A MD vaccine and its preparation method

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 them, 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; then the harvested infected cells are prepared as vaccines, packaged, and frozen, wherein a programmed cooling device is used for freezing. The program of the programmed cooling device is as follows: pre-freeze the box to 4°C, place an ampoule in it 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 it to liquid nitrogen for storage. The present application obtains a method for preparing an MD vaccine through the above design. The method has the advantages of fast preparation speed and large vaccine quantity, and is more suitable for large-scale preparation of MD vaccines. In addition, the present application also discloses an MD vaccine.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceuticals, and in particular to an MD vaccine and a preparation method thereof. Background Art

[0002] Marek's disease is a highly contagious lymphoproliferative disease of chickens caused by the cell-associated herpesvirus (MDV). Infection with Marek's virus can be devastating, severely impacting the poultry industry. With the widespread use of vaccines and the continuous breeding of chicken strains, MDV has become increasingly virulent due to both natural selection and immune pressure, repeatedly surpassing the protective effects of vaccines.

[0003] The primary strain isolated in the 1970s was virulent (vMDV). From the late 1970s to the 1980s, MDV underwent significant changes, becoming more virulent and becoming vvMDV. In the 1990s, the even more virulent vv+MDV (vv+MDV) emerged. However, HVT freeze-dried vaccines are ineffective against vv+MDV and vv+MDV.

[0004] 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 the Harbin Veterinary Biopharmaceutical Research Institute in my country from a healthy chicken flock that had not been immunized with Marek's vaccine. It belongs to the same serotype I strain as CVI988. However, strain 814 is a naturally weak strain and is non-tumorigenic. Therefore, 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 naturally weak strain like strain 814, non-tumorigenic, with good safety, rapid immune formation period, and is not affected by maternal antibodies. The vaccine can achieve a protection rate of more than 90% against super-virulent Marek's virus.

[0005] Ma Li, Zhu Xiuzhi, and Chen Ling from Beijing Veterinary Biological Pharmaceutical Factory presented a paper titled "Purification of the 814 Strain of Marek's Disease Virus and Production and Application of Live Vaccines" at the 9th Academic Symposium of the Biological Products Branch of the Chinese Society of Animal Husbandry and Veterinary Medicine. The paper documented the optimization of the 814 strain and the production and use of the 814 live vaccine.

[0006] During the optimization process of the 814 strain, the paper's specific method involved reintroducing the 814 vaccine virus into SPF chickens for several generations to restore the immunogenicity of the original virus and purify the vertically transmitted virus. This generation of virus was designated the optimized virus. The paper further tested the immune efficacy of the optimized virus and found that it reached 84%, higher than the 78% of the imported CV1988.

[0007] 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.

[0008] 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:

[0009] A. Select a cell line for vaccine production; the cell line is the chicken embryo DF-1 cell line;

[0010] B. Subculture and culture of cells for seedling production;

[0011] The above cell lines were passaged by digestion with EDTA-trypsin cell dispersion and continued to be cultured in cell growth medium. When a monolayer was formed, it was used for further passage or virus inoculation. The culture temperature was 36.5-37.5°C.

[0012] C. Propagation of cytotoxic species;

[0013] The live Marek's disease vaccine seed is diluted to a certain concentration using a seed virus diluent, inoculated onto a well-grown vaccine cell line or chicken embryo fibroblast monolayer, and adsorbed at 36.5-37.5°C for 1 hour. A maintenance solution or a secondary cell suspension is then added and cultured. When 70% or more of the monolayer cells show typical Marek's disease cytopathic changes, the cells are digested and dispersed using an EDTA-trypsin cell dispersion solution, and the harvested cell suspension is used as the vaccine seed;

[0014] D. Reproduction of seedling venom;

[0015] Dilute the seed virus for making live vaccine of Marek's disease in maintenance solution, inoculate on the cell line monolayer that has formed a monolayer and continue to culture. When 70% or more of the monolayer cells show typical Marek's disease cytopathic changes, harvest the infected cells.

[0016] E. Assort seedlings, aliquot, freeze-dry or freeze in liquid nitrogen;

[0017] The harvested infected cells are added with freeze-drying protective agent and antibiotics, quantitatively divided and then freeze-dried in a vacuum to obtain the finished product; or cryopreservation protective solution and antibiotics are added, mixed and quantitatively divided and then obtained by freezing in liquid nitrogen.

[0018] The method provides a method for producing a live chicken Marek's disease vaccine using a cell line through the above steps, which has stable production process, easy operation, high virus content, small batch-to-batch variation, and easy quality control. Further observation of the instructions for the method shows that the formula of the seed virus dilution solution or maintenance solution in the method is: adding an antibiotic at a final concentration of 100 to 400 units / ml to a DMEM / F12 culture medium containing 1 to 5% fetal bovine serum by volume, or to a mixture of equal parts of a 2-fold 199 solution and a 2-fold hydrolyzed milk protein solution containing 1 to 5% newborn bovine serum by volume, and adjusting the pH to 7.0 to 7.4;

[0019] 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 lyoprotectant is SPGA; the final concentration of the added antibiotic is 100 to 400 units / ml;

[0020] 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, transfer it into liquid nitrogen for storage. After 1 week, sample it for finished product inspection." Moreover, the program did not give much consideration and design to the impact of the freezing procedure on the potency of the vaccine.

[0021] 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

[0022] 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.

[0023] Another object of the present application is to provide a MD vaccine.

[0024] To achieve the above objectives, the present application discloses a method for preparing an MD vaccine, comprising the following steps:

[0025] Step 1: Select chicken embryos, sterilize them, obtain chicken embryo cells and culture them to obtain chicken embryo fibroblasts;

[0026] Step 2: inoculating the virus strain into the chicken embryo fibroblasts prepared in step 1 and harvesting the infected cells;

[0027] Step 3: The infected cells harvested in step 2 are prepared, packaged, and frozen;

[0028] In step 3, a programmed cooling device is used for freezing. The program of the programmed cooling device is as follows: pre-freeze the box to 4°C, place the ampoule in the box for pre-cooling for 30 minutes, and then 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;

[0029] In step 2, the chicken embryo fibroblasts inoculated with the virus strain are cultured using a second cell culture medium, wherein the second cell culture medium comprises M-199 culture medium and a buffer solution, and the mass ratio of the M-199 culture medium to the buffer solution is 100:3-4;

[0030] The buffer contains 1.8-2.2% Hepes by mass and 1.3-1.5% NaHCO3 by mass. During the culture period, cells are allowed to be cultured in a closed manner during toxin production without the need for an additional CO2 incubator or CO2 gas exchange, ensuring that the pH value of the culture solution is in the range of 7.0-7.4.

[0031] This application has improved the potency of the vaccine to a certain extent by optimizing the freezing procedure, thereby improving the protective effect of the vaccine;

[0032] The present invention utilizes a sodium bicarbonate buffer solution in the second culture medium. This buffer solution enables the cell factory culture method, effectively expanding production capacity and culture efficiency. The buffer solution uses Hepes and NaHCO₃. The CO₂ generated during the culture process can be absorbed by the buffer solution, eliminating the need for CO₂ gas exchange during the culture process and enabling large-scale culture.

[0033] Preferably, in step 1, the chicken embryo is a 9-11 day old chicken embryo.

[0034] Preferably, in step 1, the chicken embryo cells are cultured using a first cell culture medium, wherein the first cell culture medium is M-199 culture medium, and the M-199 culture medium is further supplemented with 0.5-3 mmol / L of amino acids and 1.0-1.2 μg / L of FGF;

[0035] 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.

[0036] The buffer solution contains 2% Hepes by mass, 1.4% NaHCO3 by mass, and 0.02-0.5% basic amino acids by mass; the basic amino acids are selected from at least one of arginine, lysine, and histidine.

[0037] Preferably, in step 1, the chicken embryo cells are cultured using 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, and the volume of the cell suspension in each layer of the cell factory is 200 ml.

[0038] Preferably, in step 1, the specific method of selecting chicken embryos, disinfecting, and obtaining chicken embryo cells is as follows: 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 mincing method or the magnetic bead digestion method to prepare a cell suspension.

[0039] 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%.

[0040] Preferably, during the freezing process in 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%.

[0041] In addition, the present application also discloses an MD vaccine, which is prepared by the above-mentioned MD vaccine preparation method.

[0042] Preferably, the MD vaccine is in the form of an injection.

[0043] The beneficial effects of the present application are: 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, 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 DESCRIPTION

[0044] The present invention will be described clearly and completely below in conjunction with the examples of the present invention. In the description of the present invention, it should be noted that, where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer were used. Reagents or instruments used where the manufacturer is not specified are conventional products that can be purchased commercially.

[0045] Before presenting the examples, the necessary information of the experimental consumables such as the seeds, reagents, and instruments used in the examples is first described. The specific information is shown in Table 1:

[0046] Table 1

[0047]

[0048]

[0049] It should be noted that the seed virus in the experiment was the P22 generation CVTR strain, with a virus content of 1.57×10 7 PFU / mL, identified, stored, and supplied by Guangdong Wenshi Dahuanong Biotechnology Co., Ltd.

[0050] Example 1

[0051] 1.1 Preparation of chicken embryo fibroblasts

[0052] Choose 2600 pieces of SPF chicken embryos with good development in 11 age in days, with 75% alcohol, 4% iodine tincture sterilizing chicken embryo surface, aseptic taking-up embryoid.After removing eyes, the chicken embryo that gets is put into the large beaker of 1000ml, clean three times with Hank's liquid, transfer to the magnetic force of band connecting pipe then 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 stirring apparatus room temperature digestion chicken embryo, each magnetic force digestion bottle cumulative volume is no more than 1000ml, leave standstill 1min after digestion 5min, waiting that the tissue block precipitation promptly pours out supernatant is cell suspension, add 2% serum to stop digestion and ice bath to place, disappear in bottle and add original volume pancreatin toward magnetism again, so repeatedly 5-6 time, with collected The supernatant was centrifuged at 1500 rpm for 10 minutes, the supernatant was removed and the cell suspension was resuspended in cell growth medium, the cell suspension was filtered through 4 layers of sterile gauze, mixed after filtration and a 1 ml 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 factories were cultured for 24 hours to obtain a dense monolayer of 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 alanine and 1.0 μg / L FGF were also added to the M-199 culture medium.

[0053] 1.2 Virus inoculation and culture of infected cells

[0054] The CVTR strain virus was taken out of liquid nitrogen and immediately placed in warm water at 37°C, shaken quickly and evenly, and melted within 60 seconds. 20 million PFU were inoculated in each cell factory. Cultured at 37°C, the cell pathological changes were observed for 50-60 hours: the cells were rounded and shrunken, with strong refractive index, and the diseased cells were evenly distributed. When the virus was inoculated into the cell factory, a second cell culture fluid was added to the cell factory. The second cell culture fluid included M-199 culture fluid and buffer solution, and the mass ratio of M-199 culture fluid and buffer solution was 100:3. The buffer solution contained 2% by mass of Hepes and 1.4% by mass of NaHCO3.

[0055] 1.3 Harvesting and freezing of infected cells

[0056] 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.

[0057] The cryopreservation was performed using a programmed cooling apparatus, and the specific cryopreservation method was as follows:

[0058] Prefreeze the box to 4°C, put in the ampoule and precool for 30 minutes, then 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.

[0059] Virus content assay: After the vaccine or virus is melted in 37°C warm water, it is diluted with a dedicated supporting diluent at an optimum temperature of 25 ± 2°C. For each appropriate dilution, five plates of well-grown chicken embryo fibroblasts are inoculated with 0.2 ml of each. Two uninoculated blank controls and five standard virus sample plates are also set up. The plates are incubated at 37-38°C in a 5% CO2 incubator for six days without moving. On the seventh day, characteristic cytopathic effects are observed and the average number of plaques from the five plates of the same dilution is recorded. The number of plaques per bottle of vaccine is then calculated as: the average number of plaques from the five plates of the same dilution × dilution factor × volume (ml) per bottle / 0.2 ml.

[0060] The PFU counts of the standard virus samples should not exceed ±10% between the five plates. The lowest PFU count among the three bottles should be used to determine the PFU count for each batch of vaccine. Each dose should be no less than 2000 PFU.

[0061] Example 2

[0062] The method is basically the same as Example 1, except that the first cell culture medium is M-199 culture medium, and 2 mmol / L amino acids and 1.0 μg / L 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.

[0063] Example 3

[0064] The method is basically the same as Example 1, except that the chicken embryos are 9-day-old chicken embryos.

[0065] Example 4

[0066] The method is basically the same as Example 1, except that the chicken embryo is a 10-day-old chicken embryo.

[0067] Example 5

[0068] 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.

[0069] Example 6

[0070] 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.

[0071] Example 7

[0072] 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 acids are a mixture of alanine and serine, and the mass ratio of alanine to serine is 1:1.

[0073] Example 8

[0074] 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% by mass of Hepes, 1.4% by mass of NaHCO3, and 0.2% by mass of basic amino acids;

[0075] The basic amino acid is arginine.

[0076] Example 9

[0077] 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% by mass of Hepes, 1.4% by mass of NaHCO3, and 0.2% by mass of basic amino acids;

[0078] The basic amino acid is a mixture of arginine and histidine, and the mass ratio of arginine to histidine is 1:1.

[0079] Example 10

[0080] 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%.

[0081] Example 11

[0082] 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%.

[0083] Example 12

[0084] 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.

[0085] Example 13

[0086] 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.

[0087] Example 14

[0088] 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.

[0089] Example 15

[0090] 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% NaHCO3 by mass, and 0.2% aspartic acid by mass.

[0091] Example 16

[0092] 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% NaHCO3 by mass, and 0.2% glutamic acid by mass.

[0093] Example 17

[0094] The method is basically the same as Example 1, except that the freezing method is as follows:

[0095] Prefreeze the box to 4°C, put in the ampoule and precool for 30 minutes, then 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.

[0096] Example 18

[0097] The method is basically the same as Example 1, except that the freezing method is as follows:

[0098] Prefreeze the box to 4°C, put in the ampoule and precool for 30 minutes, then 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.

[0099] Comparative Example 1

[0100] The method is basically the same as Example 1, except that the freezing method is a refrigerator freezing method. The refrigerator freezing method is 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 30 minutes, and then frozen in a -70°C freezer for 6 hours, and then transferred to liquid nitrogen for storage.

[0101] Comparative Example 2

[0102] The method is basically the same as Example 1, except that the freezing method is as follows: pre-freeze the box to 4°C, place the ampoule in the pre-cooling bottle for 30 minutes, cool the seedling temperature to -20°C at a rate of 0.5°C / min, and then cool the seedling temperature to -100°C at a rate of 1°C / min. Transfer to liquid nitrogen for storage.

[0103] Comparative Example 3

[0104] The method is basically the same as Example 1, except that the freezing method is as follows: pre-freeze the box to 4°C, put the ampoule into the box and pre-cool it for 30 minutes, then 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.

[0105] Performance testing:

[0106] 1. vaccine titer test: 3 bottles of every batch vaccine sampling, after 37 ℃ of warm water melts, dilute with special supporting diluent, optimum temperature is 25 ± 2 ℃.Get appropriate dilution, each dilution respectively inoculates 5 petri dishes that have grown into good single layer chicken embryo fibroblasts, and each petri dish is inoculated with 0.2ml. Set up 2 petri dishes and 5 petri dishes of standard virus sample that do not inoculate simultaneously. Petri dish is put 37~38 ℃, contains 5%CO Incubator culture 6 days, must not move, observe characteristic cell pathological changes and record the average plaque number of 5 petri dishes of same dilution on the 7th day, calculate the contained plaque number of every bottle of vaccine again, the average plaque number of 5 petri dishes of the contained plaque number=same dilution of every bottle of vaccine × dilution factor × every bottle of volume (ml) / 0.2ml.

[0107] The PFU counts of the five plates of the standard virus sample should not exceed ±10%. The lowest PFU count among the three bottles was used to determine the PFU count of each batch of vaccine. Each dose should be no less than 2000 PFU. The test results are shown in Table 1.

[0108] 2. cell yield and viability test: choose 11 well-developed SPF chicken embryos in age in days, with 75% alcohol, 4% iodine tincture disinfection chicken embryo surface, aseptic taking-up embryoid.After removing eyes, the chicken embryo taken is put into the magnetic force of band connecting pipe and disappears bottle (2500mL), clean three times with Hank's liquid, add trypsin by the amount of every chicken embryo 6ml, and the concentration of trypsin 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, waiting that the tissue block precipitation promptly pours out supernatant is cell suspension, add 1% serum to stop digestion and ice bath to place, in triangular flask, add original volume trypsin again, so repeatedly 5-6 time, with the centrifugal 10min of whole 1000rpm of collected supernatant, resuspend with cell growth liquid after removing supernatant, use 2 layers of sterile gauze filtered cell suspension, carry out cell counting, use trypan blue staining to measure viable cell ratio, test result is as shown in table 2;

[0109] 3. Cell yield and growth status test: According to the preparation cells of chicken embryo fibroblasts in Example 1, the growth status was observed after the cells were cultured for 24 hours. If the cells adhered to the wall, the cells were highly homogeneous and spindle-shaped without vacuoles, and the density was more than 90%, it was judged to be good. The test results are shown in Table 3;

[0110] 4. Post-inoculation cytopathic rate test: Cells were prepared and inoculated with the virus according to Example 1. The cytopathic rate was observed and recorded at different time points 24 h, 36 h, 48 h, 50 h, 55 h, 60 h, and 65 h after inoculation. The test results are shown in Table 4.

[0111] Table 1

[0112]

[0113] Result analysis:

[0114] 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 choice of freezing method and freezing rate in this scheme has an important impact on the virus titer.

[0115] Table 2

[0116]

[0117] Result analysis:

[0118] As shown in Table 2, when the pancreatin concentration in the preparation process of chicken embryo fibroblasts was reduced relative to Example 1, the yield of the cells was on a downward trend. However, after the pancreatin concentration increased, there was no obvious upward trend in the yield of the cells. In combination with the three concentrations, the percentages of weak and dead cells were similar, and the cell growth conditions were good. Therefore, for the control of production costs, the application further recommends selecting a pancreatin concentration of 0.125%.

[0119] Table 3

[0120] 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 Example 14 11 days old 4.3±0.3 good

[0121] Result analysis:

[0122] As shown in Examples 1-2 in Table 3, after the amino acids were compounded, the cell number showed a significant upward trend. We believe that the reason for this phenomenon may be that the increase in the types of amino acids makes the culture medium more suitable for cell growth and division, thereby increasing the cell number to a certain extent.

[0123] Further observation embodiment 1 and embodiment 3-4 visible, when using 9,10 chicken embryos in age in day, cell number produces decline in various degrees, and we think that the reason causing this phenomenon may be that the difference of the relative 11 chicken embryo weights in age in day of 9,10 chicken embryos in age in day causes total cell number difference;

[0124] From the observation of Examples 1-6, it can be seen 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. Therefore, it can be seen that the first cell culture medium after the amino acid compounding is more suitable for 9-day-old chicken embryo cells;

[0125] As shown in Examples 1 and 12-13, when the first cell culture medium lacks amino acids or FGF, the cell number decreases to varying degrees. We believe that this phenomenon may be caused by a decrease in the types of active ingredients in the culture medium that are beneficial to cell growth, thereby reducing the cell growth rate.

[0126] It can be seen from Examples 1 and 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.

[0127] Table 4

[0128]

[0129]

[0130] Note: Lesions cannot be observed within 24 hours, so they are indicated by -;

[0131] In Examples 8 and 9, the lesion rate was greater than 70% after 60 hours, so there was no need to continue observing and recording accurate data, so it was represented by -;

[0132] Result analysis:

[0133] As can be seen from Examples 1 and 8-9, when basic amino acids are added to the second cell culture medium, the pathological rate of cells after virus inoculation is significantly increased. This shows 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 cells.

[0134] 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

1. A method for preparing an MD vaccine, characterized in that: The following steps are involved: Step 1: Select chicken embryos, sterilize them, 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. The program of the programmed cooling device is as follows: pre-freeze the box to 4°C, place the ampoule in the box for pre-cooling for 30 minutes, and then 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, the chicken embryo fibroblasts inoculated with the virus strain are cultured using a second cell culture medium, wherein the second cell culture medium comprises M-199 culture medium and a buffer solution, and the mass ratio of the M-199 culture medium to the buffer solution is 100:3-4; The buffer solution contains Hepes with a mass fraction of 1.8-2.2%, NaHCO3 with a mass fraction of 1.3-1.5%, and basic amino acids with a mass fraction of 0.2%, wherein the basic amino acids are arginine and / or histidine.

2. The method for preparing the MD vaccine according to claim 1, characterized in that: In step 1, the chicken embryo is a 9-10 day old chicken embryo; In step 1, the chicken embryo cells are cultured in 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.

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 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, and the volume of the cell suspension in each layer of the cell factory is 200 ml.

4. The method for preparing the MD vaccine according to claim 1, characterized in that: In step 1, the specific method for selecting chicken embryos, disinfecting, and obtaining chicken embryo cells is as follows: select well-developed SPF chicken embryos of 9 to 11 days old, disinfect the surface of the chicken embryos with 75% alcohol and 4% iodine tincture, aseptically remove the embryo body, and then use the chicken embryo mincing method or magnetic bead digestion method to prepare a cell suspension.

5. The method for preparing the MD vaccine according to claim 4, characterized in that: 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%.

6. The method for preparing the MD vaccine according to claim 1, characterized in that: During the freezing process in step 3, the freezing solution contains dimethyl sulfoxide, and the concentration of dimethyl sulfoxide in the freezing solution is 5-15%.

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