CN103539847A - Cyclic synthetic peptide B, and antibacterial and antiviral application thereof - Google Patents

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

A kind of synthetic polypeptide B of ring-type and anti-bacteria and anti-virus application thereof

Technical field

The present invention relates to a kind of synthetic polypeptide B and anti-bacteria and anti-virus application thereof of ring-type, specifically the ring type polypeptide of a kind of synthetic lipopolysaccharides derived from Fenneropenaeus chinensis Anti-LPS factor FcALF2 (LPS) binding domains and anti-bacteria and anti-virus 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 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, Patgaonkar MS, 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.).

FcALF2 in Crustin is found, research shows that it responds viral infection (Li SH in prawn body, Zhang XJ, Sun Z, Li FH, Xiang JH.Transcriptome analysis on Chinese shrimp Fenneropenaeus chinensis 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 annular polypeptide 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 synthetic polypeptide B that derives from the ring-type of prawn, has significantly antibiotic and antiviral activity.

Technical scheme of the present invention is as follows:

Utilize the method for chemosynthesis to obtain the synthetic polypeptide 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 synthetic polypeptide B sequence of described ring-type has disulfide linkage structure.Its constitutional features is: between synthetic polypeptide B sequence second halfcystine of aminoterminal (C) and second halfcystine of carboxyl terminal (C), have disulfide linkage structure; The amino of the aminoterminal Y of polypeptide is acetylation (Ac-), and the carboxyl of carboxyl terminal P is amidated (NH ₂).

The synthetic polypeptide B of described ring-type is from the LPS binding domains of Fenneropenaeus chinensis Anti-LPS factor FcALF2.FcALF2 is characterised in that: have the aminoacid sequence shown in SEQ ID No.2 in sequence list.

The synthetic polypeptide B of described ring-type has obvious antimicrobial antiviral activity.Be specially: the growth of gram-positive microorganism and virus or propagation are all had to very strong restraining effect, the growth of Gram-negative bacteria is had to certain restraining effect.

The synthetic polypeptide B of described ring-type can be used as antibiotic and/or antiviral active ingredient, in antibiotic and/or antiviral drug or preparation.

The present invention has the following advantages:

1, the present invention has determined a kind of anti-bacteria and anti-virus polypeptide B and constitutional features thereof derived from Fenneropenaeus chinensis Anti-LPS factor FcALF2.

2, by the present invention, can develop the control medicine of effective prawn bacterium class and virus type disease.

Accompanying drawing explanation

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

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

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

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

Embodiment

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

There is an obviously prawn ring type polypeptide B for antibiotic and antiviral activity, its sequence and source sequence information are 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 represents with intensification black, and 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-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 ₂the carboxyl that represents 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: linearity

(b) molecule type: albumen

Sequence description: SEQ ID No.2

MRVSVFSMIIVLAVAASFAPQCQASGWEALVPAIADKLTGLWESGELELLGHYCSFNVTPKFKRWQLYFRGRMWCPGWTTIGGQAETRSRSGVVGRTTQDFVRKAFRAGLITESEAQAWLNN

Synthetic, the cyclisation of described anti-bacteria and anti-virus polypeptide B, purifying, evaluation and Analysis on Biological Activity:

By artificial chemistry route of synthesis, utilize solid phase synthesis process to obtain crude product polypeptide, through solid phase cyclization, Mass Spectrometric Identification and liquid chromatography purifying, obtain the synthetic polypeptide B that contains disulfide linkage.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-YCSFNVTPKFKRWQLYFRGRMWCP-NH ₂synthetic complete, obtain about 20mg linear polypeptide.

2) polypeptide cyclisation

After complete last amino acid of 20mg linear polypeptide coupling, 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 to solid phase synthesis device, nitrogen brushes reaction, approximately 6 hours.

3) 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 the cracking from vector resin of the polypeptide after 20mg cyclisation, 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 B is 95.29%, molecular weight is 3157.32, basically identical with predicted molecular weight (3152.78).

4) bacteriostatic test

Synthetic polypeptide B is dissolved to the PBS of 50mmol/L pH7.4 the solution that concentration is 640 μ mol/L, and the while is with the negative contrast of PBS of 50mmol/L pH7.4.Adopt minimum inhibitory concentration (MIC) method to detect synthetic polypeptide Gram-negative bacteria is comprised to intestinal bacteria (Escherichia coli), Vibrio anguillarum (Vibrio anguillarum) and gram-positive microorganism comprise the bacteriostatic activity of micrococcus luteus (Micrococcus luteus), micrococcus lysodeikticus (Micrococcus lysodeikticus).That is: by intestinal bacteria to be measured, micrococcus luteus and micrococcus lysodeikticus respectively at 37 ℃, under 200r/min culture condition, in LB substratum, be cultured to 1 * 10 ⁸cells/mL, Vibrio anguillarum culture temperature is 28 ℃, other condition is the same constant; The bacterium of cultivating joins respectively in 48 well culture plates, with fresh LB substratum, bacterium liquid to be measured is diluted to final concentration 1 * 10 ⁶cells/mL; To adding respectively polypeptide B solution to the final volume of gradient dilution in 48 well culture plates, be 200 μ l, polypeptide B final concentration is followed successively by 64 μ mol/L, 32 μ mol/L, 16 μ mol/L, 8 μ mol/L, 4 μ mol/L, 2 μ mol/L and 1 μ mol/L; With PBS, as negative control, the kantlex (Vibrio anguillarum) of the penbritin of final concentration 58 μ mol/L (intestinal bacteria, micrococcus luteus, micrococcus lysodeikticus) or 88 μ mol/L is respectively as positive control; Under 37 ℃ or 28 ℃ of conditions, cultivate after 3h, add respectively the fresh LB substratum of 300 μ l, 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 synthetic polypeptide B of 32-64 μ mol/L can effectively suppress the growth of Vibrio anguillarum; To gram-positive microorganism, the synthetic polypeptide B of 2-4 μ mol/L can effectively suppress the growth of micrococcus luteus, and the synthetic polypeptide B of 1-2 μ mol/L can effectively suppress the growth of micrococcus lysodeikticus.Illustrate that synthetic polypeptide B has the activity of obvious resisting gram-positive bacteria and negative bacterium.

5) virus neutralization experiment

Press PBS for embodiment bacteriostatic test step and dissolve synthetic polypeptide B, 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 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 synthetic polypeptide B has obvious antiviral activity.

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

Claims (6)

1. a synthetic polypeptide B for ring-type, is characterized in that: have the aminoacid sequence shown in SEQ ID No.1 in sequence list.

2. according to the synthetic polypeptide B of ring-type claimed in claim 1, its constitutional features is:

The synthetic polypeptide B of described ring-type has disulfide linkage structure, between second halfcystine of aminoterminal (C) of polypeptide B and second halfcystine of carboxyl terminal (C), has disulfide linkage structure.

3. according to the synthetic polypeptide B of the ring-type described in claim 1 or 2, its sequence signature is: SEQ ID No.1:Ac-Yc (CSFNVTPKFKRWQLYFRGRMWC) P-NH ₂.

4. according to the synthetic polypeptide B of the ring-type described in claim 1 or 2, it is characterized in that: the synthetic polypeptide B of described ring-type is derived from the LPS binding domains of Fenneropenaeus chinensis Anti-LPS factor FcALF2, and FcALF2 has the aminoacid sequence shown in SEQ IDNo.2 in sequence list.

5. an application of the synthetic polypeptide B of the arbitrary described ring-type of claim 1-4, is characterized in that:

The synthetic polypeptide B of described ring-type all has very strong restraining effect to the growth of gram-positive microorganism or virus or propagation, and the growth of Gram-negative bacteria is had to certain restraining effect, and it can be used in antibiotic and/or antiviral preparation.

6. according to the application of the synthetic polypeptide B of ring-type described in claim 5, it is characterized in that:

The synthetic polypeptide B of described ring-type can be used as antibiotic and/or antiviral active ingredient, in antibiotic and/or antiviral drug or preparation.

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