CN115873092A - Antibacterial polypeptide from banded snake, preparation method and application thereof - Google Patents

Abstract

The invention discloses Cathelicidin family antibacterial polypeptide TS-CATH from a bungarus fasciatus as well as a preparation method and application thereof, wherein the amino acid sequence of the polypeptide is shown as SEQ ID NO:1 or an amino acid sequence having more than 90% homology therewith. According to the invention, a Cathelicidin family mature peptide segment is intercepted by carrying out sequence comparison and excavation on a banded snake protein database to obtain the polypeptide TS-CATH (KRFKKFFKKIKKSVKKRVKKLFKKPRVIPISIPF). The antibacterial polypeptide TS-CATH designed and synthesized by the invention has wide antibacterial spectrum to gram-negative bacteria, high sterilization speed, low hemolysis to sheep red blood cells and small mammalian cytotoxicity, can be applied to the treatment of common bacterial infection and drug-resistant bacterial infectious diseases, and is an excellent substitute drug or adjuvant therapy drug for the existing anti-infection treatment.

Description

Antibacterial polypeptide from banded snake, preparation method and application thereof

Technical Field

The invention relates to the technical field of polypeptide medicines, in particular to Cathelicidin family polypeptide derived from a bungarus fasciatus as well as a preparation method and application thereof.

Background

Most of the traditional antibiotics are designed aiming at a special target in the bacterial body, and although the traditional antibiotics can more effectively exert the drug effect, the antibacterial spectrum of the drug is narrowed. After bacteria evolve or obtain compensatory drug resistance genes from some unknown hosts, the bacteria can quickly cause the drug resistance of the bacteria to the antibiotics, and in addition, the drug resistance genes can be quickly spread in parallel through plasmids, so that the drug-resistant bacteria can explode in a short time. However, with the emergence of drug-resistant bacteria, the development of new antibiotics is slow.

The antibacterial peptide is a kind of small molecular polypeptide, most of which are rich in positive charge and hydrophobic amino acid and generally have a secondary structure. Antimicrobial peptides are present in almost all organisms in various forms and constitute an important part of the innate immunity of organisms and are therefore also referred to as host defense peptides. The antibacterial peptide is not easy to generate drug resistance due to the unique membrane-breaking sterilization mechanism, and has no immunogenicity and lower toxicity to eukaryotic cells, so that the antibacterial peptide has great potential as a novel therapeutic drug.

However, the potential toxicity of antimicrobial peptides is a major problem that prevents their clinical use. At present, the antibacterial peptide in clinical experimental research is only used for treating skin infection due to toxicity, most of the antibacterial peptide is eliminated in clinical experiments due to overlarge toxicity, and most of the currently reported antibacterial peptides cannot meet the unification of antibacterial activity effectiveness and safety. The linear structure of the native polypeptide marks the enzymatic instability of the polypeptide. Antimicrobial peptides are sensitive to certain proteases in wounds or plasma, are highly susceptible to degradation, and have a short half-life in vivo, and thus, the stability of antimicrobial peptides is another important reason limiting their development.

There is a need to obtain polypeptides that are both safe and have good antimicrobial activity.

The invention content is as follows:

the purpose of the invention is as follows: the technical problem to be solved by the invention is to provide the polypeptide from the Cathelicidin family of the fasciola bundae, and the polypeptide gram-negative bacteria has the advantages of wide antibacterial spectrum, high sterilization speed, low hemolysis on red blood cells and low toxicity on mammalian cells.

The technical problem to be solved by the invention is to provide a preparation method of the polypeptide.

The invention also aims to solve the technical problem of providing the application of the polypeptide in the field of antibacterial drugs.

The technical scheme is as follows: in order to solve the above technical problems, the present invention provides a polypeptide TS-CATH derived from Cathelicidin family of bungarus fasciatus, the amino acid sequence of which is:

SEQ ID NO：1：

LYS-ARG-PHE-LYS-LYS-PHE-PHE-LYS-LYS-ILE-LYS-LYS-SER-VAL-LYS-LYS-ARG-VAL-LYS-LYS-LEU-PHE-LYS-LYS-PRO-ARG-VAL-ILE-PRO-ILE-SER-ILE-PRO-PHE；

or an amino acid sequence having more than 90% homology thereto.

The invention also provides a preparation method of the polypeptide TS-CATH, and the preparation method comprises a polypeptide solid phase synthesis method.

The invention also comprises the application of the polypeptide TS-CATH of Cathelicidin family derived from the zona fasciata in preparing products for preventing and/or treating diseases caused by bacterial infection.

Wherein the bacteria comprise gram-negative bacteria and/or gram-positive bacteria.

The bacteria comprise antibiotic-sensitive and/or drug-resistant bacteria, the gram-negative bacteria comprise one or more of Escherichia coli, pseudomonas aeruginosa, klebsiella pneumoniae and Acinetobacter baumannii, and the gram-positive bacteria comprise one or more of staphylococcus epidermidis and staphylococcus aureus.

Wherein the concentration of the bacteria is 1 × 10 ⁵ CFU/ml～1×10 ⁹ CFU/ml。

Wherein the product comprises a bactericidal, bacteriostatic or antibacterial product.

The invention also comprises the application of the Cathelicidin family polypeptide TS-CATH derived from the fasciola bungarus in preparing medicaments for preventing and/or treating sepsis, septicemia and related diseases thereof.

Wherein the concentration of the polypeptide is 2-1250 mug/ml, preferably, the concentration of the polypeptide is 4-64 mug/ml.

Research on bacteremia models constructed by abdominal cavity infection drug-resistant bacteria of mice shows that the antibacterial polypeptide TS-CATH prepared by the invention can obviously improve the survival rate of mice resistant to ceftazidime escherichia coli infection, has a certain protection effect on the weight of the mice, and reduces the bacterial load in blood, lung, kidney, spleen and liver tissues of the mice.

Has the beneficial effects that: compared with the prior art, the invention has the following advantages: the antibacterial polypeptide TS-CATH designed and synthesized by the invention has the advantages of wide antibacterial spectrum, high sterilization speed, small hemolysis to red blood cells and low toxicity to mammalian cells. The antibacterial polypeptide TS-CATH prepared by the invention can be applied to preparation of medicines for resisting pathogenic bacteria infection, has good in-vivo and in-vitro antibacterial activity, and is an excellent substitute medicine or adjuvant therapy medicine for the existing anti-infection treatment.

Drawings

FIG. 1 HPLC chromatogram of polypeptide TS-CATH;

FIG. 2 mass spectrum of polypeptide TS-CATH;

FIG. 3 sterilization curve of polypeptide TS-CATH against Escherichia coli;

FIG. 4 is a graph of the bactericidal profile of polypeptide TS-CATH against Klebsiella pneumoniae;

FIG. 5 bactericidal profile of polypeptide TS-CATH against Staphylococcus aureus;

FIG. 6 haemolysis of sheep erythrocytes by the polypeptide TS-CATH;

FIG. 7 cytotoxicity of the polypeptide TS-CATH on mammalian cells;

FIG. 8 shows the survival protection effect of polypeptide TS-CATH on bacteremia mouse;

FIG. 9 weight protection of the polypeptide TS-CATH against bacteremic mice;

FIG. 10 Effect of the polypeptide TS-CATH on the number of bacteria in the blood of bacteremic mice;

FIG. 11 Effect of the polypeptide TS-CATH on the number of bacteria in lung tissue of bacteremic mice;

FIG. 12 Effect of the polypeptide TS-CATH on the number of bacteria in liver tissue of bacteremia mice;

FIG. 13 Effect of the polypeptide TS-CATH on the number of bacteria in spleen tissue of bacteremia mice;

FIG. 14 Effect of the polypeptide TS-CATH on the bacterial count in the renal tissue of bacteremia mice.

Detailed Description

Example 1: design and synthesis of polypeptide TS-CATH

(1) Sequence alignment is carried out on a protein database of the band snakes by using an online BLAST tool on an NCBI website (https:// www.ncbi.nlm.nih.gov) by taking a classic snake-derived Cathelicidins family antibacterial polypeptide OH-CATH (GenBank: ACF 21002.1) as a template, and the Cathelicidins amino acid sequences in the protein database are mined.

(2) Downloading a precursor sequence of the Cathelicidin family antibacterial polypeptide from different species such as human, mouse, pig, sheep, bungarus fasciatus and the like in a UniPort protein database (https:// www.uniprot.org), introducing the precursor sequence and a sequence obtained by the previous BLAST into MEGA 7 software together for multi-sequence alignment, verifying a conserved sequence of a cathlin region, predicting an amino acid residue cleavage site of elastase, determining and intercepting a mature peptide sequence to obtain an antibacterial polypeptide TS-CATH sequence (KRFKKFFKKIKKSVKKRVKKLFKKPRVIPISIPF) of the invention, such as SEQ ID NO:1 is shown.

(3) The antibacterial polypeptide TS-CATH is synthesized in vitro by adopting a conventional polypeptide solid phase synthesis method.

Example 2 preparation of polypeptide TS-CATH:

the amino acid sequence of the prepared TS-CATH is as follows: lysine-arginine-phenylalanine-lysine-phenylalanine-lysine-lysine-isoleucine-lysine-serine-valine-lysine-arginine-valine-lysine-leucine-phenylalanine-lysine-proline-arginine-valine-isoleucine-proline-isoleucine-serine-isoleucine-proline-phenylalanine

Polypeptide synthesis: the polypeptide TS-CATH is synthesized from the C end to the N end one by one. Soaking Fmoc-Val-Wang Resin in dichloromethane for 15 minutes, and after the Resin swells, extracting the dichloromethane; piperidine/Dimethylformamide (DMF) solution (10 ml per g resin) was added at a volume ratio of 1: 4, stirred with nitrogen, reacted 2 times for 5 minutes and 15 minutes, and after the reaction was finished, the resin was washed 9 times with DMF. 20-40g of resin is added with 2-3 drops of each color testing agent ABC (liquid A is ninhydrin/absolute ethyl alcohol solution; liquid B is pyridine; liquid C is phenol/absolute ethyl alcohol solution; and 2-3 drops of A, B, C solution) and heated for 3 minutes at 100 ℃, so that the color of the solution and the resin is changed into blue, and the amino protection is removed. An excess of Fmoc-VaI-OH and HOBT, which reacted twice molar, was added, dissolved in 10ml of DMF per gram of resin, DIC and Collidine, which reacted twice molar, with nitrogen sparge, for 1h. After the reaction is finished, washing the resin with DMF for 6 times, repeatedly carrying out condensation reaction, sequentially connecting each Fmoc protected amino acid to complete the synthesis of a straight chain sequence, soaking the resin with dichloromethane and ether, and then pumping to dry. Adding TFA, and reacting for 2 hours in a constant temperature shaker at a shaker rotation speed of 110r/min and a temperature of 25 ℃. Filtering to remove resin, adding anhydrous diethyl ether into the filtrate, centrifuging to obtain solid, adding anhydrous diethyl ether, washing, centrifuging, repeating for several times, and oven drying to obtain crude polypeptide.

And (3) polypeptide purification: weighing a certain amount of crude product, adding a proper amount of acetonitrile, performing ultrasonic treatment until the mixture is clarified, and removing large-particle impurities by using a filter. Meanwhile, the samples are collected by stages through a preparative liquid chromatograph. And (4) performing gradient analysis by using an analytical chromatograph, and reserving a sample reaching the required purity. Then, freeze-drying treatment is carried out.

The synthesis and purification of the polypeptide TS-CATH are completed by Gill Biochemical (Shanghai) Co., ltd, and the purity of the obtained product reaches 95.75%.

Example 3 determination of purity of polypeptide TS-CATH (HPLC method) and results of Mass Spectrometry

The polypeptide TS-CATH of the embodiment 1 and the embodiment 2 is synthesized and then purified to obtain a finished product, and the finished product is identified by high performance liquid chromatography and mass spectrometry.

Liquid chromatography conditions: c18 column (4.6X 250mm,5 μm); the mobile phase A is acetonitrile solution containing 0.1 percent of trifluoroacetic acid; the mobile phase B is purified water containing 0.1% trifluoroacetic acid. The detection wavelength is 220nm; the flow rate is 1.0ml/min; the sample was taken in an amount of 10. Mu.l, and gradient elution was performed. The gradient elution conditions are shown in table 1.HPLC chromatogram is shown in FIG. 1, and mass spectrum is shown in FIG. 2.

TABLE 1 gradient elution conditions for the polypeptide TS-CATH

Example 4 MIC and MBC values of the polypeptide TS-CATH against gram-negative and gram-positive bacteria

Polypeptide TS-CATH is diluted into different concentration gradients by MH liquid culture medium (Beijing three medicine technology development company, cat. No. 11E 01) in multiple ratio, 100 μ l is respectively put into 96-well plates, and then inoculated in equal volume and diluted to 2 × 10 by liquid culture medium ⁵ CFU/ml bacterial suspension, the final concentration of the medicine is 128, 64, 32, 16, 8, 4, 2 and 1 mug/ml, and a blank control group without the medicine is set. The bacteria are placed at 37 ℃ for culturing for 18-22 h, and the experimental result is observed and recorded. After confirming that the bacteria without drug treatment grow normally, the lowest drug concentration of the bacteria without drug treatment is taken as the Minimum Inhibitory Concentration (MIC), and the MIC value (MIC) capable of inhibiting 50 percent of the growth of clinical strains is calculated ₅₀ . The results are shown in Table 2.

Taking 100 mul of culture without colony growth in a 96-well plate for MIC determination, adding 900 mul of sterile culture medium for dilution, transferring all the dilution liquid to a sterile plate, pouring about 10ml of corresponding agar culture medium (at the temperature of 50-55 ℃), culturing bacteria at 37 ℃ for 24h, observing and recording the number of colonies in the plate, and obtaining the lowest drug concentration (MBC) value when the number of the colonies is less than 5. The results are shown in Table 3.

TABLE 2 MIC values of the polypeptide TS-CATH against gram-negative and gram-positive bacteria

IS: clinically separating strains; MRSA: methicillin-resistant staphylococcus aureus

TABLE 3 MBC values of the polypeptide TS-CATH against gram-negative and gram-positive bacteria

IS: clinically separating strains; MRSA: methicillin-resistant staphylococcus aureus

As can be seen from the results in tables 2 and 3, the antibacterial polypeptide TS-CATH of the present invention has a broad antibacterial spectrum, and has good bacteriostatic activity and bactericidal activity against both standard strains and clinical drug-resistant strains. The MIC of the antibacterial polypeptide to gram-negative bacteria is in the range of 4-32 mu g/ml, and the MBC to the partially drug-resistant gram-negative bacteria is in the range of 4-16 mu g/ml; for gram-positive bacteria, the MIC of the antibacterial polypeptide is in the range of 8-16 mug/ml, and the MBC is in the range of 8-32 mug/ml.

Example 5 Sterilization curves of the polypeptide TS-CATH against Standard and clinically resistant strains

About 10 ⁵ The bacterial liquid to be tested (Escherichia coli, klebsiella pneumoniae, staphylococcus aureus and clinical strains with drug resistance) with the concentration of CFU/ml grows in an MHB culture medium with the concentration 4 times of MIC drug, and the co-culture samples are taken at 0, 0.5, 1, 2, 4, 8, 12 and 24 hours after the co-incubation and are subjected to gradient dilution for viable count. The results of the sterilization graphs were plotted with time as the abscissa and the log of the number of bacterial cells as the ordinate, and are shown in fig. 3 to 5.

The sterilization curve result shows that for Klebsiella pneumoniae and Pseudomonas aeruginosa, the antibacterial polypeptide TS-CATH of the invention can completely kill the Klebsiella pneumoniae, the Pseudomonas aeruginosa standard strain and the clinical drug-resistant strain within 0.5h under the drug concentration of 4 times MIC (minimal inhibitory concentration) of 16 mu g/ml. For staphylococcus aureus, under the drug concentration of 64 mu g/ml, which is 4 times MIC, the antibacterial polypeptide TS-CATH of the invention can completely kill staphylococcus aureus and methicillin-resistant staphylococcus aureus within 1h and 4h respectively.

Example 6 haemolysis of sheep Red blood cells by the polypeptide TS-CATH

The centrifugally separated sheep red blood cells were diluted to 3% (v/v) with sterile PBS (0.01 mol/L, pH 7.4) and diluted with the polypeptide TS-CATH synthesized in example 1 to terminate the drugConcentrations of 512, 256, 128, 64, 32, 16, 8, 0 μ g/ml were mixed and plated in 96-well plates, and a PBS negative control and a Triton X-100 positive control at a final concentration of 0.1% were established. CO 2 ₂ Incubate in incubator for 30 min. After centrifugation, the supernatant was measured for the OD value of absorbance at a wavelength of 450nm using a microplate reader. The hemolysis rate of sheep erythrocytes by the drug was calculated by taking the hemolysis rate of 100% for red blood cells treated with Triton X-100 and 0% for red blood cells treated with PBS, and the results are shown in FIG. 6.

The hemolytic result shows that even if the concentration of the antibacterial polypeptide TS-CATH reported by the invention reaches 512 mu g/ml, the hemolytic rate is lower than 10%, and the hemolytic property is very low.

Example 7 cytotoxicity of the polypeptide TS-CATH on mammalian cells

Culturing human liver cell L02, human umbilical vein endothelial cell Huvec and mouse fibroblast L929 to cell density of about 80-90%, digesting, centrifuging, collecting, removing supernatant, and suspending and diluting the cells to concentration of 5-10 × 10 by using DMEM complete culture medium ⁴ And (4) inoculating the cells per ml in a 96-well plate, putting the plate into an incubator for culturing for 12 hours, and administering the cells after the cells are attached to the wall. The polypeptide TS-CATH is dissolved in DMEM complete medium and diluted into 128, 64, 32, 16 and 8 mu g/ml dosing groups and one non-dosing blank group, each group is provided with 3 re-wells, and the incubation is carried out for 48h at 37 ℃. Add 10. Mu.l MTT solution (thiazole blue, 5mg/ml, i.e. 0.5% MTT) per well and continue the incubation for 4h. The supernatant was carefully aspirated off, 100. Mu.l of dimethyl sulfoxide was added to each well, and the mixture was shaken on a shaker at a low speed for 10 minutes to dissolve the crystals sufficiently. The absorbance of each well was measured by a microplate reader OD490, and the cell viability was counted, and the results are shown in FIG. 7.

According to the cytotoxicity result, the antibacterial polypeptide TS-CATH has lower cytotoxicity to mammalian cells under bacteriostatic and bactericidal concentrations. Particularly, the 48h survival rate of the polypeptide is higher than 80 percent under the concentration of 64 mu g/ml for human liver cells L02, human endothelial cells Huvec and mouse fibroblasts L929.

EXAMPLE 8 Effect of serum on the antibacterial Activity of the polypeptide TS-CATH

The polypeptide TS-CATH synthesized in example 1 was prepared in a solution of 2048. Mu.g/ml in PBS (0.01 mol/L, pH 7.4), mixed with human serum in a volume ratio of 3: 1, and incubated in an incubator at 37 ℃ while serum-free PBS was set as a control. Samples were taken at different time points 0.5h, 1h, 2h, 15% trichloroacetic acid was added at a ratio of 6: 1, incubated at 4 ℃ for 30 minutes, centrifuged at 13000rpm for 10 minutes to remove the precipitate, diluted with MHB medium at gradient, and the MIC change of the polypeptide was determined according to the method of example 4, and the results are shown in Table 4.

TABLE 4 Effect of sera on the antibacterial Activity of the polypeptide TS-CATH

IS: clinical isolation of bacterial strains

The results show that the polypeptide TS-CATH still has certain antibacterial activity after being treated by serum. The polypeptide TS-CATH still has bacteriostatic activity to clinical drug-resistant strains after being treated with serum for 2 hours.

Example 9 protective Effect of the polypeptide TS-CATH on bacteremia model mice

Healthy ICR female mice weighing 20-22 g were randomly divided into 6 groups of 10 mice each. 5 groups of mice were inoculated intraperitoneally with a minimal lethal concentration of bacterial suspension (ceftazidime-resistant Escherichia coli clinical strain, 1X 10) ⁹ CFU/ml,0.5 ml/mouse) was infected, a bacteremia model was established, and mice intraperitoneally injected with an equal volume of saline was used as a blank control. The rest model building groups were divided into 10mg/kg TS-CATH (high dose group), 5.0mg/kg TS-CATH (medium dose group), 2.5mg/kg TS-CATH (low dose group), 5mg/kg ceftazidime (control group), and physiological saline (model control). The polypeptide TS-CATH administration group and the normal saline group are intraperitoneally administered twice after 0.5h and 2h of infection respectively, and the ceftazidime group is administered once in tail vein after 2h of infection. The observation was continued for 7 days and mice were recorded for mortality and weight change.

After 8h of infection of the mice with bacteria, 3 mice were taken from each group, the eyeballs were peeled off and blood was taken into a sterile EP tube, 200. Mu.l of the mixture was gently mixed and taken into a sterile EP tube containing heparin sodium (1% heparin physiological salt solution, 50. Mu.l of the mixture was taken into a sterile EP tube, dried at 60 ℃), the blood was diluted by 10-fold gradient, 1ml of the diluted blood was taken from each tubeAdding nutrient agar culture medium, and pouring into a flat plate for viable count. Soaking the abdomen of each group of mice in 75% ethanol, dissecting the ultra-clean bench, collecting the lung, liver, spleen and kidney, placing the tissue in 5ml EP tube according to V _{Tissue of} ∶V _{Physiological saline} Adding sterile physiological saline in a ratio of 1: 9, shearing with sterile scissors, grinding with a homogenizer, performing gradient dilution by 10 times, adding 1ml of diluent into a nutrient agar culture medium, pouring into a flat plate, counting viable bacteria, placing in an incubator at 37 ℃, and observing and recording the colony number in the organ of the infected mouse.

As can be seen from figure 8, ceftazidime-resistant Escherichia coli can cause most mice in a ceftazidime control group to die, the mortality rate is up to 83.3%, the survival rate of the mice fed with the polypeptide is greatly improved compared with the survival rate of the mice in the ceftazidime control group and the mice in a model group, and the low, medium and high dosage administration of the polypeptide has dose dependence on the improvement of the survival rate of the mice, so that the polypeptide TS-CATH has a certain protection effect on the mice infected with drug-resistant bacteria. As can be seen from fig. 9, the body weight of mice in both the polypeptide-administered group and the ceftazidime group was reduced to some extent one day after the bacterial infection. The weight of the mice in the high-dose polypeptide group is recovered to the weight of the mice in the blank control group at the seventh day after bacterial infection, and compared with the mice in the low-dose polypeptide group, the weight of the mice in the high-dose polypeptide group is recovered more quickly, which shows that the polypeptide TS-CATH also has a certain protection effect on the weight of the mice.

From FIGS. 10 to 14, it can be seen that 8h after bacterial infection, the amount of bacteria in blood, lung tissue, liver tissue, spleen tissue and kidney tissue of mice was significantly reduced by administering each dose of the polypeptide, and the clearance of bacteria was dose-dependent, while the amount of bacteria in each tissue of the ceftazidime-administered group was comparable to that of the model group. The polypeptide TS-CATH shows strong bacteria clearing capacity and can obviously improve the survival rate of mice infected by drug-resistant bacteria.

Claims (8)

1. Cathelicidin family polypeptide TS-CATH derived from a bungarus fasciatus, wherein the amino acid sequence of the polypeptide is as follows:

SEQ ID NO：1：

LYS-ARG-PHE-LYS-LYS-PHE-PHE-LYS-LYS-ILE-LYS-LYS-SER-VAL-LYS-LYS-ARG-VAL-LYS-LYS-LEU-PHE-LYS-LYS-PRO-ARG-VAL-ILE-PRO-ILE-SER-ILE-PRO-PHE；

or an amino acid sequence having more than 90% homology thereto.

2. The method of claim 1, wherein the method comprises a solid phase polypeptide synthesis method.

3. Use of the Cathelicidin family polypeptide TS-CATH derived from a bungarus fasciatus as claimed in claim 1 in the manufacture of a product for the prevention and/or treatment of a disease caused by a bacterial infection.

4. Use according to claim 3, characterized in that said bacteria comprise gram-negative and/or gram-positive bacteria.

5. The use according to claim 3, wherein the bacteria comprise antibiotic-sensitive and/or drug-resistant bacteria, the gram-negative bacteria comprise one or more of Escherichia coli, pseudomonas aeruginosa, klebsiella pneumoniae, acinetobacter baumannii, and the gram-positive bacteria comprise one or more of Staphylococcus epidermidis, staphylococcus aureus.

6. Use according to claim 3, wherein the product comprises a bactericidal, bacteriostatic or antibacterial product.

7. Use of Cathelicidin family polypeptide TS-CATH derived from a bungarus fasciatus according to claim 1 for the preparation of a medicament for the prevention and/or treatment of sepsis, septicemia and diseases related thereto.

8. The use of any one of claims 3~7 wherein the polypeptide is present at a concentration of 2 to 512 μ g/ml.

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