CN109908337A - The preparation method and products thereof of chicken pox live vaccine - Google Patents
The preparation method and products thereof of chicken pox live vaccine Download PDFInfo
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Abstract
The invention discloses preparation methods of chicken pox live vaccine and products thereof.Chicken embryo fibroblasts cell is cultivated obtained adaptation serum free medium by domestication, in the passage chicken embryo fibroblasts of single-cell suspension growth conditions by the present invention；Invention further provides a kind of methods for preparing chicken pox live vaccine, it is characterised by comprising: the adaptation serum free medium is inoculated into progress cell suspension Multiplying culture in bioreactor in the passage chicken embryo fibroblasts of suspension growth by (1)；(2) harvest virus liquid after carrying out virus multiplication culture to the cell inoculation bird pox virus after suspension Multiplying culture, (3) mix virus liquid and freeze drying protectant, freeze-drying to get.Preparation method of the present invention significantly reduces pollution risk and production cost, effectively shortens the production time of chicken pox live vaccine, is convenient for expanding the scale of production；Chicken pox live vaccine safety prepared by the present invention is good, and immune protection effectiveness is high.
Description
Technical Field
The invention relates to a preparation method of a chicken pox live vaccine and a product thereof, in particular to a method for preparing the chicken pox live vaccine by adopting a serum-free suspension culture technology and an obtained chicken pox live vaccine product, belonging to the field of preparation of the chicken pox live vaccine.
Background
Fowl pox is an acute, contact infectious disease caused by Fowlpox virus (Fowlpox virus), which mainly infects chickens, turkeys and various wild rare birds. Clinically, it is mainly expressed in 3 forms. One is skin type, characterized by the appearance of pockmarks in hairless and hairless areas of the skin, which is clinically mild and has low mortality; the other two are mucosal types, commonly known as diphtheria and mixed types, which often cause acne on the mucosa of the respiratory and digestive organs and cause high mortality due to dyspnea. The mortality rate of the chicken flock infected with the disease is not high, but the growth and development of infected sick poultry are retarded, the egg yield is reduced, the hatchability of breeding poultry is reduced after the disease occurs, and once other infectious diseases and parasitic diseases are generated, the mass death of infected poultry is easy to cause, and particularly, chicks are more serious. After outbreak infection, the disease not only brings huge economic loss, but also threatens some endangered birds. The chicken pox outbreaks are prevented by the use of a pigeon pox virus attenuated vaccine and a quail pox attenuated vaccine in each large commercial farm.
DF-1 cells originated from chicken embryo fibroblasts and have now become a mature passaged cell line. DF-1 cells cultured in vitro have strong proliferation capacity, and the density of the cells can exceed CEF4 times. DF-1 cells are reverse transcriptase negative, non-tumorigenic cells. The DF-1 cell line is a global commercial cell line authorized by the united states Food and Drug Administration (FDA), and is currently widely used in the propagation of avian viruses, the expression of recombinant proteins, the study of oncoviruses, and the production of animal and human vaccines.
The traditional DF-1 culture mostly adopts a serum adherence mode, the culture mode has complex process and large floor area, needs a large amount of manual operation and needs to add serum in the culture process, increases the possibility of being polluted by exogenous viruses, mycoplasma and the like, and the serum difference between different batches can also lead to the difficulty in controlling the product quality and simultaneously increases the difficulty of downstream separation and purification. Compared with the serum adherent culture technology, the serum-free suspension culture technology does not need expensive microcarrier, saves the operation of digesting and harvesting cells, reduces the pollution risk and the production cost, and shortens the production time. Because the serum-free suspension culture technology does not need an attached matrix, the equipment space can be saved, the equipment utilization rate is improved, and the production scale is convenient to expand. Therefore, the serum-free suspension culture technology is a necessary trend for the industrial production of vaccines by cells.
Therefore, the DF-1 cells are subjected to full-suspension domestication culture to obtain cells which are adaptive to a serum-free culture medium and in a suspension growth state, and the cells are applied to the preparation of the chicken pox live vaccine, so that the cells have important significance for reducing the production cost and pollution risk of the chicken pox live vaccine, carrying out large-scale production and the like.
Disclosure of Invention
One of the purposes of the invention is to provide a passage chicken embryo fibroblast which is adapted to a serum-free culture medium and is in a single-cell suspension growth state;
the invention also aims to apply the domesticated subculture chicken embryo fibroblast (DF-1 cell) which is adapted to the serum-free culture medium and is in a suspension growth state to prepare the fowl pox live vaccine, and the preparation method adopts the suspension culture technology to reduce the production cost and the pollution risk of the fowl pox live vaccine and can carry out large-scale industrial production.
The above object of the present invention is achieved by the following technical solutions:
the invention obtains a strain of passage chicken embryo fibroblast which is adaptive to a serum-free culture medium and in a suspension growth state by carrying out a low-serum domestication process and a serum-free culture medium suspension domestication process on passage chicken embryo fibroblasts (DF-1 cells) in an adherent culture stage, wherein the cells are named as DF-1-XF.
The invention firstly adopts a method of gradually reducing serum to acclimate DF-1 cells to adapt to low concentrationAnd (3) culturing the blood serum: the invention changes the DF-1 cell which stably grows in the MEM culture solution containing 10 percent newborn calf serum into the MEM culture solution containing 5 percent newborn calf serum when the cell grows to the middle logarithmic growth phase, when the cell grows to 80 to 90 percent confluence, the cell is digested by trypsin with the cell density of 2.0 multiplied by 105cells/ml were passaged in MEM medium containing 5% newborn calf serum; after several generations, the survival rate of DF-1 cells in MEM culture solution containing 5 percent newborn calf serum is maintained to be more than 90 percent, and a faster growth rate is kept for further reducing serum acclimation culture; DF-1 cells were gradually adapted to MEM culture conditions containing 1% newborn bovine serum in the same manner. With the reduction of the use concentration of serum, the morphology of the DF-1 cell adherent growth is gradually changed, and finally, the cells show a single-cell suspension growth state after serum-free domestication and adaptation. DF-1 cells were reduced from 10% to 5% serum concentration, and the cells showed no macroscopic morphological differences and showed no maladaptation. When the serum concentration is reduced to 1%, the growth speed of the cells is reduced, after passage, the cells adapt to the nutritional condition with the serum concentration of 1%, the growth speed is recovered, but the cell morphology tends to become round, and only individual cells show a state of slightly suspending growth. The DF-1 cells show the form of cell suspension growth by passive suspension culture adaptation, but the cell agglomeration phenomenon is serious, and the individual cell mass is larger. In the late stage of suspension culture and domestication, the suspension DF-1 cells are in a better single-cell suspension growth state, the cells are in a spherical shape, the agglomeration phenomenon is less, the cell sizes are basically consistent, the growth speed is normal, and the result shows that the DF-1 cells are adapted to a serum-free culture medium and are in a single-cell suspension growth state; DF-1 cells which are adapted to a serum-free culture medium and in a single-cell suspension growth state and are obtained through a low-serum domestication process and a serum-free culture medium suspension domestication process at an adherent culture stage are named as DF-1-XF cells.
The invention submits DF-1-XF cells which are adapted to serum-free culture medium and in single cell suspension growth state obtained by domestication culture to a patent approved organization for preservation, and the preservation numbers are as follows: CGMCC No.16295, the classification name is: adapting to passage chicken embryo fibroblasts grown in full suspension; the preservation period is as follows: year 2018, month 9, day 6; the preservation unit is: china general microbiological culture Collection center; and (4) storage address: xilu No.1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences.
The invention further discloses a method for preparing the fowl pox live vaccine by applying the DF-1-XF cell which is adapted to the serum-free culture medium and is in the single-cell suspension growth state, which comprises the following steps:
(1) inoculating DF-1-XF cells into a bioreactor or a cell culture bottle for cell suspension proliferation culture; (2) inoculating chicken pox to the DF-1-XF cells after suspension multiplication culture, performing virus multiplication culture, and then harvesting virus liquid, (3) uniformly mixing the harvested virus liquid with a freeze-drying protective agent, and performing freeze drying to obtain the chicken pox live vaccine.
The invention uses DF-1-XF cells with initial density of 0.3X 106cells/ml、 0.5×106cells/ml and 0.75X 106cells/ml are inoculated into a cell culture flask for suspension culture, and sampling and counting are carried out every 24 hours, so that the result shows that the initial inoculation density of the cells is 0.75 multiplied by 106cells/ml, and the cell density can reach 3.0 multiplied by 10 after 48 hours of culture6cells/ml is more than, and the method is suitable for large-scale production requirements; thus, step (1) corresponds to 0.75X 10 DF-1-XF cells6cells/ml is the initial inoculation density of cells and is inoculated into a bioreactor or a cell culture bottle for cell suspension proliferation culture; in addition, the cell suspension proliferation culture conditions described in step (1) further comprise: the conditions of pH value, dissolved oxygen, temperature and stirring speed, etc. can be all cultured by adopting the parameters of the conventional cell suspension culture, and as reference, the pH value can be controlled to be 7.2, the dissolved oxygen value can be controlled to be 50 percent (DO), the temperature can be controlled to be 37 ℃, and the stirring speed can be controlled to be 50-500 r/min.
The invention has the serum concentration of 1 percent and the initial inoculation density of cells of 0.75 multiplied by 106Inoculating DF-1-XF cells to 7L of organisms at four stirring speeds of 60r/min, 80r/min, 100r/min and 120r/min under the conditions of cell/ml, pH 7.2, Dissolved Oxygen (DO) 50% and temperature 37 DEG CThe cell suspension culture is carried out in the reactor, and the cell number is counted by sampling every 24 hours during the culture process. As a result, it was found that when the stirring rate was 100r/min, the cells proliferated most rapidly and the cell density was 3.2X 10 at 48 hours6cells/ml; therefore, when the cell suspension growth culture is performed in step (1), the rotation speed of the stirrer used is preferably 100 r/min.
DF-1-XF cells were plated at 0.75X 106Each/ml of the initial density was inoculated in a bioreactor, and fowlpox virus was inoculated at 2 v/v% for 36 hours, 48 hours and 60 hours of cell culture, respectively, and samples were taken every 24 hours during the culture to determine the virus content, thereby determining the optimum virus dose. Other culture conditions are as follows: the pH was controlled to 7.2, Dissolved Oxygen (DO) 50%, temperature 37 ℃ and stirring speed set at 100 r/min.
The invention further discovers that the inoculation time of inoculating the fowl pox virus to the DF-1-XF cells after suspension propagation culture has larger effect on the propagation culture of the virus:
the present invention uses DF-1-XF cell in the amount of 0.75X 106Initial density of one/ml was inoculated in a bioreactor at 2X 10 per ml for 24 hours, 36 hours, 48 hours and 60 hours of cell culture, respectively3The chicken pox virus is inoculated to the inoculation dose of PFU, and the virus content is measured by sampling 48 hours after inoculation. The results show that the virus titer can reach 10.6 multiplied by 10 when the fowlpox virus is inoculated after the cell culture is carried out for 36 hours6PFU/ml. Therefore, in step (2), DF-1-XF cells are preferably cultured in a suspension culture for 36 hours and inoculated with fowlpox virus.
The invention further discovers that the inoculation amount of the virus for inoculating the fowl pox virus to the DF-1-XF cells after suspension multiplication culture and the collection time of the harvested virus liquid have very obvious influence on the multiplication culture effect of the fowl pox virus:
the present invention uses DF-1-XF cell in the amount of 0.75X 106The cells are inoculated in a 7L bioreactor at a density of one cell/ml, the fowlpox virus is inoculated according to the volume ratio of 1%, 3%, 6% and 10% respectively after the cells are cultured for 36 hours, and the virus content is measured by sampling 48 hours after the inoculation. Results tableThe virus inoculation dose is 6 percent, and the virus titer reaches 10 after 72 hours of virus inoculation7.40EID500.2 ml; therefore, the optimal inoculation amount of the DF-1-XF cells to the fowlpox virus in step (2) after the floating proliferation culture is 6%.
The present invention uses DF-1-XF cell in the amount of 0.75X 106Inoculating chicken pox virus at a density of each ml in a 7L bioreactor, inoculating the chicken pox virus at a rate of 6 v/v% in a synchronous inoculation mode, sampling every 24 hours in the culture process, and determining the virus content. The result shows that the virus titer can reach 10 after 72 hours after virus inoculation7.50EID500.2 ml; therefore, the optimal time for virus collection in step (2) is 72 hours after virus inoculation.
The lyoprotectant in step (3) can be any conventional lyoprotectant; screening tests show that the optimal freeze-drying protection effect can be achieved by adopting the following freeze-drying protective agents:
the freeze-drying protective agent consists of a liquid A and a liquid B, wherein the liquid A consists of: 5g of gelatin, 5g of cane sugar, 5g of dextrin and 2g of tryptone, and using deionized water to fix the volume to 100 ml; the preparation method comprises the following steps: and (4) carrying out high-pressure sterilization on the prepared solution A to obtain the traditional Chinese medicine.
The composition of the solution B is as follows: vitamin C0.1g, sorbitol 2g, sodium glutamate 1.5g, deionized water to 100 ml; the preparation method comprises the following steps: filtering the prepared solution B with 0.22 μm filter membrane for sterilization, and storing at 4 deg.C.
In order to better achieve the aim of uniformly mixing the protective agent A liquid and the protective agent B liquid according to the volume ratio of 1: 1 to obtain a freeze-drying protective agent; mixing the cell virus solution and the freeze-drying protective agent according to the volume ratio of 1: 1.
The preparation method of the chicken pox live vaccine adopts a serum-free suspension culture technology, and compared with a serum wall-attached culture technology, the preparation method does not need an expensive microcarrier, saves the operation of digesting and harvesting cells, obviously reduces the pollution risk and the production cost, and effectively shortens the production time; the adhesive base is not needed, so that the equipment space can be saved, the equipment utilization rate is improved, and the production scale is convenient to expand. The chicken pox live vaccine prepared by the preparation method has good safety; the experimental results of the immunoprotection efficacy show that the prepared fowl pox live vaccine only has good immunoprotection efficacy for chickens.
Drawings
FIG. 1 cellular morphological changes during DF-1 cell acclimation; a, suspension culture initial stage; and B, suspension culture later stage.
Detailed Description
The present invention is further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become more apparent as the description proceeds. These examples are only illustrative and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.
EXAMPLE 1 acclimatization culture of DF-1 cells adapted to serum-free Medium and in suspension growth
Adopting a method of gradually reducing serum to acclimate DF-1 cells to adapt to the culture of low-concentration serum. When DF-1 cells stably growing in MEM culture medium containing 10% newborn calf serum grow to the middle of logarithmic growth, the medium is replaced by MEM culture medium containing 5% newborn calf serum, when the cells grow to 80% -90% confluency, the cells are digested by trypsin with the cell density of 2.0 × 105cells/ml were passaged in MEM medium containing 5% newborn calf serum. After several generations, the survival rate of DF-1 cells in MEM culture solution containing 5% newborn bovine serum is maintained to be more than 90%, and a faster growth rate is maintained, so that the serum acclimation culture is further reduced. DF-1 cells were cultured in the same mannerGradually adapting to MEM culture condition containing 1% newborn calf serum. DF-1 cells adapting to the culture condition of 1 percent newborn calf serum are subjected to suspension culture acclimatization and adaptation in a cell triangular flask. The cell culture solution is a serum-free culture medium for culturing DF-1 from Shanghai, and the initial inoculation density of the cells is 1.0 multiplied by 106cells/ml, set at 160r/min, placed in 5% CO2Suspension culture is carried out in an incubator.
With the reduction of the serum use concentration, the morphology of DF-1 cell adherent growth is gradually changed, and finally, the cells show a single-cell suspension growth state after serum-free domestication and adaptation. DF-1 cells were reduced from 10% to 5% serum concentration, and the cells showed no macroscopic morphological differences and showed no maladaptation. When the serum concentration is reduced to 1%, the growth rate of the cells is reduced, after passage, the cells adapt to the nutritional condition with the serum concentration of 1%, the growth rate is recovered, but the cell morphology tends to become round, and the individual cells are in a slightly suspended growth state. The DF-1 cells show the form of cell suspension growth by passive suspension culture adaptation, but the cell agglomeration phenomenon is serious, and the individual cell mass is larger. In the late stage of suspension culture and domestication, the suspension DF-1 cells are in a better single-cell suspension growth state, the cells are in a spherical shape, the agglomeration phenomenon is less, the cell sizes are basically consistent, the growth speed is normal, the DF-1 cells are suitable for the growth in a serum-free suspension state, and the cells are named as DF-1-XF cells.
EXAMPLE 2 preparation of a live vaccine for fowlpox
(1) DF-1-XF cells obtained by acclimatization and culture in example 1 were cultured at a ratio of 0.5X 106cells/ml is the initial inoculation density of cells and is inoculated into a 7L bioreactor for cell suspension culture, and the culture time is 36 hours; other suspension culture conditions were as follows: controlling pH to 7.2, Dissolved Oxygen (DO) to 50%, temperature to 37 deg.C, and stirring speed to 100 r/min;
(2) DF-1-XF cells cultured for 36 hours in suspension proliferation were inoculated at an inoculum size of 6% (V/V)Performing virus proliferation culture on the chicken pox quail attenuated strain (F282E 4); virus liquid is harvested after 72 hours of inoculation; other suspension culture conditions were as follows: controlling pH to 7.2, Dissolved Oxygen (DO) to 50%, temperature to 37 deg.C, and stirring speed to 100 r/min; the virus valence is 10 by detection7.50EID50/0.2ml。
(3) Preparing seedlings and subpackaging
(3.1) preparation of lyoprotectant
Solution A: 5g of gelatin, 5g of cane sugar, 5g of dextrin, 2g of tryptone and deionized water, wherein the volume is fixed to 100ml, the mixture is sterilized at 116 ℃ under high pressure for 30min, and the mixture is stored at 15 ℃ for no more than 7 days;
and B, liquid B: vitamin C0.1g, sorbitol 2g, sodium glutamate 1.5g, deionized water to constant volume of 100ml, filtering with 0.22 μm filter membrane for sterilization, and storing at 4 deg.C for no more than 3 days;
(3.2) subpackaging
Uniformly mixing the protective agent A solution and the protective agent B solution according to the volume ratio of 1: 1 to obtain a freeze-drying protective agent, adding the qualified cell virus solution into the freeze-drying protective agent according to the volume ratio of 1: 1, and quantitatively subpackaging;
(3.3) lyophilization
And (4) quickly freezing and drying in vacuum after subpackaging to obtain the fowl pox live vaccine.
EXAMPLE 3 preparation of a live vaccine for fowlpox
(1) DF-1-XF cells obtained by acclimatization and culture in example 1 were cultured at a ratio of 0.3X 106cells/ml is the initial inoculation density of cells and is inoculated into a 7L bioreactor for cell suspension proliferation culture; other suspension culture conditions were as follows: controlling pH to 7.2, Dissolved Oxygen (DO) to 50%, temperature to 37 deg.C, and stirring speed to 60 r/min;
(2) inoculating 3% (V/V) of DF-1-XF cells cultured in suspension for 72 hours with chicken pox quail attenuated virusThe strain (F282E4) was cultured for virus growth; virus liquid is obtained after 96 hours of inoculation; the virus valence is 10 by detection6.05EID50/0.2ml；
(3) Preparing seedlings and subpackaging
(3.1) preparation of lyoprotectant
Solution A: 5g of gelatin, 5g of cane sugar, 5g of dextrin, 2g of tryptone and deionized water, wherein the volume is fixed to 100ml, the mixture is sterilized at 116 ℃ under high pressure for 30min, and the mixture is stored at 15 ℃ for no more than 7 days;
and B, liquid B: vitamin C0.1g, sorbitol 2g, sodium glutamate 1.5g, deionized water to constant volume of 100ml, filtering with 0.22 μm filter membrane for sterilization, and storing at 4 deg.C for no more than 3 days;
(3.2) subpackaging
Uniformly mixing the protective agent A solution and the protective agent B solution according to the volume ratio of 1: 1 to obtain a freeze-drying protective agent, adding the qualified cell virus solution into the freeze-drying protective agent according to the volume ratio of 1: 1, and quantitatively subpackaging;
(3.3) lyophilization
And (4) quickly freezing and drying in vacuum after subpackaging to obtain the fowl pox live vaccine.
EXAMPLE 4 preparation of a live vaccine for fowlpox
(1) DF-1-XF cells obtained by acclimatization and culture in example 1 were cultured at a ratio of 0.5X 106cells/ml is the initial inoculation density of cells and is inoculated into a 7L bioreactor for cell suspension proliferation culture; other suspension culture conditions were as follows: controlling pH to 7.2, Dissolved Oxygen (DO) to 50%, temperature to 37 deg.C, and stirring speed to 120 r/min;
(2) inoculating a chicken pox quail attenuated strain (F282E4) to DF-1-XF cells cultured for 24 hours in suspension proliferation according to the inoculation dose of 1% (V/V) for virus proliferation culture; harvesting virus liquid 120 hours after inoculation; the virus valence is 10 by detection5.15EID50/0.2ml；
(3) Preparing seedlings and subpackaging
(3.1) preparation of lyoprotectant
Solution A: 5g of gelatin, 5g of cane sugar, 5g of dextrin, 2g of tryptone and deionized water, wherein the volume is fixed to 100ml, the mixture is sterilized at 116 ℃ under high pressure for 30min, and the mixture is stored at 15 ℃ for no more than 7 days;
and B, liquid B: vitamin C0.1g, sorbitol 2g, sodium glutamate 1.5g, deionized water to constant volume of 100ml, filtering with 0.22 μm filter membrane for sterilization, and storing at 4 deg.C for no more than 3 days;
(3.2) subpackaging
Uniformly mixing the protective agent A solution and the protective agent B solution according to the volume ratio of 1: 1 to obtain a freeze-drying protective agent, adding the qualified cell virus solution into the freeze-drying protective agent according to the volume ratio of 1: 1, and quantitatively subpackaging;
(3.3) lyophilization
And (4) quickly freezing and drying in vacuum after subpackaging to obtain the fowl pox live vaccine.
EXAMPLE 5 preparation of a live vaccine for fowlpox
(1) DF-1-XF cells obtained by acclimatization and culture in example 1 were cultured at a ratio of 0.5X 106cells/ml is the initial inoculation density of cells and is inoculated into a 7L bioreactor for cell suspension proliferation culture; other suspension culture conditions were as follows: controlling pH to 7.2, Dissolved Oxygen (DO) to 50%, temperature to 37 deg.C, and stirring speed to 120 r/min;
(2) inoculating a chicken pox quail attenuated strain (F282E4) to DF-1-XF cells cultured for 48 hours in suspension proliferation according to the inoculation dose of 10% (V/V) for virus proliferation culture; virus liquid is harvested 48 hours after inoculation; the virus valence is 10 by detection5.89EID50/0.2ml；
(3) Preparing seedlings and subpackaging
(3.1) preparation of lyoprotectant
Solution A: 5g of gelatin, 5g of cane sugar, 5g of dextrin, 2g of tryptone and deionized water, wherein the volume is fixed to 100ml, the mixture is sterilized at 116 ℃ under high pressure for 30min, and the mixture is stored at 15 ℃ for no more than 7 days;
and B, liquid B: vitamin C0.1g, sorbitol 2g, sodium glutamate 1.5g, deionized water to constant volume of 100ml, filtering with 0.22 μm filter membrane for sterilization, and storing at 4 deg.C for no more than 3 days;
(3.2) subpackaging
Uniformly mixing the protective agent A solution and the protective agent B solution according to the volume ratio of 1: 1 to obtain a freeze-drying protective agent, adding the qualified cell virus solution into the freeze-drying protective agent according to the volume ratio of 1: 1, and quantitatively subpackaging;
(3.3) lyophilization
And (4) quickly freezing and drying in vacuum after subpackaging to obtain the fowl pox live vaccine.
Experimental example 1 optimization experiment of preparation Process parameters of live vaccine for fowlpox
1. Test method
1.1 optimization of cell suspension culture conditions
1.1.1 optimization of initial seeding Density of cells
DF-1-XF cells were cultured at 0.3X 10, respectively60.5X 10 pieces/ml6One/ml and 0.75X 106Three initial densities per ml were inoculated in a bioreactor for suspension culture, and the cell count was measured by sampling every 24 hours during the culture. Other suspension culture conditions: the pH is controlled to be 7.2, the Dissolved Oxygen (DO) is 50%, the temperature is 37 ℃, and the stirring speed is 100 r/min.
1.1.2 optimization of the stirring speed
DF-1-XF cells are inoculated in a 7L bioreactor at four stirring rotating speeds of 60, 80, 100 and 120r/min respectively for DF-1-XF cell suspension culture, and the number of the cells is counted by sampling every 24 hours in the culture process. Other suspension culture conditions: serum concentration of 1% and cell concentrationInitial inoculation density of 0.75X 106Piece/ml, pH 7.2, Dissolved Oxygen (DO) 50%, temperature 37 ℃.
1.2 optimization of viral suspension culture parameters
1.2.1 optimization of the inoculation time
DF-1-XF cells were plated at 0.75X 106The initial density of each/ml is inoculated in a bioreactor, the fowlpox virus is inoculated according to 3 v/v% at 0, 12, 24 and 36 hours of cell culture, and a sample is taken 72 hours after inoculation to determine the virus content so as to determine the optimal virus inoculation time. Other culture conditions were as follows: the pH was controlled to 7.2, Dissolved Oxygen (DO) 50%, temperature 37 ℃ and stirring speed set to 100 r/min.
1.2.2 optimization of the inoculation dose
DF-1-XF cells were plated at 0.75X 106The vaccine was inoculated in a 7L bioreactor at a density of one dose/ml, and the fowlpox virus was inoculated in an amount of 1 v/v%, 3 v/v%, 6 v/v% and 10 v/v%, respectively, in a simultaneous inoculation manner, and the virus content was measured by sampling 72 hours after the inoculation to determine the optimum inoculation amount. Other culture conditions were as follows: the pH was controlled to 7.2, Dissolved Oxygen (DO) 50%, temperature 37 ℃ and stirring speed set to 100 r/min.
1.2.3 optimization of detoxification time
DF-1-XF cells were plated at 0.75X 106Inoculating chicken pox virus in a 7L bioreactor at a density of one vaccine/ml, inoculating the chicken pox virus at a rate of 6 v/v% in a synchronous inoculation mode, sampling every 24 hours in the culture process, and measuring the virus content to determine the optimal virus receiving time. Other culture conditions were as follows: the pH was controlled to 7.2, Dissolved Oxygen (DO) 50%, temperature 37 ℃ and stirring speed set to 100 r/min.
2. Test results
2.1 initial inoculation Density optimization results for cells
DF-1-XF cells were each administered at an initial density of 0.3X 106cells/ml、0.5×106cells/ml and 0.75X 106cells/ml inoculated in cell culture flaskThe suspension culture is carried out, and sampling and counting are carried out every 24 hours, and the result shows that the initial inoculation density of the cells is 0.75 multiplied by 106cells/ml, the cell density can reach 3.0 multiplied by 10 after 48 hours of culture6The cells/ml is more than that, and the method is suitable for large-scale production requirements, and the results are shown in Table 1. Therefore, 0.75X 10 is selected6cells/ml is the initial seeding density of the cells.
TABLE 1 relationship between seeding density of different cells and growth rate of suspension cells
2.2 optimization of the stirring speed
DF-1-XF cells are inoculated in a 7L bioreactor for suspension culture at four stirring rotating speeds of 60, 80, 100 and 120r/min respectively, and the number of the cells is counted by sampling every 24 hours in the culture process. The results show that when the stirring speed is 100r/min, the cell proliferation is fastest, and the cell density is 3.2 multiplied by 10 in 48 hours6cells/ml, and the results are shown in Table 2. Therefore, the stirring speed is 100 r/min.
TABLE 2 relationship between different stirring speeds and growth speed of suspension cells
2.3 optimization of the inoculation time
DF-1-XF cells were plated at 0.75X 106Inoculating the strain at initial density of one/ml in a bioreactor, inoculating the strain with vaccinia virus at 3 v/v% for 0 hr, 12 hr, 24 hr and 36 hr, and sampling 72 hr after inoculation to determine virus content. The results show that the virus titer can reach 10.6 multiplied by 10 when the cells are cultured for 0h, namely the fowl pox virus is inoculated by adopting the synchronous inoculation method6PFU/ml, results are shown in Table 3. Therefore, the optimal inoculation time is selected as 36h for cell culture.
TABLE 3 Effect of different inoculation times on Virus content
2.4 optimization of the inoculation dose
DF-1-XF cells were plated at 0.75X 106Inoculating the chicken pox virus into a 7L bioreactor at a density of one dose/ml, inoculating the chicken pox virus at the inoculation amount of 1 v/v%, 3 v/v%, 6 v/v% and 10 v/v% respectively in a synchronous inoculation mode, and sampling 72 hours after inoculation to determine the virus content. The result shows that the dose of the virus inoculation is 6 v/v%, and the virus valence can reach 10 in 72 hours after virus inoculation7.40EID500.2ml, the results are shown in Table 4. Therefore, the optimal dose of the virus is selected to be 6%.
TABLE 4 Effect of different inoculation times on Virus content
2.5 optimization of detoxification time
DF-1-XF cells were plated at 0.75X 106Inoculating chicken pox virus at a density of each ml in a 7L bioreactor, inoculating the chicken pox virus at a rate of 6 v/v% in a synchronous inoculation mode, sampling every 24 hours in the culture process, and determining the virus content. The result shows that the virus titer can reach 10 after 72 hours after virus inoculation7.50EID500.2ml, the results are shown in Table 5. Therefore, the optimal time for receiving the virus is 72 hours after virus receiving.
TABLE 5 Effect of different inoculation times on Virus content
Test example 2 safety test of a fowl pox live vaccine
10 susceptible chicks of 1-2 weeks old are injected with 0.2ml of live vaccine of 10 times of dose per muscle, and the 10 chickens are all healthy and alive after observation for 7-10 days. The safety test result proves that the chicken pox live vaccine prepared by the method has good safety.
Test example 3 identification test of fowl pox live vaccine
The chicken pox live vaccine prepared in the embodiment of the invention is diluted by sterilized normal saline to contain 100EID500.1ml, adding the same amount of anti-chicken pox virus specific positive serum, neutralizing at room temperature for 1h, inoculating chicken embryo fibroblasts, and observing for 120h to ensure that no lesion appears.
Test example 4 efficacy test of fowl pox live vaccine
Diluting the chicken pox live vaccine prepared in the embodiment 1 of the invention by using normal saline, inoculating 10 chicken embryos of 11-12 days old to chorioallantoic membranes, wherein each embryo is 0.2ml, and after inoculation for 96-120h, all chicken embryo choriocapillaris is subjected to edema thickening or pockmarks appear; the test result proves that the chicken pox live vaccine prepared by the preparation method has qualified efficacy.
Claims (10)
1. A method for preparing a fowl pox live vaccine, comprising: (1) domesticating and culturing the chicken embryo fibroblasts to obtain passage chicken embryo fibroblasts which are adapted to a serum-free culture medium and in a single-cell suspension growth state; (2) inoculating the subculture chicken embryo fibroblasts obtained by the domestication and culture in the step (1) into a bioreactor or a cell culture bottle for cell suspension proliferation culture; (3) inoculating the chicken pox virus to the suspension multiplication cultured passage chicken embryo fibroblast for virus multiplication culture, and then harvesting virus liquid; (4) and uniformly mixing the harvested virus solution with a freeze-drying protective agent, and freeze-drying to obtain the fowl pox live vaccine.
2. The method according to claim 1, wherein the microorganism of the serum-free medium adapted passaged chicken embryo fibroblasts in the single cell suspension growth state in step (1) has the following preservation number: CGMCC No. 16295.
3. The method according to claim 1, wherein the passaged chicken embryo fibroblasts obtained by acclimatization culture in the step (2) are cultured at an initial density of 0.3X 106cells/ml、0.5×106cells/ml or 0.75X 106Inoculating cells/ml into a bioreactor or a cell culture bottle for suspension culture; preferably, in step (2), the domesticated and cultured passaged chicken embryo fibroblasts are cultured according to an initial density of 0.75X 106The inoculation density of cells/ml is inoculated into a bioreactor or a cell culture flask for suspension culture.
4. The method according to claim 1, wherein in step (2), the serum-free adaptive culture medium obtained through domestication culture and the passaged chicken embryo fibroblasts in the single-cell suspension growth state are inoculated into a bioreactor or a cell culture bottle for cell suspension proliferation culture under the stirring condition; wherein the stirring speed is 50-500 r/min; preferably 60-120 r/min; most preferably 100 r/min.
5. The method of claim 1, wherein the conditions of said cell suspension propagation culture of step (2) further comprise: the pH value is 7.2, the dissolved oxygen value is 50%, and the culture temperature is 37 ℃.
6. The method according to claim 1, wherein the cells cultured for 24 to 60 hours in suspension proliferation in step (3) are inoculated with fowlpox virus and subjected to virus proliferation culture; preferably, in the step (3), the cells cultured for suspension propagation for 36 hours are inoculated with the fowlpox virus and subjected to virus propagation culture.
7. The method according to claim 1, wherein in the step (3), the cells are inoculated with the fowlpox virus in an inoculation amount of 1 to 10% by volume; preferably, the cells are inoculated with fowlpox virus at a virus inoculation amount of 6%.
8. The method of claim 1, wherein the virus propagation culture conditions in step (3) further comprise: the pH value is 7.2, the dissolved oxygen is 50 percent, the temperature is 37 ℃, and the stirring speed is 100 r/min.
9. The method according to claim 1, wherein the viral solution is harvested by inoculating the fowlpox virus in the step (3) and culturing for 24 to 120 hours; preferably, the virus solution is harvested by inoculating the chicken pox virus in the step (3) and culturing for 72 hours.
10. The method according to claim 1, wherein the lyoprotectant in step (4) consists of solution A and solution B; wherein,
the composition of the solution A is as follows: 5g of gelatin, 5g of cane sugar, 5g of dextrin and 2g of tryptone; deionized water is added to the volume of 100 ml;
the composition of the solution B is as follows: 0.1g of vitamin C, 2g of sorbitol and 1.5g of sodium glutamate; deionized water was added to 100 ml.
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| CN115627259A (en) * | 2022-11-30 | 2023-01-20 | 北京赛尔富森生物科技有限公司 | Adaptation method of virus in chick embryo fibroblast |
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