KR20170054587A - Convergence antibacterial peptide P9P12 and process to synthesize it - Google Patents

Abstract

The present invention relates to antibacterial fusion peptide P9P12 and a synthesizing method thereof. The antibacterial fusion peptide P9P12 is a new substance expressed with sequence number 3, and is synthesized by combining a 3-11 amino acid region of protein transduction domain (PTD) in TAT protein derived from human immunodeficiency virus (HIV), to a front N terminal of a 1-12 amino acid region in the N terminal of papiliocin.

Description

The fusion antimicrobial peptide P9P12 and the method for synthesizing the same are also known as " Convergence antibacterial peptide P9P12 and process to synthesize it &

The present invention relates to a fusion antimicrobial peptide P9P12 and a method for synthesizing the fusion antimicrobial peptide. More particularly, the present invention relates to a fusion antimicrobial peptide P9P12, And a protein transduction domain (PDT) 3-11 amino acid region of the TAT protein derived from the TAT protein is ligated to the P9P12 fusion protein, and a method for synthesizing the fusion antimicrobial peptide.

Since penicillin, a number of antibiotics have been developed and used to combat bacterial infiltration into the body. Recently, however, strains resistant to these antibiotics have emerged and are regarded as a big problem.

Insects possess a bio-defense system that can effectively combat the invasion of external microorganisms and viruses in order to survive in various environments. Immune system protects itself by secreting innate immune factors including cellular immunity and humoral immune response System. Congenital immunity plays an important role as an effective defense against early infection in invertebrates, including insects. In the innate immune response of insects, cellular immunity includes phagocytosis, nodule formation and encapsulation of protozoa including bacteria, fungi, and nematodes. In the humoral immune response, a variety of proteins or peptides are synthesized in lipids and hemocytes and secreted into the blood or lymph, due to the invasion of pathogens, which are used as powerful defense factors for pathogens. Antibacterial proteins or peptides secreted as part of the humoral immune response of insects are highly likely to be used as natural antibiotics for the control of diseases such as livestock including humans. These antimicrobial proteins or peptides can also be useful not only for the treatment of diseases of humans and livestock but also for the prevention of crop damage caused by infections of various pathogens.

Antimicrobial proteins and peptides of insects that have been discovered to date have been isolated from insects since the first report of cecropin in the Secropia moths. They have been isolated from insects. They are largely classified according to structure and size, And peptides containing a large amount of glycine amino acid. The most common of these are antimicrobial peptides such as melittin or scheropin, which have no cysteine residues and have an amphipathic alpha-helical structure.

The present inventors have conducted studies on Papiliocin (Korean Patent No. 10-1099558) and novel antimicrobial peptides designed therefrom (Korean Patent No. 10-1465098). The papyriosin is an antimicrobial peptide isolated from a larva of a tiger, and the total size of the cDNA of the antimicrobial peptide gene is 503 bp, consisting of 62 amino acids having an encoding region initiated at the 94th base and ending at the 283rd position . The amino acid structure of the antimicrobial peptide fapyllioxin is a typical cyclopentane basic protein composed of alpha-helix-hinge-alpha-helix. On the other hand, the inventors of the present invention have repeatedly conducted researches on the above, and have completed the technology relating to the novel papilliferose-derived antimicrobial peptide of the present invention having excellent bactericidal permeability.

Korean Patent No. 10-1099558 (registered on December 21, 2011, entitled: Antimicrobial Peptide Genes and Antimicrobial Peptides Isolated from the Larva of a Tiger) Korean Patent No. 10-1150281 (filed on May 08, 2012, entitled: Novel Antibacterial Antitumor Activity Peptide Designed from Phypholysin Hyasin Peptides and Uses Thereof) Korean Patent No. 10-1465098 (Registered Date: November 19, 2014, name: PAJE of fusion antimicrobial peptide and method for synthesizing the same)

It is an object of the present invention to provide a fusion antimicrobial peptide P9P12 and a method of synthesizing the same. More specifically, the protein transduction domain (PDT) of TAT protein derived from human immunodeficiency virus (HIV) is added to the N terminus of the N-terminal region 1-12 amino acid region of papillocin, ) 4-12 amino acid region, and to provide a method for synthesizing the fusion antimicrobial peptide P9P12.

The present invention relates to the fusion antimicrobial peptide P9P12 represented by SEQ ID NO: 3.

The antimicrobial peptide P9P12 is characterized by having an antibacterial or bactericidal activity against at least one pathogenic bacterium selected from the group consisting of Gram-negative bacteria, Gram-positive bacteria and Candida-producing fungi.

The present invention also provides a composition for antibacterial or sterilizing comprising the fusion antimicrobial peptide P9P12 as an active ingredient.

In another embodiment, the present invention provides a composition for preventing or treating a bacterial disease comprising the fusion antimicrobial peptide P9P12 as an active ingredient. The bacterial disease may be a disease selected from the group consisting of bacterial enteritis, bacterial food poisoning, Salmonella disease and candidiasis.

The present invention can also provide a composition for feed comprising the fusion antimicrobial peptide P9P12 as an active ingredient.

In the present invention, introduction of a protein of the TAT protein derived from human immunodeficiency virus (HIV) represented by SEQ ID NO: 2 is introduced at the N terminus of the N-terminal region 1-12 amino acid region of Papiliocin represented by SEQ ID NO: Domain (PDT) 3-11 amino acid region, thereby providing a method for synthesizing the fusion antimicrobial peptide P9P12 represented by SEQ ID NO: 3.

Hereinafter, the present invention will be described in detail.

The fusion antimicrobial peptide P9P12 represented by SEQ ID NO: 3 of the present invention comprises the N-terminal region 1-12 amino acid region of the N-terminal region of Papiliocin represented by SEQ ID NO: 1 (RWKIFKKIEKVG) (RKKRRQRRRRWKIFKKIEKVG) in which the 3-11 amino acid region of the protein-introduced domain (PDT) 3-11 of the TAT protein derived from human immunodeficiency virus (HIV) represented by SEQ ID NO: 2 is conjugated, and the fusion antimicrobial peptide P9P12 And a molecular weight of 2851.8 Da.

 On the other hand, the core amino acid sequence of the whole amano acid sequence of papyllioxin and the protein introduction domain of HIV TAT protein is as follows.

Papillosin (SEQ ID NO: 4): RWKIFKKIEKVGRNVRDGIIKAGPAVAVVGQAATVVKG

Protein-introduced domain of TAT protein of HIV (SEQ ID NO: 5): YGRKKRRQRRR

The antimicrobial peptide P9P12 of the present invention is characterized by having an antibacterial or bactericidal activity against at least one pathogenic bacterium selected from the group consisting of gram-negative bacteria, gram-positive bacteria, and candida-producing fungi.

The Gram-negative bacteria are selected from the group consisting of Escherichia coli , Salmonella pullorum , Salmonella typhimurium and Salmonella gallinarum . The Gram positive bacteria are selected from Staphylococcus aureus and Bacillus subtilis . In addition, the fungus causing the candida can be Candida albicans .

Thus, the antimicrobial peptide P9P12 can be used as an antimicrobial peptide such as Escherichia coli , Salmonella pullorum , Salmonella typhimurium , Salmonella gallinarum , Staphylococcus aureus , Bacillus subtilis , And Candida albicans . The present invention also provides an antimicrobial or bactericidal activity against at least one pathogenic microorganism selected from the group consisting of Candida albicans and Candida albicans .

The antimicrobial peptide P9P12 of the present invention exhibits very high antibacterial activity and excellent sterilization rate in both Gram-negative and Gram-positive bacteria and Candida fungi. Thus, the antimicrobial peptide P9P12 of the present invention can be effectively used as a composition for antibacterial or sterilizing, a composition for preventing or treating a bacterial disease occurring in a human body or a plant, a composition for a feed, and the like. The antimicrobial or sterilizing composition, composition for preventing or treating a bacterial disease occurring in human or plant can be preferably used as a pharmaceutical composition.

In this case, the pharmaceutical composition may further contain a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is usually used in the present invention, and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch , Calcium carbonate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, But are not limited to, magnesium, magnesium, and mineral oil. Further, in addition to the above components, a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like may be further included. The pharmaceutical composition may be administered parenterally, and subcutaneous injection or topical administration through the skin (transdermal administration) is preferable, and a proper dosage is appropriately determined depending on the formulation method, the administration method, the age, weight, sex, , Time of administration, route of administration, rate of excretion, and responsiveness. Such a pharmaceutical composition may be prepared in a unit dosage form by formulating it with a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by those having ordinary skill in the art to which the present invention belongs. Into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

In addition, the fusion antimicrobial peptide P9P12 of the present invention can be used as a feed composition for animals such as mammals, birds, reptiles, amphibians, fishes and the like, preferably mammals. Such a feed composition is appropriately mixed with a feed material and provided as a feed. Examples of the feed material include cereals, crude steel, vegetable oils, animal feed materials, other feed materials, and refined products. When a feed composition containing the antimicrobial peptide P9P12 of the present invention is routinely ingested into an animal, it is possible to prevent infection against a pathogenic microorganism.

It is expected that the fusion antimicrobial peptide P9P12 having excellent antimicrobial activity of the present invention and the method of synthesizing the same will be able to prevent various bacterial diseases, provide a natural antibiotic for promoting animal growth, and develop a feed additive having an antibacterial function. Korean Patent No. 10-1099558 discloses a technique relating to an antimicrobial peptide Papiliocin, and Korean Patent No. 10-1150281 and Korean Patent No. 10-1465098 disclose a fusion protein derived from papilliferin Although the technology relating to the antimicrobial peptide has been disclosed, these prior art peptides are not only different in amino acid sequence from the antimicrobial peptide P9P12 of the present invention, but also have antimicrobial effects, and the antimicrobial peptide P9P12 of the present invention is superior to the peptides of the prior art Do. The difference in these effects is derived from the protein-introduced domain (PDT) 3-11 amino acid region (RKKRRQRRR) of the TAT protein derived from human immunodeficiency virus (HIV) constituting the antimicrobial peptide P9P12 of the present invention, Lt; RTI ID = 0.0 > rapidly < / RTI > permeates into bacteria without cell membrane loss. Such rapid penetration has been reported to be particularly effective for the antimicrobial activity of fungi.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a diagram showing the design and amino acid composition of the fusion antimicrobial peptide P9P12 of the present invention. Fig.
2 is a view showing the purification result of the fusion antimicrobial peptide P9P12 of the present invention by reverse phase HPLC.
3 is a diagram showing the results of molecular weight measurement of the fusion antimicrobial peptide P9P12 of the present invention using MALDI mass spectrometry.
4 is a graph showing the results of assaying the antibacterial activity of the fusion antimicrobial peptide P9P12 of the present invention against E. coli.
FIG. 5 is a graph showing the results of assaying the bactericidal activity against E. coli according to the treatment time of the fusion antimicrobial peptide P9P12 (32 μg / ml) of the present invention (control: antimicrobial peptide-free group).
6 is a graph showing the results of assaying the sterility of Staphylococcus aureus according to the treatment time of the fusion antimicrobial peptide P9P12 (32 / / ml) of the present invention (control: antimicrobial peptide-untreated group).
7 is Escherichia coli (E.coli) and Staphylococcus aureus (S.aureus) treated by the antimicrobial peptide P9P12 fusion of the present invention to the culture and the concentration after one hour from the time, LB and plated on solid medium surviving E. coli and Staphylococcus aureus (Control: anti-bacterial peptide-untreated group).
Fig. 8 is a graph showing the catalase activity of the Escherichia coli cell membrane according to the treatment with P9P12 (control: untreated group).

Hereinafter, the present invention will be described in detail with reference to examples, but the scope of the present invention is not limited thereto.

≪ Example 1: Synthesis and separation and purification of peptide &

(PDT) 3- (3-amino-5-methylpiperidin-4-yl) amino acid derived from the human immunodeficiency virus (HIV) at the N terminus of the previously developed N-terminal region 1-12 amino acid region (RWKIFKKIEKVG) of Papiliocin A novel form of the antimicrobial peptide P9P12 (RKKRRQRRRWKIFKKIEKVG), which has 21 amino acid residues and penetrates without bacterial membrane loss and exhibits antimicrobial activity, was designed by joining 11 amino acid regions (RKKRRQRRR) (Fig.

The designed fusion antimicrobial peptide P9P12 was synthesized by the Merrifield liquid phase solid phase method using an automated solid phase peptide synthesizer (Pioneer Applied Biosystems, Foster, Caclif.). The prepared peptides were purified using a reverse phase-HPLC column in an acetonitrile gradient containing 0.05% TFA, and their purity was confirmed. As a result, it was confirmed that the peptides had a purity of 95% or more 2).

 As a result of MALDI mass spectrometry, the molecular weight of P9P12 was measured at 2851.8 as shown in FIG. 3, which was confirmed to be identical with the molecular weight calculated by the amino acid sequence. Thus, it was confirmed that the precise fusion antimicrobial peptide P9P12 was synthesized.

≪ Example 2: Confirmation of antimicrobial activity of fusion antimicrobial peptide P9P12 >

Example 2-1. Cultivation of various strains for antibacterial experiments

Gram negative bacteria ( Escherichia coli, Salmonella pullorum, Salmonella typhimurium, Salmonella gallinarum ) and Gram positive bacteria ( Staphylococcus aureus, Bacillus subtilis ) used in antibacterial assays were cultured in a 3% (w / v) TSB ) Liquid culture at 37 DEG C and 200 rpm for 18 hours, and then cultured for 2 hours and 30 minutes at a concentration of 2 x 10 < ⁶ > CFU / mL under the same conditions.

Candida albicans , a fungus, was cultured in YPD medium (1% yeast extract, 2% peptone, 2% dextrose) at 30 ° C and 200 rpm for 24 hours. Followed by secondary culture in the same manner as described above.

Example 2-2. Antimicrobial activity measurement

Escherichia coli , a gram-negative bacterium, was inoculated into LB liquid medium (Difco LB Broth: 10 g of typtone, 5 g of yeast extract, 1 liter of NaCl 10 g / disilled water), and the fusion antimicrobial peptide P9P12 (Final concentration: 1, 2, 4, 8, 16, 32 and 64 / / ml) and then incubated for 18 hours with shaking culture. As a result, it was confirmed that Escherichia coli did not grow when the concentration of P9P12 was 16 占 퐂 / ml or more as shown in Fig.

Next, the antimicrobial activity of the P9P12 fusion antimicrobial peptide was measured for various pathogens. The antimicrobial activity was tested by a minimum inhibitory concentration (MIC) assay and a minimal bactericide concentration (MBC) assay. The results are shown in Table 1 below.

In order to measure the minimum growth inhibitory concentration (MIC), 90 μl of bacterial culture (2 × 10 ⁶ CFU / ml) was added to a microplate well and 10 μl of the fusion antimicrobial peptide P9P12 was added per concentration. After incubation for 18 hours, the absorbance was measured at an optical density (OD) of 600 nm using a spectrophotometer, and the MIC ₅₀ value was measured in comparison with the untreated peptide.

The minimum bactericidal concentration (MBC) was determined by adding the fusion antimicrobial peptide to the bacterial culture solution for 18 hours in the same manner as in the measurement of the minimum growth inhibitory concentration, and then the culture was added to LB solid medium (Difco LB agar: 15 g agar / The cells were cultured in a 37 ° C incubator for 18 hours, and the number of surviving colonies was counted to determine the concentration at which no colonies were formed.

Microorganisms Minimum growth inhibitory concentration
(MIC ₅₀ , 占 퐂 / ml) Minimum sterilization concentration
(MBC, 占 퐂 / ml) Gram-negative bacteria
(Gram negative bacteria) Escherichia coli 13 32 Salmonella pullorum 48 64 Salmonella typhimurium 24 32 Salmonella gallinarum 20 32 Gram-positive bacteria
(Gram positive bacteria) Staphylococcus aureus 21 32 Bacillus subtilis 11 16 Yeast fungi
Candida albicans
2
4

As shown in Table 1, the minimum inhibitory concentration (MIC ₅₀ ) of P9P12 was measured against Escherichia coli and three Salmonella strains which were gram-negative intestinal bacteria, and as a result, it was found to be 13 μg / ml in Escherichia coli in Salmonella pool room (Salmonella pullorum), Salmonella typhimurium (Salmonella typhimurium), Salmonella Galina room (Salmonella gallinarum) in 48, 24, 20㎍ / ㎖, Gram-positive bacterium Staphylococcus aureus (Staphylococcus aureus), respectively, and The minimum inhibitory concentration (MIC ₅₀ ) of Bacillus subtilis was 21 and 11 μg / ml, respectively, indicating that the antimicrobial activity of the P9P12 fusion antimicrobial peptide is excellent. For Candida albicans , the fungus that causes candidiasis, The minimum inhibitory concentration (MIC ₅₀ ) was 2 / / ml, indicating that the antifungal effect was also excellent .

The results showed that the concentration of fusion antimicrobial peptide P9P12 was 16 ~ 64 ㎍ / ㎖ in pathogens such as Escherichia coli, Salmonella and Staphylococcus, and 4 ㎍ / ㎖ in Candida albicans ( Candida albicans ) Lt; RTI ID = 0.0 > ml < / RTI >

<Example 3> Measurement of sterilizing power according to treatment time and concentration of the fusion antimicrobial peptide P9P12>

The fungicidal activity of the fused antimicrobial peptides on E. coli and Staphylococci was determined by time-kill curve assay according to the sterilization rate and the peptide concentration. Specifically, Escherichia coli and Staphylococcus aureus were mixed with 3% (w / v) TSB (Tryptic Soy Broth) (17 g of pancreatic digest of casein, 3 g of papaic digest of soybean, 2.5 g of dextrose , NaCl 5 g, dipotassium phosphate 2.5 g / distilled water 1 L) at 37 ° C and 200 rpm for 18 hours, and then cultured under the same conditions to a final concentration of 1 × 10 ⁶ CFU / ml, 90 [micro] l of bacterial culture was added to a microplate well, and 10 [mu] l of the fusion antimicrobial peptide P9P12 was added to each concentration. Thereafter, the culture solution was diluted for a predetermined time, and then plated on a LB solid medium. After culturing for 18-24 hours at 37 ° C., the number of viable colonies was determined, and the number of colony forming Unit) was measured. As a result, it can be confirmed that the sterilization rate is very fast at 32 μg / ml, which is the minimum bactericidal concentration (MBC) of P9P12, in both Gram-negative bacteria and Gram-positive bacteria (FIG. 5 - E. coli test results, FIG.

Further, in addition to the bacterial culture solution ^{(1 × 10 6 CFU / ㎖} ) the antimicrobial peptide P9P12 order to determine the fungicidal activity according to the concentration of E. coli, the antimicrobial peptide P9P12 diluted to various concentrations (Escherichia coli) and Staphylococcus aureus (Staphylococcus aureus) After 1 hour, the number of viable bacteria was determined by streaking on LB solid medium in the same manner as described above. As a result, E. coli, as shown in Figure 7 can be confirmed:: (S.aureus Staphylococcus aureus) is very high sterilization effect in 32㎍ / ㎖ concentration of at least about that (Escherichia coli E.coli), and Staphylococcus aureus. The antimicrobial peptide of the present invention inhibits the growth of various bacteria without rapidly destroying the cell membrane. Therefore, the antimicrobial peptide of the present invention can inhibit the growth of various bacteria by using the antimicrobial peptide of the present invention, Derived peptides are more excellent in sterilization speed than the peptides derived from papillian origin.

<Example 4: Catalase activity assay of cell membrane by P9P12 treatment>

The antimicrobial activity mechanism of P9P12 was confirmed by the activity assay of catalase secreted during bacterial cell membrane damage.

Escherichia coli was cultivated in a 3% (w / v) TSB (Tryptic Soy Broth) liquid medium at 37 ° C and 200 rpm for 18 hours with shaking, and then the final concentration was adjusted to 1 × 10 ⁶ CFU / Ml. &Lt; / RTI &gt; P9P12, ampicillin, and kanamycin were added to 2 ml of Escherichia coli culture, and the cells were further cultured for 2 hours at 37 DEG C and 180 rpm. Then, the cells were centrifuged at 5000 rpm for 10 minutes and the supernatant was collected. Catalase activity was measured using a Catalase Measurement Kit (CAT-100-1KT) from Sigma-Aldrich.

Catalase activity of P9P12, Ampicillin, Kanamycin, and Sonication in Escherichia coli was found to be higher than that of the existing antibiotics, ampicillin and kanamycin, as compared with the ultrasound treatment of P9P12 antimicrobial peptide, And similarly exhibited very low catalase activity (Figure 8). Therefore, it was confirmed that the P9P12 antimicrobial peptide infiltrated into the cytoplasm without the cell membrane destruction by the PTD located at the N-terminal region of the peptide.

As described above, according to the present invention, it becomes possible to prevent bacterial diseases occurring in humans and animals and to develop natural antibiotics for promoting animal growth, thereby making it possible to produce antibacterial compositions such as antibacterial agents and antifungal agents, P9P12 can be usefully used to prevent bacterial diseases such as bacterial enteritis, bacterial food poisoning, salmonellosis and candidiasis, and it is possible to produce a feed additive containing P9P12.

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Convergence antibacterial peptide P9P12 and process to          synthesize it <130> P2015-0214 <160> 5 <170> Kopatentin 2.0 <210> 1 <211> 12 <212> PRT <213> Unknown <220> <223> 1-12 amino acids of Papiliocin, Papilio xuthus <400> 1 Arg Trp Lys Ile Phe Lys Lys Ile Glu Lys Val Gly   1 5 10 <210> 2 <211> 9 <212> PRT <213> Unknown <220> <223> 3-11 amino acids of TAT core domain <400> 2 Arg Lys Lys Arg Arg Gln Arg Arg Arg   1 5 <210> 3 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> fusion peptide P9P12 <400> 3 Arg Lys Lys Arg Arg Gln Arg Arg Arg Arg Trp Lys Ile Phe Lys Lys   1 5 10 15 Ile Glu Lys Val Gly              20 <210> 4 <211> 38 <212> PRT <213> Unknown <220> <223> Papiliocin, Papilio xuthus <400> 4 Arg Trp Lys Ile Phe Lys Lys Ile Glu Lys Val Gly Arg Asn Val Arg   1 5 10 15 Asp Gly Ile Ile Lys Ala Gly Ala Val Ala Val Val Gly Gln Ala              20 25 30 Ala Thr Val Val Lys Gly          35 <210> 5 <211> 11 <212> PRT <213> Unknown <220> <223> TAT core domain, human immunodeficiency virus <400> 5 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg   1 5 10

Claims (7)

The fusion antimicrobial peptide P9P12 represented by SEQ ID NO: 3. The method according to claim 1,
Wherein said antimicrobial peptide P9P12 has antimicrobial or bactericidal activity against at least one pathogenic bacterium selected from the group consisting of Gram-negative bacteria, Gram-positive bacteria and Candida-producing fungi. A composition for antibacterial or antimicrobial use, which comprises the fusion antimicrobial peptide P9P12 of claim 1 as an active ingredient. A composition for preventing or treating bacterial diseases, which comprises the fusion antimicrobial peptide P9P12 of claim 1 as an active ingredient. The method of claim 4,
Wherein the bacterial disease is a disease selected from the group consisting of bacterial enteritis, bacterial food poisoning, Salmonella disease and candidiasis. A feed composition comprising the fusion antimicrobial peptide P9P12 of claim 1 as an active ingredient. A protein introduction domain of a TAT protein derived from Human Immunodeficiency Virus (HIV) represented by SEQ ID NO: 2 (PDT (SEQ ID NO: 2)) at the N terminus of the N-terminal region 1-12 amino acid region of Papiliocin represented by SEQ ID NO: ) 3-11, wherein the peptide represented by SEQ ID NO: 3 is prepared.

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