JP4656300B2 - Method for producing immunoregulatory protein - Google Patents

Description

The present invention relates to a method for producing an immunoregulatory protein, and more particularly to a method for producing an immunoregulatory protein such as an immunoglobulin or a cytokine.

Immunomodulating proteins such as immunoglobulins and cytokines are one of the complex proteins that are expected to be applied to the medical field such as in-vivo diagnostics and therapeutics. With the recent progress of molecular biological techniques, methods for artificially producing the immunoregulatory protein have been developed.
On the other hand, in order to apply the immunomodulating protein to in-vivo diagnostic agents, therapeutic agents, etc., it is necessary to produce the immunomodulating protein efficiently in a short time.
The immunoregulatory protein is usually produced by human lymphocytes such as human lymphocytes and human hybridoma cells, but has a problem that its production efficiency is extremely low.
In order to cope with such a problem, a method for producing the immunoregulatory protein in which citrus juice is added to the human lymphocytes has been proposed (Patent Document 1).
However, the production efficiency of the immunoregulatory protein has not been sufficient in the production method.
JP-A-6-98763

An object of the present invention is to provide a method for efficiently producing the immunoregulatory proteins such as immunoglobulins and cytokines using the human lymphocytes such as human lymphocytes and human hybridoma cells.

As a result of intensive studies, the present inventors have found that culturing the human lymphocytes such as human lymphocytes and human hybridoma cells in the presence of collagen increases the production efficiency of immune regulatory proteins such as immunoglobulins and cytokines. The headline and the present invention were completed.

That is, the present invention
[1] A method for producing an immunoregulatory protein comprising culturing human lymphocytes in the presence of collagen ,
The collagen is white type jellyfish, China type jellyfish, semi-China tie
Selected from the group consisting of jellyfish, cannonball type jellyfish and ball type jellyfish
Collagen obtained from at least one kind heated at a temperature range of 80 to 150 ° C.,
The human lymphocytes are selected from the group consisting of human lymphocytes and human hybridoma cells.
At least one kind selected,
The immunoregulatory protein is selected from the group consisting of immunoglobulins and cytokines.
At least one kind
At least the immunoglobulin is selected from the group consisting of an IgM antibody and an IgG antibody.
Is also a kind,
And whether the cytokine is interferon α, β and γ and tumor necrosis factor α.
It is at least one member selected from the group consisting of the above, and provides a method for producing an immunoregulatory protein,
[2] A method for culturing human lymphocytes using collagen,
The collagen is white type jellyfish, China type jellyfish, semi-China tie
Selected from the group consisting of jellyfish, cannonball type jellyfish and ball type jellyfish
Collagen obtained from at least one kind heated at a temperature range of 80 to 150 ° C.,
The human lymphocytes are selected from the group consisting of human lymphocytes and human hybridoma cells.
It provides a method for culturing human lymphocytes, characterized in that it is at least one kind selected ,
[3] Collagen for culturing human lymphocytes,
The collagen for culturing human lymphocytes is a collagen obtained from at least one selected from the group consisting of white type jellyfish, China type jellyfish, semi-China type jellyfish, cannonball type jellyfish and ball type jellyfish. Heated according to the temperature range,
Human collagens for culturing human lymphocytes are characterized in that the human lymphocytes using the collagen for culturing human lymphocytes is at least one selected from the group consisting of human lymphocytes and human hybridoma cells. .

According to the present invention, it is possible to provide a method for efficiently producing the immunoregulatory proteins such as immunoglobulins and cytokines using the human lymphocytes such as human lymphocytes and human hybridoma cells.

First, human lymphocytes used in the present invention will be described.
Specific examples of human lymphocytes used in the present invention include B cells, T cells, and NK.
Cells, human lymphocytes such as macrophages,
Examples include human hybridoma cells obtained by fusing cancer cells and B lymphocytes.
Examples of such human-type hybridoma cells include human lung cancer cells and B.
And human hybridoma HB4C5 cells fused with lymphocytes.
The human lymphocytes used in the present invention are not limited to those composed of a single type of cell, and may be composed of two or more types of cells.

  Moreover, the said human lymphocytes can use 1 type, or 2 or more types.

Next, the collagen used in the present invention will be described.
The collagen used in the present invention is not particularly limited, and those existing in nature, artificially synthesized ones, and the like can be used. Examples of such collagen include those derived from plants and those derived from animals. Examples of animal-derived collagen include those contained in animal bones, skin, skin, tendons and the like.

Examples of the animals include mammals such as cows, pigs, horses, sheep, sharks, sharks, and whales, birds such as chickens, fishes such as salmon, trout, and cod, and molluscs such as jellyfish.
The collagen used in the present invention is preferably derived from mammals such as cattle and rabbits, molluscs such as jellyfish, and more preferably derived from jellyfish.

Examples of the jellyfish include jellyfish such as a white type, a china type, a semi-china type, a cannonball type, and a ball type.

As a method of collecting collagen from the animal, for example, the animal and raw materials such as bone, skin, skin, and tendon collected from the animal are subjected to a treatment such as cutting in advance, and after the treatment such as the cutting. The method of extracting the thing with acidic aqueous solution is mentioned.

Examples of the acidic aqueous solution used for the extraction include aqueous solutions of inorganic acids such as hydrochloric acid, sulfuric acid, and nitric acid, and aqueous solutions of organic acids such as oxalic acid, formic acid, acetic acid, and butyric acid. Among these, an aqueous solution of inorganic acids is preferable from the viewpoint of handleability, and an aqueous hydrochloric acid solution is more preferable.
Further, the hydrogen ion concentration of the acidic aqueous solution is preferably in the range of pH 2 to 4,
More preferably, the pH is in the range of 2.5 to 3.5.

The temperature at which collagen is extracted using the acidic aqueous solution is usually in the range of 0 to 150 ° C, but preferably in the range of 0 to 130 ° C.
The pressure at the time of extracting collagen using the acidic aqueous solution is not limited to atmospheric pressure, and can be carried out under pressure.

The time for extracting collagen using the acidic aqueous solution is appropriately determined depending on various conditions such as temperature at the time of extraction, but is usually in the range of 5 minutes to 24 hours.
After extracting the collagen with the acidic aqueous solution, an operation for removing parts other than collagen of the animal subjected to the extraction operation, such as meat, bone, skin, skin, and tendon residues, and the obtained extract in water An aqueous solution containing the collagen can be obtained by performing an operation of dialysis against the collagen.

The collagen used in the present invention is not limited to a form that exists in nature, and the protein component constituting the collagen may be modified by heat. Examples of the product that has been modified by the heat include gelatin-like collagen. The gelatinous collagen can be obtained by heating collagen in a form existing in nature.
From the standpoint of promoting antibody production, the collagen used in the present invention is preferably gelatin. The gelatin-like collagen is a natural form of collagen, usually 80 ~
It can be obtained by heating to about 150 ° C.

Next, the method for producing the immunomodulating protein according to the present invention will be described.
In order to produce the immunoregulatory protein, an operation for culturing the human lymphocytes is necessary.

There is no particular limitation on the medium used for the culturing operation, and any medium can be used as long as it is a target medium for culturing cells. As such a medium, for example, Eagle
e MEM medium, McCoy 5A or 7A medium, Ham F10 or F12 medium,
199 medium and the like, and improved mediums thereof. Such a medium is generally available as a basic synthetic medium or the like.

Serum such as bovine serum may or may not be added to the medium. In the present invention, it is preferable to use a serum-free medium to which no serum is added in order to promote production of immunoregulatory proteins by serum components.

Various additives such as insulin, transferrin, ethanolamine, sodium selenite and the like can be appropriately added to the medium used in the present invention.

The temperature at which the human lymphocytes are cultured is usually in the range of 30 to 45 ° C., and 35 to 42.
It is preferable if it is in the range of ° C, and more preferable if it is in the range of 36-38 ° C.

Moreover, when culturing the human lymphocytes, the culture is usually performed in an air atmosphere containing 1 to 10% by volume of carbon dioxide. The carbon dioxide gas is preferably in the range of 3 to 8% by volume.

The humidity at which the human lymphocytes are cultured is usually in the range of 80 to 100%, and 90 to 1
It is preferably in the range of 00%, and more preferably in the range of 95 to 100%.

The time for culturing the human lymphocytes is appropriately determined according to the production efficiency of the immunomodulating protein, but is usually in the range of 5 hours to 2 weeks.

Next, in order to carry out the production method of the present invention, it is necessary to use the collagen in addition to the human lymphocytes.
By using the collagen, an immunomodulating protein can be efficiently produced.

The amount of the collagen used in the production method of the present invention is 5 × 10 ⁴ cels of human lymphocytes.
Usually, it is in the range of 5 to 3000 μg / ml based on ls / ml. The amount of the collagen to be used is preferably in the range of 5 to 2500 μg / ml, more preferably in the range of 5 to 1000 μg / ml, from the viewpoint of promoting the production of the immunomodulating protein, and 10 to 100 μg / ml. If it is the range, it is still more preferable.

By culturing the human lymphocytes using the collagen, the immunoregulatory protein can be proliferated and accumulated.

After the operation of culturing the human lymphocytes using the collagen, the immunomodulating protein can be isolated by performing a separation operation by a liquid chromatography method such as affinity chromatography.

Examples of the immunoregulatory protein obtained by the production method of the present invention include IgM antibody, Ig
Immunoglobulins such as G antibody, interferon α, β, γ, tumor necrosis factor α (TNFα)
And the like.
The immunoglobulins, cytokines and the like obtained by the production method can be widely applied to, for example, therapeutic agents for immune diseases, diagnostic agents for immune functions, and the like.

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

[Reference Example 1: Extracting collagen from white type jellyfish]
White type jellyfish before bleaching processing was finely chopped and stirred overnight at a temperature of 4 ° C. using a dilute hydrochloric acid aqueous solution whose hydrogen ion concentration was adjusted to pH 3. Thereafter, collagen was extracted at 121 ° C. for 20 minutes.
The obtained extract was dialyzed against ultrapure water, and the extract after dialysis was filtered through a filter to obtain an aqueous solution containing collagen. Hereinafter, this aqueous solution containing collagen is referred to as a white type jellyfish collagen aqueous solution.

[Reference Example 2: Collagen extraction operation from semi-china type jellyfish]
Except for using a semi-china type jellyfish in place of the white type jellyfish in Reference Example 1, the same operation was performed to obtain an aqueous solution containing collagen. Hereinafter, this aqueous solution containing collagen is referred to as a semi-china type jellyfish collagen aqueous solution.

[Reference Example 3: Extracting collagen from cannonball type jellyfish]
Except for using the cannonball type jellyfish in place of the white type jellyfish in Reference Example 1, the same operation was performed to obtain an aqueous solution containing collagen. Hereinafter, this aqueous solution containing collagen is referred to as a cannonball type jellyfish collagen aqueous solution.

[Reference Example 4: Extraction of collagen from Usiachiles tendon]
Except for using the Usiachiles tendon instead of the white type jellyfish in Reference Example 1, the same operation was performed to obtain an aqueous solution containing collagen. Hereinafter, this aqueous solution containing collagen is referred to as a Usiachiles tendon collagen aqueous solution.

As human lymphocytes, human hybridoma HB4C of 5 × 10 ⁴ cells / ml
5 and the same volume of 85 μg / ml white type jellyfish collagen aqueous solution was used.
Insulin 10 μg / ml, transferrin 20 μg / ml, ethanolamine 20 μ
M and basic synthetic medium ERDF supplemented with 25 nM sodium selenite,
The human hybridoma HB4C5 was cultured in the presence of the white type jellyfish collagen aqueous solution for 6 hours under the condition of carbon dioxide gas volume 5% -air 95 volume% and humidity 100% at a temperature of 37 ° C.
As a result, 154 ng / ml of IgM antibody was produced. This result was a production amount 193 times that in the case where the white type jellyfish collagen aqueous solution was not present. The results are shown in Table 1.
The production amount of IgM antibody was measured by the enzyme antibody method (ELISA method).

The amount of IgM antibody produced was measured in the same manner as in Example 1 using a white type jellyfish collagen aqueous solution having a varied concentration. The results are shown in FIG.

Using an aqueous solution of semi-china type jellyfish collagen, IgM as in Example 1
The amount of antibody produced was measured. The results are shown in Table 1.

The amount of IgM antibody produced was measured in the same manner as in Example 1 using a cannonball type jellyfish collagen aqueous solution having a varied concentration. The results are shown in Table 1.

The production amount of IgM antibody was measured in the same manner as in Example 1 by using an aqueous solution of Usiachiles tendon collagen with different concentrations. The results are shown in Table 1.

As human lymphocytes such, using 1 × ¹⁰ 4 cells / ml of human peripheral blood lymphocytes, were used white type jellyfish collagen solution having the same volume of 450 [mu] g / ml.
Human peripheral blood was collected from the vein of the arm of a healthy person. Separation of the human peripheral blood lymphocytes from the human peripheral blood was performed by centrifugation using a lymphocyte separation solution (LSM). The human peripheral blood diluted 2-fold with physiological phosphate buffered saline (PBS) was layered on the LSM, then centrifuged at 800 rpm (rotation / min) for 20 minutes, and the LSM and the human peripheral blood were separated. Human peripheral blood lymphocytes separated into layers at the interface were collected and used as the human peripheral blood lymphocytes.
Insulin 10 μg / ml, transferrin 20 μg / ml, ethanolamine 20
Using the basic synthetic medium ERDF to which μM and 25 nM sodium selenite were added, respectively, the white under the conditions of carbon dioxide gas volume 5% -air 95 volume% and humidity 100% at a temperature of 37 ° C. The human peripheral blood lymphocytes were cultured for 3 days in the presence of an aqueous type jellyfish collagen solution.
As a result, when using a 450 μg / ml white type jellyfish collagen aqueous solution,
Compared to the case where the white type jellyfish collagen aqueous solution was not present, IgM antibody was produced 2.8 times more and IgG antibody was produced 1.4 times more. The results are shown in FIG. 2 and FIG.
The production amounts of IgM antibody and IgG antibody were measured by the enzyme antibody method (ELISA method).

The production amount of interferon γ was reduced in the same manner as in Example 6 except that various white type jellyfish collagen aqueous solutions having different concentrations were used, and lipopolysaccharide (LPS) 10 μg / ml was further added. It was measured. The production of interferon γ was measured by the enzyme antibody method (ELISA method).
As shown in FIG. 4, interferon γ was produced 100 times more than in the case where the white type jellyfish collagen aqueous solution was not present.

As human lymphocytes, 2 × 10 ⁴ cells / ml human hybridoma HB4C
In the same manner as in Example 1, except that a white type jellyfish collagen aqueous solution having the same volume of 10 μg / ml was used, the survival rate of human hybridoma HB4C5 was set to 7
Tracked for days.
The survival rate of the human hybridoma HB4C5 on the 7th day was hardly changed as compared with the case where the white type jellyfish collagen aqueous solution was not used.
On the other hand, compared with the case where the white type jellyfish collagen aqueous solution was not used, an action of suppressing the decrease in the survival rate of the human type hybridoma HB4C5 at the later stage of culture was observed. The result is shown in FIG.
Shown in
Regarding the cell growth of human hybridoma HB4C5, the average cell doubling time from the first day to the fifth day of culture is 32.9 hours when the white type jellyfish collagen aqueous solution is not used, whereas the white type When the jellyfish collagen aqueous solution was used, it was 23.4 hours. The results are shown in FIG.

The same operation as in Example 1 was carried out except that a 10 μg / ml white type jellyfish collagen aqueous solution was used, and the time course of IgM antibody production and accumulation was examined. Accumulation increased. The results are shown in FIG.

In the case of Example 7, when the change over time of the production accumulation amount of interferon γ was examined,
The production and accumulation of interferon γ increased until the second day of culture. The results are shown in FIG.

In the case of Example 7 and when the time-dependent change in the production and accumulation amount of tumor necrosis factor α (TNFα) was examined, the production and accumulation amount of tumor necrosis factor α (TNFα) increased until the fifth day of culture.
The results are shown in FIG.
The production amount of tumor necrosis factor α (TNFα) was measured by the enzyme antibody method (ELISA method).

Comparative example

A white type jellyfish collagen aqueous solution containing 1.6 mg / ml white type jellyfish collagen is treated with the same volume of 25 μg / ml collagenase for 10 minutes.
After inactivating the collagenase activity by boiling for 5 minutes, 5 × 10 ⁴ as in Example 1.
Cells / ml of human hybridoma HB4C5 was cultured for 6 hours.
As a result, the production amount of IgM antibody was reduced to half compared to the case where the treatment with collagenase, which is a collagenolytic enzyme, was not performed.
The amount of IgM antibody produced after culturing human hybridoma HB4C5 for 6 hours under exactly the same conditions except that 250 μg / ml of collagenase was used was measured.
The production amount of M antibody was similar to the case where no white type jellyfish collagen aqueous solution was used. The results are shown in Table 2.

It is the graph which showed the relationship between the density | concentration of white type jellyfish collagen aqueous solution, and the production amount of IgM antibody (Example 2). It is the graph which showed the relationship between the density | concentration of white type jellyfish collagen aqueous solution, and the production amount of IgM antibody (Example 6). It is the graph which showed the relationship between the density | concentration of white type jellyfish collagen aqueous solution, and the production amount of IgG antibody (Example 6). It is the graph which showed the relationship between the density | concentration of white type jellyfish collagen aqueous solution, and the production amount of interferon (gamma) (Example 7). It is the graph which showed the relationship with the culture | cultivation time in the presence of white type jellyfish collagen aqueous solution, the survival rate of human type hybridoma HB4C5, and a living cell density (Example 8). It is the graph which showed the relationship between culture time in the presence of white type jellyfish collagen aqueous solution, and the production amount of IgM antibody (Example 9). It is the graph which showed the relationship between culture time and the production amount of interferon (gamma) in white type jellyfish collagen aqueous solution presence (Example 10). It is the graph which showed the relationship between culture time and the production amount of tumor necrosis factor alpha (TNF alpha) in the presence of an aqueous white type jellyfish collagen solution (Example 11).

Explanation of symbols

1, 3 Production amount of IgM antibody when white type jellyfish collagen aqueous solution is present 2, 4 Production amount of IgM antibody when white type jellyfish collagen aqueous solution is not present 5 IgG antibody production amount when white type jellyfish collagen aqueous solution is present Production amount 6 Production amount of IgG antibody in the absence of white type jellyfish collagen aqueous solution 7 Production amount of interferon γ in the presence of white type jellyfish collagen aqueous solution 8 Interferon γ in the absence of white type jellyfish collagen aqueous solution
9 Human type hybridoma HB4C5 survival rate when white type jellyfish collagen aqueous solution is present 10 Human type hybridoma HB4C5 survival rate when no white type jellyfish collagen aqueous solution is present 11 When white type jellyfish collagen aqueous solution is present Viable cell density of human hybridoma HB4C5 12 Live cell density of human hybridoma HB4C5 in the absence of white type jellyfish collagen aqueous solution 13 Production amount of IgM antibody in the presence of white type jellyfish collagen aqueous solution 14 White type jellyfish collagen aqueous solution Production and accumulation of IgM antibody in the absence of 15 White type Production and accumulation of interferon gamma in the presence of jellyfish collagen aqueous solution 16 White type Interferon in the case of jellyfish collagen aqueous solution does not exist γ
Production and accumulation of 17 tumor necrosis factor α (TN) in the presence of white type jellyfish collagen aqueous solution
Production amount of Fα) 18 Tumor necrosis factor α in the absence of white type jellyfish collagen aqueous solution (
Production amount of TNFα)

Claims (3)

A method for producing an immunoregulatory protein comprising culturing human lymphocytes in the presence of collagen ,
The collagen is white type jellyfish, China type jellyfish, semi-China tie
Selected from the group consisting of jellyfish, cannonball type jellyfish and ball type jellyfish
Collagen obtained from at least one kind heated at a temperature range of 80 to 150 ° C.,
The human lymphocytes are selected from the group consisting of human lymphocytes and human hybridoma cells.
At least one kind selected,
The immunoregulatory protein is selected from the group consisting of immunoglobulins and cytokines.
At least one kind
At least the immunoglobulin is selected from the group consisting of an IgM antibody and an IgG antibody.
Is also a kind,
And whether the cytokine is interferon α, β and γ and tumor necrosis factor α.
A method for producing an immunoregulatory protein, which is at least one member selected from the group consisting of: A method for culturing human lymphocytes using collagen,
The collagen is white type jellyfish, China type jellyfish, semi-China tie
Selected from the group consisting of jellyfish, cannonball type jellyfish and ball type jellyfish
Collagen obtained from at least one kind heated at a temperature range of 80 to 150 ° C.,
The human lymphocytes are selected from the group consisting of human lymphocytes and human hybridoma cells.
A method for culturing human lymphocytes, which is at least one selected . Collagen for culturing human lymphocytes,
The collagen for culturing human lymphocytes is white type jellyfish, china type jellyfish, semi-china thai
Selected from the group consisting of jellyfish, cannonball type jellyfish and ball type jellyfish
Collagen obtained from at least one kind heated at a temperature range of 80 to 150 ° C.,
The human lymphocytes using the collagen for culturing human lymphocytes are selected from the group consisting of human lymphocytes and human hybridoma cells.
A collagen for culturing human lymphocytes, which is at least one selected.