KR102018200B1 - Process for the production of fish pathogenic viruses using cell on microcarrier bead - Google Patents

Abstract

The present invention relates to a method for mass production of fish virus at high concentration using a cell line attached to a microcarrier, to overcome the difficulty of producing a high concentration of fish virus using a fish cell line having attachment culture characteristics, microcarrier beads Since the fish cell lines were attached and scaled up and the optimal culture conditions were derived to enable mass cultivation, the method of the present invention enables the cultivation of a large number of fish-coated cell lines even in a small space. It can be mass produced in high concentrations, it is possible to produce an efficient and stable virus and vaccine production.

Description

Production of fish virus using cell line attached to microcarrier beads {PROCESS FOR THE PRODUCTION OF FISH PATHOGENIC VIRUSES USING CELL ON MICROCARRIER BEAD}

The present invention relates to a method for mass production of fish virus at high concentration using a cell line attached to a microcarrier.

In the bio industry of the world, it is common to use fermenters or bioreactors for mass production of viruses. For large-scale culturing of host cells, which are essential for the mass production of viruses, free-cell suspension cultures (F-SCs), which can scale up to tens to hundreds of liters, are used. Although fish cell lines have been developed in close to 100 species, but there are only cell lines showing adhesion-dependency, it is difficult to mass-produce fish viruses because ocean culture is difficult to carry out by F-SC method as in the case of mammals. Recently, roller bottles have been used for the mass production of fish virus, but these methods require a very large labor force and at the same time have a very uneconomical limitation.

With the recent development of the aquaculture industry, the disease problem of fish is becoming more important, and the establishment of disease management problem is also increasing in importance. For this reason, there is an increasing demand and necessity for a management method for viral diseases, which constitute the largest part of fish diseases. The most important method of managing these fish virus diseases is the development of a vaccine. Therefore, in order to develop a virus vaccine, a new cell line mass culture method should be made first, and a large amount of virus production should be economically and efficiently made based on this.

Iridoviridae Megalocytivirus (IMV) is the causative agent of Red Sea Bream Iridovirus Disease (RSIVD). However, sea bream is much more susceptible to red snapper than about 10% cumulative mortality at aquaculture sites, resulting in high mortality of 90-100%. Mass cultivation of the virus is essential for the development of a vaccine to prevent the infection of megalocytivirus, but megalocytivirus grows only in limited fish cell lines, and infectivity is increased as the number of virus passages is increased in subculture. Is rapidly reduced (Chou eta., 1998; Imajoh et al., 2007; Nakajima and Sorimachi, 1994), indicating a very specific infection with decreased virus production resulting in decreased virus concentration in culture. There have been many difficulties in the industrial production of viruses. At present, there is no known treatment for diseases caused by Iridoviridae Megalocytivirus (IMV), and the only way to prevent it is to develop a vaccine. In particular, vaccine development is difficult in rock bream, which is the most susceptible to megalocytivirus and is very damaging when it develops.

Currently, all commercialized fish coin cells have difficulty in culturing high virus concentrations, and due to the low concentration of virus produced (industrial enrichment process of virus requires no cost, so it is economically insignificant). This is not possible (Imajoh et a., 2007; Dong et al., 2013). Therefore, current industrial vaccine development of megalocytiviruses requires a more efficient and continuous production of high concentration virus that solves these problems.

In the present invention, in order to overcome the difficulty of producing a high concentration of fish virus using fish cell lines having adhesion culture characteristics, it is possible to attach and scale up fish cell lines to microcarrier beads and derive optimal continuous culture conditions to enable mass culture. By doing so, it is easy to mass-produce fish disease-causing viruses using them, and aims to produce a high concentration of vaccine using the same.

In order to achieve the above object, the present invention comprises the steps of mixing the fish coin cell line and microcarrier beads to attach the cell line to the microcarrier beads; Culturing the fish coin cell line attached to the microcarrier beads; Infecting the virus with fish coin cells attached to the microcarrier beads; Culturing the fish coin cells infected with the virus; And obtaining a culture supernatant and isolating the virus, thereby providing a virus production method using the microcarrier beads.

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

Cell culture using the microcarrier according to the present invention enables the suspension-dependent culture of the adhesion-dependent fish cell line, can produce a large amount of cells in a narrow space, and by deriving the optimal attachment and culture conditions according to the cell line and virus cell lines and viruses It can be produced in high concentrations in large quantities, reducing labor and contamination risks, which enables efficient and stable virus production and vaccine production.

1 is a micrograph of PMF cells attached to various MCBs and 12 days after attachment:
A: immediately after PMF attachment to Cytodex 1;
B: 12 days after culture after PMF attachment to Cytodex 1;
C: immediately after PMF attachment to Cytodex 3; And
D: 12 days after incubation after attachment of PMF to Cytodex 3.
2 is a micrograph of PMF cells attached to various MCBs and 12 days after attachment:
A: immediately after PMF attachment to Cultispher;
B: 12 days after incubation after attaching PMF to Cultispher;
C: immediately after PMF attachment to Rapidcell; And
D: 12 days after incubation after attaching PMF to Rapidcell.
Figure 3 is a graph showing the number of cells produced by the culture medium (ml) according to the type of MCB (cytodex 1, cytodex 3, cultispher and Rapidcell) when the PMF cell line is attached to the MCB in culture.
4 is a graph showing the number of cells produced according to the MCB type (cytodex 1, cytodex 3 and cultispher) based on the surface area of the beads (mm ² ) when the PMF cell line is attached to the MCB and cultured.
Figure 5 is a microscopic view of the appearance after incubation for 12 days after attaching the fish cell line PMF, GF-1, EPC or E-11 to Cytodex 1:
A: PMF;
B: GF-1;
C: EPC; And
D: E-11.
FIG. 6 is a graph showing cell number growth by attaching and culturing fish coin cells PMF, GF-1, BF-2, E-11, or EPC cell lines to Cytodex 1. FIG.
FIG. 7 shows the cell numbers and virus concentrations after inoculation of Cytodex 1 with each susceptible IMV, VNNV and VHS virus after 15 days of attachment and PMF, GF-1, EPC or E-11 cell line.
FIG. 8 shows the cytopathic effect (CPE) after inoculating Cytodex 1 with PMV, GF-1, EPC or E-11 cell lines and inoculating each of the highly sensitive IMV, VNNV and VHS viruses after 15 days of culture. The picture is:
A: 15 days after incubation of PMF attachment to Cytodex 1;
B: 12 days after inoculation of IMV to PMF attached to Cytodex 1
C: 15 days after GF-1 attachment culture to Cytodex 1;
D: 12 days after immunization with GF-1 attached to Cytodex 1
E: 15 days after E-11 attachment culture to Cytodex 1;
F: 6 days after inoculation of VNNV to E-11 attached to Cytodex 1;
G: after 15 days of culture of EPC attachment to Cytodex 1; And
H: 6 days after inoculation of VHSV to EPC attached to Cytodex 1.
9 is a diagram confirming the increased cell number while transferring the fish cell line attached to the MCB to 5-fold and 10-fold culture vessel.
10 is a graph confirming the production cell number and virus concentration according to the medium exchange cycle (1 day, 3 days or 5 days) while inoculating IMV on a Cytodex 1 attached PMF cell line and semi-continuous culture.
FIG. 11 is a graph confirming production cell numbers and virus concentrations according to subgroups (subgroups II, III, and IV) of inoculated IMVs in IMV production by semi-continuous culture of PMF cell lines attached to Cytodex 1.

Hereinafter, the embodiment of the present invention will be described in detail. However, the following embodiments are presented by way of illustration of the present invention, whereby the present invention is not limited and the present invention is capable of various modifications and applications within the scope of the claims to be described later and equivalents interpreted therefrom. .

In one aspect, the present invention comprises the steps of mixing the fish coin cell line with the microcarrier beads to attach the cell line to the microcarrier beads; Culturing the fish coin cell line attached to the microcarrier beads; Infecting the virus with fish coin cells attached to the microcarrier beads; Culturing the fish coin cells infected with the virus; And obtaining a culture supernatant and isolating the virus.

In one embodiment, the fish coin cell lines of the present invention are Pargus major fin (PMF), Grouper fin cell (GF-1), Blue gill fry (BF-2), Epithelioma papulosum cyprinid (EPC) and E-11 (a clone) of the cell line SSN-1) may be any one selected from the group consisting of cell lines.

In one embodiment, the microcarrier beads may consist of dextran, gelatin or polystyrene, the diameter of the beads may be between 100 and 300 μm, the density may be between 1.02 and 1.04, and the surface area may be between 2000 and 8000 cm ² / g. Can be. Because the density of the beads is larger than water, they are allowed to settle in a state where the stirring environment is not maintained, which makes it easier to distinguish the cultured cells from the supernatant.

In one embodiment, the microcarrier beads may be commercially available Cytodex1, Cytodex3, Cultispher or Rapidcell, more preferably Cytodex1.

In one embodiment, attaching the cell line to the microcarrier beads may be performed by repeating the stirring and stopping at 30 to 60 rpm for 1 to 3 hours.

In one embodiment, fish serum, fish muscle extract or fish yolk extract in culture of the cell line, throughout the steps of attaching the cell line to the microcarrier beads, culturing it and inoculating the cell line with the virus and producing the virus. May be added to the culture medium to culture the cell lines, and may be added at concentrations of 0.5%, 1.0% and 0.1%, respectively.

In one embodiment, the fish coin cell line attached to the microcarrier beads can be cultured in a manner that exchanges 70% of the total media capacity every 1-3 days, and can be cultured at a stirring speed of 30-80 rpm.

In one embodiment, a new microcarrier bead may be subcultured to a fish coin cell attached to the microcarrier bead in a volume ratio of 1: 1 to 1: 4, and in one embodiment of the present invention, the microcarrier Bead-coated fish coin cells were divided in half and dispensed into flasks of the same volume while subcultured with new microcarrier beads added 1: 1 or 1: 4 to the volume of the existing MCB-attached cell line.

In one embodiment, fish coin cells attached to the microcarrier beads can be cultured in a small volume flask and then cultured in a scale-up subculture manner, adding new microcarrier beads while transferring to a larger volume flask. In one embodiment of the present invention, the volume of the flask was increased by 5 and 10 times to scale-up subculture.

In one embodiment, the virus is selected from the group consisting of Iridoviridae Megalocytivirus (IMV), Viral nervous necrosis virus (VNNV) and Viral Hemorrhagic Septicemia (VHSV). It may be any one and may be selected according to the sensitivity of the fish coin cells to be infected. In one embodiment, the PMF cell line and the GF-1 cell line were infected with IMV, the E-11 cell line with VNNV, and the EPC cell line with VHSV.

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

In one embodiment, the process of culturing virus-infected fish coin cells and obtaining a culture supernatant, repeating the medium exchange process of adding the culture medium in the amount obtained after obtaining the culture supernatant without passage or cell division of cells. It may be performed by a semi-continuous cell culture method.

In one embodiment, the medium exchange cycle when semi-continuous culture of infected cells is 3 days, and the medium exchange rate of replacing with fresh medium may be 70% (v / v).

Semi-continuous cultivation of the present invention is to obtain a culture solution containing the virus produced, and to perform the operation of culturing a new culture solution with the cells attached to the microcarrier beads intact in a semi-continuous manner. In one embodiment of the present invention, 70% of the cell culture supernatant (cell culture medium) was obtained every 3 days, and the optimal conditions for virus production for refilling the new medium were established, and IMV was infected with the PMF cell line attached to Cytodex1 beads. In the case of continuous culture, it was confirmed that the viral productivity was most remarkable.

As used herein, the term "coin cell" refers to a cell that has been isolated from a fish tissue and cell-cultured through continuous passage culture, and "cell line" and "cell" are used interchangeably.

As used herein, the term "passage" means delivering cells from one culture vessel to another, with or without dilution. Each time a cell is transferred from one vessel to another, certain portions of the cell may be missing, so that dilution of the cell, whether intended or unintentional, may occur. This term is synonymous with the term "subclture". The number of subcultures is the number of times the cells are cultured, which are either secondary cultured or transferred into a new vessel to grow in suspension or adherence. This term is not synonymous with the time required for the population doubling or cell population to replicate, ie the time generally required for individual cells in the cell population to replicate.

In one aspect, the invention relates to a vaccine composition comprising a virus and an excipient which inactivated a virus produced by the method of the present invention.

In one embodiment, the excipient may be any one selected from the group consisting of beta-glucan, squalene and aluminum hydroxide, imiquimod, and Montanide ISA 780 VG, with beta-glucan most desirable.

In one embodiment, the vaccine composition prevents infection of fish Megaridytivirus (IMV), viral nervous necrosis virus (VNNV) or viral hemorrhagic Septicemia (VHSV). Or as a therapeutic vaccine.

In one embodiment, the fish may be selected from the group consisting of stone bream, red snapper, gangdam bream, spot bass, flounder, black sea bream, robber leg, jerkball rock, squid and mandarin fish, more preferably rock bream or red snapper.

In one embodiment, the vaccine composition may contain inactivated megalocytivirus (IMV), viral neuronal necrosis virus (VNNV) or viral hemorrhagic sepsis virus (VHSV), wherein the megalocytivirus is sub It may be a subgroup II, subgroup III or subgroup IV isolate. Thus, in the present invention, the virus associated with such treatment is provided to the immune system of an organism to influence a specific immune response, preferably to induce an immune response, thereby vaccinating the organism to protect it from infection by the virus. do.

For the preparation of the prophylactic composition (ie vaccine) of the present invention, fish virus inactivated with formalin according to the present invention is used as a vaccine as it is or converted into a physiologically acceptable form. This can be done on the basis of experience in the preparation of chickenpox virus vaccines used for vaccination against cramps (disclosed by Stickl, H. et al. [1974] Dtsch. Med. Wschr. 99, 2386-2392). For the preparation of vaccination, for example, viral particles are lyophilized in ampoules, preferably in free ampoules. Alternatively, vaccination may be produced by sequential freeze-drying of the virus in the formulation. This formulation may contain additional 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. The glass ampoule can then be sealed and stored for several months between 4 ° C. and room temperature. However, unless otherwise required, the ampoule may preferably be stored below −20 ° C.

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

The compositions of the present invention may also include carriers, diluents, excipients or combinations of two or more commonly used in biological agents. Pharmaceutically acceptable carriers are not particularly limited so long as they are suitable for in vivo delivery of the composition, for example, Merck Index, 13th ed., Merck & Co. Inc. Compounds, saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components can be mixed and used as needed. Conventional additives can be added. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulate into main dosage forms, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Furthermore, it may be preferably formulated according to each disease or component by a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

The composition of the present invention may further comprise a pharmaceutically acceptable additive, wherein the pharmaceutically acceptable additive may be starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose , Mannitol, malt, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, opiodry, sodium starch glycolate, lead carnauba, aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate , Sucrose, dextrose, sorbitol, talc and the like can be used. The pharmaceutically acceptable additive according to the present invention is preferably included 0.1 parts by weight to 90 parts by weight based on the composition, but is not limited thereto.

The therapeutic compositions of the present invention may be administered orally (eg, intravenously, subcutaneously, intraperitoneally or topically) or orally, depending on the desired method, and the dosage may be weight, age, sex, health of the individual. The range varies depending on the condition, diet, administration time, administration method, excretion rate and severity of the disease.

In one aspect, the present invention is directed to a fish, comprising the step of administering a composition of the present invention to fish, Iridoviridae Megalocytivirus (IMV), viral nervous necrosis virus (VNNV) or viral A method for preventing Hemorrhagic Septicemia (VHSV) infection.

For vaccination or treatment, the lyophilisate is dissolved in 0.01-0.5 ml of aqueous solution, preferably physiological saline or Tris buffer and systemically or topically, i.e. peritoneal, parenteral, subcutaneous, intramuscular or known to those skilled in the art. It can be administered by any other route of administration. Dosage form, dosage and frequency of administration can be optimized by one 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 intended to embody the contents of the present invention, and the present invention is not limited thereto.

Example  1. Fish Virus Preparation

Iridoviridae Megalocytivirus (IMV), Viral nervous necrosis virus (VNNV) and Viral Hemorrhagic Septicemia (VHSV) were each isolated from field infected fish. Specifically, IMV is from sea bream (185.4 g) showing infectious symptoms in a farm in Tongyeong, Gyeongsangnam-do in 2013, VNNV is from flounder (231.2 g) showing infectious symptoms in Captain of Busan in 2014, and VHSV is from Pohang, Gyeongsangbuk-do in 2013 It was obtained from flounder (183.3 g) showing infectious symptoms. In each infected fish, spleen tissues known to have the highest IMV and VHSV and brain tissues known to have the highest VNNV were extracted and pulverized in PBS at a concentration of 10% w / v, followed by centrifugation at 10,000 g for 4 minutes. After passing through a 0.2 μm syringe filter, 10 μl were taken and each cell was injected with a concentration of 1 × 10 ⁵ cells / ml in a T-25 flask, followed by IMV and VNNV in fish coin cell lines PMF, E-11 and EPC, respectively. VHSV was inoculated, and the virus was propagated for 7 days and the supernatant was stored at 70 ° C. until use.

Example  2. of fish coin cells MCB  Attach

Parf major fin (PMF), Grouper fin cell (GF-1), Blue gill fry (BF-2), Epithelioma papμlosum cyprinid (EPC) and a clone of the cell line SSN- 1) Cell lines were cultured in T-75 flasks at 25 ° C. in L-15 medium (Sigma Aldrich) to which antibiotics (penicillin 100 IU / ml, streptomycin 100 μg / ml) and 10% FBS (Gibco) were added. It was.

MCB (micro carrier bead) was prepared using Cytodex 1, Cytodex 3, Cultispher or Rapidcell according to the manufacturer's recommendations. Each microcarrier was used at a concentration of 5 mg / ml and Cultispher 1 was used at a concentration of 1 mg / ml. The microcarriers in powder form were quantified according to the spinner flask capacity and hydrated at room temperature for more than 3 hours using PBS. During this process, MCB was precipitated to remove PBS and filled with new PBS. Repeated. After sterilization with a sterilizer and before use to remove the PBS and used after washing 2-3 times with cell culture medium (L15).

The culture medium of the fish cell line cultured in the flask was removed, and 500 µl of trypsin 0.25% (v / v) was injected into each flask to react at 25 ° C. for 5 minutes, and then all the cells were removed. It was then diluted with 10 ml of culture medium to remove trypsin and mixed in 50 ml of tube. Cells were centrifuged at 500 g using a centrifuge (Hanil, Combi-514R). After centrifugation, all the supernatant was removed and the pellet was diluted with 20 ml of PBS. The cells were counted with a hemocytometer (Supe rior, Hausser ultra plane) and injected into the spinner flask at a concentration of 1 × 10 ⁵ cells / ml. At the same time, the whole of the beads washed with the culture medium was injected with the culture medium, and fetal bovine serum was added to a final concentration of 10% (v / v). Then, the culture medium of the appropriate volume was added according to the volume of the Spinner flask and incubated in a 25 ° C. incubator at 60 rpm with a slow stirrer (Misung, MSD24), which is a method recommended by the manufacturer.

Example 3 Optimization of Fish Cell Culture Conditions Attached to MCB

Each fish coin cells attached to MCB in Example 2 were incubated at a stirring speed of 60 RPM in a 100 ml Spinner flask at a concentration of 1 × 10 ⁵ cells / ml, which is a manufacturer's recommended condition. However, when the cell culture under the above conditions, the growth of the cells did not appear due to the decrease in the nutrient content of the medium and the pH change, it was necessary to change the medium periodically. Thus, the culture conditions were optimized using the PMF cell line attached to Cytodex 1 (MCB) as follows, and whether the other fish cell lines (GF-1, BF-2, E-11 and EPC) can be cultured under the optimized conditions. It was confirmed.

3-1. Badge change cycle

In culturing each of the fish coin cells attached to the MCB in Example 2, the medium exchange cycle is divided into 1 day, 3 days and 5 days while replacing the medium with fresh medium each time 70% (v / v) Cell growth was confirmed through cell number measurement using the MTT assay. Specifically, PMF cells attached to Cytodex 1 were injected in 96-well plates by diluting stepwise at a concentration of 1/2 × from 1 × 10 ⁶ cells / ml, and the number of cells was measured using a hemocytometer after 24 hours of incubation. At the same time, the absorbance at 450 nm was measured using CCK-8 by the MTT assay method to prepare a standard curve of the cell number according to each absorbance. The cells in the beads were then measured for absorbance using CCK-8 by sampling some of the beads in the flask, and counting the cells according to the standard curve above to determine the total number of cells in the flask.

The results showed similar cell growth at 1.11x10 ⁶ cells / ml and 1.06x10 ⁶ cells / ml when the medium was changed at 1 and 3 day cycles, and the cell culture was slightly lower when the medium was changed at 5 day cycles. (Table 1). This is similar in other cell lines.

3-2. Stirring  speed

While culturing the respective fish coin cells attached to the MCB in Example 2, the number of cells according to the stirring speed was confirmed by the method of Example 3-1. Stirring speed was divided into 30, 60, 90 or 120 RPM.

As a result, each of the fish coin cells attached to the MCB showed a number of cells around 1x10 ⁶ cells / ml at 30, 60, 90 and 120 RPM, indicating that the culture was well performed, especially at 60 RPM. After 12 days, the cell number was 1.06 x 10 ⁶ cells / ml, showing the highest cell productivity (Table 1).

3-3. Spinner flask capacity

In Example 2, the respective fish coin cells attached to the MCB were cultured in 100 ml, 250 ml, and 500 ml Spinner flasks to determine the cell numbers.

As a result, it was found that cell lines attached to MCBs grow well in spinner flasks of various specifications (Table 1).

3-4. Serum Types and Concentrations

In Example 2, the growth rate of each kind and concentration of serum added to the culture medium of each fish chemotactic cell attached to MCB was confirmed. Specifically, after incubating with 0%, 1%, 5% and 10% of FBS (Fetal bovine serum), or 5% and 10% of NCS (New born carp serum), and in Example 3-1, As shown in the cell productivity.

As a result, the addition of FBS (Fetal bovine serum) showed higher cell productivity than the addition of NCS (New born carp serum), FBS grew well at 10% and 5% concentration (Table 1).

3- 5. Cell  density

The cell culture effect according to the initial cell concentration inoculated upon the cell attachment to MCB, the time to reach the maximum cell number was slightly different depending on the cell concentration, but all reached the maximum cell number (Table 1).

3-6.Culture temperature

As a result of confirming the cell culture effect according to the cell culture temperature of the PMF cell line attached to MCB, it was possible to culture at 22 ℃, 25 ℃ and 28 ℃ of the culture temperature, showed the best cell culture efficiency at 25 ℃ (Table 1).

Badge change cycle Number of cells after 12 days of culture (x10 ⁶ cells / ml) 1 day 1.11 3 days 1.06 5 days 0.89 Stirring Speed (RPM) 30 0.84 60 1.06 90 0.97 120 0.67 Spinner flask capacity 100 ml 0.87 250 ml 1.06 500 ml 1.17 Serum type and concentration FBS 10% 1.12 FBS 5% 1.06 FBS 1% 0.54 FBS 0% 0.22 NCS 10% 0.72 NCS 5% 0.43 Initial Cell Concentration (Cells / ml) 1 x 10 ⁶ 1.73 ⁵ x 10 ⁵ 1.44 1 x 10 ⁵ 1.06 5 x 10 ⁴ 0.62 Incubation temperature (℃) 22 0.77 25 1.06 28 0.65

Through Example 3-1 to 3-6, the optimum culture conditions of the fish cell line attached to MCB, the medium exchange cycle 3 days, agitation rate 60RPM, 250ml Spinner flask, LBS medium containing FBS 5% serum, initial cells The concentration was set at ¹ × 10 ⁵ cells / ml and the cell culture temperature of 25 ° C. This optimized condition showed remarkable cell culture efficiency of at least 1 × 10 ⁶ cells / ml in both PMF cell lines as well as in all fish cell lines (GF-1, BF-2, E-11 and EPC) used in the present invention.

Example  4. On MCB  Establishment conditions for adhesion and growth of attached fish cells

4-1. Early Stirring  Confirm cell adhesion and proliferation according to speed

Since high-speed agitation in the initial culture adversely affects cell adhesion, proliferation and growth, conditions for improving the adhesion and growth ability of cell lines attached to MCB are conditions for stirring and stopping the initial agitation speed for a certain time. Repeatedly confirmed the adhesion and proliferation of the cells. Control culture was carried out at 60 RPM, the optimum stirring rate in Example 3, Experimental group 1 was repeated 60RPM stirring and stopping (1 minute / 4 minutes) for 2 hours, Experimental group 2 was stirred and stopped 30RPM (1 minute / 4 minutes) was repeated for 2 hours.

As a result, in all three cell lines, the adherence rate to MVB of the cell lines of the experimental group 1 and the experimental group 2 was increased by 30% or more compared to the control group on day 1, and the cells adhered to the MCB very evenly (Table 2). In addition, the increase in cell adhesion rate affects the proliferation of cells, and compared to Test Group 1 and Control Group, the number of cells after 7 days in culture was up to 1.46-fold in PMF cell line, up to 1.38-fold in GF-1 cell line, and EPC. Cell lines showed an effect of up to 1.25 fold increase (Table 2).

Cell line Kinds Day 1
Adhesion
(%) 3rd day
Adhesion
(%) Day 6
Adhesion
(%) Number of cells after 12 days of culture
(x 10 ⁵ cells / ml) PMF Control 60 80 90 9.7 Experimental Group 1 90 96 98 14.2 Experimental Group 2 90 94 94 13.6 GF-1 Control 58 76 82 11.2 Experimental Group 1 90 94 96 15.4 Experimental Group 2 86 96 98 13.8 EPC Control 68 82 88 12.4 Experimental Group 1 94 96 98 15.5 Experimental Group 2 90 98 98 14.4

4-2. Confirmation of Cell Growth and Virus Productivity by Addition of Fish-derived Extracts

4-2-1. Cell growth check

To further improve cell culture, serum, muscle extract and yolk extract (from sea bream or red sea bream) derived from fish (rock bream, red sea bream or halibut) were added to the MCB with 5% FBS. To determine the effect of adhesion and growth enhancement. Specifically, the preparation method of each additive is as follows. Ten stone bream (210.4 ± 56.2g), red snapper (354.1 ± 83.3g), and flounder (282 ± 78g) were prepared to obtain fish-derived serum, and blood collected by nonarrhythmic blood collection using a 10ml syringe was used. Serum was separated after centrifugation (Gyrogen, Gyro 1524M) at 10,000 RPM. In addition, to prepare the muscle extract, TBS (Tris-buffered saline, 50 mM Tris, 0.9% NaCl, pH 7.6) was added to a concentration of 10% (w / v) with a homogenizer (PolytronPT 1200E) to remove muscles and scales. After that, it was ground for 3 minutes. After moving to a 50ml tube and centrifuged for 10 minutes at 3500rpm, only the supernatant was collected and centrifuged again at 10,000 RPM to extract only the supernatant. Both serum and muscle extracts were stored at −20 ° C. until passed using a 0.45 μm syringe filter. In addition, egg yolk extract was hatched from the sea bream and red snapper larvae, washed thoroughly with PBS, mixed with PBS 1: 1 (w / v), ground with a homogenizer, and extracted only with supernatant at 3,500 rpm. The supernatant was collected once more and stored at −20 ° C. until it passed through a 0.45 μm filter.

In order to confirm the effective concentration range of the fish-derived extract prepared as described above, the concentration range of 0.05%, 0.1%, 0.2%, 0.5% and 1% was set through the preceding experiment. After mixing PMF and EPC cell lines with MCB, attaching microcarrier beads and respective cell lines, and culturing the cell culture using the cell culture medium containing the additive for each concentration by the culture method optimized in Examples 3 and 4-1. The medium containing the additive of the same conditions was used also when performing medium exchange every 3 days.

As a result, the fish-derived test group of 0.5 and 1.0% concentration showed 9.19x10 ⁵ cell / ml and 12.97x10 ⁵ cell / ml after 7 days of cell culture, which showed up to 2 times more cell growth than the control group during the same period. (Table 3). In addition, the experimental group treated with muscle extract at 1.0 and 1.5% (v / v) showed cell growth of 8.07x10 ⁵ cells / ml and 9.15x10 ⁵ cell / ml after 7 days of cell culture, which showed an increase of up to 1.5 times. . In addition, the experimental group added with yolk extracts of 0.5 and 1.0% concentration showed 10.37x10 ⁵ cells / ml and 10.80x10 ⁵ cells / ml cultured for 7 days, resulting in up to 1.8 times higher cell growth. Shown (Table 3). EPC cell lines tested in the same manner showed cell growth promoting effects of up to 1.8 and 1.3 times as compared to the control group to which FBS was added.

4-2-2. Check virus productivity

Through Example 4-2-1, when cultured with the addition of 0.5% fish serum, 1.0% fish muscle extract and 0.1% yolk extract, the cell culture phenomenon was improved. It was determined as 0.5%, 1.0% and 0.1% (v / v). In addition, in order to confirm the change of virus production ability in MCB-adherent cell lines, such additives were used to identify Iridoviridae Megalocytivirus (IMV) and Viral Hemorrhagic Septicemia (VHSV), which show sensitivity to PMF and EPC cell lines attached to MCB, respectively. After each inoculation, the virus concentration was checked after 7 days. The concentration of virus was confirmed by qPCR (quantitative PCR) method. Specifically, the DNA of the virus GeneAll ^ⓡ ExgeneTM was isolated according to the manufacturer's method using a Tissue SB mini kit (GeneAll), and stored at 20 ℃ then eluted (elution) the final volume to be 100㎕. In addition, the RNA of the virus was isolated according to the manufacturer's method using the RNeasy Plus Mini Kit (Qiagen), and then suspended using 50 μl of Rnase free water. For cDNA synthesis, add 0.5 μl of M-MLV reverse transcriptase (5 μl of Promega), 2 μl of dxP, 0.5 μl of dNTP, 1 μl of specific primer and 3 μl of extracted RNA, and add Nuclease-free water so that the total volume is 10 μl. After addition, it was made to react at 45 degreeC for 60 minutes and 99 degreeC for 10 minutes. The prepared cDNA was then used as a qPCR template and stored at -80 degrees until use. For absolute qPCR of Viral DNA or RNA, 10 μl of 2 × PreMix (SYBR Green with low ROX, Enzynomics), 1 μl of 0.5 pM of each virus specific primer and 1 μl of template were used, with a final volume of 20 μl. It was made. The conditions of qPCR were 10 minutes at 95 ° C., followed by 40 cycles of reactions of 95 ° C. 10 seconds, 60 ° C. 15 seconds, and 72 ° C. 20 seconds in one cycle. The melting curve was analyzed in the region up to 95 ° C.

As a result, PMF cell line attached to MCB inoculated with IMV by treatment of fish-derived additives showed virus productivity of 4.8 times higher than that of control group (Table 3), and EPC cell line attached to MCB inoculated with VHSV. Virus productivity was 3.2 to 4.2 times higher than the control group (Table 4).

Cell Line-Virus Kinds Origin fish density(%) Cell count after 7 days of culture Concentration 7 days after virus infection (1x10 ⁵ cells / ml) (1x10 ⁷ copies / ml) PMF-Megalocytivirus FBS 0.05 5.91 0.1 5.85 1.58 0.5 6.07 1.28 1.0 6.10 1.87 1.5 6.11 serum Stone Dome 0.1 8.68 0.5 12.71 9.21 1.0 10.03 Red snapper 0.1 6.52 0.5 9.19 9.67 1.0 10.38 halibut 0.1 7.27 0.5 11.57 6.55 1.0 12.97 Muscle extract Stone Dome 0.5 7.12 1.0 9.10 3.21 1.5 9.12 Red snapper 0.5 6.58 1.0 9.15 4.51 1.5 9.10 halibut 0.5 6.19 1.0 8.14 3.10 1.5 8.07 Egg yolk extract Stone Dome 0.05 7.81 0.1 10.37 6.14 0.2 10.44 Red snapper 0.05 6.66 0.1 10.48 7.51 0.2 10.80 .

Cell Line-Virus Kinds Origin fish density(%) Cell count after 7 days of culture Concentration 7 days after virus infection (x 10 ⁵ cells / ml) (x 10 ⁷ copies / ml) EPC-VHS FBS 0.05 7.42 0.1 7.31 1.66 0.5 7.66 1.38 1.0 7.99 1.49 1.5 8.15 serum Stone Dome 0.1 8.03 0.5 10.76 5.82 1.0 9.53 Red snapper 0.1 7.65 0.5 11.66 5.56 1.0 12.42 halibut 0.1 7.20 0.5 12.24 4.85 1.0 9.10 Muscle extract Stone Dome 0.5 7.51 1.0 9.21 2.24 1.5 9.51 Red snapper 0.5 7.29 1.0 10.40 3.49 1.5 9.18 halibut 0.5 7.94 1.0 9.87 2.98 1.5 9.19 Egg yolk extract Stone Dome 0.05 7.85 0.1 9.17 4.46 0.2 8.06 Red snapper 0.05 7.24 0.1 8.08 3.22 0.2 9.25

Thus, in the subsequent experiments, the culture medium optimized in Example 3, the medium was added to the initial stirring speed and 0.5% of red sea bream serum as the basic culture conditions.

Example  5. Bead  Fish Cell Lines by Type Adhesion  Confirm

Under the optimal conditions established in the above examples, PMF cell lines were cultured in various types of MCBs, and adhesion ability was confirmed as the number of adherent cells. Specifically, MCBs are Cytodex1, Cytodex3, Cultispher and Rapidcell (Table 5). In the preliminary experiments, the increase in the concentration of beads may increase the number of cultured cells, but it was confirmed that the beads collide with each other when stirring due to the increase in the concentration of the beads, and the efficiency is lowered. , Cytodex 1, Cytodex 3 and Rapidcell were identified as 5 mg / ml and Cultispher was 1 mg / ml. PMF cell lines were attached to the appropriate concentration of beads, and the number of cells was checked at 3 days intervals while culturing. Cell attachment beads 12 days after the cell attachment were observed under a microscope (FIGS. 1 and 2).

Kinds manufacturer size
(Μm) matter
Bead Density
(g / cm ³ ) Bead surface area
(cm ² / g) Bead number
(bead / g) Min-max Average Cytodex 1 GE Healthcare 131-220 190 ± 58 Dextran 1.03 4400 4.30E + 06 Cytodex 3 GE Healthcare 133-215 175 ± 36 Collagen coated, 1.04 2700 3.00E + 06 Cultispher Percell
Biolytica 130-380 225 ± 125 Cross linked gelatin, Macroporous 1.02-1.04 7500 1.00E + 06 Rapidcell ICN
Pharmaceuticals 150-210 Polystyrene 1.02 - -

Checking the number of cells resulting, Cytodex 1, Cytodex 3, Cultispher and culture the cells produced in Rapidcell (ml) after 12 days incubation the number of cells represented by each ^{27.1x10 6 cells / ml, 21.4x10 6} cells / ml, 17.37 X10 ⁶ cells / ml and 10.96x10 ⁶ cells / ml showed the best cell adhesion and proliferation of Cytodex 1 beads (FIG. 3). In addition, Cultispher was the best in the result of displaying the produced cells based on the surface area of the beads (mm ² ) (FIG. 4). In addition, the results of calculating the number of cells according to the surface area (mm ² ) of each bead type, the average number of cells attached to one bead, and the number of cells per bead weight (mg) are shown in Table 6.

Experimental group Number of cells after 12 days of culture (10 ⁶ cells / ml) Total cell count in flask (cells / flask) Cell count per bead
(cells / bead) Number of cells per bead surface area (cells / mm ² ) Cell count per mg bead (cells / mg) Cytodex 1 Experiment 1 2.71 6.78E + 08 126.14 1.23E + 05 5.42E + 05 Experiment 2 2.59 6.48E + 08 120.56 1.18E + 05 5.18E + 05 Experiment 3 2.48 6.20E + 08 115.35 1.13E + 05 4.96E + 05 Average 2.59 6.48E +08 120.56 1.18E +05 5.18E +05 Cytodex 3 Experiment 1 2.15 5.38E + 08 143.47 1.59E + 05 4.30E + 05 Experiment 2 2.24 5.60E + 08 149.33 1.66E + 05 4.48E + 05 Experiment 3 1.97 4.93E + 08 131.47 1.46E + 05 3.94E + 05 Average 2.12 5.30E +08 141.33 1.57E +05 4.24E +05 Cultispher Experiment 1 1.73 4.34E + 08 1736.00 2.31E + 05 1.74E + 06 Experiment 2 1.54 3.86E + 08 1544.00 2.06E + 05 1.54E + 06 Experiment 3 1.88 4.72E + 08 1888.00 2.52E + 05 1.89E + 06 Average 1.72 4.31E +08 1724.00 2.30E +05 1.72E +06 Rapidcell Experiment 1 1.09 2.74E + 08 - - 2.19E + 05 Experiment 2 0.85 2.14E + 08 - - 1.71E + 05 Experiment 3 1.24 3.10E + 08 - - 2.48E + 05 Average 1.06 2.66E +08 - - 2.13E +05

Example  6. On MCB  Confirmation of Cell Growth by Attached Fish Cell Lines

Using Cytodex 1 (5 mg / ml), which showed the best cell adhesion and proliferation in Example 5, PMF, GF-1, BF-2, E-11 and EPC cell lines were attached and cultured, respectively. Each cell was cultured to be 1 × 10 ⁵ cells / ml, and cultured under optimal conditions established in the above examples. In addition, after attaching the fish cell line and incubated for 12 days, the appearance of each cell line was confirmed under a microscope (Fig. 5).

As a result, each cell line showed the same cell growth as in Figure 6 it was confirmed that the maximum concentration from about 12 days after.

Example 7. Fish Virus Production in Fish Coin Cells Cultured Using MCB

PMF, GF-1, EPC, and E-11 cells 1x10 ⁵ cells / ml were attached to Cytodex 1 and incubated in a 250 ml Spinner flask. After 15 days of cell culture, each cell line was inoculated with a susceptible virus. Specifically, PMF and GF-1 cell lines were inoculated with IMV and EPC and E-11 cells at a concentration of 1 × 10 ⁵ copies / ml of VNNV and VHSV, respectively. After virus inoculation, the cells were cultured for 15 days without changing medium, and the cells were observed under a microscope to confirm cell degeneration. In addition, the cell number was measured at intervals of 3 to 4 days during the culture, and the culture supernatant was sampled to determine the virus concentration. Since VHSV had a culture optimum of 20 ° C., EPC cell lines were cultured at 20 ° C. after virus inoculation.

As a result, as shown in Figs. 7 and 8, in the case of E-11 and EPC inoculated with VNNV and VHSV, cells dropped and lysed 7 days after infection, and the number of cells rapidly decreased, and the virus concentration was greatly increased at this time. Increased. In the case of IMV-inoculated PMF and GF-1, cell degeneration was observed, but no cell dropping was observed. Many cells were attached to MCB and virus concentration gradually increased. In particular, it was confirmed that the MCB-attached PMF showed a high concentration of virus production without decreasing the number of cells.

Example  8. MCB  Subculture and Scale-up of Adherent Fish Cell Lines

There are two methods of subculture and scale-up method for dividing cells cultured in MCB to increase the cell number of MCB-attached fish cell lines. In the present invention, the cells of existing MCB are divided into flasks of the same capacity. The process, called subculture, was defined as the scale-up of the entire cell-attached MCB from the lower volume flask to the higher volume flask.

In the case of subculture, a 250 ml flask was incubated with the PMF cell line attached to the MCB by the method optimized in the above examples, and after 15 days of culture, 125 ml of the medium containing the cell cultured MCB 5 mg / ml and the new MCB were added. 1,1 (v / v) subculture to mix 125 ml of medium containing 5 mg / ml, 50 ml containing 5 mg / ml of previously cultured cell-attached MCB and 200 ml of medium containing new MCB 5 mg / ml 1: 4 (v / v) subculture was performed respectively. After adding the new beads, the same cell culture method was repeated for 2 hours after 1 minute of 60 rpm agitation for 2 hours, and the rate of attachment of cells to MCB was measured at 3 days intervals.

In the case of scale-up, the MCB-attached cell line was transferred from a small flask to a large flask (10 ml → 50 ml → 500 ml). Specifically, 10 ml of PMF cell line attached to MCB was cultured in an optimized manner in the above examples, and 50 ml (5-fold scale-up) after 15 days of culture. 10-fold scale-up (500 ml) was performed after 21 days after 5-fold scale-up. The increase in cell number was then expressed as the total cell number in the flask (FIG. 9).

In addition, IMV, a virus showing susceptibility to PMF, was inoculated to a concentration of 1 × 10 ⁵ copies / ml in a flask in the final cultured cells in subculture and scale-up. After 15 days, the virus concentration was measured.

Cell attachment rate after subculture in microcarriers (%) Pre culture incubation period Incubation period after subculture 0 3 6 9 12 15 3 6 9 12 15 18 21 Pre culture 0 88.0 92.0 95.5 98.5 98.5 1: 1 subculture 41.0 65.7 85.7 97.9 95.9 97.7 99.0 99.2 1: 4 subculture 18.5 37.5 64.7 86.4 92.7 95.1 97.8 99.2

As a result, the rate at which 90% or more cells were attached to new MCB after subculture was confirmed as 9 and 15 days at the ratio of MCB-attached cell lines and adding new beads 1: 1 and 1: 4 (Table 7). The number of cells reached more than 2x10 ⁶ cells / ml after 15 days and 21 days in both groups. In the case of scale-up, cell adhesion rate and cell concentration continued to increase after scale-up, and after 10-fold scale-up, final 1 × ^{10 9} cells / flask or more cells were produced (FIG. 9). In addition, the virus was inoculated into MCB-PMF produced in subculture or scale-up, and confirmed after 12 days, and all of the virus produced 5 × 10 ⁹ copies / ml or more. In particular, the scale-up method allowed 350 ml of virus to be produced every three days.

Therefore, it can be seen that the culture of the microcarrier bead-attached cells using this scale-up method is suitable for mass production of cells and viruses, and the vaccine produced with the virus produced in this way is large enough to be directly used in aquaculture. .

Example  9. On MCB  Attached PMF  In cell lines IMV Semi-continuous culture

9-1. Depending on the badge change cycle Semicontinuous  culture

The PMF cell line attached to the MCB of Example 8 exhibited a characteristic of a typical continuous infection in which the virus concentration also continued to increase, and the cell maintained without degeneration for a certain period of time after the viral infection. Therefore, a titration experiment was performed to continuously perform cell growth and virus production for a long time after viral infection. Specifically, PMF cells were attached to cytodex 1 (5mg / ml) in a 250ml spinner flask and cultured for 15 days. Thereafter, the cells were infected with 1 × 10 ⁵ copies / ml of IMV to obtain an MCV-PMF cell line infected with IMV. Subsequently, continuous infection was performed by applying a semi-continuous microcarrier culturing system (SCMCS) in which the culture medium (virus) was obtained and cultured under the optimum cell culture conditions of the present invention, followed by a medium exchange in which a new medium was added. Continued production of the virus from the cell line. To this end, the medium exchange cycle after virus infection was adjusted to 1, 3, and 5 days (SCMCS-1day, SCMCS-3day, and SCMCS-5day) to confirm the change in cell number and production virus concentration.

As a result, when the medium exchange and virus harvesting is carried out every day, the host cells are maintained continuously, but the concentration of the virus did not increase more than a certain level. In the case of medium exchange every three days, the cell culture was continuously possible, and there was no significant effect on cell proliferation, and the concentration of the virus produced in the medium increased over a period of time, from 1x10 ⁹ to 1x10 ¹⁰ copies / It was kept at a high concentration of ml. The production of these viruses continued in semi-continuous cultures conducted more than 30 times, and after a long period of time, some cell degeneration and some cell dropouts were observed, but continuous compensation by growth of new cells in the beads In the meantime, host cell and virus production continued. When the medium was exchanged every 5 days, the initial virus increase was high, but since the number of host cells continued to decrease after 10 days of culture, continuous culture was difficult and the produced virus concentration also decreased (FIG. 10).

9-2. MCB  By type Semicontinuous  culture

It was confirmed whether the continuous culture of the MCB-attached PMF cell line of Example 9-1 can be attached to other types of MCBs (Cytodex1, Cytodex3, Cultispher and Rapidcell), and also compared the cell number and production virus concentration with the GF-1 cell line. It was. Cell number was measured after 12 days, the virus was inoculated after 15 days of cell culture to show the virus concentration after 12 days (Table 8).

Cell count after 12 days
(x10 ⁵ cells / ml) Virus concentration 12 days after infection
(x 10 ⁸ copies / ml) Cytodex 1 PMF 21.90 9.47 GF-1 24.50 0.25 Cytodex 3 PMF 18.00 6.08 Cultispher PMF 15.60 8.90 GF-1 17.5 0.16 Rapidcell PMF 8.54 5.39

As a result, it was confirmed that even in IMV continuous infection of PMF cells attached to various MCBs (Cytodex1, Cytodex3, Cultispher and Rapidcell), the cells were maintained without significant cytopathic effect, and in the case of Cytodex1 and 3, the number of cells was rather increased. It was. In addition, the concentrations of IMV produced by the semi-continuous culture method in all kinds of MCB-attached cell lines were up to 2x10 ⁹ copies / ml or more, especially when semi-continuously cultured MCB-attached PMF cell lines. Virus production was significantly higher than GF-1, a cell (Table 8).

9-3. By virus subgroup Semicontinuous  culture

It was confirmed whether the semi-continuous culture of the MCB-attached PMF cell line of Example 9-1 was applicable to subgroups II, III and IV viruses of IMV.

As a result, more than 1x10 ⁹ copies / ml of virus was obtained in all MCB-attached PMF cell lines infected with subgroup II, III and IV viruses, respectively, and the PMF cell lines infected with three subgroups of IMV maintained the cell number without dropping out. It was possible to continue to produce (Fig. 11). Accordingly, it was found that MCB-attached PMF cell line was able to maintain cell growth and sustain virus production in semi-continuous culture for all IMV subgroups.

Example 10: Confirmation of effect enhancement according to mass-produced vaccines and excipients using MCB

In the above embodiments of the present invention, it was confirmed that mass culture of fish coin cells was possible using MCB, and mass production of fish virus in the coin cells was confirmed. In particular, PMF cells were able to continuously produce IMV (subgroups II, III and IV) in semi-continuous culture. Thus, the vaccine SCMCS-IMV- inactivated with the formalin (0.1%) after inoculation with MCB-attached PMF was inoculated into subgroup II, which is particularly damaging to domestic bream farming, among IMV viruses. sII (hereinafter referred to as SCMCS-IMV) and Beta-glucan (Sigma, baker's yeast (S. cerevisiae)) as excipients, squalene + aluminum hydroxide. Imiquimod (Enzo, USA) and Montanide ISA 780 VG (Seppic, 36064A) were each mixed to prepare vaccines, and their effects were confirmed. Specifically, a vaccine using beta-glucan as an excipient was added to the inactivated SCMCS-IMV-sII at a concentration of 1.5 mg / ml so that 0.15 mg of glucan could be contained in a fish body, followed by PolytronPT 1200E homogenizer (Kinematica, Switzerland). Homogenized using. Vaccines using squalene + aluminum hydroxide as excipients include squalene (Sigma, 2.5%, v / v), glycerol (Amresco, 10%, v / v), Tween 80 (Sigma aldrich, 0.5%, v / v) in PBS. And mixed with aluminum hydroxide (Sigma aldrich 1% w / v) and reacted on ice for 15 minutes and homogenized with a PolytronPT 1200E homogenizer. The squalene excipient thus prepared and the inactivated SCMCS-IMV were mixed at a ratio of 1: 1 (v / v) and homogenized with a PolytronPT 1200E homogenizer. Vaccine using Imiquimod as an excipient was dissolved in distilled water at a concentration of 1 mg / ml with diminished water of 98% or more, and diluted 100-fold to 1:20 (v / v) with SCMCS-IMV inactivated with formalin. It was prepared by mixing. A vaccine using Montanide ISA 780 VG based on vegetable oil as an excipient was prepared by mixing Montanide ISA 780 VG with formalin-inactivated SCMCS-IMV at 1: 1 (v / v) and then homogenizing with PolytronPT 1200E homogenizer. It was. Vaccine mixed with each excipient was tested with 20 sea bream individuals in each group, PBS was used as a control. Each dome, inoculated with each vaccine or PBS, was challenged by diluting the SCMCS-IMV (1 × 10 ⁵ copies / fish) 4 weeks after inoculation. The water temperature was maintained at 25 ℃, the return was 100% return every day. Check the cumulative mortality rate for each group for 30 days, and immediately remove the spleen from the fish tank for the mortal and moribund experimental fish, isolate the viral DNA, and confirm the infection by PCR using the M1F / R primer set. It was.

In addition, the neutralizing antibody titers of the vaccine subjects were measured to confirm the effect of the vaccine. Specifically, three dolphin fish from each vaccination group 4 weeks after vaccination were collected by microarrhythmic blood collection and centrifuged at 10,000 rpm, and the serum was separated and stored at -80 ° C until use. The serum dissolved at room temperature was then diluted 10-fold with L-15 medium for neutralizing antibody experiments, and treated at 55 ° C. for 30 minutes for complement inactivation. After dilution, the dilution period was set from 1/10 to 1/640 through the two-fold dilution, and mixed 1: 1 with 2 × 10 ⁴ copies / ml of the virus supernatant and neutralized at 25 ° C. for 2 hours. Therefore, the final concentration for the antibody titer was set from 1/20 to 1/1280 to measure the neutralizing antibody titer. Viruses neutralized at each concentration were infected by incubating 1 × 10 ⁵ cells / ml of inoculated PMF cells for 5 days in 24 well plates, and neutralizing antibody titers were determined by observing CPE 7 days after infection.

As a result, the SCMCS-IMV-sII vaccine without the excipient showed an average survival rate of 58.33%, and survival rate of 10% or higher was higher in the groups receiving the beta-glucan, Squalene + Aluminum, and Montanide ISA 780 VG, respectively. (Table 9). In particular, the highest survival rate was 83.33% when Beta-glucan was used as an excipient.

In addition, the neutralization test showed the highest value of 266.67 in the vaccine groups using Beta-glucan or Montanide ISA 780 VG as excipients, and showed more than 200 neutralizing antibody titers in all other vaccine groups (Table 9). . It is inferred that the immunization due to vaccination resulted in high average survival rate by producing sufficient antibodies against subsequent invasion of the antigen.

Claims (12)

1) attaching the cell line to the microcarrier beads by mixing the fish carrier cell line Pargus major fin (PMF) or Grouper fin cell (GF-1) with the microcarrier beads;
2) suspending the fish coin cell line attached to the microcarrier beads;
3) infecting the virus with fish coin cells attached to the microcarrier beads;
4) suspending the fish coin cells infected with the virus of step 3); And
5) Semi-continuous production of the virus using microcarrier beads, which comprises a semi-continuous cell culture step of repeatedly carrying out a medium exchange process of obtaining a culture supernatant containing a virus and then adding fresh medium. Way. delete The method of claim 1, wherein the process of stirring and stopping at 30 to 60 rpm is repeated for 1 to 3 hours to attach the cell line to the microcarrier beads. The method for semi-continuous production of virus using microcarrier beads according to claim 1, wherein the fish cell is cultured by further adding fish serum, fish muscle extract or fish yolk extract to the culture medium. The method according to claim 1, wherein the fish coin cell line attached to the microcarrier beads is cultured by exchanging medium every 1 to 3 days. The method according to claim 1, wherein the fish coin cell line attached to the microcarrier beads is cultured by stirring at 30 to 80 rpm. The method according to claim 1, wherein semi-continuous production of virus using microcarrier beads is subcultured by adding new microcarrier beads in a volume ratio of 1: 1 to 1: 4 to fish coin cells attached to the microcarrier beads. Way. The microcarrier bead according to claim 1, wherein the fish-coated cells attached to the microcarrier beads are incubated in a small volume flask and transferred to a larger volume flask to add new microcarrier beads to scale-up subculture. Method of semi-continuous production of viruses. The virus of claim 1, wherein the virus is selected from the group consisting of Iridoviridae Megalocytivirus (IMV), Viral nervous necrosis virus (VNNV) and Viral Hemorrhagic Septicemia (VHSV). Any one of the methods, semi-continuous production of virus using microcarrier beads. delete delete delete

Images