JP2003219866A - Canine kidney-derived suspendible cultured cell line and method for producing vaccine or pathogenic microbe for infection diagnosis using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for mass-culturing/producing any of various pathogenic microbes as a raw material for vaccines for infectious animal diseases or antigens for infection diagnoses in an efficient step by using a suspendible cultured cell line capable of continuous suspension culture which is created from a normal adhesive monolayer cultured cell line sensitive to various pathogenic microbes including pathogenic virus. <P>SOLUTION: The suspendible cultured cell line is a canine kidney-derived one which is created from a canine kidney-derived adhesive monolayer cultured cell line. The canine kidney-derived suspendible cultured cell line is either a cell line deposited as an accession No. of FERM P-18443 or a cell line having biological properties equivalent to those thereof. Suspension culture of the cell line enables the efficient mass production of the aimed pathogenic microbes. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a canine kidney-derived floating culture cell line which is a suspension-culturable cell line established from a canine kidney-derived adherent monolayer culture cell line K cell line. The present invention relates to a method for producing a pathogenic microorganism using a cultured cell line, particularly as a vaccine for an infectious disease in animals or as an antigen for diagnosis of infection.

[0002]

2. Description of the Related Art Demand for vaccines and diagnostic agents for infectious diseases such as companion animals such as dogs and cats and economical animals such as poultry such as cows, pigs, horses and chickens, etc. in Japan and around the world is high. The development of expensive and efficient production / manufacturing methods therefor is desired.

Although the raw materials for vaccines and antigens for diagnostic agents for infections applied to companion animals such as dogs and cats and economical animals such as poultry such as cows, pigs, horses and chickens are various pathogenic microorganisms including viruses, A monolayer culture method is generally used for culturing and producing various pathogenic microorganisms such as viruses as the raw materials.

The industrial scale production method of pathogenic microorganisms is aimed at cells cultured in a monolayer on a substrate such as glass in an inner surface of a plate culture container or a rotary culture bottle, plastic, and plastic coated with a thin layer of collagen. A monolayer culture method of infecting various pathogenic microorganisms such as viruses and statically culturing or rotating culture to prepare virus or chlamydia in the culture supernatant, or ceramics, dextran, in a bioreactor (culture tank)
Cells are grown in a monolayer on the surface of micro spherical particles (microcarrier beads) made of various materials such as glass, silicon, plastic, collagen, gelatin, and polyacrylamide, and then the cells on the grown microcarrier beads are infected with virus. After infecting various pathogenic microorganisms such as, and then culturing while stirring to prepare various pathogenic microorganisms such as the target virus and chlamydia in the culture solution, so-called microcarrier culture method, further, the culture area A system using a hollow fiber membrane (holofiber) is commonly used to increase the number.

However, these monolayer culture methods are
(1) Peeling and dispersion work of cultured cells by treatment with enzyme such as trypsin-sodium ethylenediamine tetraacetate (EDTA) from the culture container, (2) purchase of many culture containers, (3) washing of glass rotary culture container Sterilization, (4)
It is not necessarily an efficient method in terms of production cost and production process, such as the purchase of microcarrier beads, and further (5) accurate cell attachment work to the microcarrier beads, and the productivity is low.

Under such circumstances, in the manufacturing industry for vaccines and the like, various pathogenic microorganisms such as pathogenic viruses used as raw materials for vaccines and infection diagnosis antigens can be stably produced in a large amount in an efficient process. It is desired to develop a method that can be propagated and produced at low cost.

[0007] As a method for efficiently and stably proliferating various pathogenic microorganisms such as viruses as antigens of such vaccines and infection diagnostic agents, suspension culture in which various pathogenic microorganisms such as target viruses are infected Suspension culture that can obtain various pathogenic microorganisms such as the target virus in the culture solution by a simple and efficient method of suspension culture of possible suspension culture cell lines in a culture tank while stirring The method is considered to be the preferred one.

[0008] The suspension culture method is a method consisting of a simplified process of suspension culture of a suspension-cultured cell line infected with a pathogenic microorganism while stirring in a culture tank. It is a culture method that is superior in terms of culture and production costs to the monolayer culture method that uses a plate culture vessel or a rotary culture bottle that requires a culture vessel.

However, ordinary cells cannot grow unless they adhere to a substrate, and there are few cell lines that can be suspension-cultured. Heretofore, as a method for producing a suspension culture cell line from monolayer culture cells, that is, normal adherent cells, a method for producing a suspension culture cell line from a fish-derived monolayer culture cell line (JP-A-10-66563). And a lactoferrin as an adhesion inhibitory factor in a serum-free medium, which is a mouse-derived fibroblast cell line L-9.
A method for producing a suspension-cultured cell line from 29 cell lines (Japanese Patent Laid-Open No. 8-66184) was established, and further, newborn hamster lung cell line HmLu-1 and calf kidney cell line C were established.
Only a suspension culture cell line adapted to suspension culture for KTC6-1 has been established.

As a suspension culture method, a suspension culture method of human embryonic kidney cell-derived 293 strain and baby hamster kidney-derived BHK cell line, which are human-derived monolayer culture cell lines, has been established (Japanese Patent Laid-Open No. 01- No. 296984, Japanese Patent Laid-Open No.
No. 1-296986, Japanese Patent Publication No. 08-4495).

On the other hand, Merchant and Hellman
(Proc. Soc. Exp. Biol. Med., 1962, 110: 194-198) is a mouse fibroblast cell line derived from an L cell-derived LM cell line, and Nagle et al. (Proc. Soc. Exp. Biol. . Med. 1
963, 112: 340-344), L cells, HeLa cells (human-derived cervical cancer cell line), rat spleen cell line, monkey kidney cell line, guinea pig kidney cell line, and feline kidney cell line were each subjected to shaking culture. It is reported that suspension culture can be performed by shaking culture in a medium containing methylcellulose for preventing damage to cells and formation of cell clusters.

Similarly, Nakamura and Nishizawa (Journal of Infectious Diseases, 1980, Vol. 54, No. 6: 306-312) are MDC.
It has been found that K cells (adhesive monolayer culture cell line derived from dog kidney) can be suspension-cultured without adhering the cells to the culture vessel by stirring culture in a medium containing 3% methylcellulose.

[0013]

However, even for cells that can be used for suspension culture, suspension-cultured cell lines (Japanese Patent Laid-Open No. 10-66563) produced from a fish-derived monolayer culture cell line, Cells other than the hamster kidney-derived BHK cell line, hamster lung-derived HmLu-1 cell line, bovine kidney-derived CKTC6-1 cell line and mouse-derived fibroblast cell line L-929 cell line were placed in suspension culture. It is not a cell line consisting of only acclimated cells. Therefore, among these cells, cells that have not been acclimatized to suspension culture die during the culture, and the number of dead cells in the culture medium increases, so that pathogenic microorganisms as raw materials for vaccines and diagnostic agents for infection are proliferated. Cells, that is, cells for producing a virus for vaccine and the like are not suitable.

Under these circumstances, a pathogenic virus as a raw material for a vaccine or an infection diagnostic antigen for infectious diseases of companion animals such as dogs and cats or economical animals such as poultry such as cows, pigs, horses and chickens. It is desired to establish a suspension culture cell line and a suspension culture method thereof capable of efficiently proliferating and producing various pathogenic microorganisms such as.

On the other hand, some vaccines cause serious side reactions in inoculated animals, and one of the causes of such side reactions is derived from bovine serum used in the process of growing and producing pathogenic microorganisms for vaccines. Contamination of vaccines with antigenic substances is considered. Under such circumstances, it is desired to establish a culture method using a culture solution in which the content of bovine serum components, which can be an allergen of adverse reactions, is reduced as much as possible as a method for culturing and producing pathogenic microorganisms for vaccines. It is desired to develop a vaccine that is safe for animals by using the method as one manufacturing process.

Therefore, the present invention has produced and produced a suspension culture cell line capable of continuous suspension culture from an ordinary adherent monolayer culture cell line sensitive to various pathogenic microorganisms such as pathogenic viruses. By using a floating culture cell line, companion animals such as dogs and cats, and cows, pigs,
To establish a method for culturing and producing a large amount of various pathogenic microorganisms, which are raw materials for vaccines and infectious disease diagnostic antigens for economical animals such as poultry such as horses and chickens, in an efficient process. To aim.

Further, the present invention aims to establish a method for culturing and producing a large amount of pathogenic microorganisms as a raw material of a safe vaccine in which serum components harmful to animals are reduced as much as possible in an efficient process. To aim.

[0018]

[Means for Solving the Problems] In order to achieve the above object, the present inventors have conducted various studies using various adherent monolayer culture cell lines, and as a result, found that the dog kidney-derived adherent monolayer culture cell line. In addition to producing the target floating culture cell line from
Using the canine kidney-derived floating culture cell line, a culture method for proliferating and producing pathogenic microorganisms such as various pathogenic viruses as raw materials for vaccines and infection diagnostics in an efficient manner was established.

That is, the suspension-cultured cell line of the present invention is
It is a dog kidney-derived floating culture cell line produced from a dog kidney-derived adherent monolayer culture cell line, and this dog kidney-derived floating culture cell line is the cell line deposited under accession number FERM P-18443, Alternatively, it is a cell line having biological properties equivalent to this cell line.

In the present invention, the above floating culture cell line means the following. That is, most of animal cultured cells excluding hematopoietic-derived cells and tumor cells,
When culturing, glass such as plate culture vessels and inner surfaces of rotary culture bottles, plastics, plastics coated with a thin layer of collagen, and substrates such as hollow fiber membranes (holofibers) are necessary, and monolayer cells attached to these substrates In contrast to those that form a layer and proliferate (in the present specification, such cells are referred to as “adherent monolayer cultured cells”), while the suspension-cultured cells do not have cells attached to the substrate. It is a cell that can proliferate, and means a cell that can be cultured by simply suspending it in a medium without requiring a substrate during culture. And, the floating culture cell line of the present invention, the established floating culture cells, that is,
It means a cell that has acquired the property of being able to proliferate without adhering to the substrate, and that has established cell lines that continue to maintain this property even after subculture.

In the present invention, the above floating culture cell line is used, and the floating culture cell line is infected with a pathogenic microorganism and cultured by suspension culture to grow the floating culture cell and the pathogenic microorganism. It also includes a method for efficiently producing a pathogenic microorganism useful as an antigenic substance for a vaccine or a diagnostic agent against an infectious disease of an animal and obtaining the pathogenic microorganism. That is, the method for producing a pathogenic microorganism of the present invention comprises the steps of infecting the above-mentioned canine kidney-derived floating culture cell line with a pathogenic microorganism sensitive to this dog kidney-derived floating culture cell line, and infecting with the pathogenic microorganism. And a step of culturing the canine kidney-derived suspension-cultured cell line by a suspension culture method and culturing the canine kidney-derived suspension-cultured cells and proliferating pathogenic microorganisms. The pathogenic microorganisms released inside or the pathogenic microorganisms present in the cells are then subjected to known methods, for example,
The pathogenic microorganisms released into the medium are in the supernatant after centrifugation,
Alternatively, while the pathogenic microorganisms present in the cells are recovered in the filtrate by filtration using a membrane having a pore size of 0.45 μm or 0.22 μm, the sediment after centrifugation is resuspended in a new medium or buffer solution, and then homogenized. Or sonicator (ultrasonic crusher) cells are crushed and collected in the supernatant after centrifugation, and the pathogenic microorganisms thus collected are inactivated or have an activity, and in some cases, It is purified by a density gradient centrifugation method using sucrose or cesium chloride, and after removing impurities, it is used as an antigenic substance for vaccines and diagnostic agents.

There are many viruses and chlamydia as pathogenic microorganisms that are sensitive to the canine kidney-derived floating culture cell line. Generally, viruses and chlamydia that are highly sensitive and have high titers are antigens for vaccines or diagnostic agents. It is highly available.

In the suspension culture, it is preferable to use a medium containing bovine serum having a reduced serum component harmful to animals as the medium, which causes side effects in the obtained pathogenic microorganism when used as a vaccine. It is preferable in that there is no contamination of these harmful serum components, and as a bovine serum having reduced serum components harmful to such animals, polyethylene glycol (in the description of the Japanese Pharmacopoeia, “macrogol”) is used. Therefore, polyethylene glycol 4000 (molecular weight 2700 to 3400) or polyethylene glycol 6000 (molecular weight 7) is preferable.
400 to 9000) and the like.

Furthermore, the pathogenic microorganisms obtained by the present invention can be used as various pathogenic microorganism antigens for vaccines against infectious diseases of various animals and / or as antigens for infection diagnosis against infectious diseases of animals. And a diagnostic agent for infection against animal infectious diseases.

Examples of the vaccine for animal infectious disease and the diagnostic agent for infection of animal of the present invention include canine adenovirus type 1 (also known as canine infectious hepatitis virus) and canine adenovirus type 2 (also known as dog). Infectious pharyngeal tracheitis virus), canine parainfluenza virus, chlamydia sitathy, canine distemper virus, canine calicivirus, canine parvovirus type 1, canine parvovirus type 2, canine herpes virus, rabies virus, feline panleukopenia virus , Canine coronavirus, canine reovirus type 1, canine reovirus 2
, Swine flu virus, poultry pestovirus (also known as avian influenza virus), equine influenza virus, swine fever virus, vesicular stomatitis virus, swine vesicular disease virus, and the like, but are not limited thereto. Absent.

[0026]

BEST MODE FOR CARRYING OUT THE INVENTION In general, various pathogenic microorganisms such as pathogenic viruses that infect host cells and proliferate are mass-produced and produced as raw materials for vaccines and diagnostic agents for infections. There is also a method of adhering the adherent monolayer culture cell line to the inner surface of a rotary culture bottle, and performing adherent culture while rotating at a gentle speed of about 1 rotation per minute in an incubator. There is also a method of using a hollow fiber membrane (holofiber) to increase the culture area.

Further, as another method, a method of growing a monolayer culture cell line on microcarrier beads, and then culturing the cells infected with various pathogenic microorganisms such as viruses while stirring in a culture tank Is used.

The inventors of the present invention propagated various pathogenic microorganisms such as companion animals such as dogs and cats and pathogenic viruses such as economic animals as raw materials for vaccines and infection diagnostic agents.
As a production method, various studies were carried out for the purpose of establishing a suspension culture method consisting of more efficient steps than the current adherent culture method (monolayer culture).

That is, first, Vero cells (African green monkey kidney cell line) and CrFK cells (Crandell) were used.
feline kidney cells, cat kidney cell line),
Adhesive monolayer culture cell lines such as 104C1 cells (guinea pig whole fetal cell line) and K cell line which is a canine kidney-derived cell line are each subjected to Joklik modified Eagle Minimum Essential Medium (MEM) medium (JRH Bioscience) ), A suspension culture Eagle MEM medium (manufactured by Sigma-Aldrich Corp.), and a suspension culture Eagle MEM medium "Nissui" (manufactured by Nissui Pharmaceutical Co., Ltd.) in a suspension culture medium such as a shaker (the belly dancer). Using a shaker, STOVALL Rice Science Co., Ltd.), the cells were sub-cultured while gently shaking or culturing by rotation to keep the suspension state.
As a result, Vero cells, CrFK cells and 104C
One cell died without acclimatization to suspension culture in any of the media used, but surprisingly in K cells, 8
% Of polyethylene glycol (PEG6000, Wako Pure Chemical Industries, Ltd.) treated bovine serum at a concentration of 5% in Joklik modified Eagle MEM medium, and cells acclimated to suspension culture appeared.

Therefore, the K cell line acclimated to the suspension culture thus emerged is further subcultured, and finally the KS suspension, which is a canine kidney-derived suspension culture cell line consisting of only cells acclimated to the suspension culture. A sex culture cell line (hereinafter referred to as "KS cell line") was established.

The above-mentioned K cell line, which is a canine kidney-derived cell line, is a canine kidney-derived adherent monolayer cultured cell line MDCK cell, which is a cell line having good proliferative properties by cell cloning. It was established.

Then, the proliferative properties of the KS cell line established as described above and the parental K cell line were compared. That is, 1 × 10 ⁵ cells / ml of KS cell line and K cell line were respectively put in a plastic rotary culture bottle (roller bottle).
In 250 ml of medium at 37 ° C., the KS cell line was subjected to suspension culture at 4 revolutions per minute for 14 days, while for the K cell line, monolayer culture was performed at 1 revolution per minute for 10 days. The number of cells was counted every day to prepare a cell growth curve, and the growth properties of both cell lines were compared. As a result, KS
The proliferative potential of cell lines is almost the same as that of K cell lines,
It was found that the KS cell line did not impair the growth of the parental K cell line.

The KS cell line, which is a suspension-cultured cell line having a cell growth ability equivalent to that of the adherent monolayer cell line thus produced, is an independent administrative agency, AIST.
It has been deposited at the Japan Patent Organism Depositary on August 1, 2001 under the deposit number FERM P-18443.

Next, the present inventors applied the canine parainfluenza virus (canine parainfluenza) to the KS suspension-cultured cell line produced as described above.
virus, CPIV) or canine adenovirus 2
Type (canine adenovirus type)
2, CAV-2) was infected, and the presence or absence of a difference from the parent strain (K strain) that is susceptible or infectious to pathogenic microorganisms was examined. As a result, there was no difference in susceptibility or infectivity, and as a result, these infected cells were proliferated by suspension culture and CPIV and CAV-2 were obtained in the culture solution, thereby improving the efficiency of virus multiplication and The production method was established.

In other words, CPIV or CAV-2 is used as an infectious titer (multiplicity of infection)
ion: M.A. O. I. ) 0.01-infected KS cell line with 2% of 8% polyethylene glycol treated bovine serum
Growth culture curve of both viruses by measuring the virus amount (titer) of CPIV or CAV-2 in the culture solution every 2 days in suspension culture in Joklik modified Eagle's MEM medium containing 10% concentration for 10 days. It was created. Similarly, a K cell line infected with CPIV or CAV-2 at an infectious titer of 0.01 was allowed to stand in a Dulbecco's modified Eagle MEM medium containing 2% fetal bovine serum at 37 ° C. for 10 days for monolayer culture. And CAIV released into the culture supernatant
V-2 titers were measured every two days to create growth curves for both viruses. A growth curve of CPIV and CAV-2 when suspension-cultured using the KS cell line prepared as described above and a growth curve of CPIV and CAV-2 when monolayer-cultured using the K cell line are shown. As a result of comparison, the proliferation and production ability of CPIV and CAV-2 in the suspension culture method using the KS cell line was found to be CPIV in the monolayer culture method using the K cell line.
And CAV-2 have almost the same growth and production ability, and are more efficient than the monolayer culture method in the suspension culture method, and the amount of CPIV or C is comparable to that of the monolayer culture method.
It was able to grow and produce AV-2.

As described above, the KS cell line obtained according to the present invention has the same proliferative properties and susceptibility or infectivity to pathogenic microorganisms as the parental K cell line, but unlike the parental strain, the suspension culture is Since it has the property of being possible, it is possible to produce pathogenic microorganisms by the suspension culture method of the present invention.

As the suspension culture method for the suspension culture cell line and the suspension culture method for a large amount of suspension culture cell lines infected with pathogenic microorganisms, the suspension culture cell line is prepared by rotating a plastic or glass rotary culture bottle (roller). There are a method of culturing while rotating in a bottle), a method of culturing while stirring in a spinner flask, and a method of culturing while stirring in a culture tank (bioreactor). Among these methods, large-scale spinner flasks and bioreactors are used because the process is simplified for suspension culture of a large amount of suspension culture cell lines or suspension culture cell lines infected with pathogenic microorganisms. The preferred culture method is preferred.

As the medium that can be used for the above-mentioned suspension culture, bovine serum, glutamine, sodium bicarbonate, antibiotics (penicillin, streptomycin, etc.), etc. are added.
Joklik modified Eagle minimum essential medium (ME), which is generally known as a suspension culture medium
M) medium (JRH Bioscience), Eagle MEM medium for suspension culture (Sigma-Aldrich C)
orp. Manufactured by Nissui (manufactured by Nissui Pharmaceutical Co., Ltd.) and the like. On the other hand, as the serum added to the above-mentioned Eagle MEM “Nissui” for suspension culture in the present invention, bovine serum is preferable because it is cheaper than fetal bovine serum, and further, the dispersibility of suspended cells ( It is also preferable in that it is difficult to form a cell mass) and has good proliferative properties.

In suspension culture, 1.0 × 10
⁵~ 2.0 x 10⁵Perform at a cell concentration of about 1 cell / ml
Is preferred, and generally 1.5 × by the suspension culture of the present invention.
10 ⁶~ 2.0 x 10⁶Proliferate up to about 1 / ml
You can

In the above suspension culture method, it is particularly preferable that the bovine serum added to the medium is a medium containing bovine serum in which serum components harmful to animals are reduced. As a result, a companion animal such as dogs and cats, and pathogenic viruses and various pathogenic microorganisms such as economic animals such as cows, pigs, horses, and poultry using a medium containing bovine serum reduced in harmful components to animals. By suspension culture of the infected KS cell line, various pathogenic microorganisms such as virus can be prepared in the culture supernatant as a raw material for a vaccine that is less likely to cause side reactions in animals and has increased safety.

That is, since the lipoprotein, serum albumin, serum globulin, etc. contained in bovine serum show antigenicity when inoculated into an animal, the content of serum components harmful to the inoculated animal is 8%. ~ 12% (may be 25% in some cases) molecular weight of about 500 to 9000 (eg, PEG1500, 4000, 6000, etc.), preferably about 2400 to 9000 (eg, PEG4000, 6000, etc.) polyethylene glycol treatment. It is preferable that a harmful serum component such as lipoprotein is reduced, and a suspension culture method using a Joklik modified Eagle MEM medium containing 5% of bovine serum reduced in such a harmful serum component is used. Could be established.

By producing pathogenic microorganisms using this suspension culture method, various pathogenic microorganisms such as viruses as raw materials for vaccines that are less likely to cause side reactions in animals and have improved safety are contained in the culture supernatant. It can be prepared.

Furthermore, the present invention is to produce a vaccine for animals and a diagnostic agent for infectious diseases by a known method using the pathogenic microorganism prepared as described above as a raw material. As a method for producing a vaccine, for example, infected cells are suspension-cultured to recover pathogenic microorganisms released in the supernatant, or pathogenic microorganisms existing in cells are recovered by destroying the cells. At the same time, the vaccine can be prepared by inactivating the pathogenic microorganism by formalin treatment or the like and adding an adjuvant to the inactivated pathogenic microorganism. On the other hand, by attenuating the attenuated virus or the like by a suspension culture method, a live attenuated vaccine can be produced using the amplified attenuated virus or the like. The amount of pathogenic microorganisms in the vaccine is an amount sufficient to confer immunity in the vaccinated animal to prevent the infection of the targeted pathogenic microorganisms, typically 1 × 10 ³ when the pathogenic microorganism is a virus. ^.5 TCID ₅₀ / ml to 1 ×
The virus amount is 10 ^5.0 TCID ₅₀ / ml or more. In addition, examples of adjuvants that can be used include adjuvants that can impart systemic protective immunity or local protective immunity to vaccinated animals.

When used as a diagnostic agent for infectious diseases,
The infectious agent can be prepared by using the pathogenic microorganism inactivated as described above as an antigen or fixing the antigen isolated from the pathogenic microorganism on a support such as an ELISA plate or a nitrocellulose membrane. Such an infection diagnostic agent is known as a reaction with a secondary antibody having horseradish peroxidase, alkaline phosphatase or the like bound thereto after binding with an antibody present in the serum of a test animal, and a known color reaction thereafter. It is possible to visualize by carrying out the reaction, and it is possible to detect and confirm the presence or absence of antibody in the blood of animals infected with various pathogenic microorganisms, that is, the presence or absence of infection in test animals.

In the following, establishment of a canine kidney-derived floating culture cell line, that is, KS cell line, and proliferation of the KS cell line,
And, the case of using CAV-2 and CPIV as an example of the proliferative property of a pathogenic microorganism will be described in detail and specifically as an example of the suspension culture method of the present invention, but the present invention is not limited thereto. Not something.

[0046]

EXAMPLES Example 1. Canine kidney-derived floating cell line (K
S cell line) established Vero cells, CrFK cells, 104C1 cells and K
The cells were adapted to suspension culture. The medium is J
oklik modified Eagle MEM medium (manufactured by JRH Bioscience), Eagle MEM medium for suspension culture (Sig
ma-Aldrich Corp. (Manufactured by Nissui Pharmaceutical Co., Ltd.) was used. In addition, bovine serum added to each medium,
8% polyethylene glycol (PEG60 at 4 ° C overnight)
00, manufactured by Wako Pure Chemical Industries, Ltd.)
Remove the serum components precipitated by centrifugation at 00 xg for 30 minutes,
Further, a filter sterilized with a membrane filter (manufactured by Sartorius KK) having a pore size of 0.22 μm was used at a final concentration of 5%. A 25 ml tissue culture bottle (Sumitomo Bakelite Co., Ltd.) was used as the incubator.

2 × 10 ⁶ cells / 10 ml of each of the above cell lines
The suspension culture was carried out at 37 ° C. with static culture and gently shaking on The Belly Dancer Shaker (manufactured by STOVALL Life Science), and subculture was continued while exchanging the entire amount of the medium every 3 to 4 days. As a result of carrying out such suspension culture 20 times for each cell line, Vero cells, CrFK cells and 104C1 cells did not acclimate to suspension cultures and died in any of the used media. On the other hand, with respect to the K cell line, the appearance of cells growing in a floating state was observed only in the culture system using the Joklik modified Eagle MEM medium. Therefore, K cells acclimated to this suspension culture were further subcultured by the above-mentioned suspension culture method, and finally a suspension culture cell line consisting of only cells acclimated to suspension culture was established.

The dog kidney-derived floating culture cell line (KS cell line) thus obtained is named "KS floating culture cell line", and the administrative agency, National Institute of Advanced Industrial Science and Technology, Patent Depository Center And the consignment number F as of August 1, 2001
Deposited as ERM P-18443.

Example 2. Proliferation of KS cell line Canine kidney-derived floating culture cell line established in Example 1 (KS
The cell growth curve of the cell line) was prepared and the cell growth ability was examined. As the medium, 250 ml of Joklik modified Eagle MEM medium containing 5% of bovine serum treated with 8% of the above PEG6000 was used. The incubator has a culture area of 850c.
m ² plastic roller bottle (Becton,
Dickinson and Company) was used.

1 × 10 ⁵ cells / ml of KS cell line was added to 37
Suspension culture was performed at 4 ° C. while rotating 4 times per minute. Every day until the 14th day after the start of the culture, a part of the cultured cells was collected, and 0.
Dead cells were stained by mixing equal amounts of 3% trypan blue-PBS solution, and the number of living cells not stained with trypan blue was counted. A cell growth curve of the KS cell line was prepared based on the number of cells thus obtained and shown in FIG.

On the other hand, as a control, the proliferative ability of the parental canine kidney-derived adherent monolayer cell line (K cell line) was examined by the following method.

250 ml of Dulbecco's modified Eagle MEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.) containing 10% fetal bovine serum was used as the medium, and 1 × 10 ⁵ cells / ml of the cells were placed in the roller bottle at 37 ° C. Using 1 per minute
Monolayer culture was performed while rotating. Monolayer culture under these conditions was performed simultaneously on 10 roller bottles, and cells were detached from each roller bottle daily with trypsin-EDTA until 10 days after the start of culture, and cells washed with a new medium were prepared. Equal volumes of 0.3% trypan blue-PBS solution were mixed to stain dead cells, and the number of unstained live cells was counted. A cell growth curve of the K cell line was prepared based on the number of viable cells thus obtained and shown in FIG.

Regarding the above-mentioned KS cell line and K cell line proliferative tests, two lines (A
Series and B series) tests were conducted at the same time, and the presence or absence of error in each test was examined.

As a result of comparing the proliferative properties of the KS cell line shown in FIG. 1 with the proliferative properties of the K cell line shown in FIG. 2, the KS cell line was found to be 1.5 × 1 ml / ml by the 6th day after the start of culture. 10 ⁶
, To 1.6 × 10 ⁶ , whereas the K cell line grew from 1.6 × 10 ⁶ to 1.7 × 10 ⁶ per ml by the fourth day after the start of culture. As a result, it was clarified that the KS cell line maintained a proliferative capacity almost equal to that of the parental K cell line.

Example 3. CAV-2 using KS cell line
The proliferative property of the CAV-2 V197 strain in the KS cell line (suspension culture) was compared with that of the CAV-2 V197 strain in the control K cell line (monolayer culture).

(1) CAV-2 Proliferation of CAV-2 Infected KS Cell Line by Suspension Culture A total of 2.5 × 10 ⁸ KS cell lines were infected with CAV-2 at an infectivity of 0.01 and the infection. Replace the cells with 8% PEG
Jokli containing 2% bovine serum treated with 6000
Suspension culture was carried out in 250 ml of modified eagle MEM medium at 37 ° C. using a roller bottle at 4 revolutions per minute.
The culture supernatant collected every 2 days was used as a test sample, and CAV-2
The amount was quantified by the following virus titration method to prepare a CAV-2 growth curve. The prepared CAV-2 growth curve is shown in FIG.

(2) Proliferation of CAV-2 by monolayer culture of CAV-2-infected K cell line.
^Eight K cell lines were monolayer-cultured in 250 ml of Dulbecco's modified Eagle's MEM medium containing 2% fetal bovine serum at 37 ° C. using the roller bottle while rotating once per minute. Using the culture supernatant collected every 2 days as a test sample, the amount of CAV-2 was quantified by the following virus titration method,
A growth curve was created. The prepared CAV-2 growth curve is shown in FIG.

The CAV-2 titer determination method was performed as follows. That is, the titer of CAV-2 was 2 × 10 ⁵ cells / ml in each well of a 96-well tissue culture plate.
MDCK (Madin Darby kindey
cell) (ATCC CL-34 strain) (100 μl) was dispensed and cultured for 2 days to form a monolayer. Then, the culture supernatant is removed, and 1:10 ¹ to 1: 1 with Eagle MEM medium.
0 ⁸ to 10-fold serial diluted aforementioned test sample 50μl was added to each well for one hour virus at 37 ° C. After adsorption infect cells, the addition of Eagle's MEM medium 100μl containing 2% fetal calf serum The cells were cultured at 37 ° C for 6 days. For the measurement of virus titer, the well in which the cytopathic effect (CPE) due to CAV-2 was observed was regarded as positive, and the TCID ₅₀ / ml was calculated using the Behren's-Kelber method.

Regarding the above-mentioned CAV-2 growth curve preparation, two strains (A series and B series) were tested simultaneously for both the KS cell line and the K cell line, and there was an error in each test. It was investigated.

Increased CAV-2 of FIG. 3 created as described above.
CAV-2-infected KS cell line as evidenced by the growth curve
In case of suspension culture of
Rus grows most and its amount is 1 x 10^6.3 TC
ID_Fifty/ Ml to 1 x 10 ^6.7 TCID_Fifty/ M
It was in the range of 1. On the other hand, a single CAV-2 infected K cell line
In the case of layer culture, as shown in FIG.
Irus grows the most and its amount is 1 x 10^6.0 T
CID_Fifty/ Ml to 1 x 10^6.4 TCID _Fifty/
It was in the ml range. As a result, KS suspension culture cell line
CAV-2 has the ability to grow in suspension culture of K.
Compared to the growth ability of CAV-2 by monolayer culture of cell lines
It became clear that it was comparable to the vaccine and
Increasing CAV-2 as an antigen for dye diagnostics on an industrial scale
Compared to the monolayer culture method, the production process is
Floating culture method that is efficient and efficient in terms of production cost
Was established.

Example 4. CPIV using KS cell line
Of the proliferative CPIV 91880 strain was examined in comparison with the proliferative ability of the K cell line (monolayer culture) as a control.

(1) CPIV Proliferation by Suspension Culture of CPIV-Infected KS Cell Lines 2.5 × 10 ⁸ KS cell lines were infected with CP at an infectivity of 0.01
IV was infected, and the infected cells were treated with the above 8% PEG600.
Suspension culture was performed in 250 ml of Joklik modified Eagle's MEM medium containing 2% of 0-treated bovine serum at 37 ° C. using a roller bottle at 4 revolutions per minute. The amount of CPIV in the culture solution was quantified every two days by the virus titration method according to the following method, and a growth curve of CPIV was prepared. The prepared CPIV growth curve is shown in FIG.

(2) Proliferation of CPIV by monolayer culture of CPIV-infected K cell line Dulbecco containing 2 × 10 ⁸ K cell lines infected with CPIV at a titer of 0.01 and containing 2% fetal bovine serum Monolayer culture was carried out in 250 ml of modified Eagle MEM medium at 37 ° C. using a roller bottle while rotating once per minute.
The culture supernatant collected every two days was used as a test sample, and the amount of CPIV was quantified by the following virus titration method to prepare a CPIV growth curve. The growth curve of the prepared CPIV is shown in FIG.

CPIV titer determination was performed as follows. That is, the titration of CPIV was performed by MD of 2 × 10 ⁵ cells / ml in each well of a 96-well tissue culture plate.
100 μl of CK cells (ATCC CL-34 strain) was dispensed and cultured for 2 days to form a monolayer. Then, the culture supernatant was removed, and 50 μl of the above-described test sample, which was serially diluted 10 times from 1:10 ¹ to 1:10 ^8, was added to each well, and the mixture was added at 37 ° C.
After adsorbing and infecting cells with the virus for an hour, 100 μl of Eagle's MEM medium containing 2% fetal bovine serum was added to the cells, and 3
It was cultured at 7 ° C for 6 days. Next, 50 μl of the culture supernatant of the above-mentioned infected cells was added to each well of a V-bottom 96-well microtiter plate, and 50 μl of 0.4% (V / V) guinea pig red blood cell suspension PBS solution was added and mixed. The presence or absence of hemagglutination ability by CPIV was examined, and the well in which hemagglutination was confirmed was determined to be positive, and TCID ₅₀ / ml was calculated by the Behrens-Kelber method.

Regarding the above-mentioned CPIV growth curve, two strains were tested simultaneously for both the KS cell line and the K cell strain, and the presence or absence of error in each test was examined.

The CPIV of FIG. 5 created as described above.
As is clear from the growth curve, in the suspension culture of the CPIV-infected KS cell line, the virus was most proliferated on the 2nd or 4th day of the culture, and the amount thereof was 1 × 10 ^8.7 TCI.
D ₅₀ / ml to 1 × 10 ^{8 . 5} TCID ₅₀ / ml
Was in the range. On the other hand, in the case of monolayer culture of CPIV-infected K cell line, as shown in FIG. 6, the virus was most proliferated on the second day of culture, and the amount was 1 × 10 ^8.7 TCID.
The range was from ₅₀ / ml to 1 × 10 ^9.0 TCID ₅₀ / ml.

As a result, C by suspension culture of the KS cell line was obtained.
Proliferative ability of PIV is comparable to that of CPIV by monolayer culture of parental K cell line, and as a method for proliferating and producing CPIV as an antigen of vaccine or infection diagnostic agent on an industrial scale, The production process was made more efficient than the monolayer culture method, and an efficient floating culture method was established in terms of production cost.

Example 5. Method for Culturing KS Suspended Culture Cell Line in the Presence of Hazardous Substance-Reduced Bovine Serum A KS cell line infected with CAV-2 or CPIV is suspension-cultured, and CAV-as an antigen of a vaccine or an infection diagnostic agent is cultured in the culture. 2 and CPIV were prepared, the serum protein which was harmful when the animal was inoculated from the bovine serum added to the medium was reduced.

More specifically, bovine serum has a P concentration of 12%.
EG4000 (Macrogol 4000, manufactured by NOF CORPORATION)
Was added and the mixture was allowed to stand at 4 ° C. overnight, and the serum components precipitated by centrifugation at 11,900 × g for 30 minutes were removed, and the supernatant was centrifuged at 0.4
It was sterilized by filtration through a membrane filter (made by Corning Inc.) of 5 μm and then 0.22 μm.

Next, the serum component content in the above 12% polyethylene glycol-treated bovine serum was analyzed. Specifically, bovine serum before treatment with polyethylene glycol and 1
For 2% polyethylene glycol-treated bovine serum, the total protein amount was measured by the Burett method and the albumin amount was measured by BCG.
The amount of globulin was calculated by subtracting the amount of albumin from the amount of total protein by each method, and it was examined how these serum proteins were reduced by treatment with 12% PEG4000. The results are shown in Table 1 including the case of treatment with other types and concentrations of polyethylene glycol.

[0071]

[Table 1] As is clear from Table 1, the serum not treated with polyethylene glycol had a total protein content of 9.
4 g, albumin 3.2 g, and globulin 6.2
The total amount of protein in 100 ml of serum treated with polyethylene glycol was 3.1 to 100 g.
The amount of albumin was 7.5 g, the amount of albumin was 2.4 to 3.0 g, and the amount of globulin was 0.7 to 4.4 g, respectively.

On the other hand, the total amount of protein, the amount of albumin and the amount of globulin in the serum were all significantly reduced by the PEG6000 treatment as compared with the PEG1500 treatment and the PEG4000 treatment. However, considering that polyethylene glycol-treated serum is used in a culture system for propagating viruses and the like to be vaccinated in animals, and that PEG4000 is generally used as a drug, PEG4000 is used as a KS cell line. It is considered preferable to use it in a culture system.

Regarding the concentration of polyethylene glycol in the polyethylene glycol treatment, as shown in Table 1, PEG1500 and PEG400 having a concentration of 8% were used.
The amount of total protein, globulin, and albumin in bovine serum decreased even when treated with 0 or PEG6000, but the reduction was less than with polyethylene glycol treatment with a concentration of 12%. The bovine serum treated with was preferable as a growth system for producing a vaccine that is expected to reduce side effects in inoculated animals.

Further, PEG1 is used at least when the concentration of polyethylene glycol for treating serum is 12%.
The dispersibility of the KS cell line is better and the formation of cell clusters is less likely to occur in the culture in the medium containing bovine serum treated with PEG4000 than in the culture medium containing the bovine serum treated with 500 or PEG6000. It was a thing. That is, the treatment with polyethylene glycol not only removes the protein in the serum that causes side effects when vaccinated, but it is unconfirmed,
This polyethylene glycol treatment (particularly preferably at a concentration of 12%) is made possible because the factor that promotes the attachment of cells to a substrate or the like, which is considered to be a proteinaceous substance, is also removed to make the suspension culture of the present invention more reliable. Or 25
The use of bovine serum by PEG4000 treatment up to about 10% is not only the production of pathogenic microorganisms for vaccine by suspension culture using KS cell line, but also the side effect of inoculation is not necessary to be considered as an antigen for diagnostic reagents. The production of pathogenic microorganisms as above was also preferable.

Next, 12% PE prepared as described above.
The culturing ability of the KS cell line in the suspension culture method using a medium containing 5% of G4000-treated bovine serum was examined. Specifically, at 37 ° C. in a Joklik modified Eagle MEM medium containing 12% PEG4000-treated serum at a final concentration of 5%.
Then, KS cells having a cell concentration of 1.0 × 10 ⁵ cells / ml were subjected to suspension culture for 14 days while rotating 4 times for 1 minute using a roller bottle. As a result, as shown in FIG.
The cells proliferated from 1.8 × 10 ⁶ cells / ml to 2.0 × 10 ⁶ cells / ml by the 7th day of culture, and the cells were 8% as shown in FIG.
Jokli containing 5% PEG6000 treated bovine serum
Proliferation of the KS cell line in suspension culture using k-modified Eagle MEM medium and cell proliferation equal to or higher than that in the monolayer culture of the parent K cell line shown in FIG.

Polyethylene glycol other than the above, that is, 12% PEG 1500 or 12% PEG 60
The cell proliferation when using Joklik modified Eagle MEM medium containing 5% of bovine serum treated with 00 was shown in FIG. 8 and FIG. 9, respectively. 8 and 9
As shown in (1), it can be seen that almost the same results can be obtained by changing the polyethylene glycol used for the treatment of bovine serum.

As a result of the above, the KS cell line is a culture system in which, when inoculated into an animal, bovine serum proteins that are harmful, such as allergic reactions due to their antigenicity, are significantly reduced, and are the parental K cell lines. It was found to show comparable or higher cell proliferative properties compared to that of the above, and even when the vaccine prepared in this way was ingested in animals, allergic reactions such as paraface swelling, pruritus and urticaria were observed. No side effects such as collapse, anemia, hypotension, respiratory distress, hypothermia, salivation, tremor, convulsions and anaphylactic reactions such as urinary incontinence were not observed. As a result, a suspension culture method of the KS cell line for producing a virus vaccine or the like with improved safety could be established.

[0078]

INDUSTRIAL APPLICABILITY According to the present invention, a floating culture cell line derived from dog kidney is provided. This cell line has a cell proliferation property equivalent to that of the canine kidney adherent monolayer culture strain which is a parent strain, and further, the proliferation property of a pathogenic microorganism used as a raw material of a vaccine or an infectious agent is also equivalent to that of the parent cell strain, The present invention has the effect that the target pathogenic microorganism can be grown and produced by an efficient process of only suspension culture of the cell line infected with various pathogenic microorganisms as antigens of vaccines or infection diagnostic agents.

Further, the development of a suspension culture method of the suspension culture cell line in a medium containing bovine serum in which serum components harmful to animals are reduced was developed by various methods for producing an animal vaccine in which side reactions are minimized. It was useful for the growth and production of pathogenic microorganisms.

[Brief description of drawings]

FIG. 1 is a graph showing the proliferative properties of the established canine kidney-derived floating culture cells (KS cell line) of the present invention. In the figure, ■
And ◯ indicate the results of each of the two tested lines (A series and B series).

FIG. 2 is a graph showing the proliferative property of an adherent monolayer cell line derived from dog kidney (K cell line), which is a parent strain of the KS cell line.
In the figure, ▪ and ○ show the results of each of the two tested lines (A series and B series).

FIG. 3 is a graph showing the proliferative activity of canine adenovirus type 2 (CAV-2) infected with the established KS cell line of the present invention. In the figure, ▪ and ○ show the results of each of the two tested lines (A series and B series).

FIG. 4 is a graph showing the proliferative activity of canine adenovirus type 2 (CAV-2) infected with a K cell line, which is a parent strain of the KS cell line. In the figure, ■ and ○ are the two tested lines (A
The results of each of the series and the series B) are shown.

FIG. 5 is a graph showing the proliferative activity of canine parainfluenza virus (CPIV) infected with the established KS cell line of the present invention. In the figure, ▪ and ○ show the results of each of the two tested lines (A series and B series).

FIG. 6 is a graph showing the proliferative activity of canine parainfluenza virus (CPIV) infected with a K cell line, which is a parent strain of the KS cell line. In the figure, ▪ and ○ show the results of each of the two tested lines (A series and B series).

FIG. 7 shows polyethylene glycol (P
3 is a graph showing the proliferative properties of KS cell lines when a suspension medium supplemented with bovine serum treated with EG4000) was used. In the figure, ▪ and ○ show the results of each of the two tested lines (A series and B series).

FIG. 8 shows polyethylene glycol (P
3 is a graph showing the proliferation of KS cell lines when a suspension medium supplemented with bovine serum treated with EG1500) was used. In the figure, ▪ and ○ show the results of each of the two tested lines (A series and B series).

FIG. 9 shows polyethylene glycol (P
3 is a graph showing the proliferation of KS cell lines when a suspension medium supplemented with bovine serum treated with EG6000) was used. In the figure, ▪ and ○ show the results of each of the two tested lines (A series and B series).

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 39/15 A61K 39/175 39/175 39/187 39/187 39/205 39/205 39/215 39 / 215 39/23 39/23 39/235 39/235 A61P 31/12 171 A61P 31/12 171 31/16 31/16 31/22 31/22 C12N 1/20 A C12N 1/20 7/02 7 / 02 5/00 E (72) Inventor Susumu Furuuchi 2-9-22 Takamihara, Kukizaki-cho, Inashiki-gun, Ibaraki Kyoritsu Pharmaceutical Co., Ltd. Tsukuba Central Research Laboratory F-term (reference) 4B065 AA01X AA90X AA95X BA30 BB25 CA45 4C085 AA03 BA45 BA51 BA52 BA55 BA57 BA60 BA64 BA71 BA75 BA77 BA78 CC04 CC08

Claims (14)

[Claims] 1. A suspension culture cell line derived from canine kidney, which can be suspension-cultured. 2. The dog according to claim 1, wherein the floating culture cell line derived from canine kidney is the cell line deposited under accession number FERM P-18443 or a cell line having biological properties equivalent to this cell line. Kidney-derived floating culture cell line. 3. A step of infecting a canine kidney-derived floating culture cell line with a pathogenic microorganism sensitive to the canine kidney-derived suspension culture cell line, and a method of subjecting the infected dog kidney-derived suspension culture cell line to a suspension culture method. A method of producing a pathogenic microorganism, which comprises culturing and proliferating the pathogenic microorganism. 4. The method for producing a pathogenic microorganism according to claim 3, wherein the pathogenic microorganism is a virus or chlamydia. 5. The medium for culturing the canine kidney-derived floating culture cell line by the suspension culture method is a medium containing bovine serum reduced in serum components harmful to animals.
Alternatively, the method for producing a pathogenic microorganism according to claim 4. 6. The method for producing a pathogenic microorganism according to claim 5, wherein the bovine serum having reduced serum components harmful to animals is bovine serum treated with polyethylene glycol. 7. The method for producing a pathogenic microorganism according to claim 3, wherein the pathogenic microorganism is a pathogenic microorganism as various pathogenic microorganisms for vaccines against infectious diseases of animals. 8. The method for producing a pathogenic microorganism according to claim 3 or 4, wherein the pathogenic microorganism is a pathogenic microorganism as an antigen for diagnosing infection against animal infectious diseases. 9. An animal vaccine prepared by using the pathogenic microorganism produced by the production method according to any one of claims 3 to 7 as an antigen. 10. The animal vaccine according to claim 9, wherein the pathogenic microorganism is a pathogenic microorganism that infects a companion animal or a pathogenic microorganism that infects an economic animal. 11. The pathogenic microorganism is canine adenovirus 1
Type, canine adenovirus type 2, canine parainfluenza virus, Chlamydia sittaci, canine distemper virus, canine calicivirus, canine parvovirus 1
Type, canine parvovirus type 2, canine herpesvirus,
Rabies virus, feline panleukopenia virus, canine coronavirus, canine reovirus type 1, canine reovirus type 2, swine influenza virus, poultry plague virus, equine influenza virus, swine fever virus, vesicular stomatitis virus, swine vesicle. The animal vaccine according to claim 9 or 10, which is a disease virus. 12. An infection diagnostic agent prepared by using the pathogenic microorganism produced by the production method according to claim 3, 4, or 8 as an antigen. 13. The infection diagnostic agent according to claim 12, wherein the pathogenic microorganism is a pathogenic microorganism that infects a companion animal or a pathogenic microorganism that infects an economic animal. 14. The pathogenic microorganism is canine adenovirus 1
Type, canine adenovirus type 2, canine parainfluenza virus, Chlamydia sittaci, canine distemper virus, canine calicivirus, canine parvovirus 1
Type, canine parvovirus type 2, canine herpesvirus,
Rabies virus, feline panleukopenia virus, canine coronavirus, canine reovirus type 1, canine reovirus type 2, swine influenza virus, poultry plague virus, equine influenza virus, swine fever virus, vesicular stomatitis virus, swine vesicle. The diagnostic agent for infection according to claim 12, which is a disease virus.