CN104357406A - Rabies virus SNK-CTN strain and application thereof - Google Patents
Rabies virus SNK-CTN strain and application thereof Download PDFInfo
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Abstract
The invention relates to the field of microbes and biological pharmacy, and particularly relates to an adapted strain of a rabies virus strain (a CTN-1V strain) in a diploid cell MRC-5 strain. The preparation method of the rabies virus strain comprises the following steps: 1) taking MRC-5 cells in a cell bank for resuscitation, cultivating for 3 days for forming single layer cells, pouring away a cell culture fluid in an MRC-5 cell culture flask, adopting a 0.25% of trypsin digestive juice for digestion, dispersing into uniform cells, inoculating a rabies virus CTN-1V5 strain suspension according to the dosage of 0.01-1.0 MOI, cultivating at 37 DEG C for 2-3 days, replacing the suspension with a maintenance medium containing 2-4% of bovine serum, cultivating at 33-35 DEG C for 3-5 days to obtain a virus liquid, and cryopreserving at -70 DEG C to prepare a virus suspension; 2) adopting the method, and continuously subculturing for 30-32 generations on MRC-5 cells. The preservation code of the rabies vaccine virus strain (an SNK-CTN strain) is CGCC No8887.
Description
Technical field
The present invention relates to microorganism and field of biological pharmacy, specifically, relate to the adapted strain of a kind of rabies virus strain CTN-1V strain in diploid cell MRC-5 strain.
Background technology
Rabies are a kind of diseases caused by rabies virus, once morbidity, 100% is dead.China in recent years Rabies continues to rise, and sickness rate now occupies second place of the world, is only second to India, and the rabies number of dying of illness occupies first of 37 kinds of notifiable infectious diseases.Rabies cannot be cured so far but can effectively be prevented, and therefore the rabic prevention work of China just seems particularly important.
Mainly contain primary hamster kidney cell, primary chick embryo cell and African green monkey kidney passage cell (Vero) cell for the production of the culture medium cell of Rabies Vaccine in the world at present) etc. animal derived cell.Animal derived primary cell can not get rid of the pollution of the external source virulence factors such as bacterium, virus, parasite, and have industrial scale not easily to expand, environmental protection is difficult to resolve the shortcoming such as certainly; And Vero cell is owing to being continuous cell line, have certain tumorigenicity, in vaccine, cell rests DNA must reach standard.Human diploid cell is humanized's normal karyotype cell, non-carcinogenesis, and without different DNA, its security is subject to consistent accreditation.Though the Rabies Vaccine kind that current China uses is many, but quality is very different, so far there is no and cultivated vaccine by the human diploid cell of the tissue affirmatives such as WHO, FDA and recommendation, and be substantially the vaccine by animal derived cell cultures such as hamster kidney cell, Vero cell and chick-embryo cells.
The human diploid cell rabies vaccine of external production, as the diploid vaccine that Wyeth factory of the U.S., French Merieux institute and German Behring factory produce, has confirmed that it has good immunogenicity and security.After vaccine confirms inoculation after not commensurability's anthroposcopy, side reaction is very light, good immune effect, and can subtract pin injection for exposing rear treatment, generally acknowledging at present can as a kind of standard control vaccine of new development vaccine.
About the report of diploid cell Rabies Vaccine comprises in prior art:
The present inventor finds through research, human diploid cell rabies virus strain in prior art, when preparing vaccine, problem is many, as: titre is low, and output is unstable, vaccine extration difficulty, impurity is many, and the present invention screens diploid cell rabies virus strain for this reason, obtains a kind of diploid cell rabies virus strain of excellent property, and for the production of, achieve good technique effect.
Summary of the invention
The object of this invention is to provide a kind of new rabies virus strain.By the rabies virus strain obtained after the upper adaptation of human diploid cell (MRC-5 cell), this cell strain has been deposited in China General Microbiological culture presevation administrative center, be numbered CGMCC8887, preservation date on 06 16th, 2014, Classification And Nomenclature: rabies virus (CTN) human diploid cell (MRC-5) adapted strain, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, address).SNK-CTN strain is called in this patent.
Rabies virus strain of the present invention, is prepared by following methods:
1) the MRC-5 cell got in cell bank is recovered, cultivate after within 3 days, becoming monolayer cell, cell culture fluid in MRC-5 Tissue Culture Flask is outwelled, digests with 0.25% tryptic digestive juice, be dispersed into uniform cell, inoculation rabies viruses CTN-1V5 strain suspension is carried out according to 0.01 ~ 1.0MOI dosage, cultivate 2 ~ 3 days, change the maintenance medium containing 2 ~ 4% bovine serums into for 37 DEG C, cultivate after 3 ~ 5 days for 33-35 DEG C and gather in the crops virus liquid, frozen in-70 DEG C, be prepared into viral suspension;
2) with above-mentioned method, uploaded for 30 ~ 32 generations continuously at MRC-5 cell, gained cell strain is called SNK-CTN strain, and this cell strain, can as the production strain preparing vaccine on MRC-5 cell through detecting.
Wherein, MRC-5 cell derived is in ATCC.
Wherein, rabies virus strain CTN-1V5 strain obtains in National Institute for Food and Drugs Control.
Wherein, the preparation of described 0.25% tryptic digestive juice: measure Earle ' s liquid 8000ml with graduated cylinder and join in bottle, taking 25g trypsinase with electronic balance again joins in same bottle, outstanding shaking makes it dissolve, add Earle ' s liquid again to mix to 10000ml, send in 2 ~ 8 DEG C of freezers carry out cold cut (cold cut time can not more than 20 hours) or at room temperature by stirring, outstanding shake make it dissolve, clarify, transparent (water temperature can not more than 40 DEG C, and the room-temperature dissolution time is no more than 4 hours).
Wherein, described maintenance medium is that MEM series or 199 serial culture bases add 2 ~ 4% bovine serums.
The present invention also provides the application of SNK-CTN strain in preparation Rabies Vaccine.
The present invention also provides the application of SNK-CTN strain in preparation rabies diagnostic reagent.
The screening process that SNK-CTN strain of the present invention is through screening acquisition is as follows:
(1) CTN-1V5 strain is in the experiment of MRC-5 cell different generation gained virus strain virus infection titer.
Rabies viruses CTN-1V strain adapts to go down to posterity at human diploid cell
(2) morphological observation
Electricity Microscopic observation SNK-CTN strain is the same with CTN-1V5 strain form, be bullet shape, be about 180nm, diameter 75nm, virion is made up of cyst membrane and nucleocapsid, cyst membrane surface is thick with glycoprotein projection (glycoprotein antigen), and internal layer is the lipoprotein antigen of lipoprotein composition, and virus central authorities are nucleocapsid (nuclear antigen).(electron microscopic examination with China Sickness Prevention Control Center Virus Disease Prevention Control Institute Electron Microscopy Room cooperated).Have no morphology of virus to change.
(3) telling test
SNK-CTN strain through in rabies specific antisera and after carry out telling test, neutralization index is 4000, higher than regulation 500 units more than, there is identical antigenicity, meet the requirement of Chinese Pharmacopoeia version production of vaccine strain in 2010.
Rabies virus SNK-CTN strain has with the characteristic of original seed culture of viruses fixed virus pathogenic more weak to mouse, rabbit, and has good immunogenicity, therefore can be used as the vaccine strain of Rabies Vaccine production.
(4) neutralization index measures
Sample abdominal injection body weight is 12 ~ 14g mouse, every 0.5ml, and immunity 2 times, 7 days, interval is test group.The same batch of mouse without immunity is control group.The 14th day after just exempting from, test group and control group are attacked with the CVS virus encephalocoele of 10 times of serial dilutions respectively, often only inject 0.03ml, each extent of dilution injects 10 mouse, day by day observe, in 3 days, died is disregarded (animal dead quantity should be no more than 20% of experimental animal sum), observes 14 days.
CTN-1V-M strain virus is to the immunogenicity of small white mouse
The present invention also provides the method preparing Rabies Vaccine with SNK-CTN strain of the present invention, and described method steps is as follows:
Step 1, diploid cell loads SNK-CTN virus
Get the human embryonic lung diploid fibroblast (MRC-5) of the cell bottle cultivation of covering with individual layer, through 0.25% trypsin digestion and cell, amplifying cells, on cell factory or bio-reactor, adds MEM cell culture fluid, and enchylema is pressed 0.05-0.IMOI and inoculated SNK-CTN rabies virus;
Step 2, cultivates and results
Put 37 DEG C to cultivate after 3 days, discard nutrient solution and change to maintenance medium.Put 35 DEG C of quiescent culture 3 ~ 15 days; Results virus liquid, harvesting method is: cell factory results are gathered in the crops 2 ~ 3 times according to the pathology of cell; Bio-reactor results carry out Multiple harvests virus for virus harvest liquid according to the index of reactor monitoring and the pathology of cell; Harvest liquid aseptically adds the beta-propiolactone deactivation 24 hours of 1:4000;
Step 3, Isolation and purification method is as follows:
After molecular weight cut-off is ultra-filtration membrane ultrafiltration and concentration 40-80 times of 300KD, carries out purifying with ultracentrifuge, obtain the rabies virus stoste of purifying.
Wherein carry out purifying with ultracentrifuge, method is as follows:
Sucrose density gradient centrifugation 10%, 20%, 30%, 40%, 50% (W/V) sucrose solution makes density gradient, 34000rpm ultracentrifugation is after 4 hours, through the antigenic content to ultraviolet absorption peak, the analysis of protein concentration, determines virus antigen position.Method purification process of the present invention is simple above, only uses ultracentrifuge, once centrifugal, and equipment is simple, and fast, cost is low, and obtained vaccine product is stablized, and impurity is few, and content is high.
Vaccine product of the present invention, high through detecting titre, stable yield, vaccine extration is simple, and impurity is few.
Relevant contrast experiment is as follows:
It is as follows that Impurity removal of the present invention and antigen reclaim detected result:
Protovirus CTN-1V strain reaches on human diploid cell MRC-5 cell and adapts to cultivate by the present invention, obtains SNK-CTN strain.This SNK-CTN strain proves rabies virus through the method such as gene sequencing, neutralization test.In adaptive process, early generation culture cycle is 7-10 days, and after reaching for 8 generations, culture cycle foreshortens to 5-8 days.Continue cultivation 30 generation, virus infection titer is stabilized in after the 6th generation can reach 6.5-7.5lgL D
50/ ml.Show through immunogenicity and cross-protection test; with rabies inactivated vaccine prepared by this strain, there is good immunogenic protected effect; tire and reach 2.5IU/ agent; being suitable for the rabies inactivated vaccine of large-scale industrial production, is the desirable strain producing diploid cell rabies inactivated vaccine.And this strain has the advantages that proliferating cycle is short, reproductive efficiency is high on MRC-5 cell, be suitable as the rabies inactivated vaccine seed culture of viruses of large Regulations mould suitability for industrialized production.
The present invention finds to use virus strain of the present invention unexpectedly in addition, can greatly reduce the step of Isolation and purification, reduces production cost, improves output, also reduces the content of impurity simultaneously.
Accompanying drawing explanation
Fig. 1 is that SNK-CTN strain and CTN-1V5 strain electronic image compare
Embodiment
By following specific embodiment, the present invention is further illustrated, but not as restriction.
The adaptation of embodiment 1 rabies viruses CTN strain on MRC-5 cell
Get the cryopreservation tube containing MRC-5 cell, put into rapidly 40 DEG C of water-baths to melt, then pre-temperature is joined in the MEM cell culture fluid of 37 DEG C, be placed in 37 DEG C of cultivations, after within 3-4 days, growing up to individual layer after 0.25 tryptic digestion according to 0.01 ~ 1.0MOI dose inoculation CTN-1V5 strain suspension, cultivate 2 ~ 3 days for 37 DEG C, change the maintenance medium containing 2 ~ 4% bovine serums into, cultivate after 3 ~ 5 days for 33-35 DEG C and gather in the crops virus liquid, frozen in-70 DEG C, be prepared into viral suspension.
By above method, the viral suspension of results is uploaded 30 generations at MRC-5 cell continuously, the rabies virus SNK-CTN strain that final acquisition one strain MRC-5 cell adaptation is good.
Following observation and test are carried out in the rabies virus SNK-CTN strain that the present invention obtains:
(1) SNK-CTN strain is well-adjusted on MRC-5 cell, virus titer can reach 7.0 ~ 7.5LogLD50/ml, virus multiplication peak was at 5 ~ 7 days, the most applicable propagation temperature 33-35 DEG C, 0.1 ~ 0.01MOI measured by best poison of planting, and CTN-1V-M strain is bred to hold time and is reached 29 days most on MRC-5 cell, 5 ~ 11 days inner virus harvest liquid titres all at more than 6.0LogLD50/ml, therefore can realize Multiple harvests virus liquid.
Compared with the cell adapted situation of Vero, each corresponding generation virus titer is lower slightly, and virus titer peak and antigen peak occur for slightly slow 1-2 days.This situation illustrates that CTN strain virus there are differences different cell adaptation.
The application of diploid cell Rabies Vaccine is prepared in embodiment 2 SNK-CTN strain
Get the human embryonic lung diploid fibroblast (MRC-5) of the cell bottle cultivation of covering with individual layer, through 0.25% trypsin digestion and cell, amplifying cells is on cell factory or bio-reactor, add MEM cell culture fluid, enchylema is pressed 0.05-0.IMOI and is inoculated SNK-CTN rabies virus, put 37 DEG C to cultivate after 3 days, discard nutrient solution and change to maintenance medium.Put 35 DEG C of quiescent culture 3 ~ 15 days, cell factory results virus liquid is gathered in the crops 2 ~ 3 times according to the pathology of cell; Bio-reactor results carry out Multiple harvests virus for virus harvest liquid according to the index of reactor monitoring and the pathology of cell.
Harvest liquid aseptically adds the beta-propiolactone deactivation 24 hours of 1:4000.The rabies venom of deactivation through deactivation after the assay was approved, after molecular weight cut-off is ultra-filtration membrane ultrafiltration and concentration 40-80 times of 300KD, purifying is carried out with ultracentrifuge, obtain the rabies virus stoste of purifying, 1.0IU/ml is diluted to according to virus antigen contents level, be mixed with work in-process, work in-process be sub-packed in aseptic cillin bottle, make finished product vaccine through freeze-drying.
The quality test of diploid cell Rabies Vaccine is prepared in example 3SNK-CTN strain
1) virus titer measures
Sample 10 times of serial dilutions, attack mouse in brain, every 0.03ml each extent of dilution intracranial inoculation body weight is 11 ~ 13g mouse at least 6, and observe 14 days, virus titer should be not less than 7.0lg LD
50/ ml.
2) antigenic content measures
Adopt purified anti-rabies virus polyclonal antibody bag by enzyme-linked reaction plate and mark horseradish peroxidase (HRP), application double-antibody sandwich principle, adopt the reaction pattern of two-step approach, the antigenic content in detection by quantitative Rabies Vaccine, should be not less than 0.4IU/ agent.
Antirabic Vaccine's antigenic content and corresponding virus harvest liquid virus titer result
| Interventions Requested | SNK-CTN harvest liquid 1 | SNK-CTN harvest liquid 2 | SNK-CTN harvest liquid 3 |
| Virus titer (logLD50/ml) | 7.3 | 7.5 | 7.05 |
| Antigenic content (IU/ml) | 2.0 | 2.0 | 1.7 |
3) titration
Sample does 5 times of serial dilutions, get different extent of dilution sample and reference, vaccine is immune 12-14g mouse 16 respectively, every peritoneal immunity 0.5ml, interval second time immunity in a week, CVS brain inside fire attack poison is used in first time immunity after 14 days, every 0.03ml, observe 14 days, add up dead quantity, calculate relative effectivenes.
| Calibrating project | Norm standard | SNK-CTN-sample 1 | SNK-CTN-sample 2 | SNK-CTN-sample 3 |
| Efficacy determinations | >=3.5IU/ agent | 5.3. | 5.6 | 5.0 |
The application of example 3 SNK-CTN strain in related immunological field
With SNK-CTN strain immune mouse (rabbit), can adjuvant be added with SNK-CTN strain virus suspension or not add adjuvant, respectively at the 0th, 7,14,21 day mouse peritoneal immunity 0.5ml, in blood sampling in 28-42 days, in 2nd ~ 4 immunity blood sampling in latter 7 days, separation of serum, namely obtains rabies virus-immune serum.Adopt marked by fluorescein isothiocyanate serum antibody, can be used for preparing immunofluorescence detection agent.
The application of example 4 SNK-CTN strain in related immunological field
With SNK-CTN strain immune mouse (rabbit), can adjuvant be added with SNK-CTN strain virus suspension or not add adjuvant, respectively at the 0th, 7,14,21 day mouse peritoneal immunity 0.5ml, strengthen every other week once, each 0.5ml/ only, in 2nd ~ 4 immunity blood sampling in latter 7 days, separation of serum, namely obtains rabies virus-immune serum.Adopt Over-voltage protection horseradish peroxidase (HRP) to mark serum antibody, can be used for preparation ELISA reagent.ELISA kit can be prepared by coated elisa plate with rabies virus-immune serum.
The application of example 5 SNK-CTN strain in related immunological field
With SNK-CTN strain immune mouse, can adjuvant be added with SNK-CTN strain virus suspension or not add adjuvant, respectively at the 0th, 7,14,21 day mouse peritoneal immunity 0.5ml, in blood sampling in 28-42 days, measure serum titer, extracting spleen cell, with SP/20 myeloma cell fusion, through cloning selection can stably excreting to the hybridoma cell strain of Rables virus glycoprotein gene high-titer mono-clonal neutralizing antibody.
With SNK-CTN virus antigen abdominal injection immunization. Female BABL/c mouse, only, immune programme for children is 1,14,28,35,37 day to 0.5ml/, and each 0.5ml/ only, got spleen in the 39th day.Before each booster immunization, afterbody gets hematometry immunizing potency.Extracting spleen cell, by splenocyte: SP2/0 cell=1:5 ~ 10, merges at 50%PAGE, selects to cultivate with HAT, and the 48 orifice plate clones cultivated there being feeder layer cells cultivate, and 37 DEG C of 5%CO2 cultivate.Merge the 7th day, measure antibody titer by ELISA method.Merge latter 9th day, select the high hole of positive antibody to carry out continuous three time clonings, screen.First time cloning 50% is positive, and second time cloning 80% is positive, and third time cloning 100% is positive.Carry out the corresponding antigen immunogenicity qualification of viral protein of clone strain, antibody typing qualification, antibody purification and conservation.
The application of example 6 SNK-CTN strain in related immunological field
With SNK-CTN strain immunization. Female BABL/c mouse, can adjuvant be added with SNK-CTN strain virus suspension or not add adjuvant, respectively at the 0th, 7,14,21 day mouse peritoneal immunity 0.5ml, in blood sampling in 28-42 days, measure serum titer, extracting spleen cell, with SP/20 myeloma cell fusion, through cloning selection can stably excreting to the hybridoma cell strain of rabies virus glycoprotein high-titer mono-clonal neutralizing antibody.Carry out the corresponding antigen immunogenicity qualification of viral protein of clone strain, antibody typing qualification, antibody purification and conservation.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can do some amendment or improve, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (10)
1. a human diploid cell rabies virus strain, is characterized in that, prepares by the following method:
1) the MRC-5 cell got in cell bank is recovered, cultivate after within 3 days, becoming monolayer cell, cell culture fluid in MRC-5 Tissue Culture Flask is outwelled, digests with 0.25% tryptic digestive juice, be dispersed into uniform cell, inoculation rabies viruses CTN-1V5 strain suspension is carried out according to 0.01 ~ 1.0MOI dosage, cultivate 2 ~ 3 days, change the maintenance medium containing 2 ~ 4% bovine serums into for 37 DEG C, cultivate after 3 ~ 5 days for 33 ~ 35 DEG C and gather in the crops virus liquid, frozen in-70 DEG C, be prepared into viral suspension;
2) with above-mentioned method, uploaded for 30 ~ 32 generations at MRC-5 cell continuously.This Rabies Vaccine seed culture of viruses (SNK-CTN strain), its preserving number is CGCC No8887.
2. virus strain according to claim 1, is characterized in that, described maintenance medium is that MEM series or 199 serial culture bases add 2 ~ 4% calf serums.
3. virus strain according to claim 1, is characterized in that, rabies viruses CTN-1V5 strain continuous passage on human diploid cell MRC-5 adapts to; The human diploid cell rabies virus strain virus obtained can reach the infection titer of 6.5lgLD50/ml after the 6th generation.
4. virus strain according to claim 1, is characterized in that, the rabies poison antigenic content obtained measures to reach through enzyme linked immunosorbent assay and is greater than 0.4IU/ml.
5. virus strain according to claim 1, is characterized in that, the virus strain obtained can reach and be greater than 100 to the protection neutralization index of rabies virus CVS strain after the 6th generation.
6. a preparation method for Rabies adapted virus strain (SNK-CTN), is characterized in that, comprise the following steps:
1) the MRC-5 cell got in cell bank is recovered, cultivate after within 3 days, becoming monolayer cell, cell culture fluid in MRC-5 Tissue Culture Flask is outwelled, digests with 0.25% tryptic digestive juice, be dispersed into uniform cell, inoculation rabies viruses CTN-1V5 strain suspension is carried out according to 0.01 ~ 1.0MOI dosage, cultivate 2 ~ 3 days, change the maintenance medium containing 2 ~ 4% bovine serums into for 37 DEG C, cultivate after 3 ~ 5 days for 33 ~ 35 DEG C and gather in the crops virus liquid, frozen in-70 DEG C, be prepared into viral suspension;
2) with above-mentioned method, uploaded for 30 ~ 32 generations at MRC-5 cell continuously.
7. preparation method according to claim 6, is characterized in that, comprises the following steps:
Get the cryopreservation tube that MRC-5 cell is housed, put into rapidly 40 DEG C of water-baths to melt, then pre-temperature is joined in the MEM cell culture fluid of 37 DEG C, be placed in 37 DEG C of cultivations, after within 3 ~ 4 days, growing up to individual layer after 0.25 tryptic digestion according to 0.01 ~ 1.0MOI dose inoculation CTN-1V5 strain suspension, cultivate 2 ~ 3 days for 37 DEG C, change the maintenance medium of 2 ~ 4% bovine serums into, cultivate after 3 ~ 5 days for 33 ~ 35 DEG C and gather in the crops virus liquid, frozen in-70 DEG C, be prepared into viral suspension; By above method, the viral suspension of results is uploaded 30 generations at MRC-5 cell continuously, obtain rabies virus SNK-CTN strain.
8. the application of virus strain according to claim 1 in preparation Rabies Vaccine, wherein said preparation Rabies Vaccine method is as follows:
Step 1, diploid cell loads SNK-CTN virus
Get the human embryonic lung diploid fibroblast (MRC-5) of the cell bottle cultivation of covering with individual layer, through 0.25% trypsin digestion and cell, amplifying cells, on cell factory or bio-reactor, adds MEM cell culture fluid, and enchylema is pressed 0.05-0.IMOI and inoculated SNK-CTN rabies virus;
Step 2, cultivates and results
Put 37 DEG C to cultivate after 3 days, discard nutrient solution and change to maintenance medium.Put 35 DEG C of quiescent culture 3 ~ 15 days; Results virus liquid, harvesting method is: cell factory results are gathered in the crops 2 ~ 3 times according to the pathology of cell; Bio-reactor results carry out Multiple harvests virus for virus harvest liquid according to the index of reactor monitoring and the pathology of cell; Harvest liquid aseptically adds the beta-propiolactone deactivation 24 hours of 1:4000;
Step 3, Isolation and purification method is as follows:
After molecular weight cut-off is ultra-filtration membrane ultrafiltration and concentration 40-80 times of 300KD, carries out purifying with ultracentrifuge, obtain the rabies virus stoste of purifying.
Wherein carry out purifying with ultracentrifuge, method is as follows:
Sucrose density gradient centrifugation 10%, 20%, 30%, 40%, 50% (W/V) sucrose solution makes density gradient, 34000rpm ultracentrifugation is after 4 hours, through the antigenic content to ultraviolet absorption peak, the analysis of protein concentration, determines virus antigen position.
9. the application of virus strain according to claim 1 in preparation rabies diagnostic reagent.
10. a rabies diagnostic kit, containing virus strain according to claim 1.
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| CN109666653B (en) * | 2019-01-15 | 2022-10-04 | 吉林惠康生物药业有限公司 | Subculture method of rabies vaccine virus and preparation method of rabies vaccine |
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