JPS631960B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for producing alloctin A by affinity chromatography, and more specifically, a method for producing alloctin A by affinity chromatography. It relates to a simple and efficient novel method for producing the above, which is characterized by contacting the adsorbent with the bound adsorbent, followed by elution treatment.

The present inventor discovered a new substance from the medicinal plant aloe vera that has anticancer properties and has not been described in literature, and named it alloctin A. Completed (see Japanese Patent Application Publication Nos. 54-73109 and 54-84018).

Alloctin A has the following properties.

The molecular weight is approximately 18,000.

The isoelectric point is PH6.1.

It is a glycoprotein with a protein to sugar ratio of approximately 8:2 (by weight), and contains 10 sugars, only one type of sugar, galactose.

Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (hereinafter referred to as "SDS") gives a single band, which is treated with a reducing agent to cleave the S-S bond, resulting in the formation of α-subunits and β-subunits. - creates a subunit and gives two bands.

It has lymphocyte rejuvenation effect and lymphocyte membrane fluidity promoting effect.

Has red blood cell and tumor cell agglutination activity.

It has the effect of forming a sedimentation line on an agarose plate by reacting with certain serum proteins.

It has a complement third component activation effect.

Alloctin A has various physiological activities, and is particularly effective in treating inflammation, burns, skin diseases, etc. in addition to having anticancer effects.

Until now, alloctin A has been produced by the following method called gel filtration (gel chromatography). That is, a dialyzed product of aloe or its acidic precipitate is dissolved in a buffer solution, and this solution is placed on a column filled with Cephaadeix G200 gel,
Then it is eluted with a buffer (gel chromatography). After the start of elution, alloctin A was obtained as a fraction of 27.3 ml to 44.2 ml, and this product was purified by performing gel chromatography again using Sephadix G200 gel (Japanese Patent Application Laid-open No. 73109/1983).
(see publication). Although this gel filtration method achieves the purpose of collecting alloctin A, this method is complicated, time-consuming, and requires special skill. The degree of purification of alloctin A obtained by filtration is not necessarily high enough, and therefore, repurification is required, resulting in poor efficiency.

In view of the current situation, the present inventor has conducted extensive research to resolve the above-mentioned drawbacks, and has found that anti-α-subunit antibodies bind to alloctin A with immunological specificity, and when utilizing this property, The present invention was achieved by discovering that alloctin A can be selectively produced by affinity chromatography.

That is, the present invention provides for easily producing alloctin A by affinity chromatography, and
In order to achieve this objective, an alloctin A-containing substance, particularly a dialyzed product of aloe vera or an acidic precipitate thereof, is brought into contact with an adsorbent bound to an anti-α-subunit antibody, and then, Perform elution treatment.

In the present invention, it is an essential condition to use an anti-α-subunit antibody. Alloctin A is α
-subunit and β-subunit, but even though they are the same constituents of alloctin A, β-subunit
Allocin A cannot be selectively obtained using anti-β-subunit antibodies prepared from -subunits. This is because when an anti-β-subunit antibody is used, the β-subunit, which is present in free form in dialyzed aloe products, is bound to the antibody together with alloctin A. This is because it is not possible to obtain only A alone. Incidentally, the α-subunit does not exist in free form in dialyzed products of aloe.

The present invention selects one α-subunit from among the constituent components of alloctin A, which is the target substance, and
Allocin A is selectively collected using an anti-α-subunit antibody prepared from this antibody, and this has never been done before.
In this respect, the present invention is novel.

Hereinafter, the configuration of the present invention will be described in more detail.

The term "alloctin A-containing material" used in the present invention roughly means anything that contains alloctin A and in which collection or purification of alloctin A becomes a problem, but particularly allocin A-containing products include dialyzed aloe vera and Mention may be made of its acidic precipitates. The dialyzed product and acidic precipitate of aloe can be obtained by the following method. That is,
Separate the sap from the aloe plant, such as the leaf, and collect the precipitate by subjecting it to ammonium sulfate fractionation, completely remove the salt by dialysis, and then add carbonate/bicarbonate buffer (PH9). .4) to obtain the dialyzed product. Add a small amount of acetic acid to this and adjust the pH
If adjusted to 4.4, a precipitate will form. The acidic precipitate is recovered by centrifuging this precipitate. The acidic precipitate is also dissolved in a buffer solution in the same way as the dialyzed material.
(Refer to Japanese Unexamined Patent Publication No. 1973-73109).

Next, the adsorbent to which the anti-α-subunit antibody is bound will be explained.

The present inventor previously discovered that the molecule of alloctin A is 1
It is composed of two α-subunits (molecular weight approximately 7,500) and one β-subunit (molecular weight approximately 10,500), and these two subunits are linked by an S-S bond, and allocin A can be combined with 2-mercaptoethanol, etc. It has been found that when treated with a reducing agent, the S--S bond is cleaved and an α-subunit and a β-subunit are obtained (see JP-A-54-73109).

When living organisms such as rabbits and goats are immunized with this α-subunit as an antigen, IgG antibodies, ie, anti-α-subunit antibodies, are produced in the antiserum. The anti-α-subunit antibody separated from the antiserum is bound to an adsorbent as a support. The adsorbent may be one commonly used for this type of immunoadsorption;
Particularly suitable is Cephalose 4B (trademark of Palmacia, Sweden). In addition, polyamino acids,
Polystyrene, maleic anhydride ethylene copolymer, methacrylic anhydride polymer, etc. can also be used as long as they are compatible with the purpose of the present invention.

To bind an anti-α-subunit antibody to an adsorbent, particularly Sepharose 4B, the anti-α-subunit antibody dissolved in a buffer solution and the adsorbent gel treated with a buffer solution are mixed and subjected to a binding reaction with stirring. The binding reaction is carried out at 0°C to room temperature, particularly at about 4°C.
is the preferred temperature. Since it is a low temperature reaction, the reaction takes a long time, about 10 to 20 hours, preferably about 12 hours.
Allow time to react. In this way, approximately 60 to 70% of the anti-α-subunit antibody is bound to the adsorbent gel. When the bound adsorbent gel is packed into a column and subjected to post-treatment such as washing, an adsorbent gel to which the anti-α-subunit antibody is bound can be obtained.

Now, the present invention utilizes the adsorbent gel bound to the anti-α-subunit antibody obtained herein and allocin A.
Alloctin A is obtained by affinity chromatography by contacting the substance with the contained substance, but since affinity chromatography can be considered a type of adsorption chromatography, the operation is usually also performed in the case of adsorption chromatography. This is done in the same way as in the column method. Therefore, in the present invention, the operation is usually carried out according to the technique of column chromatography, but the method is not limited to the column method, and a batch method may also be adopted. The contact between the alloctin A-containing material and the adsorbent gel can be achieved by dissolving the alloctin-containing material, particularly the dialyzed product of aloe vera or its acidic precipitate, in a buffer solution, and mixing and stirring this solution with the adsorbent gel. It will be done. After the anti-α-subunit antibody and alloctin A are adsorbed and bonded with immunological specificity on the adsorbent gel, the gel is washed with a buffer solution.

This gel was then loaded into a column and eluted with a solution containing sodium thiocyanate (NaSCN).
Alloctin A is obtained by collecting the sections showing a peak in the absorption curve of OD ₂₈₀ mμ.

Since the present invention utilizes immunological specificity to produce alloctin A, the operation is simpler and the purity of the obtained alloctin A is higher than that of the conventional method using gel filtration. According to the present invention, since the operation is simple as described above, a homogeneous product can be stably provided by an ordinary worker without requiring special skill, and the present invention also allows highly purified products to be provided. Therefore, it is sufficient to perform the operation only once, and this is a remarkable effect compared to the conventional method, which requires repeating the operation two or three times to obtain a highly purified product. It can be said that. Furthermore, there is an advantage that the adsorbent after alloctin A elution can be used repeatedly several to 10 times. However, in the present invention, it is necessary to prepare the anti-α-subunit antibody and its bound adsorbent in advance, but it is not necessary to prepare it each time. Therefore, this inconvenience can be avoided.

As explained above, the present invention utilizes immunological specificity to produce highly purified alloctin A simply, efficiently, and stably, and is industrially extremely useful. It can be said to be of high value.

Examples of the present invention are shown below, but the present invention is not limited thereto.

Example 1 (1) Preparation of dialyzed Aloe leaves 1 kg of aloe leaves were thoroughly crushed and filtered through gauze to collect 720 ml of liquid juice. This liquid juice was centrifuged (10,000 rpm) for 30 minutes to remove impurities, and 700 ml of supernatant liquid was collected. This supernatant was subjected to ammonium sulfate fractionation (40% solution) to form a precipitate 6.
10ml of 0.05M sodium carbonate monobicarbonate buffer (PH9.4) was added to this,
It was dialyzed, completely desalted, and then concentrated by freeze-drying to obtain 0.4 g of dialyzed product.

(2) Preparation of anti-α-subunit antibody First, 1 mg of alloctin A or 30 mg of acidic precipitate
was treated with 2-mercaptoethanol to obtain S-S
The bonds were cleaved and the α- and β-subunits were separated electrophoretically using a preparative polyacrylamide gel electrophoresis device (FriCobaler, a trademark of Fuji Riken Co., Ltd.) using a 10% gel containing 1% sodium dodecyl sulfate (SDS). - fractionated into subunits. Thus obtained α-subunit 1
Dissolve 0.5 mg of saline in 0.5 ml of physiological saline and mix with 0.5 ml of Complete, Freund, Ajiyuband (manufactured by Kyokuto Seiyaku Co., Ltd.) to make an emulsion. After weekly injections, anti-α-subunit antibodies were produced in the blood, and this was confirmed by blood sampling 10 days after the final injection. Anti-α obtained from this rabbit blood
- The subunit antiserum was subjected to salting out with ammonium sulfate and DEAE cellulose column chromatography to detect IgG antibodies, i.e., anti-α-
The subunit antibodies were purified and separated. The anti-α-subunit antibody thus obtained was adjusted to a concentration of 3 mg/ml by adding 0.1 M sodium bicarbonate.

(3) Binding to adsorbent Sepharose 4B activated with bromic cyanide (CNBr) was washed with 10 ^-3 M hydrochloric acid to remove dextran and lactose mixed in as stabilizers, and then washed with 0.1 M sodium bicarbonate solution. Processed with.

Anti-α-subunit antibody at a concentration of 3 mg/ml.
40 ml of 0.1 M sodium bicarbonate solution and 7.5 g of the above Sepharose 4B gel were mixed and stirred gently overnight at 4°C to perform a binding reaction. After the binding reaction was completed, the gel was packed into a column and washed with 0.05M phosphate buffer - 0.14M sodium chloride solution until the absorption at OD ₂₈₀ mμ disappeared, the excess anti-α-subunit antibody was completely washed away, and the column was washed with 1M ethanolamine. After allowing the mixture to stand at room temperature for 2 hours to block the trace amount of agarose active groups remaining, the ethanolamine was washed off with a 0.05M phosphate buffer-0.5M sodium chloride solution (PH7.4). In this way, Sepharose 4B to which the anti-α-subunit antibody was bound was obtained.

(4) Production of alloctin A 50 ml of the dialyzed aloe prepared in (1) above
of 0.05M phosphate buffer-0.5M sodium chloride solution. This solution was mixed with the anti-α-
Sepharose 4B conjugated with subunit antibodies
After mixing with the gel and stirring, 0.05M phosphate buffer - 0.5M sodium chloride solution (PH7.4) was added to OD ₂₈₀ m
The unadsorbed dialysate was washed away until μ was no longer absorbed.

Pack the gel obtained here into a column.
Elution was performed with a 3.5M sodium thiocyanate-0.15M sodium chloride solution, and 1.0 to 1.5 ml portions were collected using a fraction collector and the OD ₂₈₀ mμ was measured.

The fractions showing a peak in the absorption curve at OD ₂₈₀ mμ were collected and dialyzed until SCN ^- was no longer detected, and a trace amount of precipitate was removed by centrifugation, yielding 20 mg of highly pure alloctin A. It was confirmed that it was alloctin A by forming a single band on SDS polyacrylamide electrophoresis.

The thus obtained alloctin A had the same or higher activity in the hemagglutination reaction and lymphocyte blastogenesis than that obtained by the conventional gel filtration method.

Example 2 The dialyzed aloe prepared in (1) of Example 1 above was dissolved in a sodium carbonate-sodium bicarbonate solution (PH
9.4) and adjust the pH by adding 1M acetic acid.
Adjust to 4.4 and centrifuge this solution (10000 rpm)
An acidic precipitate was obtained. 50ml of this
Use by dissolving in 0.05M phosphate buffer-0.5M sodium chloride solution.

20 mg of alloctin A was obtained in the same manner as in Example 1, except that this acidic precipitate solution was used instead of the dialyzed product in Example 1.

Claims (1)

[Scope of Claims] 1. A method for producing alloctin A by affinity chromatography, which comprises bringing a material containing alloctin A into contact with an adsorbent to which an anti-α-subunit antibody is bound, and then subjecting it to elution treatment. 2. The production method according to claim 1, wherein a dialyzed product of aloe or an acidic precipitate thereof is used as the alloctin A-containing material.