CN103539844B - A kind of improvement on synthesis C of ring-type and anti-bacteria and anti-virus application thereof - Google Patents
A kind of improvement on synthesis C of ring-type and anti-bacteria and anti-virus application thereof Download PDFInfo
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Abstract
The present invention relates to a kind of improvement on synthesis C and anti-bacteria and anti-virus application thereof of ring-type.Does the improvement on synthesis C of ring-type involved in the present invention have SEQ in sequence list? ID? aminoacid sequence shown in No.1 and constitutional features; Is the improvement on synthesis C of this ring-type derived from SEQ in sequence list? ID? the LPS binding domains of the FcALF1 of Fenneropenaeus chinensis Anti-LPS factor shown in No.2; Through carrying out biological function checking to the improvement on synthesis C of the ring-type that the present invention relates to, confirm that they to the growth of gram-positive microorganism, Gram-negative bacteria all or have certain restraining effect, have very strong restraining effect to the propagation of virus.
Description
Technical field
The present invention relates to a kind of improvement on synthesis C and anti-bacteria and anti-virus application thereof, the ring type polypeptide of the lipopolysaccharides derived from Fenneropenaeus chinensis Anti-LPS factor FcALF1 (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-lipopolysaccharidefactor, ALF) is exactly wherein a kind of important antibacterial peptide.Nineteen eighty-two, Tanaka etc. are separated first and obtain ALF from Tachypleus tridentatus (Tachypleustridentatus) and the hemocyte of North America king crab (Limuluspolyphemus), 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 (Penaeusmonodon), Crustin (Fenneropenaeuschinensis), Marsupenaeus japonicus (Marsupenaeusjaponicus), Environment of Litopenaeus vannamei Low (Litopenaeusvannamei), pink shrimp (Farfantepenaeusduorarum), 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 to inject copying of shrimp body WSSV.
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 SachinSharma etc. have studied tool 24 amino-acid residues synthesized according to the LPS binding domains of Young Crab (Scyllaserrata) 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(SharmaS, YederyRD, PatgaonkarMS, SelvaakumarC, ReddyKV.Antibacterialactivityofasyntheticpeptidethatmimi cstheLPSbindingdomainofIndianmudcrab, Scyllaserrataanti-lipopolysaccharidefactor (SsALF) alsoinvolvedinthemodulationofvaginalimmunefunctionsthrou ghNF-kBsignaling.Microbialpathogenesis2011, 50:179-191.).
FcALF1 in Crustin is found, research shows that it responds the infection (LiSH of virus in prawn body, ZhangXJ, SunZ, LiFH, XiangJH.TranscriptomeanalysisonChineseshrimpFenneropenae uschinensisduringWSSVacuteinfection.PLoSONE, 8 (3): e58627.).Based on existing result of study, the present invention is according to the aminoacid sequence of Crustin FcALF1, utilize the method for chemosynthesis to obtain the circular polypeptides C 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 C 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 C of ring-type, have the aminoacid sequence shown in SEQIDNo.1, its sequence signature is:
SEQIDNo.1：Ac-Qc(CTYSVTPTVKSFELYFKGRMSC)P-NH
2
The improvement on synthesis C sequence of described ring-type has disulfide bond pattern.Its constitutional features is: have disulfide bond pattern between improvement on synthesis C sequence aminoterminal second halfcystine (C) and carboxyl terminal second halfcystine (C); The amino of the aminoterminal Q of polypeptide is acetylation (Ac-), and the carboxyl of carboxyl terminal P is amidated (-NH
2).
The improvement on synthesis C of described ring-type is from the LPS binding domains of Fenneropenaeus chinensis Anti-LPS factor FcALF1.The feature of FcALF1 is: have the aminoacid sequence in sequence list shown in SEQIDNo.2.
The improvement on synthesis C of described ring-type has obvious antimicrobial antiviral activity.Be specially: to the growth of gram-positive microorganism, Gram-negative bacteria, all there is certain restraining effect, to the propagation of virus, there is very strong restraining effect.
The improvement on synthesis C of described ring-type can be used as antibacterial and/or antiviral active ingredient, can be used in the antibacterial and/or antiviral drug of preparation or preparation.
The present invention has the following advantages:
1, the present invention determines a kind of anti-bacteria and anti-virus peptide C derived from Fenneropenaeus chinensis Anti-LPS factor FcALF1 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 C;
The space structure figure of Fig. 2 improvement on synthesis C;
The HPLC purity detecting figure of Fig. 3 improvement on synthesis C;
The MS Mass Spectrometric Identification figure of Fig. 4 improvement on synthesis C.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail.
A prawn ring type polypeptide C with obviously antibacterial and antiviral activity for chemosynthesis, its sequence and derived sequences information as follows:
(1) information of SEQIDNo.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: SEQIDNo.1
Ac-Qc(CTYSVTPTVKSFELYFKGRMSC)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 Q is acetylation;-NH
2represent that the carboxyl of amino acid P is acetylation.
(2) information of SEQIDNo.2
(a) sequence signature
* length: 123 amino acid
* type: amino acid
* chain: strand
* topological framework: linear
(b) molecule type: albumen
Sequence description: SEQIDNo.2
MKLSFVVGVVTVLAAVALFATPCQGQVWEALVPLITQQVVGLWKNGEREFFGHQCTYSVTPTVKSFELYFKGRMSCPSLSSVRGEALTRSRSGVEVKTVEDYVKKVLAQGVITEEEAKAWITK
The synthesis of described anti-bacteria and anti-virus peptide C, 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 C 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 10mgRink-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; SerafimPapas, VassiliosTsikaris.C-terminalN-alkylatedpeptideamidesresu ltingfromthelinkerdecompositionoftheRinkamideresin.Anewc leavagemixturepreventstheirformation.JournalofPeptideSci ence2006; 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, resin is rinsed with 5mLNMP vibration, 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-QCTYSVTPTVKSFELYFKGRMSCP-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 C is 95.11%, and molecular weight is 2814.12, basically identical with predicted molecular weight (2812.31).
4) bacteriostatic test
The PBS of improvement on synthesis C 50mmol/LpH7.4 is dissolved to the solution that concentration is 640 μm of ol/L, simultaneously with the PBS of 50mmol/LpH7.4 for negative control.Adopt minimum inhibitory concentration (MIC) method to detect improvement on synthesis and the bacteriostatic activity that intestinal bacteria (Escherichiacoli), Vibrio anguillarum (Vibrioanguillarum) and gram-positive microorganism comprise micrococcus luteus (Micrococcusluteus), micrococcus lysodeikticus (Micrococcuslysodeikticus) 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; Cultivate bacterium join in 48 well culture plates respectively, with fresh LB substratum by bacterium liquid dilute to be measured to final concentration 1 × 10
6cells/mL; Peptide C solution to the final volume adding gradient dilution in 48 well culture plates is respectively 200 μ l, and peptide C 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 C of 16-32 μm of ol/L can effectively suppress colibacillary growth; To gram-positive microorganism, the improvement on synthesis C of 16-32 μm of ol/L effectively can suppress the growth of micrococcus luteus.Illustrate that improvement on synthesis C has the activity of obvious resisting gram-positive bacteria and negative bacterium.
5) viral Neutralizing test
Dissolve improvement on synthesis C by embodiment bacteriostatic test step PBS, with the improvement on synthesis of final concentration 50 μm of ol/L at room temperature with WSSV(whitespotsyndromevirus, white spot syndrome virus) hatch 2h, hatch WSSV with the irrelevant polypeptide (the green fluorescent protein GFP peptide section of equal length) of same concentrations under the same conditions as negative control, the WSSV after hatching injects dusky white prawn (Exopalaemoncarinicauda) 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: YumiaoSun of VP28 gene in standard substance and prawn DNA sample to be measured, FuhuaLi, JianhaiXiang.AnalysisonthedynamicchangesoftheamountofWSS VinChineseshrimpFenneropenaeuschinensisduringinfection.A quaculture2013, 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 C treatment group is 1.47 × 10
4/ ngDNA, and the WSSV copy number of GFP negative control group is 1.67 × 10
5/ ngDNA, in treatment group, the copy number of WSSV is starkly lower than control group, illustrates that improvement on synthesis C has obvious antiviral activity.
The discovery of this peptide C and Biological Activity Identification, the exploitation of novel antibacterial antiviral has important application prospect.
Claims (2)
1. an improvement on synthesis C for ring-type, its constitutional features is:
The improvement on synthesis C of described ring-type to the growth of gram-positive microorganism, Gram-negative bacteria all or have certain restraining effect, has very strong restraining effect to the propagation of virus;
The improvement on synthesis C of described ring-type has disulfide bond pattern, has disulfide bond pattern between aminoterminal second halfcystine (C) of peptide C and carboxyl terminal second halfcystine (C);
The aminoacid sequence of the improvement on synthesis C of described ring-type is the aminoacid sequence in sequence list shown in SEQIDNo.1, it is derived from the LPS binding domains of Fenneropenaeus chinensis Anti-LPS factor FcALF1, and the aminoacid sequence of FcALF1 is the aminoacid sequence in sequence list shown in SEQIDNo.2.
2., according to the application of the improvement on synthesis C of ring-type described in claim 1, it is characterized in that:
The improvement on synthesis C of described ring-type can be used as antibacterial and/or antiviral activeconstituents, for the preparation of in antibacterial and/or antiviral drug or preparation.
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| CN109180794B (en) * | 2018-09-21 | 2021-09-10 | 深圳大学 | Novel penaeus monodon ALFpm12 antibacterial peptide, preparation method and application thereof |
| 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 |
Citations (3)
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|---|---|---|---|---|
| CN1459506A (en) * | 2003-05-30 | 2003-12-03 | 山东大学 | Recombination expression and application of Chinese prawn antibacterial peptide gene |
| CN1740320A (en) * | 2004-08-27 | 2006-03-01 | 中国科学院海洋研究所 | Fenneropenaeus chinensis Anti-LPS factor gene and cloning process thereof |
| CN102417539A (en) * | 2010-09-27 | 2012-04-18 | 中国科学院海洋研究所 | Antibacterial protein and its coding gene, expression system, refolding system and application |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1459506A (en) * | 2003-05-30 | 2003-12-03 | 山东大学 | Recombination expression and application of Chinese prawn antibacterial peptide gene |
| CN1740320A (en) * | 2004-08-27 | 2006-03-01 | 中国科学院海洋研究所 | Fenneropenaeus chinensis Anti-LPS factor gene and cloning process thereof |
| CN102417539A (en) * | 2010-09-27 | 2012-04-18 | 中国科学院海洋研究所 | Antibacterial protein and its coding gene, expression system, refolding system and application |
Non-Patent Citations (2)
| Title |
|---|
| Shrimp (Penaeus monodon) anti-lipopolysaccharide factor reduces the lethality of Pseudomonas aeruginosa sepsis in mice;Pan C.等;《International Immunopharmacology》;20070531;第7卷(第5期);第687-700页 * |
| Transcriptome Analysis on Chinese Shrimp Fenneropenaeus chinensis during WSSV Acute Infection;Li S.等;《PLOS ONE》;20130531;第8卷(第3期);e58627 * |
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