KR101956440B1 - Process for the production of virus using semi-continuous cell culture system - Google Patents

Abstract

The present invention relates to a method for producing a virus by a semi-continuous culture method and a vaccine using the virus prepared using the method, and more particularly, to a method for producing a virus using a conventional continuous infection cell line (Batch culturing system, BCS) By using a semi-continuous culture method (SCCCS method) using a fish cell line that has been continuously infected with megalochoti virus to continuously produce virus at a high concentration while overcoming the complexity and inefficiency of subculturing a new cell line and incubating the virus, Even long-term semi-continuous cultivation can produce viruses of high concentration without decreasing or decreasing the number of host cell lines. Thus, it is possible to easily and continuously produce a high concentration of megalochotivirus, which makes it easy to prepare vaccines and industrialize them.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for producing a virus using a semi-continuous culture method,

The present invention relates to a method for producing a virus by a semi-continuous culture method and a vaccine using the virus prepared using the same.

Conventionally, a BCS (Batch Culture system) has been used in order to produce a virus after infecting a cell with a virus, and a virus infection supernatant is obtained and the infected cell line is discarded together with a culture container. Therefore, in order to obtain an additional amount of viral solution, a new continuously infected cell line is divided into a culture container and cultured (subculturing, subculture), and if it is not a continuous infectious cell line, Is not high. Furthermore, because of the characteristics of the closed system BCS method, the culture of the host cells is only performed for 5-7 days, and the intracellular accumulated virus does not regenerate the virus. There has been a problem that it is not achieved.

IMV (Iridoviridae Megalocytivirus) is the causative agent of Red Sea Bream Iridovirus Disease (RSIVD). However, in the aquaculture field, the dolomite is much more susceptible than the red sea bream which has a cumulative mortality rate of around 10%, and when the infection occurs at high temperature, 90-100% mass mortality occurs. In order to develop a vaccine for preventing the infection of such a megalopoietic virus, it is necessary to cultivate a large amount of virus, but the megalochoti virus propagates only in limited fish cell lines, and as the virus passage increases in the subculture, (Chou et al., 1998; Imajoh et al., 2007; Nakajima and Sorimachi, 1994), a very specific infection with decreased virus production resulting in decreased virus concentration in the culture medium , There was much difficulty in industrial production of viruses.

The only known countermeasure currently available for MVC, which is devastating to marine aquaculture, is vaccine development. In particular, it causes high incidence rate and many mortalities every year in the dolomite. However, the vaccine has not been effective in the development until now, and the reason is that it is difficult to produce a high concentration vaccine because the concentration of the culture virus to be used is low.

Among the commercially available cell lines, RTG-2 (chinook salmon embryo), FHM (fathead minnow), BF-2 (blue gill fry), and the like are known to show megalovirus susceptibility. , And KRE-3 (belp and reed spotted grouper embryo), which are known to proliferate as cytotoxic effects (CPE) occur (Nakajima and Sorimachi, 1994a; Chou et al., 1998). However, all of these coin cells have been known to decrease in viral titer after passage of megalochotivirus, and thus it has been difficult to economically supply large quantities of megalocytaviruses required for vaccine production.

Since all the commercialized coin cells have difficulties in cultivating these high-concentration viruses continuously, and since the concentration of the produced viruses is low (industrial enrichment of viruses requires high cost, it is not economically meaningful) (Imajoh et al., 2007; Dong et al., 2013). Therefore, in order to develop an industrial vaccine of megalochotivirus, a method of producing a more efficient and continuous high concentration virus which solves these problems is needed. In the present invention, various coin cells are used to produce various megarotic viruses We have developed a method to increase the virus productivity while eliminating the reduction of infectivity by the number of culture and subculture.

In the present invention, in order to continuously produce virus at a high concentration while overcoming the complexity and inefficiency of subculturing a new cell line and culturing the virus in a batch culture system (BCS) using a conventional continuous infectious cell line, (SCCCS method) using a fish cell line continuously infected with megalochoti virus.

In order to achieve the above object, the present invention provides a method for producing a virus, comprising infecting a fish coin cell with a virus; Culturing the infected cells; A semi-continuous cell culture step in which a culture supernatant is obtained and replaced with fresh medium; And separating the virus from the resulting culture supernatant.

In addition, the present invention provides a vaccine composition comprising the virus produced by the virus production method of the present invention.

The semi-continuous culture method (SCCCS) of the present invention is a method in which a culture supernatant of an infected cell line is taken without subculturing a new cell line and a new infection process, and then a new medium is added again. In particular, It is possible to produce a high concentration of virus without a decrease in the number of hosts or decrease in the number of host cells, and thus it is possible to easily and continuously produce a high concentration of megalochotivirus, and a very high concentration of virus can be obtained in a short cycle So that it is possible to mass-produce, and the possibility of contamination that may occur from the outside is low, and it is easy to prepare a vaccine and industrialize it.

Brief Description of the Drawings Fig. 1 is a diagram showing the virus concentration in the supernatant obtained daily after IMV infection in PMF, GF-1 and BF-2 cell lines.
FIG. 2 is a microscopic view showing cell degeneration after 10 times of continuous culture:
A: PMV-infected PMF cell line (0 times culture);
B: IMV infected GF-1 cell line (0 times culture);
C: IMV-infected BF-2 cell line (0 times incubation);
D: IMV-infected PMF cell line 10 times cultured by SCCCS culture method (10 times with addition of medium after obtaining supernatant);
E: IMV-infected GF-1 cell line 10 times cultured by SCCCS culture method (10 times culture medium after supernatant was obtained); And
F: IMV-infected BF-2 cell line cultured 10 times by SCCCS culture method (10 times with addition of culture medium after obtaining supernatant).
FIG. 3 is a graph for confirming the productivity of virus according to medium exchange cycles (1 day, 3 days, and 5 days) in producing IMV in the PMF cell line by the SCCCS culture method.
FIG. 4 is a graph showing the virus productivity according to the ratio (30%, 70%, and 100%) of medium to be exchanged with a fresh medium when the medium was changed every 3 days in the production of IMV by the SCCCS culture method in the PMF cell line .
FIG. 5 is a graph showing the virus productivity according to the culture medium (L-15, MEM, and RPMI1640) in the production of IMV by the SCCCS culture method in the PMF cell line.
6 is a graph showing virus productivity according to FBS concentration (1%, 5% and 10%) in the culture medium when producing IMV in the PMF cell line by the SCCCS culture method.
FIG. 7 is a graph showing virus productivity according to culture temperatures (20 ° C, 23 ° C, 25 ° C, 28 ° C and 30 ° C) in the production of IMV by the SCCCS culture method in the PMF cell line.
FIG. 8 is a graph showing the cell number and virus productivity at the time of producing IMV in the PMF cell line using the optimized SCCCS method according to the number of times of exchanging the medium.
FIG. 9 shows the morphology and cell degeneration of cells in a PMF cell line in which IMV was continuously infected using an optimized SCCCS method:
A: IMV-infected PMF cell line before virus culture;
B: IMV-infected PMF cell line 20 times after medium exchange;
C: IMV-infected PMF cell line 50 times after medium exchange; And
D: IMV-infected PMF cell line after 100 times of medium exchange.
Figure 10 shows the cell changes after 20 times of medium exchange with the optimized SCCCS method for PMF infected with the IMV isolate subgroup II, III or IV:
A: PMF before virus infection;
B: PMF infected with subgroup II isolates;
C: PMF infected with subgroup III isolate; And
D: PMF infected with subgroup IV isolate.
11 is a graph showing the continuous productivity of virus according to the IMV isolate subgroup II, III or IV in the PMF cell line using the optimized SCCCS method.

Hereinafter, the present invention will be described in detail with reference to embodiments of the present invention. It should be understood, however, that the invention is not limited thereto and that various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined by the appended claims and their equivalents. .

In one aspect, the present invention provides a method of treating a fish coin cell comprising infecting a virus with a virus; Culturing the infected cells; A semi-continuous cell culture step in which a culture supernatant is obtained and replaced with fresh medium; And separating the virus from the resulting culture supernatant.

In one embodiment, the virus of the present invention may be the Iridoviridae family virus, preferably megalocytivirus, which is a disease causing virus in fish.

In one embodiment, the viruses produced in the present invention may be megalocytiviruses selected from the group consisting of megalochotivirus subgroup II, subgroup III, and subgroup IV.

In one embodiment, the virus may be inoculated into fish coin cells and the medium may be continuously infected with viruses by more than 20 times of medium exchange every 3 days.

In one embodiment, the infected cells can be cultured at 25 占 폚 in a medium containing 5% fetal bovine serum (FBS).

In one embodiment, the medium change period when the infected cells are semi-continuous incubated is 3 days, and the medium exchange rate to replace the fresh medium may be 70% (v / v).

The semi-continuous culture of the present invention means that a culture containing viruses produced from virus-infected cells is obtained, and cultivation is carried out by inserting a new culture solution while leaving the cells intact. In one embodiment of the present invention, virus production optimum conditions were established in which 70% of the cell culture supernatant (cell culture medium) was obtained every 3 days and the new medium was replenished.

In one embodiment, the fish-derived coin cell may be a coin cell of a fish (susceptible) that is infected with the virus, for example, a dolomite, a sea bream, an angel fish dome, a pointed sea bass, a flounder, , A group consisting of pagrus major fin (PMF) cell line, grouper fin (GF-1) cell line and bluegill fry (BF-2) cell line , And the PMF cell line (Accession No. KCLRF-BP-00324, Korea Cell Line Bank) which is most optimized for the semi-continuous culture method of the present invention is most preferable.

The term "coin cell" of the present invention refers to a cell that has been cell-cultured through continuous subculture by separating the cells from fish tissue, and the term " cell line "

In one embodiment of the present invention, the PMF cell line produced 100 times more viruses than GF-1 and BF-2 when cultured by the semi-continuous culture method of the present invention, And thus it was confirmed that the cell line was optimized for the semi-continuous culture method of the present invention. Generally, when the cell lines infected with the virus are cultured semi-continuously or continuously, due to denaturation or death of the cells, virus production can not be performed and the cells have to be subcultured.

In one embodiment of the present invention, when the semi-continuous culture method and the conventional in vitro subculture method were compared with each other, the yield of megalochotivirus production was compared with that of the conventional subculture, Of the total virus production and the average amount of virus produced during one day, it was confirmed that viruses with a high concentration of up to 10 times or more can be obtained.

The term "passage" of the present invention means delivering the cells from one culture vessel to another without dilution or without dilution. Every time a cell is transferred from one container to another, certain parts of the cell may become defective, resulting in dilution of the cell, whether intentionally or unintentionally. This term has the same meaning as the term " subclture ". The number of subculture cultures is the number of times the cells are cultured and is then cultured or transferred into a new container to grow into a suspension or adherent state. This term is not synonymous with the population doubling time or the time required for the cell population to replicate, that is, the time it takes for an individual cell to replicate in a cell population.

In one aspect, the present invention relates to a vaccine composition comprising a virus inactivated virus prepared by the virus production method of the present invention and a pharmaceutically acceptable carrier or excipient.

In one embodiment, the vaccine composition can be used as a vaccine for the prevention or treatment of fish megalocytovirus infection.

In one embodiment, the fish may be selected from the group consisting of a red sea bream, a red sea bream, a red sea bream, a point bass, a halibut, a rhododendron, a strong leg, a rockfish, a fish and a fish.

In one embodiment, the vaccine composition may contain an inactivated megalochotivirus. The megalopoietic virus may be a subgroup II, subgroup III, or subgroup IV dissociative strain. Therefore, in the present invention, the virus related to such treatment is preferably administered to the immune system of an organism to induce an immune response to affect a specific immune response, thereby preventing the organism from being infected with the virus do.

For the production of the preventive composition (i. E., Vaccine) of the present invention, the megalocytivirus inactivated with formalin according to the present invention may be used as a vaccine or transformed into a physiologically acceptable form. This can be done based on experience in the production of chickenpox virus vaccine used for vaccination against the dorsal root (described by Stickl, H. et al. [1974] Dtsch. Med. Wschr. 99, 2386-2392). For the preparation of vaccines, for example, viral particles are lyophilized in ampoules, preferably in glass ampoules. Alternatively, vaccination may be produced by sequential freeze-drying of the virus in the preparation. The formulation may contain further additives such as mannitol, dextran, sugar, glycine, lactose or polyvinylpyrrolidone or other adjuvants such as antioxidants or inert gases, stabilizers or recombinant proteins suitable for in vivo administration. Glass ampoules can then be sealed and stored for several months between 4 ° C and room temperature. However, unless otherwise specified, the ampoule can preferably be stored at less than -20 < 0 > C.

The therapeutically effective amount of the therapeutic composition of the present invention may vary depending on various factors, such as the method of administration, the site of administration, the condition of the individual, and the like. Therefore, when used in fish, the dose should be determined at an appropriate level considering safety and efficiency. It is also possible to determine the effective amount through animal experiments. Such considerations in determining the effective amount are described, for example, in Hardman and Limbird, eds., Goodman and Gilman ' s Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; And E.W. Martin ed., Remington ' s Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

Compositions of the present invention may also include carriers, diluents, excipients, or a combination of two or more thereof commonly used in biological formulations. The pharmaceutically acceptable carrier is not particularly limited as long as the composition is suitable for in vivo delivery, for example, Merck Index, 13th ed., Merck & Inc. A buffered saline solution, a buffer solution, a dextrose solution, a maltodextrin solution, glycerol, ethanol, and one or more of these components may be mixed and used, and if necessary, an antioxidant, a buffer, Conventional additives may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into main dosage forms such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules or tablets. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990) in a suitable manner in the art.

The composition of the present invention may further comprise a pharmaceutically acceptable additive, wherein pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose Starch glycolate, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, calcium stearate, , White sugar, dextrose, sorbitol and talc. The pharmaceutically acceptable additives according to the present invention are preferably included in the composition in an amount of 0.1 to 90 parts by weight, but are not limited thereto.

The therapeutic composition of the present invention can be administered orally or non-orally (for example, intravenously, subcutaneously, intraperitoneally or topically) or orally administered in accordance with a desired method, and the dose may be appropriately determined depending on the body weight, The range varies depending on the condition, diet, administration time, administration method, excretion rate, and severity of disease.

In one aspect, the invention relates to a method of preventing a megalocytovirus infection in a fish, comprising the step of administering the composition of the present invention to a fish.

For vaccination or treatment, the lyophilizate is dissolved in an aqueous solution of 0.01 to 0.5 ml, preferably physiological saline or Tris buffer, and administered systemically or topically, i. E., Intraperitoneally, parenterally, subcutaneously, intramuscularly, May be administered by any other route of administration. The dosage form, dose and frequency of administration can be optimized by those skilled in the art in a known manner.

The present invention will be described in more detail with reference to the following examples. However, the following examples are only for the purpose of illustrating the present invention, and thus the present invention is not limited thereto.

Example  One: Irido Bebrie Megalocyte virus  detach

1-1. IMV  Separation of subgroup II, subgroup III and subgroup IV

IMID (Iridoviridae Megalocytivirus) subgroup Ⅱ, subgroup Ⅲ and subgroup Ⅳ for infecting fish coin cells were cultivated in 1853 grams (185.4 grams), which is a bloomed state showing IMV infection at a farm in Tongyeong, Gyeongsangnam-do in 2013, (3.2g), and a flounder (323.4g) showing infection symptoms in a farm in Geoje, Gyeongsangnam-do, The virus was harvested from the spleen tissues of each infected fish, ground in a 10-fold volume of PBS, centrifuged at 10,000 g for 5 minutes at 4 ° C, and filtered through a 0.2 μm syringe filter. Each virus was identified by PCR using MCP (Major capsid protein) gene and confirmed by base sequence analysis. For viral amplification, 10 μl of the virus sample filtered in the above-described manner was inoculated into a GF-1 coin cell line cultured in a T-25 flask for 3 days. After incubation for 7 days, the supernatant was taken and stored at 70 ° C. until use.

1-2. Quantitative analysis of isolated viruses

QPCR (quantitative PCR) analysis was performed for quantitative analysis of isolated viruses. Specifically, the viral DNA was isolated according to the manufacturer's method using GeneAll ^ⓡ Exgene 占 Tissue SB mini kit (GeneAll), and the final volume was separated to 100 에서 in the elution step. The viral DNA was separated at -20 캜 Respectively. For the absolute qPCR of the viral DNA, 10 μl of 2 × PreMix (SYBR Green with low ROX, Enzynomics), 0.5 pM of primer and 1 μl of template DNA were used, resulting in a final volume of 20 μl. The conditions of qPCR were 10 minutes at 95 ° C, 40 cycles of 95 ° C for 10 seconds, 60 ° C for 15 seconds, and 72 ° C for 20 seconds as one cycle. After the final cycle, Melting curve analysis was performed in the region up to < RTI ID = 0.0 >< / RTI > To prepare a standard curve of qPCR, qM1F (5'-GGC GAC TAC CTC ATT AAT GT-3 ') and qM1R (5') prepared by targeting a base sequence of MCP gene with megalocytovirus viral DNA as a template, (Invitrogen, USA) was inserted into Escherichia coli DH5α, which was then mass-cultured. Then, the recombinant plasmid was transformed into Escherichia coli DH5α Separately, 10-fold stepwise dilution (1 x 10 ⁸ to ¹ x 10 ¹ copies / μl) was used to create a standard curve.

Example  2. Semicontinuous  Culture method ( SCCCS ) In the coin cell IMV  production

As coin cells, pegrus major fin (PMF) cell line (KCLRF-BP-00324), Grouper fin (GF-1) cell line (ECACC, Australia) and Bluegill After the cells were inoculated into T-25 flasks at a concentration of 1 × 10 ⁵ cells / ml each of bluegill fry (BF-2) cell line (ATCC, USA) 1 ml of the IMV subgroup II virus supernatant of Example 1-1 ( ⁵ × 10 ⁵ copies / ml) was injected and sufficiently adsorbed by Twister (Vision Scientific co., LTD., VS-96 TW) in the incubator for 2 hours. Followed by incubation at 25 ° C. The viral concentration of the supernatant taken daily was measured with the semi-continuous cell culture system (SCCCS) by taking the supernatant from the flask daily and replacing with 100% fresh medium after IMV inoculation. In addition, after 10 consecutive cultures, cytopathic changes were confirmed with a microscope (Nikon, Eclipse TS100).

As a result, the highest concentration of IMV was produced in the PMF cell line and the mean virus concentration was 1.46x10 ⁵ , 4.14x10 ⁵ and 9.49x10 ⁶ copies / ml in BF-2, GF-1 and PMF, . In particular, PMF produced IMV up to 100 times higher than GF-1 and BF-2, and up to 7 × 10 ⁷ copies / ml on day 15 of SCCCS culture. In addition, the denaturation of the cell line by the microscope was confirmed. As a result, the cell degeneration after 10 times of continuous cultivation was most frequently observed in the PMF cell line. PMF denaturation due to the IMV infection maintained the cells in the continuous medium exchange, (Fig. 2). Therefore, the optimization experiment of IMV production through the subsequent SCCCS culture method was carried out using PMF cell line as a representative coin cell line.

Example  3. Semicontinuous  culture( SCCCS ) Optimization of exchange of media

3-1. Medium exchange cycle

To produce IMV in the PMF cell line using the SCCCS culture method, 100% medium exchange (100% addition of fresh supernatant after addition of whole supernatant) was carried out at intervals of 1 day, 3 days and 5 days And the obtained virus was quantitatively analyzed by the q-PCR method of Example 1-2.

Medium exchange cycle
(Work) Number of badges exchanged
(time) Total amount of virus produced
(viral copies) One 30 4.87E + 09 3 10 6.04E + 10 5 6 4.80E + 09

As a result, normal cell culture was possible and viruses were produced when the culture medium was changed every 1 day, 3 days or 5 days, but when the medium was changed every 3 days, virus productivity was 10 times higher than other cycles (Table 1). The total amount of virus produced showed the amount of virus in all the virus supernatants obtained for 30 days immediately after the viral infection, and the amount of the supernatant of the virus produced was 150 ml, 50 ml and 30 ml Respectively. In addition, the 5-day cycle of medium exchange showed a somewhat higher concentration at the beginning, but thereafter the virus production gradually decreased due to the cell disappeared (Fig. 3). In other words, it was shown that the virus productivity was highest when cultivated in a medium exchange period of 3 days, and then the medium exchange period was 3 days in the SCCCS culture method.

3-2. Badge exchange rate

The productivity of IMV was determined by SCCCS culture method. Specifically, when the medium is exchanged at the medium exchange period (three days) established under the condition of Example 3-1, the medium is changed to 30%, 70% or 100% by replacing the medium to be replaced with the new medium Then, the amount of the obtained virus was quantitatively analyzed by the q-PCR method of Example 1-2.

As a result, the cells were maintained at all ratios and the increase of the virus concentration was observed. However, at the medium exchange ratio of 30%, the virus concentration rapidly increased at the early stage but after 15 days, the cell gradually disappeared and the virus concentration continuously decreased. In addition, although the virus concentration was continuously increased at the medium exchange ratio of 100%, it was higher than the 70% exchange It took a long time to reach the maximum virus concentration by 70% and 30% exchange rate. At a medium exchange rate of 70%, the virus concentration continued to increase for 15 days, after which the virus concentration was maintained without cell detachment (FIG. 4). Therefore, during the subsequent experiments, the medium exchange ratio was optimized to 70% for SCCCS culture.

Example  4. Semicontinuous  culture( SCCCS ) Culture conditions

4-1. Type of culture medium

The virus productivity according to the kind of medium, FBS concentration and culture temperature was compared in the IMV production using the SCCCS culture method in which the medium was replaced with 70% of the medium in the 3-day cycle established in Examples 2 and 3, Respectively. Among them, L-15, MEM and RPMI1640 were compared with each other. First, PMF cells were exchanged with each medium for adaptation to each medium, and then cultured for more than 10 times. Each of the PMF cells adapted to the new medium was cultured by injecting into a T-25 flask at 1 × 10 ⁵ cells / ml after the last passage. When the cells occupied more than 80% of the bottom of the flask, Virus supernatant was used to inoculate 1 × 10 ⁵ copies / ml. Three days after the inoculation, 70% of the virus supernatant was obtained and cultured in the same manner as described above (70% exchange of medium in 3-day cycle). The amount of virus obtained daily in this manner was quantitatively analyzed by the q-PCR method of Example 1-2.

As a result, virus productivity was similar in all three kinds of media (Fig. 5).

4-2. U feta  Serum concentration

The virus productivity according to the kind of medium, FBS concentration and culture temperature was compared in the IMV production using the SCCCS culture method in which the medium was replaced with 70% of the medium in the 3-day cycle established in Examples 2 and 3, Respectively. In order to set the appropriate concentration of fetal bovine serum (FBS) used as an additive of the culture medium in the SCCCS, the cells were cultured in the same manner as in Example 4-1, The medium was exchanged 70%. At this time, FBS was added to the medium at 1%, 5% and 10%, and the concentration of the virus supernatant was analyzed by q-PCR.

As a result, low concentration of virus was produced in 1% FBS group and cell confluency decreased to less than 50% after 15 days of culture. In addition, all of the groups of 5% and 10% of the FBS concentrations showed normal cell culture conditions, and it was confirmed that viruses with similar concentrations were produced (FIG. 6). Therefore, in the present invention, 5% FBS was selected as the optimal culture condition in consideration of the economical aspect.

4-3. Incubation temperature

The virus productivity according to the kind of medium, FBS concentration and culture temperature was compared in the IMV production using the SCCCS culture method in which the medium was replaced with 70% of the medium in the 3-day cycle established in Examples 2 and 3, Respectively. In order to confirm the optimal cell culture temperature, the cells were incubated in the same manner as in Example 4-1, and the virus was inoculated. The culture medium was incubated at 20, 23, 25, 28 And 30 [deg.] C, respectively. Other cell culture conditions were cultured under conditions optimized in Example 4-2.

As a result, it was confirmed that SCCCS culture of IMV-infected PMF cells was possible at all temperature conditions, and the virus yield was remarkably low at 20 ° C. When the culture was carried out at 30 ° C, Showed a decrease in virus concentration, and showed the highest virus productivity at 25 ° C and 28 ° C (FIG. 7). The subsequent cell culture was carried out at 25 캜.

Example  5. Semicontinuous  culture( SCCCS ) To determine the long-term productivity of the virus

The optimum conditions (culture medium: L-15, fetal bovine serum (FBS): 5%, and culture medium) established in the above Examples in the production of IMV using a culture method of SCCCS (Semi- The culture temperature was 25 ° C, the medium exchange period was 3 days, and the medium exchange rate was 70%). Specifically, the IMV subgroup Ⅱ virus supernatant 1ml of Examples 1-1 after the 5th of the inoculated cells inoculated the PMF 1x10 ⁵ cells / ml concentration in T-flask, and the cells accounted for more than 80% of the bottom of the flask 5x10 ⁵ The cells were incubated at 25 ° C for 2 h with addition of 4 ml of Twister (Vision Scientific co., LTD, VS-96 TW). For periodic cell counts, cells were removed from one flask each time in 10 T-flasks inoculated with the same amount of cells in the same manner. Cells were desensitized with 0.25% trypsin every 15 cycles of culture medium, diluted and counted with a hemocytometer to make 80-200 cells in 0.1 mm ^{3, and} the number of cells in 1 ml was counted Respectively. The viral concentration was also sampled by sampling the supernatant at the same intervals and DNA was isolated and quantitated by q-PCR analysis.

As a result, in PMF cultured with SCCCS, IMV showed a minimum concentration of 4.99 × ^{10 9} copies / ml and a maximum of 8.47 × ^{10 9} copies / ml, and the average virus amount per day was 1.09 × ^{10 10} copies / day (FIG. 8) . At this time, the cell concentration was 3.86x10 ⁶ cells / ml on average (FIG. 8), and it was confirmed that the cells were maintained without loss or detachment even after 20, 50, and 100 times of medium exchange (FIG. 9). Subsequently, the virus was also produced in the subsequent culture without cell detachment.

Example 6. Confirmation of semi-continuous culture productivity by IMV subgroup

In order to confirm the productivity according to the SCCCS of the viruses in each subgroup of IMV, the isolates of subgroups II, III and IV obtained in Example 1 were inoculated into the PMF cell line and cultured in the optimized manner in Examples 3 and 4 The virus productivity was confirmed. In addition, denaturation of the cells inoculated with each virus subgroup was confirmed by microscopy.

As a result, viruses of the respective subgroups showed the same degree of cell degeneration (FIG. 10). In addition, the cells remained without disruption in all groups, and no conspicuous differences in the successive productivity of viruses against subgroup II were found regardless of the IMV subgroup (FIG. 11). From this, it can be seen that the present invention can be applied to high efficiency production of all IMV subgroups.

Example 7. Semi-continuous incubation according to container

Virus production by cell culture vessels was confirmed in the production of IMV using a culture of semi- batch cell culture system (SCCCS) in the PMF cell line. T-25 flasks, T-75 flasks, 55 mm dishes, and Roller Bottles (corning, 850 cm ² ) were used for culture. In addition, the medium was added to 15 ml of the T-75 flask, 5 ml of the 55 mm dish, and 200 ml of the roller bottles, and the cells were cultured under the optimal conditions established in the above examples. Roller bottles were incubated at 2 RPM using a Low Profile Roller Mixer (2.3 inches in Height). To the cells cultured in T-25 flask and 55m dish, 1 ml of virus supernatant was added. After adsorption for 2 hours, 4 ml of medium was added. To the cells cultured in T-75 flask, 3 ml was added and adsorbed for 2 hours. Was added. Twister (Vision Scientific Co., LTD, VS-96 TW) was used for adsorption. To the cells cultured in Roller bottles, 100 ml of virus supernatant was added, followed by adsorption at 2 RPM for 2 hours, followed by addition of 100 ml. The viral supernatants in all vessels were exchanged 70% of the medium every three days to obtain viruses, and the average virus concentration during the 30 day culture period was measured in each vessel.

Amount of medium (ml) Average virus production concentration
(x10 ⁹ copoies / ml) T-25 Flask 5 6.52 T-75 Flask 15 7.01 55mm Dishes 5 0.95 850cm ² Roller Bottles 200 6.81

As a result, it was possible to confirm the normal maintenance of the cells in all the vessels, but the 55 mm dish showed a low CPE continuously from the initial stage after the virus inoculation. In addition, when the 850 cm ² Roller bottles were used, the maximum virus concentration and the average virus production were similar, confirming that this method was utilized irrespective of the area of host cell attachment space (Table 2).

Example 8 . Comparison of virus productivity between subculture ( BCS ) and semi-continuous culture ( SCCCS )

The productivity of IMV through the Batch culturing system (BCS) and the productivity of IMV using the semi - continuous culture (SCCCS) method of the present invention were compared. Specifically, IMV was inoculated to 10 ⁵ copies / ml of the PMF cell line in the flask, and the culture medium was exchanged every 3 days so that the number of times was more than 20 (P20), so that sufficient continuous infection was induced. After that, cells that were judged to be sufficiently consecutively infected for accurate comparison were used. Specifically, as a test group using the BCS method, 30 or 60 passages of cell lines (BCS-P30 or P60 ), And subcultured once every 7 days. As the SCCCS test group, the cell line (SCCCS-W50 or W100) subjected to the medium exchange 50 times or 100 times under the optimum conditions set in the above examples was administered once every 3 days After incubation for 21 days in a semi - continuous culture method, the virus productivity was confirmed.

virus
Production method division Production of virus for 21 days (ml) Average concentration of virus
(copies / ml) BCS BCS-P30 15.0 6.17E + 08 BCS-P60 15.0 5.93E + 08 SCCCS SCCCS-W50 24.5 5.55E + 09 SCCCS-W100 24.5 6.97E + 09

As a result, the amount of virus supernatant produced by BCS for 21 days was 15 ml, the amount produced by SCCCS was 24.5 ml, which was 1.6 times larger than that of BCS, and the SCCCS method was compared with that of BCS Concentrations are also more than 10 times higher, suggesting that the absolute amount of total virus produced is 15 times higher (Table 3). Thus, it can be seen that the production of virus using the SCCCS method can efficiently produce a high dose of megalochoti virus.

Example  9. Semicontinuous  Production of inactivated vaccine using virus produced by culture method and confirmation of its effect

High Dose Iridovirus Inactivated Vaccine (HDIV) was prepared using the IMID (Iridoviridae Megalocytivirus) subgroup II isolated from infected dead dams in Example 1 above. Specifically, the IMV subgroup II was produced under the optimal conditions established in the above examples and diluted to 2 × 10 ⁹ copies / ml (SCCCS-IMV-SII). As a control, the virus supernatant produced by the subculture method under the same medium and conditions was used at a concentration of 5 × 10 ⁸ copies / ml (BCS-IMV-SII). The viruses prepared and produced by each method were centrifuged at 12,000 rpm for 10 minutes and then inactivated with formalin 0.1% (v / v) for 2 weeks at 4 ° C to prepare a vaccine (BCS-IMV-SII and HDIV SCCCS-IMV-SII). The inactivated vaccine (0.1ml) was injected intraperitoneally into 20 healthy dogs (6.98 ± 1.54g in fish body) for 4 weeks. As a result, both vaccines did not have any dead organisms and both vaccines were stable.

(2 × 10 ⁹ copies / ml), BCS-IMV-SII vaccine (5 × 10 ⁸ copies / ml) and PBS (pH 7.2) were administered to healthy (7.94 ± 1.37 g) and red sea bream (9.28 ± 2.21 g). Two weeks after the vaccination, the vaccine effect was confirmed by intraperitoneal injection of SCCCS-IMV-SII virus (1x10 ⁴ copies / fish) and the average survival rate was confirmed by repeating the above procedure three times.

As a result, the antiviral effect of anti-viral vaccine (HDIV) produced by the semi-continuous culture method (SCCCS) was 25% and 15% larger than that of the BCS-IMV-SII vaccine 4). In the HDIV (SCCCS-IMV-SII vaccine) diluted 1/10, the survival rate was similar to that of the BCS-IMV-SII vaccine using the undiluted solution, and compared with the virus produced by the conventional submerged culture method, It was useful.

Fish species Vaccine type Survival rate (%) Average survival (%) Dolomo SCCCS-HDIV 60 68.33 + - 7.64 75 70 1/10 diluted
SCCCS-HDIV 40 46.67 + - 7.64 55 45 BCS-IMV 50 43.33 + - 16.07 55 25 1/10 diluted

BCS-IMV 30 23.33 + - 5.77 20 20 Control 0 0.00 ± 0.00 0 0 Red sea bream SCCCS-HDIV 90 93.33 ± 2.89 95 95 1/10 diluted
SCCCS-HDIV 75 81.67 ± 5.77 85 85 BCS-IMV 70 78.33 + - 7.64 85 80 1/10 diluted
BCS-IMV 60 68.33 + - 7.64 70 75 Control 25 48.33 + 2.89 30 30

Claims (9)

1) continuous infectious stage in which viruses are inoculated into papyrus major fin (PMF) derived from red sea bream and the medium is exchanged 20 times or more; And
2) culturing continuously infected cells; And
3) a semi-continuous cell culture step of obtaining a culture supernatant containing the virus and adding fresh medium as much as the obtained culture supernatant. delete The method according to claim 1, wherein the virus is an Iridoviridae family virus. 4. The method according to claim 3, wherein the iridoviride and the virus are megalocytis. 5. The method according to claim 4, wherein the megalochoti virus is any one selected from the group consisting of subgroup II, subgroup III and subgroup IV. The method according to claim 1, wherein the infection is a continuous infection in which the virus is inoculated and the medium is changed every three days. The method according to claim 1, wherein the culture condition is a culture at 25 占 폚 in a medium containing 5% fetal bovine serum (FBS). The method according to claim 1, wherein the medium change period of the semi-continuous incubation step is 3 days, and the medium exchange ratio to replace the new medium is 70% (v / v). A vaccine composition comprising a viral agent inactivated in culture supernatant containing virus prepared by the method of claim 1 and a pharmaceutically acceptable carrier or excipient.

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