CN102380093A - Method for producing newcastle disease living vaccines by utilizing passage fibroblasts - Google Patents
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Abstract
The invention discloses a method for producing newcastle disease living vaccines by utilizing passage fibroblasts, which is characterized in that the defects of unstable virus titer and greater differences among batches of I-series virus stock solution produced by existing primary fibroblasts can be overcome, and the passage fibroblasts are used for replacing the primary fibroblasts to be used as virus inoculation cells for performing virus multiplication. By adopting the method for producing newcastle disease living vaccines by utilizing the passage fibroblasts, newcastle disease I-series virus can be inoculated under a relatively consistent cell environment, thereby providing stable growth conditions for the virus multiplication, reducing the differences among the batches of products, upgrading the product quality and ensuring the stability in the quality of the vaccines.
Description
Technical field
The present invention relates to a kind of method for preparing of chicken Newcastle disease live vaccine, particularly a kind of through the utilization method that fibroblast produces chicken Newcastle disease live vaccine that goes down to posterity, belong to the vaccine production field.
Background technology
Newcastle (Newcastle disease, ND) be by NDV (Newcastle disease virus, a kind of acute, the height contact birds infectious disease that NDV) cause, very harmful to aviculture, classified as the category-A infectious disease by World Organization for Animal Health.
NDV has only 1 serotype, but according to the difference of its virulence, can be divided into virulent strain, mesogenic strains and low virulent strain.Virulent strain can be described as the anaphylactic type strain again, and mesogenic strains is middle hair style strain, and low virulent strain is the delayed type strain.According to its to the different tissues organ invade preferendum again can with the strain that virulence is arranged be divided into have a liking for internal organs with have a liking for the nervous system type strain, the former mainly causes infringements such as digestive tract hemorrhage, the disease that the latter causes mainly shows as nervous symptoms and respiratory tract injury.
China was separated to NDV first in 1948, had controlled the popular of typical newcastle for many years and basically although China's newcastle immunity inoculation has been popularized, and atypia ND still happens occasionally, and the sound development of poultry husbandry in serious threat.Vaccine is the important means that the control primary disease takes place at present.
Since 1940 beginnings, just use newcastle disease vaccine in the world, the newcastle live-virus vaccine that the most successful vaccine is made up of hair style Strain in delayed type Strain and meticulous some of screening of process.
Inactivated vaccine began to use from the seventies, divided simple inactivated vaccine and oil-emulsion inactivated vaccine, and using more at present is oil-emulsion inactivated vaccine.Oil-emulsion inactivated vaccine can produce strong and persistent immunity, can not spread cause of disease through the use of vaccine.
China's attenuated vaccine commonly used at present is an I system, La Sota vaccine, V4 vaccine.
Recent years, along with the development of cell culture technology, cell vaccine is come out one after another with clone's Seedling.It is single that clone's Seedling has antigenicity, the characteristics that immunogenicity is strong.The clone N-79 plant type LaSota low virulent strain that China introduces from the U.S., clone-30 who from I system, picks out and clone the-83rd have the highly stable attenuated vaccine of good immunogenicity and heritability.
Cell vaccine has the low characteristics of producing easily of cost, and passage cell can be avoided the infection of chicken source property potential disease.
Fibroblast is the topmost cell component of connective tissue; It is that mesenchymal cell by embryo period differentiates; Playing the part of crucial role at secretory cell epimatrix composition with making up in the extracellular matrix, in the tissue injury repair process, also playing an important role.Because fibroblast relatively easily obtains, and multiplication capacity is strong, adaptability is strong, has good tolerability, and character is more stable, the difficult conversion.Possessing above characteristics just, make fibroblast except being applied to cell and molecular biological research, also be widely used in the virological investigation field, also is the important cells resource of production of vaccine simultaneously.
But at present commercially available I is that the live vaccine overwhelming majority derives from Embryo Gallus domesticus and organizes Seedling, is virus stock solution used because utilize existing former generation I of being produced of fibroblast production technology, and virus titer is unstable, and differences between batches are bigger.So to unstable this present situation of former generation fibroblast product poison, the present invention has drawn the present invention on the basis of having carried out lot of experiments.
Summary of the invention
To the problem of above-mentioned existence, this experiment is through adopting the CEF technology that goes down to posterity, and is applied to production practices, and the fibroblast of will going down to posterity substitutes former generation fibroblast.
The objective of the invention is to realize through following technical scheme:
A kind of utilization of the present invention method that fibroblast produces chicken Newcastle disease live vaccine that goes down to posterity is characterized in that carrying out as the Strain inoculating cell with the fibroblast after going down to posterity the preparation of cell venom.
A kind of utilization of the present invention method that fibroblast produces chicken Newcastle disease live vaccine that goes down to posterity is characterized in that may further comprise the steps:
(1) digestion: the former generation CEF to growing up to monolayer carries out digestion process;
(2) frozen: as to preserve in the former generation CEF immigration liquid nitrogen container after step (1) digestion process;
(3) recovery: the cell with step (2) after frozen is regularly recovered, and the survival rate of inspection cell adds in the DMEM culture fluid and cultivates, and treats cell adherent can the use fully;
(4) go down to posterity: the cell that step (3) the is obtained cultivation of going down to posterity;
(5) seedling is with the preparation of cell venom: with the Strain inoculation through step (4) CEF after cultivating that goes down to posterity; Connecing poison continues to cultivate for rearmounted 36～37 ℃; The observation of cell pathological changes when pathological changes such as the appearance of the cell more than 75% circle contracts, index of refraction grow, is put sharp freezing with Tissue Culture Flask; Melt back harvesting culture again in sterilization container, put preserve below-15 ℃ subsequent use;
(6) seedling: the cell venom that the step (5) of effective dose is obtained adds adjuvant and processes described chicken Newcastle disease live vaccine.
In a specific embodiment of the present invention, digestion process specifically may further comprise the steps in the described step (1):
(1) examine under a microscope the former generation CEF that grows up to monolayer, treating 80%, be paved with just can be through row digestion;
(2) discard culture fluid in the cell bottle, add 0.01mol/L PBS in the ratio of cell bottle capacity 10%, the parallel gently culture bottle that rocks, the culture fluid that flush away is residual, sucking-off cleanout fluid is lentamente abandoned it again, repeats this operation 3 times;
(3) the trypsinization liquid of adding 0.25% in the cell bottle that cleaned, addition is 1% of a cell bottle capacity.Put into 37 ℃ of water-baths after adding immediately and digest 0.5～2min.After treating that cell comes off fully, add the hyclone of 1% volume of cell bottle capacity immediately to it, mixing is to end the Digestion of pancreatin.
(4) at the DMEM culture fluid that contains 10% hyclone that in culture bottle, adds cell bottle capacity 10% volume, mixing is poured in the centrifuge tube of sterilization, and the centrifugal 15min of 2000r/min abandons supernatant.
(5) repeating step is (4) 3 times.
In a specific embodiment of the present invention; Frozen in the described step (2) is meant the former generation CEF after step (2) digestion process suspended with the DMEM culture fluid that contains 10%DMSO again; Be sub-packed in the lyophilizing pipe, the 1ml/ pipe, cell density will be not less than 1～2 * 10
6/ ml places 20min for 4 ℃ ,-80 ℃ spend the night after, move in the liquid nitrogen container and preserve.
In a specific embodiment of the present invention; From liquid nitrogen container, take out during recovery in the described step (3) and put into 37 ℃ of water-baths rapidly behind the lyophilizing pipe of former generation CEF and melt; Melting the back takes out from water-bath on the placement ice chest; Add then in the DMEM culture fluid and cultivate, treat cell adherent can the use fully.
In a specific embodiment of the present invention, the cultivation of going down to posterity of cell specifically may further comprise the steps in the described step (4):
(1) examines under a microscope the CEF that grows up to monolayer, treat that 80% is paved with just and can digests through row;
(2) discard culture fluid in the cell bottle, add 0.01mol/L PBS in 10% ratio of cell bottle capacity, the parallel gently culture bottle that rocks, the culture fluid that flush away is residual, sucking-off cleanout fluid is lentamente abandoned it again, repeats this operation 3 times;
(3) the trypsinization liquid of adding 0.25% in the cell bottle that cleaned, addition is 1% of a cell bottle capacity.Put into 37 ℃ of water-baths after adding immediately and digest 0.5～2min.After treating that cell comes off fully, add the hyclone of 1% volume immediately to it, mixing is to end the Digestion of pancreatin;
(4) at the DMEM culture fluid that contains 10% hyclone that in culture bottle, adds cell bottle capacity 30% volume, mixing, other gets 2 same size culture bottles, adds the cell suspension of cell bottle capacity 10% volume in every culture bottle;
(5) to 37 ℃ of cultivation 48h～72h, treat that cell attachment grows up to monolayer and can use.
In a specific embodiment of the present invention, in the described step (5) Strain done suitable dilution with cell maintenance medium after, through step (4) CEF after cultivating that goes down to posterity, pH is transferred to 7.2 by 0.1%～1% inoculation of Cell sap volume.
In a specific embodiment of the present invention, described Strain is a low virulent strain for newcastle disease I.Preferred, described Strain is newcastle disease virus mesogenic strains Mukteswar strain.
A kind of utilization of the present invention method that fibroblast produces chicken Newcastle disease live vaccine that goes down to posterity; Guaranteed under the consistent relatively situation of cellular environment; Inoculation newcastle disease I system virus for the propagation of virus provides stable growth conditions, has been dwindled the differences between batches of product; Promote product quality, guaranteed the stability of vaccine quality.
The specific embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage of the present invention and characteristics will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment with form or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall within the scope of protection of the present invention the details of technical scheme of the present invention.
The preparation of embodiment 1 chicken Newcastle disease live vaccine
One, material:
Bacterial strain: newcastle disease virus mesogenic strains Mukteswar strain (I system) is available from China Veterinery Drug Inspection Office, and CEF prepares, goes down to posterity and preserves for research and development centre of Harbin Pharmaceutical Group Biological Vaccine Co., Ltd.
Reagent: hyclone and DMEM available from Invitrogen GIBCO, pancreatin available from Amresco, dimethyl sulfoxide available from sigma
Two, the method for preparing of chicken Newcastle disease live vaccine:
1. the preparation of cell
1.1 digestion
1.1.1 examine under a microscope the CEF that grows up to monolayer, treat that 80% is paved with just and can digests through row.
1.1.2 discard the culture fluid in the cell bottle, add 0.01mol/L PBS in the ratio of cell bottle capacity 10%, the parallel gently culture bottle that rocks, the culture fluid that flush away is residual, sucking-off cleanout fluid is lentamente abandoned it again.Repeat this operation 3 times.
1.1.3 the trypsinization liquid of adding 0.25% in the cell bottle that cleaned, addition is 1% of a cell bottle capacity.Put into 37 ℃ of water-baths after adding immediately and digest 0.5～2min.After treating that cell comes off fully, add the hyclone of 1% volume of cell bottle capacity immediately to it, mixing is to end the Digestion of pancreatin.
1.1.4 at the DMEM culture fluid that contains 10% hyclone that in culture bottle, adds cell bottle capacity 10% volume, mixing is poured in the centrifuge tube of sterilization, the centrifugal 15min of 2000r/min abandons supernatant.
1.1.5 repeat 1.1.4 step 3 time.
1.2 it is frozen
Deposition suspends with the DMEM culture fluid that contains 10%DMSO (dimethyl sulfoxide) again, is sub-packed in the lyophilizing pipe, and the 1ml/ pipe, cell density will be not less than 1～2 * 10
6/ ml places 20min for 4 ℃ ,-80 ℃ spend the night after, move in the liquid nitrogen container and preserve.
1.3 recovery
Recovery regularly behind the cell cryopreservation; The survival rate of inspection cell is put into 37 ℃ of water-baths rapidly behind the taking-up lyophilizing pipe during recovery and is melted from liquid nitrogen container, melt back taking-up from water-bath and place on the ice chest; Add then in the DMEM culture fluid and cultivate, treat cell adherent can the use fully.
1.4 go down to posterity
1.4.1 examine under a microscope the CEF that grows up to monolayer, treat that 80% is paved with just and can digests through row.
1.4.2 discard the culture fluid in the cell bottle, add PBS in 10% ratio of cell bottle capacity, the parallel gently culture bottle that rocks, the culture fluid that flush away is residual, sucking-off cleanout fluid is lentamente abandoned it again.Repeat this operation 3 times.
1.4.3 the trypsinization liquid of adding 0.25% in the cell bottle that cleaned, addition is 1% of a cell bottle capacity.Put into 37 ℃ of water-baths after adding immediately and digest 0.5～2min.After treating that cell comes off fully, add the hyclone of 1% volume immediately to it, mixing is to end the Digestion of pancreatin.
1.4.4 at the DMEM culture fluid that contains 10% hyclone that in culture bottle, adds cell bottle capacity 30% volume, mixing, other gets 2 same size culture bottles, adds the cell suspension of cell bottle capacity 10% volume in every culture bottle.
1.4.5, treat that cell attachment grows up to monolayer and can use to 37 ℃ of cultivation 48h～72h.
2 seedlings are with the preparation of cell venom
2.1 inoculation is that mesogenic Mukteswar strain is diluted 100 times with cell maintenance medium with newcastle disease I, connects poison by 0.1%～1% of cell liquid measure, and pH is transferred to 7.2.
2.2 connecing poison, cultivation and observation continue to cultivate the observation of cell pathological changes for rearmounted 37 ℃.
2.3 when pathological changes such as circle contracts, index of refraction grow appears in results when the cell 75% or more, Tissue Culture Flask is put sharp freezing, melt back harvesting culture again in sterilization container, every some bottles is one group, it is subsequent use to put preservation below-15 ℃.Holding time is no more than 2 months.
3 inspections of semifinished product
3.1 steriling test Embryo Gallus domesticus venom or cell venom are taken a sample respectively for every group, answer asepsis growth.
3.2 Embryo Gallus domesticus liquid RCA valency is measured every group and is taken a sample respectively, measures the RCA valency, should >=1: 64 (micromethods).
3.3 viral level is measured and taken a sample respectively from every group, mixed in equal amounts is diluted to 10-6～10-8 with 0.01mol/L PBS, and each inoculates 5 of 10 age in days SPF Embryo Gallus domesticus, inoculation 0.1ml in every embryo, the allantoic cavity.The record Embryo Gallus domesticus has obvious lesion person 24～72 hours death, fetuses, calculates ELD50, and every 0.1ml viral level >=107.0ELD50 person can be used to join Seedling.
4 join Seedling and packing
Some groups of cell venom that are up to the standards are mixed in the same container; Add a certain proportion of freeze drying protectant (glycine 0.1% (w/w), sodium thiosulfate 1% (w/w), sucrose 5% (w/w), skimmed milk 5% (w/w)) by regulation plumage part; After fully mixing; Quantitatively packing, every plumage part viral level is answered >=105.0ELD50.Carry out lyophilisation after the packing rapidly.
The immune effect of embodiment 2 chicken Newcastle disease live vaccines
In order to verify the fertility performance of new method, so when continuing to use " rules " and preparing newcastle disease I with Embryo Gallus domesticus and be live vaccine, also adopting new method to prepare 10 crowdes of I with CEF is live vaccine, existing comparative result with the some of them index is summed up as follows:
Following by " rules " production method:
Seedling is with the preparation of cell venom
1, inoculation is a low virulent strain with 100 times of cell maintenance medium dilutions with newcastle disease I, by former generation CEF cell liquid measure 0.1%～1% connect poison, pH is transferred to 7.2.
2, cultivation and observation connect rearmounted 36～37 ℃ of continuation of poison and cultivate the observation of cell pathological changes.
When 3, pathological changes such as circle contracts, index of refraction grow appears in results when the cell 75% or more, Tissue Culture Flask is put sharp freezing, melt back harvesting culture again in sterilization container, every some bottles is one group, and it is subsequent use to put preservation below-15 ℃.Holding time is no more than 2 months.
4 inspections of semifinished product
4.1 steriling test Embryo Gallus domesticus venom or cell venom are taken a sample respectively for every group, answer asepsis growth.
4.2 Embryo Gallus domesticus liquid RCA valency is measured every group and is taken a sample respectively, measures the RCA valency, should >=1: 64 (micromethods).
4.3 viral level is measured and taken a sample respectively from every group, mixed in equal amounts is diluted to 10-6～10-8 with sterile saline, and each inoculates 5 of 10 age in days SPF Embryo Gallus domesticus, inoculation 0.1ml in every embryo, the allantoic cavity.The record Embryo Gallus domesticus has obvious lesion person 24～72 hours death, fetuses, calculates ELD50, and every 0.1ml viral level >=107.0ELD50 person can be used to join Seedling.
5 join Seedling and packing
Some groups of cell venom that are up to the standards are mixed in the same container, add a certain proportion of 5% sucrose skim milk by regulation plumage part and make stabilizing agent.The antibiotic that adding simultaneously suits, after fully mixing, quantitatively packing, every plumage part viral level is answered >=105.0ELD50.Carry out lyophilisation after the packing rapidly.
Compare by new production method and the vaccine of producing by " rules " production method, shown in table 1-5:
Two kinds of production method semi-finished product of table 1 RCA valency comparison sheet
Two kinds of production method semi-finished product of table 2 EID
50Comparison sheet
| Batch | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| " rules " | 7.2 | 7.8 | 7.6 | 8.2 | 8.6 | 8.6 | 8.0 | 6.8 | 6.8 | 8.2 |
| New method | 7.6 | 7.8 | 8.6 | 8.0 | 7.8 | 8.2 | 8.2 | 8.4 | 8.2 | 8.2 |
The assorted inspection of two kinds of production method semi-finished product of table 3 positive rate comparison sheet
| Batch | 1 | 2 | 3 | 4 | 5 | 6 | 7 | ?8 | 9 | 10 |
| " rules " | 1.3％ | 0.2％ | 0 | 2.4％ | 0 | 0.7％ | 3.3％ | ?2.4％ | 1.8％ | 2.8％ |
| New method | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ?0 | 0 | 0 |
Two kinds of production method product percents of pass of table 4 comparison sheet
Two kinds of production method comparative statement of product costs of table 5 (unit: unit/ml)
| Batch | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| " rules " | 1.25 | 1.23 | 1.23 | 1.25 | 1.25 | 1.24 | 1.27 | 1.28 | 1.24 | 1.24 |
| New method | 0.65 | 0.65 | 0.65 | 0.65 | 0.65 | 0.65 | 0.65 | 0.65 | 0.65 | 0.65 |
As above shown in the table, by the vaccine that new method is produced, the vaccine of " rules " production is compared semi-finished product RCA valency, viral level (EID
50) and product percent of pass all increase significantly; And the production cost of positive assorted inspection rate of semi-finished product and unit volume all has tangible reduction, so new method more can promote product quality, practices thrift producing cost, and the method does not still have the record of using aborning at home, is on the leading domestic level.
Claims (9)
1. the utilization method that fibroblast produces chicken Newcastle disease live vaccine that goes down to posterity is characterized in that carrying out as the Strain inoculating cell with the fibroblast after going down to posterity the preparation of cell venom.
2. the method for claim 1 is characterized in that may further comprise the steps:
(1) digestion: the former generation CEF to growing up to monolayer carries out digestion process;
(2) frozen: as to preserve in the former generation CEF immigration liquid nitrogen container after step (1) digestion process;
(3) recovery: the cell with step (2) after frozen is regularly recovered, and the survival rate of inspection cell adds in the DMEM culture fluid and cultivates, and treats cell adherent can the use fully;
(4) go down to posterity: the cell that step (3) the is obtained cultivation of going down to posterity;
(5) seedling is with the preparation of cell venom: with the Strain inoculation through step (4) CEF after cultivating that goes down to posterity; Connecing poison continues to cultivate for rearmounted 36～37 ℃; The observation of cell pathological changes when pathological changes such as the appearance of the cell more than 75% circle contracts, index of refraction grow, is put sharp freezing with Tissue Culture Flask; Melt back harvesting culture again in sterilization container, put preserve below-15 ℃ subsequent use;
(6) seedling: the cell venom that the step (5) of effective dose is obtained adds adjuvant and processes described chicken Newcastle disease live vaccine.
3. method as claimed in claim 2 is characterized in that digestion process specifically may further comprise the steps in the described step (1):
(1) examine under a microscope the former generation CEF that grows up to monolayer, treating 80%, be paved with just can be through row digestion;
(2) discard culture fluid in the cell bottle, add 0.01mol/L PBS in the ratio of cell bottle capacity 10%, the parallel gently culture bottle that rocks, the culture fluid that flush away is residual, sucking-off cleanout fluid is lentamente abandoned it again, repeats this operation 3 times;
(3) the trypsinization liquid of adding 0.25% in the cell bottle that cleaned, addition is 1% of a cell bottle capacity; Put into 37 ℃ of water-baths after adding immediately and digest 0.5～2min; After treating that cell comes off fully, add the hyclone of 1% volume of cell bottle capacity immediately to it, mixing is to end the Digestion of pancreatin;
(4) at the DMEM culture fluid that contains 10% hyclone that in culture bottle, adds cell bottle capacity 10% volume, mixing is poured in the centrifuge tube of sterilization, and the centrifugal 15min of 2000r/min abandons supernatant;
(5) repeating step is (4) 3 times.
4. method as claimed in claim 2; It is characterized in that frozen in the described step (2) is meant suspends the former generation CEF after step (2) digestion process with the DMEM culture fluid that contains 10%DMSO again; Be sub-packed in the lyophilizing pipe, the 1ml/ pipe, cell density will be not less than 1～2 * 10
6/ ml places 20min for 4 ℃ ,-80 ℃ spend the night after, move in the liquid nitrogen container and preserve.
5. method as claimed in claim 2; From liquid nitrogen container, take out when it is characterized in that recovery in the described step (3) and put into 37 ℃ of water-baths rapidly behind the lyophilizing pipe of former generation CEF and melt; Melting the back takes out from water-bath on the placement ice chest; Add then in the DMEM culture fluid and cultivate, treat cell adherent can the use fully.
6. method as claimed in claim 2 is characterized in that the cultivation of going down to posterity of cell specifically may further comprise the steps in the described step (4):
(1) examines under a microscope the CEF that grows up to monolayer, treat that 80% is paved with just and can digests through row;
(2) discard culture fluid in the cell bottle, add 0.01mol/L PBS in 10% ratio of cell bottle capacity, the parallel gently culture bottle that rocks, the culture fluid that flush away is residual, sucking-off cleanout fluid is lentamente abandoned it again, repeats this operation 3 times;
(3) the trypsinization liquid of adding 0.25% in the cell bottle that cleaned, addition is 1% of a cell bottle capacity; Put into 37 ℃ of water-baths after adding immediately and digest 0.5～2min; After treating that cell comes off fully, add the hyclone of 1% volume immediately to it, mixing is to end the Digestion of pancreatin;
(4) at the DMEM culture fluid that contains 10% hyclone that in culture bottle, adds cell bottle capacity 30% volume, mixing, other gets 2 same size culture bottles, adds the cell suspension of cell bottle capacity 10% volume in every culture bottle;
(5) to 37 ℃ of cultivation 48h～72h, treat that cell attachment grows up to monolayer and can use.
7. method as claimed in claim 2 after it is characterized in that in the described step (5) Strain done suitable dilution with cell maintenance medium, through step (4) CEF after cultivating that goes down to posterity, transfers to 7.2 with pH by 0.1%～1% inoculation of Cell sap volume.
8. like claim 1,2 or 7 described methods, it is characterized in that described Strain is a low virulent strain for newcastle disease I.
9. method as claimed in claim 8 is characterized in that described Strain is newcastle disease virus mesogenic strains Mukteswar strain.
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| CN102766603A (en) * | 2012-04-26 | 2012-11-07 | 秦卓明 | Chicken Newcastle disease virus and its separation method |
| CN107058245A (en) * | 2017-03-22 | 2017-08-18 | 广西医科大学 | It is a kind of to strains of HIV 1 of RTI resistance and its preparation method and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107058245A (en) * | 2017-03-22 | 2017-08-18 | 广西医科大学 | It is a kind of to strains of HIV 1 of RTI resistance and its preparation method and application |
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| CN102380093B (en) | 2013-10-09 |
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