JP2016053003A - Polypeptide derived from coccoidea - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a polypeptide derived from Coccoidea [Ceroplastes ceriferus Fabricius.] having hemolytic activity and cytotoxicity.SOLUTION: The invention provides a polypeptide derived from Ceroplastes ceriferus Fabricius. containing the amino acid sequence represented by GSGSVLVRNSGGYVATFSMA, and a hemolytic composition containing the polypeptide. The invention relates to a hemolysis method comprising contacting the polypeptide or the composition to a liquid sample containing red blood cells, and to a cytotoxic composition containing the polypeptide, as well as to an antitumor composition containing the polypeptide. The method of obtaining the polypeptide comprises fractioning a sample obtained by homogenizing entire Ceroplastes ceriferus Fabricius. by a suitable chromatography to separate the fraction representing hemolytic activity.SELECTED DRAWING: Figure 3

Description

  The present invention relates to a scale-derived polypeptide. The present invention further relates to a hemolytic agent composition comprising a scale-derived polypeptide and a hemolysis method using the scale-derived polypeptide. The invention further relates to a cytotoxic composition comprising a polypeptide from scales and an anti-tumor composition comprising a polypeptide from scales.

  A hemolytic agent is a substance that breaks down the cell membrane of red blood cells and leaks protoplasm. The hemolytic agent is used for measuring the amount of hemoglobin, removing red blood cells, and classifying white blood cells (Patent Document 1). The hemolytic agent can also be used for testing and diagnosis of various diseases (Non-patent Document 1), and the possibility of therapeutic application in, for example, thrombosis-related diseases is also considered.

  Hemolysis can be achieved in various ways. For example, hemolysis is physically achieved by mechanically damaging red blood cells or exposing them to hypotonic solutions. In addition, hemolysis can be induced chemically with an organic solvent or a surfactant.

  As an example of biological hemolysis, hemolysis in vivo via an antibody against red blood cells is known. It is also known that bacteria such as streptococci produce a factor having hemolytic activity (hemolysin). Furthermore, it is known that some lectins (sugar binding proteins) derived from sea cucumber animals and mushrooms have hemolytic activity (Non-patent Document 2). However, the biological origin of hemolysin is often not understood in detail, and industrial applications are still scarce.

JP2003-344387

Abstracts of the 52nd Tohoku Medical Examination Society (2011) Biophysics 49 (2), pp. 075-079 (2009)

  As described above, hemolytic agents have various application possibilities, and the need for new hemolytic agents continues to exist. In particular, there is a need for hemolytic agents that can be easily obtained from inexpensive materials. In particular, hemolytic agents derived from biological sources, especially polypeptides, can be developed by applying various biochemical and genetic engineering techniques, and are expected to provide high utility as new tools in clinical fields and other fields. Is done.

  The scale insect is an insect belonging to the superfamily of the order of the order of the order of the order of the order of the order of the order of the bugs of the order of the order of the order of the order of the order of the order of the order of the order of the order of the order of the order of the order of the order of the order of the order of the order of the stinkbug In the present invention, a scale insect called a hornworm (also known as a hornworm scale, Ceroplastes ceriferus) is used as a source of a hemolytic agent.

  The horned weevil is a gray-white insect with a length of 6 to 9 mm, a large wax shell, a thickness, and a slight pale red color (Fig. 1). The mature shell is rounded like a dome. The horned weevil occurs once a year, the overwintering adults lay eggs in May, the larvae hatch from mid to late June, and become adults from September to October. The horned weevil parasitizes many plants such as mandarin orange, tea, camellia, zelkova, etc., causing soot disease and causing great harm. The horned weevil is distributed all over Japan in Miyagi and Yamagata prefectures in Honshu and throughout the world.

  The inside of the hornworm wax husk is red (Fig. 1), and this red pigment cochineal is used as a food additive. In addition, the wax part is used as a floor wax or a coating agent. Thus, it is very meaningful and efficient from an industrial point of view to collect and use useful substances from weevil, a pest that is widely inhabited and to be controlled.

  The present inventors have surprisingly discovered that there is a hemolytic activity among polypeptides produced by hornworms and succeeded in isolating the polypeptide. The inventors have further discovered that the polypeptide is cytotoxic to non-erythroid cells. The present invention has been completed based on these findings.

Accordingly, the present invention includes the following aspects.
[1] A polypeptide derived from weevil, comprising the amino acid sequence represented by SEQ ID NO: 1.
[2] A hemolytic agent composition comprising the polypeptide of [1].
[3] A hemolysis method comprising contacting a liquid sample containing erythrocytes with the polypeptide of [1] or the composition of [2].
[4] The hemolysis method according to [3], wherein the liquid sample contains blood.
[5] A cytotoxic composition comprising the polypeptide of [1] above.
[6] An antitumor composition comprising the polypeptide according to [1].

FIG. 1 shows a taxonomic position of the hornworm and a photograph of its appearance. FIG. 2 shows a chromatogram obtained by gel filtration chromatographic separation of a homogenized liquid of weevil. The elution position of CC-IV polypeptide is indicated by an arrow. FIG. 3 shows the results of an experiment in which each fraction of gel filtration chromatography is mixed with blood. A well containing a fraction containing CC-IV polypeptide is shown surrounded by a black frame. FIG. 4 shows the results of SDS-PAGE of each sample in the purification process. From left, 1. Size marker, 2. Sample after dialysis, 3. Sample of flow-through after anion chromatography, and 4. Sample of CC-IV fraction of gel filtration chromatography are electrophoresed. The position of the 14 kDa size marker is indicated by an arrow. FIG. 5 shows the results of the pH stability test. The numbers 32 and 64 indicate that hemolytic activity was confirmed in the 32-fold and 64-fold diluted CC-IV polypeptide fractions, respectively. FIG. 6 shows the results of the temperature stability test. The CC-IV polypeptide fraction was incubated at the temperature indicated on the horizontal axis, and then the hemolytic activity was measured. FIG. 7 shows the results of an experiment in which various monosaccharides, disaccharides, glycoproteins, or proteins were added to wells and examined for inhibition of the hemolytic activity of CC-IV polypeptide. “NC” is a negative control with PBS added instead of CC-IV polypeptide. FIG. 8 shows a comparative experiment of hemolytic activity against blood of different animal species. The numbers indicate the dilution rate of the CC-IV polypeptide fraction. “Agglutination” is a positive control for aggregation using agglutinin RCA120, “NC” is a negative control with PBS added instead of CC-IV polypeptide, and “Hemolysis” is the detergent Triton X-100 Is a positive control for hemolysis using FIG. 9 shows the results of a cytotoxicity test using Vero cells. The horizontal axis represents the concentration of the added CC-IV hemolytic polypeptide, and the vertical axis represents the survival rate of Vero cells. + And − represent the presence or absence of addition of FBS to the medium. FIG. 10 shows the results of a cytotoxicity test using U937 cells. The horizontal axis represents the concentration of the added CC-IV hemolytic polypeptide, and the vertical axis represents the survival rate of U937 cells. + And − represent the presence or absence of addition of FBS to the medium.

  The present invention provides a weevil-derived polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1. The polypeptide of the present invention can be obtained by collecting and purifying from weevil. The number or weight of hornworms to be used is not particularly limited, but for efficient collection and purification, for example, at least 10 g by wet weight, more preferably at least 100 g, more preferably about 150 g (about 3200 animals). Equivalent) weevil can be used. Further, a larger amount of polypeptide can be obtained using a large number of individuals as starting materials.

  The polypeptide of the present invention can be obtained by fractionating a sample obtained by homogenizing the whole weevil by appropriate chromatography and separating the fraction showing hemolytic activity. Specifically, each fraction of the above chromatography is mixed with an erythrocyte-containing liquid sample (usually blood or a diluted solution thereof) to obtain a fraction having hemolytic activity, that is, a fraction containing the polypeptide of the present invention. Can be identified. When the hemolytic activity fraction is mixed with the red blood cell-containing liquid sample, the red blood cells are lysed and the components inside the cell, including hemoglobin, are released into the liquid mixture. It is visually recognized that On the other hand, in the case of a fraction having no hemolytic activity, blood cells are precipitated at the bottom of the mixed solution, or blood cells are aggregated, and can be distinguished from the above-mentioned lysate macroscopically or microscopically. A suspension is obtained.

  The erythrocyte-containing liquid sample used for the purpose of identifying hemolytic activity may be blood derived from various animals such as humans, sheep, cows, horses, chickens, etc., or diluted solutions thereof. For example, hemolysis activity can be screened by diluting the blood with phosphate buffered saline or the like so that the blood cell component is about 2% by volume and mixing the same volume of the chromatographic fraction into the diluted solution. . For example, since the detection of hemolytic activity is affected by osmotic pressure, such as blood cells spontaneously hemolyze in hypotonic solutions, it is preferable that the dilute solution and the mixed solution be isotonic solutions. Mixing of the chromatographic fraction and the erythrocyte-containing liquid sample can be easily performed, for example, in a well of a 96-well plate, and the hemolytic activity-containing fraction can be confirmed by the naked eye or by a microscope.

  Examples of such chromatography include, but are not limited to, anion exchange chromatography, gel filtration chromatography, and combinations thereof. Examples of columns for anion exchange chromatography include those having a cross-linked agarose as a matrix and a quaternary ammonium group as a functional group. Specifically, GE Healthcare's Q-Sepharose Fast Flow is suitable. Can be used. An example of a column for gel filtration chromatography is a column composed of dextran and highly crosslinked agarose. Specifically, Superdex 75 (1.6 × 60 cm, open column) manufactured by GE Healthcare can be preferably used. Gel filtration chromatography using a column having a resolution in the molecular weight range of 3,000 to 70,000 is particularly preferable because the polypeptide of the present invention can be separated in a substantially pure state. After performing anion exchange chromatography, the flow-through fraction is used for gel filtration chromatography, and each fraction of the gel filtration chromatography is assayed for hemolytic activity as described above, thereby containing the polypeptide of the present invention. Most preferably, the fraction is identified. Before and after the chromatography, additional purification steps such as centrifugation and dialysis may be appropriately performed.

  The purification methods described above and in the examples are merely examples, and those skilled in the art will appropriately select the type of chromatography and appropriate purification steps to separate proteins, and indicate the presence of hemolytic activity in the separated fractions. Thus, the polypeptide of the present invention can be purified and isolated.

  The polypeptide of the present invention has a molecular weight of about 10 to 11 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Therefore, in identifying the fraction containing the polypeptide of the present invention, this molecular weight can be used as an index in addition to the hemolytic activity.

  The polypeptide of the present invention comprises the amino acid sequence GSGSVLVRNSGGYVATFSMA (SEQ ID NO: 1). This amino acid sequence is found at the N-terminus of the mature polypeptide. However, since the most terminal amino acid is not methionine, it is understood that in the polypeptide immediately after translation, the amino acid sequence is not in the N-terminal but in the polypeptide, that is, on the C-terminal side with respect to the signal peptide. . Techniques for determining the N-terminal or internal sequence of a polypeptide are well known to those skilled in the art. A method for obtaining a polypeptide immediately after translation by inhibiting cleavage of the signal peptide is also known to those skilled in the art. The polypeptide of the present invention was isolated by determining the partial or full-length amino acid sequence of the polypeptide contained in the separated fraction and confirming that the amino acid sequence of SEQ ID NO: 1 was included. Can be confirmed.

  Furthermore, as understood by those skilled in the art, an approach is also possible in which an antibody against the peptide having the amino acid sequence of SEQ ID NO: 1 is prepared, and the polypeptide of the present invention is purified and isolated based on the binding to the antibody. is there. Specific examples of such approaches include, but are not limited to, antibody affinity chromatography, immunoprecipitation, and expression library screening.

  In another aspect of the present invention, a hemolytic composition comprising the above polypeptide is provided. The concentration of the polypeptide contained in the composition depends on the degree of hemolysis desired, and can be provided as a form premised on dilution or concentration before use. It is not limited. Typically, the composition may comprise 0.1-99.9%, 1-99%, or 10-90% by weight of the polypeptide. Alternatively, the present composition comprises 10-20% by weight, 20-30% by weight, 30-40% by weight, 40-50% by weight, 50-60% by weight, 60-70% by weight, 70-70% by weight. You may contain 80weight% or 80-90weight%.

  The hemolytic agent composition of the present invention can be provided as a liquid, solid, or semi-solid composition. In the case of a liquid composition, the component other than the polypeptide is typically water, but may be other suitable solvents known to those skilled in the art or a mixture of a plurality of solvents. The liquid composition of the present invention may contain other components known to those skilled in the art, such as salts, buffers, stabilizers, preservatives and the like. The pH of the liquid composition of the present invention is preferably in the range of pH 4.0 to 11.5. Component compositions known to cause polypeptide denaturation should not be used.

  The solid composition can be provided in a form known to those skilled in the art, such as powders, tablets, capsules and the like, and as components other than the above-mentioned polypeptides, excipients, binders, coating agents known to those skilled in the art A desiccant and the like can be used as appropriate. By lyophilizing an aqueous solution of the polypeptide of the present invention, a solid state polypeptide can be obtained, which can be included in the solid composition of the present invention.

  In another aspect of the present invention, there is provided a method of hemolysis comprising contacting a liquid sample comprising red blood cells with a polypeptide of the present invention or a composition of the present invention. The liquid sample containing erythrocytes is typically blood or a diluted solution thereof, and can be, for example, blood derived from various animals such as humans, sheep, cows, horses, rabbits, chickens, or diluted solutions thereof. This method is carried out by mixing or dissolving the polypeptide of the present invention or the composition of the present invention in a liquid or solid state in a liquid sample. The amount ratio of erythrocytes to the present polypeptide in the contact reaction mixture is not particularly limited because it can be appropriately adjusted by those skilled in the art according to the desired degree of hemolysis and the like. For example, in the same manner as the screening of the chromatographic fraction described above, a liquid sample is prepared so that the blood cell component is about 2% by volume, and a polypeptide solution having a concentration of, for example, 1 to 100 μg / ml is added to the liquid sample. Hemolysis can be caused by volume mixing.

  As the reaction pH in the hemolysis method of the present invention, a wide range of pH can be used. For example, hemolysis can be achieved in the range of pH 4.0 to 11.5. In order to maintain these pHs, various buffer solutions such as citrate buffer, phosphate buffer, Tris buffer, and glycine buffer can be used as the reaction solution. Appropriate buffer types and concentrations corresponding to the desired pH can be appropriately selected by those skilled in the art.

  The polypeptide of the present invention is inactivated at a temperature of 50 ° C. or higher. Accordingly, the reaction temperature in the hemolysis reaction of the present invention is preferably 40 ° C. or lower. The reaction temperature is preferably 4 ° C. or higher. The hemolysis reaction of the present invention can be preferably carried out at room temperature.

  The polypeptide of the present invention exhibits cytotoxicity against cells other than erythrocytes in addition to hemolytic activity against erythrocytes. Accordingly, in another aspect of the invention, there are provided cytotoxic compositions comprising the polypeptides of the invention. As used herein, “cytotoxic” means the ability of a substance to kill cells. The concentration of the polypeptide in the cytotoxic composition of the present invention, the components other than the polypeptide, the liquid / solid type, etc. are not particularly limited and are not particularly limited because they are the same as in the case of the hemolytic agent composition. It can be adjusted as appropriate.

  Since the polypeptide of the present invention has strong cytotoxicity against tumor cells, it is useful as an anti-tumor agent, that is, a cytotoxic agent for use in tumor treatment, examination, research and the like. Accordingly, in another aspect of the invention, an anti-tumor composition comprising a polypeptide of the invention is provided. As used herein, a tumor can be an epithelial tumor or a non-epithelial tumor, such as a lymphoma, and can be a benign tumor or a malignant tumor. The concentration of the polypeptide in the antitumor composition of the present invention, the components other than the polypeptide, the liquid / solid type, etc. are not particularly limited because they are the same as in the case of the hemolytic composition. It can be adjusted as appropriate.

  EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to these Examples.

[Fractionation of homogenized solution of hornworms]
We collected hornworm scale insects parasitizing the snowy willows in Toyo University Itakura Campus (Ikura, Gunma Prefecture). In order to prepare a homogenized solution of collected hornworm scales, homogenize using about 3200 animals (approx. 150 g in wet weight) with PBS (phosphate buffered saline, 0.15 M NaCl, pH 7.4). did. The volume of the homogenizing solution in a PBS suspension was about 200 ml. This homogenized solution was centrifuged (4 ° C., 8,000 G, 20 minutes) to separate and remove insoluble wax and the like as precipitates. The supernatant (about 120 ml) was collected in a cellulose tube (Sanko Junyaku Co., Ltd.) and dialyzed against PBS to remove low molecular impurities and the like. The dialyzed sample was subjected to purification by anion chromatography using a 500 ml glass buchner type filter (manufactured by Sansho). That is, a strong anion exchange resin (Q-Sepharose Fast Flow, GE Healthcare) placed in the filter was equilibrated with PBS, and the dialyzed sample was gently applied. Elution was performed with PBS, and the flow-through fraction was collected. The collected flow-through solution is centrifuged (4 ° C, 5,000G, 30 minutes) using a spin column tube (50 ml Macrosep, 10K MWCO), and the resulting supernatant is used as a sample for the next step. did. This sample was subjected to ultrafiltration concentration using an ultrafilter equipped with an ultrafiltration filter (NMWL: 10,000, Millipore). The sample after ultrafiltration concentration was applied to a HiLoad 16/60 Superdex 75 prep grade column (0.5 × 0.5 × 60 cm, GE Healthcare). Gel filtration chromatography was performed using PBS as the buffer at a flow rate of 1.0 ml / min. A 2 ml aliquot was taken per test tube and fractionated into 96 fractions, and the fractions near the 60th were collected.

  FIG. 2 shows a chromatogram of gel filtration chromatography. The elution position corresponding to the CC-IV fraction (fraction containing the polypeptide of the present invention) described later is indicated by an arrow. It can be seen that when a Superdex 75 column having a resolution in the molecular weight range of 3,000 to 70,000 is used, the polypeptide of the present invention is contained in the last eluted peak.

[Identification of fractions having hemolytic activity]
As shown in FIG. 3, sheep blood diluted to 2% (v / v) blood cells with PBS was placed in each well of a 96-well plate, and the same volume of gel filtration chromatography fraction was added from the upper left according to the elution order. Added to the lower right well. Here, the horizontal arrangement of the 96-well plate is called “row”, and the vertical arrangement is called “column”.

  In the first and eighth lines, it can be seen that the red blood cells have settled at the bottom of the well without being affected by the fraction. In the fractions before and after the third row, blood agglutinating activity is present, and erythrocytes aggregate to form a network structure in the well, causing suspension of the liquid. On the other hand, in the 2nd to 7th columns of the 6th row, hemolytic activity was observed in which the red blood cells were lysed to produce a uniform red solution (in the photograph in FIG. Are difficult to distinguish from wells that are present, but in practice they can be clearly distinguished by the naked eye or by a microscope). A combination of the fractions in the range of the sixth row and the second to seventh columns is referred to as a CC-IV fraction, which corresponds to the peak indicated by the arrow in FIG.

  Figure 4 shows a size marker (1st lane), a sample after dialysis (2nd lane), a flow-through sample after anion chromatography (3rd lane), and a sample of CC-IV fraction (4th lane) The result of having analyzed by SDS-PAGE is shown. CC-IV has been shown to contain a single polypeptide of approximately 10-11 kDa in a substantially pure state. Further, by performing mass spectrometry (MALDI-TOF / MS) on the sample, it was confirmed that the molecular weight of the polypeptide of the present invention was 10.9 kDa (data not shown). In FIG. 4, the concentration between lanes is not uniform, and a concentrated sample is applied particularly to the CC-IV lane.

[PH stability test]
To examine the pH stability for the hemolytic activity of CC-IV samples, the following buffers were prepared at a concentration of 0.1 M for each pH: citrate buffer (pH 4.0, 5.0, 5.8), phosphate buffer Solution (pH 5.7, 6.0, 7.0, 8.0), Tris buffer (pH 7.2, 8.0, 9.0), glycine buffer (pH 8.6, 9.0, 10.0, 10.6, 11.0, 11.5). These buffers were made the same volume as the sample (buffer solution 25 μl: sample 25 μl), added to 2% (v / v) sheep blood in a 2-fold dilution series, and left at room temperature for 1 hour.

  FIG. 5 shows the results of the pH stability test. The numerical values in the table represent the dilution factor. Over a wide pH range, hemolytic activity was detected with samples diluted to 32-fold or 64-fold.

[Temperature stability test]
To examine the temperature stability of the CC-IV sample for hemolytic activity, the CC-IV sample diluted 2 times was left for 10 minutes at a temperature from 0 ° C. to 70 ° C. (10 ° C. interval). Then, immediately after placing at 4 ° C, as in the above pH stability test, a sample (25 µl) was prepared in a 2-fold dilution series in a 96-well U bottom plate (dilution solvent was PBS), and 2% v / v) The same volume (25 μl) of erythrocyte suspension was added, the plate was shaken, and allowed to stand at room temperature for 1-2 hours. Thereafter, the hemolysis state was confirmed.

  Figure 6 shows the results of the temperature stability test. The vertical axis represents AU (Activity Unit) and corresponds to the dilution rate. Although the hemolytic activity was stably maintained after incubation at a temperature of 0 to 40 ° C., it was shown that the hemolytic activity was inactivated at a temperature of 50 ° C. or higher. These results are consistent with the fact that the CC-IV polypeptide (protein) provides hemolytic activity and that the polypeptide is heat denatured at temperatures above 50 ° C. Although data is not shown, protease activity is not detected in CC-IV polypeptide, and CC-IV polypeptide appears to have achieved hemolysis with a function different from protease enzyme activity.

[Verification of hemolysis inhibition by sugar]
It is known that a lectin (sugar binding protein) derived from a certain organism has hemolytic activity (Non-patent Document 2). In order to verify the hypothesis that the CC-IV polypeptide is a lectin, an inhibition experiment with sugar was conducted. The results are shown in FIG. A variety of monosaccharides, disaccharides, and glycoproteins were added at different concentrations shown at the top of FIG. 7 (however, for BSA (bovine serum albumin), fetuin, asialofetuin, mucin, and egg albumin) The left well is added at a final concentration of 0.5 mg / ml, and each well on the right is a 2-fold serial dilution). However, in any case, inhibition of the hemolytic activity of CC-IV by sugar was not detected (FIG. 7). This result suggests that CC-IV polypeptide is not a lectin.

[Evaluation of animal species specificity]
The hemolytic activity of CC-IV was examined using blood from different animal species (sheep, cow, horse, chicken). The point that a 2-fold dilution series CC-IV solution was added to 2% (v / v) blood dilution was the same as in the above experiment. As shown in FIG. 8, the CC-IV polypeptide showed hemolytic activity against blood of any animal species, but it was confirmed that there was a difference in hemolysis strength depending on the animal species. This suggests that this hemolytic polypeptide may recognize the difference in cell membrane structure between animal species.

[Evaluation of cytotoxicity]
Non-Patent Document 2 describes an example in which a lectin having hemolytic activity also has cytotoxicity against non-erythroid cells through a mechanism similar to that of hemolytic activity. Standards known to those skilled in the art using Vero cells (1 × 10 ⁴ cells / well) to verify the potential of CC-IV polypeptides to be toxic to cells other than erythrocytes Cytotoxicity tests were performed by a novel MTT assay. Vero cells are adherent immortalized cell lines derived from African green monkey kidney epithelial cells and are commonly used for toxin screening. FIG. 9 shows the results of the cytotoxicity test. The horizontal axis indicates the concentration of the added CC-IV polypeptide, and the vertical axis indicates the survival rate of Vero cells. Plus (+) represents a medium supplemented with FBS (fetal bovine serum) (that is, a normal medium for cell culture), and minus (-) represents a medium without FBS. For example, when evaluating the cytotoxicity of a lectin, it is known that various sugar components contained in FBS affect the assay, and therefore testing with FBS minus is common. In the case of CC-IV polypeptide, cytotoxicity was detected at a concentration of 20 μg / ml against Vero cells in FBS minus medium. This is a general toxicity as a toxin.

[Evaluation of cytotoxicity against hematological malignancies]
Cytotoxicity was examined using U937 cells (1 × 10 ⁴ cells / well) in the same manner as in the Vero cell experiment. U937 cells are malignant tumors (cancers) derived from human blood cells (lymphoid system) and are floating cell lines. The results are shown in FIG. As in FIG. 9, the horizontal axis represents the concentration of the added CC-IV polypeptide, and the vertical axis represents the survival rate of U937 cells. For U937 cells, even stronger toxicity than Vero cells was detected.

[Determination of N-terminal amino acid sequence]
As in the example shown in Fig. 4, from the SDS-PAGE gel obtained by electrophoresis of CC-IV sample, the band portion was cut out to obtain about 14 μg of polypeptide, which was then subjected to Edman degradation using a protein sequencer. The N-terminal amino acid sequence was determined. As a result, a sequence called GSGSVLVRNSGGYVATFSMA (SEQ ID NO: 1) was obtained.

  A homology search using the BLAST algorithm available on the NCBI (The National Center for Biotechnology Information) website was used to check whether there was a sequence similar to SEQ ID NO: 1 in the GenBank database. The same sequence was not present in the database. On the other hand, only the first 15 amino acids of SEQ ID NO: 1 have 88% identity with the C-terminal internal sequence of 1-phosphofructokinase of Leifsonia xyli (bacteria causing sugarcane dwarf disease) It was found. Many other sequences were found that were less similar (ie, have a lower percentage of identity or a shorter alignable sequence). These were mainly bacterial sequences and did not contain insect sequences.

  Although a certain number of sequences showing “hits” by the BLAST algorithm were found as described above, their sequence similarity to SEQ ID NO: 1 is very limited, and such limited sequence similarity It was impossible to determine whether or not has biological significance. In any case, protein sequences that clearly suggest hemolytic activity or cytotoxicity were not found in the results of homology searches with the BLAST algorithm.

  The present invention can be used in the fields of medicine, pharmacy, veterinary medicine, livestock and the like that utilize hemolytic activity or cytotoxicity. The active ingredient in the present invention is a polypeptide, and therefore there is a possibility of functional regulation and mass production by application of various chemical modifications or analogs known in the field of polypeptides, or by application of genetic recombination techniques. . For example, the cell binding ability of the polypeptides of the present invention can provide cytotoxicity targeting specific cells by conferring cell specificity through chemical modification or sequence modification. Therefore, the present invention has a wider range of applicability as agricultural chemicals and fungicides.

Claims (6)

A polypeptide derived from a hornworm, comprising the amino acid sequence represented by SEQ ID NO: 1. A hemolytic agent composition comprising the polypeptide according to claim 1. A hemolysis method comprising contacting the polypeptide of claim 1 or the composition of claim 2 with a liquid sample containing erythrocytes. 4. The hemolysis method according to claim 3, wherein the liquid sample contains blood. A cytotoxic composition comprising the polypeptide of claim 1. An antitumor composition comprising the polypeptide of claim 1.