 | 2012-02-01 | 长春生物制品研究所有限责任公司 | Production method for tick-borne encephalitis virus horse antiserum |
| CN109432413A (en) * | 2018-12-29 | 2019-03-08 | 长春生物制品研究所有限责任公司 | A kind of russian spring-summer encephalitis virus inactivated vaccine and preparation method thereof |
| CN110669738A (en) * | 2019-09-27 | 2020-01-10 | 长春生物制品研究所有限责任公司 | Preparation method of forest encephalitis virus based on cell factory |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1228085C (en) | 2005-11-23 |
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