JPS63188699A - Cell growth promoting substance extracted from bovine mamma, extraction thereof and inactivation treatment of virus - Google Patents

Abstract

NEW MATERIAL:A cell growth promoting substance which is a water-soluble protein, extracted from a bovine mamma and having 55,000 and 9,900 estimated molecular weights as well as about 6.0-6.5 and 7.0-7.5 isoelectric points thereof for the former and latter. USE:A hepatotonic agent, drug for gastric and duodenal ulcer, damage reparative agent and culture medium additive. PREPARATION:For example, a bovine mamma is finely cut and homogenized at a low temperature. Water or sodium chloride solution, etc., is added to allow the homogenate to stand for 6hr-a whole day and night. The homogenate is then centrifuged to separate and collect the obtained supernatant liquid. Ammonium sulfate is subsequently added to the supernatant liquid so as to provide 40% saturated concentration and centrifugation is carried out to separate and collect the resultant supernatant liquid. Ammonium sulfate is then added to the supernatant liquid so as to provide 60% saturated concentration. Formed precipitates are then purified by gel filtration and ion exchange chromatography is combination and heat-treated at 60 deg.C for 10hr to carry out inactivation of viruses and afford the aimed cell growth promoting substance.

Description

DETAILED DESCRIPTION OF THE INVENTION [A] Object of the Invention The present invention relates to a novel water-soluble protein ζ extracted from cow udder, a method for its extraction, and a virus inactivation treatment method.

(Field of Industrial Application) The novel water-soluble protein according to the present invention has a molecular weight of 55.0.
00 and 9,900, and the effect of activating cell metabolism is remarkable. That is, the water-soluble protein according to the present invention is! I Promotes proliferation and expansion of fibroblasts. Therefore, the novel water-soluble protein according to the present invention can be used directly or as a combination agent, for example, as a human or veterinary drug, such as a liver tonic, a gastric or duodenal ulcer agent, or a wound repair agent. It can also be used directly or in combination as an external preparation or in cosmetics. It is also useful as a medium additive in tissue culture.

(Prior art) Organs of various primitive species (animal species) are used as starting materials, and their extracts obtained by employing various extraction methods or their active ingredients are separated to produce animal medicines, It has been known for a long time to be used in cosmetics.

However, there are no other examples of the application or extraction method for cell proliferation-promoting substances from the breast.Therefore, upon further investigation, K. lagsbrun found that, for example, regarding milk-derived growth factors, Molecular weight 14,000-18,000, isoelectric point 4,4 in human breast milk
reported ~4.7 peptides (K, lags
brun, M:Proc, Natl, Ac1d,
sci, 75.5057-5061.1978)
, Carpenter also states that the main cell growth factor in breast milk has a molecular weight of 6.000. reported that EGF has an isoelectric point of 4.5 (Carpentar, G.

5science, 210.198=199,1980)
-On the other hand, in recent years, fibroblast growth factor (molecular weight 13.300±1.200, isoelectric point 9.6) has been found in the bovine brain and bovine pituitary gland.
e+249, 123-127.1974).

(Problems to be Solved by the Invention) The present inventors have focused on the above-mentioned tissue metabolism activation effect or tissue regeneration and repair ability of milk extracts, and have discovered that they can be used in animal medicines or cosmetics. In consideration of the fact that
and 9,900, and succeeded in separating proteins whose isoelectric points are around 6.0 to 6.5 for the former and 7.0 to 7.5 for the latter.

(Comments regarding measurement of cell proliferation-promoting effect, etc.) There was no method to accurately express cell proliferation-promoting effect, even for growth factors that are generally known today.

The present inventors have developed a method for quantitatively expressing the cell proliferation-promoting effect of a target substance by controlling the cell proliferation rate of a control to zero. This is the fetal bovine serum (FB) to be added.
This was achieved by adjusting the amount of S).

On the other hand, the present inventors have determined that the cell proliferation promoting effect of the active substance is
We have investigated various T cell lines, but they have different morphology,
They are functionally and genetically different from normal cells.

Therefore, here we decided to conduct an investigation using primary cells isolated from normal skin. The method is described below.

The main cells of the skin include epidermal cells (keratinocytes), 1
For example, fibroblasts collected from guinea pigs (Hartley strain) actively divide and proliferate in Eagle's MEM medium supplemented with 5% FBS, and their multiple proliferation time is shown in Figure 1. As shown in , it takes about 48 hours.

To measure the cell proliferation rate, first, add 4 to s
Cells are seeded at 1o' and the Eagle's MEM medium supplemented with low concentration of FBS is changed every 2-3 days.

Cell counts are determined every 2-3 days using a Bürkertürk hemocytometer.

The target substance was adjusted in terms of protein content, and one-tenth the amount per medium was added each time the medium was replaced, and as a control, only the same amount of physiological saline was added.

[Mouth] Structure of the Invention The present invention provides a cell growth promoting substance, which is a water-soluble protein extracted from a cow's udder and has a molecular weight of 55,000 and 9,900, an extraction method thereof, and a process of the extraction operation. , 60% precipitate precipitates at 80% saturation concentration of ammonium sulfate.
'C. A method for virus inactivation of water-soluble proteins, which is characterized by heat treatment for 10 hours.

(Means for Solving the Problems) The gist of the present invention is as described above, but in order to show it more specifically, examples and effects will be described below.

Incidentally, in presenting the embodiments, the basic points according to the present invention are as follows.

(B) The starting material for the extraction method is cow's milk5.Also, if necessary, the udder of livestock animals such as pigs can be used, but this is not suitable for the treatment or treatment of the target animal species. It may be selected depending on the use such as pharmaceutical use, cosmetics, etc.

(b) In the examples, the simplest method is shown under the specified extraction conditions, but the key points in the extraction process are not to apply heat and to avoid extraction operations using organic solvents.
It is necessary not to use strong acids, strong alkaline decomposition, or various proteolytic enzymes.

That is, these processing operations deactivate the activity of the target substance. According to this, it can be obtained by various combinations of conventional methods for extracting biological components, such as centrifugation, salting-out fractionation, gel filtration, and freeze-drying.

[Example-1] Cow's udder is cut into small pieces without pretreatment of washing, and homogenized using a homogenizer or the like at low temperature. To this, water, sodium chloride solution, or a known buffer solution as shown in Table 1 is added, stirred, left for 6 hours to overnight, and then centrifuged to obtain a supernatant. After adding ammonium sulfate to the supernatant to a concentration of 40% saturation, centrifugation was performed to collect the supernatant, and then,
To this supernatant, ammonium sulfate is added again to a concentration of 80% saturation, and the precipitate is recovered by centrifugation.゛After dissolving the precipitate in a small amount of water or physiological saline,
Perform desalting operation.

Desalination operations include gel filtration, ultrafiltration, and dialysis, and any of these methods may be used. A liquid containing water-soluble protein having a molecular weight of 55,000 and 9,900, which was made from cow udder as a starting material, was obtained by dialysis for more than one day and one night.

[Example-2] When considering the use of the above-mentioned extract obtained in Example-1, sufficient consideration must be given to the inactivation of viruses. For example, in order to inactivate hepatitis viruses, 6
Heat treatment at 0° C. for 10 hours is said to be effective. When the liquid obtained in Example 1 was heat-treated at 60° C. for 10 hours, it was found that the cell proliferation promoting effect was 100% inactivated.

Therefore, the present inventors investigated a stabilization method against long-term high heat treatment as a means to maintain the cell growth promoting effect of this substance and to inactivate only the virus.

As a means for this purpose, for example, monosaccharides such as glucose and mannose, 2Ws such as sucrose and maltose, etc. were added and heat treatment was performed, and studies were conducted, but it was found that they were similarly inactivated. They believe that in a dissolved state, the target substance, the protein, undergoes irreversible denaturation of its higher-order structure, resulting in its inactivation. state-
Based on the idea that it is possible to suppress denaturation by heating for 30 minutes, a novel heat treatment method as shown in Example 3 was completed.

[Example-3] During the process of Example-1, ammonium sulfate was added again to a concentration of 80% saturation, and the precipitate was collected by centrifugation or the precipitate obtained in Example-1. The resulting liquid was freeze-dried, suspended in an 80% ammonium sulfate saturated solution, or its precipitate was collected and heat-treated at 60° C. for 10 hours.

According to this method, it was found that the virus was inactivated and 80 to 90% of the growth promoting effect on cells remained.

In addition, according to Example 3, the test was conducted under the assumption that the temperature was 60°C for 10 hours, but there is no particular concern about the relationship between this temperature and time, and for example, depending on the target virus, The processing time can also be shortened by changing the processing time or increasing the temperature.

[Example 4] Here, the protein obtained in Example 1 or 3 is an extract that has a direct metabolic activation effect on cells, which is the object of the present invention, and as it is, it can be used as is. being given,
It can be used in the field of application of extracts derived from various animals. However, if necessary, it may be purified by fractionation using a combination of gel filtration, ion chromatography, etc.

Carriers used for gel filtration include Sephadex,
Agarose, Sephacryl, etc. can be mentioned, and using Sephacryl as an example, water-soluble proteins obtained from cow udder can be fractionated by the following processing operations.

[1] Oil: Sephacryl S-200 Super Fine Column size: 26X9561111 Solvent: O, 1M-0,05N) Lith-hydrochloric acid buffer PH 8,0 Flow rate: 3,3 m/an''/h Above [1]
The active fraction obtained is further fractionated using an ion exchange resin. Examples of carriers used for ion exchange include cationic and anionic exchangers such as Sephadex, Sepharose, and Sephacryl.
Fractionation is carried out using EAE-Sepharose CL-6B as an example.

[2] Fat: DEAE-Sepharose CL-6B Solvent: O,1M-0,05N Tris-HCl buffer PH
8,0 Elution conditions: NaCl concentration 0-0.3M The two active fractions obtained in [2] above were further gel-filtered with Sephacryl S-200, the active fractions obtained thereby were fractionated, and the molecular weight 55,000 and 9,900 soluble proteins are obtained. Furthermore, in this purification method, the hepatitis virus was not inactivated in the one obtained in Example 1, so the molecular weight s s, o o o obtained here and the aqueous solubility of 9,900 Viruses can be inactivated and the desired cellular metabolic activation effect can be stably maintained by precipitating the biological proteins in an 80% ammonium sulfate saturated solution and heating the precipitate. be done.

Table 1: Relationship between main buffers that can be used in Example 1 and ion exchangers used in purification (Example 4) In addition, it is a new water-soluble protein. This protein has a high direct metabolic activation effect on cells.

Conventionally known extraction extracts or extracted substances have used organic solvents, strong acids, strong alkalis, and proteolytic enzymes in the process, but the results have been determined by these agents and the starting materials used. The cell growth-active substances contained in the organ were deactivated and turned into inactive extracts. Of course, it was also deactivated by heat treatment.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the growth curve of cells used in the present invention at a concentration of 5% FBS. FIG. 2 shows the cell proliferation promoting effect of the water-soluble protein with a molecular weight of 55,000 extracted from cow udder according to the present invention. FIG. 3 shows the cell proliferation promoting effect of the water-soluble protein with a molecular weight of 9,900 extracted from cow udder according to the present invention. In Figures 2 and 3, A is the growth curve at an addition amount of 1 μs/mjl, A is a growth curve at an addition amount of 0.1 μg/−, and C is a growth curve of the control group. FIG. 4 is an elution curve with DEAE-Sepharose CL-6B. Figure 5 shows blue dextran: A1 in the figure and bovine serum albumin (molecular weight 67.000'): B1α- in the figure.
Chymotrypsinogen A (molecular weight 25°000): Elution curve obtained by gel filtration using Sephacryl S-200 Super Fine with C in the figure as a marker protein. Figure 6 shows the elution curve of the protein corresponding to the elution curve shown in Figure 5.
This shows a standard curve based on molecular weight and Kav value. Figure 7.8 shows gel filtration of two active fractions of water-soluble proteins extracted from cow udders, fractionated with DEAE-Sepharose CL-6B, using Sephacryl S-200 superfine. , is the elution curve.

Claims (3)

[Claims] (1) The estimated molecular weights of water-soluble proteins extracted from cow udders are 55,000 and 9,900, and their isoelectric points are 6.0-6.5 for the former and 7.0-7 for the latter. A cell growth-promoting substance characterized by having a value around .5. (2) The starting material is cow's udder, which is cut into small pieces, homogenized at low temperature, water, sodium chloride solution, or various known buffers are added, and the mixture is left for 6 hours to overnight. Collect the supernatant obtained by centrifugation, then add ammonium sulfate to a concentration of 40% saturation with respect to the supernatant, and collect the supernatant obtained by centrifugation. , further to the supernatant solution to a concentration of 80% saturation,
The estimated molecular weight of the water-soluble protein purified by adding ammonium sulfate and precipitating the precipitate by combining gel filtration and ion exchange chromatography was 5.
5,000 and 9,900, and the isoelectric point of the former is 6.0.
6.5, and the latter is around 7.0 to 7.5, the method for producing a cell growth promoting substance according to claim 1. (3) The starting material is cow's udder, which is cut into small pieces, homogenized at low temperature, water, sodium chloride solution, or various known buffers are added, and the mixture is left for 6 hours to overnight. Collect the supernatant obtained by centrifugation, then add ammonium sulfate to a concentration of 40% saturation with respect to the supernatant, and collect the supernatant obtained by centrifugation. , further to the supernatant solution to a concentration of 80% saturation,
Ammonium sulfate is added and the resulting precipitate is heated at 60°C for 10 hours, or the precipitate is further purified by a combination of gel filtration and ion exchange chromatography. The activity of the cell growth promoting substance according to claim 1, characterized in that it is added to a solution at a concentration of 80% saturation to precipitate it, and the precipitate is heat-treated at 60° C. for 10 hours. A stable virus inactivation treatment method.