CN101862334A

CN101862334A - Application of evodiamine in preparation of preparation for resisting white spot syndromevirus

Info

Publication number: CN101862334A
Application number: CN201010198342A
Authority: CN
Inventors: 章晓波; 智斌
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2010-06-11
Filing date: 2010-06-11
Publication date: 2010-10-20
Anticipated expiration: 2030-06-11
Also published as: CN101862334B

Abstract

The invention provides application of evodiamine in the preparation of a preparation for resisting white spot syndromevirus. Evodiamine can act on the protein essential for the apoptosis pathway of prawns, so the small molecules can be utilized to prepare the preparation for resisting the white spot syndromevirus so as to greatly enhance the resistance of the prawns to the white spot syndromevirus (WSSV) and reduce the fatality rate. The advantages of the invention are mainly embodied in that: the evodiamine can effectively induce activation of the PjCaspase, greatly enhance the resistance of the prawns to the white spot syndromevirus (WSSV) and reduce the fatality rate; and because the evodiamine is the medicament which is widely used in human body for improving the immunity, the evodiamine has no toxicity to the human body, and the prawns using the evodiamine can be enjoyed with safety no matter whether the medicament is completely metabolized.

Description

The application of rutaecarpin in the preparation of the anti-prawn white spot syndrome of preparation

(1) technical field

The present invention relates to the application of rutaecarpin (Evodiamine) in the preparation of the anti-prawn white spot syndrome of preparation.

(2) background technology

Prawn is the most dominant crustacean in tropical and subtropical zone shallow sea.Because its kind is many, population quantity is big, and reproductive capacity is strong, and growth is rapid, and the meat flavour deliciousness has very high economic worth.Reason owing to aspects such as environmental pollution, cultural technique, disease controls, prawn, particularly Penaeus monodon (Penaeus monodon), Penaeus vannamei (Penaeus vannamei), Chinese prawn (Penaeuschinensis) and japonicus (Marsupenaeus japonicus) etc., very easily be subjected to virus infraction, and cause a large amount of losses.(White spot syndrome virus WSSV), is the main cause of disease that causes most serious harm in the prawn culturing to white spot syndrome virus (WSSV).Make full use of innate immune system and the immunologic mechanism of prawn, resist virus by the disease-resistant mechanism that stimulates prawn targetedly, for prevention, the treatment of shrimp disease, the economic benefit that improves prawn culturing has great importance.

Prawn is one of most important sea-farming product, but since generation nineteen ninety, be subjected to the serious threat of disease always, especially white spot syndrome virus (WSSV) (white spot syndromevirus, WSSV) be the main cause of disease of harm prawn culturing, its pathogenecity is strong, and spread scope is wide, cause tremendous loss for world's shrimp culture industry, yet still do not have the effectively preventing method so far.

Many researchs at shrimp disease only rest on application owing to it, not the disease-resistant mechanism of resultant medicine are carried out more deep discussion, so the application of its medicine has been subjected to certain restriction.Owing to can't analyze at its mechanism of action, the problem that produces in these drug use processes also can not get answer, and then can't effectively solve simultaneously.Therefore, it is very necessary to study the disease-resistant medicine of known pharmacology.

Prawn is mainly resisted disease by processes such as its physical barriers, stress, congenital humoral immunization, cell physiological adjustings as a member of invertebrates.Apoptosis is as removing old and feeble and paracytic main mechanism, prawn catch an illness cell removing and prevent that virus has important function in spreading, be the important means of prawn disease resistance.And be the important weapon that to resist viral infringement.

Two subfamilies in the Caspase protein family are served as the initial sum effect protein respectively in apoptosis, be apoptotic key protein.Serve as the caspase-8,9 etc. of initiation, receive apoptotic signal after, can cause the caspase cascade reaction then by activating from montage; Serve as the caspase-3,6 of effect effect, structural protein and the functional protein in the born of the same parents that can directly degrade such as 7, cause apoptosis.

(3) summary of the invention

The object of the invention provides the application of rutaecarpin (Evodiamine) in the preparation of the anti-prawn white spot syndrome of preparation.

The technical solution used in the present invention is:

The application of rutaecarpin (Evodiamine) in the preparation of the anti-prawn white spot syndrome of preparation.

Rutaecarpin, extraction is from the fruit of rutaceae Fructus Evodiae [Evodia rutaecarpa (Juss.) Benth.] and congener stone tiger [E.rutaecarpa (Juss.) Benth.var.officinalis (Dode) Huang], CAS:518-17-2, structural formula is as follows:

Rutaecarpin has and is good for the stomach, eases pain, ends and retch and end effect such as acid eructation; Diuresis is arranged; Escherichia coli there is powerful inhibitory action; Ascaris suum there is remarkable insecticidal action; Shrink uterus and boosting in addition; Treatment degenerative brain disorder and apoplexy have certain curative effect.The present application people finds that this micromolecule can act on prawn apoptosis pathway key protein, therefore can utilize this micromolecule to prepare the preparation of anti-prawn white spot syndrome, can greatly strengthen the resistance of prawn to prawn white spot syndrome (WSSV), reduces fatality rate.

Described rutaecarpin can be used for preparing the preparation of inducing the PjCaspase protein activation.

Preferably, described preparation is an intramuscular dose, and wherein the injected dose of Evodiamine is 1～5 μ g/g shrimp (about preferred 3.5 μ g/g shrimps).

Evodiamine of the present invention promptly acts on the relevant micromolecule of the anti-white spot syndrome of prawn of prawn apoptosis pathway key protein PjCaspase.By direct injection Evodiamine or carry out feeding and can strengthen the opposing of prawn by activating the proteic activity of PjCaspase to virus, greatly strengthen the resistance of prawn to prawn white spot syndrome (WSSV), reduce fatality rate.

Beneficial effect of the present invention is mainly reflected in: Evodiamine of the present invention can effectively induce the proteic activation of PjCaspase, greatly strengthens the resistance of prawn to prawn white spot syndrome (WSSV), reduces fatality rate; Because Evodiamine is the medicine of widely used raising immunity in human body, and human body is not had toxicity, use the prawn of Evodiamine, no matter whether it can relieved eat drug metabolism fully.

(4) description of drawings

Fig. 1 be Evodiamine in cell with the checking of Caspase protein binding ability;

Fig. 2 is the influence of Evodiamine to apoptosis activity; A is that the activity of apoptosis index effect enzyme Caspase3 detects; B is an apoptosis index TUNEL activity;

Fig. 3 is the influence of Evodiamine to virus replication;

Fig. 4 is the influence of Evodiamine to the mortality rate of infection WSSV prawn; Abscissa is represented natural law, and vertical coordinate is represented mortality rate.

(5) specific embodiment

The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:

Embodiment 1:

1, the checking of the binding ability of Evodiamine and PjCaspase:

1.1 amplification PjCaspase gene.

1.1.1 the prawn tissue cDNA is synthetic:

1.1.1.1 the extraction of prawn total tissue RNA:

1) gets the fresh and alive 200mg of organizing of prawn (japonicus is available from food market, Hangzhou, Zhejiang province city), in 800 μ L Trizol (BBI company), grind rapidly.

2) put 5min on ice.

3) (phenol and chloroform equal-volume mix the back and use 0.1mol/LTris.HCl (pH7.6) extracting several times with this mixture of balance to add 200 μ L phenol/chloroformic solutions, put in the Brown Glass Brown glass bottles and jars only, cover isopyknic 0.01mol/l Tris.HCl (pH7.6) liquid layer above, be stored in 4 ℃; Draw lower floor's solution with suction pipe during use), behind the vibration mixing 12, the centrifugal 10min of 000rpm.

4) pipette supernatant to the equivalent isopropyl alcohol.

5) vibration mixing after 12, the centrifugal 10min of 000rpm.

6) precipitation does not have RNA enzyme distilled water dissolving back adding 1 μ LDNase I (RNaseFree) (TaKaRa) with 50 μ L, digests 1 hour.

7) repeating step 3)～5).

8) precipitation is washed twice with 300 μ L80% (v/v) ethanol.

9) 12000rpm, after 2min dried ethanol, 50 μ L did not have the dissolving of RNA enzyme distilled water.

1.1.1.2 the reverse transcription of total RNA obtains cDNA:

Following compositions is put into the Eppendolf pipe of a RNase-free:

Total RNA 4g

Oligo(dT)(500μg/mL) 1μL

75 ℃ of degeneration 5min, ice bath, adding subsequently immediately:

5 * reverse transcriptase buffer, 4 μ L

4 kinds of dNTPs mixed liquors, the 2 μ L of each 10mmol/L

M-MLV?RT(RNase?H-)(200U/μL)(TaKaRa) 1μL

Water complements to 20 μ L

25 ℃ of reaction 10min, behind 42 ℃ of reaction 30min, 70 ℃ of heating 15min cessation reactions.

1.1.2PCR amplification:

Synthesize following primer (being depicted as the restriction enzyme site of the line part of introducing in the bracket) according to the gene reading frame sequence of having determined:

5’-AT CCCGGGATGGACGAGGTAATCCAG-3’(SmaI)

5’-TT CTCGAGTCACTGTGGGGCGGAG-3’(XhoI)

With prawn cDNA is template, the genetic fragment that pcr amplification is special.In the Eppendorf of 0.5mL pipe, add:

10 * PCR reactant liquor, 5 μ L

dNTP(2.5mmol/L?each) 4μL

Template (about 1 μ g/ μ L) 2 μ L

Each 1 μ L of 20mol/L gene-specific primer

Taq enzyme (TaKaRa) 1U

Add H ₂O is to cumulative volume 50 μ L

Carry out pcr amplification by the following method: 95 ℃ of 5min; 94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 1.5min, 35 circulations; 72 ℃ are extended 10min.

1.1.3 fragment reclaims:

Reclaim dna fragmentation according to a conventional method from agarose gel, the glue recovery test kit of producing with omega is an example:

1) cuts off the agar caked sugar that contains DNA, make it as far as possible little, put into the 1.5mL centrifuge tube.If it is agarose weight less than 100mg, is mended to 100mg with distilled water, unlikely too little to guarantee the system cumulative volume;

2) ratio that adds 300 μ L S1 liquid in every 100mg agarose adds S1 liquid (test kit carries), puts 50 ℃ of water-bath 10min, and the agar caked sugar is dissolved fully, and every 2min puts upside down mixing once;

3) agarose solution after will dissolving moves into adsorption column, and the centrifugal 30s of 7000rpm outwells the liquid in the collecting pipe, adsorption column is put into same collecting pipe again;

4) add 500 μ L W1 liquid (test kit carries) in adsorption column, the centrifugal 15s of 7000rpm outwells the liquid in the collecting pipe, and adsorption column is put into same collecting pipe;

5) add 500 μ L W1 liquid in adsorption column, leave standstill 1min, the centrifugal 15s of 7000rpm outwells the liquid in the collecting pipe, and adsorption column is put into same collecting pipe;

7) the centrifugal 1min of 7000rpm;

8) adsorption column is put into the centrifuge tube of a clean 1.5mL, central authorities add 10 μ L sterilized water at adsorbed film, leave standstill 1min, and the centrifugal 1min of 10000rpm is stored in-20 ℃ with the EP pipe.

Transform carrier 1.2 be cloned into PIZ:

1.2.1 endonuclease reaction:

In 0.5mL Eppendorf pipe, add successively:

Restriction endonuclease buffer (10 *) is 2 μ L (TaKaRa)

The about 0.5 μ g of DNA

Restriction endonuclease (TaKaRa) 0.5 μ L

Add water to cumulative volume 20 μ L

37 ℃ of water-bath 2～3h.70 ℃ of 15min inactivations.

Reclaim according to a conventional method

Be transformed into competent escherichia coli cell 1.2.2 connect

1) (original pIZ/V5-His carrier is connected into EGFP albumen and is transformed available from Invitrogen 6 μ L purpose fragment+1.5 μ L in advance with the transformation pIZ/V5-His carrier of enzyme double digestion.The EGFP fragment can obtain by buying pEGFP (BD Biosciences Clontech))+1 μ L T4buffer (TaKaRa)+0.25 μ L T4Ligase (TaKaRa)+1.5 μ LH ₂16 ℃ of connections of O are spent the night.

2) get the DH5 α competent cell of-70 ℃ of preservations, make it melt the back with hand rubbing and flick with finger, the mixing thalline adds an amount of plasmid or connects product, places 30min on ice.

3) 42 ℃ of water-bath thermal shock 90s, ice bath 1～2min rapidly.

4) add 400 μ L LB fluid mediums, low speed (100rpm) shakes 45min on 37 ℃ of shaking tables.

5) draw bacterium liquid 100～200 μ L, evenly coat on LB (the containing 100 μ g/mL Amp) flat board, dull and stereotyped room temperature leaves standstill 20～30min, treat that bacterium liquid immerses culture medium after, be inverted in 37 ℃ of overnight incubation.

(6) bacterium colony PCR primary dcreening operation recon, enzyme action are identified the back order-checking.

1.3 High Five cell is advanced in transfection

Sequence verification is inserted fragments sequence correct (its sequence is shown in SEQ ID NO:1) back:

A) mix 100 μ L ExpressFiveSFM culture medium (Invitrogen) that contain 1 μ g clone and 100 μ L ExpressFiveSFM culture medium (Invitrogen) culture medium that contain 1 μ g cellinfectin (Invitrgen).

B) leave standstill 45min.

C) add 800 μ L ExpressFiveSFM culture medium (Invitrogen), mixing.

D) join in the clear HighFive cell (Invitrogen) of collapse due to massive hemorrhage, room temperature leaves standstill 4h.

E) change clean ExpressFiveSFM culture medium (Invitrogen) overnight incubation.

1.4 after adding Evodiamine cultivates 48h together in ExpressFiveSFM culture medium (Invitrogen), with the proteic cell of single expression EGFP in contrast, the cell of expressing PjCaspase and EGFP fusion rotein is as experimental group, with Flex Station IImicroplate reader (Molecular Devices, USA) fluorescence intensity, the results are shown in Figure 1, abscissa is represented the consumption of Evodiamine, the longitudinal axis is represented the fluorescence intensity (being the binding ability of Evodiamine and PjCaspase) of cell, and as seen from the figure: Evodiamine can combine with Caspase albumen in cell effectively.

2, Evodiamine is to the influence of apoptosis activity:

2.1 sample process:

Get 20 one group of big or small well-balanced prawn, respectively injecting normal saline (100 μ L), WSSV virus (10 ⁴Copy), WSSV virus (10 ⁴Copy)+Evodiamine (dosage is that 3.5 μ g/g body weight shrimps are each, with the equivalent physiological saline solution), Evodiamine (dosage is that 3.5 μ g/g body weight shrimps are each, with the equivalent physiological saline solution).Wherein micromolecule Evodiamine injects preceding 12 hours, the injection in 0,12,24 hour of injection back at WSSV, and other group after-teemings are simultaneously penetrated equal normal saline and handled in contrast.Prawn after the processing was at 24,36,48 hours blood samplings.

2.2Caspase active the detection:

(1) extracts prawn blood at 1: 1 with the 500U heparin sodium.

(2) with caspase3/7 reaction substrate powder with the reaction Buffer (

3/7assay kit Promega) mixes.

(3) with 100ul shrimp blood and 100ul reaction mixture mixing (

3/7 assaykit, Promega).

(4) 25 ℃ of following lucifuge incubations 3 hours.

(5) detect chemiluminescence with the chemiluminescence microplate reader

2.3Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method detects apoptosis activity

(1) on microscope slide, evenly is coated with the shrimp hemocyte.

(2) microscope slide is immersed in 4% paraformaldehyde (worker is given birth in the Shanghai) solution, fix 25 minutes for 4 ℃.

(3) give a baby a bath on the third day after its birth time each 5 minutes under the room temperature with PBS.

(4) 2%Triton X-100 oozed 5 minutes in advance.

(5) PBS washes twice.

(6) with 100 μ L level pad (TUNEL test kit, Promega) balances.

(7) (the TUNEL test kit, Promega), 37 ℃ of lucifuge incubations are 1 hour in the wet box (be paved with the gauze that gets wet in the plastic lunch box and get final product, also can adopt the sharp clever product in Shanghai) to add 50 μ L rTdT.

(8) add 2 * SSC (TUNEL test kit, Promega) cessation reaction.

(9) PBS gives a baby a bath on the third day after its birth inferior.

(10) with 1 μ g/ml PI (Shanghai give birth to worker) solution-dyed 15 minutes.

(11) PBS gives a baby a bath on the third day after its birth inferior.

(12) microscopy under the fluorescence microscope.

Fig. 2 adopts the prawn of normal saline, WSSV virus, WSSV virus+Evodiamine, Evodiamine processing at 24,36,48 hours detected apoptosis activities.As seen from the figure, Evodiamine has greatly improved apoptosis activity.

3, Evodiamine is to the influence of virus replication:

3.1 the prawn genome extracts

Extract according to the total DNA of prawn after the processing of 2.1 methods with SQ Tissue Kit (Promega).

3.2Real-time PCR measures viral copy number

Synthesize the Taqman probe and compare 50 ℃ of 4min, (95 ℃ of 45s, 52 ℃ of 45s, 72 ℃ of 45s) 45 circulations of * with b-actin.Standard curve is made by the WSSV plasmid of concentration known.

Fig. 3 is for adopting WSSV virus, WSSV virus+Evodiamine (3.5 μ g/g, with physiological saline solution), detected WSSV virus copy number changed WSSV virus+Evodiamine (0.35 μ g/g is with physiological saline solution) prawn of handling at 36,48 hours.As seen from the figure, Evodiamine has effectively suppressed WSSV and has duplicated in that prawn is intravital.

3, mortality rate is measured and is got 20 one group of big or small well-balanced prawn, respectively injecting normal saline (100 μ L), WSSV virus (10 ⁴Copy), WSSV virus (10 ⁴Copy)+Evodiamine (dosage is that 3.5 μ g/g body weight shrimps are each, with the equivalent physiological saline solution), Evodiamine (dosage is that 3.5 μ g/g body weight shrimps are each, with the equivalent physiological saline solution).Wherein micromolecule is injected preceding 12 hours, the injection in 0,12,24 hour of injection back at WSSV, and other group after-teemings are simultaneously penetrated equal normal saline and handled in contrast.The record mortality rate.

The mortality rate variation of prawn in six days that Fig. 4 handles for adopting normal saline, WSSV virus, Evodiamine, WSSV virus+Evodiamine.As seen from the figure, Evodiamine effectively reduces the mortality rate of infection WSSV prawn and prawn is not had toxicity, can be extensive use of in prawn culturing.

Claims

1. the application of rutaecarpin (Evodiamine) in the preparation of the anti-prawn white spot syndrome of preparation.

2. application as claimed in claim 1 is characterized in that described rutaecarpin is used to prepare the preparation of inducing the PjCaspase protein activation.

3. application as claimed in claim 1 or 2 is characterized in that described preparation is an intramuscular dose, and wherein the injected dose of Evodiamine is 1～5 μ g/g shrimp.