CN103554232A - Linear synthetic peptide G and antiviral application thereof - Google Patents

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

A kind of synthetic polypeptide G and antiviral application thereof of linearity

Technical field

The present invention relates to a kind of synthetic polypeptide G and antiviral application thereof of linearity, specifically linear polypeptide and the antiviral application thereof of a kind of synthetic lipopolysaccharides derived from Fenneropenaeus chinensis Anti-LPS factor ALFFc (LPS) binding domains.

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 it is fundamentally controlled that the deterioration of antibiotic abuse and environment is difficult to the disease of marine cultured animal.

Antibacterial peptide (albumen) is considered to the important effect molecule of the external source pathogen infections such as the defense against bacterial such as fish, shrimp, shellfish, virus, in the congenital immunity process of animal, plays a significant role.The antibacterial peptide of finding from crustacean is at present of a great variety, and coagulogen (anti-lipopolysaccharide factor, ALF) is exactly a kind of important antibacterial peptide wherein.Nineteen eighty-two, Tanaka etc. are the separated ALF that obtains from the hemocyte of Tachypleus tridentatus (Tachypleus tridentatus) and North America king crab (Limulus polyphemus) first, and finds that it can suppress the activation of the blood coagulation reaction of LPS mediation.1985, it is active that the discovery ALF such as Morita also have very strong anti-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 the recombinant protein that confirms ALF has the activity of anti-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 in advance after hatching together with recombinant expressed ALF albumen again, can suppress copying of WSSV in shrimp body.

Different from the antibiotic mechanism of action of tradition, ALF directly in and the lipopolysaccharides of bacteria cell wall, dissolution of bacteria, is therefore difficult for diseaseful resistance.ALF is still unclear at present to the viral mechanism of action.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 have been opened up wide prospect.

Research is found, between two conservative halfcystines in ALF aminoacid sequence, form disulfide linkage, and and between aminoacid sequence common form one section of negatively charged ion polypeptide, there is the ability of being combined with LPS, this section of conservative structure called after LPS binding domains, is considered to the functional domain of ALF degraded gram-negative bacteria cell wall lipopolysaccharides.2011, Sachin Sharma etc. has studied and has acted in antibacterial immunity process according to the polypeptide of synthetic 24 amino-acid residues of tool of the LPS binding domains of Young Crab (Scylla serrata) ALF, find that synthetic polypeptide SsALF24 has in conjunction with the active of LPS and intestinal bacteria are all had to obvious restraining effect, 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 mimics the LPS binding domain of Indian mud crab, Scylla serrata anti-lipopolysaccharide factor (SsALF) also involved in the modulation of vaginal immune functions through NF-kB signaling.Microbial pathogenesis2011, 50:179-191.).

Interestingly, kind and the active size thereof of different types of ALF cause of disease that polypeptide is resisted all have obvious difference, point out us to obtain the peptide molecule with high resistance or special resistance by changing indivedual amino acid whose kinds in LPS binding domains.Based on this imagination; we are the FS of the Crustin ALFFc(Liu based on having found; Liu YC; Li FH; Dong B, Xiang JH.Molecular cloning and expression profile of putative antilipopolysaccharide factor in Chinese shrimp (Fenneropenaeus chinensis) .Marine Biotechnology2005; 7:600-608.) the change of the partial amino-acid residue that carries out of LPS binding domains, utilize the method for chemosynthesis to obtain linear polypeptide G, this linear polypeptide has significantly special antiviral biologic activity, has important application prospect.

Summary of the invention

The present invention aims to provide a kind of linear synthetic polypeptide 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 synthetic polypeptide 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 synthetic polypeptide G aminoacid sequence are the cysteine residues C(Me that methylates and modify); The amino of the aminoterminal E of polypeptide is acetylation (Ac-), and the carboxyl of carboxyl terminal P is amidated (NH ₂).

Described linear synthetic polypeptide G, described linear synthetic polypeptide 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, has the aminoacid sequence shown in SEQ ID No.2 in sequence list, and concrete sequence is: ECKFTVKPYIKRFQLYYKGRMWCP.

Described linear synthetic polypeptide G has obvious antiviral activity.Be specially: viral propagation is had to very strong restraining effect.

The synthetic polypeptide G of described linearity 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 has determined a kind of antiviral polypeptide G and constitutional features thereof derived from Fenneropenaeus chinensis Anti-LPS factor ALFFc.

2, by the present invention, can develop the control medicine of effective prawn ' s virus class disease.

Accompanying drawing explanation

The skeleton structure diagram of the synthetic polypeptide G of Fig. 1;

The space structure figure of the synthetic polypeptide G of Fig. 2;

The HPLC purity detecting figure of the synthetic polypeptide G of Fig. 3;

The MS Mass Spectrometric Identification figure of the synthetic polypeptide G of Fig. 4.

Embodiment

Below in conjunction with embodiment, the present invention is described in further detail.

A prawn linear polypeptide G with obvious antiviral activity, its sequence and source sequence information as follows:

(1) information of SEQ ID No.1

(a) sequence signature

* length: 24 amino acid

* type: amino acid

* chain: strand

* topological framework: linearity, the modification that methylated of two halfcystines

* space structure: have 1) skeleton structure shown in Fig. 1: the halfcystine in linear polypeptide sequence (C) modifications that methylated, the sulfydryl of the modification that methylates represents with intensification black, C skeleton and other atom represent by grey; With 2) space structure shown in Fig. 2: peptide sequence forms two β-pleated sheet structure structures that are 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 ₂the carboxyl that represents 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: linearity

(b) molecule type: albumen

Sequence description: SEQ ID No.2

ECKFTVKPYIKRFQLYYKGRMWCP

Synthetic, the purifying of described antiviral polypeptide G, evaluation and Analysis on Biological Activity:

By artificial chemistry route of synthesis, utilize solid phase synthesis process to obtain crude product polypeptide, through Mass Spectrometric Identification and liquid chromatography purifying, obtain synthetic polypeptide G.Be specially:

1) polypeptide is synthetic

Adopt 9-fluorenylmethyloxycarbonyl (Fmoc) synthesis strategy, synthetic to N extreme direction from C end.With 10mg Rink-Amide-Resin resin (AAPPTec, article No. RRZ001), as carrier, rely on active group and the 5mg of carrier itself by Fmoc, to be carried out first amino acid (Fmoc-Pro-NH of amido protecting ₂) carboxyl (the method detailed reference: Panagiotis Stathopoulos that is connected; Serafim Papas, Vassilios Tsikaris.C-terminal N-alkylated peptide amides resulting from the linker decomposition of the Rink amide resin.A new cleavage mixture prevents their formation.Journal of Peptide Science2006; 12:227-232.).

With N-methyl, adjoin and cough up Anhui intoxicated (NMP) and rinse resin and remove redundant protection amino acid, in reactor (solid phase synthesis device), add 20% piperidines/nmp solution (volume fraction) to remove Fmoc group, reaction 20min, emptying reactor, with 5mL NMP vibration, rinse resin, repeat 3 times, remove the Fmoc protection of first amino-acid residue; The active amino group exposing is connected with the carboxyl that the next one is carried out the amino acid (5mg) of amido protecting by Fmoc, forms first peptide bond (Pro-Cys).The above step cycle of this section is carried out (difference is: just at every turn need to adopt the corresponding amino acid of amido protecting that undertaken by Fmoc) until peptide sequence Ac-EC (Me) KFTVKPYIKRFQLYYKGRMWC (Me) P-NH ₂synthetic complete, wherein in building-up process, halfcystine C residue used is the halfcystine C(Me that methylates and modify), the linearity that finally obtains about 20mg is synthesized polypeptide.

2) peptide purification and evaluation

With 50mL, cut peptide reagent trifluoroacetic acid: thioanisole: phenol: dithioglycol: distilled water (volume ratio 82.5:5:5:2.5:5) gets off synthetic 20mg linear polypeptide cracking from vector resin, after 2 hours, add the ether 100ml of 4 ℃ of precoolings to make polypeptide precipitation, centrifugal collecting precipitate, and wash 3 times with ether, vacuum is drained, and the polypeptide crude product obtaining, 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 μ m; Mobile phase A: 0.1%TFA/ acetonitrile, Mobile phase B: 0.1%TFA/H ₂o; Linear elution gradient: 15%A-100%A; Flow velocity is 1ml/min, and detection wavelength is 220nm; Single injected sampling amount is 10 μ l.HPLC and the demonstration of MS detected result, the purity of synthetic polypeptide G is 95.76%, molecular weight is 3158.84, basically identical with predicted molecular weight (3154.82).

3) virus neutralization experiment

Press PBS for embodiment bacteriostatic test step and dissolve synthetic polypeptide G, with the synthetic polypeptide of final concentration 50 μ mol/L at room temperature with WSSV(white spot syndrome virus, white spot syndrome virus) hatch 2h, with the irrelevant polypeptide (the green fluorescent protein GFP peptide section of equal length) of same concentrations, as negative control, hatch under the same conditions WSSV, WSSV after hatching injects respectively dusky white prawn (Exopalaemon carinicauda), and the injection volume of every tail shrimp is 10 ⁴copy WSSV particle, after injection 24h, get dusky white prawn swimmeret, adopt the DNA extraction test kit (article No.: DP324) extract total DNA of Tian Gen biochemical technology company limited, gradient dilution WSSV is as standard substance, utilize WSSV copy number to detect primer pair VP28rF(5 '-AAACCTCCGCATTCCTGTGA-3 ') and VP28rR(5 '-TCCGCATCTTCTTCCTTCAT-3 '), adopt increase respectively expression amount (the detection method reference in detail: Yumiao Sun of VP28 gene in standard substance and prawn DNA sample to be measured of Real-Time Fluorescent Quantitative PCR Technique, Fuhua Li, Jianhai Xiang.Analysis on the dynamic changes of the 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 synthetic polypeptide 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 synthetic polypeptide G has obvious antiviral activity.

Discovery and the Biological Activity Identification of this polypeptide G have important application prospect in the exploitation of novel antiviral medicine.

Claims (7)

1. a linear synthetic polypeptide G, is characterized in that: have the aminoacid sequence shown in SEQ ID No.1 in sequence list.

2. according to the synthetic polypeptide G of linearity claimed in claim 1, its sequence signature is: SEQ ID No.1:Ac-EC (Me) KFTVKPYIKRFQLYYKGRMWC (Me) P-NH ₂.

3. according to the linear synthetic polypeptide G described in claim 1 or 2, its constitutional features is:

The cysteine residues C(Me that two halfcystine C residues in described linear synthetic polypeptide G aminoacid sequence are modified for methylate (Me)).

4. according to the linear synthetic polypeptide G described in claim 1 or 2, it is characterized in that: described linear synthetic polypeptide G is derived from the LPS binding domains of Fenneropenaeus chinensis Anti-LPS factor ALFFc.

5. according to the synthetic polypeptide G of linearity claimed in claim 4, it is characterized in that: the LPS binding domains of described Fenneropenaeus chinensis Anti-LPS factor ALFFc, there is the aminoacid sequence shown in SEQ ID No.2 in sequence list, be specially: ECKFTVKPYIKRFQLYYKGRMWCP.

6. an application of the synthetic polypeptide G of the arbitrary described linearity of claim 1-4, is characterized in that:

The synthetic polypeptide G of described linearity is inhibited to viral propagation, and it can be used in antiviral preparation.

7. according to the application of synthetic polypeptide G linear described in claim 6, it is characterized in that:

The synthetic polypeptide G of described linearity can be used as antiviral active ingredient, in antiviral drug or preparation.

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