Antimicrobial peptide Scyreprocin of <i>Scylla paramamosain </i>and method thereof

Abstract

The present disclosure discloses an antimicrobial peptide Scyreprocin of Scylla paramamosain and a method thereof, wherein an amino acid sequence of the antimicrobial peptide Scyreprocin comprises a sequence shown in SEQ ID NO 01, and the antimicrobial peptide is expressed and purified by using genetic engineering technology. The recombinant antimicrobial peptide Scyreprocin has advantages of wide antimicrobial spectrum, good antimicrobial effect, and rapid germicidal rate, shows great application significance, and has good application in preparation of antimicrobial agents. The recombinant antimicrobial peptide Scyreprocin has no cytotoxicity to mouse hepatocytes AML12, human liver cells L02, and can be safely used for medical treatment or can be used as a feed composition.

Claims

1. A method for preparing an antimicrobial composition to be used to inhibit growth of microorganisms, wherein the method comprises recombination expression and purification of an antimicrobial peptide Scyreprocin of Scylla paramamosain with a His tag, wherein an amino acid sequence of the antimicrobial peptide Scyreprocin consists of SEQ ID NO: 01, and wherein the recombination expression and purification comprises: cloning an open reading frame of the antimicrobial peptide Scyreprocin of Scylla paramamosain and constructing into a vector pET28a to obtain a recombinant vector; and transferring the recombinant vector into Escherichia coli BL21 to obtain a recombinant expression strain configured to express the antimicrobial peptide Scyreprocin of Scylla paramamosain with a protein purification solution.

2. The method according to claim 1, wherein the antimicrobial composition is a feed composition.

3. The method according to claim 1, wherein the antimicrobial composition is an anti-mildew and antiseptic composition.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1 and 2 illustrates a resulting view of an antimicrobial peptide Scyreprocin of Scylla paramamosain obtained by a recombinant expression and purification of Embodiment 1 of the present disclosure. FIG. 1 illustrates a sodium dodecyl sulphate-polyacrylamide gel (SDS-PAGE) electrophoresis showing a view of the recombinant Scyreprocin obtained by expression and purification, wherein M represents a protein marker 26616, 1 represents a total protein before expression induction, 2 represents a total protein after a 24-hour induction, 3 represents a supernatant obtained by ultrasonication, and 4 shows a purified recombinant Scyreprocin.

(2) FIG. 2 illustrates a graph of a purification process of the recombinant Scyreprocin, wherein an arrow indicates a peak of the purified recombinant Scyreprocin.

(3) FIG. 3 illustrates a resulting view of a spore germination of Aspergillus niger inhibited by the recombinant Scyreprocin.

(4) FIGS. 4A-4D illustrate a bactericidal kinetic curve of the recombinant Scyreprocin against Cryptococcus neoformans, Candida albicans, Pseudomonas schneideri, and Micrococcus lysoleikticus, respectively. An abscissa represents time (min), and an ordinate represents a percentage of survival colonies relative to original colonies.

(5) FIG. 5 illustrates a resulting view of a proliferation effect of the recombinant Scyreprocin by a method MTS ([3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]) against mouse hepatocytes (AML12) and human liver cells (L02).

DETAILED DESCRIPTION OF THE EMBODIMENTS

(6) The present disclosure will be further described below in combination with the accompanying drawings and embodiments.

(7) A microbe of the following embodiments comprises Escherichia coli, Escherichia coli BL21, Staphylococcus aureus, Vibrio fluvialis, Pseudomonas stutzeri, Micrococcus lysoleikticus, Candida albicans, Candida krusei, Candida tropicalis, Cryptococcus neoformans, Aspergillus niger, etc., which were all purchased from China General Microbiological Culture Collection Center (CGMCC, belonging to Institute of Microbiology, Chinese Academy of Sciences). Escherichia coli MC1061 was purchased from ShenZhen Gene Power Tech. Co., Ltd., and Pichia pastoris GS115 was purchased from Invitrogen.

Embodiment 1: A Preparation of an Antimicrobial Peptide Scyreprocin of Scylla paramamosain

(8) A sequence (i.e., a nucleotide sequence) of an open reading frame of an antimicrobial peptide Scyreprocin of Scylla paramamosain is as follows:

(9) TABLE-US-00001 (SEQ ID NO 02) ATGAAGGAAGACAGCAACATTCTAGACAAGACCGCCAAGATGACCAAAC AGAACAAGGCCCTGCTCTTCACTGCAGGCGGCGCCGCGGCGTTCATGGC AGGATACTACTACTATCACTGCAATTACAGAAATCCTGCACCCAAGAAA AGTGGCAGTACTACTTCACAAGACAAGACTGATGCTCAGGCGGTTCAGT CTATCCCCTCACCCAGTGGCAACAAGGGCAAAGAAAGCAAGGACCCAAA AGTAAAATAA

(10) A full-length of a cDNA sequence of a gene of the peptide Scyreprocin was obtained and verified by Rapid Amplification of cDNA ends (RACE). The open reading frame of the gene of the peptide Scyreprocin is 255 bp (comprising a termination codon TAA), and a GenBank Accession number is MH488960.

(11) According to the cDNA sequence of the gene of the peptide Scyreprocin, the gene specific primers were designed and repeatedly verified by the RACE method (Table 1).

(12) TABLE-US-00002 TABLE 1 A primer list for Scyreprocin sequence amplification Primer name Sequence 5′-3′ Scyreprocin 3′-1 GCAAAGAAAGCAAGGACCCAAAA (SEQ ID NO 03) Scyreprocin 3′-2 TGCTCAGGCGGTTCAGTCTATCC (SEQ ID NO 04) Scyreprocin 3′-3 ACAAGACCGCCAAGATGACCAAA (SEQ ID NO 05) Scyreprocin 5′-1 TCTGTTTGGTCATCTTGGCGGTCTT (SEQ ID NO 06) Scyreprocin 5′-2 CCACTGGGTGAGGGGATAGACTGAA (SEQ ID NO 07) Scyreprocin 5′-3 ACTTTTGGGTCCTTGCTTTCTTTGC (SEQ ID NO 08) Scyreprocin full-length F TTCACGCCACTCAGTACAAATCTA (SEQ ID NO 09) Scyreprocin full-length R  CAAACATAAGTAAAGCTGAAGGTA (SEQ ID NO 10) 5′RACE Outer Primer CATGGCTACATGCTGACAGCCTA (SEQ ID NO 11) 5′RACE Inner Primer CGCGGATCCACAGCCTACTGATGATCAGTCGATG (SEQ ID NO 12) 3′RACE Outer Primer TACCGTCGTTCCACTAGTGATTT (SEQ ID NO 13) 3′RACE Inner Primer CGCGGATCCTCCACTAGTGATTTCACTATAGG (SEQ ID NO 14)

(13) A noncoding sequence of the gene of the peptide Scyreprocin of Scylla paramamosain was amplified by the RACE method (i.e., a 5′UTR amplification method):

(14) The cDNA prepared by method RACE (i.e., by the applicant) was taken as a polymerase chain reaction (PCR) template, and a specific reaction system is as follows:

(15) 1) Outer PCR reaction:

(16) TABLE-US-00003 cDNA 2 μL 10 × LA PCR Buffer II (Mg.sup.2+ plus) 5 μL dNTP Mixture (2.5 mM each) 8 μL 5′ RACE Outer Primer (10 μM) 2 μL Scyreprocin 5′-3 (10 μM) 2 μL LA Taq (5 U/μL) 0.25 μL Milli-Q water 30.75 μL Total reaction volume 50 μL

(17) A reaction process is as follows:

(18) 1) Pre-denaturizing at 95° C. for 3 minutes;

(19) 2) Denaturizing at 95° C. for 30 seconds, annealing at 60° C. for 30 seconds, and elongating at 72° C. for 2 minutes as a cycle, and cycling for 20 times in total;

(20) 3) Elongating at 72° C. for 10 minutes; and

(21) 4) Stopping at 4° C. to obtain a first round PCR product.

(22) The first round PCR product was diluted for 50-100 times to obtain a diluted outer PCR reaction solution, and the diluted outer PCR reaction solution was then used as a template for nested PCR amplification. A reaction system was as follows:

(23) {circle around (2)} Inner PCR Reaction:

(24) TABLE-US-00004 Diluted outer PCR reaction solution 2 μL 10 × LA PCR Buffer II (Mg.sup.2+ plus) 5 μL dNTP Mixture (2.5 mM each) 8 μL 5′ RACE Outer Primer (10 μM) 2 μL Scyreprocin 5′-2 (10 μM) 2 μL LA Taq (5 U/μL) 0.25 μL Milli-Q water 30.75 μL Total reaction volume 50 μL

(25) The aforementioned reactants were well mixed to carry out a PCR reaction. A reaction process is as follows:

(26) 1) Pre-denaturizing at 95° C. for 5 minutes;

(27) 2) Denaturizing at 95° C. for 30 seconds, annealing at 60° C. for 30 seconds, and elongating at 72° C. for 2 minutes as a circle, and cycling for 30 times in total;

(28) 3) Elongating at 72° C. for 10 minutes; and

(29) 4) Stopping at 4° C. to obtain amplification products.

(30) A 3′UTR amplification method of the gene of the peptide Scyreprocin is similar to the 5′ UTR amplification method. The amplification products were sequenced and were then spliced to obtain the full length of the cDNA sequence of the gene of the peptide Scyreprocin. Proper primers were designed, the cDNA sequence of Scylla paramamosain was taken as a template, and a full length of the gene of the peptide Scyreprocin obtained by the splicing was verified. A PCR reaction system is as follows:

(31) TABLE-US-00005 cDNA 2 μL 10 × LA PCR Buffer II (Mg.sup.2+ plus) 5 μL dNTP Mixture (2.5 mM each) 8 μL Scyreprocin full-length F (10 μM) 2 μL Scyreprocin full-length R(10 μM) 2 μL LA Taq (5 U/μL) 0.25 μL Milli-Q water 30.75 μL Total reaction volume 50 μL

(32) The aforementioned reactants were well mixed to carry out a PCR reaction. A reaction process is as follows:

(33) 1) Pre-denaturizing at 95° C. for 5 minutes;

(34) 2) Denaturizing at 95° C. for 30 seconds, annealing at 55° C. for 30 seconds, and elongating at 72° C. for 1 minute as a cycle, and cycling for 30 times in total;

(35) 3) Elongating at 72° C. for 5 min; and

(36) 4) Stopping at 4° C.

(37) The gene of the peptide Scyreprocin is derived from Scylla paramamosain, and an amino acid sequence of the peptide Scyreprocin is as follows:

(38) TABLE-US-00006 (SEQ ID NO 01) MKEDSNILDKTAKMTKQNKALLFTAGGAAAFMAGYYYYHCNYRNPAPKKS GSTTSQDKTDAQAVQSIPSPSGNKGKESKDPKVK.

(39) A molecular formula of the peptide Scyreprocin is C.sub.396H.sub.636N.sub.106O.sub.127S.sub.4, and a molecular weight of the peptide Scyreprocin is 9107.258 Dalton. As predicted by SignalP4.1 software, there is no signal peptide in the peptide Scyreprocin, and there are 84 amino acids in total comprising 15 amino acid residues with positive charge and 7 amino acid residues with negative charge. According to an amino acid residue charge, an isoelectric point of the peptide Scyreprocin is predicted to be 9.61. An average hydrophilicity coefficient of the peptide Scyreprocin is −0.968, a water solubility of the peptide Scyreprocin is relatively high. When the pH is 7.0, a net charge of the peptide Scyreprocin is +7, indicating it a cationic peptide.

(40) A recombinant product of Scyreprocin with a purity of more than 85% was obtained by a gene engineering expression technology. In this embodiment, the antimicrobial peptide Scyreprocin of Scylla paramamosain was expressed and purified by the gene engineering technology in a laboratory (the results are shown in FIGS. 1 and 2).

Embodiment 2: An Expression Product Comprising the Antibacterial Peptide Scyreprocin of Scylla paramamosain was Obtained by Gene Engineering

(41) (a) The cDNA template of Scylla paramamosain was cloned to obtain the open reading frame sequence of the antimicrobial peptide Scyreprocin of Scylla paramamosain, and the open reading frame was cloned and recombined to a vector pET28a to obtain a recombinant vector. The recombinant vector with a correct open reading frame was verified by base sequencing and was transferred into Escherichia coli BL21 to obtain a recombinant expression strain configured to express the antimicrobial peptide Scyreprocin of Scylla paramamosain with a His tag.

(42) (b) A clone of the recombinant expression strain was selected and cultured in a liquid medium Luria-Bertani (LB) at 37° C. for 10-14 hours at a speed of 180 revolutions per minute (RPM) in a shake flask, then transferred to a liquid medium LB comprising 0.5% glucose with a ratio of 1:1000, and was then cultured until OD.sub.600=0.3. Isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM (mmol/L) to induce expression, and the strain was cultured at 16° C. for 24 hours at a speed of 160 RPM. The strain was collected by centrifugation, and suspended with a 50 mM phosphate buffer (pH 8.0) at a volume ratio of 1:10.

(43) (c) Phenylmethylsulfonyl fluoride (PMSF) was added to the suspensions to inhibit protease activity. The strain was ultrasonicated and centrifuged at a speed of 12000 RPM for 30 minutes to obtain a strain lysate supernatant comprising the recombinant Scyreprocin. After the strain lysate supernatant was filtered by a 0.22 μM filter membrane, recombinant Scyreprocin with the His tag was purified by a Ni column affinity chromatography.

(44) (d) The recombinant Scyreprocin with the His tag (i.e., a protein purification solution) was dialyzed in a dialysate (50 mM Tris HCl, 50 mM NaCl, pH 8.0) for 16-18 hours to remove imidazole from the protein purification solution, and the protein purification solution was transferred to Milli-Q water and was then dialyzed for 10-12 hours.

(45) (e) The dialyzed recombinant Scyreprocin was concentrated by ultrafiltration through an ultrafiltration tube, and a protein concentration was measured by a Bradford method. The concentrated recombinant Scyreprocin (i.e., the purified protein) was stored at −80° C. before use. The purified protein was detected by a denaturing gradient gel electrophoresis (i.e., an SDS-PAGE electrophoresis) and mass spectrometry, and the purified protein was identified as the antimicrobial peptide Scyreprocin of Scylla paramamosain.

Embodiment 3: A MIC (Minimum Inhibition Concentration) of the Recombinant Scyreprocin was Detected

(46) (a) The preserved Escherichia coli, Escherichia coli MC1061, Pseudomonas stutzeri, Pseudomonas hydrophila, Staphylococcus aureus, and Micrococcus lysoleikticus were streaked on a nutrient broth plate, the Vibrio fluvialis was streaked on a marine broth 2216E plate. The microbes were cultured at their corresponding optimum temperature for 12-16 hours. Candida albicans, Pichia pastoris, Candida krusei, Candida tropicalis, and Cryptococcus neoformans were streaked on yeast extract peptone glycerol (YPG) plates and cultured at 28° C. for 1-2 weeks; Aspergillus niger spores were coated on a plate potato dextrose agar (PDA) and cultured at 28° C. for 3-7 days. Colonies of each plate were selected and inoculated on a slant surface of the corresponding medium. For bacteria, the colonies were cultured for 10-16 hours. For fungi, the colonies were cultured for 1-3 days. For molds, the colonies were cultured for 3-7 days. The slant surface of the corresponding medium was washed with a 10 mM phosphate buffer (pH 7.4). Bacteria were diluted liquid medium Mueller-Hinton (MH) (diluted to OD.sub.600=0.003). Vibrio were diluted marine broth 2216E (diluted to OD.sub.600=0.0006). Yeasts were diluted by yeast extract peptone dextrose (YPD) (diluted to OD.sub.600=0.00067). Molds were diluted by PDA (diluted to a spore concentration of 5×10.sup.4 spores/mL) (Embodiment 4 describes a specific operation). The prepared microbe solutions were used within 20 minutes.

(47) (b) The recombinant Scyreprocin was filtered by a 0.22 μM filter film. A protein concentration was then assayed by a Bradford method, and the protein concentration was diluted to 0.5, 1, 2, 4, 8, 16, and 32 μM. The antimicrobial peptide was stored at 4° C.

(48) (c) A minimum inhibitory concentration test was carried out on 96-well plates. Each microbe to be tested comprised a blank control group, a negative control group, and a test group. In accordance with the following operations, each group of the blank control group, the negative control group, and the test group comprised 3 parallel samples.

(49) The negative control group: 50 μL sterile ultra-pure water and 50 μL microbe suspension were added.

(50) The blank control group: 50 μL protein sample to be tested and 50 μL phosphate buffer were added.

(51) The test group: 50 μL protein sample to be tested and 50 μL microbe suspension were added.

(52) For bacteria, the 96-well plates were placed at an optimum temperature for each microbe and cultured for 18-24 hours, and results were observed.

(53) For fungi, vibrio, and molds, the 96-well plates were placed at 28° C. for 1-2 days and results were observed.

(54) The liquid medium in the test group where the growth of bacteria was not observed by naked eyes was evenly mixed to obtain a mixture medium, 2 μL of the mixture medium was inoculated to a corresponding plate and cultured at the optimum growth temperature for 24-48 hours. Minimum bactericidal concentration (MBC) results were observed.

(55) MIC and MBC results of the recombinant Scyreprocin are shown in Table 2.

(56) TABLE-US-00007 TABLE 2 Determination of antimicrobial activity of Scyreprocin Microorganism CGMCC No. MIC(μM) MBC(μM) Gram-negative bacteria Escherichia coli 1.2389 2-4 >15 Pseudomonas stutzeri 1.1803 0.5-1   1-2 Vibrio fluvialis 1.1609 1-2 2-4 Escherichia coli MC1061 — 2-4 4-8 Gram-positive bacteria Staphylococcus aureus 1.363  <0.5 2-4 Micrococcus lysoleikticus 1.0634 <0.5 1-2 Bacillus subtilis 1.108  1-2 4-8 Fungi Candida krusei 2.1857  8-16 >32 Candida albicans 2.2411 2-4 16-32 Cryptococcus neoformans 2.1563 1-2  8-16 Candida tropicalis 2.1975 16-32 >32 Pichia pastoris (GS115) 2.2238 Invitrogen 4-8 >30 Aspergillus niger 3.0316 4-8 >32 Note: MIC: minimum inhibitory concentration (μM) and a-b represents MIC, wherein a represents a highest concentration of the recombinant Scyreprocin in which microbial growth was observed by naked eyes, and b represents a lowest concentration of the recombinant Scyreprocin in which no microbe growth was observed. MBC: minimum bactericidal concentration (μM), a minimum concentration of the recombinant Scyreprocin configured to kill 99.9% of bacteria.

Embodiment 4: Spore Germination of Aspergillus niger Inhibited by the Recombinant Scyreprocin was Detected

(57) (a) A preserved Aspergillus niger was coated on a plate PDA and was cultured at 28° C. for 3-7 days to obtain spores.

(58) (b) The spores were collected from the plate PDA, inoculated on a slant surface PDA, and cultured at 28° C. for 3-7 days. The slant surface was washed with 10 mM phosphate buffer (pH 7.4), the hypha were removed by filtration with a cell strainer (an aperture of the cell strainer is 100 nm), and the fungal spores (i.e., a spore suspension) were collected. A PDA liquid medium and 10 mM phosphate buffer with a ratio of 1:1 were used, and a spore concentration was adjusted to 5×10.sup.4 spores/mL.

(59) (c) The recombinant Scyreprocin was filtered with a 0.22 μM filter membrane, diluted to a concentration of 0.5, 1, 2, 4, 8, 16, 32 μM, and placed on ice for use.

(60) (d) 96-well plates were set with a positive control group and a test group as follows, and each group comprised three parallel samples.

(61) The positive control group: 50 μL spore suspension and 50 μL 10 mM phosphate buffer were added.

(62) The test group: 50 μL spore suspension and 50 μL recombinant Scyreprocin were added.

(63) The 96-well plates were cultured in a 28° C. incubator for 24 hours, and spore germination of Aspergillus niger was observed by an optical microscope. Referring to FIG. 3, as a result, the 4 M recombinant Scyreprocin significantly inhibited the spore germination of Aspergillus niger.

Embodiment 5: Bactericidal Kinetic Curve of the Recombinant Scyreprocin

(64) In this embodiment, a Gram-negative bacterium (Pseudomonas stutzeri), a Gram-positive bacterium (Micrococcus lysoleikticus), and two fungi (Candida albicans and Cryptococcus neoformans) were selected as bacteria to be tested, and a germicidal kinetic curve of the recombinant Scyreprocin was generated.

(65) (a) Pseudomonas stutzeri and Micrococcus lysoleikticus were maintained on nutrient broth plates. Candida albicans and Cryptococcus neoformans were maintained on YPD plates. The aforementioned microbes were cultured at an optimum temperature.

(66) (b) Colonies were selected from the plates (i.e., the nutrient broth plates or the YPD plates), were inoculated on a slant surface, and cultured. The slant surface was washed with a 10 mM phosphate buffer (pH 7.4) to obtain microbial suspensions. A concentration of the microbial suspensions was adjusted to OD.sub.600=3.3×10.sup.4 cfu/mL with a mixed medium comprising a liquid medium MH (or a liquid medium YPD) and a 10 mM phosphate buffer with a ratio of 2:3.

(67) (c) The recombinant Scyreprocin was filtered by a 0.22 m filter membrane, diluted to 0.5, 1, 2, 4, 8, 16, 32 μM, and placed on ice for use.

(68) (d) 96-well plates were set with a positive control group and a test group as follows, and each group comprised three parallel samples.

(69) The positive control group: 100 μL microbe suspension and 100 μL 10 mM phosphate buffer were added.

(70) The test group: 100 μL microbe suspension and 100 μL recombinant Scyreprocin were added.

(71) The 96-well plates were cultured at 28° C. A proper amount of culture medium was sampled after 0, 15, 30, 60, 90, 120, 180, and 240 minutes, diluted, and coated on nutrient broth plates or YPD plates. The 96-well plates were cultured and the number of clones on each plate was counted. A bactericidal kinetics curve was generated (referring to FIG. 4) due to statistical analysis. As a result, the recombinant Scyreprocin with 2×MBC killed 50% of Cryptococcus neoformans and Candida albicans in 100 minutes, and a fungicidal index reached 100% in 500 minutes. After the recombinant Scyreprocin with 2×MBC acted on Micrococcus lysozyme for 60 minutes, a bactericidal index reached 100%. After the recombinant Scyreprocin with 2×MBC acted on the Pseudomonas stutzeri for 180 minutes, a bactericidal index reached 100%.

Embodiment 6: The Effect of the Recombinant Scyreprocin on the Proliferation of Animal Cell Line

(72) In this example, human liver cells L02 and mouse hepatocyte AML12 were selected as test cell lines, and the anti-proliferation effect against different cells of recombinant Scyreprocin.

(73) (a) The human liver cells L02 and the mouse hepatocyte AML12 were maintained in a corresponding cell culture medium, digested with trypsin comprising ethylenediaminetetraacetic acid (EDTA), and resuspended in the corresponding cell culture medium. The cell density was adjusted, and 2×10.sup.5 cells were inoculated in each well of a 96-well plate and cultured overnight at 37° C. in 5% CO.sub.2.

(74) (b) The medium was removed, and the recombinant Scyreprocin was diluted to 0.5, 1, 2, 4, 8, and 16 μM with the corresponding cell culture medium. The corresponding cell culture medium was used as a control group, and each group comprised three parallel samples.

(75) The 96-well plate was placed at 37° C. in 5% CO.sub.2 for 48 hours, then a 20 μL MTS reagent ([3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]) was added to each well, and incubated for 2 hours. The absorption value at 490 nm was measured by a microplate reader. Referring to FIG. 5, as a result, the recombinant Scyreprocin had no inhibitory effect on the growth of the mouse hepatocyte AML12 and human liver cells L02.

(76) The aforementioned embodiments are merely some embodiments of the present disclosure, and the scope of the disclosure is not limited thereto. Thus, it is intended that the present disclosure cover any modifications and variations of the presently presented embodiments provided they are made without departing from the appended claims and the specification of the present disclosure.