CN110551191A - Improved melittin with low hemolytic activity and application thereof - Google Patents

Abstract

The invention relates to an improved melittin with low hemolytic activity and application thereof. Melittin is one of the best substitutes of antibiotics as an excellent biological antibacterial agent, but melittin has stronger erythrocyte hemolysis, is not suitable for being directly injected into blood, has a plurality of constraints for local use, and the problem that how to reduce the hemolysis capability of melittin while keeping the antibacterial capability of melittin is urgently to be solved in the clinical application of melittin. The inventor obtains another novel melittin by sequence improvement according to the sequence of the wild melittin, and simultaneously obtains low hemolytic energy on the basis of keeping the antibacterial function of the wild melittin. The novel melittin and the variant thereof provided by the invention have good clinical application prospects.

Description

Improved melittin with low hemolytic activity and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to improved melittin with low hemolytic activity and application thereof.

Background

With the abuse of antibiotics, the problem of drug-resistant bacteria has become a serious problem which seriously threatens the national health. Such as Methicillin-resistant Staphylococcus aureus (MRSA), have now possessed broad-spectrum resistance to all β -lactam and cephalosporin antibiotics with similar structures to Methicillin, as well as aminoglycosides, macrolides, tetracyclines, fluoroquinolones, sulfonamides, and rifames, which are resistant to varying degrees on average. MRSA is also a common pathogenic bacterium of nosocomial infection, the infection is frequently seen in trauma and operation, the average detection rate of MRSA in hospitals in major areas of China is 38.4 percent in 2016 years, the detection rate of partial hospitals is as high as 53.5 to 75.3 percent, and the recovery process of patients is seriously influenced, which puts urgent requirements on the development of novel antibacterial drugs and materials.

melittin (Melittin) is the main component of bee venom, and is also the main component with pharmacological action and biological activity in bee venom. The specific amino acid sequence enables the melittin to target bacterial cell walls and generate a membrane penetrating effect on bacterial cell membranes, so the melittin has broad-spectrum antibacterial property, has better killing effect on gram-positive bacteria and gram-negative bacteria, and is a good antibiotic substitute product. However, melittin has a strong red blood cell hemolytic property, is not suitable for direct injection into blood, and has many constraints for local use, which greatly limits the clinical use of melittin. How to reduce the hemolytic ability of melittin while maintaining its antibacterial ability is an urgent problem to be solved in clinical application of melittin.

The invention aims to obtain another novel melittin by repeatedly improving the sequence according to the sequence of the wild melittin and obtaining low hemolytic capacity on the basis of keeping the antibacterial function of the wild melittin.

In addition, in a tumor cell toxicity test, the novel melittin provided by the invention has a good killing effect on tumor cells. The novel melittin provided by the invention has good clinical application prospect.

disclosure of Invention

The invention aims to provide a novel melittin, the sequence of the melittin is a sequence which has homology of more than 75% with SEQ NO ID.1, the sequence of the SEQ NO ID.1 is as follows: GIGAVLKVLTTGLPALISWIKRKRQQ are provided.

The invention aims to provide a novel melittin, the sequence of which has homology of more than 75 percent with SEQ NO ID.1, and the novel melittin is characterized in that one or more amino acids are substituted, deleted and added on the amino acid in SEQ NO ID.1 to form a novel melittin variant with the same function as the melittin.

Further, the variant sequence of the novel melittin is a sequence having more than 75% homology with GIGAVLKVLTTGLPALISWIKKKKQQ (SEQ ID. 2).

At present, melittin has various modification forms, such as attachment of fatty acid, fluorination and acylation of amino acid, replacement of L-amino acid with D-amino acid, etc., and the activity of polypeptide is improved, the toxicity is reduced, the preparation process is simplified, etc. by different modifications.

Further, the N-terminal of the novel melittin is modified by acetylation and/or the C-terminal is modified by amidation.

a nucleotide fragment, wherein the nucleotide fragment codes the novel melittin.

A recombinant vector, wherein the vector contains the nucleotide fragment.

A recombinant vector, which expresses the novel melittin.

Further, the vector is a prokaryotic expression vector or a eukaryotic expression vector.

Furthermore, the vector is an escherichia coli expression vector, a chlorella expression vector, a baculovirus-mediated insect expression vector and a yeast expression vector.

A recombinant cell comprising the above recombinant vector.

Expression vector, recombinant microorganism or transgenic cell line containing the nucleotide fragment.

A composition comprising the novel melittin or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carrier includes ion exchange materials, alumina, aluminum stearate, lecithin, Self Emulsifying Drug Delivery Systems (SEDDS), surfactants for pharmaceutical formulations such as d-tocopheryl polyethylene glycol 1000 succinate, tween or other similar polymeric media, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, saturated vegetable fatty acid partial glyceride mixtures, water, salts, electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, silica gel, magnesium silicate, and the like. Polyvinylpyrrolidone, cellulosic materials, polyvinyl alcohol, sodium carboxymethylcellulose, polyacrylates, ethylene-polyoxyethylene-block polymers and lanolin, cyclodextrins such as alpha-, beta-, gamma-cyclodextrins or chemically modified derivatives thereof such as hydroxyalkyl cyclodextrins of 2-and 3-hydroxypropyl-beta-cyclodextrin and soluble derivatives thereof.

The novel melittin, the variant of the novel melittin, the nucleotide fragment, the composition, the recombinant vector or the recombinant cell are applied to the preparation of products or medicines with bacteriostatic and/or bactericidal properties.

Further, the product may be a medical consumable.

Further, the drug is a drug for preventing or treating infection, and preferably, the interference is caused by bacteria, fungi, viruses, parasites, or the like.

Further, the bacteria are gram-positive bacteria, gram-negative bacteria, fungi, viruses or parasites.

Further, the bacteria are staphylococcus aureus, escherichia coli, vibrio parahaemolyticus, vibrio enteritis, pseudomonas aeruginosa, methicillin-resistant staphylococcus aureus, acinetobacter baumannii or full drug-resistant bacteria isolated from baumannii clinics.

Staphylococcus aureus, an important pathogenic bacterium in humans, belongs to the genus Staphylococcus, is a representative of gram-positive bacteria, and causes many serious infections. Escherichia coli is gram-negative brevibacterium, is generally not pathogenic, is a common bacterium in intestinal tracts of human beings and animals, can cause parenteral infection under certain conditions, and has strong pathogenicity of certain serotype strains, causing diarrhea, causing severe diarrhea and septicemia. Vibrio parahaemolyticus, also known as Vibrio enteritis, belongs to the genus Vibrio and is a common pathogenic bacterium. Vibrio enteritis is a halophilic gram-negative bacterium, the main habitat is in seawater and is one of the main pathogenic bacteria causing food poisoning, and in coastal areas, a great number of patients suffer from food poisoning due to eating marine products polluted by vibrio enteritis every year. Methicillin-resistant staphylococcus aureus is a common pathogenic bacterium of hospital infection, and the infection is often seen after trauma or operation. Acinetobacter baumannii, also known as acinetobacter baumannii, belongs to gram-negative bacteria, which are one of the bacteria of the genus acinetobacter, commonly found in nosocomial infections, and are also pathogenic bacteria of aquaculture animals, which commonly cause bacteremia, pneumonia, meningitis, peritonitis, endocarditis, and urinary tract and skin infections. Acinetobacter baumannii has become a main source of nosocomial infections, especially in intensive care units, and the pathogenic bacteria cause the Acinetobacter baumannii to generate drug resistance due to the abuse of antibiotics, so that the Acinetobacter baumannii becomes 'multi-drug-resistant Acinetobacter baumannii' (full-drug-resistant bacteria isolated in the Acinetobacter baumannii clinic).

The novel melittin, the variant of the novel melittin, the nucleotide fragment, the composition, the recombinant vector or the recombinant cell are applied to the preparation of antitumor drugs.

The medicament further comprises pharmaceutical excipients including fillers (such as anhydrous lactose, starch, lactose beads and glucose), binders (such as microcrystalline cellulose), disintegrants (such as cross-linked sodium carboxymethyl starch, cross-linked sodium carboxymethyl cellulose, low-substituted hydroxypropyl cellulose and cross-linked PVP), lubricants (such as magnesium stearate), absorption enhancers, flavoring agents, sweeteners, diluents, excipients, wetting agents, solvents, solubilizers, colorants and the like.

The administration preparation of the medicament can comprise injection, cream, ointment, patch, spray and the like.

The drug administration routes include subcutaneous, intradermal, intraarterial, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional, intracranial injection or infusion, oral, topical, rectal, nasal, buccal, vaginal, sublingual, intradermal, mucosal, tracheal, urethral administration, administration by inhalation of aerosol, accumulation of implants and acupuncture.

Drawings

FIG. 1 is an antibacterial potency plot of wild-type melittin and melittin-K against SA29213 at different concentrations;

FIG. 2 is an antibacterial potency plot of wild-type melittin and melittin-K against USA300 at different concentrations;

FIG. 3 is an antibacterial potency plot of wild-type melittin and melittin-K against AbBAA747 at different concentrations;

FIG. 4 is an antibacterial potency plot of wild-type melittin and melittin-K against Ab1814516 at different concentrations;

FIG. 5 is a plot of the hemolytic potency of wild-type melittin and melittin-K against erythrocytes at different concentrations;

FIG. 6 is a graph comparing the hemolytic capacity of wild type melittin and melittin-K against erythrocytes at a concentration of 4 ug/ml;

FIG. 7 is a graph of tumor cell killing by wild-type melittin and melittin-K at different concentrations.

Detailed Description

The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention. Those of ordinary skill in the art will understand that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents. The following examples are examples of experimental methods not indicating specific conditions, and the detection is usually carried out according to conventional conditions or according to the conditions recommended by the manufacturers.

Example 1 polypeptide design and antibacterial testing

1. Design of polypeptides

Based on the polypeptide sequence of melittin, a melittin-K sequence (K-Meli) was designed:

Melittin (Melittin) sequence: GIGAVLKVLTTGLPALISWIKRKRQQ-NH2

melittin-K (K-Meli) sequence: GIGAVLKVLTTGLPALISWIKKKKQQ-NH2

2. antibacterial testing

melittin-K was tested for Minimum Inhibitory Concentration (MIC) using broth dilution.

2.1 MIC plate preparation

Dropping the sample solution with the concentration of 256mg/mL into a first row of holes of a 96-hole polystyrene plate, wherein each sample is 100ul, repeating for 3 times, adding 50ul of LB liquid culture medium into each hole of the next row, sucking 50ul of the sample solution from the first row by using a row gun, fully blowing and mixing with a second row, sucking 50ul, adding into and blowing and mixing with a third plate, and carrying out twice dilution in sequence till the last row discards the mixed 50ul solution, so that 8 samples with concentration gradients of 256 ug, 128 ug, 64 ul, 32 ul, 16, 8, 4ug and 2ug/mL are obtained, and additionally arranging 1 row of blank groups only adding 100ul of LB culture medium and a row of blank groups with the bacteria concentration of 5 × 10 ⁵ CFU/mL and without samples.

2.2 inoculum formulation

Gram-positive staphylococcus aureus SA29213 strain, methicillin-resistant staphylococcus aureus USA300 strain, gram-negative acinetobacter baumannii AbBAA747 standard strain and clinically-isolated full-drug-resistant strain Ab1814516 of acinetobacter baumannii (present in Anzhen hospital laboratory of Beijing) are used for the experiment. Bacteria are cultured overnight to prepare bacterial suspension with 0.5 McLeod turbidity standard, 50ul is added into each hole of the sample experimental group after dilution is carried out at a ratio of 1:100, and the sample concentrations of 1-8 holes are respectively 128, 64, 32, 16, 8, 4, 2 and 1 ug/ml.

2.3 Observation of statistical Experimental data

And (5) performing incubator culture for 24h, measuring the absorbance value of each hole in the 96-well plate by using an enzyme-labeling instrument OD600, and drawing a graph.

Bacterial growth rate (OD experimental-OD blank)/(OD control-OD blank)

3. Results of the experiment

The data shown in the figure show that the two melittins have good antibacterial and bactericidal effects on gram-positive bacteria and gram-negative bacteria, when the melittin concentration is 32ug/ml, the killing effect on the gram-positive bacteria is close to 100%, and when the melittin concentration is 8ug/ml, the killing effect on the gram-negative bacteria is close to 100%. In the aspect of antibacterial capacity, the two antibacterial peptides are not changed greatly, and the antibacterial capacity of the melittin-K to the Bowman immortal drug-resistant strain is slightly better than that of the wild type melittin at low concentration.

EXAMPLE 2 erythrocyte hemolysis assay

1. Experimental reagent:

TBS buffer 605mg Tris (final concentration 10mM TRIS), 4.4g NaCl (150mM NaCl), 500ml ddH ₂ O, pH 7.2;

Erythrocyte lysate: 0.2% Triton X-100 diluted in TBS buffer.

2. The experimental steps are as follows:

1) Blood samples were taken from normal human blood, centrifuged at 1000g for 5min, the supernatant removed, equal amount of TBS buffer added, gently mixed, centrifuged again at 1000g for 5min, repeated 3-5 times until the supernatant cleared.

2) the resulting pellet was collected, diluted 1:50 in TBS buffer, and mixed by gently inverting back and forth to obtain a suspension of red blood cells.

3) Loading a 96-well plate: using a gun-arranging sample-adding dilution method to enable each sample solution of the experimental group to form 8 concentration gradients of 128, 64, 32, 16, 8, 4, 2 and 1ug/ml, wherein each well has 100ul, and then adding 100ul of erythrocyte suspension; the blank group was not added with the polypeptide sample, 100ul TBS buffer +100ul erythrocyte suspension; the control group was added with 100u l erythrocyte lysate and 100ul erythrocyte suspension. The mixture was shaken gently and mixed, and cultured at 37 ℃ for 1 hour.

4) The 96-well plate was prepared by flat centrifugation at 3700rpm for 5 min.

5) And (3) taking a new 96-well plate, sucking supernatant of the 96-well plate after 70ul centrifugation, sequentially adding the supernatant into the new plate, measuring the OD600 content by using an enzyme labeling instrument, calculating the hemolytic capacity of each sample, and drawing a chart.

Cell survival rate ═ (OD experimental group-OD blank)/(OD control group-OD blank)

3. Results of the experiment

As can be seen from the data in FIGS. 5 and 6, melittin-k is less hemolytic than wild type melittin and has a half lower hemolysis rate than wild type at 4 ug/ml. In general, melittin-K has the potential to be a novel broad-spectrum antibacterial drug.

Example 3 toxicity test of melittin-K to tumor cells

1. Experimental reagent:

MTS cytotoxic staining reagent, purchased from Promega corporation. DMEM high-glucose cell culture medium, 10% fetal bovine serum and 90% DMEM, purchased from the cell resource center of Chinese academy of medicine science.

Tumor cells: SW620 human colon cancer cells.

2. the experimental steps are as follows:

1) Recovering SW620 cells, passing for more than 2 generations, after the cells grow stably, taking a dish of well-grown cells for digestion, counting a flat plate, adjusting the concentration of cell suspension according to a statistical value, adding a 96-well plate for plating, adding only a culture medium and not adding the cells in a row as a blank group, treating the cells in each other hole at about 20000 at 37 ℃ and 5% carbon dioxide in an incubator overnight, and finishing the adherence of the cells after 12 hours.

2) After the cells adhere to the wall, old culture medium in a 96-well plate is carefully sucked out, new culture medium containing specific polypeptide concentration is added according to experiment requirements, and the mixed solution is prepared in an EP tube before being added with the sample. The control group contained no polypeptide and the experimental group had concentration gradients of 128, 64, 32, 16, 8, 4, 2, 1ug/ml, with two replicates per group. Incubate in incubator for 24 h.

3) And (3) selecting a wavelength of 490nm, measuring the light absorption value of each hole on an enzyme-linked immunosorbent assay detector, recording the result, and drawing a chart.

Cell activity ═ (OD experimental group-OD blank)/(OD control group-OD blank)

3. Results of the experiment

As can be seen from the figure, the killing effect of the two melittins on the tumor cells is obvious, when the concentration of the wild-type melittin is 16ug/ml, the death rate of the tumor cells SW620 is less than 60%, when the concentration of the melittin-K is 16ug/ml, the death rate of the tumor cells SW620 exceeds 80%, and when the concentration of the melittin-K is 32ug/ml, the death rate of the tumor cells SW620 is 100%.

sequence listing

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Claims (10)

1. A novel melittin, the melittin sequence is a variant of the novel melittin with the same function as the above melittin by substituting, deleting and adding one or more amino acids on the amino acid in SEQ NO ID.1, and the sequence has homology of 75%, 80%, 85%, 90%, 95% or more than 100% with SEQ NO ID.2.

2. The novel melittin according to claim 1, wherein the N-terminus of the novel melittin is modified by acetylation and/or the C-terminus is modified by amidation.

3. A nucleotide fragment encoding the novel melittin of claim 1.

4. An expression vector, recombinant microorganism or transgenic cell line comprising the nucleotide fragment of claim 3, preferably the vector is an E.coli expression vector, a Chlorella expression vector, a baculovirus-mediated insect expression vector or a yeast expression vector.

5. a composition comprising melittin or a pharmaceutically acceptable salt thereof according to any one of claims 1-2 and a pharmaceutically acceptable carrier.

6. The melittin of any one of claims 1-2, the nucleotide fragment of claim 3, the expression vector of claim 4, the recombinant microorganism or transgenic cell, the composition of claim 5 for use in the preparation of a product or medicament with bacteriostatic and/or bactericidal properties.

7. Use according to claim 6, wherein the bacteria are gram-positive bacteria, gram-negative bacteria, fungi, viruses or parasites.

8. the use of claim 6, wherein the bacteria are clinically isolated full-drug-resistant bacteria of Staphylococcus aureus, Escherichia coli, Vibrio parahaemolyticus, Vibrio enteritis, Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, Acinetobacter baumannii, or Acinetobacter baumannii.

9. The melittin according to any one of claims 1-2, the nucleotide fragment according to claim 3, the expression vector according to claim 4, the recombinant microorganism or the transgenic cell, the composition according to claim 5 for use in the preparation of an anti-tumor medicament.

10. The use according to any one of claims 6 to 9, wherein the medicament further comprises pharmaceutical excipients including fillers, binders, disintegrants, lubricants, absorption enhancers, flavoring agents, sweeteners, diluents, excipients, wetting agents, solvents, solubilizers and coloring agents.