CN110551191B

CN110551191B - Melittin with low hemolytic activity and application thereof

Info

Publication number: CN110551191B
Application number: CN201910854177.0A
Authority: CN
Inventors: 王晨轩; 邓准; 于兰兰; 张文博; 祖瑞涓; 郑明睿
Original assignee: Institute of Basic Medical Sciences of CAMS
Current assignee: Institute of Basic Medical Sciences of CAMS
Priority date: 2019-09-10
Filing date: 2019-09-10
Publication date: 2020-04-28
Anticipated expiration: 2039-09-10
Also published as: CN110551191A

Abstract

The invention relates to 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 melittin by sequence improvement according to the sequence of the wild type melittin, and simultaneously obtains low hemolytic energy on the basis of keeping the antibacterial function of the wild type melittin. The melittin and the variant thereof provided by the invention have good clinical application prospects.

Description

Melittin with low hemolytic activity and application thereof

Technical Field

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

Background

The MRSA is also a common pathogenic bacterium of hospital infection, the infection of MRSA is mostly seen in trauma and operation in 2016, the average detection rate of MRSA in hospitals in major areas in China is 38.4%, the detection rate of MRSA in some hospitals is as high as 53.5% -75.3%, the recovery process of patients is seriously influenced, and the requirement is brought to 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 a good 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 melittin by repeated experiment sequence improvement according to a wild type melittin sequence, and simultaneously obtain low hemolytic ability on the basis of keeping the antibacterial function of the wild type melittin.

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

Disclosure of Invention

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

The invention aims to provide melittin, the sequence of which is a sequence with homology of more than 75 percent with SEQ ID.1, and the melittin is characterized in that one or more amino acids are substituted, deleted and added on the amino acid in SEQ ID.1 to form a melittin variant with the same function as the melittin.

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

At present, melittin has various modification forms, such as connecting fatty acid, fluorinating amino acid, acylating, replacing L-type amino acid with D-type 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 melittin is modified by acetylation and/or the C-terminal is modified by amidation.

A nucleotide fragment encoding the melittin.

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

A recombinant vector expressing the 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 melittin or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

Such pharmaceutically acceptable carriers include 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, mixtures of partial glycerides of saturated vegetable fatty acids, water, salts, electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, silica gel, magnesium silicate, polyvinyl pyrrolidone, cellulosic substances, polyvinyl alcohol, sodium carboxymethylcellulose, polyacrylates, ethylene-polyoxyethylene-block polymers and lanolin, cyclodextrins such as α -, β -, gamma-cyclodextrin or chemically modified derivatives thereof such as hydroxyalkyl cyclodextrins such as 2-and 3-hydroxypropyl- β -cyclodextrin and soluble derivatives thereof.

The melittin, melittin variant, nucleotide fragment, composition, recombinant vector or recombinant cell can be used for preparing 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 a gram-negative short bacillus, is generally not pathogenic, is a normally inhabitant bacterium in human and animal intestines, 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 major source of nosocomial infections, especially in intensive care units, and the disease causes the Acinetobacter baumannii to generate drug resistance due to the abuse of antibiotics, so that the disease becomes 'multi-drug resistant Acinetobacter baumannii' (full-drug resistant bacteria isolated in the immobilized clinic of the Acinetobacter baumannii).

The melittin, melittin variant, nucleotide fragment, composition, recombinant vector or recombinant cell can be used for preparing 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 sodium croscarmellose, low substituted hydroxypropylcellulose and cross-linked PVP), lubricants (such as magnesium stearate), absorption enhancers, flavoring agents, sweeteners, diluents, excipients, wetting agents, solvents, solubilizers and colorants, etc.

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, implantation accumulation 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

The concentration is 256mg/ml of the sample solution was spotted into the first row of wells of a 96-well polystyrene plate, 100ul per well, and each sample was repeated 3 times. Adding 50ul LB liquid culture medium into each hole of the back row, sucking 50ul sample solution from the first row by using a row gun, fully blowing and mixing with the second row, sucking 50ul, adding into the third row, blowing and mixing, and diluting twice in sequence until the mixed 50ul solution in the last row is discarded to obtain samples with 8 concentration gradients of 256, 128, 64, 32, 16, 8, 4 and 2 ug/ml. 1 column of blank group containing 100ul LB medium, and one column of bacteria concentration of 5X 10⁵CFU/mL without sample control.

2.2 inoculum formulation

Gram-positive staphylococcus aureus SA29213 strain, methicillin-resistant staphylococcus aureus USA300 strain, gram-negative acinetobacter baumannii AbBAA747 standard strain and acinetobacter baumannii clinical separation full-drug-resistant strain Ab1814516 (present in Anzhen hospital clinical laboratory of Beijing city) 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 with 1:100, and the sample concentration of 1-8 holes is 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

As can be seen from the data shown in FIGS. 1-4, both melittin and melittin have good antibacterial and bactericidal effects on gram-positive bacteria and gram-negative bacteria, and when the melittin concentration is 32ug/ml, the killing effect on gram-positive bacteria is close to 100%, and when the melittin concentration is 8ug/ml, the killing effect on 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), 500mlddH₂O，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 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 plates, 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 other holes about 20000 at 37 ℃ overnight in a 5% carbon dioxide incubator, and completing 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 FIG. 7, the killing effect of both melittin on tumor cells is evident, the death rate of the tumor cells SW620 is less than 60% when the concentration of the wild type melittin is 16ug/ml, the death rate of the tumor cells SW620 exceeds 80% when the concentration of melittin-K is 16ug/ml, and the death rate of the tumor cells SW620 is 100% when the concentration of melittin-K is 32 ug/ml.

Sequence listing

<110> institute of basic medicine of Chinese academy of medical sciences

<120> an improved melittin with low hemolytic activity and its application

<160>2

<170>SIPOSequenceListing 1.0

<210>1

<211>26

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>1

Gly Ile Gly Ala Val Leu Lys Val Leu Thr Thr Gly Leu Pro Ala Leu

1 5 10 15

Ile Ser Trp Ile Lys Arg Lys Arg Gln Gln

20 25

<210>2

<211>26

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>2

Gly Ile Gly Ala Val Leu Lys Val Leu Thr Thr Gly Leu Pro Ala Leu

1 5 10 15

Ile Ser Trp Ile Lys Lys Lys Lys Gln Gln

20 25

Claims

1. Melittin is characterized in that the amino acid sequence of melittin is shown in SEQ NO. 2.

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

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

4. An expression vector, recombinant microorganism or transgenic cell line comprising the nucleotide fragment of claim 3.

5. An expression vector comprising the nucleotide fragment of claim 3, wherein the vector is an E.coli expression vector, a Chlorella expression vector, a baculovirus-mediated insect expression vector, or a yeast expression vector.

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

7. Use of melittin according to any of claims 1-2, the nucleotide fragment according to claim 3, the expression vector according to claim 4, a recombinant microorganism or a transgenic cell, the expression vector according to claim 5, the composition according to claim 6 for the preparation of a product with bacteriostatic and/or bactericidal properties.

8. Use of the melittin according to any of claims 1-2, the nucleotide fragment according to claim 3, the expression vector according to claim 4, a recombinant microorganism or a transgenic cell, the expression vector according to claim 5, the composition according to claim 6 for the preparation of a medicament with bacteriostatic and/or bactericidal properties.

9. Use according to any one of claims 7 or 8, wherein the bacteria are gram-positive or gram-negative bacteria.

10. The use according to any one of claims 7 or 8, wherein the bacterium is Staphylococcus aureus, Escherichia coli, Vibrio parahaemolyticus, Vibrio enteronitis, Pseudomonas aeruginosa, or Acinetobacter baumannii.

11. The use of any one of claims 7 or 8, wherein the bacteria are methicillin-resistant staphylococcus aureus or acinetobacter baumannii clinically isolated full-drug-resistant bacteria.

12. 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 expression vector according to claim 5, the composition according to claim 6 for use in the preparation of an anti-tumor medicament, wherein the tumor is colon cancer.

13. The use according to any one of claims 8 to 12, wherein the medicament further comprises pharmaceutical excipients comprising fillers, binders, disintegrants, lubricants, absorption enhancers, flavoring agents, sweeteners, wetting agents, solubilizers and colorants.