CN118356484A - A MD vaccine and preparation method thereof - Google Patents

Abstract

The present application belongs to the field of biopharmaceutical technology, and discloses a method for preparing an MD vaccine. The method first selects chicken embryos, disinfects, takes chicken embryo cells and cultures them to obtain chicken embryo fibroblasts; then inoculates the virus strain into the chicken embryo fibroblasts for culture and harvests the infected cells, and then prepares the harvested infected cells for seedling, packages them, and freezes them, wherein a programmed cooling device is used for freezing, and the program of the programmed cooling device is as follows: pre-freeze the box to 4°C, put an ampoule in for pre-cooling for 30 minutes, and reduce the seedling temperature to -40°C at a rate of 0.8-1.2°C/min; then reduce the seedling temperature to -100°C at a rate of 8-12°C/min, and transfer them into liquid nitrogen for storage. The present application obtains a method for preparing an MD vaccine through the above design, and the method has the advantages of fast preparation speed, large vaccine quantity, etc., and is more suitable for large-scale preparation of MD vaccines. In addition, the present application also discloses an MD vaccine.

Description

A MD vaccine and preparation method thereof

Technical Field

The present invention relates to the technical field of biopharmaceuticals, and in particular to an MD vaccine and a preparation method thereof.

Background technique

Marek's disease is a highly contagious lymphoproliferative disease of chickens caused by cell-associated herpesvirus (MDV). Chickens infected with Marek's virus are very harmful and cause serious damage to the poultry industry. With the widespread use of vaccines and the continuous selection of chicken strains, the virulence of MDV has been continuously enhanced under the dual effects of natural selection and immune pressure, repeatedly breaking through the protection provided by vaccines.

The MDV isolated in the 1970s was mainly virulent (vMDV). From the late 1970s to the 1980s, MDV underwent a major change, with its virulence enhanced to become super virulent (vvMDV). In the 1990s, the even more virulent super virulent (vv+MDV) appeared. However, the HVT freeze-dried vaccine has poor immune effects against super virulent and super virulent MD.

At present, the commonly used vaccine strains that can resist super-virulent viruses are mainly strains 814 and CVI988. Among them, strain 814 is a non-tumorigenic Marek's virus isolated by Mr. Tong Kunzhou of Harbin Veterinary Biological Pharmaceutical Research Institute from a healthy chicken flock that had not been immunized with Marek's vaccine. It belongs to the same serum type I strain as CVI988. However, strain 814 is a natural weak strain and is non-tumorigenic, so there is no problem of virulence reversion. The chicken Marek's disease live vaccine (CVTR strain) independently developed by Zhaoqing Dahuanong Biopharmaceutical Co., Ltd. obtained a Class III new veterinary drug certificate in 2021. Its vaccine strain CVTR strain is also a domestic natural weak strain like strain 814, non-tumorigenic, with good safety, fast immune formation period, and is not affected by maternal antibodies. The vaccine can protect against super-virulent Marek's virus with a protection rate of more than 90%.

Ma Li, Zhu Xiuzhi and Chen Ling from Beijing Veterinary Biological Pharmaceutical Factory presented an article entitled "Purification of Chicken Marek's Disease Virus "814" Strain and Production and Application of Live Vaccine" at the 9th Academic Symposium of the Biological Products Branch of the Chinese Society of Animal Husbandry and Veterinary Medicine. The article recorded the optimization of the 814 strain and the production and use of the 814 live vaccine.

In the process of optimizing the 814 strain, the specific method of this article is to restore the 814 vaccine virus to SPF chickens for several generations to restore the immunogenicity of the original virus and purify the vertically transmitted virus. The virus of this generation is the optimized virus. This article further tests the immune efficacy of the optimized virus and finds that the immune efficacy of the optimized virus reaches 84%, which is higher than the 78% of the imported CV1988.

It can be seen that the above text provides corresponding records on the optimization of the 814 strain, and the optimized strain has good immune efficacy.

Chinese patent application 201410233335.8 discloses a method for producing a live vaccine for Marek's disease in chickens using a cell line, the method comprising the following steps:

A. Select a cell line as a vaccine cell; the cell line is a chicken embryo DF-1 cell line;

B. Subculturing and cultivation of cells for seedling production;

The above cell lines are digested and subcultured with EDTA-trypsin cell dispersion solution, and then continued to be cultured with cell growth medium. When a monolayer is formed, it is used for further subculture or virus inoculation; the culture temperature is 36.5-37.5°C;

C. Reproduction of cytotoxic species;

Dilute the chicken Marek's disease live vaccine virus seed to a certain concentration with a seed virus diluent, inoculate it on a well-growing cell line or chicken embryo fibroblast monolayer for vaccine production, adsorb it at 36.5-37.5°C for 1 hour, then add a maintenance solution or a secondary cell suspension, continue culturing, and when 70% or more of the monolayer cells show typical Marek's disease cell lesions, digest and disperse the cells with an EDTA-trypsin cell dispersion solution, and the harvested cell suspension is used as the virus seed for vaccine production;

D. The reproduction of seedling poison;

Dilute the virus seed for making chicken Marek's disease live vaccine with maintenance solution, inoculate on the cell line monolayer that has formed a monolayer and continue to culture, and when 70% or more of the monolayer cells show typical Marek's disease cytopathic changes, harvest the infected cells;

E. Assemble seedlings, pack, freeze-dry or freeze in liquid nitrogen;

The harvested infected cells are added with freeze-drying protective agent and antibiotics, quantitatively divided into packages and then freeze-vacuum dried into finished products; or cryopreservation protective solution and antibiotics are added, mixed and then quantitatively divided into packages into finished products and frozen in liquid nitrogen.

The scheme obtains a method for producing a live vaccine of Marek's disease in chickens by using a cell line through the above steps, which has the advantages of stable production process, easy operation, high virus content, small batch-to-batch difference and easy quality control. Further observation of the instructions of the scheme shows that the formula of the seed virus dilution solution or maintenance solution in the scheme is: in a DMEM/F12 culture medium with a volume ratio of 1-5% fetal bovine serum, or in a mixed solution of equal amounts of 2 times 199 solution and 2 times hydrolyzed milk protein solution containing a volume ratio of 1-5% newborn bovine serum, add an antibiotic with a final concentration of 100-400 units/ml, and adjust the pH to 7.0-7.4;

On the other hand, the formula of the cryopreservation protection solution is: 15% by volume of newborn calf serum, 10% by volume of dimethyl sulfoxide 199 solution; the lyophilization protection agent is SPGA; the final concentration of the added antibiotic is 100-400 units/ml;

At the same time, observing paragraph 36 of the program instructions, it can be seen that the specific freezing process of the virus in the program is: "Immediately place the packaged vaccine in a liquid nitrogen program cooling system. After reaching -70°C, move it into liquid nitrogen for storage. After 1 week, take samples for finished product inspection." Moreover, the program did not give too much consideration and design to the impact of the freezing procedure on the potency of the vaccine.

The problem that this program needs to solve: How to improve the vaccine potency of MD vaccine by optimizing the freezing procedure during the production process.

Summary of the invention

The purpose of the present invention is to provide a method for preparing an MD vaccine. By optimizing the freezing procedure, the present application improves the potency of the vaccine to a certain extent, thereby improving the protective effect of the vaccine.

Another object of the present application is to provide a MD vaccine.

To achieve the above objectives, the present application discloses a method for preparing an MD vaccine, comprising the following steps:

Step 1: Select chicken embryos, sterilize, obtain chicken embryo cells and culture them to obtain chicken embryo fibroblasts;

Step 2: inoculating the virus strain into the chicken embryo fibroblasts prepared in step 1 and harvesting the infected cells;

Step 3: The infected cells harvested in step 2 are prepared, packaged, and frozen;

In step 3, a programmed cooling device is used for freezing, and the program of the programmed cooling device is as follows: pre-freeze the box to 4°C, put the ampoule bottle in for pre-cooling for 30 minutes, and reduce the seedling temperature to -40°C at a rate of 0.8-1.2°C/min; then reduce the seedling temperature to -100°C at a rate of 8-12°C/min, and transfer to liquid nitrogen for storage;

In step 2, chicken embryo fibroblasts inoculated with the virus strain are cultured using a second cell culture fluid, wherein the second cell culture fluid comprises an M-199 culture fluid and a buffer solution, and the mass ratio of the M-199 culture fluid to the buffer solution is 100:3-4;

The buffer contains 1.8-2.2% Hepes by mass and 1.3-1.5% NaHCO ₃ by mass. During the culture period, the cells are allowed to be cultured in a closed manner during the period of toxin production, without the need to use an additional CO ₂ incubator or CO ₂ for gas exchange, ensuring that the pH value of the culture solution is in the range of 7.0-7.4.

This application improves the potency of the vaccine to a certain extent by optimizing the freezing procedure, thereby improving the protective effect of the vaccine;

The present invention uses a sodium bicarbonate buffer solution in the second culture medium. The use of the buffer solution enables the cell factory culture method to be implemented, which can effectively expand the production capacity and culture efficiency. The buffer solution uses Hepes and NaHCO _3. When CO ₂ generated during the culture process can be absorbed by the buffer solution, there is no need to perform CO ₂ gas exchange during the culture process, making large-scale culture possible.

Preferably, in step 1, the chicken embryo is a 9-11 day old chicken embryo.

Preferably, in step 1, the chicken embryo cells are cultured using a first cell culture medium, wherein the first cell culture medium is an M-199 culture medium, and the M-199 culture medium is further supplemented with 0.5 to 3 mmol/L of amino acids and 1.0 to 1.2 μg/L of FGF;

The amino acid is selected from at least one of alanine, aspartic acid, glutamic acid, glycine, proline, serine, threonine, tyrosine, arginine, cystine, histidine, isoleucine, leucine, lysine, tryptophan and valine.

The buffer contains 2% Hepes by mass, 1.4% NaHCO ₃ by mass, and 0.02-0.5% basic amino acid by mass; the basic amino acid is selected from at least one of arginine, lysine, and histidine.

Preferably, in step 1, the chicken embryo cell culture method is a cell factory culture method, and the cell density in the cell suspension during the cell factory culture method is 2 million cells per milliliter, the number of cell factory layers in the cell factory culture method is 10 layers, and the volume of the cell suspension in each layer of the cell factory is 200 ml.

Preferably, in step 1, the specific method of selecting chicken embryos, disinfecting, and obtaining chicken embryo cells is: selecting well-developed SPF chicken embryos of 9 to 11 days old, disinfecting the surface of the chicken embryos with 75% alcohol and 4% iodine tincture, aseptically removing the embryos, and then using the chicken embryo shearing method or the magnetic bead digestion method to prepare a cell suspension.

Preferably, when the cell suspension is prepared by magnetic bead digestion in step 1, the amount of trypsin used during the magnetic bead digestion is 6 ml per chicken embryo, and the concentration of trypsin is 0.05-0.5%.

Preferably, during the freezing process of step 3, the freezing solution is a freezing solution containing dimethyl sulfoxide, and the concentration of dimethyl sulfoxide in the freezing solution is 5-15%.

In addition, the present application also discloses an MD vaccine, which is prepared by the above-mentioned MD vaccine preparation method.

Preferably, the dosage form of the MD vaccine is an injection form.

The beneficial effects of the present application are as follows: the present application improves the potency of the vaccine to a certain extent by optimizing the freezing procedure, thereby improving the protective effect of the vaccine, and the preparation method of the MD vaccine disclosed in the present application has the advantages of fast preparation speed and large vaccine quantity, and is more suitable for large-scale preparation of MD vaccines.

Detailed ways

The present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. In the description of the present invention, it should be noted that, if no specific conditions are specified in the embodiments, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

Before presenting the examples, the necessary information of the experimental consumables such as the seeds, reagents, instruments, etc. used in the examples is described first. The specific information is shown in Table 1:

Table 1

It should be noted that the seed virus used in the experiment was the P22 generation CVTR strain with a virus content of 1.57×10 ⁷ PFU/mL, which was identified, stored and supplied by Guangdong Wenshi Dahuanong Biotechnology Co., Ltd.

Example 1

1.1 Preparation of chicken embryo fibroblasts

Choose 2600 well-developed SPF chicken embryos of 11 days, sterilize the chicken embryo surface with 75% alcohol, 4% iodine tincture, aseptically take out embryoid. After removing eyes, the chicken embryo taken is put into the large beaker of 1000ml, clean three times with Hank's liquid, then transfer to the magnetic force of band connecting pipe and disappear in the bottle (2500mL), add pancreatin by the amount of every chicken embryo 6ml, and the concentration of pancreatin is 0.125%, use magnetic agitator room temperature digestion chicken embryo, each magnetic force digestion bottle cumulative volume is no more than 1000ml, leave standstill 1min after digestion 5min, wait tissue block precipitation and promptly pour out supernatant to be cell suspension, add 2% serum to stop digestion and ice bath to place, in the magnetic force disappearing bottle, add original volume pancreatin again, so repeatedly 5-6 time, by collected The supernatant was centrifuged at 1500 rpm for 10 min, and after removing the supernatant, it was resuspended with cell growth medium, and the cell suspension was filtered through 4 layers of sterilized gauze. After filtering, the mixture was mixed and 1 ml of the volume was taken for cell counting, and the cells were inoculated into 10 layers of cell factories at a density of 2 million cells per ml. 200 ml of cell suspension was cultured in each factory. The cell factory was cultured for 24 hours to obtain a single layer of dense chicken embryo fibroblasts, wherein the first cell culture medium used in the cell factory culture process was M-199 culture medium, and 2 mmol/L of alanine and 1.0 μg/L of FGF were also added to the M-199 culture medium.

1.2 Virus inoculation and culture of infected cells

Take out the CVTR strain virus from liquid nitrogen, immediately put it into warm water at 37°C, shake it quickly and evenly, and melt it within 60 seconds. Inoculate 20 million PFU in each cell factory. Culture at 37°C, observe cell lesions for 50-60 hours: the cells are rounded, have strong refractive index, and the diseased cells are evenly distributed. When the virus is inoculated into the cell factory, a second cell culture fluid is added to the cell factory, and the second cell culture fluid includes M-199 culture fluid and buffer solution, and the mass ratio of M-199 culture fluid and buffer solution is 100:3, and the buffer solution contains 2% Hepes by mass and 1.4% NaHCO ₃ by mass.

1.3 Harvesting and freezing of infected cells

When the diseased cells in the cell factory accounted for more than 70% of the cell monolayer area, they were harvested, prepared, packaged and frozen. A total of 6,940 bottles of vaccine were harvested;

The cryopreservation was performed by using a programmed cooling instrument, and the specific cryopreservation method was as follows:

Prefreeze the box to 4°C, put in the ampoule for precooling for 30 minutes, and lower the seedling temperature to -40°C at a rate of 1°C/min; then lower the seedling temperature to -100°C at a rate of 10°C/min, and transfer to liquid nitrogen for storage.

Virus content determination: After the vaccine or virus is melted in 37°C warm water, it is diluted with a special supporting diluent, and the suitable temperature is 25±2°C. Take an appropriate dilution, and inoculate 5 plates of chicken embryo fibroblasts that have grown into a good monolayer for each dilution, and inoculate 0.2ml for each plate. At the same time, set up 2 plates of blank controls without inoculation and 5 plates of standard virus samples. The plates are placed in a 37-38°C, 5% _CO2 incubator for 6 days and must not be moved. On the 7th day, the characteristic cell lesions are observed and the average number of plaques of 5 plates with the same dilution is recorded, and then the number of plaques contained in each bottle of vaccine is calculated, and the number of plaques contained in each bottle of vaccine = the average number of plaques of 5 plates with the same dilution × dilution factor × volume of each bottle (ml)/0.2ml.

The PFU error among the 5 plates of the standard virus sample should not exceed ±10%. The PFU of each batch of vaccine should be determined based on the lowest PFU count among the 3 bottles. Each dose should not be less than 2000 PFU.

Example 2

It is basically the same as Example 1, except that the first cell culture medium is M-199 culture medium, and 2 mmol/L of amino acids and 1.0 μg/L of FGF are added to the M-199 culture medium; the amino acids are a mixture of alanine and glycine, and the mass ratio of alanine to glycine is 1:1.

Example 3

The method is basically the same as Example 1, except that the chicken embryo is a 9-day-old chicken embryo.

Example 4

The method is basically the same as Example 1, except that the chicken embryo is a 10-day-old chicken embryo.

Example 5

The method is basically the same as Example 1, except that the chicken embryo is a 9-day-old chicken embryo, and the first cell culture medium is M-199 culture medium, and the M-199 culture medium is supplemented with 2 mmol/L of amino acids and 1.0 μg/L of FGF; the amino acid is a mixture of alanine and glycine, and the mass ratio of alanine to glycine is 1:1.

Example 6

The method is basically the same as Example 1, except that the chicken embryo is a 10-day-old chicken embryo, and the first cell culture medium is M-199 culture medium, and the M-199 culture medium is supplemented with 2 mmol/L of amino acids and 1.0 μg/L of FGF; the amino acid is a mixture of alanine and glycine, and the mass ratio of alanine to glycine is 1:1.

Example 7

The method is basically the same as Example 1, except that the chicken embryo is an 11-day-old chicken embryo, and the first cell culture medium is M-199 culture medium, and the M-199 culture medium is supplemented with 2 mmol/L of amino acids and 1.0 μg/L of FGF; the amino acid is a mixture of alanine and serine, and the mass ratio of alanine to serine is 1:1.

Example 8

The method is substantially the same as Example 1, except that the second cell culture fluid comprises an M-199 culture fluid and a buffer solution, and the mass ratio of the M-199 culture fluid to the buffer solution is 100:3, and the buffer solution contains 2% by mass of Hepes, 1.4% by mass of NaHCO ₃ , and 0.2% by mass of basic amino acids;

The basic amino acid is arginine.

Example 9

The method is substantially the same as Example 1, except that the second cell culture fluid comprises an M-199 culture fluid and a buffer solution, and the mass ratio of the M-199 culture fluid to the buffer solution is 100:3, and the buffer solution contains 2% by mass of Hepes, 1.4% by mass of NaHCO ₃ , and 0.2% by mass of basic amino acids;

The basic amino acid is a mixture of arginine and histidine, and the mass ratio of arginine to histidine is 1:1.

Example 10

The method is basically the same as Example 1, except that during the preparation of chicken embryo fibroblasts, the concentration of trypsin is 0.25%.

Embodiment 11

The method is basically the same as Example 1, except that during the preparation of chicken embryo fibroblasts, the concentration of trypsin is 0.05%.

Example 12

The method is basically the same as Example 1, except that the first cell culture medium includes M-199 culture medium, and 2 mmol/L alanine is added to the M-199 culture medium.

Example 13

The method is basically the same as Example 1, except that the first cell culture medium includes M-199 culture medium, and 1.0 μg/L FGF is added to the M-199 culture medium.

Embodiment 14

The method is basically the same as Example 1, except that the first cell culture medium includes M-199 culture medium, and 2 mmol/L alanine and 1.0 μg/L EGF are added to the M-199 culture medium.

Embodiment 15

The method is basically the same as Example 1, except that the second cell culture medium includes M-199 culture medium and buffer solution, and the mass ratio of M-199 culture medium to buffer solution is 100:3, and the buffer solution contains 2% Hepes by mass, 1.4% NaHCO ₃ by mass, and 0.2% aspartic acid by mass.

Example 16

The method is basically the same as Example 1, except that the second cell culture medium includes M-199 culture medium and buffer solution, and the mass ratio of M-199 culture medium to buffer solution is 100:3, and the buffer solution contains 2% Hepes by mass, 1.4% NaHCO ₃ by mass, and 0.2% glutamic acid by mass.

Embodiment 17

The method is basically the same as Example 1, except that the freezing method is as follows:

Prefreeze the box to 4°C, put in the ampoule for precooling for 30 minutes, and lower the seedling temperature to -40°C at a rate of 0.8°C/min; then lower the seedling temperature to -100°C at a rate of 8°C/min, and transfer to liquid nitrogen for storage.

Embodiment 18

The method is basically the same as Example 1, except that the freezing method is as follows:

Prefreeze the box to 4°C, put in the ampoule for precooling for 30 minutes, and lower the seedling temperature to -40°C at a rate of 1.2°C/min; then lower the seedling temperature to -100°C at a rate of 12°C/min, and transfer to liquid nitrogen for storage.

Comparative Example 1

The method is basically the same as Example 1, except that the freezing method is a refrigerator freezing method, and the refrigerator freezing method is specifically as follows: the ampoule is placed in a 4°C freezer for pre-freezing for 30 minutes, then placed in a -20°C refrigerator for freezing for 30 minutes, and then frozen in a -70°C freezer for 6 hours, and then transferred to liquid nitrogen for storage.

Comparative Example 2

The method is basically the same as Example 1, except that the cryopreservation method is as follows: pre-freeze the box to 4°C, put the ampoule in for pre-cooling for 30 minutes, reduce the seedling temperature to -20°C at a rate of 0.5°C/min, and then reduce the seedling temperature to -100°C at a rate of 1°C/min. Transfer to liquid nitrogen for storage.

Comparative Example 3

It is basically the same as Example 1, except that the freezing method is specifically as follows: pre-freeze the box to 4°C, put in the ampoule bottle for pre-cooling for 30 minutes, reduce the temperature to -4°C at a rate of 1°C/min; reduce the temperature to -40°C at a rate of 8°C/min; increase the temperature to -12°C at a rate of 5°C/min; reduce the temperature to -40°C at a rate of 1°C/min; reduce the temperature to -100°C at a rate of 10°C/min, and transfer to liquid nitrogen for storage.

Performance Testing:

1. Vaccine titer test: 3 bottles of each batch of vaccine were sampled, and after melting in 37 ℃ of warm water, they were diluted with special supporting diluent, and the optimum temperature was 25 ± 2 ℃. Take appropriate dilution, and each dilution was inoculated with 5 plates that had grown into good single-layer chicken embryo fibroblasts, and each plate was inoculated with 0.2ml. At the same time, 2 plates of blank controls and 5 plates of standard virus samples that were not inoculated were set up. The plates were placed at 37～38 ℃, containing 5% CO ₂ incubators for 6 days, and must not be moved. On the 7th day, characteristic cell pathological changes were observed and the average number of plaques of 5 plates of the same dilution was recorded, and the number of plaques contained in each bottle of vaccine was calculated again, and the number of plaques contained in each bottle of vaccine = the average number of plaques of 5 plates of the same dilution × dilution factor × volume (ml)/0.2ml per bottle.

The PFU error between the 5 plates of the standard virus sample does not exceed ±10%. The PFU of each batch of vaccine is determined based on the lowest PFU number in the 3 bottles. Each dose should not be less than 2000 PFU. The test results are shown in Table 1.

2. Cell yield and viability test: choose 11 well-developed SPF chicken embryos in days, sterilize the chicken embryo surface with 75% alcohol, 4% iodine tincture, aseptically take out embryoid. After removing eyes, the chicken embryo taken is put into the magnetic force disappearing bottle (2500mL) with connecting pipe, clean three times with Hank's liquid, add pancreatin by the amount of every chicken embryo 6ml, and the concentration of pancreatin is 0.125%, each magnetic force digestion bottle cumulative volume is no more than 1000ml, use magnetic bead room temperature digestion chicken embryo, leave standstill 1min after digestion 5min, wait tissue block precipitation and promptly pour out supernatant to be cell suspension, add 1% serum to stop digestion and ice bath to place, in triangular flask, add original volume pancreatin again, so repeatedly 5-6 time, by the centrifugal 10min of whole 1000rpm of collected supernatant, resuspend with cell growth liquid after removing supernatant, filter cell suspension with 2 layers of sterile gauze, carry out cell counting, use trypan blue staining to measure viable cell ratio, test result is as shown in table 2;

3. Cell yield and growth status test: Prepare cells according to Example 1 chicken embryo fibroblasts, observe the growth status after 24 hours of cell culture, if the cells adhere to the wall, the cells are highly homogeneous and spindle-shaped without vacuoles, and the density is more than 90%, it is judged to be good, and the test results are shown in Table 3;

4. Post-inoculation cytopathic rate test: cells were prepared and virus was inoculated according to Example 1, and the cytopathic rate was observed and recorded at different time points of 24h, 36h, 48h, 50h, 55h, 60h, and 65h after inoculation. The test results are shown in Table 4;

Table 1

Result analysis:

It can be seen from Example 1 and Comparative Examples 1-3 that when the type of freezing method or the freezing rate during the freezing process is modified, the titer of the virus decreases to varying degrees. This shows that the selection of the freezing method and freezing rate in this scheme has an important influence on the virus titer.

Table 2

Result analysis:

It can be seen from Table 2 that when the pancreatin concentration in the preparation process of chicken embryo fibroblasts is reduced relative to Example 1, the cell yield shows a downward trend, and when the pancreatin concentration is increased, the cell yield does not show an obvious upward trend. Combined with the fact that the percentages of weak and dead cells at the three concentrations are not much different, and the cell growth conditions are all good, therefore, for the control of production costs, the present application further recommends selecting a pancreatin concentration of 0.125%.

table 3

Group Chicken embryo age Number of cells (100 million/embryo) Cell growth status Example 1 11 days old 4.5±0.3 good Example 2 11 days old 4.8±0.3 good Example 3 9 days old 1.3±0.1 good Example 4 10 days old 2.2±0.2 good Example 5 9 days old 1.6±0.1 good Example 6 10 days old 2.7±0.2 good Example 7 11 days old 4.7±0.3 good Example 12 11 days old 4.2±0.3 good Example 13 11 days old 4.1±0.3 good Embodiment 14 11 days old 4.3±0.3 good

Result analysis:

It can be seen from Examples 1-2 in Table 3 that after the amino acids were compounded, the number of cells showed an obvious upward trend. We believe that the reason for this phenomenon may be that increasing the types of amino acids makes the culture medium more suitable for cell growth and division, thereby increasing the number of cells to a certain extent.

Further observation of Example 1 and Example 3-4 shows that when using 9- and 10-day-old chicken embryos, the number of cells decreases to varying degrees. We believe that the reason for this phenomenon may be that the difference in the embryo weight of 9- and 10-day-old chicken embryos relative to 11-day-old chicken embryos causes the difference in the total number of cells;

It can be seen from Examples 1-6 that after the amino acids are compounded, the growth promotion effect on 9-day-old chicken embryo cells is significantly better than that on 10- and 11-day-old chicken embryo cells. It can be seen that the first cell culture medium after the amino acids are compounded is more suitable for 9-day-old chicken embryo cells.

It can be seen from Example 1 and Examples 12-13 that when the first cell culture medium lacks amino acids or FGF, the number of cells will decrease to varying degrees. We believe that the reason for this phenomenon may be that the types of effective ingredients beneficial to cell growth in the culture medium are reduced, thereby reducing the growth rate of cells;

It can be seen from Example 1 and Example 14 that when EGF is used to replace FGF, the number of cells shows a downward trend. We believe that the reason for this phenomenon may be that EGF, as a member of the epidermal growth factor receptor family, has a significantly lower promoting effect on fibroblast growth than FGF, thereby reducing the number of cells. This shows that not all cell growth factors can effectively increase cell growth rate by replacing FGF.

Table 4

Note: Lesions cannot be observed for 24 hours, so they are indicated by -;

In Examples 8 and 9, after 60 hours, the pathological changes were greater than 70%, and no further observation and recording of accurate data were required, so they were indicated by -.

Result analysis:

It can be seen from Examples 1 and 8-9 that when basic amino acids are added to the second cell culture fluid, the pathological rate of cells after virus inoculation is significantly increased. It can be seen that the addition of basic amino acids can increase the pathological rate of cells after virus inoculation to a certain extent. Furthermore, when the basic amino acids are compounded in Example 9, the pathological rate of Example 9 is further accelerated. We believe that, on the one hand, the addition of basic amino acids can make the pH value of the system more stable, and on the other hand, the addition of multiple nutrients can also increase the replication rate of the virus in the cell.

Further observation of Example 1 and Examples 15-16 shows that when acidic amino acids are used instead of basic amino acids, the rate of cell pathological changes after virus inoculation shows a certain downward trend. We believe that the reason for this phenomenon may be that the alkaline environment is conducive to the pathological changes of cells after virus inoculation, and the addition of acidic amino acids causes the pH value of the system to change or become unstable, which in turn causes the rate of cell pathological changes after virus inoculation to show a certain downward trend.

Claims (10)

1. A method for preparing an MD vaccine, comprising the following steps: Step 1: Select chicken embryos, sterilize, obtain chicken embryo cells and culture them to obtain chicken embryo fibroblasts; Step 2: inoculating the virus strain into the chicken embryo fibroblasts prepared in step 1 and harvesting the infected cells; Step 3: The infected cells harvested in step 2 are prepared, packaged, and frozen; In step 3, a programmed cooling device is used for freezing, and the program of the programmed cooling device is as follows: pre-freeze the box to 4°C, put the ampoule bottle in for pre-cooling for 30 minutes, and reduce the seedling temperature to -40°C at a rate of 0.8-1.2°C/min; then reduce the seedling temperature to -100°C at a rate of 8-12°C/min, and transfer to liquid nitrogen for storage; In step 2, chicken embryo fibroblasts inoculated with the virus strain are cultured using a second cell culture fluid, wherein the second cell culture fluid comprises an M-199 culture fluid and a buffer solution, and the mass ratio of the M-199 culture fluid to the buffer solution is 100:3-4; The buffer solution contains 1.8-2.2% by mass of Hepes and 1.3-1.5% by mass of NaHCO ₃ . 2. The method for preparing the MD vaccine according to claim 1, characterized in that in step 1, the chicken embryo is a 9-11 day old chicken embryo. 3. The method for preparing the MD vaccine according to claim 1, characterized in that, in step 1, the chicken embryo cells are cultured using a first cell culture medium, wherein the first cell culture medium is an M-199 culture medium and the M-199 culture medium is further added with 0.5 to 3 mmol/L of amino acids and 1.0 to 1.2 μg/L of FGF; The amino acid is selected from at least one of alanine, aspartic acid, glutamic acid, glycine, proline, serine, threonine, tyrosine, arginine, cystine, histidine, isoleucine, leucine, lysine, tryptophan and valine. 4. The method for preparing MD vaccine according to claim 1, characterized in that the buffer contains 2% by mass of Hepes, 1.4% by mass of _NaHCO3 , and 0.02-0.5% by mass of basic amino acids; the basic amino acids are selected from at least one of arginine, lysine, and histidine. 5. The method for preparing the MD vaccine according to claim 1, characterized in that, in step 1, the culture method of the chicken embryo cells is a cell factory culture method, and the density of the cells in the cell suspension during the cell factory culture method is 2 million cells per milliliter, the number of layers of the cell factory in the cell factory culture method is 10 layers, and the volume of the cell suspension in each layer of the cell factory is 200 ml. 6. The method for preparing the MD vaccine according to claim 1 is characterized in that, in step 1, the specific method of selecting chicken embryos, disinfecting, and taking chicken embryo cells is: selecting well-developed SPF chicken embryos of 9 to 11 days old, disinfecting the surface of the chicken embryos with 75% alcohol and 4% iodine tincture, aseptically taking out the embryo body, and then using the chicken embryo shearing method or the magnetic bead digestion method to prepare a cell suspension. 7. The method for preparing the MD vaccine according to claim 6 is characterized in that, when the magnetic bead digestion method is used to prepare the cell suspension in step 1, the amount of pancreatin used during the magnetic bead digestion process is 6 ml per chicken embryo, and the concentration of pancreatin is 0.05-0.5%. 8. The method for preparing the MD vaccine according to claim 1, characterized in that, during the freezing process of step 3, the freezing solution is a freezing solution containing dimethyl sulfoxide, and the concentration of dimethyl sulfoxide in the freezing solution is 5-15%. 9. An MD vaccine, characterized in that it is prepared by the preparation method of the MD vaccine according to any one of claims 1 to 8. 10. The MD vaccine according to claim 9, characterized in that the dosage form of the MD vaccine is an injection dosage form.