CN101862444B - Application of interleukin 2 in preparing preparation for resisting prawn white spot syndrome virus - Google Patents

Abstract

The invention provides the application of interleukin 2 (Interleukin-2) in the preparation of anti-prawn white spot syndrome preparation. Interleukin-2 can act on the key protein of shrimp apoptosis pathway, so this small molecule can be used to prepare anti-white spot syndrome preparations, which can greatly enhance the resistance of shrimp to white spot syndrome (WSSV) and reduce the lethality rate. The beneficial effects of the present invention are mainly reflected in: Interleukin-2 of the present invention can effectively induce the activation of PjCaspase protein, greatly enhance the resistance of prawns to prawn white spot syndrome (WSSV), and reduce the lethal rate; Immunity-enhancing drugs widely used in the human body are non-toxic to the human body, and the prawns using Interleukin-2 can be safely eaten regardless of whether the drug is completely metabolized or not.

Description

Application of Interleukin 2 in Preparation of Anti-White Spot Syndrome of Shrimp

(1) Technical field

The invention relates to the application of interleukin-2 in the preparation of preparations against white spot syndrome of prawns.

(2) Background technology

Shrimp is the most dominant crustacean in tropical and subtropical shallow seas. Because of its many types, large population, strong fecundity, rapid growth, and delicious meat, it has high economic value. Due to environmental pollution, breeding technology, disease control and other reasons, shrimp, especially Penaeus monodon, Penaeus vannamei, Penaeuschinensis and Marsupenaeus japonicus, etc. Infected by viruses and caused a lot of damage. White spot syndrome virus (WSSV) is the most serious pathogen in shrimp farming. Making full use of the innate immune system and immune mechanism of prawns and stimulating the disease-resistant mechanism of prawns to resist viruses is of great significance for the prevention and treatment of prawn diseases and improving the economic benefits of prawn farming.

Shrimp is one of the most important mariculture products, but since the 1990s, it has been seriously threatened by diseases, especially white spot syndrome virus (white spot syndrome virus, WSSV) is the most important pathogen that harms cultured shrimp. It has strong ability and wide spread, which has caused huge losses to the shrimp farming industry in the world, but there is still no effective control method so far.

Many studies on shrimp diseases are only at the application level, and the disease resistance mechanism of the obtained drugs has not been further explored, so the application of the drugs is limited to a certain extent. At the same time, due to the inability to analyze its mechanism of action, the problems generated during the use of these drugs cannot be answered, and thus cannot be effectively resolved. Therefore, it is necessary to study anti-disease drugs with known pharmacology.

As a member of invertebrates, shrimp mainly resists diseases through its physical barrier, stress response, innate humoral immunity, and cell physiological regulation. Apoptosis, as the main mechanism to remove senescent and abnormal cells, plays an important role in the removal of infected cells in shrimp and prevents the spread of viruses, and is an important means of disease resistance in shrimp. And it is an important weapon that can resist viral attacks.

The two subfamilies of the Caspase protein family act as initiation and effector proteins in apoptosis, and are key proteins in apoptosis. Caspase-8, 9, etc., which act as the initiator, can be activated by self-splicing after receiving the apoptotic signal, and then cause a caspase cascade reaction; caspase-3, 6, 7, etc. that act as the effector can directly degrade intracellular Structural and functional proteins that cause apoptosis.

(3) Contents of the invention

The purpose of the present invention is to provide the application of interleukin 2 (Interleukin-2, IL-2) in the preparation of the preparation against white spot syndrome of prawns.

The technical scheme adopted in the present invention is:

Application of interleukin 2 (Interleukin-2, IL-2) in the preparation of anti-prawn white spot syndrome preparation.

The interleukin 2 can be used to prepare a preparation for inducing PjCaspase protein activation.

Preferably, the preparation is an intramuscular injection, wherein the injection dose of Interleukin-2 is 5-10 ng/g shrimp (preferably about 7 ng/g shrimp).

The Interleukin-2 of the present invention is a small molecule related to shrimp anti-white spot syndrome that acts on the key protein PjCaspase of the shrimp apoptosis pathway. Direct injection of Interleukin-2 or feeding can activate the activity of PjCaspase protein, strengthen the resistance of prawns to viruses, greatly enhance the resistance of prawns to prawn white spot syndrome (WSSV), and reduce the lethality rate.

The beneficial effects of the present invention are mainly reflected in: Interleukin-2 of the present invention can effectively induce the activation of PjCaspase protein, greatly enhance the resistance of prawns to prawn white spot syndrome (WSSV), and reduce the lethal rate; Immunity-enhancing drugs widely used in the human body are non-toxic to the human body, and the prawns using Interleukin-2 can be safely eaten regardless of whether the drug is completely metabolized or not.

(4) Description of drawings

Figure 1 is the verification of the ability of Interleukin-2 to bind to Caspase protein in cells;

Figure 2 is the effect of Interleukin-2 on apoptosis activity; a is the activity detection of the apoptosis index effector enzyme Caspase3; b is the activity of the apoptosis index TUNEL;

Figure 3 is the effect of Interleukin-2 on viral replication;

Fig. 4 is the effect of Interleukin-2 on the mortality of shrimp infected with WSSV; the abscissa indicates the number of days, and the ordinate indicates the mortality.

(5) Specific implementation methods

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

Example 1:

1. Verification of the binding ability of IL-2 and PjCaspase:

1.1 Amplify the PjCaspase gene.

1.1.1 cDNA synthesis of shrimp tissue:

1.1.1.1 Extraction of total RNA from shrimp tissue:

1) Take 200 mg of live shrimp tissue (Penaeus japonicus, purchased from a vegetable market in Hangzhou, Zhejiang Province), and grind it quickly in 800 μL Trizol (BBI company).

2) Place on ice for 5 minutes.

3) Add 200 μL of phenol/chloroform solution (phenol and chloroform are mixed in equal volumes and then extracted several times with 0.1 mol/LTris.HCl (pH 7.6) to balance the mixture. mol/l Tris.HCl (pH7.6) liquid layer, stored at 4°C; use a pipette to absorb the lower layer solution), shake and mix, and then centrifuge at 12,000rpm for 10min.

4) Pipette the supernatant to an equal volume of isopropanol.

5) After shaking and mixing, centrifuge at 12,000 rpm for 10 minutes.

6) Dissolve the precipitate in 50 μL RNase-free double distilled water, add 1 μL DNase I (RNase Free) (TaKaRa), and digest for 1 hour.

7) Repeat steps 3) to 5).

8) The precipitate was washed twice with 300 μL of 80% (v/v) ethanol.

9) 12000rpm, after 2min to dry the alcohol, dissolve in 50μL RNase-free double distilled water.

1.1.1.2 Reverse transcription of total RNA to obtain cDNA:

Place the following components into an RNase-free Eppendolf tube:

Total RNA 4g

Oligo(dT)(500μg/mL) 1μL

Denature at 75°C for 5 minutes, immediately ice-bath, then add:

5×reverse transcriptase buffer 4μL

4 kinds of dNTPs mixture of 10mmol/L each 2μL

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

Make up to 20μL with water

After reacting at 25°C for 10 minutes, and at 42°C for 30 minutes, heat at 70°C for 15 minutes to terminate the reaction.

1.1.2PCR amplification:

Synthesize the following primers according to the determined gene reading frame sequence (shown in parentheses is the restriction site of the introduced underlined part):

5'-AT CCCGGG ATGGACGAGGTAATCCAG-3'(SmaI)

5'-TT CTCGAG TCACTGTGGGGGCGGAG-3'(XhoI)

Specific gene fragments were amplified by PCR using prawn cDNA as a template. Add to a 0.5 mL Eppendorf tube:

10×PCR reaction solution 5μL

dNTP(2.5mmol/L each) 4μL

Template (about 1μg/μL) 2μL

20mol/L gene-specific primers 1 μL each

Taq enzyme (TaKaRa) 1U

Add H ₂ O to a total volume of 50 μL

Carry out PCR amplification as follows: 95°C for 5min; 94°C for 30s, 58°C for 30s, 72°C

1.5min, 35 cycles; 72°C extension for 10min.

1.1.3 Fragment recycling:

Recover DNA fragments from agarose gel according to conventional methods, taking the gel recovery kit produced by Omega as an example:

1) Cut off the agarose block containing DNA, make it as small as possible, and put it into a 1.5mL centrifuge tube. If the weight of agarose is less than 100mg, make up to 100mg with double distilled water to ensure that the total volume of the system is not too small;

2) Add S1 solution (included with the kit) at the ratio of 300 μL S1 solution per 100 mg of agarose, place in a 50°C water bath for 10 minutes to completely dissolve the agarose block, and mix by inversion every 2 minutes;

3) Transfer the melted agarose solution into the adsorption column, centrifuge at 7000rpm for 30s, pour off the liquid in the collection tube, and then put the adsorption column into the same collection tube;

4) Add 500 μL W1 solution (included in the kit) to the adsorption column, centrifuge at 7000rpm for 15s, pour off the liquid in the collection tube, and put the adsorption column into the same collection tube;

5) Add 500 μL of W1 solution to the adsorption column, let it stand for 1 min, centrifuge at 7000 rpm for 15 s, pour off the liquid in the collection tube, and put the adsorption column into the same collection tube;

7) Centrifuge at 7000rpm for 1min;

8) Put the adsorption column into a clean 1.5mL centrifuge tube, add 10 μL of sterile water to the center of the adsorption membrane, let it stand for 1 min, centrifuge at 10,000 rpm for 1 min, and store the EP tube at -20°C.

1.2 Cloning into PIZ transformation vector:

1.2.1 Enzyme digestion reaction:

In a 0.5mL Eppendorf tube add:

Endonuclease buffer (10×) (TaKaRa) 2 μL

DNA about 0.5μg

Endonuclease (TaKaRa) 0.5μL

Add water to a total volume of 20μL

37 ℃ water bath for 2 ~ 3h. Inactivate at 70°C for 15 minutes.

Recycle by conventional method

1.2.2 Ligation transformation into Escherichia coli competent cells

1) 6 μL target fragment + 1.5 μL modified pIZ/V5-His vector (the original pIZ/V5-His vector was purchased from Invitrogen, pre-linked into EGFP protein for modification. EGFP fragment can be purchased through pEGFP (BD Biosciences Clontech) + 1 μL T4buffer (TaKaRa) + 0.25 μL T4Ligase (TaKaRa) + 1.5 μL H ₂ O were ligated at 16° C. overnight.

2) Take the DH5α competent cells stored at -70°C, rub them with your hands to melt them, flick them with your fingers, mix the cells, add an appropriate amount of plasmids or ligation products, and place them on ice for 30 minutes.

3) Heat shock in a water bath at 42°C for 90 seconds, and quickly ice-bath for 1-2 minutes.

4) Add 400 μL LB liquid medium and shake at a low speed (100 rpm) on a shaker at 37°C for 45 minutes.

5) Take 100-200 μL of the bacterial solution, spread it evenly on the LB (containing 100 μg/mL Amp) plate, and let the plate stand at room temperature for 20-30 minutes. After the bacterial solution is immersed in the culture medium, place it upside down at 37°C and incubate overnight.

(6) Recombinants were screened by colony PCR, sequenced after identification by enzyme digestion.

1.3 Transfection into High Five cells

After sequencing to verify that the sequence of the inserted fragment is correct (its sequence is shown in SEQ ID NO: 1):

a) Mix 100 μL ExpressFiveSFM medium (Invitrogen) containing 1 μg clone and 100 μL ExpressFiveSFM medium (Invitrogen) medium containing 1 μg cellinfectin (Invitrgen).

b) Stand still for 45 minutes.

c) Add 800 μL ExpressFiveSFM medium (Invitrogen) and mix well.

d) adding to serum-depleted HighFive cells (Invitrogen), and standing at room temperature for 4 hours.

e) Replace with clean ExpressFiveSFM medium (Invitrogen) and cultivate overnight.

1.4 After adding IL-2 to ExpressFiveSFM medium (Invitrogen) and culturing together for 48h, the cells expressing EGFP protein alone were used as the control, and the cells expressing PjCaspase and EGFP fusion protein were used as the experimental group. Flex Station II microplatereader (Molecular Devices, USA ) to detect the fluorescence intensity, the results are shown in Figure 1, the abscissa indicates the amount of IL-2, and the ordinate indicates the fluorescence intensity of the cells (ie, the binding ability of IL-2 and PjCaspase), as can be seen from the figure: IL-2 can interact with Caspase proteins bind efficiently.

2. The effect of IL-2 on apoptosis activity:

2.1 Sample processing:

Take a group of 20 prawns with uniform size and inject them with normal saline (100μL), WSSV virus ( ¹⁰⁴ copies), WSSV virus ( ¹⁰⁴ copies)+IL-2 (the dose is 7ng/g body weight of shrimp each time, with an equal amount of Dissolved in normal saline), IL-2 (the dose is 7ng/g body weight shrimp each time, dissolved with an equal amount of normal saline). Among them, the small molecule IL-2 was injected 12 hours before WSSV injection, 0, 12, and 24 hours after the injection, and the other groups were injected with the same normal saline at the same time as the control treatment. Blood samples were taken from the treated prawns at 24, 36, and 48 hours.

2.2 Caspase activity detection:

(1) Extract blood from prawns with 500 U heparin sodium 1:1.

(2) mix caspase3/7 reaction substrate powder with reaction Buffer ( 3/7 assay kit, Promega) mixed.

3/7 assaykit，Promega)。(3) Mix 100ul shrimp blood with 100ul reaction mixture (

3/7 assaykit, Promega).

(4) Incubate at 25° C. in the dark for 3 hours.

(5) Detection of chemiluminescence with a chemiluminescence microplate reader

2.3 Detection of apoptotic activity by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method

(1) Evenly spread shrimp hemocytes on a glass slide.

(2) Immerse the glass slide in 4% paraformaldehyde (Shanghai Sangong) solution, and fix at 4°C for 25 minutes.

(3) Wash three times with PBS at room temperature, 5 minutes each time.

(4) 2% Triton X-100 pre-infiltration for 5 minutes.

(5) Wash twice with PBS.

(6) Equilibrate with 100 μL of equilibration buffer (TUNEL kit, Promega).

(7) Add 50 μL rTdT (TUNEL kit, Promega), and incubate for 1 hour at 37°C in the dark in a wet box (the plastic lunch box can be covered with water-soaked gauze, or Shanghai Ruicong products can also be used).

(8) Add 2×SSC (TUNEL kit, Promega) to stop the reaction.

(9) Wash with PBS three times.

(10) Stain with 1 μg/ml PI (Shanghai Sangong) solution for 15 minutes.

(11) Wash with PBS three times.

(12) Examination under a fluorescent microscope.

Fig. 2 adopts normal saline, WSSV virus (10 ⁴ copies), WSSV virus (10 ⁴ copies)+IL-2 (the dose is 7ng/g body weight shrimp every time, dissolves with equal amount of normal saline), IL-2 (dose The apoptotic activity detected in 24, 36, and 48 hours of prawns treated with 7 ng/g body weight shrimp each time (dissolved with an equal amount of physiological saline). It can be seen from the figure that IL-2 greatly enhanced the apoptotic activity.

3. The effect of IL-2 on virus replication:

3.1 Shrimp genome extraction

The total DNA of the prawns treated according to the method 2.1 was extracted with SQ Tissue Kit (Promega).

3.2 Real-time PCR determination of virus copy number

Synthesize Taqman probe and use b-actin as a control, 50°C for 4min, (95°C for 45s, 52°C for 45s, 72°C for 45s)*45 cycles. A standard curve was made with known concentrations of WSSV plasmid.

Fig. 3 shows the use of WSSV virus (10 ⁴ copies), WSSV virus (10 ⁴ copies)+IL-2 (7ng/g prawns, dissolved in physiological saline), WSSV virus+IL-2 (0.7ng/g prawns, dissolved in physiological saline) Dissolved in saline), WSSV virus+IL-2 (70pg/g prawns, dissolved in saline) treated prawns detected changes in the copy number of WSSV virus in 36 and 48 hours. It can be seen from the figure that IL-2 effectively inhibits the replication of WSSV in prawns.

3. Mortality determination

Take a group of 20 prawns with uniform size and inject them with normal saline (100μL), WSSV virus ( ¹⁰⁴ copies), WSSV virus ( ¹⁰⁴ copies)+IL-2 (the dose is 7ng/g body weight of shrimp each time, with an equal amount of Dissolved in normal saline), IL-2 (the dose is 7ng/g body weight shrimp each time, dissolved with an equal amount of normal saline). Among them, small molecules were injected 12 hours before WSSV injection, 0, 12, and 24 hours after injection, and the other groups were injected with the same normal saline at the same time as the control treatment. Mortality was recorded.

Fig. 4 is the change of mortality of prawns treated with physiological saline, WSSV virus, IL-2, WSSV virus+IL-2 within six days. It can be seen from the figure that IL-2 effectively reduces the mortality of prawns infected with WSSV and has no toxicity to prawns, so it can be widely used in prawn farming.

Claims (2)

1. the application of interleukin 2 (Interleukin-2) in the preparation preparation anti-prawn white spot syndrome, it is characterized in that described interleukin 2 is used for the preparation induction PjCaspase protein activation preparation. 2. application as claimed in claim 1, is characterized in that described preparation is intramuscular injection, and wherein the injection dosage of interleukin 2 is 5～10ng/g shrimp.

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