JP3420582B2 - New lectin-like substance - Google Patents

Description

TECHNICAL FIELD The present invention relates to cell aggregation activity, specific binding to immunoglobulin G (IgG), activation activity of macrophage phagocytosis, extracted from silkworm feces, The present invention relates to a novel lectin-like substance having an antiviral action and the like, and a pharmaceutical composition containing the lectin-like substance as an active ingredient.

"Background Art" Lectins are sugar-binding proteins that recognize and bind to specific sugar chain structures. Lectin was first discovered by H. Stillmark in 1888 as a protein that aggregates red blood cells in castor bean seeds. Lectins, also called cell agglutinins or hemagglutinins, are known to be present not only in plants but also in surface antigens of influenza virus and animal liver. The main physiological actions of lectins are blood group-specific agglutination of red blood cells, specific agglutination of certain cancer cells only, promotion of lymphocyte division (mitogenic activity), toxicity to cells, in liver, etc. Specific uptake of obsolete proteins is known. That is, the lectin was originally used only as a blood group search reagent by utilizing the binding property with the sugar chain that determines the blood group existing on the membrane surface of red blood cells.
After that, some lectins such as concanavalin A and kidney bean lectin were found to have various immunological effects such as blastogenesis of lymphocytes at resting phase and induction of differentiation into proliferating cells. It has come to be used as a model for elucidation or as a stimulant for the production of physiologically active substances in immune reactions. In addition, it was revealed that lectin shows strong aggregating property to malignant cells, and this is due to the binding property of lectin to specific sugar chains present on the surface of malignant cells. It is also used for diagnosis to detect cancer cells.

Development, differentiation, immunity, which occur in vivo in multicellular organisms in recent years,
From the viewpoint of elucidating complex biological phenomena such as cancer in terms of cell-cell cooperation or cell information recognition, lectins, which are characterized by specifically binding to a certain structure of carbohydrates on the cell membrane, have attracted attention. , Lectin-based research such as the search for cell surface glycoconjugates and the search for the molecular structure of cell membranes has become popular. Therefore, lectins are currently used as research reagents in a wide range of fields such as serology, biochemistry, immunology, cell biology, and molecular biology.

Therefore, lectins, and in particular, lectins derived from animal body fluids and tissues, or lectin-like substances having similar activities, initiate intercellular recognition reaction in animal development, differentiation, nervous system, immune system, immune reaction and protein. It is expected that new research and application will be carried out in the future as it plays various important roles such as metabolism.

Therefore, an object of the present invention is to provide a novel lectin-like substance.

DISCLOSURE OF THE INVENTION In order to solve the above-mentioned problems, the present inventors have focused on silkworm feces, which have been fresh and have been used as folk medicines since ancient times, and used this silkworm feces extract as a muscle differentiation cell (QM-RSV). It was found that the cells that had adhered to the culture dish peeled off and aggregated when added to the cells). The present inventors succeeded in purifying this active substance singly, clarified its biochemical characteristics (molecular weight, isoelectric point, thermal stability, etc.), and investigated the aggregation of cells caused by the active substance. Upon detailed investigation, they found that it has specific binding property to immunoglobulin G (IgG), activation action of phagocytic action of macrophages, and antiviral action, and completed the present invention.

Lectin is a sugar-binding protein, has a cell aggregation action, precipitates polysaccharides and complex sugars, and its binding property is blocked by monosaccharides and oligosaccharides. On the other hand, the active substance purified by the present invention is similar to the lectin in the cell membrane sugar chain,
Alternatively, although it is a substance that binds to a protein and has an action of aggregating cells, the main sugar does not inhibit its activity. However, since only mannose inhibits its activity, it can be said that the active substance recognizes mannose. Therefore, the active substance in the present invention is a novel lectin-like substance which is considered to be different from the known lectin, and was named NUE.

That is, the NUE of the present invention relates to a novel lectin-like substance having the following biochemical characteristics.

  It can be obtained from silkworm dung.

Estimated molecular weight of 60kD by SDS-PAGE under non-reducing conditions
a, estimated molecular weights by SDS-PAGE under reducing conditions are 50 kDa and 17 kDa.

  The isoelectric point is pH 4.8.

  It is a glycoprotein.

  Has cell aggregation activity.

  It specifically binds to immunoglobulin G (IgG).

  Activates the phagocytic effect of macrophages.

  Has an antiviral effect.

First, the method for manufacturing the NUE of the present invention will be described in detail. The NUE of the present invention can be isolated from, for example, a water-soluble substance extracted from silkworm feces.

NUE of the present invention, silkworm feces extracted with 10 times volume of PBS (-), the supernatant obtained by removing insoluble substances from the extract by filtration and centrifugation, physicochemical of NUE. It can be obtained by utilizing various properties and purifying by a combination of various separation means. In the present invention, a method combining a salting-out method, a dialysis method, an isoelectric point precipitation and a reverse phase HPLC can be exemplified. Specifically, ammonium sulfate was added to the above extraction supernatant so that the concentration was 50%, the obtained precipitate was dialyzed with PBS (-), and further the precipitate was obtained by isoelectric focusing at pH 3.5. The reverse phase HPL
It can be adjusted by applying to C etc.

In the present invention, it was determined that NUE was a glycoprotein by testing whether or not it was recognized by the lectin blotting method. Concanavalin A, peanut lectin, and wheat embryo lectin-peroxidase are used as reagents used in the lectin blotting method.

The NUE of the present invention has an SDS-PAGE of about 60 kD under non-reducing conditions.
Shows a single broad band near a, and SDS-
On PAGE, it migrates as two sharp bands of about 50 kDa and about 17 kDa.

In the present invention, examples of cells that cause aggregation by NUE include Ehrlich ascites tumor cells, QM-RSV cells, and Hela cells. In the present invention, for the assay of its agglutination activity, myoblasts (Quail myob
last, QM) and Rous sacoma virus, R
Transformed QM-RSV cells were used which were infected with a temperature-sensitive mutant strain of SV). QM-RSV cells continue to proliferate as undifferentiated myoblasts at the RSV permissive temperature of 35.5 ° C, but at 41 ° C they begin to differentiate and form multinucleated myotubes. That is, it is a cell that can control the muscle differentiation process by temperature.

In the present invention, the binding property between NUE and immunoglobulin G can be assayed by Western blotting using rabbit IgG. The NUE of the present invention is IgG Fab, F
It binds to all of c, H chain and L chain. Since the sugar chain exists only in the Fc portion of IgG, it is considered that NUE also recognizes a site other than the sugar chain. In addition, IgG that has been reduced to destroy the higher-order structure
Also in, the binding with NUE suggests that the site recognized by NUE is the primary structure of IgG. In addition, IgG
The binding of NUE and IgG is not seen by the trypsin treatment of. Therefore, it is considered that the site recognized by NUE contains an amino acid sequence cleaved by trypsin.

NUE of the present invention, by having a cell aggregation activity,
It may be useful for analysis of the phenomenon in which cells adhere to each other and aggregate during the developmental process in which cells proliferate and differentiate to form complex tissues. Further, it can be applied to various clinical diagnoses including cancer diagnosis by utilizing the binding property between the substance and tissue (particularly malignant cells). In addition, since the substance has a specific binding property with immunoglobulin G, it is expected to be used as a research reagent in the field of immunology. Further, the substance activates the phagocytic action of macrophages, and thus has the effect of preventing infection by pathogens (viruses, bacteria) or improving immunity to them. In addition, the substance is recognized to have an antiviral action, particularly an anti-Sendai virus (HVJ), an anti-HIV action, an anti-hashika virus action, and an anti-herpesvirus action. , Shows strong antiviral activity against enveloped viruses including hepatitis B virus. Therefore, the NUE of the present invention is effective as an immune stimulant from its macrophage phagocytic activity activating action, and as an antiviral action as an antiviral agent. Is effective as.

The prophylactic / therapeutic agent for infectious diseases containing NUE as an active ingredient in the present invention is administered orally or by injection after lowering the molecular weight. At the time of administration, it is formulated and administered in an appropriate form depending on each administration route, for example, an oral preparation or an injection. Oral preparations (eg, tablets, capsules) and injectable preparations are each manufactured by a conventional method. Each formulation usually contains a pharmaceutically acceptable carrier or additive according to each dosage form. In the case of transdermal administration, an ointment is typically used as the dosage form, and the preparation can be prepared by conventional means using conventional carriers such as excipients, emulsifiers, stabilizers, etc., if necessary. It can be prepared.

The content of the active ingredient NUE in the prophylactic / therapeutic agent for infectious diseases is not particularly limited because it can be appropriately changed depending on the administration method, dosage form, indication disease and the like.

The dose of the prophylactic / therapeutic agent for infectious diseases varies depending on the disease to be applied, the degree of the disease, the age of the patient, etc. / ml to the affected area several times a day.

[Brief Description of Drawings] FIG. 1 shows an elution pattern of a purified silkworm feces extract by reverse phase HPLC.

FIG. 2 shows the state of detachment of myoblasts (QM-RSV cells) from the culture vessel wall (morphology of living cells) by "NUE" fraction.

FIG. 3 shows SDS-polyacrylamide electrophoresis patterns of purified fractions of silkworm feces extract under reducing and non-reducing conditions.

FIG. 4 shows a lectin blot image (electrophoresis pattern) with Concanavalin A of “NUE” fraction.

  FIG. 5 shows an absorption curve of “NUE” fraction.

FIG. 6 shows the aggregated state (morphology of living cells) of myoblasts (QM-RSV cells) in the culture at 35.5 ° C. with the addition of “NUE” fraction.

FIG. 7 shows the aggregation state (morphology of biological cells) of myoblasts (QM-RSV cells) in the culture at 41 ° C. with the addition of “NUE” fraction.

FIG. 8 shows the adhesion state (morphology of biological cells) of the myoblasts (QM-RSV cells) untreated with “NUE” fraction, pretreated, and removed after pretreatment to the incubator wall at 35.5 ° C.

FIG. 9 shows the adhesion state (morphology of biological cells) of the myoblasts (QM-RSV cells) untreated with “NUE” fraction, pretreated, and removed after pretreatment to the incubator wall at 41 ° C.

FIG. 10 shows the degree of aggregation due to “NUE” fraction in cells in a floating state.

FIG. 11 shows a Western blot image (electrophoresis pattern) using “NUE” fraction rabbit IgG.

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in more detail below with reference to Reference Examples and Examples, but the present invention is not limited thereto.

Example 1 Silkworm feces extraction and purification (i) Extraction method Silkworm feces were obtained in a dry state from the Nagano Prefectural Silkworm Experiment Station.
10 times the volume of PBS (-) for the weight of silkworm feces (g)
(Phosphate buffered saline; 1 L H ₂ O in NaCl 8 g, Na ₂ HPO
₄ 1.15 g, KCl 0.1 g, KH ₂ PO ₄ 0.1 g) was added, and the mixture was extracted overnight at 60 ° C. with occasional stirring. The extract was filtered through gauze and centrifuged at 10 ⁴ × g for 15 minutes. Ammonium sulfate was added to the supernatant so that the concentration became 50%, the mixture was allowed to stand at 4 ° C. for 1 hour, and then centrifuged at 10 ⁴ × g for 15 minutes to dissolve the precipitate in an appropriate amount of PBS (-). This was dialyzed against PBS (-) for 3 days, freeze-dried, and stored at -20 ° C.

(Ii) Purification method The freeze-dried silkworm feces extract was dissolved in distilled water to 10 mg / ml, and the pH was lowered to 3.5 with 0.5 M acetic acid.
The mixture was allowed to stand at 4 ° C for 1 hour and then centrifuged at 10 ⁴ × g for 15 minutes. 1M Na
The pH was returned to 7 with OH and the precipitate was dissolved in an appropriate amount of PBS (-).

Furthermore, reverse phase HPLC was performed to proceed with the purification. Reverse phase
HPLC is U6K pump, Waters 600E system controller, Wat
ers 484 tunable absorbance detector, Waters 741 dat
a module (above Waters), model 201 fraction collecto
It was performed using r (Gilson). Column A Protein C ₄ column (Vydac 10 mm × 25 cm, particle size 5 μm, pore size 300 A) was used and performed at room temperature. Eluent A was distilled water containing 0.1% trifluoroacetic acid, eluent B was acetonitrile containing 0.1% trifluoroacetic acid, and elution was performed at a flow rate of 3 ml / min. The ratio of eluents A and B is 0-20 minutes after injection of the sample, 90:10, 20-
As a result of stepwise change to 30 minutes, 70:30, 30-45 minutes, 50:50, two fractions (Fr.1 and Fr.2) were obtained. The elution pattern is shown in FIG. When the aggregation activity of each fraction was examined by the method described in Example 2 below, the activity was found in the fraction (Fr. 1) that was first eluted. The active fraction (Fr.1) was designated as "NUE" fraction.

Example 2 Cell aggregation activity (i) Cells used; QM-RSV cells Pectoral muscles were taken out from quail embryos on the 10th day, the cells were suspended in 0.25% trypsin (Difco Labolatories), and the cells were suspended in a growth medium at 1 × 10 ^6. It was soaked into pieces / 100 mm glass culture dish. Trypsin was dissolved in PBS (-) and the growth medium was 20
% Fetal bovine serum (FBS), 2m
DMEM (Dulbecoo's modific) containing M glutamine (Nissui Pharmaceutical Co., Ltd.), 0.2% sodium hydrogen carbonate, 100 units / ml penicillin G potassium, 100 μg / ml streptomycin sulfate
ation of Eagle's medium, Flow Laboratories). In order to select myoblasts and fibroblasts, the culture dish was incubated at 37 ° C for 1.5 hours with gentle shaking every 10 minutes.
This operation was repeated once more in order to perform the selection sufficiently. At this time, cells that had not adhered to the culture dish were made into myoblasts, and the cells were seeded in a 60 mm plastic culture dish (CORNING) so as to give 5.9 × 10 ⁵ cells / 3 ml growth medium. The plastic culture dish was pretreated with collagen (Type-IP, Nitta Gelatin Co., Ltd.). This was infected with temperature-sensitive Rous sarcoma virus (RSV ts NY68) on ice for 30 minutes, and then cultured in a growth medium for use in the experiment.

(Ii) Method QM-RSV cells were added to 2 × 10 ⁵ cells / 2 ml growth medium,
Plated in a collagen-treated 35 mm plastic culture dish. After culturing at 35.5 ° C for 24 hours, a differentiation medium containing silkworm feces extract at 200 μg / ml (5% PBS, 2 mM glutamine, 0.2% sodium hydrogen carbonate, 100 units / ml penicillin G potassium, 100
The medium was replaced with a DMEM medium containing μg / ml streptomycin sulfate), and the cells were cultured at 35.5 ° C. and observed at regular intervals. The time until the cells that had adhered to the culture dish peeled off and agglutination occurred was confirmed at 6 hours after the addition of the silkworm feces extract, so the presence or absence of activity was judged at 6 hours after the addition (FIG. 2). reference).

Example 3 Biochemical characteristics of "NUE" fraction (i) Analysis by electrophoresis SDS-PAGE was performed by the method of Laemmli et al. [Laemmli, UK, Clearavag.
e of Structual Proteins during the Assembly of the
Head of Bacteriophage T4, Nature, 227 , 680−685 (197
0)]. All gels for separation were prepared by adding 12.5% acrylamide (manufactured by Wako Pure Chemical Industries, Ltd.). 20
After the sample was electrophoresed with a constant current of mA, it was stained with Coomassie Brilliant Blue R250 (fluka Chemie AG).

The results are shown in Fig. 3. Under non-reducing condition, it appeared as one broad band around 60 kDa (Fig. 3, lane 1). Under reduction, two sharp bands were given at around 50 kDa and 17 kDa, respectively (Fig. 3, 4 lane).

(Ii) Confirmation by lectin-blotting method The lectin-blotting method was performed as follows. SDS
− After PAGE, separate 50 protein bands on the gel.
It is energized with a constant current of mA for 2 hours, and nitrocellulose paper (Bio
Rad Laboratories). The nitrocellulose paper was washed twice with distilled water for 5 minutes each and immersed in 25 mM H ₂ SO ₄ at 80 ° C. for 1 hour. Then, twice with distilled water for 5 minutes each in T buffer (0.15M NaCl-0.05% Tween20-10mM Tris.HCl, pH 7.
After washing twice with 4) for 5 minutes each, it was reacted with a concanavalin A-peroxidase reagent (Toyoen Oil Co., Ltd.) at 4 ° C. for 1 hour. Concanavalin A-peroxidase reagent is T
After diluting 100 times with the buffer, 1 mM CaCl ₂ .MnCl ₂ .MgCl ₂ was added. After the reaction, the nitrocellulose paper was washed 4 times with T buffer for 15 minutes each and twice with 15 mM phosphate buffer pH 6.7 for 5 minutes each.
mg / ml 4-chloro-1-naphthol (Bio Rad Laborator
ies) and 0.02% H ₂ O ₂ were added to develop color. 4-chloro-
1-Naphthol was added to methanol, and H ₂ O ₂ was diluted with 15 mM phosphate buffer. This color development was stopped by washing with distilled water. The result of lectin-blotting using the present peroxidase-conjugated concanavalin reagent is shown in FIG. Under non-reduction,
That is, it was confirmed that sugar was bound to the 60 kDa protein (Fig. 4, lane 2). In addition, the peanut lectin and wheat-peroxidase reagents were also used for the 60 kDa
Protein was recognized.

(Iii) Isoelectric focusing: Isoelectric focusing is performed using Phast Gel IFF4-6.5 (Pharmaci
a) was used in the Phast system (Pharmacia). A wide band appeared near the isoelectric point of 4.8. Therefore, "NUE" fract
The isoelectric point of ion was shown to be about 4.8.

(Iv) Other characteristics When the thermal stability of the aggregation activity of "NUE" fraction was examined, it was stable at 70 ° C for 1 hour,
It became inactive when heated for 5 minutes.

Moreover, when the absorbance curve of "NUE" fraction was created by a DU-64 absorbance meter (Beckmann Co., Ltd.), it showed an absorption maximum at 230 nm (FIG. 5).

Example 4 Characteristics of aggregation activity of “NUE” fraction (1) Difference in aggregation activity of “NUE” fraction depending on temperature (method) 2 × 10 ⁵ QM-RSV cells / 2 ml to a growth medium of 35 mm
The mixture was spread on a culture dish and cultured at 35.5 ° C for 24 hours. Replace with differentiation medium containing 200 μg / ml or 400 μg / ml silkworm faecal extract and
Cultures were performed at 5.5 ° C and 41 ° C, respectively.

(Results) At 35.5 ° C., aggregation certainly occurred at 6 hours after the addition of “NUE” fraction. However, at 41 ° C, such aggregation was not observed even after 10 hours from the addition (Fig. 6-
a, FIG. 7-f). After adding "NUE" fraction at 41 ℃,
After 24 hours, mononuclear cells began to aggregate, but myotube formation was not inhibited (Fig. 7-g). Thus, 35.5
There was a difference in the action of the "NUE" fraction between ° C and 41 ° C, which suggests that the properties of the QM-RSV cell surface differ between 35.5 ° C and 41 ° C.

In addition, if the concentration of "NUE" fraction is 400 μg / ml,
At both 5.5 ° C. and 41 ° C., aggregation occurred 6 hours after the addition of “NUE” fraction, and eventually the cells died (FIG. 6-d, FIG. 7-h). Myotube formation at 41 ° C also did not occur (Fig. 7-i).

(2) Effect of "NUE" fraction on QM-RSV cells before adhesion (Method) QM-RSV cells were recovered with 0.02% EDTA / PBS (-), and silkworm feces extract was included or not (400 μg / ml) The differentiation medium was adjusted to 2 × 10 ⁵ cells / ml. After leaving it on ice for 15 minutes, it was diluted 2-fold with a differentiation medium, and 2 ml each was poured into a 35 mm culture dish and cultured at 35.5 ° C and 41 ° C. When removing the "NUE" fraction added to the cells from the cells, after diluting 2 times with the differentiation medium and centrifuging at 1000 rpm for 5 minutes to remove the supernatant,
The remaining cells are 35 mm so that 2 × 10 ⁵ cells / 2 ml differentiation medium may be used.
It was seeded in a culture dish and cultured in the same manner.

(Results) The cells to which the “NUE” fraction was not added adhered to the culture dish when observed 24 hours after the culture. But,
Those to which "NUE" fraction was added did not adhere at either 35.5 ° C or 41 ° C (Fig. 8-a, b and Fig. 9-d,
e). After applying "NUE" fraction in ice for 15 minutes,
35.5% when the UE "fraction was removed from the cells and cultured in the same manner
The cells adhered to the culture dish at both C and 41 C (Fig. 8-c and Fig. 9-f).

Thus, since the activity of "NUE" fraction is not seen only by centrifugation, "NUE" fraction and its binding site are considered to be very weak.

(3) "NUE" fraction for QM-RSV cells in suspension
Effect (method) QM-RSV cells were added to 1.6 x 10 ⁶ cells / 5 ml growth medium 100
It was seeded in a mm culture dish and cultured at 35.5 ° C. for 24 hours. This QM
-RSV cells were recovered with 0.02% EDTA and suspended in PBS (-) so that the concentration was 5 x 10 ⁶ cells / ml. 0.5 ml each was dispensed into a test tube with a lid, to which 200 μg / ml silkworm feces extract was added, and cells were adsorbed on ice for 15 minutes. Shake at a rate of 100 times / min at 35.5 ° C, take out cells at regular intervals, and add 5 times with PBS (-).
After doubling the dilution, it was placed on a Fuchs-Rosenthal hemocytometer and a photograph was taken to measure the degree of aggregation according to the following formula.

Formula: Aggregation degree (%) = (1-number of cells when aggregated mass is 1 / total number of cells) x 100 (Result) 35.5 ° C for cells not subjected to "NUE" fraction
Aggregation occurred only slightly after shaking for 30 minutes. When the "NUE" fraction was applied, the cells aggregated, and the degree of aggregation increased over time (Fig. 10).

In this way, even cells in a floating state that are not adhered to the culture dish are aggregated by "NUE" fraction, so "NUE" fr
It seems that action binds to the cell surface and shows cell aggregation activity.

(4) "NUE" fraction for cells other than QM-RSV cells
(Method) According to the method described in Example 2, each cell was examined for aggregation by "NUE" fraction.

MDCK cells were seeded in a 100 mm glass culture dish so as to be 3 × 10 ⁶ cells / 5 ml 10% CS-MEM, and cultured at 37 ° C. for 24 hours. 0.2% with 0.25% trypsin (Difco Laboratories)
Aggregation was observed by collecting with a 1: 1 mixed solution of% EDTA. Incidentally, trypsin and EDTA were dissolved in PBS (-). Hela cells also showed agglutination activity in the same manner as MDCK cells. As chicken erythrocytes, those collected from the white leghorn wing vein were used, and after washing with PBS (-), the agglutinating activity was similarly observed. Ehrlich ascites tumor cells (ETC) were grown in the peritoneal cavity of ddy male mice and
After washing with (-), the aggregation activity was similarly observed. Also QM-RSV
Cells were harvested with 0.25% trypsin and also showed aggregation. The results are shown in Table 1. All cells are "NUE" fracti
Aggregated by on. In particular, the Ehrlich ascites tumor cells were strongly aggregated, but the other cells did not show the stronger aggregation than the QM-RSV cells.

(5) Comparison between known lectin and "NUE" fraction 0.5M glucose, mannose, galactose (above, Wako Pure Chemical Industries, Ltd.), N-acetyl glucose,
N-acetylgalactose (Nakarai Tesque Co., Ltd.) and 20 mg / ml silkworm feces extract were mixed at a ratio of 1: 1,
It was left at room temperature for 2 hours. About this mixed solution, Example 2
The agglutination activity was examined according to the method described. The results are shown in Table 2.
It was shown to. All sugars except "mannose" are "NUE" fracti
did not block on. From this, the "NUE" fraction seems to be a novel one, which is different from known lectins.

Example 5 Binding Specificity of "NUE" Fraction with Immunoglobulin G Western blotting was performed as follows. SDS
-After PAGE, the proteins separated on the gel were energized at 50 mA for 2 hours and transferred to nilotcellulose paper (Bio Rad Laboratories). Use this nitrocellulose paper with TBS (Tis buffere
d saline; 20 mM Tris.HCl pH7.4-0.5M NaCl) for 10 minutes, blocking solution [3% gelatin (Bio Rad Labora
tories / TBS] for 1 hour and then T ・ TBS (0.05% Tween20
-TBS), the cells were washed twice with 5 minutes each and reacted with the primary antibody overnight. Rabbit IgG was used as the primary antibody. Rabbit I
gG was purified from serum obtained from Japanese white rabbits using Ampure (registered trademark) PA kit. After reacting with the primary antibody, T.
The cells were washed twice with TBS for 5 minutes each and reacted with a secondary antibody. Two
The secondary antibody was anti-rabbit Ig conjugated with horseradish peroxidase.
G goat antibody (Bio Rad Laboratories) antibody solution (1%
It was used after being diluted 1500 times with gelatin / T / TBS). Wash with T / TBS twice for 5 minutes each and twice for 5 minutes each with TBS, then 3 mg / ml 1
-Chloro-4-naphthol and 0.02% hydrogen peroxide solution were added for color development. 1-Chloro-4-naphthol was dissolved in methanol and hydrogen peroxide solution was diluted with TBS. Color development was stopped by washing with H ₂ O. The results are shown in Fig. 11.

Example 6 Activation of macrophage phagocytic activity of "NUE" fraction Proteose peptone was intraperitoneally administered to ddy male mice to allow macrophages to migrate intraperitoneally. 2,3 after administration
Since the day, MEM containing heparin and 10% calf serum (CS)
Ascites was collected with (Minimum essential medium) to collect macrophages. After washing the macrophages with the same culture solution, the macrophages were seeded at a concentration of 2 × 10 ⁶ cells / ml 10% CS-MEM on a 35 mm glass culture dish containing a coverslip. After culturing at 37 ℃ for 4 hours, remove unadhered cells and continue at 37 ℃
It was cultured for 24 hours. Contains 200 μg / ml “NUE” fraction 10
Replace the culture solution with 2 ml of% CS-MEM and incubate for 24 hours at 37 ℃.
E "fraction was applied to the cells. 2 latex beads
Replace the culture medium with 10% CS-MEM containing × 10 ⁶ cells / ml and store at 37 ℃.
The cells were allowed to incorporate the beads for 20 minutes. After fixing the cells with glutaraldehyde, the number of beads incorporated was measured. The number of beads incorporated into 100 macrophages was counted, and the result was expressed by the number of beads incorporated per cell. The control was 3.16 ± 1.51 cells / macrophage, whereas 11.2 ± 2.6 when the “NUE” fraction was applied.
There were 4 cells / macrophage, and there was a 3-fold or more difference.
Therefore, it was shown that macrophage phagocytic activity was activated by "NUE" fraction.

Example 7 Sendai virus (HVJ) of "NUE" fraction
Inhibitory action of HVJ 1HAu (3 x 10 ⁷ virus / ml) against "NUE" f
raction is applied at a concentration of 400 μg / ml for 3 minutes, and then PBS
It was diluted 3-fold with (-). After serially diluting this solution 10-fold,
100 μl of each diluted solution was infected to 5 hatched chicken eggs on the 10th day. On the third day after the infection, a urine collecting solution was collected, and the agglutination value of chicken erythrocytes was examined for each to determine the presence or absence of virus infection. All control (“NUE” fraction non-acting) hatched eggs were infected, whereas “NU
When E "fraction was applied, no virus infection was observed, suggesting that" NUE "fraction inhibits HVJ infection.

Example 8 Anti-HIV action of "NUE" fraction 20 µl of 200 µg / ml of "NUE" fraction was added to a 96-well culture dish.
20 μl of 10-fold serially diluted HIV (JMH-1 strain)
l were added and mixed. After incubating at 37 ℃ for 1 hour, 2 × 10 ⁵
Cells / ml 10% fetal bovine serum (FBS) -RPMI MT-4 cells (HLV
100 μl of human T4 lymphocyte suspension containing the genome of
Each was dispensed into each hole and mixed. After culturing at 37 ° C. in 5% CO ₂ , the infectious titer was measured using cell degeneration as an index. The infectious titer of the control was 1 × 10 ^6.45 TCID ₅₀ / ml, whereas “NUE” fract
When treated with ion, the infectious titer was suppressed to 1 × 10 ^5.45 TCID ₅₀ / ml (1/10 times compared to the control), and "NUE" fraction
Has been shown to have anti-HIV activity.

Formulation Example 1 Ointment White petrolatum 60% Polyethylene glycol 1000 40% To 100 g of the above base, 1 g of "NUE" fraction of the present invention was mixed and mixed at 60 ° C to give an ointment.

“Industrial Applicability” The novel lectin-like substance of the present invention is useful not only for analysis of cell aggregation mechanism important in cell biology but also in immunology field by utilizing its specific binding property with IgG. Expected to be used as a research reagent. Furthermore, since it has an action of activating the phagocytic action of macrophages and an antiviral action, it is aimed at prevention, treatment, alleviation of symptoms, prevention or suppression of exacerbation of various infectious diseases including viral infection. It is effective as a preventive and therapeutic agent for infectious diseases.

Claims (3)

(57) [Claims] 1. A novel lectin-like substance having the following characteristics: It can be obtained from silkworm feces. Estimated molecular weight of 60kD by SDS-PAGE under non-reducing conditions
a, estimated molecular weights by SDS-PAGE under reducing conditions are 50 kDa and 17 kDa. The isoelectric point is pH 4.8. It is a glycoprotein. Has cell aggregation activity. It specifically binds to immunoglobulin G (IgG). 2. A novel lectin-like substance according to claim 1, which has the following characteristics: It activates the phagocytic action of macrophages. Has an antiviral effect. 3. An infectious disease therapeutic agent comprising the novel lectin-like substance according to claim 1 or 2 as an active ingredient.