Epinephelus lanceolatus anti-nervous necrosis virus gene ackr3 and its eukaryotic expression vector and application

Abstract

The application discloses an ackr3 gene of Epinephelus lanceolatus against nervous necrosis virus and a protein coded by the ackr3 gene, and a eukaryotic expression vector containing the ackr3 gene; and the nucleotide sequence of the ackr3 gene is shown as SEQ ID NO. 1. The application further discloses an application of the ackr3 gene or the protein coded by the ackr3 gene or the eukaryotic expression vector containing the ackr3 gene in preparing an anti-nervous necrosis virus drug and in anti-nervous necrosis virus breeding.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, and more specifically, relates to an anti-neural necrosis virus gene ackr3 in saddleback grouper and its eukaryotic expression vector and applications. Background Technology

[0002] Saddleback grouper ( Epinephelus lanceolatus Also known as giant grouper or giant rockfish, it belongs to the order Perciformes (… Perciformes ), Serranidae ( Serranidae ), Grouper genus ( Epinephelus The grouper (Platycodon grandiflorus) is the largest of the grouper species and is also known as the "King of Groupers." It has become a major species for marine aquaculture in coastal areas of southern my country, including Guangdong, Hainan, Fujian, and Guangxi, and has enormous economic value.

[0003] Currently, with the continuous increase in grouper farming, disease and pest problems during the farming process are becoming increasingly prominent, especially viral nervous necrosis (VNN), caused by nerve necrosis virus (NVV). NNV belongs to the β-Nodavirus genus and is a type of small RNA virus, which can be divided into four genotypes: Striped jack NNV (SJNNV), Tiger puffer NNV (TPNNV), Barfin flounder NNV (BFNNV), and Red spotted grouper NNV (RGNNV). RGNNV is the most harmful to grouper, mainly causing damage to the nervous system and retina. Larvae and juveniles are highly susceptible to infection, with mortality rates exceeding 90%, and sometimes reaching 100%. Currently, there is no effective vaccine for nerve necrosis virus, and the virus begins to cause disease in juvenile fish, making it difficult to administer a vaccine.

[0004] Chemokines and chemokine receptors play crucial roles in resisting pathogen invasion and are closely related to the occurrence and development of inflammation in the immune environment, thus becoming potential targets for antiviral treatment. Atypical chemokine receptor 3 (ACKR3) is the chemokine receptor for chemokines Cxcl11 and Cxcl12. ACKR3 can promote the expression of Cxcr4 and Ackr3 in cells by activating HIF-1α; ACKR3 activation can also upregulate phosphorylated ERK, protein kinase B (Akt), and nuclear factor κB (NF-κB), thereby mediating inflammation and exerting antiviral effects. ACKR3 can bind to Cxcl12 to participate in regulating the migration and activation of microglia, and can also mediate the polarization type of macrophages in bony fish by binding to specific ligands, thus regulating the antiviral capacity of fish by modulating macrophage polarization. In other words, ACKR3 plays an important role in antiviral treatment and the regulation of immune cell function, therefore, ACKR3 has broad application prospects in fish immunotherapy. Summary of the Invention

[0005] The purpose of this invention is to provide an ackr3 gene for resisting nerve necrosis virus in saddle-banded grouper and its eukaryotic expression vector.

[0006] A second objective of the present invention is to provide the application of the above-mentioned ackr3 gene or the protein it encodes, or a eukaryotic expression vector containing the above-mentioned ackr3 gene.

[0007] To achieve the aforementioned first objective, the present invention adopts the following technical solution:

[0008] An ackr3 gene for resisting nerve necrosis virus in saddleback grouper, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0009] The amino acid sequence of the protein encoded by the ackr3 gene is shown in SEQ ID NO.2.

[0010] The protein encoded by the ackr3 gene has a structure comprising seven transmembrane domains, such as... Figure 1 As shown.

[0011] The present invention also provides a eukaryotic expression vector containing the ackr3 gene.

[0012] Preferably, the eukaryotic expression vector is pcDNA3.1-Flag or pEGFP-C1.

[0013] The second objective of this invention is achieved through the following technical solution:

[0014] The use of the ackr3 gene or the protein it encodes, or a eukaryotic expression vector containing the ackr3 gene, in the preparation of anti-neural necrosis virus drugs.

[0015] Application of the ackr3 gene or its encoded protein or a eukaryotic expression vector containing the ackr3 gene in breeding against neuronecrosis virus.

[0016] Specifically, the nerve necrosis virus is the red spot nerve necrosis virus.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] This invention provides for the first time the nucleotide sequence of the ackr3 gene, an anti-RGNNV gene, found in grouper. The ackr3 gene can inhibit RGNNV virus replication and increase the expression of several inflammatory factors, including TNFα, IL1β, IL10, and TGFβ. This invention also provides the application of the ackr3 gene, its encoded protein, and a eukaryotic expression vector containing the ackr3 gene in the preparation of anti-RGNNV drugs and in RGNNV breeding, which is beneficial for the prevention and treatment of viral neuronecrosis in grouper and other perchidian fishes, representing a significant and substantial advancement. Attached Figure Description

[0019] Figure 1 A predicted transmembrane topology map of the protein encoded by the ackr3 gene shows seven transmembrane regions.

[0020] Figure 2 This is an electrophoresis image of PCR amplification of the full-length cDNA of the ackr3 gene.

[0021] Figure 3 Electrophoresis images of recombinant expression vectors pcDNA3.1-ACKR3 and pEGFP-C1-ACKR3 and their transformation into Escherichia coli DH5α for PCR verification.

[0022] Figure 4 The effect of overexpressing ackr3 using the recombinant expression vectors pEGFP-C1-ACKR3 and pcDNA3.1-ACKR3 was investigated.

[0023] Figure 5 This shows the mRNA expression levels of TNFα, IL1β, IL10 and TGFβ after overexpression of ackr3.

[0024] Figure 6 This shows the expression levels of CP and RdRp after overexpression of ackr3.

[0025] Figure 7 This shows the expression levels of CP and RdRp after knocking down ackr3. Detailed Implementation

[0026] The application method of the present invention is further illustrated below with reference to specific embodiments. The following embodiments and accompanying drawings are for illustrative purposes only and should not be construed as limiting the present invention. Unless otherwise specified, the reagent raw materials used in the following embodiments are commercially available or conventionally obtained biological reagent raw materials; unless otherwise specified, the methods and equipment used in the following embodiments are methods and equipment conventionally used in the art.

[0027] Example 1: Cloning of the ackr3 gene in the saddleback grouper

[0028] (1) Extraction of total RNA from spleen of grouper with saddleback fin

[0029] Healthy grouper were anesthetized on ice for 3 minutes, and spleen tissue was separated. Total RNA was extracted using Trizol reagent.

[0030] (2) Synthesis of the first strand of cDNA

[0031] A total RNA sample of 1 μg from the spleen of a grouper was treated with DNase to remove genomic DNA contamination. The sample was then mixed with RNAOligodT and reverse transcribed to obtain the first strand of cDNA. The resulting product was stored at -20℃ for later use.

[0032] (3) Cloning of the full cDNA sequence of the ackr3 gene from the oblique-banded grouper

[0033] Specific primers ackr3-F and ackr3-R were designed as shown in SEQ ID NO.3 and SEQ ID NO.4. Using the first strand of the above cDNA as a template, amplification was performed to obtain the ORF fragment of the ackr3 gene (as shown in SEQ ID NO.1), with a size of 1137 bp. The electrophoresis results are attached. Figure 2 As shown, the target band was recovered by gel excision and the product was purified. The product was ligated into the pMD18-T vector, transformed into DH5α E. coli, and positive clones were selected for sequencing. BLAST homology analysis confirmed that the target product was indeed a cDNA sequence fragment of the ackr3 gene.

[0034] The cDNA sequence of the ackr3 gene is shown in SEQ ID NO.1, which is:

[0035]

[0036] The amino acid sequence of the protein ACKR3 encoded by the ackr3 gene is shown in SEQ ID NO.2, which is:

[0037] MSLSTSELEDLWESFGELNFSETFSNISSVDAMVCATAFNRSALLYSMCVLYTFIFIVGLAANALVLWVNIRAQRDSAPRHETHMYIAHLAVADLCVCATLPVWVSSLAQHGHWPFGEVACKLTHLLFSVNLFSSIFFLACMSVDRYLSVTQHRDNEGGVRRKLIRRGVCVGVWLLALVASLPDTYFLR TVKSTHGDTMMCRPVYPGDNPREWMVGVQLSFILLGFVLPFPIIAVFYALLASAFARSSTSSSSTVEQERRVSRRMILAYIVVFLGCWGPYHAVLLADALSQLGLLPLTCGLENVIYVALHLTQCLSLLHCCFNPILYNFINRNYRYDLMKAFIFKYSTRTGLARLIEAPNMSETEYSPVAVENPPQI.

[0038] Example 2: Construction and validation of recombinant expression vectors pEGFP-C1-ACKR3 and pcDNA3.1-ACKR3

[0039] Based on the full-length cDNA sequence of the ackr3 gene, a pair of primers were synthesized at both ends of the coding gene. The upstream primer contained an EcoRI cleavage site, and the downstream primer contained a KpnI cleavage site, as shown in Table 1. Using the pMD18-T plasmid containing the ackr3 coding gene as a template, PCR amplification was performed using the primers shown in Table 1, resulting in a single, specifically amplified band with a product size of approximately 1155 bp. The PCR amplification products were cloned into the expression vectors pEGFP-C1 and pcDNA3.1-Flag, respectively, to obtain the recombinant expression vectors pEGFP-C1-ACKR3 and pcDNA3.1-ACKR3. The PCR verification results of the two recombinant expression vectors are attached. Figure 3 As shown, lane 1 represents the validation result of pEGFP-C1-ACKR3, and lane 2 represents the validation result of PcDNA3.1-ACKR3.

[0040] Table 1 Primer sequences and descriptions for vector construction

[0041]

[0042] Example 3: Overexpression effect of recombinant expression vectors pEGFP-C1-ACKR3 and pcDNA3.1-ACKR3

[0043] Add an appropriate amount of 293T cells (approximately 1 × 10⁻⁶ cells per well). 6 The cells were seeded into 96-well plates. The next day, when the cells reached 60%–80% confluency, they were transfected. Before transfection, the medium was replaced with serum-free Opti-MEM. Each well was transfected with 150 ng of the recombinant expression vector pEGFP-C1-ACKR3, pcDNA3.1-ACKR3, and their blank vectors as controls. The transfection solutions were: Solution A (10 μL Opti-MEM medium, added with the expression vector, mixed well), and Solution B (10 μL Opti-MEM medium, added with 0.25 μL Lipo3000, mixed well, and incubated at room temperature for 5 min). Solution A and Solution B were gently mixed to obtain the transfection solution, incubated at room temperature for 20 min, and then added to the corresponding transfection wells. The cells were incubated for 6 hours. Finally, the medium was replaced with DMEM containing 10% fetal bovine serum.

[0044] Cells were collected 36 hours after transfection, total RNA was extracted, reverse transcribed, and quantitative real-time PCR was performed to detect the mRNA expression level of ackr3. The amplification primers used were qACKR3-F and qACKR3-R. The internal reference gene was β-actin, and the amplification primers used were qβ-actin-F and qβ-actin-R. The results were analyzed based on the CT values ​​obtained, and the relative expression level of ackr3 was calculated using the 2-ΔΔct method. The experimental results are shown in the attached figure. Figure 4 As shown in Table 2, the primer sequences used in the detection are listed below:

[0045] Table 2 Primers for Real-Time PCR

[0046]

[0047] From the appendix Figure 4 The results show that transfection with the recombinant expression vectors pEGFP-C1-ACKR3 or pcDNA3.1-ACKR3 significantly increased the expression level of ackr3 mRNA in cells. These results indicate that the recombinant expression vectors were successfully constructed, and transfection significantly increased ackr3 expression in cells.

[0048] Example 4: Overexpression of ackr3 upregulates the mRNA transcription of multiple inflammatory factors

[0049] Appropriate amounts of saddleback grouper spleen cells (GS cells) were prepared at a density of approximately 4 × 10⁻⁶ cells per well. 6Seed the cells into 6-well plates. The next day, when the cells reach 50%–60% confluency, transfect them. Before transfection, replace the medium with serum-free Opti-MEM. Transfect each well with 2000 ng of pcDNA3.1-ACKR3 or the pcDNA3.1-Flag blank vector control. The transfection solutions are: Solution A (50 μL Opti-MEM medium, add the expression vector, mix well), Solution B (50 μL Opti-MEM medium, add 4 μL Lipo2000, mix well, incubate at room temperature for 5 min); gently mix Solution A and Solution B to obtain the transfection solution, incubate at room temperature for 20 min, add to the corresponding transfection wells, and incubate for 12 hours; finally, replace with L15 medium containing 10% fetal bovine serum.

[0050] Cells were collected 48 hours after transfection, total RNA was extracted, reverse transcribed, and quantitative real-time PCR was performed to detect the mRNA levels of TNFα, IL1β, IL10, and TGFβ. β-actin was selected as the internal reference gene, and qβ-actin-F and qβ-actin-R primers were used for amplification. The results were analyzed based on the CT values ​​obtained, and the relative expression levels of the above inflammatory factors were calculated using the 2-ΔΔct method. The experimental results are shown in the attached figure. Figure 5 As shown in Table 3, the primer sequences used in the detection are listed below:

[0051] Table 3 Primers for Real-Time PCR

[0052]

[0053] From the appendix Figure 5 The results show that overexpression of ackr3 in GS cells significantly increases the expression of several inflammatory factors, including TNFα, IL1β, IL10, and TGFβ. These findings indicate that the ackr3 gene or ACKR3 protein can regulate the expression of inflammatory factors.

[0054] Example 5: Effect of ackr3 overexpression on RGNNV replication

[0055] Apply an appropriate amount of GS cells at a density of approximately 4 × 10⁶ cells per well. 6Seed the cells into 6-well plates. The next day, when the cells reach 50%–60% confluency, transfect them. Before transfection, replace the medium with serum-free Opti-MEM. Transfect each well with 2000 ng of pcDNA3.1-ACKR3 or the pcDNA3.1-Flag blank control. The transfection solutions are: Solution A (50 μL Opti-MEM medium, add the expression vector, mix well), Solution B (50 μL Opti-MEM medium, add 4 μL Lipo2000, mix well, incubate at room temperature for 5 min); gently mix Solution A and Solution B to obtain the transfection solution, incubate at room temperature for 20 min, add to the corresponding transfection wells, and incubate for 12 hours; finally, replace with L15 medium containing 10% fetal bovine serum.

[0056] Forty hours after transfection, cells were challenged with RGNNV. Twenty-four hours after challenge, cells were collected, total RNA was extracted, reverse transcribed, and quantitative real-time PCR was performed to detect the mRNA levels of capsid protein gene (CP) and RNA-dependent RNA polymerase (RdRp). β-actin was selected as the internal reference gene, and qβ-actin-F and qβ-actin-R primers were used for amplification. The results were analyzed based on the CT values, and the relative expression levels of CP and RdRp were calculated using the 2-ΔΔct method. The experimental results are shown in the attached figure. Figure 6 As shown in Table 4, the primer sequences used in the detection are listed below:

[0057] Table 4 Primers for Real-Time PCR

[0058]

[0059] From the appendix Figure 6 The results show that overexpression of ackr3 in GS cells significantly inhibits the expression of the RGNNV core gene capsid protein gene (CP) and RNA-dependent RNA polymerase (RdRp). These results indicate that the ackr3 gene or ACKR3 protein can inhibit RGNNV replication.

[0060] Example 6: Effect of knocking down ackr3 on RGNNV replication

[0061] Apply an appropriate amount of GS cells at a density of approximately 1 × 10⁻⁶ cells per well. 4Seed the cells into 96-well plates. Transfect the cells the following day when they reach 50%–60% confluency. Before transfection, replace the medium with serum-free L15 medium. The siRNA sequences used are: siACKR3 (siACKR3-sense: 5'-GAGCUCAACUUCUCCGAAATT-3'; siACKR3-antisense: 5'-UUUCGGAGAAGUUGAGCUCTT-3'), and Negative Control sequences (Negative Control-sense: 5'-UUCUCCGAACGUGUCACGUT-3'; Negative Control-antisense: 5'-AUGGAGACAGCUGAUAGGCT-3'), diluted to 20 nM. The transfection solutions were: Solution A (5 μL of Opti-MEM culture medium, add 0.3 μL of siRNA and mix well), and Solution B (5 μL of Opti-MEM culture medium, add 0.4 μL of lipo3000 and mix well, incubate at room temperature for 5 min). The transfection solutions were then gently mixed and incubated at room temperature for 15 min. The mixture was then added to the corresponding transfection wells and incubated for 6 h. Finally, the solution was replaced with L15 culture medium containing 10% fetal bovine serum.

[0062] Forty hours after transfection, cells were challenged with RGNNV at a concentration of 2 μg / mL. An equal volume of RNA was digested with RNase A (20 mg / mL) at 37°C for 1 hour as a control. Six hours after stimulation, cells were harvested, total RNA was extracted, reverse transcribed, and quantitative real-time PCR was performed to detect the expression levels of CP and RdRp. The experimental results are attached. Figure 7 As shown.

[0063] From the appendix Figure 7 As can be seen, knocking down ackr3 significantly increases the replication of RGNNV in GS cells.

[0064] The gain-of-function and loss-of-function experiments in Examples 5-6 above collectively demonstrate that ACKR3 can significantly inhibit OGNNV replication in GS cells. Therefore, the ackr3 gene and its encoded protein can be encapsulated in nanolipid materials to prepare an anti-RGNNV drug, which can exert an anti-RGNNV effect after injection into fish. Alternatively, ackr3 can be used as an antiviral target, and its expression can be increased through transgenic methods to achieve an anti-RGNNV effect, thus preparing RGNNV-resistant grouper breeding materials.

Claims

1. An ackr3 gene for resisting nerve necrosis virus in saddleback grouper, characterized in that, The nucleotide sequence is shown in SEQ ID NO.

1.

2. A eukaryotic expression vector comprising the ackr3 gene of claim 1.

3. The protein encoded by the ackr3 gene according to claim 1, characterized in that, The amino acid sequence is shown in SEQ ID NO.

2.

4. The use of the ackr3 gene of claim 1 or the protein it encodes, or a eukaryotic expression vector containing the ackr3 gene, in the preparation of a drug against red spot neuronecrosis virus.

5. The application of the ackr3 gene of claim 1 or the protein it encodes, or a eukaryotic expression vector containing the ackr3 gene, in the breeding of anti-erythrodermic nerve necrosis virus.

Images