CN103539847B - Cyclic synthetic peptide B, and antibacterial and antiviral application thereof - Google Patents
Cyclic synthetic peptide B, and antibacterial and antiviral application thereof Download PDFInfo
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- CN103539847B CN103539847B CN201310534912.2A CN201310534912A CN103539847B CN 103539847 B CN103539847 B CN 103539847B CN 201310534912 A CN201310534912 A CN 201310534912A CN 103539847 B CN103539847 B CN 103539847B
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Abstract
The invention relates to a cyclic synthetic peptide B, and antibacterial and antiviral application thereof. The cyclic synthetic peptide B related to the invention has an amino acid sequence and structural characteristics shown in SEQ ID No.1 in a sequence list; the cyclic synthetic peptide B is derived from an LPS binding structure domain of a fenneropenaeus merguiensis anti-lipopolysaccharide factor FcALF2 shown in SEQ ID No.2 in the sequence list. The cyclic synthetic peptide B is verified to have a strong inhibiting effect on growth or multiplication of a gram-positive bacterium or a virus, and has a certain inhibiting effect on growth of a gram-negative bacterium by carrying out biological functional verification on the cyclic synthetic peptide B related to the invention.
Description
Technical field
The present invention relates to a kind of improvement on synthesis B and anti-bacteria and anti-virus application thereof, the ring type polypeptide of the lipopolysaccharides derived from Fenneropenaeus chinensis Anti-LPS factor FcALF2 (LPS) binding domains of specifically a kind of synthesis and anti-bacteria and anti-virus application thereof of ring-type.
Background technology
The cultivation of prawn occupies critical role in the culture fishery of China, in the last few years, disease problem in prawn culturing process has seriously hindered the sound development of its breeding production, and the deterioration of antibiotic abuse and environment makes the disease of marine cultured animal be difficult to fundamentally be controlled.
Antibacterial peptide (albumen) is considered to the important effect molecule that the exogenous pathogen such as defense against bacterial, virus such as fish, shrimp, shellfish infect, and plays a significant role in the congenital immunity process of animal.The antibacterial peptide found from crustacean is at present of a great variety, and coagulogen (anti-lipopolysaccharide factor, ALF) is exactly wherein a kind of important antibacterial peptide.Nineteen eighty-two, Tanaka etc. are separated first and obtain ALF from Tachypleus tridentatus (Tachypleus tridentatus) and the hemocyte of North America king crab (Limulus polyphemus), and find the activation of its Coagulation test that LPS can be suppressed to mediate.1985, it is active that Morita etc. find that ALF also has very strong against gram-negative bacteria.Afterwards, people find the existence of ALF gene in succession in the multiple crustaceans such as tigar prawn (Penaeus monodon), Crustin (Fenneropenaeus chinensis), Marsupenaeus japonicus (Marsupenaeus japonicus), Environment of Litopenaeus vannamei Low (Litopenaeus vannamei), pink shrimp (Farfantepenaeus duorarum), and confirm that the recombinant protein of ALF has the activity of against gram-negative bacteria and Gram-positive bacteria growing widely.In addition, research also finds that shrimp white spot syndrome virus (WSSV) is injected in shrimp body after hatching together with recombinant expressed ALF albumen in advance again, can suppress copying of WSSV in shrimp body.
Different from the mechanism of action of conventional antibiotic, ALF directly in and the lipopolysaccharides of bacteria cell wall, dissolution of bacteria, therefore not easily diseaseful resistance.ALF is still unclear at present to the mechanism of action of virus.Along with antibiotic application, many pathogenic bacterias progressively produce resistance to existing microbiotic, and the discovery of new antibiotic is extremely difficult, and therefore, the research of ALF is that development of new antibacterials open wide prospect.
Research finds, disulfide linkage is formed between two Conserved cysteines in ALF aminoacid sequence, and and between aminoacid sequence jointly form one section of anionic polypeptides, there is the ability be combined with LPS, this section of conservative structure called after LPS binding domains, is considered to ALF and degrades the functional domain of gram-negative bacteria cell wall lipopolysaccharides.2011, the polypeptide that Sachin Sharma etc. have studied tool 24 amino-acid residues synthesized according to the LPS binding domains of Young Crab (Scylla serrata) ALF acts in antibacterial immunity process, find that improvement on synthesis SsALF24 has the activity in conjunction with LPS and has obvious restraining effect to intestinal bacteria, its minimum inhibition concentration is 16.16 ~ 32.32uM(Sharma S, Yedery RD, PatgaonkarMS, Selvaakumar C, Reddy KV.Antibacterial activity of a synthetic peptide that mimicsthe LPS binding domain of Indian mud crab, Scylla serrata anti-lipopolysaccharidefactor (SsALF) also involved in the modulation of vaginal immune functions throughNF-kB signaling.Microbial pathogenesis2011, 50:179-191.).
FcALF2 in Crustin is found, research shows that it responds infection (the Li SH of virus in prawn body, ZhangXJ, Sun Z, Li FH, Xiang JH.Transcriptome analysis on Chinese shrimp Fenneropenaeuschinensis during WSSV acute infection.PLoS ONE, 8 (3): e58627.).Based on existing result of study, the present invention is according to the derivation aminoacid sequence of Crustin FcALF2, utilize the method for chemosynthesis to obtain the circular polypeptides B of its LPS binding domains, this ring type polypeptide has obvious anti-bacteria and anti-virus biologic activity, has important application prospect.
Summary of the invention
The present invention aims to provide a kind of improvement on synthesis B deriving from the ring-type of prawn, has significantly antibacterial and antiviral activity.
Technical scheme of the present invention is as follows:
Utilize the method for chemosynthesis to obtain the improvement on synthesis B of ring-type, have the aminoacid sequence shown in SEQ ID No.1, its sequence signature is:
SEQ ID No.1：Ac-Yc(CSFNVTPKFKRWQLYFRGRMWC)P-NH
2
The improvement on synthesis B sequence of described ring-type has disulfide bond pattern.Its constitutional features is: have disulfide bond pattern between improvement on synthesis B sequence aminoterminal second halfcystine (C) and carboxyl terminal second halfcystine (C); The amino of the aminoterminal Y of polypeptide is acetylation (Ac-), and the carboxyl of carboxyl terminal P is amidated (-NH
2).
The improvement on synthesis B of described ring-type is from the LPS binding domains of Fenneropenaeus chinensis Anti-LPS factor FcALF2.The feature of FcALF2 is: have the aminoacid sequence shown in SEQ ID No.2 in sequence list.
The improvement on synthesis B of described ring-type has obvious antimicrobial antiviral activity.Be specially: to the growth of gram-positive microorganism and virus or propagation, all there is very strong restraining effect, to the growth of Gram-negative bacteria, there is certain restraining effect.
The improvement on synthesis B of described ring-type can be used as antibacterial and/or antiviral active ingredient, for the preparation of in antibacterial and/or antiviral drug or preparation.
The present invention has the following advantages:
1, the present invention determines a kind of anti-bacteria and anti-virus polypeptide B derived from Fenneropenaeus chinensis Anti-LPS factor FcALF2 and constitutional features thereof.
2, the protective agents of effective prawn bacterium class and viral diseases can be developed by the present invention.
Accompanying drawing explanation
The skeleton structure diagram of Fig. 1 improvement on synthesis B;
The space structure figure of Fig. 2 improvement on synthesis B;
The HPLC purity detecting figure of Fig. 3 improvement on synthesis B;
The MS Mass Spectrometric Identification figure of Fig. 4 improvement on synthesis B.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail.
A prawn ring type polypeptide B with obviously antibacterial and antiviral activity for chemosynthesis, its sequence and derived sequences information as follows:
(1) information of SEQ ID No.1
(a) sequence signature
* length: 24 amino acid
* type: amino acid
* chain: strand
* topological framework: annular, forms disulfide linkage between two halfcystines
* space structure: have 1) skeleton structure shown in Fig. 1: be connected by disulfide linkage between two halfcystines, form ring texture, wherein disulfide linkage intensification black represents, C framework and other atom grey represent; With 2) space structure shown in Fig. 2: peptide sequence forms two β-pleated sheet structure structures be connected.
(b) molecule type: albumen
Sequence description: SEQ ID No.1
Ac-Yc(CSFNVTPKFKRWQLYFRGRMWC)P-NH
2
Amino acid in its bracket is into the amino acid of ring, and the small letter c in the outer left side of bracket represents that the amino acid in bracket is formation ring texture amino acid; Ac-represents that the amino of amino acid Y is acetylation;-NH
2represent that the carboxyl of amino acid P is acetylation.
(2) information of SEQ ID No.2
(a) sequence signature
* length: 122 amino acid
* type: amino acid
* chain: strand
* topological framework: linear
(b) molecule type: albumen
Sequence description: SEQ ID No.2
MRVSVFSMIIVLAVAASFAPQCQASGWEALVPAIADKLTGLWESGELELLGHYCSFNVTPKFKRWQLYFRGRMWCPGWTTIGGQAETRSRSGVVGRTTQDFVRKAFRAGLITESEAQAWLNN
The synthesis of described anti-bacteria and anti-virus polypeptide B, cyclisation, purifying, qualification and Analysis on Biological Activity:
By artificial chemistry route of synthesis, utilize solid phase synthesis process to obtain crude product polypeptide, obtain the improvement on synthesis B containing disulfide linkage through solid phase cyclization, Mass Spectrometric Identification and liquid chromatography purification.Be specially:
1) Peptide systhesis
Adopt 9-fluorenylmethyloxycarbonyl (Fmoc) synthesis strategy, synthesize to N extreme direction from C end.With 10mg Rink-Amide-Resin resin (AAPPTec, article No. RRZ001) as carrier, the active group of carrier itself and 5mg is relied on to be carried out first amino acid (Fmoc-Pro-NH of amido protecting by Fmoc
2) carboxyl be connected (method detailed reference: PanagiotisStathopoulos; Serafim Papas, Vassilios Tsikaris.C-terminal N-alkylated peptideamides resulting from the linker decomposition of the Rink amide resin.A new cleavagemixture prevents their formation.Journal of Peptide Science2006; 12:227-232.).
Adjoin with N-methyl and cough up Anhui intoxicated (NMP) and rinse resin removing redundant protection amino acid, in reactor (solid phase synthesis device), add 20% piperidines/nmp solution (volume fraction) remove Fmoc group, reaction 20min, emptying reactor, vibrate with 5mL NMP and rinse resin, repeat 3 times, remove the Fmoc protection of first amino-acid residue; The active amine groups exposed is connected by the carboxyl that Fmoc carries out the amino acid (5mg) of amido protecting with the next one, forms first peptide bond (Pro-Cys).The above step cycle of this section carries out (difference is: just need adopt the corresponding amino acid being carried out amido protecting by Fmoc at every turn) until peptide sequence Ac-YCSFNVTPKFKRWQLYFRGRMWCP-NH
2synthesize complete, obtain about 20mg linear polypeptide.
2) polypeptide cyclisation
After 20mg linear polypeptide coupling last amino acid complete, the I of configuration 0.1mol/L
2solution (I
2be dissolved in the methyl alcohol of volume ratio 1:1: in DMF mixing solutions), add 10mL in solid phase synthesis device, nitrogen brushes reaction, about 6 hours.
3) peptide purification and qualification
Peptide reagent trifluoroacetic acid is cut: thioanisole: phenol: dithioglycol: the polypeptide after 20mg cyclisation gets off from cracking vector resin by distilled water (volume ratio 82.5:5:5:2.5:5) with 50mL, after 2 hours, the ether 100ml adding 4 DEG C of precoolings makes polypeptide precipitate, centrifugal collecting precipitate, and with washed with diethylether 3 times, vacuum is drained, and the polypeptide crude product obtained, through reverse phase liquid chromatography purifying, carries out HPLC purity detecting (Fig. 3) and Mass Spectrometric Identification (Fig. 4) after the polypeptide freeze-drying after purifying.Detecting HPLC chromatographic column is 250*4.6mm, Kromasil-C18-5 μm; Mobile phase A: 0.1%TFA/ acetonitrile, Mobile phase B: 0.1%TFA/H
2o; Linear eluent gradient: 15%A-100%A; Flow velocity is 1ml/min, and determined wavelength is 220nm; Single injected sampling amount is 10 μ l.HPLC and MS detected result shows, and the purity of improvement on synthesis B is 95.29%, and molecular weight is 3157.32, basically identical with predicted molecular weight (3152.78).
4) bacteriostatic test
The PBS of improvement on synthesis B 50mmol/L pH7.4 is dissolved to the solution that concentration is 640 μm of ol/L, simultaneously with the PBS of 50mmol/L pH7.4 for negative control.Adopt minimum inhibitory concentration (MIC) method to detect improvement on synthesis and the bacteriostatic activity that intestinal bacteria (Escherichia coli), Vibrio anguillarum (Vibrio anguillarum) and gram-positive microorganism comprise micrococcus luteus (Micrococcus luteus), micrococcus lysodeikticus (Micrococcus lysodeikticus) is comprised to Gram-negative bacteria.That is: by intestinal bacteria to be measured, micrococcus luteus and micrococcus lysodeikticus respectively at 37 DEG C, in LB substratum, be cultured to 1 × 10 under 200r/min culture condition
8cells/mL, Vibrio anguillarum culture temperature is 28 DEG C, and other condition is the same constant; The bacterium cultivated joins in 48 well culture plates respectively, with fresh LB substratum, bacterium liquid to be measured is diluted to final concentration 1 × 10
6cells/mL; Polypeptide B solution to the final volume adding gradient dilution in 48 well culture plates is respectively that 200 μ l, polypeptide B final concentration is followed successively by 64 μm of ol/L, 32 μm of ol/L, 16 μm of ol/L, 8 μm of ol/L, 4 μm of ol/L, 2 μm of ol/L and 1 μm ol/L; With PBS as negative control, the penbritin (intestinal bacteria, micrococcus luteus, micrococcus lysodeikticus) of final concentration 58 μm of ol/L or the kantlex (Vibrio anguillarum) of 88 μm of ol/L are respectively as positive control; Cultivate 3h under 37 DEG C or 28 DEG C of conditions after, add 300 μ l fresh LB respectively, continue to cultivate 18h, measure the OD600 light absorption value of bacterium in every hole, calculate bacterial concentration.
Result shows, to Gram-negative bacteria, the improvement on synthesis B of 32-64 μm of ol/L effectively can suppress the growth of Vibrio anguillarum; To gram-positive microorganism, the improvement on synthesis B of 2-4 μm of ol/L can effectively suppress the growth of micrococcus luteus, the improvement on synthesis B of 1-2 μm of ol/L effectively can suppress the growth of micrococcus lysodeikticus.Illustrate that improvement on synthesis B has the activity of obvious resisting gram-positive bacteria and negative bacterium.
5) viral Neutralizing test
Dissolve improvement on synthesis B by embodiment bacteriostatic test step PBS, with the improvement on synthesis of final concentration 50 μm of ol/L at room temperature with WSSV(white spot syndrome virus, white spot syndrome virus) hatch 2h, WSSV is hatched under the same conditions as negative control with the irrelevant polypeptide (the green fluorescent protein GFP peptide section of equal length) of same concentrations, WSSV after hatching injects dusky white prawn (Exopalaemon carinicauda) respectively, and the injection volume of every tail shrimp is 10
4copy WSSV particle, get dusky white prawn swimmeret after injection 24h, adopt the DNA extraction kit (article No.: DP324) of Tian Gen biochemical technology company limited to extract STb gene, gradient dilution WSSV is as standard substance, WSSV copy number is utilized to detect primer pair VP28rF(5 '-AAACCTCCGCATTCCTGTGA-3 ') and VP28rR(5 '-TCCGCATCTTCTTCCTTCAT-3 '), Real-Time Fluorescent Quantitative PCR Technique is adopted to increase respectively expression amount (the detailed detection method reference: Yumiao Sun of VP28 gene in standard substance and prawn DNA sample to be measured, Fuhua Li, Jianhai Xiang.Analysis on the dynamic changes ofthe amount of WSSV in Chinese shrimp Fenneropenaeus chinensis during infection.Aquaculture2013, 376-379:124-132.), be then converted into the copy number of WSSV in prawn DNA sample to be measured.
Result shows, the WSSV copy number of improvement on synthesis B treatment group is 4.74 × 10
3/ ng DNA, and the WSSV copy number of GFP negative control group is 1.67 × 10
5/ ng DNA, in treatment group, the copy number of WSSV is starkly lower than control group, illustrates that improvement on synthesis B has obvious antiviral activity.
The discovery of this polypeptide B and Biological Activity Identification, the exploitation of novel antibacterial antiviral has important application prospect.
Claims (3)
1. the improvement on synthesis B of ring-type is for the preparation of the application in antibacterial and/or antiviral preparation, it is characterized by:
The improvement on synthesis B of described ring-type to Vibrio anguillarum, intestinal bacteria, micrococcus luteus, micrococcus lysodeikticus, and WSSV growth or propagation all inhibited;
The improvement on synthesis B of described ring-type has disulfide bond pattern, has disulfide bond pattern between aminoterminal second amino-acid residue (halfcystine, C) of polypeptide B and carboxyl terminal second amino-acid residue (halfcystine, C),
The improvement on synthesis B sequence of described ring-type is the aminoacid sequence shown in SEQ ID No.1 in list.
2. according to the application of the improvement on synthesis B of ring-type according to claim 1, it is characterized in that: it is derived from the LPS binding domains of Fenneropenaeus chinensis Anti-LPS factor FcALF2, FcALF2 is the aminoacid sequence shown in SEQ ID No.2 in sequence table.
3., according to the application of the improvement on synthesis B of ring-type described in claim 1, it is characterized in that:
The improvement on synthesis B of described ring-type as antibacterial and/or antiviral active ingredient, for the preparation of in antibacterial and/or antiviral drug or preparation.
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| CN106749586A (en) * | 2016-11-22 | 2017-05-31 | 中国科学院海洋研究所 | One kind transformation ring type polypeptide PV and its application and antibiotic preparation |
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| CN112694525B (en) * | 2020-12-29 | 2022-09-27 | 中国科学院海洋研究所 | Small molecular polypeptide and antibacterial and antiviral application thereof |
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| CN106749586A (en) * | 2016-11-22 | 2017-05-31 | 中国科学院海洋研究所 | One kind transformation ring type polypeptide PV and its application and antibiotic preparation |
| CN106749586B (en) * | 2016-11-22 | 2020-08-11 | 中国科学院海洋研究所 | Modified cyclic polypeptide PV and application and antibacterial preparation thereof |
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