WO1993017046A1 - Novel lectinlike substance - Google Patents

Abstract

A novel lectinlike substance characterized in that: (1) it is obtained from silkworm feces, (2) it has an estimated molecular weight of 60 kDa according to SDS-PAGE under nonreducing conditions and of 50 kDa and 17 kDa under reducing conditions, (3) it has an isoelectric point of pH 4.8, (4) it is a glycoprotein, (5) it has a cytoagglutinin activity, and (6) it specifically combines with immunoglobulin G. This substance serves to analyze the phenomena of cell adhesion and agglutination in the process of cell development wherein cells grow and differentiate into complicated tissues. Also it is promising as a clinical diagnostic reagent for especially cancer and a reagent for the research in the immunological field in virtue of its capability of combining with malignant cells or the immunoglobulin. In addition, it is efficacious in preventing, treating and alleviating various infectious diseases represented by viral infection and inhibiting or suppressing the aggravation of the diseases, because it has the effect of activating the phagocytosis of macrophages and an antiviral activity.

Description

 Specification

 New lectin-like substance

 `` Technical field J

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

 "Background technology"

 Lectins are sugar-binding proteins that recognize and bind to specific sugar chain structures. In 1888, H. Stillraark first discovered lectin as a protein that aggregates red blood cells in castor seeds. Lectin, also called hemagglutinin or hemagglutinin, is known to be present not only in plants but also in influenza virus surface antigens and animal liver. The main physiological actions of lectins are: 1) Blood cells specifically agglutinate red blood cells, 2) Specific agglutination of only certain types of cancer cells, 3) Promote lymphocyte division (mitogenic activity), 4) Toxicity to cells取 り 込 み Specific uptake of waste proteins in the liver and the like is known. In other words, lectins were initially used only as blood group search reagents by utilizing their ability to bind to sugar chains that determine the blood group present on the membrane surface of erythrocytes. Some lectins, such as lectins, have been found to cause various immunological phenomena, such as the blastogenesis of quiescent lymphocytes and the induction of differentiation into proliferating cells. Alternatively, it has come to be used as a stimulator of the production of a physiologically active substance in an immune response. In addition, it was revealed that lectin showed strong cohesiveness to malignant cells.Based on the fact that lectin binds to specific sugar chains present on the surface of malignant cells, lectin was used with labeled lectin. It is also used for diagnosis to detect cancer cells.

From the perspective of trying to elucidate the complex living phenomena such as development, differentiation, immunity, and cancer that occur in the living body of multicellular organisms in recent years from the aspect of cell-cell cooperation or cell information recognition, Lectins characterized by specific binding to certain structures Research using lectins, such as searching for glycoconjugates on the cell surface and molecular structure of cell membranes, has become popular. For this reason, lectins are currently used as research reagents in a wide range of fields such as serology, biochemistry, immunology, cell biology, and molecular biology. Thus, lectins, especially lectins derived from animal body fluids and tissues, or lectin-like substances having activities similar to them, are involved in the initiation of animal development, differentiation, nervous system, immune system, New research and applications are expected to play various important roles, such as reactions and protein metabolism.

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

 "Disclosure of Invention J

 The present inventors have focused on silkworm dung, which has been used as a folk medicine in Korea since ancient times, and added this silkworm dung extract to muscle differentiation cells (QM-RSV cells) to solve the above problems. They found that the cells that had adhered to the culture dish were detached and aggregated. The present inventors have succeeded in purifying this active substance singly, clarified its biochemical characteristics (molecular weight, isoelectric point, thermal stability, etc.), and agglutinated cells caused by the active substance. In addition to investigating in detail, the present inventors have found that it has specific binding to immunoglobulin G (IgG), activation of macrophage phagocytosis, and antiviral activity, and completed the present invention. Was.

 Lectins are sugar-binding proteins that have a cell-aggregating effect, precipitate polysaccharides and complex saccharides, and their binding properties are blocked by monosaccharides and oligosaccharides. On the other hand, the active substance purified according to the present invention binds to cell membrane sugar chains or proteins like lectin and has the action of aggregating cells, but 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 that is considered to be different from known lectins, and was named NUE.

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

① It can be obtained from silkworm dung. (2) Estimated molecular weight by SDS-PAGE under non-reducing conditions is 60 kDa, and estimated molecular weights by SDS-PAGE under reducing conditions are 50 kDa and 17 kDa.

 ③ The isoelectric point is pH 4.8.

 で It is a glycoprotein.

 有 す る Has cell aggregation activity.

 特異 Specifically binds to immunoglobulin G (IgG).

 活性 Activates the phagocytic activity of macrophages.

 を Has antiviral effect.

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

 The NUE of the present invention is obtained by extracting silkworm feces with a 10-fold volume of PBS (-), removing the insoluble substance from the extract by filtration and centrifugation, and removing the supernatant obtained by NUE physical chemistry. It can be obtained by purifying by a combination of various separation means utilizing the characteristic properties. In the present invention, a method combining salting out method, dialysis method, isoelectric point precipitation, and reverse phase HPLC can be exemplified. Specifically, ammonium sulfate was added to the above extraction supernatant to a concentration of 50%, and the obtained precipitate was dialyzed against PBS (-), and further subjected to isoelectric point precipitation at pH 3.5. The precipitate can be prepared by subjecting it to reverse phase HP LC or the like.

 In the present invention, NUE was determined to be a glycoprotein by testing whether or not it was recognized by lectin blotting. As reagents used in the lectin blot method, concanavalin VIII, peanut lectin, wheat embryo lectin-peroxidase are used.

 The NUE of the present invention shows a single broad band at about 60 kDa on SDS-PAGE under non-reducing conditions, and about 5 kDa and about 17 kDa on SDS-PAGE under reducing conditions. Move as a sensitive band in the book.

In the present invention, examples of cells that cause aggregation by NUE include Ehrlich ascites cancer cells, QM-RSV cells, and He1a cells. In the present invention, myoblasts (Quail myoblast, QM) prepared from pea pectoralis muscle were used for the assay of their agglutinating activity. Were infected with a temperature-sensitive mutant of Rous sacoma virus (RSV), and transformed QM-RSV cells were used. At the permissive RSV temperature of 35.5, QM-RSV cells continue to grow as undifferentiated myoblasts, but at 41 ° C they begin to differentiate and form multinucleated myotubes. In other words, it is a cell whose muscle differentiation process can be controlled by temperature.

 In the present invention, the binding characteristic between NUE and immunoglobulin G can be assayed by the stantam blot method using egos IgG. The NUE of the present invention binds to all of IgG Fab, Fc, H chain and L chain. Since no sugar chain exists only in the Fc portion of IgG, NUE is considered to recognize sites other than sugar chains. In addition, even in IgG that has been reduced to a higher-order structure by being reduced, it binds to ΝϋΕ, suggesting that the site recognized by NUE is the primary structure of IgG. Furthermore, the binding of NUE to IgG is not seen by IgG trypsin 她 S. Therefore, the site recognized by ΝϋΕ is thought to contain an amino acid sequence that is cleaved by trypsin.

Since the NUE of the present invention has a cell aggregation activity, it has a possibility of being useful for analyzing a phenomenon in which cells adhere and aggregate to each other in a growth process in which cells proliferate and differentiate to form a complex tissue. In addition, it can be applied to various clinical diagnoses, such as cancer diagnosis, by utilizing the binding properties between the substance and tissues (particularly, malignant cells). In addition, since the substance has specific binding properties to immunoglobulin G, it is expected to be used as a research reagent in the field of immunology. Furthermore, the substance activates the phagocytic activity of macrophages, thereby preventing infection by pathogens (viruses and bacteria) or improving immunity against them. In addition, the substance has been found to have antiviral activity, especially anti-Sendai virus (HVJ), anti-HIV activity, anti-Hashika virus activity, and anti-herpes virus activity. It has strong antiviral activity against envelope viruses including influenza virus and hepatitis B virus. Therefore, the NUE of the present invention is effective as an immunostimulant because of its phagocytic activity of the macula phage, and as an antiviral agent because of its antiviral effect, and has both effects. In other words, it is effective as an agent for preventing and treating infectious diseases.

 The prophylactic / therapeutic agent for infectious diseases comprising NUE as an active ingredient in the present invention is administered orally or by injection after depolymerization. Upon administration, the composition is administered in an appropriate form according to each administration route, for example, formulated as an oral preparation or an injection. Oral formulations (eg, tablets, capsules) and injectable formulations are each manufactured by conventional methods. Each formulation usually contains a pharmaceutically acceptable carrier or additive depending on the dosage form. In the case of transdermal administration, an ointment is typically used as a dosage form, and the preparation may be prepared by a conventional method using a conventional carrier such as an excipient, an emulsifier, a stabilizer, if necessary. Can be prepared.

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

 The dosage of the infectious disease prophylactic / therapeutic agent varies depending on the applicable disease, the degree of the disease, the patient's annual meeting, etc.

 For example, when used as an ointment for the treatment of herpes virus herpes, 50-50 mg / m1 is usually applied to the affected area several times a day.

 "Brief description of the drawings"

 FIG. 1 shows the elution pattern of the silkworm dung extract purified by reverse phase HPLC.

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

 FIG. 3 shows the SDS-polyacrylamide electrophoresis pattern of each purified fraction of the silkworm feces extract under reducing and non-reducing conditions.

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

 FIG. 5 shows the absorbance curve for the "NUE" fraction.

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

FIG. 7 shows the aggregation state (biological cell morphology) of myoblasts (QM-RSV cells) in the culture at 41 ° C. with the addition of the “NUE” fraction. Figure 8 shows the adherence of the myoblasts (QM-RSV cells) untreated, pre-treated, and removed after pre-treatment to the culture vessel wall (organism of the biological cells) at 35.5 ° C. . Fig. 9 shows the state of adhesion (biological cell morphology) of myoblasts (QM-RSV cells) untreated, pretreated, and removed after pretreatment to the culture vessel wall at 41 ° C. FIG. 10 shows the degree of aggregation by the “NUE” fraction in the cells in suspension. FIG. 11 shows a western blot image (electrophoretic pattern) using a “NUE” fraction of egret IgG.

 "Best mode for carrying out the invention J

 Hereinafter, the present invention will be described in more detail with reference to Reference Examples and Examples, but the present invention is not limited thereto.

Example 1 Silkworm dung extraction and purification

 (i) Extraction method

Silkworm dung was obtained in a dry state from the Nagano Prefectural Silkworm Experimental Station. The weight (g) of Kotsubasa, 10 volumes of (ml) PBS (-) ( phosphate buffered saline; 1L H 2 0 in NaCl

_{8g, Na 2 HP0 4 1.15g,} C10. Lg, KH 2 P0 4 O.lg) was added, issued one晚抽at 60 with occasional stirring. The extract was filtered through gauze, and centrifuged for 15 min at 10 ⁴ xg. Ammonium sulfate was added to the supernatant to 50%, left at 4 ° C for 1 hour, and centrifuged at 10 ⁴ xg 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 120 ° C.

 (ii) Purification method

The freeze-dried silkworm feces extract was dissolved in distilled water to a concentration of 10 mg / ml, and the pH was lowered to pH 3.5 with 0.5 M acetic acid. The mixture was left at 4 for 1 hour, and centrifuged at 10 ⁴ Xg for 15 minutes. The pH was raised to 7 with 1M NaOH, and the precipitate was dissolved in an appropriate amount of PBS (-).

In addition, reverse-phase HPLC was performed in order to proceed with the purification. Reversed phase HPLC was performed using a U6K pump, a Waters 600B system cotroller, a Waters 484 tunable absorbance detector, a Waters 741 data module (above Waters), and a model 201 fraction collector (Gilson). Column The procedure was carried out at room temperature using a Protein C ₄ column (Vydac 10 hidden x 25 cm, particle size 5 mm,? L size 300 A). Eluate A contains 0.1% trifluoroacetic acid- Distilled water and eluent B were eluted with acetonitrile containing 0.1% trifluoroacetic acid at a flow rate of 3 ml / min. The result of changing the ratio of eluents A and B stepwise so that the ratio becomes 0-20 minutes, 90:10, 20-30 minutes, 70:30, 30-45 minutes, 50:50 after injection of the sample. And two fractions (Fr. 1 and Fr. 2) were obtained. Figure 1 shows the elution pattern. The agglutination activity of each fraction was determined by the method described in Example 2 below, and the activity was observed in the fraction (Fr. 1) eluted first. The active fraction (Fr. 1) was defined as "NUE" fraction.

Example 2 Cell aggregation activity

(0 cells used; QM-RSV cells

The pectoral muscle was removed from the Pezula embryo on day 10, and the cells were suspended in 0.25% trypsin (Difco Labolatories), and then spread in a growth medium so as to form 1 × 10 ⁶ cells / lOOram glass culture dish. Trypsin is dissolved in PBS (-), and the growth medium is 20% fetal bovine serum (FBS), 2mM glutamate (Nissui Pharmaceutical Co., Ltd.), 0.2% sodium bicarbonate, 100 units / DMEM (Dulbecoo's modification of Eagle's medium, Flow Laboratories) containing ml penicillin G potassium and 100 g / ml streptomycin sulfate was used. The culture dish was cultured at 37 for 1.5 hours at 37 with gentle shaking every 10 minutes to select for myoblasts and fibroblasts. This operation was performed once more in order to perform the screening more sufficiently. The cells that had not adhered to the culture dish at this time were used as myoblasts and spread on a 60-mm plastic culture dish (CORNING) so as to obtain 5.9 × 10 ⁵ cells / 3 ml growth medium. The plastic culture dishes were pretreated with collagen (Type-IP, Nitta Gelatin Co., Ltd.). This is the temperature-sensitive Rous sarcoma virus

After infecting (RSV ts NY68) on ice for 30 minutes, the cells cultured in a growth medium were used for the experiment.

(ii) Method

QM-RSV cells were seeded into collagen-treated 35 thigh plastic culture dishes to give 2 x 10 ⁵ Z2 ml growth media. After culturing at 35.5 ° C for 24 hours, a differentiation medium containing 200 zg / ml silkworm feces extract (5% FBS, 2 mM glumin, 0.2% sodium bicarbonate, 100 units / ml potassium penicillin G, 100 ug / ml streptomycin sulfate Was replaced with DMEM medium, and the cells were cultured at 35.5 ° C and observed at regular intervals. The time required for the cells adhered to the culture dish to separate and to agglomerate is assured at 6 hours after the addition of the silkworm feces extract. 2) o

Example 3 Biochemical properties of "NU E" fraction

(i) Analysis by electrophoresis

 SDS-PAGE was performed according to the method of Lae Thighli et al. [Laemmli, U.K., Cleavage of Structure Proteins during the Assembly of the Head of Bacteriophage T4, Nature, 227, 680-685 (1970)]. All separation gels were prepared by adding 12.5% acrylamide (manufactured by Wako Pure Chemical Industries, Ltd.). After electrophoresis of the sample at a constant current of 20 mA, the sample was stained with Coomassie Brilliant Blue R250 (fluka Chemie AG).

 The results are shown in Figure 3. Under non-reducing conditions, it appeared as a single broad band around 60 kDa (Fig. 3, lane 1). Under reduction, two sensitive bands were given near 5 OkDa and 17 kDa, respectively (Fig. 3, lane 4).

Cii) Confirmation by lectin-blot method

The lectin-plot method was performed as follows. After SDS-PAGE, the protein band separated on the gel was passed for 2 hours at a constant current of 50 mA, and transferred to Nitrocell mouth paper (Bio Rad Laboratories). The nitrocellulose paper was washed five minutes twice with distilled water, soaked for 1 hour at 80 ° C in 25πιΜ H ₂ S0 _4. Then, wash twice with distilled water twice for 5 minutes each with T buffer (0.15 NaCl-0.05¾ Tween20-10mM Tris · HC1, pH 7.4) for 5 minutes each. Then, concanapalin A-peroxidase reagent ( (Honen Oil Co., Ltd.) at 4 ° C for 1 hour. Concanavalin A-peroxidase reagent was diluted 100-fold with T buffer, and lmM CaCl ₂ .nCh.gCh was added. After the reaction, the nitrocell mouth paper was washed four times with T buffer for 15 minutes and twice for 5 minutes each with 15 mM phosphate buffer pH 6.7, and 3 mg / ml 4-chloro-1-naphthol (Bio Rad Laboratories) ) and 0.02% H ₂ 0 ₂ was added and allowed to develop. 4 one chloro 1 one naphthol was added to methanol, H ₂ 0 ₂ was diluted with 15mM phosphate buffer. The color development was stopped by washing with distilled water. Figure 4 shows the results of a plot of lectin using this peroxidase-conjugated concanapalin reagent. Shown in Under non-reducing conditions, that is, sugar binding to the 60 kDa protein was confirmed (Fig. 4, lane 2). Similarly, the 60 kDa protein was also recognized by the peanut lectin and wheat beoxidoxidase reagents.

 (iii) Isoelectric focusing

 Isoelectric focusing was performed using Phast Gel IFF4-6.5 (Pharmacia) with a Phast system (Pharmacia). A broad band appeared near the isoelectric point 4.8. Therefore, the isoelectric point of the "N UE" fraction was shown to be about 4.8.

 (iv) Other characteristics

When the thermal stability of the agglutinating activity in the "NUE" fraction was examined, it was stable at 70 ^eC for 1 hour, but was inactivated when heated at 100 for 15 minutes.

 When the absorbance curve of the "NUE" fraction was prepared using a DU-64 absorbance meter (Beckmann), the absorption maximum was shown at 230 rounds (Fig. 5).

Example 4 Characteristics of Aggregation Activity in "NUE" Fraction

 (1) Difference in aggregation activity of "NUE" fraction by temperature

 (Method)

QM-RSV cells were seeded on a 35 mm culture dish so as to give 2 × 10 ⁵ cells / 2 ml growth medium, and cultured at 35.5 ° C. for 24 hours. The medium was replaced with a differentiation medium containing 200 ug / nd or 400 / g / ml silkworm dung extract, and cultured at 35.5 and 41 ° C, respectively.

 (Result)

 At 35.5, agglutination was evident at 6 hours after adding the "NUE" fraction. However, at 41 ° C, such aggregation was not observed even after 10 hours from the addition (Fig. 6-a, Fig. 7-: f). Even at 41 ° C, mononuclear cells aggregated 24 hours after the addition of the “NUE” fraction, but myotube formation was not inhibited (Fig. 7-g). Thus, there is a difference in the effect of the "NUE" fraction between 35.5 ° C and 41 ° C, suggesting that the properties of the QM-RSV cell surface differ between 35.5 ° C and 41. is there.

If the concentration of the "NUE" fraction is 400 ug / ml, it is 35.5. Aggregation occurs 6 hours after addition of the "NUE" fraction at both C and 41 ° C. He died (Fig. 6-d, Fig. 7-h). Neither did myotube formation occur at 41 ° C (Fig. 7-i) o

 (2) Effect of "NUE" fraction on QM-RSV cells before adhesion

 (Method)

QM-RSV cells were collected with 0.02% EDTA / PBS (-), and adjusted to 2 x 10 ⁵ cells / ml in a differentiation medium with or without 400 g / ral silkworm feces extract. After standing on ice for 15 minutes, the cells were diluted 2-fold with the differentiation medium, and cultured in a 35 咖 culture dish at 21111: 35.5 and 41. To remove the "NUE" fraction added to the cells from the cells, dilute twice with the differentiation medium, centrifuge at lOOOOrpm for 5 minutes to remove the supernatant, and remove the remaining cells into 2 x 10 ⁵ cells / 2ml differentiation medium Was spread on a 35 mm culture dish as described above, and cultured in the same manner.

 (Result)

 Cells without the "NUE" fraction added adhered to the culture dish when observed 24 hours after culturing. However, the addition of the "NUE" fraction did not adhere at either 35.5 ° C or 41 ° C (Fig. 8-a, b and Fig. 9-d, e). After applying a simple "NUE" fraction for 15 minutes in ice, the "NUE" fraction was removed from the cells, and the cells were attached to the culture dish at both 35.5 and 41 in the same culture (Fig. 8-c, and Figure 9-1f).

 Thus, the activity of the "NUE" fraction disappeared only by centrifugation, indicating that the "NUE" fraction and its binding site are very weak.

(3) Effect of "NUE" fraction on QM-RSV cells in suspension

 (Method)

QM-RSV cells were spread on a 100-fold culture dish so as to have 1.6 × 10 ⁶ cells / 5 ml of growth medium, and cultured at 35.5 ° C. for 24 hours. The QM-RSV cells were collected with 0.02% EDTA and suspended in PBS (-) at a concentration of 5 × 10 ⁶ cells / ml. 0.5 ml each was dispensed into a test tube with a lid, and a 200 / zg / ml silkworm dung extract was added thereto, and the cells were adsorbed on ice for 15 minutes. Shake at 35.5 ° C 100 times at the speed of Z minutes, take out the cells at regular intervals, dilute 5 times with PBS (1), and take a picture on Fuchs' Rosenthal hemocytometer, The degree of aggregation was measured according to the following equation.

 Formula: Degree of agglutination () = (1 number of cells when one aggregate is 1) Total number of cells X100

(Result)

 In cells without the "NUE" fraction, only slight aggregation occurred when shaken at 35.5 for 30 minutes. When the "NUE" fraction was applied, the cells aggregated, and the degree of aggregation increased over time (Figure 10).

 Thus, even cells in suspension, which are not adhered to the culture dish, are aggregated by the "NUE" fraction. Therefore, it is considered that the "NUE" fraction exhibits cell aggregation activity by binding to the cell surface.

 (4) Effect of "NUE" fraction on cells other than QM-RSV cells

 (Method)

 According to the method described in Example 2, each cell was examined for aggregation by the "NUE" fraction.

^^ 10101 ^ cells were seeded into a 100 讓 glass culture dish so as to be 3 10 ⁶ / 5ml 10¾ CS-MEM and cultured at 37 ° C for 24 hours. The cells were collected with a 1: 1 mixture of 0.25% tribcine (Difco Laboratories) and 0.02% EDTA, and aggregation was observed. Tribcine and EDTA were dissolved in PBS (1). He1a cells also showed agglutinating activity in the same manner as MDCK cells. Chicken erythrocytes were collected from white leghorn wing vein, washed with PBS (1), and examined for agglutinating activity in the same manner. Ehrlich ascites carcinoma cells (ETC) were grown in the peritoneal cavity of ddy male mice, and after washing with PBS (-), showed similar agglutinating activity. In addition, QM-RSV cells were collected with 0.25% trypsin and aggregated similarly. The results are shown in Table 1. All cells aggregated in a "NUE" fraction. In particular, Ehrlich ascites carcinoma cells aggregated strongly, but other cells did not show as strong agglutination as QM-R SV cells. Table 1 Incubation time (min)

 Cells used 0 5 10 15 30

QM-RSV + + + + + + + +

 MDCK + + + +

 He 1 a Sat + +

 Erythrocyte soil +

 ETC + + + + Aggregation activity 1 <± <+ <++

 (5) Comparison of known lectins with "NUE" fraction

0.5M glucose, mannose, galactose (Wako Pure Chemical Industries, Ltd.), N-acetyl glucose, N-acetyl galactose (Nakarai Tesque, Inc.) and 20mg / nil silkworm dung extract The mixture was mixed at a ratio of 1: 1 and left at room temperature for 2 hours. _C. The mixed solution was examined for aggregation activity according to the method described in Example 2. Table 2 shows the results. None of the sugars except mannose inhibited the "NUE" fraction. This suggests that the "NUE" fraction is new and distinct from known lectins.

 Table 2 Added sugar aggregation activity

 Glucose + +

 Mannose

 Galactose + +

 N-acetylglucose + +

N-acetyl galactose + + Example 5 Binding specificity with "NUE" fraction of immunoglobulin G

 The Western plot method was performed as follows. After SDS-PAGE, the proteins separated on the gel were energized at 50 mA for 2 hours and transferred to nitrocellulose paper (Bio Rad Lab-oratories). This nitrocellulose paper was placed in TBS (Tis buffered saline; 20m Tris-HC1 pH 7.4-0.5M NaCl) for 10 minutes with a blocking solution (3% gelatin).

(Bio Rad Laboratories ZTBS) for 1 hour, washed twice with T · TBS (0.05% Tween20-TBS) twice for 5 minutes, and reacted with the primary antibody overnight.

IgG was used. Egret IgG was purified from serum obtained from Japanese White Egret using Ampure (registered trademark) PA kit. After the reaction with the primary antibody, the cells were washed twice with T'TBS for 5 minutes each, and then reacted with the secondary antibody. The secondary antibody used was a horseradish peroxidase-conjugated anti-pawn heron IgG goat antibody (Bio Rad Laboratories) diluted 1,500-fold with an antibody solution (1% gelatin / T.TBS). After washing twice with T · TBS twice for 5 minutes and twice with TBS for 5 minutes each, SmgZml 1-chloro-41-naphthol and 0.02% hydrogen peroxide solution were added to develop color. 1-chloro-41-naphthol was dissolved in methanol and aqueous hydrogen peroxide was diluted with TBS. Color development was stopped by washing with H ₂ 0. The results are shown in FIG.

Example 6 Activation of macrophage phagocytosis by "NUE" fraction

Proteose peptone was administered intraperitoneally to ddy male mice, and macrophages were allowed to migrate into the intraperitoneal cavity. A few days after administration, ascites was collected using MEM (Minimum essential medium) containing heparin and 10% calf serum (CS), and macrophages were collected. After washing the macrophages with the same culture solution, they were spread at a concentration of 2 × 10 ⁶ 2 ml 10% CS-MEM in a 35 mm glass culture dish containing a forcepour.

After culturing at 37 ° C for 4 hours, non-adherent cells were removed, followed by culturing at 37 ° C for 24 hours.

The culture medium was replaced with 2 ml of 10% CS-MEM containing 200 igl of the "NUE" fraction, and "NUE" fraciion was allowed to act on the cells at 37 for 24 hours. The culture medium was exchanged for 10% CS-MEM containing 2 × 10 ⁶ Zml of latex beads, and the beads were incorporated into the cells for 20 minutes at 37. After fixing the cells with glutaraldehyde, the number of incorporated beads was determined. Count the number of beads incorporated into 100 macrophages The results were expressed as the number of beads taken up per cell. The control yielded 3.16 ± 1.51 cells / macrophage, while the “NUE” fraction produced 11.02 ± 2.64 cells / macrophage, which was a three-fold or more difference. Therefore, it was shown that the macrophage phagocytic activity was activated by the "NUE" fraction.

Example 7 Inhibitory effect of Sendai virus (HVJ) on "NUE" fraction

HVJ 1 HAu (3 × 10 ⁷ viruses Zm 1) was allowed to act on the “NUE” fraction at a concentration of 400 ngl for 3 minutes, and then diluted 3-fold with PBS (−). After serially diluting this solution 10 times, each diluted solution was used to infect 100 embryos of 5 embryonated chicken eggs on day 10. On the third day after the infection, urine fluid was collected, and the agglutination value of chicken erythrocytes was examined for each to determine the presence or absence of virus infection. When all the eggs of the control (without "NUE" fraction) were infected, the infection was established, but when the "NUE" fraction was applied, no virus infection was observed, and the "NUE" fraction was infected with HVJ. Was suggested to inhibit

Example 8 Anti-HIV action of "NUE" fraction

200 ag / ml "NUE"& (^ 011 was dispensed in 20〃1 portions into a 96-well culture dish, and HIV (JMH-1 strain 1), which was serially diluted 10-fold, was added and mixed in 20 1 portions. after incubation for 1 hour at ^e C, a 2 X10 ⁵ cells / ml 10% fetal bovine serum (FBS) -RPMI of MT- 4 cells (human T4 lymphocytes containing the genome of HLV) suspension, each by 100 \ dispensed into the holes, mixed. 37 ° C, after 5¾ C0 ₂ middle column nutrient, was infectivity titer was measured infectivity cytopathic an indicator. control is 1 Χ10 ⁶ · ⁴⁵ TClD ₅ ./ml If contrast, allowed to act "NUE" fraction, infectivity 1 Χ10 ⁵ · ⁴⁵ is inhibited until the TCID ₅₀ / ml (1/10 times that of the control), "NUE" the fraction anti-HIV It was shown to work.

Formulation Example 1 Ointment

 White Pserin 60%

 Polyethylene glycol 1000 4%

 Mix 1 g of the "NUE" fraction of the present invention with the base 100 described above. C. The mixture was mixed to prepare an ointment.

"Industrial applicability" The novel lectin-like substance of the present invention is useful not only for analyzing cell aggregation mechanisms important in cell biology, but also as a research reagent in the field of immunology by utilizing its specific binding to IgG. The use of is expected. Furthermore, since it has macrophage phagocytic activating activity and antiviral activity, it aims to prevent, treat, reduce symptoms, and prevent or suppress symptom exacerbation against various infections including virus infection. It is effective as an agent for preventing and treating infectious diseases.

Claims

The scope of the claims  1. a novel lectin-like substance having the following characteristics:  ① It can be obtained from silkworm dung.  (2) The estimated molecular weight by SDS-PAGE under non-reducing conditions is 60 kDa, and the estimated molecular weight by SDS-PAGE under reducing conditions is 5 OkDa and 17 kDa.  ③ The isoelectric point is pH 4.8.  で It is a glycoprotein.  有 す る Has cell aggregation activity.  特異 Binds specifically to immunoglobulin G (IgG).  2. The novel lectin-like substance according to claim 1 having the following features;  ① Activates the phagocytic action of macrophages.  ② Has antiviral effect.  3. A pharmaceutical composition comprising the novel lectin-like substance according to claim 1 or 2 as an active ingredient.  4. The pharmaceutical composition according to claim 3, wherein the pharmaceutical composition is an infectious disease therapeutic agent.