KR102228267B1 - Methods for virus production using BHK-21 cell - Google Patents

Abstract

The present invention relates to a method for mass-producing various types of viruses using BHK-21 cells. Since the present invention has established the optimal conditions for effectively producing a large amount of virus, and can be applied to the production of various types of viruses, it is expected that it can be usefully used in related pharmaceutical fields such as viral drug production.

Description

Methods for virus production using BHK-21 cells {Methods for virus production using BHK-21 cells}

The present invention relates to a method for mass-producing various types of viruses using BHK-21 cells.

Viruses are infectious particles of several hundred μm or less that are parasitic in living cells such as animals, plants, bacteria, and radioactive bacteria and can only grow within cells. It has the characteristics of living organisms that replicate the same thing as itself, but some determine it as a nucleoprotein outside the cell. According to the type of nucleic acid, it is classified into DNA virus and RNA virus, and it is classified into animal virus, plant virus, and bacteriophage depending on the host. Viruses do not have their own metabolic system, so they synthesize necessary enzyme proteins through the metabolic system of host cells using viral nucleic acids as a template, replicate viral nucleic acids, and at the same time make antigen proteins, assemble them to complete new viruses and release them out of the cell. do. At this time, the virus contains sugar lipids, kills cells and shows pathogenicity.

On the other hand, oncolytic virus (OV) is a replication therapeutic agent designed or selected to specifically grow and kill tumor cells. Several groups are in the early stages of commercialization based on pre-clinical animal models and early clinical data in humans. However, a challenge still facing in the art is the large-scale production of pharmaceutically usable viruses for delivery to patients. Since viruses are real organisms rather than synthetic drugs, the manufacturing process should include removing contaminating cellular debris while maintaining the viral production and infectious effects within the cells.

In general, for vaccination applications, patients are given low doses of viruses in a local area (often intramuscularly) where the contaminating cellular protein can actually act as an adjuvant, thereby increasing the immunogenicity of the agent. In contrast, in the case of oncolytic viral therapeutics, the dose of virus required is up to a million times greater than the dose for vaccine application and can be administered by intravenous, intracranial, intraperitoneal or direct tumor injection. Therefore, in the case of OV, it is important to be able to prepare a large amount of virus from which contaminating non-human cell debris has been removed, but related studies on an improved method for large-scale oncolytic virus production are insufficient.

Republic of Korea Published Publication No. 10-2014-0058601

Accordingly, the present inventors have repeatedly researched the method for mass production of viruses, and as a result, select BHK-21 cells as the optimal virus-producing cell line, and establish the optimal conditions (MOI, infection period, etc.) for economically producing the virus. By doing this, the present invention was completed.

Accordingly, it is an object of the present invention to provide a virus production method.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems that are not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention, the present invention provides a virus production method comprising the following steps:

(a) contacting BHK (Baby Hamster Kidney)-21 cells with a virus at 0.0001 to 0.5 MOI (multiplicity of infection) to generate infected BHK-21 cells;

(b) culturing the BHK-21 cells infected in step (a) in a medium; And

(c) separating the virus from the culture of step (b).

In addition, the present invention provides a virus produced by the virus production method.

In one embodiment of the present invention, the virus of step (a) may be a mammalian reovirus, but is not limited thereto.

In another embodiment of the present invention, the mammalian reovirus may be a human reovirus, but is not limited thereto.

In another embodiment of the present invention, the human reovirus may be a serotype 3 reovirus, but is not limited thereto.

In another embodiment of the present invention, the reovirus of the serotype 3 may be a Dearing main (reovirus type 3 Dearing), but is not limited thereto.

In another embodiment of the present invention, the step (a) may be to infect BHK-21 cells with a virus of 0.001 to 0.5 MOI, but is not limited thereto.

In another embodiment of the present invention, the step (a) may be to infect BHK-21 cells with a virus of 0.005 to 0.5 MOI, but is not limited thereto.

In another embodiment of the present invention, the step (a) may be to infect BHK-21 cells with a virus of 0.005 to 0.1 MOI, but is not limited thereto.

In another embodiment of the present invention, the step (a) may be to infect BHK-21 cells with a virus of 0.01 to 0.5 MOI, but is not limited thereto.

In another embodiment of the present invention, the step (a) may be to infect BHK-21 cells with a virus of 0.01 to 0.1 MOI, but is not limited thereto.

In another embodiment of the present invention, the step (b) may be cultured for 1 to 10 days after virus infection, but is not limited thereto.

In another embodiment of the present invention, step (b) may be cultured for 2 to 8 days after virus infection, but is not limited thereto.

In another embodiment of the present invention, the step (b) may be cultured for 3 to 8 days after virus infection, but is not limited thereto.

In another embodiment of the present invention, the step (b) may be cultured for 4 to 7 days after virus infection, but is not limited thereto.

In another embodiment of the present invention, the step (b) may be cultured for 5 to 7 days after virus infection, but is not limited thereto.

In another embodiment of the present invention, the step (b) may be cultured for 5 to 6 days after virus infection, but is not limited thereto.

In another embodiment of the present invention, step (a) infects BHK-21 cells with a virus of 0.001 to 0.1 MOI, and step (b) can be cultured for 5 to 6 days after virus infection. , But is not limited thereto.

In another embodiment of the present invention, step (a) infects BHK-21 cells with a virus of 0.01 to 0.1 MOI, and step (b) can be cultured for 5 to 6 days after virus infection. , But is not limited thereto.

In another embodiment of the present invention, the medium in step (b) may be selected from the group consisting of DMEM, MEM, EMEM, RPMI-1640, F-12, and DMEM/F12 medium, but is not limited thereto. .

In another embodiment of the present invention, the virus in the step (c) is a supernatant obtained by centrifuging the medium of (i) step (b) during or after the culturing, and (ii) the medium After centrifugation, the resulting pellet may be mixed with the infected cells remaining in the culture obtained in step (b), crushed the cells in the mixture, and then separated from the supernatant obtained by centrifugation, but is not limited thereto. .

In another embodiment of the present invention, the cell disruption may be performed by performing sonication, but is not limited thereto.

According to another embodiment of the present invention, the step (c) comprises the steps of crushing the cells in the culture of step (b); And performing CsCl gradient centrifugation of the cell disruption result, but is not limited thereto.

According to another embodiment of the present invention, the CsCl oblique centrifugation may be performed two or more times, but is not limited thereto.

The virus production method of the present invention can be mass-produced using a cell factory system using BHK-21 cells, and the production yield is high as not only the medium but also the residual virus present in the cells is recovered. Since the present invention has established the optimal conditions for effectively producing a large amount of virus, and can be applied to the production of various types of viruses, it is expected that it can be usefully used in related pharmaceutical fields such as viral drug production.

1A shows BHK-21 cells were infected with reovirus at various MOIs (0.01, 0.1, 1, 10, 30, 100 MOI) and then cultured for 3 or 5 days, and then collected and centrifuged to collect the cultured medium. It is a diagram showing the production _{amount (TCID 50} ) and production yield of the reovirus present in the supernatant (Sup alone).
1b is obtained by incubating BHK-21 cells with reovirus at various MOIs (0.01, 0.1, 1, 10, 30, 100 MOI) for 3 or 5 days, and then collecting and centrifuging the cultured medium. It is a diagram showing the production _{amount (TCID 50} ) and production yield of the reovirus present in the pellet and infected cells (Pellet alone).
Figure 1c is a diagram showing the sum of the amount of reovirus production (TCID ₅₀ ) and production yield present in Sup alone and Pellet alone (Sup+Pellet).
Figure 2a shows BHK-21 cells in various MOIs (0.0001, 0.001, 0.01, 0.1, 1, 10 MOI) after infection with the reovirus and cultured for 1 to 10 days, and the culture of the reovirus present in Sup + Pellet by day It is a diagram showing the production volume (TCID _{50 ).}
Figure 2b shows BHK-21 cells in various MOIs (0.0001, 0.001, 0.01, 0.1, 1, 10 MOI) after infection with the reovirus and cultured for 1 to 10 days, and the reovirus present in Sup + Pellet produced by culture day It is a diagram showing the production yield.

The present invention provides a method for producing a virus comprising the following steps:

(a) contacting BHK (Baby Hamster Kidney)-21 cells with a virus at 0.0001 to 0.5 MOI (multiplicity of infection) to generate infected BHK-21 cells;

(b) culturing the BHK-21 cells infected in step (a) in a medium; And

(c) separating the virus from the culture of step (b).

In addition, the present invention provides a virus produced by the virus production method.

Specifically, the present inventors compared and evaluated the virus productivity according to the isolation location in the case of producing a virus using BHK-21 cells through Examples (see Example 1), and additional experiments based on the results of Example 1. The optimal virus production conditions (virus MOI, number of days of infection) were established through (see Example 2).

In the term "BHK-21 cells" as used herein , BHK stands for B aby H amster K idney, and BHK cells were originally isolated by polyoma transformation of hamster cells. , Can be used as a substrate for viral transmission and viral mediated expression for vaccines. In addition, BHK cells are useful as host cell lines for the stable expression of various recombinant proteins. The BHK-21 (BHK Strain 21) cell line was derived from baby hamster kidneys of five unsexed, 1-day-old hamsters in March 1961 by IA Macpherson and MGP Stoker. The hamster was used to generate BHK-21 cells, and is generally known as a Syrian golden hamster (Mesocricetus auratus).

As used herein, the term “virus” refers to a virus capable of infecting the BHK-21 cells and replicating within the cells, and is not limited to a specific virus.

In the present invention, the virus may be a mammalian reovirus, but is not limited thereto.

As used herein, the term “reovirus” refers to a virus having a double-stranded segmented RNA genome, and refers to any virus classified as Reoviridae. The virion of the reovirus has a diameter of 60 to 80 nm and has two concentric capsid shells. This genome is composed of 10-12 discrete segments, double-stranded RNA, and has a total genome size of 16-27 kbp, and each RNA segment has a different size.

In the present invention, the reovirus includes not only a naturally occurring reovirus, but also a modified or recombinant reovirus. The reovirus can be isolated from nature and is said to be "naturally occurring" when no artificial alteration has been made by humans. For example, the reovirus can be from a “field source”, ie from a person infected with this reovirus.

The reovirus can be modified, but lytically infect mammalian cells with an activated ras pathway. In addition, the reovirus may be pretreated chemically or biochemically (eg, by treatment with a protease such as chymotrypsin or trypsin) before administration to proliferating cells. By pretreatment using a protease, the outer membrane or capsid of the virus can be removed, thereby increasing the infectivity of the virus. The reovirus may be coated with liposomes or micelles, and for example, virion may be treated with chymotrypsin in the presence of an alkyl sulfate detergent having a micelle-forming concentration to generate new infectious subviral particles. have.

In the present invention, the mammalian reovirus may be a human reovirus, but is not limited thereto.

Human reovirus can be composed of three serotypes: type 1 (Lang strain or T1L), type 2 (Jones strain, T2J) and type 3 (Dearing strain, T3D or Abney strain, T3A). The three serotypes can be easily identified based on a neutralization reaction and an assay for inhibiting hemagglutination.

In the present invention, the human reovirus may be a serotype 3 reovirus, but is not limited thereto.

In the present invention, the human reovirus may be a reovirus serotype 3 Dearing (Reovirus type 3 Dearing, T3D), but is not limited thereto.

As used herein, the term "infected BHK-21 cells" refers to a state in which the virus invades into BHK-21 cells and then the virus is replicated within the cell.

As used herein, the term “MOI” stands for multiplicity of infection, and is the ratio of the amount of inoculated virus to the number of cells inoculated with the virus (the amount of inoculated virus/number of cells). It indicates how many infectious viruses can be obtained per one. In practice, the MOI does not indicate the number of virus particles that have invaded into the cell as it is, because a lot of virus particles that do not invade remain in the inoculum without being adsorbed or even if adsorbed.

In the present invention, the step (a) may infect BHK-21 cells with a virus of 0.001 to 0.5 MOI. For example, the step (a) is 0.001 to 0.4, 0.001 to 0.3, 0.001 to 0.2 or 0.001 to 0.1 MOI; 0.002 to 0.5, 0.002 to 0.4, 0.002 to 0.3, 0.002 to 0.2 or 0.002 to 0.1 MOI; 0.003 to 0.5, 0.003 to 0.4, 0.003 to 0.3, 0.003 to 0.2 or 0.003 to 0.1 MOI; Alternatively, BHK-21 cells can be infected with a virus of 0.004 to 0.5, 0.004 to 0.4, 0.004 to 0.3, 0.004 to 0.2, or 0.004 to 0.1 MOI.

In the present invention, the step (a) may infect BHK-21 cells with a virus of 0.005 to 0.5 MOI.

In the present invention, the step (a) may infect BHK-21 cells with a virus of 0.005 to 0.1 MOI.

In the present invention, the step (a) may infect BHK-21 cells with a virus of 0.01 to 0.5 MOI.

In the present invention, the step (a) may infect BHK-21 cells with a virus of 0.01 to 0.1 MOI.

For example, the step (a) is 0.005 to 0.4, 0.005 to 0.3, 0.005 to 0.2 or 0.005 to 0.1; 0.006 to 0.5, 0.006 to 0.4, 0.006 to 0.3, 0.006 to 0.2 or 0.006 to 0.1; 0.007 to 0.5, 0.007 to 0.4, 0.007 to 0.3, 0.007 to 0.2 or 0.007 to 0.1; 0.008 to 0.5, 0.008 to 0.4, 0.008 to 0.3, 0.008 to 0.2 or 0.008 to 0.1; 0.009 to 0.5, 0.009 to 0.4, 0.009 to 0.3, 0.009 to 0.2 or 0.009 to 0.1; Alternatively, BHK-21 cells can be infected with a virus of 0.01 to 0.5, 0.01 to 0.4, 0.01 to 0.3, 0.01 to 0.2, or 0.01 to 0.1 MOI.

As used herein, the term “culture” refers to any action performed to propagate a virus while growing a virus-infected cell in an appropriately artificially controlled environmental condition.

In the present invention, the cell culture may be performed using a cell factory system. According to the above method, there is an advantage that mass production is possible by scaling up the virus during production.

In the present invention, any medium or culture medium commonly used for cell culture may be used for viral growth or production in this field.

In the present invention, the medium in step (b) may be a medium selected from the group consisting of DMEM, MEM, EMEM, RPMI-1640, F-12, and DMEM/F12 medium, but is not limited thereto. For example, the present invention may use the DMEM/F12 medium in step (b).

In the present invention, the step (b) may be cultured for 1 to 10 days after virus infection, but is not limited thereto. For example, the step (b) may be 1 to 9, 1 to 8, 1 to 7, 1 to 6, or 1 to 5 days after virus infection; 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, or 2 to 5 days; 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6 or 3 to 5 days; 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, or 4 to 5 days; 5 to 10, 5 to 9, 5 to 8, 5 to 7 or 5 to 6 days; Alternatively, it may be cultured for 6 to 10 days, 6 to 9 days, 6 to 8 days, or 6 to 7 days.

In the present invention, the step (b) may be cultured for 2 to 8 days after virus infection, but is not limited thereto.

In the present invention, the step (b) may be cultured for 3 to 8 days after virus infection, but is not limited thereto.

In the present invention, the step (b) may be cultured for 4 to 7 days after virus infection, but is not limited thereto.

In the present invention, the step (b) may be cultured for 5 to 7 days after virus infection, but is not limited thereto.

In the present invention, the step (b) may be cultured for 5 to 6 days after virus infection, but is not limited thereto.

In the present invention, in the step (a), the BHK-21 cells are infected with a virus having an MOI selected from the above numerical range, and in the step (b), the cells may be cultured for a culture day selected from the number of culture days described above after virus infection. . For example, the present invention can be practiced as follows.

In the present invention, step (a) infects BHK-21 cells with a virus of 0.001 to 0.1 MOI, and step (b) may be cultured for 4 to 7 days after virus infection, but is limited thereto. no.

In addition, step (a) infects BHK-21 cells with a virus having an MOI of 0.001 to 0.1 MOI, and step (b) may be cultured for 5 to 6 days after virus infection, but is not limited thereto.

In addition, the step (a) infects BHK-21 cells with a virus of 0.01 to 0.1 MOI, and the step (b) may be cultured for 4 to 7 days after virus infection, but is not limited thereto.

In addition, the step (a) infects BHK-21 cells with a virus of 0.01 to 0.1 MOI, and the step (b) may be cultured for 5 to 6 days after virus infection, but is not limited thereto.

The method of separating the virus from the culture in step (c) of the present invention may be used without limitation through a method known in the art capable of separating and purifying the virus.

In the present invention, the virus may be separated in the middle of culture, or may be separated at a time after the desired number of culture days is completed.

In the present invention, the virus in step (c) is a supernatant obtained by centrifuging the medium of step (b) during or after (i) culturing, and (ii) generated after centrifugation of the medium. After mixing the infected cells remaining in the culture obtained in step (b) to the pellet, the cells in the mixture may be crushed and then separated from the supernatant obtained by centrifugation, but is not limited thereto.

In the present invention, the virus isolation in step (c) comprises the steps of disrupting the cells in the culture of step (b); And performing CsCl gradient centrifugation of the cell disruption result, but is not limited thereto.

For example, the virus separation in step (c) is during or after the culture is completed, the supernatant obtained by centrifuging the medium of step (b) ((i) supernatant) and after centrifugation of the medium Obtaining a supernatant ((ii) supernatant) obtained by mixing the resulting pellet with the infected cells remaining in the culture obtained in step (b), crushing the cells in the mixture, and then centrifuging; And (i) and (ii) decanting the supernatant with CsCl, but is not limited thereto.

In the present invention, the cell disruption may be performed by performing sonication, but is not limited thereto. The sonication treatment is not a conventional cell freeze-thawing method or a surfactant (detergent) method, but the sonication treatment, which wakes up the cells and recovers the residual virus present in the cells without discarding the cells, and thus has a high virus yield.

In the present invention, the CsCl gradient centrifugation may be performed two or more times. Higher purity virus can be obtained through two or more decanter centrifugation of CsCl.

Hereinafter, a preferred embodiment is presented to aid the understanding of the present invention. However, the following examples are provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[Example]

Example 1. Reovirus productivity comparison according to the separation site (pellet vs sup)

BHK (Baby Hamster Kidney)-21 cells (1 x 10 ⁶ cells/dish) are infected with Reovirus type 3 Dearing of 0.01, 0.1, 1, 10, 30 and 100 MOI (multiplicity of infection, PFU standard) CPE (Cytopathic effect) 80% or more at the time point where the cells were cultured (DMEM: F-12, Ham's nutrient mixture F-12 and Dulbecco's modified Eagle's essential medium, ThermoFisher) was added to each 15 ml bottle for centrifugation ( centrifuge bottle) and centrifuged (3,270±20 g, 10±0.5 minutes). After centrifugation, only the supernatant, excluding the cell pellet, was separated and collected in a 50 ml bottle (Sup, about 8.5 ml).

In addition, after adding 2 ml of fresh medium to the remaining cell pellet, sonication (3 minutes) is performed to break the cells, collect the same amount in a 15 ml centrifuge bottle, and centrifuge (3,270±20 g). , 10±0.5 minutes) and the supernatant was collected again (Pellet, about 4.5 ml).

For gradient centrifugation of cesium chloride (CsCl), 9 ml of Sol'A (1.40 CsCl, 62 g in 100 ml PBS), Sol'B (1.25 CsCl, 36 g in) in an Ultra tube. 100 ml PBS) 9 ml and 18 ml of a sample subjected to the sonication for 3 minutes were sequentially added, and a first ultracentrifuge was performed at 26,000 rpm for 2 hours. Then, after removing the supernatant by suction, leaving only the lower reovirus band, take only the reovirus and transfer it to a new 50 ml tube, and then add PBS (phosphate buffered saline) equal to the volume of the reovirus to mix well. I got it. Then, 15 ml of Sol'C (1.38 CsCl, 60 g in 100 ml PBS) and 20 ml of a sample mixed with PBS were sequentially added to an ultra tube, and a second ultracentrifugation was performed at 26,000 rpm for 2 hours. After removing the supernatant by suction, leaving only the reovirus band, only the reovirus was taken and transferred to a new 50 ml tube.

For viral dialysis, the reovirus is put in a dialysis tubing and well sealed with a closure, and then dialysis is carried out by placing it in a 2.4 L dialysis reservoir containing 2 L PBS and a magnetic bar. It was performed, and PBS was exchanged three times in total (PBS was exchanged at intervals of 16 hours for the first time and 3 hours from the second time). When the dialysis was finished, it was stored at 4°C.

As a result of the experiment, as shown in FIGS. 1A and 1B, when the virus is isolated from Sup alone and Pellet alone when producing a reovirus using BHK-21 cells, the optimal production conditions are 30 MOI and 5 days incubation period, respectively. Was. In addition, as shown in Figure 1c, in the case of separating the virus from Sup + Pellet, a much lower MOI (10-100 MOI), the greatest amount of reovirus was produced when the culture period was 5 days. These results show that producing virus from Sup + Pellet can produce a large amount of virus at a low MOI, so it is excellent in economics.

Example 2. Comparison of reovirus productivity according to infection period

BHK-21 cells (1 x 10 ⁶ cells/dish) were infected with reovirus of 0.0001, 0.001, 0.01, 0.1, 1 and 10 MOI (based on PFU), and then cultured for 10 days. -12, Ham's nutrient mixture F-12 and Dulbecco's modified Eagle's essential medium, ThermoFisher) were collected in equal amounts in a 15 ml centrifuge bottle, and centrifuged (3,270±20 g, 10±0.5 minutes) for the supernatant. Only collected (Sup, 8 ml). After centrifugation, the remaining pellets are combined with the infected cells and subjected to sonication (sonication 10 minutes), collected in an equal amount in a 500 ml centrifuge bottle, and centrifuged (3,270 ± 20 g, 10 ± 0.5 minutes), and then only the supernatant. Collected (Pellet, about 4.5 ml). Thereafter, cesium chloride decanter centrifugation and virus dialysis were performed as in Example 1.

As a result of the experiment, it was confirmed that the present invention is an effective virus production method, such as having a high yield since it is possible to replicate a large amount of virus and recover residual virus in cells by improving over the existing virus production method. Specifically, as shown in Fig. 2a, the ^{maximum amount of reovirus that 1 x 10 6} BHK-21 cells could produce was about +10 ¹⁰ CID ₅₀ /ml, and when cultured for more than 4 days (4 days post infection) Treatment of more than 0.001 MOI produced almost similar amounts of reovirus, and when cultured for more than 8 days, the reovirus titer tended to decrease. In addition, when the reovirus was treated at a high concentration (1 or 10 MOI), the amount of reovirus production was high until the initial stage (3 days), but after that, it showed a tendency to decrease rather than that of the low MOI treatment group.

Taken together, when both production (Fig. 2a) and production rate (Fig. 2b) are considered, it is possible to efficiently produce a large amount of virus by treating the virus at an MOI of 0.001-0.1 and culturing it for 5 to 6 days. Treatment with 0.01 to 0.1 MOI, and incubation for 5 to 6 days, the virus can be produced more efficiently.

The above description of the present invention is for illustrative purposes only, and those of ordinary skill in the art to which the present invention pertains will be able to understand that other specific forms can be easily modified without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative and non-limiting in all respects.

Claims (23)

Virus production method comprising the following steps:
(a) contacting BHK (Baby Hamster Kidney)-21 cells with a virus at 0.005 to 0.1 MOI (multiplicity of infection) to generate infected BHK-21 cells;
(b) culturing the BHK-21 cells infected in step (a) in a medium; And
(c) separating the virus from the culture of step (b),
The virus in step (c) is added to a supernatant obtained by centrifuging the medium of step (b) during or after the culturing (i) and (ii) a pellet produced after centrifugation of the medium in step (b). ) After mixing the infected cells remaining in the culture obtained in), crushing the cells in the mixture, and then separating the cells from the supernatant obtained by centrifugation. The method of claim 1,
The virus of the step (a) is characterized in that the mammalian reovirus, virus production method. The method of claim 2,
The mammalian reovirus is a human reovirus, characterized in that, virus production method. The method of claim 3,
The human reovirus is characterized in that the reovirus of serotype 3, virus production method. The method of claim 4,
The reovirus of the serotype 3 is characterized in that Dearing main (reovirus type 3 Dearing), virus production method. delete delete delete delete The method of claim 1,
The step (a) is characterized in that infecting BHK-21 cells with a virus of 0.01 to 0.1 MOI, virus production method. The method of claim 1,
The step (b) is characterized in that the culture for 1 to 10 days after the virus infection, virus production method. The method of claim 1,
The step (b) is characterized in that the culture for 2 to 8 days after the virus infection, virus production method. The method of claim 1,
The step (b) is characterized in that the culture for 3 to 8 days after the virus infection, virus production method. The method of claim 1,
The step (b) is characterized in that the culture for 4 to 7 days after the virus infection, virus production method. The method of claim 1,
The step (b) is characterized in that the culture for 5 to 7 days after the virus infection, virus production method. The method of claim 1,
The step (b) is characterized in that the culture for 5 to 6 days after the virus infection, virus production method. delete The method of claim 1,
The step (a) is infecting BHK-21 cells with a virus of 0.01 to 0.1 MOI, and the step (b) is a virus production method, characterized in that incubation for 5 to 6 days after virus infection. The method of claim 1,
In the step (b), the medium is selected from the group consisting of DMEM, MEM, EMEM, RPMI-1640, F-12, and DMEM/F12 medium. delete The method of claim 1,
The step (c), the step of disrupting the cells in the culture of step (b); And CsCl gradient centrifuge the cell disruption result. The method of claim 21,
The CsCl decanter centrifugation is characterized in that to perform two or more times, virus production method. The method of claim 21,
The cell disruption is characterized in that by performing ultrasonic treatment (sonication), virus production method.

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