A vitiligo syndrome virus-encoded protein wsv156 and its application

Abstract

This invention relates to a WSSV-encoded protein wsv156 and its applications. The amino acid sequence of wsv156 is shown in SEQ ID NO:2, and the nucleotide sequence encoding the protein is shown in SEQ ID NO:1. This invention verifies the function of wsv156 in WSSV infection, showing that wsv156 targets host mitochondria. Inhibition of wsv156 significantly suppresses host cell mitophagy and reduces viral replication in hematopoietic tissue (Hpt) cells. Therefore, wsv156 has important research value in exploring how mitophagy promotes WSSV infection, and it has great potential application prospects in the development of new anti-WSSV drugs targeting mitophagy.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a white spot syndrome virus-encoded protein wsv156 and its applications. Background Technology

[0002] White spot syndrome virus (WSSV) is one of the most serious viral pathogens affecting the aquaculture of important aquatic crustaceans such as crayfish and shrimp, causing huge economic losses to the shrimp farming industry. To date, effective prevention and drug treatment measures are still lacking. WSSV is an enveloped rod-shaped double-stranded DNA virus with a genome of approximately 300 kbp, making it one of the largest known animal viruses. Previous studies have shown that WSSV infection can cause significant structural damage to host mitochondria, including cristae destruction and vacuolation, and can significantly enhance the production of reactive oxygen species (ROS) in the hemocytes of Litopenaeus vannamei, leading to an imbalance in redox homeostasis.

[0003] Mitochondria are highly dynamic organelles that play a central role in cellular energy metabolism, redox balance, apoptosis regulation, and innate immune responses. Many viruses have evolved the ability to manipulate mitochondrial function to facilitate replication and evade host immunity. For example, DNA viruses such as hepatitis B virus (HBV) and porcine circovirus type 2 (PCV2) can promote Drp1-dependent mitochondrial fission and utilize Parkin-mediated mitophagy to inhibit apoptosis and antiviral signaling. Furthermore, various viral proteins can directly target mitochondria, disrupting their function and dynamics: for instance, the Agno protein of JC polyomavirus inhibits mitochondrial protein import by acting on the TOM / TIM translocase complex and interacts with the electron transport chain complex to impair ATP production, leading to mitochondrial dysfunction; the ORF4 protein of PCV2 can directly interact with mitochondrial adenine nucleotide translocase 3 (ANT3), inducing mitochondrial membrane depolarization and increased ROS levels; and the PB1-F2 protein of influenza virus translocates to the inner mitochondrial membrane via Tom40, inducing mitochondrial depolarization and activating Drp1, thereby driving mitochondrial fission. However, it remains unclear whether and how WSSV infection affects mitochondrial dynamics and leads to their dysfunction.

[0004] Existing WSSV-related research mainly focuses on the functional identification of other viral proteins such as wsv220 and wsv051. For example, wsv220 promotes viral replication by regulating the Nrf2 antioxidant pathway, and wsv051, as a very early gene, participates in viral transcriptional regulation. However, none of these studies have addressed the functional identification and application development of the wsv156 protein. Summary of the Invention

[0005] This invention aims to at least partially solve one of the technical problems in related technologies. Therefore, the object of this invention is to provide a vitiligo syndrome virus-encoded protein wsv156 and its applications.

[0006] Therefore, in a first aspect of the present invention, the present invention provides a white spot syndrome virus encoding protein wsv156, the amino acid sequence of which is shown in SEQ ID NO:2.

[0007] According to an embodiment of the present invention, the wsv156 protein consists of 62 amino acids and specifically targets mitochondria during WSSV infection of host cells, promoting WSSV replication by inducing mitophagy.

[0008] In a second aspect of the invention, the invention proposes a gene encoding the above-mentioned vitiligo syndrome virus-encoded protein wsv156, the nucleotide sequence of which is shown in SEQ ID NO: 1.

[0009] According to an embodiment of the present invention, wsv156 The gene ORF is 186 bp long and is a functional protein gene encoded in the WSSV genome.

[0010] In a third aspect, the present invention proposes a recombinant expression vector containing the above-mentioned genes.

[0011] According to an embodiment of the present invention, the recombinant expression vector is used to express... wsv156 pET28a-wsv156, obtained by inserting the gene into the pET28a vector, can efficiently express the recombinant His-wsv156-His fusion protein.

[0012] In a fourth aspect, the present invention proposes to include the above-described... wsv156 The host cell of the gene or recombinant expression vector.

[0013] According to an embodiment of the present invention, the host cell is Escherichia coli Transetta strain, which can efficiently express soluble wsv156 recombinant protein.

[0014] In a fifth aspect, the present invention provides a polyclonal antibody. According to an embodiment of the present invention, the polyclonal antibody is prepared by immunizing BALB / c mice with a recombinant protein expressed in host cells as an immunogen, and can specifically recognize both natural and recombinant wsv156 protein.

[0015] In a sixth aspect of the invention, the invention proposes the application of the above-mentioned WSSV-encoded protein wsv156 as a target in screening drugs against WSSV infection.

[0016] According to embodiments of the present invention, WSSV replication in host cells can be significantly inhibited by interfering with the expression or function of wsv156.

[0017] In a seventh aspect, the present invention makes the above-mentioned provision. wsv156 Application of gene inhibitors in the preparation of drugs against WSSV infection.

[0018] According to an embodiment of the present invention, the inhibitor is wsv156 The double-stranded RNA (dsRNA) of the gene can specifically knock down the expression of wsv156, thereby inhibiting WSSV replication.

[0019] In an eighth aspect of the invention, the invention proposes the use of the above-described polyclonal antibody in the preparation of WSSV detection reagents or kits.

[0020] According to embodiments of the present invention, the polyclonal antibody can be used in experiments such as Western blot and immunofluorescence to detect WSSV infection.

[0021] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0022] Figure 1 According to an embodiment of the present invention, an SDS-PAGE analysis of the purified His-wsv156-His recombinant protein showed a His-wsv156-His protein band of approximately 15 kDa. Figure 2 The image shows a Western blot detection of the wsv156 polyclonal antibody according to an embodiment of the present invention. The corresponding wsv156 band can be detected in WSSV-infected Hpt cells and in recombinant cells overexpressing wsv156-GFP protein. Figure 3 To verify the localization of wsv156 protein in mitochondria by Western blot detection after mitochondrial component separation according to embodiments of the present invention; Figure 4 To knock down Hpt cells after WSSV infection according to an embodiment of the present invention using RNA interference technology wsv156 Genes; WSSV infection for 12 hours reduces WSSV transcriptional and replication capacity. Figure 5 To verify the effect of wsv156-induced mitophagy using a western blot experiment according to an embodiment of the present invention. Detailed Implementation

[0023] The technical solution of the present invention is illustrated below through specific examples. It should be understood that the one or more method steps mentioned in the present invention do not preclude the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, unless otherwise stated, the numbering of each method step is merely a convenient tool for identifying each method step, and not for limiting the order of the method steps or defining the scope of the present invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the present invention.

[0024] To better understand the above technical solutions, exemplary embodiments of the present invention are described in more detail below. While exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the invention to those skilled in the art.

[0025] The test materials used in this invention are all commercially available products and can be purchased on the market; unless otherwise specified, the experiments involved are all conventional experimental methods.

[0026] The present invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the present invention in any way.

[0027] Example 1 wsv156 Gene cloning Hematopoietic tissue (Hpt) cells from redclaw crayfish were prepared and diluted in L15 medium before being added to cell culture plates for culture under sterile conditions at 20°C. After the Hpt cells were stably adhered, they were infected with WSSV at an MOI of 1. The cell culture plates were incubated at 26°C for 12 h. Total RNA was extracted from the WSSV-infected Hpt cells and reverse transcribed to synthesize cDNA, which served as a template for gene amplification. Based on the WSSV genome sequence (GenBank: AF332093.3), cDNA was generated. wsv156Gene-specific primers were designed based on the gene's ORF sequence for amplification. The upstream primer F was 5'-ATGAGGTTGTCACATCAAT-3' SEQ ID NO: 3, and the downstream primer R was 5'-CATTAGATTCCGTGTTGGTTTG-3' SEQ ID NO: 4. The gene sequence was amplified using PrimeSTAR® Max DNA Polymerase. The PCR reaction mixture was prepared as follows: PrimeSTAR Max Premix 15 μL; upstream primer F (10 μM) 0.5 μL; downstream primer R (10 μM) 0.5 μL; cDNA template 1 μL; RNase-Free H2O 13 μL. The PCR amplification program was: 98 ℃ pre-denaturation for 5 min; 98 ℃ denaturation for 10 s, 55 ℃ annealing for 15 s, 72 ℃ extension for 5 s, repeated 30 times; and a final extension at 72 ℃ for 10 min.

[0028] After recovering the PCR product, it was ligated into the pMD18-T vector and transformed into Escherichia coli DH5α. Positive single clones were selected for sequencing, and comparative analysis was performed to determine the obtained gene sequence. The nucleotide sequence is shown in SEQ ID NO: 1. wsv156 The gene's ORF is 186 bp long and encodes 62 amino acids.

[0029] Example 2: Recombinant expression vector pET28a-wsv156 in Escherichia coli E. coli Transetta-induced expression and protein purification 1. Construction of the recombinant expression vector pET28a-wsv156: The above-amplified encoding... wsv156 Addition to the 5' end of the gene ORF-specific upstream primer F BamH I restriction site, downstream primer R has a 5' end added Sal The I restriction site was identified, and its ORF was amplified by PCR. The PCR amplification system and reaction conditions are described in Example 1. The PCR product was recovered using the kit, and then the PCR product and the prokaryotic expression vector pET28a were separately processed. BamH I / Sal Double digestion with enzyme I was performed at 37 °C for 3 h, followed by recovery of the digestion products. The two digestion products were then ligated at 16 °C for 6 h to obtain the recombinant expression vector pET28a-wsv156. Sequencing confirmed accurate reading frames.

[0030] 2. Transform the constructed recombinant expression vector pET28a-wsv156 E. coli Transetta, incubated at 37 ℃ for 12 h.

[0031] 3. Pick a single colony and inoculate it into 1 mL of LB medium containing kanamycin. Incubate at 37 °C and 220 rpm for 12 h.

[0032] 4. Inoculate 1:100 into 250 mL of LB medium containing kanamycin and incubate at 37 °C with a shaker at 220 rpm until OD (dose elapsed). 600 It ranges from 0.4 to 0.6.

[0033] 5. Add IPTG to a final concentration of 0.1 mM and induce expression at 28 ℃ and 200 rpm for 16 h.

[0034] 6. Centrifuge at 5000×g for 10 min to collect the bacterial cells.

[0035] 7. Resuspend the bacterial cells in Tris-HCl buffer containing 50 mL of 8M urea (to dissolve and denature the wsv156 protein), add PMSF protease inhibitor, sonicate to break up the resuspended solution until the bacterial solution is clear, then centrifuge at 10000×g for 30 min at 4℃ and collect the supernatant.

[0036] 8. Add 500 μL of Ni Beads to the column with a gasket, wash with pure water, and then equilibrate the column using Tris-HCl buffer containing 8 M urea. Pour the collected supernatant into the column and repeat the process 3-4 times.

[0037] 9. Elute with Tris-HCl elution buffer containing 20 mM imidazole and 8 M urea, then elute the target protein with Tris-HCl elution buffer containing 500 mM imidazole and 8 M urea. Collect the eluent and perform SDS-PAGE electrophoresis on a small amount for identification.

[0038] 10. Cut a dialysis bag to a suitable length, boil it in pure water for 10 minutes, and store it in a bottle of RO water at 4°C. Put the protein solution with high concentration and purity detected into the dialysis bag, fix and seal it with a clip, and dialyze it in Tris-HCl dialysate containing 4 M urea. Change the dialysate every 4 hours. After dialysis 3 times, aspirate the protein from the dialysis bag, run it on a gel for identification, and store it at -80°C.

[0039] 11. The purified recombinant protein His-Wsv156-His was used for antibody preparation.

[0040] The results are as follows Figure 1 The recombinant protein His-Wsv156-His is a relatively single protein band, which is the correct size and is approximately 15 kDa.

[0041] Example 3: Preparation of wsv156 polyclonal antibody 1. Pre-collection of blood: Blood is collected by tail clipping. 100 μL of blood is placed in a sterile centrifuge tube, left to stand at room temperature for 30 min, then left to stand at 4℃ for 2 h, and centrifuged at 6000 g for 5 min at room temperature. The supernatant is then transferred to a new centrifuge tube. The resulting negative serum is stored at -80℃.

[0042] 2. Animal Immunization: ① Primary Immunization: Order three female BALB / c mice and stably house them for one week. Using a syringe, draw recombinant protein His-wsv156-His and Freund's complete adjuvant in a 1:1 ratio and emulsify the protein using a protein emulsifier. Using a 1 mL syringe, administer 200 μL of the emulsified protein subcutaneously to each mouse using the three-point method. ② Second and Third Immunizations: One week apart, administer the same volume of protein as above. Collect blood 3-5 days after the third immunization and use Western blot to detect wsv156 expression in Hpt cells. ③ Booster Immunization: Without adjuvant, administer 50-100 μg of protein directly, subcutaneously using a fine-tipped syringe (or intramuscularly, via the footpads, peritoneally, intravenously, or via the spleen), a slower process. Collect blood 3-5 days after the booster immunization to detect antibody titers. ④ Blood collection from mouse eyeballs: Gently grasp the mouse's neck skin using the thumb, index, and middle fingers of your left hand, and use the little and ring fingers of your left hand to hold the mouse's tail. Gently press the skin around the eye on the side where the eyeball needs to be removed, causing the eyeball to protrude further due to congestion. Quickly remove the eyeball and collect the blood in a sterile centrifuge tube. Then, euthanize the mouse using the cervical dislocation method. Process the serum using the same method described above.

[0043] 3. Validation of wsv156 polyclonal antibody: Results are as follows Figure 2 Antibody verification using Hpt cells infected with WSSV revealed a specific WSSV156 band.

[0044] Construction of recombinant expression vector pCDNA3.1-wsv156-GFP: Amplification of the encoding vector in Example 1 above. wsv156 Addition to the 5' end of the gene ORF-specific upstream primer F BamH I restriction site, downstream primer R has a 5' end added EcoR1 The I restriction site and vector construction steps are described in Example 2. The wsv156-GFP expression plasmid was processed using Lipo293... TM HEK293T cells were transfected using the transfection kit. 36 h after transfection, cells were lysed with 1×SDS loading buffer and collected. The samples were boiled for 10 min, and Western blot analysis showed that the wsv156-GFP recombinant protein also showed bands of the correct size.

[0045] Example 4: Localization detection of wsv156 polyclonal antibody in HPT cells of red swamp crayfish infected with WSSV Red swamp crayfish (Hpt) cells were cultured in vitro, diluted in L15 medium, and then added to cell culture plates under sterile conditions at 20 °C. After the Hpt cells were stably adhered, they were infected with WSSV. Cells were harvested after 12 h, and mitochondria were extracted using the TransGen® Cell Mitochondria Isolation Kit. Mitochondria were lysed with 1×SDS loading buffer, and the samples were boiled for 10 min. Western blot was used to detect the localization of wsv156 protein.

[0046] The results are as follows Figure 3 The wsv156 protein was detected in the mitochondrial components of red swamp crayfish Hpt cells infected with WSSV, indicating that the wsv156 protein targets mitochondria.

[0047] Example 5 wsv156 Effects of WSSV knockdown on Hpt cells wsv156 double-stranded RNA (dsRNA) was synthesized using the MEGAscript® T7 Transcription Kit for gene interference experiments. The nucleotide sequence of the double-stranded template is SEQ ID NO: 1. The upstream primer F: 5'-GCGTAATACGACTCACTATAGGGatgaggttgtcacatcaatt-3' SEQ ID NO: 5, and the downstream primer R: 5'-GCGTAATACGACTCACTATAGGGGCACGAGGAAGAAATGTGCT-3' SEQ ID NO: 6. Red swamp crayfish Heptagram cells were cultured, diluted in L15 medium, and added to cell culture plates under sterile conditions at 20 °C. After Hpt cells were stably adhered to the culture plate, WSSV was infected with MOI=1. The cell culture plate was placed in an incubator at 26 ℃ and cultured for 3 h. Then, an appropriate amount of wsv156 dsRNA, cellfectin transfection reagent and DEPC water were mixed and incubated at room temperature for 15 min. The mixture was then added to the cultured cells. The control dsRNA was GFP dsRNA. The nucleotide sequence of GFP dsRNA is referenced (GenBank: U55761.1). The template was obtained from the plasmid obtained in Example 3. The upstream primer F: 5'-TAATACGACTCACTATAGGGCGACGTAAACGGCCACAAGT-3'SEQ ID NO: 7, and the downstream primer R: 5'-AATACGACTCACTATAGGGTTCTTGTACAGCTCGTCCATGC-3'SEQ ID NO: 8. Twelve hours after infection, cells were lysed and collected using 1×SDS loading buffer. The samples were boiled for 10 minutes, and Western blot was used to detect changes in wsv156 and viral VP28 proteins. Twelve hours after infection, RNA was extracted using a cell RNA extraction kit, and viral transcription and replication were detected after reverse transcription.

[0048] See results Figure 4 After the wsv156 gene was knocked down, the expression level of the wsv156 gene was significantly reduced compared with the control group, the transcriptional replication level of WSSV was reduced, and the level of viral envelope protein VP28 protein was also reduced.

[0049] Example 6: wsv156 expression induces mitophagy Construction of recombinant expression vector pCDNA3.1-wsv156-Myc: Amplification of the encoding in Example 1 wsv156 Addition to the 5' end of the gene ORF-specific upstream primer F BamH I restriction site, downstream primer R has a 5' end added EcoR1The steps for I restriction site cleavage, vector construction, and HEK 293T cell transfection are described in Example 3. After transfecting the wsv156-Myc expression plasmid for 36 h, the cells were lysed with 1×SDS loading buffer and collected. The samples were boiled for 10 min, and the expression of mitochondrial proteins TOMM20, COXIV, VDAC, and wsv156 was detected by Western blot.

[0050] See results Figure 5 The expression of mitochondrial proteins TOMM20, COX IV, and VDAC decreased in a gradient with wsv156 expression, indicating that wsv156 induces mitophagy.

[0051] In summary, according to embodiments of the present invention, after amplification... wsv156 Based on genes, according to wsv156 The recombinant protein His-wsv156-His, purified from its gene sequence, was used for antibody preparation. dsRNA was designed to interfere with its expression, and its function in WSSV infection was verified. The results showed that wsv156 targets host mitochondria; inhibition of wsv156 significantly suppressed host cell mitophagy and reduced viral replication in hematopoietic tissue (Hpt) cells. Therefore, wsv156 has significant research value in exploring how mitophagy promotes WSSV infection and shows great potential for development of novel anti-WSSV drugs targeting mitophagy.

[0052] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. The illustrative expressions of the above terms in this specification should not be construed as necessarily referring to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. In addition, those skilled in the art can combine and integrate the different embodiments or examples described in this specification.

[0053] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A vitiligo syndrome virus encoding protein wsv156, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

2.

2. The gene encoding the wsv156 protein of the vitiligo syndrome virus according to claim 1, characterized in that, Its nucleotide sequence is shown in SEQ ID NO:

1.

3. A recombinant expression vector, characterized in that, It contains the gene described in claim 2.

4. A host cell, characterized in that, The invention comprises the recombinant expression vector of claim 3, wherein the host cell is Escherichia coli Transetta strain.

5. A polyclonal antibody, characterized in that, The polyclonal antibody was prepared by immunizing BALB / c mice with the recombinant protein expressed in the host cells as described in claim 4 as an immunogen.

6. The use of the WSSV156 protein encoded by the vitiligo syndrome virus as described in claim 1 as a target in screening drugs against WSSV infection.

7. The use of the gene inhibitor according to claim 2 in the preparation of an anti-WSSV infection drug, characterized in that, The inhibitor is a double-stranded RNA of the wsv156 gene.

8. The use of the polyclonal antibody according to claim 5 in the preparation of WSSV detection reagents or kits.

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