Application of fatty acid binding protein of penaeus vannamei

Abstract

The invention discloses an application of a penaeus vannamei fatty acid binding protein (FABP) as an immunologic stimulant and an application of the FABP in preparation of drugs for enhancing the immune response capability of crustacean, and aims to enhance the vibrio parahaemolyticus infection resistance of the crustacean by improving the expression level of intracellular antibacterial peptides. Large-area death of crustacean caused by acute hepatopancreatic necrosis (AHPND) is avoided. The amino acid sequence number is E2IH93, the molecular weight is 15495 Dalton, no toxic or side effect exists, the synthesis cost is low, a new way is provided for treatment of crustacean bacterial infection, the ecological balance in the breeding process is not damaged, and the bacillus subtilis is an environment-friendly and efficient bacterial infection defense substance and has important application value.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of a recombinant protein of Penaeus vannamei fatty acid binding protein (FABP for short) in resisting Vibrio parahaemolyticus that causes AHPND. Background Art

[0002] my country is the world's largest shrimp farming country and major trading country. According to statistics, my country's total shrimp farming output reached 2.42 million tons in 2022, accounting for more than 40% of the world's total shrimp farming output. However, in recent years, while my country's shrimp farming industry has achieved rapid development, it has faced problems such as frequent diseases (acute hepatopancreatic necrosis disease, white spot syndrome, and white stool syndrome, etc.) and seedling degeneration, which has increased the risks and difficulties of shrimp farming operations, reduced profitability, and worrying prospects for sustainable development. Among them, acute hepatopancreatic necrosis syndrome (AHPND) is one of the most serious diseases affecting shrimp farming in recent years. The disease has a high mortality rate and a wide range, causing huge losses to the shrimp farming industry. At present, it mainly focuses on ecological prevention and control, and lacks direct and effective means. Farmers often use large amounts of antibiotics for feeding, but this is often counterproductive, and the use of large amounts of antibiotics may promote the development of drug-resistant bacteria and cause environmental pollution. In recent years, scholars have studied the pathogenic mechanism of AHPND and the immune defense mechanism of shrimp, including feeding antagonistic antibodies against PirA, phage therapy, probiotics, Chinese herbal medicine, etc., but the effects are unstable.

[0003] In the prior art, the treatment of bacterial diseases in crustaceans is mainly limited to drugs such as antibiotics, disinfectants and antibacterial compounds. However, on the one hand, such drugs will indiscriminately disinfect beneficial and harmful microorganisms, causing damage to the homeostasis of the flora in the organism and the external environment. On the other hand, the long-term use of antibiotics will accelerate the occurrence of bacterial resistance, making the treatment of diseases more difficult. Therefore, in crustacean breeding systems, the use of traditional methods such as antibiotics to control bacterial diseases is not a green and efficient prevention and control measure. In view of this, finding stimulants that effectively activate shrimp immunity is an effective way to solve the problem of shrimp prevention and control of diseases such as AHPND. Summary of the invention

[0004] The purpose of the present invention is to solve the shortcomings of currently commonly used antibiotics and other drugs that are easy to induce drug resistance and increase the body load, and to propose a new way to enhance the immune response ability of crustaceans.

[0005] The following technical solutions are adopted:

[0006] An application of a fatty acid binding protein of Penaeus vannamei, and an application of the fatty acid binding protein of Penaeus vannamei in the preparation of a drug for enhancing the immune response ability of crustaceans.

[0007] The present invention aims to provide a recombinant protein of FABP gene of Penaeus vannamei and its effect on resistance to AHPND-causing Vibrio parahaemolyticus (Vp AHPND ) in the application. The recombination of the FABP gene of the present invention can effectively inhibit Vp AHPND Proliferation in the body of Penaeus vannamei, improving the survival rate of shrimp, so as to solve the problem of Vannamei infection with Vp AHPND The problem of death.

[0008] A recombinant protein of a fatty acid binding protein gene of Penaeus vannamei, the sequence of which is shown in Seq_1.

[0009] A recombinant protein of the fatty acid binding protein gene of Penaeus vannamei and its application in the preparation of anti-Vp AHPND Application in preparations. First, an expression plasmid with the FABP gene of Penaeus vannamei was constructed. After the injection of VpAHPND, the differential proteins in the plasma of Penaeus vannamei were analyzed by LC-MS, and the highest abundance was found to be FABP. Therefore, FABP was constructed on the expression plasmid. The purified FABP protein (rFABP) expressed by the prokaryotic system was injected into the shrimp, and Vp AHPND Compared with the control group, the Vp AHPND The decreased copy number expression and increased shrimp survival rate indicated that rFABP helped to resist Vp AHPND The recombinant protein of the PvFABP gene of the present invention is Vp AHPND This provides a potential drug for the prevention and treatment of Vp AHPND The FABP recombinant protein of the present invention is non-toxic and has no side effects, has low synthesis cost, provides a new approach for the treatment of bacterial infection in crustaceans, and does not destroy the ecological balance in the breeding process. It is an environmentally friendly and efficient bacterial infection defense substance with important application value.

[0010] The present invention also provides a recombinant bacterium containing the FABP gene.

[0011] The present invention also provides a recombinant protein of the FABP gene.

[0012] Preferably, the fatty acid binding protein of Penaeus vannamei is used in the preparation of a drug for enhancing the immune response of crustaceans to pathogen infection.

[0013] Preferably, the immune response capability includes inhibiting the proliferation of the pathogen.

[0014] Preferably, the immune response capability includes reducing mortality caused by the pathogen.

[0015] Preferably, the duration of enhancement of the immune response capability exceeds 7 days.

[0016] After research, it was unexpectedly discovered that the mechanism of action of the present invention is mainly to activate shrimp immunity through FABP, which significantly increases the clearance rate of shrimp against bacteria such as Vibrio, thereby reducing the mortality rate of shrimp infected with Vibrio. It is currently found that after injection of FABP, the immune response ability is significantly enhanced, and its protection period is at least 7 days.

[0017] Preferably, the pathogen comprises AHPND Vibrio parahaemolyticus.

[0018] Preferably, the crustacean comprises shrimp.

[0019] Preferably, the ratio of the added amount of the vannamei fatty acid binding protein to the body weight of the crustacean is 1 nmol / 6g.

[0020] Preferably, the dosage form of the drug includes an injection, especially an injection.

[0021] A medicine obtained by applying the above-mentioned Penaeus vannamei fatty acid binding protein.

[0022] The drug comprises fatty acid binding protein as the only active ingredient and also comprises pharmaceutically acceptable excipients or auxiliary ingredients.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention adopts the method of injecting FABP protein at Vp AHPND The function and mechanism of action of FABP protein in infection are explored. Injection of FABP protein can enhance the ability of crustaceans to resist Vibrio parahaemolyticus infection by increasing the expression level of intracellular antimicrobial peptides, thereby avoiding large-scale death of crustaceans due to acute hepatopancreatic necrosis disease (AHPND). Its amino acid sequence number is E2IH93, with a molecular weight of 15495 Daltons. It is non-toxic and has no side effects. It has a low synthesis cost and provides a new way to treat bacterial infections in crustaceans. It will not destroy the ecological balance in the breeding process. It is an environmentally friendly and efficient bacterial infection defense substance with important application value. Vp after injection of FABP protein AHPND Compared with the control group, Vp AHPND The copy number decreased, the shrimp AHPND symptoms decreased and the survival rate increased, indicating that the recombinant protein of the PvFABP gene inhibited Vp AHPND Therefore, the recombinant protein of PvFABP gene can be used as an anti-Vp AHPND New drugs for Vp AHPNDin the prevention and treatment work. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the structure of FABP protein.

[0026] Figure 2 This is the PvFABP expression plasmid map.

[0027] Figure 3 The figure shows the results of purification and endotoxin detection of FABP recombinant protein. A shows the purification process of FABP recombinant protein, and B shows the endotoxin content detection of FABP protein.

[0028] Figure 4 Vp after injection of FABP recombinant protein AHPND After infection, shrimp survival rate was analyzed.

[0029] Figure 5 For injection of rFABP, Vp AHPND infection, reducing AHPND symptoms and hemolymph Vp AHPND Among them: A is the infection status of AHPND symptoms of shrimps in different groups; B is the statistics of AHPND symptoms of shrimps in different groups; C is the Vp of shrimps in different groups AHPND Load detection; D is the survival rate statistics of shrimps in different groups. DETAILED DESCRIPTION

[0030] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings.

[0031] Example 1

[0032] Prokaryotic expression and purification of FABP protein

[0033] pET-N-His-TEV (D2905) was purchased from Bio-Technology Co., Ltd. FABP is a fatty acid binding protein with an amino acid sequence number of E2IH93 and a molecular weight of 15495 Daltons. The amino acid sequence is shown in Seq_1 and the structure is shown in Figure 1 First, the EGFP gene and PvFABP gene were constructed into the pET-N-His-TEV plasmid using a homologous recombination kit and specific primers.

[0034] (1) Prokaryotic expression of His fusion protein

[0035] a. Activation: Take 100 μL of the recombinant expression plasmid strain pET-N-His-TEV-EGFP / PvFABP in (1) and add them to 50 mL of LB liquid culture medium (containing 1‰ Kan+, i.e. 50 μg / mL), and shake and culture at 16°C and 180 rpm for 12 h;

[0036] b. Expansion culture: Take 100 μL of the activated bacterial solution in a and add 50 mL of LB liquid medium (containing 1‰ Kan+, i.e. 50 μg / mL), and shake and culture at 37°C and 180 rpm for 4 to 6 hours;

[0037] c. Induce expression: add IPTG (final concentration 0.1 mM) to the bacterial solution and induce at 16°C and 180 rpm for 12 h;

[0038] d. Transfer the induced bacterial solution to a centrifuge tube and centrifuge at 5000g for 1 min at 4°C to collect the bacteria;

[0039] e. Add 10 mL of 0.01 M PBS (pH 7.4) to resuspend the bacteria, transfer the bacterial solution to a vial, and place it on ice and ultrasonically disrupt the bacteria (power: 400 W, disruption for 3 seconds, interval of 5 seconds, a total of 20 minutes);

[0040] f. Dispense the broken cells into 2 mL centrifuge tubes, centrifuge at 20,000 g for 15 min at 4°C, and collect the supernatant.

[0041] (2) Purification of His fusion protein

[0042] a. Gently shake the reagent bottle containing nickel column material (cytiva 17531806) to mix the column material evenly, take 50 μL of the column material into a clean 1.5 mL centrifuge tube, centrifuge at 500 g for 5 min at 4°C, carefully aspirate the supernatant and discard it;

[0043] b. Add 5 times the volume of PBS to resuspend the nickel column material, and mix by inverting (e.g., if you take 50 μL of nickel column material, add 250 μL of PBS); centrifuge at 4°C and 500 g for 5 min, carefully aspirate the supernatant and discard; repeat this step once;

[0044] c. Take 1 mL of cell disruption solution or inclusion body refolding solution from (1) and add it to the treated nickel column material, and incubate with rotation at 4°C for 2 h;

[0045] d. Use a pipette to transfer the mixture to a new 1.5 mL centrifuge tube;

[0046] e. Add 1 mL of PBS (containing 0.5% Triton 100) to resuspend the nickel column and rotate to mix for 5 minutes;

[0047] f. Centrifuge at 4°C and 500 g for 5 min. Carefully aspirate and discard the supernatant. Repeat this step 5 times.

[0048] g. Add 1 mL of PBS (containing 0.5% Triton 114) to resuspend the nickel column and rotate to mix for 5 minutes;

[0049] h. Centrifuge at 4°C and 500 g for 5 min. Carefully aspirate and discard the supernatant. Repeat this step 5 times.

[0050] i. Prepare the eluent according to the instructions of the nickel column (cytiva 17531806) for elution, take an appropriate amount of the eluent and use conventional SDS-PAGE to detect the purification effect, and temporarily store the purified protein at -80°C.

[0051] (3) Determination of endotoxin content of recombinant protein

[0052] According to the instructions of ToxinSensor Chromogenic LAL Endotoxin Assay Kit, the endotoxin content of purified rFABP and rEGFP was measured, and it was found that their endotoxin contents were 0.012 ng / μg and 0.031 ng / μg, which were less than the commercial standard of 0.1 ng / μg and could be used for subsequent injection treatment and other experiments.

[0053] Since shrimp have an open circulatory system, FABP recombinant protein can be used as an injectable preparation, circulates in the body cavity, and has a wide range of action.

[0054] Example 2

[0055] Shrimp survival rate determination

[0056] The experimental group was injected with 1 nmol rFABP / shrimp into the shrimp muscle, and the control group was injected with 100 μL PBS / shrimp and 1 nmol rEGFP / shrimp into the shrimp muscle. 24 hours after the injection, each group was divided into two batches.

[0057] Using VPN AHPND Single colonies were activated in LB medium at 37°C. The bacterial solution activated to the logarithmic growth phase was centrifuged and resuspended in PBS. After repeated centrifugation and resuspending twice, OD560 was detected using an ELISA reader and calculated and diluted to 1×10 6 CFU / mL bacterial suspension. 100 μL bacterial suspension / shrimp was injected into the shrimp muscle as Vp AHPND Disease model. 100 μL PBS / shrimp was injected into the shrimp muscle as a healthy shrimp model. Vp AHPND Infection and PBS injection treatment, and the survival rate of shrimp after infection with pathogens was recorded. AHPNDThe mortality of shrimp was recorded every 12 hours after infection. The results showed that the survival rate of shrimp injected with 1 nmol rFABP / shrimp was significantly higher than that of the control group (P < 0.05). Therefore, rFABP can enhance the immune response ability of crustaceans ( Figure 5 ).

[0058] Example 3

[0059] Hemolymph Vibrio load determination

[0060] The experimental group was injected with 1 nmol rFABP / shrimp into the shrimp muscle, and the control group was injected with 100 μL PBS / shrimp into the shrimp muscle. After 24 hours of injection, each group was divided into two batches.

[0061] Using VPN AHPND Single colonies were activated in LB medium at 37°C. The bacterial solution activated to the logarithmic growth phase was centrifuged and resuspended in PBS. After repeated centrifugation and resuspending twice, OD560 was detected using an ELISA reader and calculated and diluted to 1×10 6 CFU / mL bacterial suspension. 9 CFU / L Vp AHPND As shrimp Vp AHPND Infection disease model. On the one hand, the occurrence of AHPND symptoms in shrimps after immersion treatment was observed. The results showed that the number of shrimps injected with 1 nmol rFABP / shrimp that developed AHPND symptoms was far less than that in the control group. AHPND Seven days after infection, shrimp hemolymph was collected and DNA was extracted. The load of Vibrio parahaemolyticus in shrimp hemolymph was detected by qPCR. The load of Vibrio parahaemolyticus in shrimp hemolymph injected with 1 nmol rFABP / shrimp was significantly lower than that in the control group (P < 0.0001). Therefore, rFABP can enhance the ability of crustaceans to eliminate pathogens. It can be seen from the experimental results that after 7 days of r-FABP injection, the Vp AHPND Not only did the survival rate of the treated experimental group increase, but the load of Vibrio parahaemolyticus detected in the shrimp hemolymph was significantly lower than that of the control group, that is, the enhancement of immune response ability lasted for more than 7 days.

[0062] The above disclosure is only the preferred embodiment of the present invention, which certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

[0063] Sequence Listing

[0064] MAKIEGKFKMESSENFDEFMKALGVGLVMRKMGNAATPTVEITKDGDTYTMKTTTFTFKTTEIKFKLGEEFEETTADGRVVKSTITLDGNKLVHKQVGDKEKKEKDSELLREFTDDKMLMECKVDDVVCKRVYSRLE

Claims

1. An application of a fatty acid binding protein of Penaeus vannamei, characterized in that: Application of fatty acid binding protein of Penaeus vannamei in the preparation of drugs for enhancing the immune response of crustaceans.

2. The use of the vannamei fatty acid binding protein according to claim 1, characterized in that: Application of fatty acid binding protein of Penaeus vannamei in the preparation of drugs for enhancing the immune response of crustaceans to pathogen infection.

3. The use of the vannamei fatty acid binding protein according to claim 2, characterized in that: The immune response capability includes inhibiting the proliferation of the pathogen.

4. The use of the vannamei fatty acid binding protein according to claim 2, characterized in that: The immune response capability includes reducing mortality caused by the pathogen.

5. The use of the vannamei fatty acid binding protein according to any one of claims 2 to 4, characterized in that: The pathogens include AHPND Vibrio parahaemolyticus.

6. The use of the vannamei fatty acid binding protein according to claim 1, characterized in that: The enhancement duration of the immune response capability exceeds 7 days.

7. The use of the vannamei fatty acid binding protein according to claim 1, characterized in that: The crustaceans include shrimps.

8. The use of the vannamei fatty acid binding protein according to claim 1, characterized in that: The ratio of the added amount of the vannamei fatty acid binding protein to the body weight of the crustacean is 1 nmol / 6g.

9. The use of the vannamei fatty acid binding protein according to claim 1, characterized in that: The dosage form of the drug includes injection.

10. A medicine obtained by using the vannamei fatty acid binding protein according to claim 1.

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