CN113999821B - Large-scale preparation method for mumps virus - Google Patents
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Abstract
The invention relates to a large-scale preparation method for mumps virus, belonging to the technical field of biological medicines. The invention provides a large-scale preparation method for mumps virus, which uses a cell factory as a carrier for large-scale preparation of mumps virus, and obtains an optimal harvest and exchange solution combination for mumps virus culture by systematic comparison, wherein the optimal harvest and exchange solution combination is obtained by harvesting virus culture solutions at intervals of 22-26 h, the virus culture solutions are harvested for three-four times in total, and new cell maintenance solution is required to be injected into the cell factory after the first two-three times of harvest; compared with the bottle-rotating process, the method provided by the invention has the advantages that the harvesting times are multiple, the virus titer of the single virus harvesting liquid and the mixed virus harvesting liquid is high, the cytopathic effect is stable, and the like.
Description
Technical Field
The invention relates to a large-scale preparation method for mumps virus, belonging to the technical field of biological medicines.
Background
Mumps is an acute respiratory infectious disease caused by Mumps virus (MuV). The main clinical symptoms are fever, parotid non-suppurative swelling and pain; in some cases, aseptic meningitis, pancreatitis, orchitis, etc. are also secondary; severely symptomatic patients can lead to disability or death. Epidemic parotitis belongs to third class infectious diseases managed by law in China, specific medicines are not available at present, and the antibiotic treatment has no obvious effect. At this stage, vaccination with vaccines remains the only effective means of prevention and control of mumps.
The mumps attenuated live vaccine is an important product in the vaccine for controlling the mumps, and has the advantages of good immune effect, long immune action time, no need of adjuvant stimulation and convenient preparation process. Before the mumps attenuated live vaccine is used in a large amount, the mumps in all parts of the world are flooded; however, with the widespread use of live attenuated mumps vaccines, the incidence of mumps is greatly controlled.
The preparation process of the mumps attenuated live vaccine mainly comprises two steps of large-scale production of mumps virus attenuated strains (cell preparation, virus inoculation, cell liquid exchange and virus harvesting) and clarification. The high-titer mumps virus attenuated strain harvest obtained by large-scale production is a precondition for batch preparation of mumps attenuated live vaccines, and can directly influence the quality and yield of the mumps attenuated live vaccines.
At the present stage, the large-scale production of the mumps virus attenuated strain is mainly realized by a bottle rotating process. However, the spinner flask process still has many defects, for example, the cell feeding area of a single spinner flask is limited, a large number of spinner flasks are needed for large-scale production, and the risk of aseptic control is higher; the effect of the large-scale preparation method cannot be guaranteed due to possible leakage caused by factors such as operation methods; the space used by the factory building is larger, and the harvest liquid with the same volume needs larger space compared with the cell factory; the cleaning and sterilization of the rotary bottle require a large amount of labor force, and the automatic replacement difficulty of operations such as liquid collection and replacement is high. Overcoming the defects is very important for further improving the quality and the yield of the mumps attenuated live vaccine.
Disclosure of Invention
In order to solve the problems of the existing large-scale preparation method of the mumps attenuated live vaccine, the invention provides a large-scale preparation method for mumps virus, which comprises the following steps:
cell preparation: preparing a chicken embryo fibroblast suspension;
virus inoculation: after the mumps virus is inoculated into the chicken embryo fibroblast suspension, the suspension is added into a cell factory containing virus growth liquid for primary culture;
cell liquid change: after the first culture, leading out the virus growth liquid in the cell factory, and washing the cell factory by using a buffer solution; after the cell surface is cleaned, injecting the cell maintenance liquid into a cell factory for secondary culture;
and (3) harvesting viruses: after the second culture is finished, harvesting virus culture solution in the cell factory; the harvesting is totally three or four times, and the harvesting interval is 22-26 h.
In one embodiment of the invention, the harvesting comprises the steps of:
harvesting for the first time: after the second culture is finished, the virus culture solution in the cell factory is led out, and the cell maintenance solution is injected into the cell factory for the third culture;
and (3) harvesting for the second time: after the third culture is finished, the virus culture solution in the cell factory is led out, and the cell maintenance solution is injected into the cell factory for the fourth culture;
and (3) harvesting for the third time: after the fourth culture is finished, leading out the virus culture solution in the cell factory, and combining the virus culture solution with the virus culture solution obtained by the first harvest and the second harvest to obtain a virus harvest solution;
the time of the third culture and the fourth culture is 22-26 h.
In one embodiment of the invention, the harvesting comprises the steps of:
harvesting for the first time: after the second culture is finished, the virus culture solution in the cell factory is led out, and the cell maintenance solution is injected into the cell factory for the third culture;
and (3) harvesting for the second time: after the third culture is finished, the virus culture solution in the cell factory is led out, and the cell maintenance solution is injected into the cell factory for the fourth culture;
and (3) harvesting for the third time: after the fourth culture is finished, the virus culture solution in the cell factory is led out, and the cell maintenance solution is injected into the cell factory for the fifth culture;
and (4) harvesting for the fourth time: after the fifth culture is finished, leading out the virus culture solution in the cell factory, and combining the virus culture solution with the virus culture solution obtained by the first harvesting, the second harvesting and the third harvesting to obtain a virus harvest solution;
the time for the third culture, the fourth culture and the fifth culture is 22-26 h.
In one embodiment of the invention, the time for the first culture is 22-26 h; the time of the second culture is 66-80 h.
In one embodiment of the present invention, the cell preparation step is: and (3) cutting chicken embryos incubated for 9-11 days into pieces, adding pancreatin for digestion, and then beating by blowing to obtain chicken embryo fibroblast suspension.
In one embodiment of the present invention, the cell preparation step is: removing the head and the internal organs of 9-11 days old chick embryos, and shearing the chick embryos into 1-3 mm by using scissors3Adding pancreatin in the tissue blocks in an amount of 4-6 mL/chick embryo, digesting at 36-38 ℃ for 15-25 min, blowing and beating after digestion to prepare the chicken tissue blocks with the concentration of 1.7-2.5 multiplied by 106individual/mL of chicken embryo fibroblast suspension.
In one embodiment of the present invention, the virus inoculation step is: inoculating the mumps virus into the chicken embryo fibroblast suspension according to the inoculation amount of 0.001-0.01 MOI, adding the cell suspension inoculated with the mumps virus into a cell factory according to the addition amount of 150-250 mL/layer, and performing primary culture at 33-35 ℃; 150-250 mL of virus growth solution is added to each layer of the cell factory.
In one embodiment of the present invention, the cell exchange step is: after the first culture is carried out for 22-26 h, the virus growth solution in the cell factory is led out, the cell factory is washed by buffer solution, 100-200 mL of solution is added into each layer, and the solution is led out after the cell surface is washed by shaking; after the cell surface is cleaned, injecting the cell maintenance liquid into a cell factory, adding 150-250 mL of liquid into each layer, and culturing at 33-35 ℃ for the second time.
In one embodiment of the invention, the buffer is 0.01M, pH 7.4.4 PBS buffer.
In one embodiment of the present invention, the first harvesting step is: after the second culture is carried out for 66-80 h, the virus culture solution in the cell factory is led out, the cell maintenance solution is injected into the cell factory, 150-250 mL of liquid is added into each layer, and the third culture is carried out at 33-35 ℃.
In one embodiment of the present invention, the second harvesting step is: after the third culture for 22-26 h, the virus culture solution in the cell factory is taken out, the cell maintenance solution is injected into the cell factory, 150-250 mL of solution is added into each layer, and the fourth culture is carried out at 33-35 ℃.
In one embodiment of the present invention, the third harvesting step is: and after the fourth culture for 22-26 h, leading out the virus culture solution in the cell factory, and combining the virus culture solution with the virus culture solution obtained by the first harvest and the second harvest to obtain a virus harvest solution.
In one embodiment of the present invention, the third harvesting step is: after the fourth culture for 22-26 h, the virus culture medium in the cell factory is taken out, the cell maintenance medium is injected into the cell factory, 150-250 mL of liquid is added to each layer, and the fifth culture is performed at 33-35 ℃.
In one embodiment of the present invention, the fourth harvesting step is: and after the fifth culture for 22-26 h, leading out the virus culture solution in the cell factory, and combining the virus culture solution with the virus culture solution obtained by the first harvest, the second harvest and the third harvest to obtain a virus harvest solution.
The invention also provides a preparation method of the mumps attenuated live vaccine, which comprises the following steps:
the method comprises the following steps: carrying out large-scale preparation on the mumps virus attenuated strain by using the large-scale preparation method to obtain a virus harvest solution;
step two; and separating and purifying the virus harvest liquid to obtain the mumps attenuated live vaccine.
The invention also provides application of the large-scale preparation method in preparation of mumps virus vaccines.
In one embodiment of the invention, the mumps virus vaccine is a mumps attenuated live vaccine.
The technical scheme of the invention has the following advantages:
the invention provides a large-scale preparation method for mumps virus, which takes a cell factory as a carrier for large-scale preparation of mumps virus, and obtains an optimal collection and exchange solution combination for mumps virus culture by systematic comparison, wherein the optimal collection and exchange solution combination is obtained by harvesting virus culture solution at intervals of 22-26 h, the virus culture solution is harvested for three-four times in total, and new cell maintenance solution is required to be injected into the cell factory after the first two-three times of harvest; compared with the bottle rotating process, the large-scale preparation method provided by the invention has the advantages that the harvesting times are multiple, the virus titer of the single virus harvesting liquid and the mixed virus harvesting liquid is high, the cytopathic effect is stable, and the like.
Drawings
FIG. 1: the virus harvesting protocol in example 1 and comparative examples 1-2.
FIG. 2: growth curves (10 layers) for the virus harvesting protocols in example 1 and comparative examples 1-2.
FIG. 3: growth curves for the virus harvesting protocol in example 2 (40 layers).
Detailed Description
The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.
The following examples do not show specific experimental procedures or conditions, and can be performed according to the procedures or conditions of the conventional experimental procedures described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.
The buffers referred to in the following examples are as follows:
PBS buffer: first, 8.0g NaCl, 0.2g KCl and 1.44g Na are weighed2HPO4、0.24g KH2PO4Dissolving in 800mL of distilled water, adjusting pH to 7.4 with HCl, and adding distilled water to a constant volume of 1L to obtain 0.01M, pH 7.4.4 PBS buffer solution.
Example 1: large-scale preparation method for mumps virus
The embodiment provides a large-scale preparation method for mumps virus, which comprises the following steps:
cell preparation: removing head and viscera of 9-11 days old chick embryo (purchased from Lihua, Zhejiang Co.), and shearing into 1.5mm with scissors3The tissue blocks were digested with pancreatin (purchased from Gibco, Inc., cat # 27250018) in an amount of 5 mL/chick embryo for 20min at 37 deg.C, and then blown into a mass of 2.0X 106one/mL of chicken embryo fibroblast suspension;
virus inoculation: after mumps virus (preservation number: CCTCC No: V201950, described in patent publication No. CN 111019910A) was inoculated to the chicken embryo fibroblast suspension at an inoculum size of 0.001MOI, the cell suspension inoculated with mumps virus was added to a cell factory (purchased from Thermo, 10 layers) at an addition level of 200mL per layer, and the first culture was carried out at 34 ℃; 200mL of virus growth medium (Bacto, product number 259962, from Gibco) was added to each layer of the cell factoryTMTC whey protein hydrolysate);
cell liquid change: after 24h of first culture, introducing virus growth solution in the cell factory into a waste solution tank, washing the cell factory with 0.01M, pH 7.4.4 PBS buffer solution, adding 150mL of solution into each layer, shaking to wash the cell surface, introducing the solution into the waste solution tank, and repeating the operation for 3 times; after the cell surface was washed, a cell-maintaining solution (medium 199 available from Gibco under the trade name of 12350039) was poured into the cell factory, 200mL of the solution was added to each layer, and the cells were cultured at 34 ℃ for the second culture;
harvesting for the first time: after the second culture for 72 hours, the virus culture medium in the cell factory was removed, and a cell-maintaining solution (medium No. 12350039 199 available from Gibco Co.) was injected into the cell factory, 200mL of which was added per layer, and cultured at 34 ℃ for the third culture;
and (3) harvesting for the second time: after the third culture for 24 hours, the virus culture medium in the cell factory was removed, and a cell maintenance solution (which was culture medium 199 purchased from Gibco under the product number of 12350039) was injected into the cell factory, 200mL of which was added to each layer, and the cell factory was cultured at 34 ℃ for the fourth culture;
and (3) harvesting for the third time: after the fourth culture for 24 hours, the virus culture medium in the cell factory was removed, and a cell-maintaining solution (medium No. 12350039 199 available from Gibco Co.) was poured into the cell factory, to which 200mL of the solution was added, and the cell-maintaining solution was cultured at 34 ℃ for the fifth culture;
and (4) harvesting for the fourth time: after the fifth culture for 24h, the virus culture fluid in the cell factory is led out and combined with the virus culture fluid obtained from the first harvest, the second harvest and the third harvest to obtain the virus harvest fluid (the harvest scheme is shown as # 2 in figure 1).
Comparative example 1: large-scale preparation method for mumps virus
The present comparative example provides a large-scale preparation method for mumps virus, comprising the steps of:
cell preparation: removing head and viscera of 9-11 days old chick embryo (purchased from Lihua, Zhejiang Co.), and shearing into 1.5mm with scissors3The tissue blocks were digested with pancreatin (purchased from Gibco, Inc., cat # 27250018) in an amount of 5 mL/chick embryo for 20min at 37 deg.C, and then blown into a mass of 2.0X 106one/mL of chicken embryo fibroblast suspension;
virus inoculation: after mumps virus (CCTCC No: V201950, described in patent publication No. CN 111019910A) was inoculated to a suspension of chicken embryo fibroblasts at an inoculation amount of 0.001MOI, the suspension of cells inoculated with mumps virus was added to a cell factory (10 layers, model: CF10, available from Thermo) at an addition amount of 200 mL/layer, and the suspension was cultured in a culture mediumPerforming first culture at 34 deg.C; 200mL of virus growth medium (Bacto, product number 259962, from Gibco) was added to each layer of the cell factoryTMTC whey protein hydrolysate);
cell liquid change: after 24h of first culture, introducing virus growth solution in the cell factory into a waste solution tank, washing the cell factory with 0.01M, pH 7.4.4 PBS buffer solution, adding 150mL of solution into each layer, shaking to wash the cell surface, introducing the solution into the waste solution tank, and repeating the operation for 3 times; after the cell surface was washed, a cell-maintaining solution (medium 199 available from Gibco under the trade name of 12350039) was poured into the cell factory, 200mL of the solution was added to each layer, and the cells were cultured at 34 ℃ for the second culture;
harvesting for the first time: after the second culture for 48 hours, the virus culture medium in the cell factory was removed, and a cell-maintaining solution (medium No. 12350039 199 available from Gibco Co.) was injected into the cell factory, 200mL of which was added per layer, and cultured at 34 ℃ for the third culture;
and (3) harvesting for the second time: after 48 hours of the third culture, the virus culture medium in the cell factory was removed, and a cell maintenance solution (medium 199 available from Gibco under the reference 12350039) was poured into the cell factory, 200mL of which was added to each layer, and the cell factory was cultured at 34 ℃ for the fourth culture;
and (3) harvesting for the third time: after the fourth culture for 48h, the virus culture fluid in the cell factory is led out and combined with the virus culture fluid obtained from the first harvest and the second harvest to obtain the virus harvest fluid (the harvest scheme is shown as # 1 in figure 1).
Comparative example 2: large-scale preparation method for mumps virus
The present comparative example provides a large-scale preparation method for mumps virus, comprising the steps of:
cell preparation: removing head and viscera of 9-11 days old chick embryo (purchased from Lihua, Zhejiang Co.), and shearing into 1.5mm with scissors3After the tissue mass, 5mL of the composition was added per chick embryoPancreatin (from Gibco, cat # 27250018) was digested at 37 deg.C for 20min, and blown to a concentration of 2.0X 106one/mL of chicken embryo fibroblast suspension;
virus inoculation: after mumps virus (preservation number: CCTCC No: V201950, described in patent publication No. CN 111019910A) was inoculated to the chicken embryo fibroblast suspension at an inoculum size of 0.001MOI, the cell suspension inoculated with mumps virus was added to a cell factory (purchased from Thermo, 10 layers) at an addition level of 200mL per layer, and the first culture was carried out at 34 ℃; 200mL of virus growth medium (Bacto, product number 259962, from Gibco) was added to each layer of the cell factoryTMTC whey protein hydrolysate);
cell liquid change: after 24h of first culture, introducing virus growth solution in the cell factory into a waste solution tank, washing the cell factory with 0.01M, pH 7.4.4 PBS buffer solution, adding 150mL of solution into each layer, shaking to wash the cell surface, introducing the solution into the waste solution tank, and repeating the operation for 3 times; after the cell surface was washed, a cell-maintaining solution (medium 199 available from Gibco under the trade name of 12350039) was poured into the cell factory, 200mL of the solution was added to each layer, and the cells were cultured at 34 ℃ for the second culture;
harvesting for the first time: after the second culture for 72 hours, the virus culture medium in the cell factory was removed, and a cell-maintaining solution (medium No. 12350039 199 available from Gibco Co.) was injected into the cell factory, 200mL of which was added per layer, and cultured at 34 ℃ for the third culture;
and (3) harvesting for the second time: after 48h of the third culture, the virus culture fluid in the cell factory is led out and is combined with the virus culture fluid obtained by the first harvest, and then the virus harvest fluid is obtained (the harvest scheme is shown as # 3 in figure 1).
Experimental example 1: experiment for influence of harvesting mode on total virus harvest amount of virus harvest liquid
This experimental example provides an experiment of the influence of the harvest mode on the total virus harvest amount of the virus harvest solution, and the experimental process is as follows:
the results of examining the infectious mumps virus titer in the virus harvest at different harvest times of example 1 and comparative examples 1 to 2 by the cell pathology method (third pharmacopoeia) are shown in table 1 and fig. 2.
As can be seen from Table 1 and FIG. 2, the harvest protocol of example 1 was harvested once a day for a total of four times starting on day four, wherein the viral titers of the virus fluids harvested the first three times were all higher than 6.5 lgCCID50mL, first harvest titer exceeded 7.0 lgCCID50mL, fourth titer drop very rapidly; the harvest protocol of comparative example 1 started on day three with an intervening day, and was harvested three times in total, with the virus titer of the second harvest being approximately 7.0 lgCCID50mL, but the titer reduction of the first and third harvests was significant, both less than 6.5 lgCCID50mL, from which it can be seen that example 1 is clearly superior to comparative example 1; the harvest protocol of comparative example 2 was also harvesting from day four, separated by one day, for a total of two harvests, wherein the titer of the first harvest also exceeded 7.0 lgCCID50mL, second harvest exceeds 6.5 lgCCID50mL, both harvests were high in titer average. The titer of the first harvest and the second harvest of comparative example 2 and example 1 are equivalent, but the number of harvests of example 1 is two more than that of comparative example 2, and the harvest titer is higher than the pharmacopoeia standard, so that the harvest scheme of example 1 can obtain more volumes and higher titer of harvest liquid, and is the best choice.
TABLE 1 Effect of different harvest styles on viral titer of the viral harvest (lgCCID)50/mL）
Example 2: large-scale preparation method for mumps virus
The embodiment provides a large-scale preparation method for mumps virus, which comprises the following steps:
on the basis of example 1, the cell factories with 10 layers are replaced by cell factories with 40 layers, and the virus harvest obtained by the first harvest, the second harvest and the third harvest are combined to obtain the virus harvest.
Experimental example 2: stability test of harvesting mode
This experimental example provides a stability experiment of the harvesting mode, and the experimental process is as follows:
three batches of virus harvests (lot numbers 05001, 05002 and 05003, respectively) were continuously produced using the large-scale preparation method of example 2, and infectious mumps virus titers at different harvest times of the three batches of virus harvests were detected using the cell pathology method (third pharmacopeia), and the results are shown in fig. 3.
As can be seen from FIG. 3, the virus titer of the triple virus harvest has a higher agreement with the data of the 10-layered cell factory despite the use of the 40-layered cell factory with a larger specification, indicating that the process has excellent stability and does not significantly decrease with the scale-up of the production scale. At the same time, the total amount of virus in each batch was counted to 4.18 × 1011CCID50，4.49*1011CCID50And 7.26 x 1011CCID50The statistical result shows that the three-batch production process is stable, achieves higher yield and can be used for producing the vaccine stock solution.
Comparative example 3: large-scale preparation method for mumps virus
The present comparative example provides a large scale preparation method for mumps virus, the large scale preparation method being:
cell preparation: removing head and viscera of 9-11 days old chick embryo (purchased from Lihua, Zhejiang Co.), and shearing into 1.5mm with scissors3The tissue blocks were digested with pancreatin (from Gibco) in an amount of 5 mL/chick embryo for 20min at 37 deg.C, and prepared into a mass of 2.0X 106one/mL of chicken embryo fibroblast suspension;
cell inoculation: adding the cell suspension into a cell transfer bottle (purchased from Sichuan cattle) at an addition amount of 200 mL/bottle, and performing primary culture at 34 ℃; 200mL of virus growth solution (the virus growth solution isBacto, cat # 259962 from GibcoTMTC whey protein hydrolysate);
virus inoculation: after the first culture for 48 hours, the mumps virus (with the preservation number of CCTCC No: V201950, which is described in the patent publication with the publication number of CN 111019910A) is inoculated into the cell suspension of the cell spinner bottle according to the inoculation amount of 0.001MOI, and the second culture is carried out at 34 ℃;
cell liquid change: after 24h of second culture, introducing the virus growth solution in the cell roller bottle into a waste solution tank, washing the cell roller bottle with 0.01M, pH 7.4.4 PBS buffer solution, adding 1000mL of solution into each roller bottle, shaking to wash the cell surface, introducing the solution into the waste solution tank, and repeating the operation for 3 times; after the cell surface was washed, a cell-maintaining solution (medium 199 available from Gibco under the trade name of 12350039) was poured into each of the flasks, 200mL of the solution was added to each flask, and the flask was cultured at 34 ℃ for the third culture;
harvesting: and after the third culture for 72 hours, leading out the virus culture solution in the cell spinner bottle to obtain the virus harvest solution.
Experimental example 3: experiment for influence of different scale preparation method modes on total virus harvest amount and total protein amount of virus harvest liquid
The experimental example provides an experiment for the influence of different large-scale preparation methods on the total virus harvest amount and the total protein amount of the virus harvest liquid, and the experimental process is as follows:
experiment one: infectious mumps virus titers in the virus harvest of example 1 and comparative example 3 were measured using the cell pathology method (third pharmacopoeia), and the results are shown in table 2.
Experiment two: after the virus harvest of example 2 and comparative example 3 was stirred at 500rpm for 20min, a high-speed refrigerated centrifuge (from Hitachi, model CR 22N) was used to perform continuous flow centrifugation at a fixed centrifugal force of 8000g, a loading speed of 700mL/min, and a temperature of 4 ℃ to obtain clarified virus harvest of example 2 and comparative example 3; the results of examining the total protein amount in the clarified virus harvest of example 2 and comparative example 3 using Lowry method (reference pharmacopoeia three), using the unclarified virus harvest as a blank control, are shown in table 2.
As can be seen from table 2, the culture conditions of the same surface area (20 310cm roller bottles equivalent to 1 CF10(6320 cm)) had higher titer of the virus harvested by the cell factory, almost consistent total protein amount of the two harvests, and more virus harvests available to the cell factory compared to roller bottle culture. In summary, the higher titer of virus fluid obtained from the cell factory process for the same total protein, the lower total protein content and higher quality when diluted to the same virus titer. Meanwhile, more virus harvesting liquid can be harvested in a single operation, the yield is better improved, the operation steps are simplified, the risk of pollution is reduced, and the method has the advantage of smaller occupied space.
TABLE 2 influence of different scale preparation methods on various detection indexes of the virus harvest
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (7)
1. A large-scale preparation method for mumps virus is characterized by comprising the following steps:
cell preparation: preparing a chicken embryo fibroblast suspension;
virus inoculation: after mumps virus with a preservation number of CCTCC No. V201950 is inoculated into chicken embryo fibroblast suspension according to the inoculation amount of 0.001-0.01 MOI, adding the cell suspension inoculated with the mumps virus into a cell factory in an addition amount of 150-250 mL/layer, and carrying out primary culture at 33-35 ℃; 150-250 mL of virus growth solution is added to each layer of the cell factory;
cell liquid change: after the first culture is carried out for 22-26 h, the virus growth solution in the cell factory is led out, the cell factory is washed by buffer solution, 100-200 mL of solution is added into each layer, and the solution is led out after the cell surface is washed by shaking; after the cell surface is cleaned, injecting cell maintenance liquid into a cell factory, adding 150-250 mL of liquid into each layer, and culturing at 33-35 ℃ for the second time;
a first harvesting step: after the second culture is carried out for 66-80 hours, the virus culture solution in the cell factory is led out, the cell maintenance solution is injected into the cell factory, 150-250 mL of liquid is added into each layer, and the third culture is carried out at 33-35 ℃;
a second harvesting step: after the third culture is carried out for 22-26 h, the virus culture solution in the cell factory is led out, the cell maintenance solution is injected into the cell factory, 150-250 mL of liquid is added into each layer, and the fourth culture is carried out at 33-35 ℃;
and a third harvesting step: after the fourth culture is carried out for 22-26 h, the virus culture solution in the cell factory is led out and is combined with the virus culture solution obtained by the first harvesting and the second harvesting to obtain a virus harvest solution;
or, the large-scale preparation method comprises the following steps:
cell preparation: preparing a chicken embryo fibroblast suspension;
virus inoculation: after mumps virus with a preservation number of CCTCC No. V201950 is inoculated into chicken embryo fibroblast suspension according to the inoculation amount of 0.001-0.01 MOI, adding the cell suspension inoculated with the mumps virus into a cell factory in an addition amount of 150-250 mL/layer, and carrying out primary culture at 33-35 ℃; 150-250 mL of virus growth solution is added to each layer of the cell factory;
cell liquid change: after the first culture is carried out for 22-26 h, the virus growth solution in the cell factory is led out, the cell factory is washed by buffer solution, 100-200 mL of solution is added into each layer, and the solution is led out after the cell surface is washed by shaking; after the cell surface is cleaned, injecting cell maintenance liquid into a cell factory, adding 150-250 mL of liquid into each layer, and culturing at 33-35 ℃ for the second time;
a first harvesting step: after the second culture is carried out for 66-80 hours, the virus culture solution in the cell factory is led out, the cell maintenance solution is injected into the cell factory, 150-250 mL of liquid is added into each layer, and the third culture is carried out at 33-35 ℃;
a second harvesting step: after the third culture is carried out for 22-26 h, the virus culture solution in the cell factory is led out, the cell maintenance solution is injected into the cell factory, 150-250 mL of liquid is added into each layer, and the fourth culture is carried out at 33-35 ℃;
and a third harvesting step: the third harvesting step is as follows: after the fourth culture is carried out for 22-26 h, the virus culture solution in the cell factory is led out, the cell maintenance solution is injected into the cell factory, 150-250 mL of liquid is added into each layer, and the fifth culture is carried out at 33-35 ℃;
and a fourth harvesting step: and after the fifth culture for 22-26 h, leading out the virus culture solution in the cell factory, and combining the virus culture solution with the virus culture solution obtained by the first harvest, the second harvest and the third harvest to obtain a virus harvest solution.
2. The method of claim 1, wherein the cell preparation step is: and (3) cutting chicken embryos incubated for 9-11 days into pieces, adding pancreatin for digestion, and then beating by blowing to obtain chicken embryo fibroblast suspension.
3. The method of claim 2, wherein the cell preparation step is: removing the head and the internal organs of 9-11 days old chick embryos, and shearing the chick embryos into 1-3 mm by using scissors3Adding pancreatin in the tissue blocks in an amount of 4-6 mL/chick embryo, digesting at 36-38 ℃ for 15-25 min, blowing and beating after digestion to prepare the chicken tissue blocks with the concentration of 1.7-2.5 multiplied by 106individual/mL of chicken embryo fibroblast suspension.
4. The method of claim 1, wherein the buffer is 0.01M, pH 7.4.4 PBS buffer.
5. A method for preparing a mumps attenuated live vaccine, which comprises the following steps:
the method comprises the following steps: carrying out large-scale preparation on the mumps virus attenuated strain by using the large-scale preparation method of any one of claims 1-4 to obtain a virus harvest;
step two; and separating and purifying the virus harvest liquid to obtain the mumps attenuated live vaccine.
6. The use of the large-scale production method of any one of claims 1 to 4 in the production of mumps virus vaccines.
7. The use of claim 6 wherein the mumps virus vaccine is a live attenuated mumps vaccine.
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