CN103554232B - Linear synthetic peptide G and antiviral application thereof - Google Patents
Linear synthetic peptide G and antiviral application thereof Download PDFInfo
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- CN103554232B CN103554232B CN201310534388.9A CN201310534388A CN103554232B CN 103554232 B CN103554232 B CN 103554232B CN 201310534388 A CN201310534388 A CN 201310534388A CN 103554232 B CN103554232 B CN 103554232B
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Abstract
The invention relates to a linear synthetic peptide G and an antiviral application thereof. The linear synthetic peptide G provided by the invention has an amino acid sequence shown as SEQ ID No.1 in a sequence list and a structural characteristic. The linear synthetic peptide G is derived from an LPS (Lipopolysaccharide) binding domain of a Fenneropenaeus chinensiss lipopolysaccharide factor ALFFc shown as SEQ ID No.2. Through biological function in-vitro verification of the linear synthetic peptide G provided by the invention, the linear synthetic peptide G provided by the invention has a strong inhibiting effect to proliferation of viruses.
Description
Technical field
The present invention relates to a kind of linear improvement on synthesis G and antiviral application thereof, the linear polypeptide of the lipopolysaccharides derived from Fenneropenaeus chinensis Anti-LPS factor ALFFc (LPS) binding domains of specifically a kind of synthesis and antiviral application thereof.
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.).
Interestingly, different types of ALF polypeptide resist the kind of cause of disease and active size all has obvious difference, point out us can be obtained by the kind changing Individual amino acids in LPS binding domains and have peptide molecule that is resistance or specific resistance.Based on this imagination; we are based on the Crustin ALFFc(Liu FS found; Liu YC; Li FH; DongB, Xiang JH.Molecular cloning and expression profile of putativeantilipopolysaccharide factor in Chinese shrimp (Fenneropenaeus chinensis) .MarineBiotechnology2005; The change of the partial amino-acid residue that LPS binding domains 7:600-608.) carries out, utilizes the method for chemosynthesis to obtain linear polypeptide G, and this linear polypeptide has obvious special antiviral biologic activity, has important application prospect.
Summary of the invention
The present invention aims to provide a kind of linear improvement on synthesis G of the LPS binding domains derived from Fenneropenaeus chinensis Anti-LPS factor ALFFc, has obvious antiviral activity.
Technical scheme of the present invention is as follows:
Utilize the method for chemosynthesis to obtain linear improvement on synthesis G, have the aminoacid sequence shown in SEQ ID No.1, its sequence signature is:
Ac-EC(Me)KFTVKPYIKRFQLYYKGRMWC(Me)P-NH
2。
Two cysteine residues C in described linear improvement on synthesis G aminoacid sequence are the cysteine residues C(Me modified that methylates); The amino of the aminoterminal E of polypeptide is acetylation (Ac-), and the carboxyl of carboxyl terminal P is amidated (-NH
2).
Described linear improvement on synthesis G, described linear improvement on synthesis G is derived from the LPS binding domains of Fenneropenaeus chinensis Anti-LPS factor ALFFc.
The LPS binding domains of described Fenneropenaeus chinensis Anti-LPS factor ALFFc, have the aminoacid sequence shown in SEQ ID No.2 in sequence list, concrete sequence is: ECKFTVKPYIKRFQLYYKGRMWCP.
Described linear improvement on synthesis G has obvious antiviral activity.Be specially: to the propagation of virus, there is very strong restraining effect.
Described linear improvement on synthesis G can be used as antiviral active ingredient, can be used for preparing in antiviral drug or preparation.
The present invention has the following advantages:
1, the present invention determines a kind of antiviral polypeptide G derived from Fenneropenaeus chinensis Anti-LPS factor ALFFc and constitutional features thereof.
2, the protective agents of effective prawn ' s virus class disease can be developed by the present invention.
Accompanying drawing explanation
The skeleton structure diagram of Fig. 1 improvement on synthesis G;
The space structure figure of Fig. 2 improvement on synthesis G;
The HPLC purity detecting figure of Fig. 3 improvement on synthesis G;
The MS Mass Spectrometric Identification figure of Fig. 4 improvement on synthesis G.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail.
A prawn linear polypeptide G with obvious 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: linear, two methylated modifications of halfcystine
* space structure: have 1) skeleton structure shown in Fig. 1: halfcystine (C) the methylated modification in linear peptide sequence, the sulfydryl modified that methylates represents with intensification black, and 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-EC(Me)KFTVKPYIKRFQLYYKGRMWC(Me)P-NH
2
Me in its bracket represents that the halfcystine before it is methylated halfcystine C; Ac-represents that the amino of amino acid E is acetylation;-NH
2represent that the carboxyl of amino acid P is acetylation.
(2) information of SEQ ID No.2
(a) sequence signature
* length: 24 amino acid
* type: amino acid
* chain: strand
* topological framework: linear
(b) molecule type: albumen
Sequence description: SEQ ID No.2
ECKFTVKPYIKRFQLYYKGRMWCP
The synthesis of described antiviral polypeptide G, purifying, qualification and Analysis on Biological Activity:
By artificial chemistry route of synthesis, utilize solid phase synthesis process to obtain crude product polypeptide, obtain improvement on synthesis G through 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-EC (Me) KFTVKPYIKRFQLYYKGRMWC (Me) P-NH
2synthesize complete, cystine C residue wherein used in building-up process is the halfcystine C(Me modified that methylates), finally obtain the linear improvement on synthesis of about 20mg.
2) peptide purification and qualification
Peptide reagent trifluoroacetic acid is cut: thioanisole: phenol: dithioglycol: the 20mg linear polypeptide of synthesis 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 G is 95.76%, and molecular weight is 3158.84, basically identical with predicted molecular weight (3154.82).
3) viral Neutralizing test
Dissolve improvement on synthesis G 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 G treatment group is 5.07 × 10
3/ ng DNA, and the WSSV copy number of GFP negative control group is 1.50 × 10
5/ ng DNA, in treatment group, the copy number of WSSV is starkly lower than control group, illustrates that improvement on synthesis G has obvious antiviral activity.
The discovery of this polypeptide G and Biological Activity Identification, the exploitation of novel antiviral medicine has important application prospect.
Claims (5)
1. a linear improvement on synthesis G, is characterized in that: meet the aminoacid sequence shown in SEQ ID No.1 in sequence list;
The cysteine residues C (Me) that two cystine C residue in described linear improvement on synthesis G aminoacid sequence are modified for methylate (Me).
2. according to linear improvement on synthesis G according to claim 1, it is characterized in that: described linear improvement on synthesis G is derived from the LPS binding domains of Fenneropenaeus chinensis Anti-LPS factor ALFFc.
3. according to linear improvement on synthesis G according to claim 2, it is characterized in that: the LPS binding domains of described Fenneropenaeus chinensis Anti-LPS factor ALFFc, the aminoacid sequence of its aminoacid sequence as shown in SEQ ID No.2 in list, is specially: ECKFTVKPYIKRFQLYYKGRMWCP.
4., according to the application of the arbitrary described linear improvement on synthesis G of claim 1-2, it is characterized in that:
Described linear improvement on synthesis G is for the preparation of the application of anti-virus formulation or antiviral, and the propagation of described preparation or drug on viral has the effect of suppression, and described virus is white spot syndrome virus.
5., according to the application of improvement on synthesis G linear described in claim 4, it is characterized in that:
Described linear improvement on synthesis G is as the active ingredient of anti-white spot syndrome virus.
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| CN201310534388.9A CN103554232B (en) | 2013-11-01 | 2013-11-01 | Linear synthetic peptide G and antiviral application thereof |
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| CN201310534388.9A CN103554232B (en) | 2013-11-01 | 2013-11-01 | Linear synthetic peptide G and antiviral application thereof |
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| CN103554232A CN103554232A (en) | 2014-02-05 |
| CN103554232B true CN103554232B (en) | 2015-04-08 |
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Non-Patent Citations (3)
| Title |
|---|
| Modification of a synthetic LPS-binding domain of anti-lipopolysaccharide factor from shrimp reveals strong structure-activity relationship in their antimicrobial characteristics;Shuyue Guo等;《Developmental and comparative immunology》;20140321;第45卷(第2期);第227-232页 * |
| 中国明对虾抗脂多糖因子在毕赤酵母中的高效表达;张继泉等;《中国甲壳动物学会第十一届年会暨学会研讨会论文摘要集》;20110325;第219页 * |
| 中国明对虾抗脂多糖因子基因克隆及其在大肠杆菌中的重组表达;周文杰;《中国优秀硕士学位论文全文数据库》;20070915;第2007卷(第3期);第D052-5页 * |
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