CN1336383A - Giant web bombinin and its prepn. and pharmaceutical application - Google Patents

Abstract

The invention belongs to the field of biomedicine. Bombina grandis antimicrobial peptide is a single-chain polypeptide isolated from the skin secretion of Bombina grandis, a Chinese amphibian, with a molecular weight of 2697.6 and an isoelectric point of _10.2 . -NH ₂ . The preparation method is to clean the living Bombina grandis with water, put it in a glass container with a cover, add anhydrous ether dropwise, and seal the container for 3-5 minutes. A large amount of foamy substances can be seen secreted from the skin of the Bombina grandis. The secretion is collected, centrifuged to remove the precipitate, freeze-dried, separated and purified by ion exchange, gel filtration, and high-pressure liquid phase phase column chromatography. The bombesin antibacterial peptide has broad-spectrum and significant antibacterial and fungal growth effects, and is used as a broad-spectrum antimicrobial substance in the preparation of drugs for treating microbial infection diseases.

Description

Bombinain antimicrobial peptide and its preparation method and application in pharmacy

The invention provides a bombesin antimicrobial peptide and its preparation method and application in pharmacy, belonging to the field of biomedicine.

Since the invention of antibiotics, humans have made great achievements in the control and treatment of microbial infection diseases, but with the continuous use of drugs, microbial resistance has become a major problem in the treatment of clinical microbial infection diseases, so that some pathogenic bacteria There is no first-line drug for clinical treatment. Vancomycin-resistant staphylococci, enterococci, and other Gram-negative infections are currently a worldwide clinical problem. The three types of bacteria that mainly cause meningitis also have strong drug resistance clinically. Penicillin-resistant, chloramphenicol-resistant meningococcus, pneumococcus, and pneumococcus resistant to new cephalosporin antibiotics also widely appear. Therefore, the development of a new class of antibiotics has become a top priority and an international hotspot. Compared with the widely used antibiotics at present, polypeptide antibiotics have many advantages: such as rapid and broad-spectrum killing of microorganisms (including current clinical drug-resistant bacteria) at the minimum effective concentration, and inhibitory effect on fungi, and the ability to generate resistant strains Small, effective for both local infection and systemic infection, it is becoming a new class of antibiotics, and its development is currently receiving extensive attention.

Many amphibians are widely used in traditional Chinese medicine and ethnic medicine in China, such as Chinese toad (Bufo gargarizans), large webbed bell toad (Bombina maxima), black-spotted frog (pelophylax nigromaculata), marsh frog (Hylarana guentheri) and marsh frog ( Euphlyctis limnocharis) etc. The skin and viscera of these amphibians have a wide range of pharmacological activities and clinical curative effects. It has been reported that the pharmacological activities include broad-spectrum antimicrobial effects, anti-tumor effects, local anesthesia, analgesia, immune regulation, and cardiovascular system effects. In foreign countries, the search for specific pharmacologically active monomeric compounds in amphibian skin has become a hot spot for new drug inventions. According to foreign literature reports, the antibacterial peptide Magainin obtained from the skin secretion of Xenopus laevis has broad-spectrum antimicrobial effects and has Anti-tumor activity, has been approved as a broad-spectrum antibacterial drug in the United States, and its genetic engineering products have entered Phase III clinical trials; recently, Magainin Pharmaceuticals announced that the Xenopus skin antimicrobial peptide Magainin analog MSI-78 they produced was used to treat 926 cases Phase III clinical results of diabetic patients with foot ulcers caused by various bacterial infections showed that MSI-78 had the same effect as ofloxacin, but produced fewer side effects. The antibacterial peptide IB-367 produced by Intrabiotics has been approved for the first phase of clinical trials in the treatment of oral ulcers caused by various bacterial infections in cancer patients; the clinical trial of the antimicrobial peptide nisin in the treatment of gastric ulcers in cooperation between Applied Microbiology and Astra has also achieved good results Efficacy. Foreign scholars have also reported that several polypeptides with broad-spectrum antimicrobial activity have been isolated from Bombina orientalis and Bombina variegata, such as Bombinin and BLP-like polypeptides.

The application of amphibian drugs has a long history in my country, but the research on their active ingredients and pharmacological properties mainly focuses on small organic molecules such as alkaloids, and there are few researches on their skin active peptides. The research on amphibian skin antimicrobial peptides has not been reported yet. Bombina maxima is mainly distributed in Yunnan and Sichuan provinces in my country. It is one of the characteristic resource animals in my country and also a characteristic folk medicine.

The inventor searched and compared the full sequence structure of the bombesin antimicrobial peptide of the present invention through protein databases, and found no identical polypeptides.

The object of the present invention is based on above-mentioned prior art basis, provides a kind of broad-spectrum antimicrobial (comprising Gram-positive bacteria, Gram-negative bacteria, fungus) active bombesin and preparation method thereof applications in pharmaceuticals.

In order to realize the purpose of the present invention, the present invention provides following technical scheme:

Bombina grandis antimicrobial peptide is a single-chain polypeptide isolated from the skin secretion of the Chinese amphibian Bombina grandis for the first time, with a molecular weight of 2697.6 and an isoelectric point of 10.2. The primary complete sequence structure of the polypeptide is: glycine- Isoleucine-Glycine-Glycine-Valine-Leucine-Leucine-Serine-Alanine-Glycine-Lysine-Alanine-Alanine-Leucine-Lysine-Glycine -Leucine-Alanine-Lysine-Valine-Leucine-Alanine-Alanine-Lysine-Tyrosine-Alanine-Asparagine-Amidated Asparagine (NH ₂ -GIGGVLLSAGKAALKGLAKVLAAKYANN-NH ₂ ).

The preparation method of Bombina grandis antimicrobial peptide is to clean the living Bombina grandis with water, place it in a glass container with a cover, add anhydrous ether dropwise (1 liter of volume adds 1 milliliter of anhydrous ether), and seal the container 3-5 minutes, it can be seen that a large amount of foamy substance is secreted from the skin of Bombina maxima, collect the secretion, centrifuge (10000rpm, 20 minutes), freeze-dry, and collect the obtained peak after ion exchange with DEAE-Sephadex A-50 I gets peak IV after Sephadex G-50 gel filtration, and peak IV is ion-exchanged with CM-Sephadex C-25 again, gets the peak II that the third step column chromatography separates, and then reacts through high-performance liquid chromatography (HPLC) Phase C18 column separation and purification, take the peak II separated by the fourth step of HPLC reverse phase C18 column chromatography (as shown by the arrow in Figure 4). Then use high-performance liquid chromatography (HPLC) to identify its purity, determine its molecular weight by fast atom bombardment mass spectrometry (Fast atom bombardment mass spectrometry, FAB-MS), determine its isoelectric point by isoelectric focusing electrophoresis, and use an automatic amino acid sequencer to determine its amino acid sequence structure .

The use of the bombesin antimicrobial peptide as a broad-spectrum antimicrobial drug.

Below the pharmacological experiment result of bombesin of the present invention is used to illustrate the drug effect and beneficial effect of the present invention:

1. The effect of bombesin on inhibiting bacterial growth:

The antibacterial activity was detected by the cup and saucer method, and the medium was ordinary agar medium. Inject 20ml of heated and melted medium into the plate as the bottom layer, spread it evenly in the bottom of the plate, after solidification, take another appropriate amount of medium and heat and melt, add 5ml of bacterial suspension to each plate, shake well, Spread it evenly on the bottom layer as a bacterial layer. After cooling, put 6 sterilized stainless steel cups evenly in the plate at equal distances. Add 0.1ml of the test compound solution at a concentration of 0.3mg/ml to the first steel cup, add the sample solution to the remaining steel cups by doubling dilution method, incubate at 37°C, and observe the size of the inhibition zone. The bacteriostatic zone above 10mm was regarded as the minimum inhibitory concentration (minimal inhibitory concentration, MIC). The bacterial strains were obtained from the First Affiliated Hospital of Kunming Medical College. The test was repeated four times and the average value was taken. The results are shown in Table 1.

2. The effect of bombesin on inhibiting the growth of fungi:

Antifungal activity was detected by the cup and saucer method. The culture medium is improved Sabousand culture medium, inject 20ml of heated and melted culture medium into the plate as the bottom layer, spread it evenly in the bottom of the plate, after solidification, take another appropriate amount of culture medium and heat and melt it, respectively Add 5ml of bacterial suspension to each dish, shake well, and spread it evenly on the bottom layer as a bacterial layer. After cooling, put 6 sterilized stainless steel cups evenly in the plate at equal distances. Add 0.1ml of the test compound solution at a concentration of 0.3mg/ml to the first steel cup, add the sample solution to the remaining steel cups by doubling dilution method, incubate at 37°C, and observe the size of the inhibition zone. The bacteriostatic zone above 10mm was regarded as the minimum inhibitory concentration (minimal inhibitory concentration, MIC). The fungal strain comes from the Institute of Microbiology, Yunnan University. This test was repeated three times, and the average value was taken. The results are shown in Table 2.

Table 1, the effect of bombesin on inhibiting bacterial growth: strain Minimal inhibitory concentration (μg/ml) Crude sample bombesin Escherichia coli ATCC25922 (Escherichia coli ATCC25922) Staphylococcus aureus ATCC2592 (Staphylococcus auneus ATCC2592) Pseudomonas aeruginosa CMCCB10104 (Pseudomonas aeruginosa CMCCB10104) Bacillus megaterium (Bacillus dysenteriae) siella pneumococcus ( 150 1.275 3.675 14.4150 1.237.5 1.2150 3.6

As can be seen from Table 1, the first step of bombesin antimicrobial peptide DEAE-Sephadex A-50 ion exchange crude product and purified bombesin antimicrobial peptide all have significant inhibition of bacteria (comprising gram-positive bacteria, gram-negative bacteria ) growth, and compared with other sources of antibacterial peptides reported in the literature, the antibacterial activity of bombesin antibacterial peptides is 5-20 times higher on average.

Table 2, the effect of bombesin on inhibiting fungal growth: strain Minimal inhibitory concentration (μg/ml) Crude sample bombesin Candida albicans ATCC2002 (Candida albicans ATCC2002) Aspergillus flavus IFFI4015 (Aspergillus flavus sp IFFI4015) Penicillium IFFI2001 (Penicillium sp IFFI2001) Mucor pusillus Yeast: Saccharomyces cerervisiae 25 3.675 12.8 > 300 5050 5.280 10.4

It can be seen from Table 2 that, in addition to inhibiting bacterial growth, the first step of bombesin antimicrobial DEAE-SephadexA-50 ion-exchange crude product and purified bombesin antibacterial peptide all have a significant effect on inhibiting fungal growth, and compared with other sources Compared with antibacterial peptides, the antifungal activity is also 5-20 times higher on average.

Can draw from above-mentioned experimental result that the beneficial effect of bombesin macrobrain antimicrobial peptide of the present invention is:

Both the crude product and the purified bombesin antimicrobial peptide have a significant effect on inhibiting the growth of bacteria and fungi. The microbial strains used include: Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Shigella, Candida albicans , Penicillium, Aspergillus oryzae, etc., the results showed that bombesin had broad-spectrum antimicrobial activity (including Gram-positive bacteria, Gram-negative bacteria, fungi; MIC was in the range of 1-15μg/ml), and Compared with other sources of antibacterial peptides, the antibacterial activity is also 5-20 times higher on average. Therefore, the bombesin of the present invention is a biologically active polypeptide with broad-spectrum antimicrobial activity developed from Chinese characteristic medicinal biological resources for the first time.

The substantive content of the present invention will be further described below with reference to the accompanying drawings, but the content of the present invention is not limited thereto.

Fig. 1 is the DEAE-Sephadex A-50 ion exchange chromatogram of bombesin of the present invention.

Fig. 2 is a Sephadex G-50 gel filtration chromatogram of bombesin of the present invention.

Fig. 3 is the CM-Sephadex C-25 ion-exchange chromatogram of bombesin of the present invention.

Fig. 4 is an HPLC reversed-phase C18 column chromatogram of bombembin of the present invention.

Example:

1. Preparation of bombesin antimicrobial peptide:

Clean the living Bombina grandis with water, put the Bombina grandis in a glass container with a cover, add anhydrous ether (1 liter of volume plus 1 ml of anhydrous ether), and seal the container for 3-5 minutes. The foamy substance was secreted from the skin of Bombina maxima, and the secretion was collected, centrifuged (10000rpm, 20 minutes), freeze-dried, and stored at low temperature. The first step: DEAE Sephadex A-50 ion exchange, according to the above method to obtain the lyophilized powder of the raw material Bombina grandis secretion, the lyophilized powder was dissolved in 0.05M Tris-HCl, containing 10mM EDTA, pH7.3 buffer, packed in 3.5KDa dialysis bag, dialyzed in the same buffer solution for 12 hours; DEAESephadex A-50 (Pharmacia product) anion exchange column (500mm long, 26mm diameter) equilibrated with 0.05MTris-HCl, containing 10mM EDTA, pH7.3 buffer solution, dialyzed A good sample is put on the column, washed with two column volumes of the same buffer until the breakthrough peak is completely eluted, and then gradient eluted with a buffer containing NaCl to collect the breakthrough peak I.

The second step, Sephadex G-50 gel filtration: the active ingredient breakthrough peak I obtained in the first step was lyophilized, dissolved in 0.1M Na ₂ HPO ₄ -NaH ₂ PO ₄ , pH 6.0 buffer, and loaded on Sephadex G-50 (Pharmacia product) gel filtration column (1000 mm long, 26 mm diameter), eluted with the same buffer, and collected peak IV.

The third step, CM Sephadex C-25 ion _exchange : the sample on _the active _ingredient peak IV obtained by gel filtration was placed _in CH Sephadex C-25 ( Pharmacia product) cation exchange column (300mm long, 26mm diameter), carry out gradient elution with the buffer solution containing NaCl again, collect peak II.

The fourth step, reversed-phase high-pressure liquid chromatography (RP-HPLC): CM Sephadex C-25 ion exchange obtained active ingredient peak II with water (containing 0.1% trifluoroacetic acid): acetonitrile (containing 0.1% trifluoroacetic acid ) constitutes an elution system for gradient elution, and collects high-pressure liquid chromatography HPLC reverse-phase C18 column chromatography peaks (marked by arrows in Figure 4) that are bombesin.

2. The molecular weight was determined by Fast atom bombardment mass spectrometry (FAB-MS), using glycerol: m-nitrobenzyl alcohol: dimethyl sulfoxide (1:1:1, V:V:V, volume ratio) As the substrate, Cs ⁺ as the bombardment particles, the current is 1μA, and the emission voltage is 25Kv.

3. The purity of the purified bombesin was identified by high performance liquid chromatography (HPLC), the molecular weight was determined by fast atom bombardment mass spectrometry, the isoelectric point was determined by isoelectric focusing electrophoresis, and the amino acid sequence structure was determined by an automatic amino acid sequencer.

The Bombina grandis antimicrobial peptide prepared by the above method is a single-chain polypeptide isolated from the skin secretion of the Chinese amphibian Bombina major for the first time, with a molecular weight of 2697.6, an isoelectric point of 10.2, and a complete sequence of the polypeptide at the first level The structure is: glycine-isoleucine-glycine-glycine-valine-leucine-leucine-serine-alanine-glycine-lysine-alanine-alanine-leucine- Lysine-Glycine-Leucine-Alanine-Lysine-Valine-Leucine-Alanine-Alanine-Lysine-Tyrosine-Alanine-Asparagine- Amidated asparagine (NH ₂ -GIGGVLSAGKAALKGLAKVLAAKYANN-NH ₂ ). The bombesin antimicrobial peptide has a broad-spectrum antimicrobial activity and a significant effect of inhibiting the growth of bacteria and fungi, and is used as a broad-spectrum antimicrobial substance in the preparation of drugs for treating microbial infection diseases.

Claims (3)

1. Bombina grandis antimicrobial peptide, which is characterized by a single-chain polypeptide isolated from the skin secretion of the Chinese amphibian Bombina grandis, with a molecular weight of 2697.6 and an isoelectric point of 10.2. The primary structure of the complete sequence of the polypeptide is: Glycine-Isoleucine-Glycine-Glycine-Valine-Leucine-Leucine-Serine-Alanine-Glycine-Lysine-Alanine-Alanine-Leucine-Lysine -Glycine-Leucine-Alanine-Lysine-Valine-Leucine-Alanine-Alanine-Lysine-Tyrosine-Alanine-Asparagine-Amidated Day Paragine. 2. The preparation method of Bombina grandis antimicrobial peptide is characterized in that the living Bombina grandis is cleaned with water, placed in a glass container with a cover, anhydrous ether is added dropwise, and the container is sealed for 3-5 minutes, and a large amount of The foamy substance is secreted from the skin of Bombina maxima, and the secretion is collected, centrifuged to remove the precipitate, freeze-dried, separated and purified by ion exchange, gel filtration, and high-pressure liquid phase phase column chromatography. 3. The application of bombesin antimicrobial peptide as a broad-spectrum antimicrobial substance in the preparation of drugs for the treatment of microbial infection diseases.