KR100286366B1 - Lectin genes from Viscum album - Google Patents

Abstract

The present invention relates to a lectin protein having an immuno-enhancing function isolated from Korean mistletoe plants and its coding genes. The two lectin proteins isolated from the Korean mistletoe plants have an amino acid sequence and its coding gene from lectin proteins isolated from European mistletoe. There was a difference in the nucleotide sequence of the present invention, which has a very excellent effect of enhancing the antigen-specific immune response is a very useful invention in the biopharmaceutical industry.

Description

Lectin genes isolated from Korean mistletoe extract {Lectin genes from Viscum album}

The present invention relates to a lectin protein having an immune enhancing function isolated from a Korean mistletoe plant and its coding gene.

Mistletoe (Misttletoe.Viscum album) is a semi-parasitic plant that grows as a host of various kinds of trees and is known to have 30 plants and 1,500 species. Mistletoe, which is currently used as a medicinal herb among various kinds of mistletoe, is a mistletoe of the genus Viscum, a European mistletoe (Viscum album Loranthacea) mainly in Europe. Mistletoe, which belongs to the European loranthacea family, has been used as a folk medicine for a long time as a mysterious drug that is effective against hypertension, arteriosclerosis, and cancer. Since 1921, it has been recognized as an anticancer activity and used as an adjunct to the treatment of tumors. Therefore, studies on the biological activity of mistletoe have been carried out mainly on the mistletoe inhabiting Europe.

Mistletoe living in Europe has been reported to have an immune enhancing effect on the humoral and cellular immune systems, and clinical trials in animals and humans have shown that macrophages and natural killing of tumor cells directly and indirectly. Increasing cell activity has been reported to inhibit the growth of tumor cells to increase the survival rate of cancer patients (USP 5547674). These effects are known to be due to the immune enhancing action of mistletoe and direct cytotoxic effects on tumor cells. Representative active substances are lectins, which are classified into three types of lectins I, II, and III according to the specificity and molecular weight of sugar chains. Differentiation, mainly immunological and biochemical studies of lectin I are in progress.

Meanwhile, Korean mistletoe, which is distinguished from European mistletoe, has also been used as a medicine for low back pain, hypertension, miscarriage, and toothache in private and oriental medicines. It was suggested that there are different effects on the host tree and the difference in the components resulting therefrom. In fact, European mistletoe has been reported in lectin components according to the host, so the possibility that there may be a change in the component according to the host in Korea mistletoe can not be excluded. Based on this fact, Khwaja et al. Reported the antitumor effect and active ingredient of Korean mistletoe for the first time in the scientific study on Korean mistletoe (V. album, Coloratum) (USP No. 5,565,200). This study reports that the anti-tumor effect of Korean mistletoe is not caused by lectin, but rather by cytotoxic effect by strong toxic alkaloids, unlike the case of European mistletoe, and distinguishes the antitumor activity and mechanisms of European mistletoe and Korean mistletoe. However, there is no follow-up study yet. However, recent studies have shown that Korean mistletoe also directly activates macrophage, inducing Interleukin-1 (IL-1) and Turmor necrosis factor-α (TNF-α), and the active ingredients that induce these cytokines are precipitated by ammonium sulphate. It was found to be a protein component, and it was predicted that lectin was present among Korean mistletoe components, and then lectins were isolated from Korean mistletoe.

We isolated two lectin components (KML-IIU and KML-IIL) with different characteristics from Korean mistletoe plants using chromatography and monoclonal antibody screening, and then cloned the nucleotide sequence of the lectin protein coding gene by gene cloning. Determining the amino acid sequence deduced therefrom and comparing the two isolated lectin proteins with European lectin proteins, the present invention was completed by confirming the apparent difference between Korean and European and confirming the immunopotentiating effect of the lectin protein. It is an object of the present invention to provide a lectin protein and its coding gene having immuno-enhancing function which is purified from Korean mistletoe.

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

1 is a photograph showing the SDS-PAGE analysis of KML-C under reducing and non-reducing conditions.

Figure 2 is a photograph showing the Western blot analysis of KML-C using a monoclonal (9H7D10).

Figure 3 is a SDS-PAGE analysis of KML-C, KML-IIU, KML-IIL under reducing conditions.

Figure 4 shows the N-terminal amino acid sequence of Korean mistletoe lectin.

5 shows a schematic of the recombinant plasmid vector.

Figure 6 compares the amino acid sequence of some of the present invention Korean mistletoe lectin KML-IIUA and KML-ULB and European mistletoe lectin.

Figure 7 shows the response to mitogen addition of splenocytes of mice injected with KML-IIU.

8 is a diagram showing the IL-1 induced activity of KML-IIU of macrophages.

9 is a diagram showing the effect of KML-IIU on the induction of NK-cell activity.

10 is a diagram showing the effect of KML-IIU on the inducibility of antibodies specific for antigen KLH.

Figure 11 shows the analysis of subisotypes of antibodies against KLH induced by immunization with antigen KLH alone or simultaneously with KML-IIU.

12 shows antigen specific differentiation of splenocytes of mice immunized with antigen KLH.

Figure 13 shows the production of IL-2 and IL-4 against KLH from splenocytes of mice sensitized by KLH antigen.

14 shows the effect of KML-IIU on DTH induction.

The present inventors have isolated, extracted and cloned two lectin proteins from Korean mistletoe. As with the lectin proteins of conventional plants, we found that there is a possibility that several isoforms may exist due to the slight differences in nucleotide sequences between clones even in Korean mistletoe. Could know. In addition, the amino acid sequence was deduced from the DNA sequence and compared with the known amino acid sequence of the European mistletoe lectin protein and the related plant lectin protein.

To investigate the characteristics of isolated Korean mistletoe lectin, the cytotoxic effect on various cell lines was investigated in vitro using European mistletoe lectin (EML-1) as a control. The lectin protein KML-IIU was 6-BL6 melanoma, Meth A. While it showed relatively high resistance in Fibrosarcoma cell line, it showed relatively high sensitivity to 3LL casinoma and Raji lymphoma, and it was thought to have some specificity for tumor cells. It was found to be stronger than European lectin, and the cell line showed more than 10 times higher cytotoxic effect, indicating that the activity of lectin protein isolated from Korean mistletoe was very good. Comparing the cytotoxic effects of KML-IIU and KML-IIL in colon26-M3.1 casinoma, B16-BL6 melanoma, and L1210 Lucemia cells, KML-IIU showed a relatively high cytotoxic effect in all cases.

Hereinafter will be described the specific configuration and effect of the present invention through the examples. However, the following examples are only for the purpose of illustrating the present invention and the scope of the present invention is not limited to the following examples.

Example 1 Preparation of Mistletoe and Lectin Separation

Mistletoe grown with oak as a host to separate the lectin components was collected in December-February, washed three times with distilled water, frozen at -80 ℃, finely chopped, and chopping again in a blender. Extracted at 8 ° C. for 8-12 hours with a volume of 0.15 M NaCl solution, centrifuged at 10,000 × g for 20 minutes, and the supernatant was taken. In addition, ammonium sulfate powder was added to the supernatant to precipitate the protein component, and a saturated ammonium sulfate solution of 70% was made and stirred gently at 4 ° C. Centrifuge again at 10,000 × g for 20 minutes, discard the supernatant, take the pallet, resuspend in phosphate buffer (PBS) containing 0.15 M NaCl in the minimum volume, and change buffer two or three times in the same solution for 24 hours. Dialysis as it went. After centrifugation at 10,000 × g, only the supernatant was collected, filtered through a 0.45um membrane filter, and the filtrate was passed through a column filled with Sepharose 4B, hydrolyzed with 0.2M HCl at 50 ° C. for 3 hours, followed by PBS of 5-fold column volumn. The material obtained by washing with the column and eluting with PBS containing 50 mM Galactose was named KML-C.

Example 2. Investigation of Purity and Molecular Weight of KML-C

To determine the molecular weight and purity of KML-C, electrophoresis was performed on 13% polyacrylamide gels containing 0.1% SDS to compare with standard proteins. After completion of development, protein bands were stained with coomassie blue dye. As a result of the experiment, as shown in FIG. 1, four bands having a molecular weight of 33.2, 31, 28.6, and 25.5 KD were observed in the reduced state treated with 2-mercaptoethanol. Therefore, it was confirmed that two proteins with two subunits exist in KML-C. Each protein was named as KML-IIU (61.8KD) and KML-IIL (56.4KD).

Example 3. Production and Characterization of Monoclonal Antibodies Against KML-C

For production of monoclonal antibodies, KML-C dissolved in PBS in Balb / c mice at a level of 20 μg / 100 μl was emulsified in the same amount of complete Freund's adjuvant to immunize the abdominal cavity. After another immunization 14 days later, 10 days later, a small amount of blood was collected from mouse eyes, serum was obtained, and titer was confirmed by indirect ELISA immobilized with antigen. Then, 20 μg of antigen was injected into the abdominal cavity without adjuvant. One week after the final immunization, splenocytes of Balb / c mice and P4U1 myeloma were fused using PEG, hybridomas were selected with HAT medium, and cells producing antibodies against KML-C were cloned in 96 well plates. Then, screening and cloning were repeated to select hybridomas producing monoclonal antibodies against KML-C. In order to mass-produce the antibody, selected hybridomas were injected with pristane and injected into the abdominal cavity of mice to obtain ascitic fluid. The antibody was isolated by passing the ascitic fluid through a protein-G column to purify the antibody. Western blotting was performed to select antibodies that specifically react with KML-C from the purified monoclonal antibodies. First, KML-C was subjected to electrophoresis in the presence of SDS, transfer of the protein portion to the Nitrocellulose membrane, reaction of each monoclonal antibody, and reaction of the secondary antibody conjugated with alkaline phosphatase, followed by color development with BCIP-NBT solution. As a result of the experiment, as shown in FIG. 2, a single clone (9H7D10) having strong specificity was selected for the band of the upper portion (33KD) of KML-IIU without binding to the European lectin used as a control.

Example 4 Preparation of Immuno-affinity Columns and Isolation of KML-IIU and KML-IIL

In order to separate the two proteins KML-IIU and KML-IIL, an immuno-affinity column was immobilized with 9H7D10 antibody activated Sepharose according to the manufacturer's (Pharmacia Biotech's) instructions. Was separated. First, KML-C was dialyzed in PBS to remove galactose, then passed through an immuno affinity column, and the parts eluted with PBS (KML-IIL) and the parts eluted with glycine-HCl buffer (pH 2.7) (KML-IIU) were collected, respectively. Purity was measured by electrophoresis and compared with European mistletoe lectin (EML) as a control. Experimental results showed that KML-IIU and KML-IIL could be separated from KML-C as shown in Figure 3, and they were identified as protein bands with different molecular weight from EML.

Example 5 Hemagglutination and Glycolysis Reaction Test against KML-IIU and KML-IIL

In order to investigate whether isolated Korean mistletoe lectins function as agalutinin and which sugars have specificity, erythrocyte aggregation and glucose-inhibition reactions were performed. Agglutination and inhibition reactions were performed by serial double dilution using a U-Type 96 well microtiter plate. First, B type red blood cells were suspended in 2% PBS, and then cultured for 1 hour in a predetermined concentration of lectin solution and 96 well microtiter plate to confirm the minimum concentration required for aggregation (Table 1). In addition, the lectin solution of this concentration was incubated with sugar of various kinds and various concentrations for 30 minutes, and then reacted with the red blood cell suspension again to observe the inhibition of aggregation by sugar (Table 2). As a result, agglutination reaction was observed at 8μg / mL level for both KML-C, KML-IIU and KML-IIL, and the aggregation effect was inhibited by Lactose, galactose and N-acetylgalactoseamine. Therefore, Korean lectins isolated from the present invention were identified as lectins having specipicity in Lactose, galactose, and N-acetylgalactoseamine.

Hemagglutination Effects of KML-ⅡU and KML-IIL Lectin Concentration (µg / ml) KML-C 8 KML-ⅡU 8 KML-ⅡL 8 EML 2 Note: Concentration (μg / ml) is the minimum concentration required for erythrocyte aggregation.

Inhibitory Effects of KML-IIU and KML-IIL by Sugars Party Concentration (mM) Galactose 6 Lactose 3 N-acetylgalactoseamine 3 Mannose 100 Glucose 100 N-acetylglucoseamine 100 Note: Concentration (mM) is the minimum concentration of several types of sugars that inhibit erythrocyte aggregation activity.

Example 6. Amino Acid Sequence Determination for KML-IIU and KML-IIL

After performing SDS-PAGE on each sample and transferring to PVDF membrane, the sequencing of each chain in the amino acid sequence is shown in FIG. 4.

Example 7. Partial Cloning of Korean Mistletoe lectin Gene

Based on the amino acid sequence performed in Example 6 for purified KML-IIU, KML-IIL, two oligonucleotide primers were designed (Primer1: 5'-GTIACICATCAIACIGG-3 ', Primer2: 5'). -ACIATICGCACIGTIGGTTC-3 ') Polymerase Chain Reaction (PCR) was performed from the total genomic DNA of Korean mistletoe using these two primers. Thermal cycler (Perkin-Elmer) was prepared using 1 µg of genomic DNA extracted by CTAB method, 100 pmol of each primer, 200 μM of dNTP, 1.5 mM MgCl ₂ , and Amplitaq (Perkin-Elmer) DNA polymerase 2.5units. 9600) was performed (denaturation at 94 ° C 1min, annealing at 45 ° C 2min, primer extension at 72 ° C 2min, repeated 35 times). About 800bp of DNA obtained as a result of PCR was cloned by connecting to EcoR V position (suitable for cloning of PCR product because T is attached to 3'-overhang) of pGEM-T vector manufactured by Promega. A schematic diagram of the recombinant plasmid vector is shown in FIG. 5.

Example 8. Partial base sequence and deduced amino acid sequence of Korean mistletoe lectin gene

Two clones of the cloned lectin gene were determined using an automated sequencer for DNA sequencing, as shown in the following nucleotide sequence 1 and nucleotide sequence 2. Conventional plant lectins have been reported to have several isoforms together. In the case of Korean mistletoe, the sequencing results showed some differences in nucleotide sequences among the clones, suggesting the possibility of multiple isoforms. In addition, the amino acid sequence is deduced from the DNA base sequence and compared with the known European mistletoe lectin and the sequence of the lectin of other related plants as shown in FIG.

Example 9. Cytotoxicity Test of Cancer Cells of KML-IIU and KML-UUL

For the characterization of isolated Korean mistletoe lectins, the cytotoxic effect on various cell lines was observed in vitro using European mistletoe lectin (EML-1) as a control. First, put a certain amount of each cell line into a 96 well plate, add various concentrations of lectin, and after 48 hours, examine the growth of cells by MTT method and measure the concentration of the sample that inhibited the growth of each tumor cell line by 50% (ED ₅₀ ). 3 is shown. As a result, KML-IIU showed relatively high resistance to 6-BL6 melanoma and Meth A Fibrosarcoma cell lines, while it showed relatively high sensitivity to 3LL carcinoma and Raji lymphoma. In addition, the cytotoxic effect of KML-IIU was stronger than that of European lectin in most tumor cells, and the cell line showed more than 10 times higher cytotoxic effect. Comparing the cytotoxic effects of KML-IIU and KML-IIL in colon26-M3.1 carcinoma, B16-BL6 melanoma and L1210 leukemia cell lines, KML-IIU showed relatively high cytotoxic effects in all cases.

Cytotoxic Effects of KML and EML-1 on Various Tumor Cells Cell lines Origin ED ₅₀ to tumor cells (mg / mL) KML-ⅡU KML-ⅡL EML-1 Murine cells Colon26-M3.1 Carcinoma 19.0 2.0 0.8 B16-BL6 Melanoma 125.0 100.0 310.0 3LL Carcinoma 0.08 NT 0.03 Meth A Fibrosarcoma 210.0 NT 1200.0 L1210 Leukemia 10.0 0.2 2.0 L5178Y Lymphoma 5.0 NT 100 L929 Fibroblast 2.0 NT NT Human cells 4 NT 50 U937 Leukemia 3 NT NT HL60 Leukemia 0.12 NT NT Raji Lymphoma 0.9 NT NT

Example 10. Reactivity of mitogen to immune related cells

Three 6-week-old Balb / c (female) mice were injected (iv) with 50 ng of KML-IIU as a group, and splenocytes were separated 2 days later. Flatbottoned 96-well culture plate into the respective cells in the spleen (Nunc, Denmark) a density of ⁵ × 10 5 / 100㎕ each well, a known mitogen for T cells and B cells Con. A and LPS were incubated in each well for 100 μl at concentrations of 0.5 and 5 μg / ml, respectively, and then incubated at 5% CO ₂ and 37 ° C. for 3 days. After 6 hours of incubation, 0.5 μCi of [ ³ H] -TdR was added to 50 μl / well, and the cultured cells were adsorbed onto the glass filter by Filter mate 196 (Packard Instrument, Meriden, CT), followed by Matrix 96 ^TM Direct. Radioactivity was measured at Beta Counter (Packard). As shown in FIG. 9, splenocytes of mice administered with KML-IIU showed increased DNA synthesis compared to untreated samples even when mitogen was not added. It was found to be stimulated in vitro, and the addition of mitogen was thought to increase the reactivity of mitogen, which was proportional to the concentration of KML-IIU. Taken together, KML-IIU has the effect of enhancing the cellular reactivity of mature host immune cells for an effective immune response. By amplifying the number of effector cells for a specific immune response, an effective immune response to the antigen was induced.

Example 11 Induction of Cytokines from Macrophages

4 ml of 1% thioglycollate was intraperitoneally injected in Balb / c mice, and after 4 days, the mice were sacrificed by the cervical dislocation method. Peritoneal exaudative cells (PEC) were collected. The harvested PEC was plated by adjusting the concentration to 1.5 × 10 ⁶ on a 24 well culture plate. After culturing for 2 hours to attach macrophages, the cells were washed with culture to remove non-attached cells, and then, various concentrations of KML-IIU were added as samples to stimulate macrophages. IL-1 activity was confirmed by ELISA kit (ENDOGEN) induction of secretion of cytokine (IL-1), TNF-α activity was investigated by biological quantification using L929 cells. At this time, after stimulating the macrophages for 1 hour to exclude the direct effect on the sample L929 cells, the cells were washed four times using a culture medium, and then cultured for 24 hours to prepare a culture supernatant. TNF-α activity was examined by inhibition of proliferation of L929 cells by macrophage culture supernatant. TNF-α secretion was confirmed by rabbit-anti-TNF-α neutralization experiment. The experimental results are shown in FIG. 8, and IL-1 induced activity of macrophages by KML-IIU was significantly increased from 50ng / mL to 1ng / mL of KML-IIU, and was dependent on KML-IIU concentration. As a result of the investigation of TNF-α activity, KML-IIU-stimulated macrophage culture supernatants killed L929 cell line depending on the concentration of the sample, and its cytotoxic effect was inhibited by rabbit anti-TNF-α. -IIU was thought to be capable of inducing TNF-α by stimulating macrophages. KML-IIU, which induces TNF-α in vitro, was effective at a concentration of 2 ~ 0.4ng / mL.

Example 12 Measurement of Activity of NK-Cells

After 3 days of intravenous injection of KML-IIU in Balb / c mice, the mice were incubated for 18 hours after controlling the ratio of splenocytes and YAC-1 cells to 100: 1, 50: 1 and 25: 1. Six hours before the end of the culture, 0.5 [mu] Ci of [ ³ H] -TdR was added, and the amount of [ ³ H] -TdR used by the cultured cells was measured using the Beta Counter. Obtained by

Cytostatic activity (%) = (YAC -1 cells in a total of 1-group ^[3 H] -TdR uptake amount / total of the YAC-1 cells in the control group ^[3 H] -TdR uptake quantity) × 100

Experimental results showed effective NK-cell tumor cell killing ability between 50 ~ 10ng KML-IIU administration as shown in FIG.

Example 13. Effect of Enhancing Antibody Induction of KML-IIU to KLH Antigen

After immunizing KLH alone or simultaneously with KML-IIU as an antigen, the inducibility of KLH-specific antibodies was examined by ELISA. As shown in FIG. 10, the synergistic effect of KL-IIU 20ng compared with the KLH antigen alone was statistically significant, but the booster injection resulted in marked effects. The antibody titer was 60 times higher than the antibody titer of KLH alone, and the effect was significant up to 10 weeks of challenge. Therefore, KML-IIU had an adjuvant effect of activating the humoral immune system to the antigen. As a result of investigating the subisotype of the enhanced antibody, as shown in FIG. 10, the IgG1, IgG2a and IgG2b types were elevated.

Example 14. Lymphocyte Stimulation Experiment

Three Balb / c mice per group were lysed with 20 μg of KLH in PBS, followed by immunization with KLH alone or KML-IIU (20 ng) twice a total of 2 subcutaneous injections (100 μl / mouse) at 2 week intervals. . Two weeks after the last immunization, spleens were taken from immunized mice or from normal mice and suspended in RPMI-1640 medium containing 10% FBS to give a concentration of 1.5 × 10 cells.⁵Of The wells were adjusted and dispensed into 96-well culture plates. In each well of the splenocytes, antigen KLH was diluted 4 times from 40 µg / ml, adjusted to a concentration of 0.039 µg / ml, and LPS or Con-A was adjusted to a final concentration of 0.2 µg / ml. 37 ℃, 5% CO₂Incubated for 72 hours under the conditions of. 0.5 μCi of [5 hours before the end of culture.³H] -TdR (50 μl / well) was added to each well and the radioactivity of each well was measured. As shown in FIG. 14, splenocytes of mice injected with KML-IIU simultaneously showed effective proliferation activity of cells against KLH used as antigen, compared to splenocytes of mice injected with KLH alone. The differentiation activity of splenocytes sensitized against KLH tends to be dependent on the concentration of KLH restimulating in vitro, indicating that the adjuvant activity of KML-IIU against antigen in vitro is antigen specific.

Example 15 Secretion Induction Effect of Antigen-Specific Cytokines

The effect of KML-IIU as an adjuvant on KLH antigen was mainly related to cytokine secreted from T helper cell subunits, and IL- secreted from Th1 and Th2 types using splenocyte culture supernatants of mice sensitized to KLH. 2 and IL-4 secretion was investigated using the ELISA Kit results are as shown in FIG. PBS-treated mice, which were the control group, showed cytokine activity below the limit of detection in both cytokines, but were induced when the splenocytes of a mouse subcutaneously injected with antigen KLH were restimulated with antigen in vitro. IL-2 and IL-4 had an effective secretory effect at levels of 105 and 10 pg / mL, respectively. When KML-IIU was simultaneously administered to KLH as adjuvant, both cytokines showed more than two-fold induction activity compared to KLH alone. These results suggest that KM-110 or KML-IIU can act on any type of CD ⁺ T cells, either Th1 or Th2, so that subsequent immunization can increase not only humoral but also cellular immunity.

Example 16 Effect on Delayed Hypersensitivity Response

Delayed type hypersensitivity (DTH) to the antigen was immunized with Keyhole lympet hemocyanine (KLH; 20 ㎍) alone or KML-IIU (20 ng) to the antigen twice a week. After 14 weeks of final immunization, the subcutaneous injection of KLH (50 μg), an antigen, was applied to the paw of a host animal previously sensitized to the antigen. Induction of delayed and sensitive responses was measured as a ratio. As shown in FIG. 14, delayed hypersensitivity reactions of mice co-administered with 20 ng of KML-IIU had a significant enhancement effect up to 2 days after antigen re-administration compared to mice injected with antigen alone, thereby enhancing delayed hypersensitivity reactions. It was recognized to induce an effect.

As described in the above examples, the two lectin proteins isolated from the Korean mistletoe plants of the present invention differed from the lectin proteins isolated from the European mistletoe in the amino acid sequence and the nucleotide sequence of the coding gene. There is a very good effect of enhancing the present invention is a very useful invention in the biomedical industry.

Claims (2)

The lectin KML-IIU gene, which has the same base sequence as SEQ ID NO: 1 encoding the lectin protein isolated from the Korean mistletoe extract. Lectin KML-IIL gene, characterized in that it has the same base sequence as SEQ ID NO: 2 encoding the lectin protein isolated from Korean mistletoe extract.