CN102988974A - Method for preparing porcine pseudorabies live vaccine by using passage CEF (chicken embryo fibroblast) - Google Patents
Method for preparing porcine pseudorabies live vaccine by using passage CEF (chicken embryo fibroblast) Download PDFInfo
- Publication number
- CN102988974A CN102988974A CN2012105417958A CN201210541795A CN102988974A CN 102988974 A CN102988974 A CN 102988974A CN 2012105417958 A CN2012105417958 A CN 2012105417958A CN 201210541795 A CN201210541795 A CN 201210541795A CN 102988974 A CN102988974 A CN 102988974A
- Authority
- CN
- China
- Prior art keywords
- prv
- pseudorabies
- cell
- embryo fibroblast
- virus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses a method for preparing a porcine pseudorabies live vaccine by using the passage CEF (chicken embryo fibroblast), comprising the following steps: firstly, inoculating the primary CEF in the porcine PRV (pseudorabies virus) low virulent strain to obtain a low porcine PRV; then, preparing a DF-1 cell monolayer; next, inoculating the DF-1 cell monolayer in the prepared low porcine PRV; subsequently, putting the DF-1 cell monolayer in a constant-temperature incubator to incubate; and finally, observing the cytopathy, harvesting a virus solution at the right time, freezing and drying the harvested virus solution in vacuum to obtain the porcine pseudorabies live vaccine. According to the method, the passage CEF (DF-1) replaces the primary CEF to culture the PRV, and the process for culturing is optimized systematically, the PRV is easier to culture, the obtained PRV has higher viral titer, the vaccine has more stable quality, and the production cost is lower.
Description
Technical field
The present invention relates to belong to veterinary biologics and cultivate technical field, be specifically related to a kind of usefulness chick embryo fibroblast that goes down to posterity and prepare the live method of Seedling of pseudorabies.
Background technology
Pseudorabies claims again AujeszkyShi sick, it is a kind of acute infectious disease take heating, miscarriage, very itch (except pig), encephalomyelitis as cardinal symptom by the caused multiple domestic animal of pseudorabies virus (Pseudora-bies virus, PRV), poultry and wild animal.This virus ownership herpetoviridae (Herpesviridae) first type herpesvirus subfamily (Alphaherpesvirinae of alphavirinae) therefore also becomes herpesvirus suis type (Suid herpesvirus), the sick virus of infectiousness bulbar paralysis or the syndrome virus of very itching.This Viral infection spectrum is extremely wide, has 40 kinds of animals can be infected to show, pig is most important reservoir and the carrier of this virus, thereby the propagation of this virus is played an important role.Because infected animal species is extremely many, virus can exist at the nature iterative cycles, belongs to typical disease of natural focus, also is one of infectious disease of extremely difficult anti-system.
After the pig generation pseudorabies, its clinical symptoms depends on the age of infected pigs, virulence route of infection and the dosage of Strain.Adult and sow main manifestations is respiratory symptom, is one more and crosses property, is the main source of band poison and toxin expelling.Can cause farrowing sow miscarriage, produce the comprehensive syndromes such as stillborn fetus, mummy fetus and boar be sterile.More up to 100%, the ablactational baby pig sickness rate can reach 40% to 15 ages in days with interior piglet death rate of the onset, and dead pig is increasing.
Primary disease there is no the specific treatment medicine at present, the immunity inoculation of vaccine is the essential measure of prevention and control pseudorabies, conventional attenuated vaccine, inactivated vaccine and the gene-deleted vaccine of pseudorabies have been developed at present both at home and abroad, these vaccines can both alleviate or prevent and treat the clinical symptoms of pseudorabies to a certain extent, thereby reduce the economic loss that causes.
But generally use at present former generation chick embryo fibroblast (CEF) to cultivate the method for the weak poison of PRV in the preparation method of porcine pseudorabies virus live vaccine, there is the supply that loaded down with trivial details, the malicious valency of operating process complexity is low, difference between batch is large, production cost is high, a large amount of production is limited by the SPF egg in this method, and introduces easily exogenous virus or other polluter, is easy to cause the defective such as vaccine quality is unstable.
In addition, DF-1 cell line is formed in improvement in 1998 by Department of Animal Science Douglas doctor Foster of Univ Minnesota-Twin Cities USA the earliest, the ELL-0(that originates from 10 ages in days navigates) chick embryo fibroblast (CEF), be spontaneous immortality, can infinitely breed.It has fibroblastic typical cellular morphology.DF-1 cell reversal record enzyme is negative, does not have the endogenous gene sequence of birds sarcoma and leucovirus, so be non-tumorigenic.DF-1 cell line is the global commerce cell line of authorizing through FDA (FDA), has been widely used in the propagation of birds virus, the expression of recombiant protein, the research of oncovirus and the production of animal and human's class vaccine at present.The DF-1 cell is to infections chicken cloacal bursa virus, reovirus, avian leukosis virus, rous sarcoma virus, the equal susceptible of herpes turkey virus, the pathological changes of birds virus on the DF-1 cell is more violent, more obvious, can copy at the DF-1 cell, and can form obvious cytopathy.The DF-1 cell has good multiplication potentiality, compares with chick embryo fibroblast, and the density of DF-1 cell can be than large more than 4 times.Therefore, be a present main direction of studying with DF-1 cells produce pig PRV live vaccine.
Summary of the invention
The objective of the invention is, propose a kind of usefulness chick embryo fibroblast that goes down to posterity for the deficiencies in the prior art and prepare the method for pseudorabies live vaccine, the method utilization is gone down to posterity, and PRV is weak malicious for the former generation chick embryo fibroblast CEF cultivation of chick embryo fibroblast (DF-1) replacement, and culture process is carried out the optimization of system, overcoming complicated loaded down with trivial details, high cost, and cause easily the unsettled defective of vaccine quality.
Technical scheme of the present invention realizes as follows:
(1) prepares first former generation chick embryo fibroblast (CEF), 20-28h after the preparation of CEF cell, PRV (Pseudorabies virus) (PRV) low virulent strain is inoculated former generation chick embryo fibroblast (CEF), obtain the weak seed culture of viruses poison of PRV (Pseudorabies virus), survey according to a conventional method the median infective dose (TCID of virus behind the virus liquid of the weak seed culture of viruses poison of the PRV (Pseudorabies virus) of results, multigelation 3 times
50) should reach 10
7.0TCID
50More than/the ml.
(2) prepare chick embryo fibroblast (DF-1) cell monolayer that goes down to posterity, the DF-1 cell is tamed with the DMEM culture medium that contains 8% serum, be positioned over 37 ℃, CO
2Content is to cultivate in 5% the constant incubator.Contain in the wherein said DMEM culture medium 1.5g sodium bicarbonate/liter, PH is transferred to 7.0-7.2.
(3) about 40～48h behind the DF-1 passage, when cell density is the 90-100% monolayer, the malicious dosage of inoculation by 0.8%～1%(v/v) of seed culture of viruses a little less than the PRV of preparation is inoculated into the DF-1 cell monolayer, adds the DMEM culture medium that contains 2% serum, continue to be positioned over 37 ℃, CO
2Content is to continue in 5% the constant incubator to cultivate.Contain in the wherein said DMEM culture medium 1.5g sodium bicarbonate/liter, PH is transferred to 7.0-7.2.
(4) the DF-1 cell can produce cytopathy behind the virus inoculation, the observation of cell pathological changes, and when cytopathy reaches 85%, the results virus liquid.
(5) with results virus liquid mix with freeze drying protectant in the ratio of 1:1, through the vacuum freezing lyophilizing, the pseudorabies live vaccine.Every 100ml contains the defatted milk powder of 5g sucrose and 10g in the wherein said freeze drying protectant.
Serum described in the wherein above step 2,3 is domestic serum, elects as in Hangzhou Ilex purpurea Hassk.[I.chinensis Sims hyclone, Wuhan three sharp hyclones, Lanzhou Min Hai top grade new-born calf serum and the Jinan strength cattle new-born calf serum at least a.
Wherein the pseudorabies low virulent strain described in the present invention is Bartha K61 strain, available from Harbin Veterinary Medicine Inst., China Academy of Agriculture.
A kind of usefulness of the present invention chick embryo fibroblast that goes down to posterity prepares the method for pseudorabies live vaccine, utilization is gone down to posterity, and PRV is weak malicious in chick embryo fibroblast (DF-1) replacement CEF cultivation, and culture process is optimized, 40～48h behind the DF-1 passage for example, cell be 90% when just being paved with monolayer virus inoculation, the virus liquid that obtains poison valency is higher, when the malicious valency of kind of poison 10
7.0TCID
50/ ml～10
7.5TCID
50In the time of between/the ml, when dosage of inoculation was 0.8%～1%(v/v), the malicious valency that obtains was more high.The method is had cultivate the operation of PRV virus easier, the advantage such as the virus titer that obtains is higher, and vaccine quality is more stable, production cost is lower.
The specific embodiment
In order to make technical scheme of the present invention clearer, the present invention is further illustrated below in conjunction with specific embodiment.
Embodiment:
Used pseudorabies low virulent strain is Bartha K61 strain in the present embodiment, available from Harbin Veterinary Medicine Inst., China Academy of Agriculture.The chick embryo fibroblast that goes down to posterity (DF-1) is available from Shanghai Inst. of Life Science, CAS cell resource center.
When the DF-1 cell is tamed with domestic serum and culture medium, select the serum of domestic several producers when cultivation and domestication DF-1 cell, be respectively Hangzhou Ilex purpurea Hassk.[I.chinensis Sims hyclone, Wuhan three sharp hyclones, Lanzhou Min Hai top grade new-born calf serum and Jinan strength cattle new-born calf serum.Can not well adapt to domestic serum and domestic culture medium at domestication initial stage DF-1 cell, increase and the phenomenon that cell produces a large amount of cavitys occurs.To all add a certain amount of import serum in these four kinds of domestic serum, carry out continuous passage, finally make the DF-1 cell adapt to gradually domestic serum, the DF-1 cell of four kinds of serum free culture system domestications is carried out the contrast of cell state, comprise cellular morphology, the speed of growth, adherent speed, the factors such as edge index of refraction are judged, find that 4 kinds of serum free culture system DF-1 cell state difference are little, because the new-born calf serum cost of Jinan strength cattle is lower, be fit to reduce in the large-scale production requirement of cost, use the new-born calf serum of Jinan strength cattle when therefore, this scheme is finally determined to cultivate the DF-1 cell.
Selected domestic clear large day one DMEM culture medium replacement when cultivation and domestication DF-1 cell.Use the sodium bicarbonate of a clear large day DMEM culture medium powder 13.78 gram and 1.5 grams to be dissolved in the 1L water, transfer PH to 7.0 between 7.2, the Jinan strength cattle new-born calf serum of adding 80ml in every liter of culture medium of DF-1 cell forward direction of cultivation.
The implementation step is as follows:
1, the weak seed culture of viruses poison of preparation PRV, with the SPF Embryo Gallus domesticus of 9～11 ages in days decaptitate, extremity and internal organs, prepare chick embryo fibroblast.PRV (Pseudorabies virus) (PRV) low virulent strain is inoculated former generation chick embryo fibroblast (CEF), cultivate about 72h, results virus liquid when treating that cytopathy reaches 80% with the virus liquid multigelation of results 3 times, obtains the weak seed culture of viruses poison of PRV (Pseudorabies virus).Measure simultaneously the TCID of the weak seed culture of viruses poison of PRV
50, the malicious valency of the PRV kind poison of mensuration is 10
7.4TCID
50/ ml.
2, after the DF-1 cell covers with monolayer, carry out passage.Use first the PBS washed twice, add an amount of tryptic digestive juice, keep flat about 10s, observation of cell digestion situation, if it is smudgy to find that cellular layer occurs, then discard pancreatin, add an amount of culture fluid (DMEM that contains 8% serum), blow and beat gently to cell with glass pipette and be separated into individual cells.Seed cell bottle number be vaccinated cell bottle number and can go down to posterity in the ratio of 1:2.DF-1 cell after going down to posterity is put 37 ℃ 5%CO
2Constant incubator in cultivate;
3, the DF-1 cell is cultivated about 48h in incubator, takes out cell observation cell state secret degree.The DF-1 cell state that goes down to posterity is good, and cell density is about 90%～100%.Discard culture fluid with the DF-1 cell this moment, with PBS washing 2 times, then seed culture of viruses poison a little less than the PRV of preparation inoculated the DF-1 cell monolayer with 0.8% concentration, puts 37 ℃ the 5%CO that contains
2Constant incubator in adsorb 1h, discard virus liquid, add cell maintenance medium, put 37 ℃ the 5%CO that contains
2Constant incubator in continue to cultivate.When cultivating about 96h, the observation of cell pathological changes reaches 85% results virus approximately, and cell shows as cell rounding, comes off.
4, the virus liquid of results is through steriling test, and viral level is measured, and after safety verification is qualified, adds 5% sucrose skimmed milk and an amount of antibiotic quantitative separating in the ratio of 1:1, through the vacuum freezing lyophilizing, the pseudorabies live vaccine.
Claims (6)
1. one kind prepares the method for pseudorabies live vaccine with the chick embryo fibroblast that goes down to posterity, and it is characterized in that cultural method and step are as follows:
(1) prepare first former generation chick embryo fibroblast (CEF), 20-28h after the preparation of CEF cell inoculates former generation chick embryo fibroblast (CEF) with PRV (Pseudorabies virus) (PRV) low virulent strain, obtains the weak seed culture of viruses poison of PRV (Pseudorabies virus);
(2) prepare chick embryo fibroblast (DF-1) cell monolayer that goes down to posterity, with the DMEM of DF-1 cell with 8% serum
The DMEM culture medium is tamed, and is positioned over 37 ℃, CO
2Content is to cultivate in 5% the constant incubator;
(3) 40～48h behind the DF-1 passage when cell density is the 90-100% monolayer, is inoculated into the DF-1 cell monolayer with the malicious dosage of inoculation by 0.8%～1%(v/v) of seed culture of viruses a little less than the PRV of preparation, adds the DMEM culture medium that contains 2% serum, is positioned over 37 ℃, CO
2Content is to continue in 5% the constant incubator to cultivate;
(4) the DF-1 cell can produce cytopathy behind the virus inoculation, the observation of cell pathological changes, and when cytopathy reaches 85%, the results virus liquid;
(5) with results virus liquid mix with freeze drying protectant in the ratio of 1:1, through the vacuum freezing lyophilizing, the pseudorabies live vaccine.
2. a kind of usefulness according to claim 1 chick embryo fibroblast that goes down to posterity prepares the method for pseudorabies live vaccine, it is characterized in that: the median infective dose (TCID that surveys according to a conventional method virus behind the virus liquid of the weak seed culture of viruses poison of the PRV (Pseudorabies virus) of step (1) results, multigelation 3 times
50) reach 10
7.0TCID
50More than/the ml.
3. a kind of usefulness according to claim 1 chick embryo fibroblast that goes down to posterity prepares the method for pseudorabies live vaccine, it is characterized in that: described DMEM culture medium is domestic clear large day one DMEM culture medium, wherein contain the 1.5g sodium bicarbonate/liter, DMEM cultivates keynote PH to 7.0-7.2.
4. the chick embryo fibroblast that goes down to posterity of a kind of usefulness described in according to claim 1 prepares the method for pseudorabies live vaccine, it is characterized in that: wherein the serum described in step (2), (3) is domestic serum, elects as in Hangzhou Ilex purpurea Hassk.[I.chinensis Sims hyclone, Wuhan three sharp hyclones, Lanzhou Min Hai top grade new-born calf serum and the Jinan strength cattle new-born calf serum at least a.
5. a kind of usefulness according to claim 1 chick embryo fibroblast that goes down to posterity prepares the method for pseudorabies live vaccine, and it is characterized in that: in the step (5), every 100ml contains the defatted milk powder of 5g sucrose and 10g in the described freeze drying protectant.
6. the chick embryo fibroblast that goes down to posterity of a kind of usefulness described in according to claim 1 and 2 prepares the method for pseudorabies live vaccine, and it is characterized in that: described pseudorabies low virulent strain is Bartha K61 strain.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210541795.8A CN102988974B (en) | 2012-12-14 | 2012-12-14 | Method for preparing porcine pseudorabies live vaccine by using passage CEF (chicken embryo fibroblast) |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210541795.8A CN102988974B (en) | 2012-12-14 | 2012-12-14 | Method for preparing porcine pseudorabies live vaccine by using passage CEF (chicken embryo fibroblast) |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102988974A true CN102988974A (en) | 2013-03-27 |
| CN102988974B CN102988974B (en) | 2015-05-20 |
Family
ID=47918408
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210541795.8A Active CN102988974B (en) | 2012-12-14 | 2012-12-14 | Method for preparing porcine pseudorabies live vaccine by using passage CEF (chicken embryo fibroblast) |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102988974B (en) |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103865889A (en) * | 2014-04-03 | 2014-06-18 | 深圳市卫光生物制品股份有限公司 | Rabies virus CTN chick-embryo cell adaptive strain |
| CN104689311A (en) * | 2014-12-31 | 2015-06-10 | 瑞普（保定）生物药业有限公司 | Method for producing avian encephalomyelitis virus inactivated vaccine |
| CN105521487A (en) * | 2014-09-30 | 2016-04-27 | 广东永顺生物制药股份有限公司 | Method for preparing pseudorabies live vaccines from DF1 continuous cells and product prepared by method |
| CN105602909A (en) * | 2016-03-29 | 2016-05-25 | 四川海林格生物制药有限公司 | Method for culturing porcine pseudorabies virus |
| CN105624123A (en) * | 2016-03-29 | 2016-06-01 | 四川海林格生物制药有限公司 | Cell affinity agent for improving porcine pseudorabies virus in-vitro infection efficiency and use method thereof |
| US20160279231A1 (en) * | 2015-03-20 | 2016-09-29 | Pulike Biological Engineering, Inc. | A Method of Attenuating Porcine Pseudorabies Virus, Attenuated Strains of Porcine Pseudorabies Virus, Vaccine Composition and Use Thereof |
| CN106282098A (en) * | 2016-08-31 | 2017-01-04 | 天津瑞普生物技术股份有限公司 | A kind of utilize relatively low serum content nutrient cultivate DF1 cell with the method preparing infections chicken cloacal bursa virus |
| CN109234239A (en) * | 2018-07-13 | 2019-01-18 | 广东永顺生物制药股份有限公司 | A kind of cultural method of duck plague virus |
| CN110387354A (en) * | 2019-08-16 | 2019-10-29 | 江苏省农业科学院 | Pseudorabies virus Attenuation strain and its application |
| CN114350601A (en) * | 2021-12-21 | 2022-04-15 | 广东省华晟生物技术有限公司 | Cherry valley duck fibroblast line and construction method and application thereof |
| US11604026B2 (en) | 2019-03-14 | 2023-03-14 | Terumo Bct Biotechnologies, Llc | Lyophilization loading tray assembly and system |
| US11634257B2 (en) | 2017-10-09 | 2023-04-25 | Terumo Bct Biotechnologies, Llc | Lyophilization container and method of using same |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101457215A (en) * | 2008-12-01 | 2009-06-17 | 华中农业大学 | Recombinant porcine pseudorabies virus-porcine propagate and breath complex virus-porcine circovirus genetic engineering strain and application |
| CN101730544A (en) * | 2007-07-03 | 2010-06-09 | 卡迪拉保健有限公司 | Adaptation of pitman moore strain of rabies virus to primary chick embryo fibroblast cell cultures |
-
2012
- 2012-12-14 CN CN201210541795.8A patent/CN102988974B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101730544A (en) * | 2007-07-03 | 2010-06-09 | 卡迪拉保健有限公司 | Adaptation of pitman moore strain of rabies virus to primary chick embryo fibroblast cell cultures |
| CN101457215A (en) * | 2008-12-01 | 2009-06-17 | 华中农业大学 | Recombinant porcine pseudorabies virus-porcine propagate and breath complex virus-porcine circovirus genetic engineering strain and application |
Non-Patent Citations (2)
| Title |
|---|
| MAAS R ET AL: "Replacement of primary chicken embryonic frbroblasts（CEF）by the DF-1 cell line for detection of avian leucosis viruses", 《BIOLOGICALS》 * |
| 王岩等: "PK15 、Vero、BHK、CEF细胞增殖猪伪狂犬病病毒的比较", 《安徽农业科学》 * |
Cited By (25)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103865889A (en) * | 2014-04-03 | 2014-06-18 | 深圳市卫光生物制品股份有限公司 | Rabies virus CTN chick-embryo cell adaptive strain |
| CN103865889B (en) * | 2014-04-03 | 2015-02-25 | 深圳市卫光生物制品股份有限公司 | Rabies virus CTN chick-embryo cell adaptive strain |
| CN105521487A (en) * | 2014-09-30 | 2016-04-27 | 广东永顺生物制药股份有限公司 | Method for preparing pseudorabies live vaccines from DF1 continuous cells and product prepared by method |
| CN104689311A (en) * | 2014-12-31 | 2015-06-10 | 瑞普（保定）生物药业有限公司 | Method for producing avian encephalomyelitis virus inactivated vaccine |
| US20160279231A1 (en) * | 2015-03-20 | 2016-09-29 | Pulike Biological Engineering, Inc. | A Method of Attenuating Porcine Pseudorabies Virus, Attenuated Strains of Porcine Pseudorabies Virus, Vaccine Composition and Use Thereof |
| JP2017512455A (en) * | 2015-03-20 | 2017-05-25 | プリケ生物工程股▲ふん▼有限公司 | Herpesvirus attenuation method, attenuated virus strain, vaccine composition and application |
| EP3103866A4 (en) * | 2015-03-20 | 2017-10-04 | Pulike Biological Engineering, Inc. | Porcine pseudorabies virus attenuating method, viral strains attenuated thereby, vaccine composition and application thereof |
| US9795667B2 (en) * | 2015-03-20 | 2017-10-24 | Pulike Biological Engineering, Inc. | Method of attenuating porcine pseudorabies virus, attenuated strains of porcine pseudorabies virus, vaccine composition and use thereof |
| CN105624123A (en) * | 2016-03-29 | 2016-06-01 | 四川海林格生物制药有限公司 | Cell affinity agent for improving porcine pseudorabies virus in-vitro infection efficiency and use method thereof |
| CN105602909A (en) * | 2016-03-29 | 2016-05-25 | 四川海林格生物制药有限公司 | Method for culturing porcine pseudorabies virus |
| CN106282098A (en) * | 2016-08-31 | 2017-01-04 | 天津瑞普生物技术股份有限公司 | A kind of utilize relatively low serum content nutrient cultivate DF1 cell with the method preparing infections chicken cloacal bursa virus |
| CN106282098B (en) * | 2016-08-31 | 2019-10-29 | 天津瑞普生物技术股份有限公司 | A method of using lower serum content nutrients culture DF1 cell to prepare infections chicken cloacal bursa virus |
| US11634257B2 (en) | 2017-10-09 | 2023-04-25 | Terumo Bct Biotechnologies, Llc | Lyophilization container and method of using same |
| CN109234239A (en) * | 2018-07-13 | 2019-01-18 | 广东永顺生物制药股份有限公司 | A kind of cultural method of duck plague virus |
| CN109234239B (en) * | 2018-07-13 | 2021-09-14 | 广东永顺生物制药股份有限公司 | Method for culturing duck plague virus |
| US11609042B2 (en) | 2019-03-14 | 2023-03-21 | Terumo Bct Biotechnologies, Llc | Multi-part lyophilization container and method of use |
| US11604026B2 (en) | 2019-03-14 | 2023-03-14 | Terumo Bct Biotechnologies, Llc | Lyophilization loading tray assembly and system |
| US11609043B2 (en) | 2019-03-14 | 2023-03-21 | Terumo Bct Biotechnologies, Llc | Lyophilization container fill fixture, system and method of use |
| US11740019B2 (en) | 2019-03-14 | 2023-08-29 | Terumo Bct Biotechnologies, Llc | Lyophilization loading tray assembly and system |
| US11747082B2 (en) | 2019-03-14 | 2023-09-05 | Terumo Bct Biotechnologies, Llc | Multi-part lyophilization container and method of use |
| US11815311B2 (en) | 2019-03-14 | 2023-11-14 | Terumo Bct Biotechnologies, Llc | Lyophilization container fill fixture, system and method of use |
| US11994343B2 (en) | 2019-03-14 | 2024-05-28 | Terumo Bct Biotechnologies, Llc | Multi-part lyophilization container and method of use |
| CN110387354A (en) * | 2019-08-16 | 2019-10-29 | 江苏省农业科学院 | Pseudorabies virus Attenuation strain and its application |
| CN114350601B (en) * | 2021-12-21 | 2022-12-27 | 广东省华晟生物技术有限公司 | Cherry valley duck fibroblast line and construction method and application thereof |
| CN114350601A (en) * | 2021-12-21 | 2022-04-15 | 广东省华晟生物技术有限公司 | Cherry valley duck fibroblast line and construction method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102988974B (en) | 2015-05-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102988974B (en) | Method for preparing porcine pseudorabies live vaccine by using passage CEF (chicken embryo fibroblast) | |
| CN102220287B (en) | Avian infectious bronchitis cold adaptation attenuated vaccine strain and application thereof | |
| CN109439634A (en) | Pseudorabies virus genetic engineering attenuated vaccine strain and its application | |
| CN111632137A (en) | Triple vaccine for feline calicivirus disease, feline infectious rhinotracheitis and feline panleukopenia as well as preparation method and application thereof | |
| CN104258385A (en) | Applications of BHK-21 cell full-suspension culture technology in production of newcastle disease vaccine | |
| CN101792739A (en) | Optimum condition for proliferating Guangxi epidemically representative strains of infectious bursal disease virus (IBDV) in chicken embryo | |
| CN105582533B (en) | Avian influenza virus and avian adenovirus bivalent inactivated vaccine | |
| CN106075429B (en) | Goat pox, sheep pox and aphtha triple cell attenuated vaccine and preparation method and application thereof | |
| CN113384692A (en) | Duck reovirus and duck circovirus bivalent inactivated vaccine and preparation method thereof | |
| CN103952377A (en) | Cell line used for separation culture and multiplication of Orf virus (ORFV) as well as preparation method and application of cell line | |
| CN103977400B (en) | Method for producing marek disease live vaccine of chicken by using cell line | |
| CN103864931B (en) | A kind of preparation of pseudoabies standard positive serum and freeze-drying store method thereof | |
| CN110404063A (en) | Using the non-method exempted from egg and prepare avian infectious bronchitis virus antigen | |
| CN114107171B (en) | Goose retinal epithelial cell line and construction method and application thereof | |
| CN103127496B (en) | Type III herpes turkey virus freeze dried vaccine | |
| CN106139141B (en) | Sheep pox and orf bivalent cell attenuated vaccine and preparation method and application thereof | |
| CN104293736A (en) | Telomerase immortal cattle thyroid cell line and purpose thereof | |
| CN101380470B (en) | Pig parvovirus live vaccine | |
| CN105727277A (en) | Method for preparation of swine pseudorabies virus vaccine and vaccine product | |
| CN112080478B (en) | Efficient propagation method and application of H5 subtype avian influenza virus | |
| CN104450630A (en) | Method for culturing goose parvovirus by using goose embryo fibroblast line | |
| CN103721253A (en) | Live avian encephalomylitis and henpox combined vaccine | |
| CN104740627B (en) | A kind of large-scale method for producing of pseudo- mad dog attenuated live vaccines for animals | |
| CN103316334B (en) | Infectious bursal disease live vaccine and production method thereof | |
| CN102978167A (en) | Method for preparing nephropathogenic avian infectious bronchitis viruses and live vaccines by using cell lines |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C53 | Correction of patent of invention or patent application | ||
| CB03 | Change of inventor or designer information |
Inventor after: Yang Lingzhi Inventor after: Niu Chengming Inventor after: Zhang Jing Inventor after: Ma Yanbin Inventor before: Xu Mingming Inventor before: Yang Lingzhi Inventor before: Zhang Jing |
|
| COR | Change of bibliographic data |
Free format text: CORRECT: INVENTOR; FROM: XU MINGMING YANG LINGZHI ZHANG JING TO: YANG LINGZHI NIU CHENGMING ZHANG JING MA YANBIN |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |