DsRNA, recombinant vector, recombinant cell or recombinant strain thereof and application of dsRNA

Abstract

The invention discloses dsRNA, a recombinant vector, a recombinant cell or a recombinant strain thereof and application of the dsRNA. The invention also discloses a construction method of the recombinant vector or the recombinant strain. The recombinant vector constructed by the invention can stably express the dsRNA in the bacillus subtilis, so that the synthesis cost of the dsRNA is greatly reduced, and as important biocontrol bacteria and probiotics, the bacillus subtilis has sufficient potential of serving as a vector to deliver the dsRNA, so that a full-chain scheme is provided for solving the problem of production-delivery of the dsRNA.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering and relates to a dsRNA, a recombinant vector, a recombinant cell or a recombinant strain and applications thereof, and in particular to a plasmid capable of stably expressing target dsRNA in Bacillus, a construction method and applications thereof. Background Art

[0002] In the RNA interference (RNAi) system, double-stranded RNA (dsRNA) serves as the core molecule that induces gene silencing. Small interfering RNA (siRNA) is generated through the cleavage of the Dicer nuclease. These small interfering RNAs (siRNAs) bind to the RNA-induced silencing complex (RISC) to specifically degrade target mRNAs, thereby precisely regulating gene expression. Notably, dsRNA not only plays a key role in gene silencing but also possesses significant antiviral activity. By inducing an innate immune response within host cells, dsRNA can target RNA intermediates produced during viral replication and utilize the RNAi mechanism to effectively inhibit viral gene expression, reducing the viral load in the host and achieving antiviral capabilities. Current dsRNA synthesis methods, such as chemical synthesis and in vitro transcription, generally suffer from high cost, low yield, and insufficient stability, which limits their application in areas such as agricultural pest control and viral disease prevention.

[0003] Bacillus subtilis ( Bacillus subtilis Bacillus subtilis is a Gram-positive, aerobic, spore-forming bacterium widely found in plants and soil. It belongs to the phylum Firmicutes, class Bacillus, order Bacilales, family Bacilaceae, and genus Bacillus. Due to its excellent genetic operability, safety, and stress resistance, Bacillus subtilis is often used as a research tool and production platform for bioactive substances such as exogenous proteins and secondary metabolites. Furthermore, Bacillus subtilis is used to express antigenic sites such as viral membrane proteins, making it a candidate for the preparation of live bio-vector vaccines.

[0004] Bacillus subtilis, with its mature genetic manipulation platform and excellent industrial fermentation performance, is a key platform for synthetic biology and is widely used in the expression of exogenous proteins and the production of secondary metabolites. However, its application in RNA production is limited. Due to the abundant presence of RNases within its system, the single-stranded RNA (ssRNA) produced by traditional single-promoter transcription is unstable and is cleaved and degraded by bacterial RNases, which severely limits the application of Bacillus subtilis in RNA production. Furthermore, its ability to serve as a vector for delivering biologically active RNA to animals has not been verified, which is a key reason why Bacillus subtilis is currently rarely used for RNA production. Summary of the Invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a dsRNA that can be stably expressed in Bacillus.

[0006] The technical problem to be solved by the present invention is to provide a recombinant vector, a recombinant cell or a recombinant strain, and in particular to an Escherichia coli-Bacillus subtilis shuttle plasmid vector capable of stably expressing target dsRNA in Bacillus.

[0007] The technical problem that the present invention also aims to solve is to provide a method for constructing a recombinant vector and a recombinant strain.

[0008] The technical problem that the present invention also aims to solve is to provide the use of the dsRNA, the recombinant vector, the recombinant cell or the recombinant strain in the preparation of vaccines or drugs against viral infection.

[0009] The final technical problem to be solved by the present invention is to provide an antiviral vaccine or medicine.

[0010] Technical solution: In order to solve the above technical problems, the present invention provides a dsRNA, the DNA sequence of which is shown in SEQ ID NO.1.

[0011] The present invention also includes a recombinant vector, a recombinant cell or a recombinant strain containing the dsRNA.

[0012] Wherein, the recombinant vector is an Escherichia coli-Bacillus shuttle vector.

[0013] The recombinant vector further includes an Escherichia coli replication origin, a Bacillus subtilis thermosensitive replication origin, a resistance gene for Escherichia coli screening, a resistance gene for Bacillus subtilis screening, a fluorescent protein gene for screening positive transformed colonies, and a xylose-inducible promoter for expressing fluorescent protein.

[0014] Wherein, the nucleotide sequence of the recombinant vector is shown as SEQ ID NO.2.

[0015] The method for constructing the recombinant vector comprises the following steps: (1) synthesizing the DNA sequence of the dsRNA and amplifying or synthesizing the constitutive promoter Pylb; (2) Amplify or synthesize the gene fragments of the xylose-inducible promoter PxylA and the red fluorescent protein mCherry; (3) The dsRNA DNA sequence and Pylb from step (1) and the gene fragments of PxylA and mCherry from step (2) are seamlessly connected to obtain the product.

[0016] The method for constructing the recombinant strain comprises the following steps: transforming the recombinant vector into Bacillus by chemical transformation or electroporation, culturing the strain in LB solid medium containing xylose and spectinomycin, and selecting colonies emitting red fluorescence using fluorescence excitation at an excitation wavelength of 580 nm.

[0017] The present invention also includes the use of the dsRNA, the recombinant vector, the recombinant cell or the recombinant strain in the preparation of vaccines or drugs against viral infections.

[0018] Wherein, the virus includes avian influenza H9N2 virus.

[0019] The present invention also includes an antiviral vaccine or drug, which includes the dsRNA or the recombinant vector, recombinant cell or recombinant strain.

[0020] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The present invention adds a pair of bidirectional promoters at both ends of the sequence encoding the target RNA and simultaneously initiates the transcription of the double-stranded DNA to obtain dsRNA, thereby enhancing the stability of the target RNA production. At the same time, dsRNA, as an important factor in activating the RNAi pathway, can activate the RNAi pathway and exert an antiviral effect.

[0021] 2. The present invention constructs the target dsRNA containing a bidirectional promoter into an E. coli-Bacillus shuttle vector, allowing the production of a high-copy plasmid using E. coli, which is then transformed into Bacillus. Because the plasmid constructed in the present invention is an integrative plasmid of Bacillus, it can be integrated into the Bacillus genome for stable expression, resolving the problem of conventional Bacillus plasmids being easily lost during passage. After the plasmid has completed its function, it can be eliminated in Bacillus by high temperature. This genetically modified engineered Bacillus strain no longer contains exogenous plasmids, preventing the drift of resistance genes carried by the plasmid and promoting environmental friendliness.

[0022] 3. The present invention also constructs a resistance gene and a xylose-induced red fluorescent protein mCherry gene on the basis of a plasmid containing a bidirectional constitutive promoter Pylb, so that when picking positive transformed colonies after transformation, colonies with good expression effects can be picked by detecting the red fluorescence intensity of the positive colonies.

[0023] In summary, the plasmid constructed in the present invention can stably express dsRNA in Bacillus subtilis, greatly reducing the cost of dsRNA synthesis and significantly improving the yield and stability of dsRNA. At the same time, delivering dsRNA through Bacillus subtilis can effectively protect dsRNA from degradation by the environment and a large number of RNases in the animal body, helping it to be successfully delivered into the animal body, activating the animal's RNAi system, and achieving an antiviral effect. This provides a full-chain solution for solving the production and delivery of dsRNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the construction process of plasmid pDGds-AAD for stably expressing dsRNA targeting AIV in Bacillus subtilis; Figure 2 This is the plasmid map of plasmid pDGds-AAD; Figure 3 Bacillus subtilis transformed with plasmid pDGds-AAD Bacillus subtilis 168 Fluorescence image of LB solid plates containing 1.5% xylose and 100 μg / mL spectinomycin grown at 37°C for 10 h; Figure 4 Agarose gel electrophoresis of the cDNA amplification product of the recombinant bacteria AAD transformed with the plasmid pDGds-AAD; Lane M is a marker; Lane 1 is a positive control for in vitro transcribed dsRNA; Lane 2 is the cDNA amplification product of the recombinant bacteria AAD; Lane 3 is the wild-type Bacillus subtilis Bacillus subtilis cDNA amplification product of 168; Figure 5 The figure shows the daily weight changes of chicks. The PBS group refers to chicks challenged with PBS after intranasal inoculation; the RNA group refers to chicks challenged with PEI-encapsulated H9N2 dsRNA after intranasal inoculation; the Bs168 group refers to chicks challenged with wild-type Bacillus subtilis after intranasal inoculation. Bacillus subtilis 168 refers to chickens challenged with the virus after intranasal administration of recombinant Bacillus subtilis AAD expressing H9N2-targeting dsRNA (subsequent grouping is not repeated here). Graphpad Prism 9 was used for image plotting, difference significance analysis, and statistical testing. Figure 6 The following are the pathological examination pictures of the chicken lungs. Blank is the lung of the chicken that was not treated and not challenged with poison. The rest of the groups are the same. Figure 5 illustrate; Figure 7 HE staining of chicken lung pathological sections, grouped in the same Figure 6 illustrate;

[0025] Figure 8The figure shows the detection of H9N2 virus load in chicken lungs. β-Actin was used as the internal reference gene. The expression level of H9N2-HA mRNA was detected by SYBR method. -ΔΔct The data were processed by the method, the logarithm of the vertical axis data was calculated, and Graphpad Prism 9 was used to draw the graph and perform difference significance analysis and statistical test;

[0026] Figure 9 The figure shows the detection of H9N2 virus load in chicken trachea. β-Actin was used as the internal reference gene. The expression level of H9N2-HA mRNA was detected by SYBR method. -ΔΔct The data were processed by the method, the logarithm of the vertical axis data was calculated, and Graphpad Prism 9 was used to draw the graph and perform difference significance analysis and statistical test. DETAILED DESCRIPTION

[0027] The technical solutions of the present invention are further described below in conjunction with the accompanying drawings. The present invention is further described below through specific examples and accompanying drawings. Unless otherwise specified, the materials, reagents, etc. used in the examples of the present invention are commercially available. The methods used in the following examples are conventional methods unless otherwise specified. The resistance genes and synthetic genes in the present invention are all known genes.

[0028] The sources of materials and reagents in the examples of the present invention are as follows: pDG1663 (Shanghai Zeye Biotechnology Co., Ltd., Catalog No.: ZY1866), pAX01-mCherry (Shanghai ELISA Biotechnology Co., Ltd., Catalog No.: ml-G13175), Bacillus subtilis Bacillus subtilis 168 (gift from Professor Gao Xuewen of the College of Plant Protection, Nanjing Agricultural University; also available through commercial channels) DMEM medium (Nanjing Senbega Biotechnology Co., Ltd.); 10% protein precast gel (Shanghai Yazyme Biopharmaceutical Technology Co., Ltd.); 5× SDS-PAGE (Wuhan Gaiyuntian Biotechnology Co., Ltd.); 4× denaturing protein loading buffer (Shanghai Yisheng Company); ClonExpress II One Step Cloning Kit (Nanjing Novozymes Biotechnology Co., Ltd.).

[0029] The reagents used for the conversion in the examples of the present invention are as follows: SP solution (2 mg / mL ammonium sulfate, 14 mg / mL potassium dihydrogen phosphate trihydrate, 6 mg / mL potassium dihydrogen phosphate, 1 mg / mL trisodium citrate), solution 1 (50 mg / mL magnesium sulfate heptahydrate), solution 2 (20 mg / mL casein hydrolysate, 100 mg / mL yeast extract), solution 3 (500 mg / mL glucose), solution 4 (5.549 mg / mL anhydrous calcium chloride), solution 5 (23.802 mg / mL magnesium chloride), and solution 6 (3.804 mg / mL EGTA, adjusted to pH = 8.0 using NaOH).

[0030] Example 1 Construction and transformation of pDGds plasmid like Figure 1 As shown, using plasmid pDG1663 as a template, primers pDG-F and pDG-R were designed to amplify the vector backbone pDG; using plasmid pAX01-mCherry as a template, primers PxylA-F and PxylA-R were designed to amplify the xylose-inducible promoter PxylA; using plasmid pAX01-mCherry as a template, primers mCherry-F and mCherry-R were designed to amplify the red fluorescent protein mCherry; using Bacillus subtilis as a template, primers Bacillus subtilis 168 genome as a template, and primers Pylb-F and Pylb-R were designed to amplify the constitutive promoter Pylb; the primer sequences are shown in Table 1, the amplification system is shown in Table 2, and the amplification conditions are shown in Table 3. DNA encoding dsRNA targeting AIV was synthesized, and the DNA sequence of dsRNA is shown in SEQ ID NO.1. The amplified fragments were handed over to General Bio (Anhui) Co., Ltd. in the order of PxylA-mCherry-Pylb (forward)-anti AIV RNA-Pylb (reverse) and connected to the backbone pDG vector using seamless connection technology to obtain a plasmid vector pDGds-AAD capable of expressing dsRNA targeting AIV. The plasmid map is shown in Figure 2 As shown, the nucleotide sequence of the plasmid pDGds-AAD is shown as SEQ ID NO.2.

[0031] Table 1 Primer sequences

[0032] Table 2 Amplification system

[0033]

[0034] Table 3 Amplification conditions

[0035] Example 2 Transformation of pDGds plasmid vector 1. Preparation of Bacillus subtilis competent cells and transformation with pDGds-AAD plasmid 1) Pick the host bacteria (Bacillus subtilis Bacillus subtilis 168) were inoculated into 20 mL of LB liquid medium and cultured at 37°C, 200 rpm overnight.

[0036] 2) Take 800 μL of the overnight culture and inoculate it into 25 mL of SP I medium (24 mL of SP solution + 250 μL of solution 1 + 250 μL of solution 2 + 250 μL of solution 3). Incubate at 37°C with shaking at 200 rpm. After 2 h, measure the OD600. When the culture reaches the late logarithmic growth phase (OD600 = 1.0), quickly inoculate 2.5 mL of the bacterial culture into 25 mL of SP II medium (24 mL of SP solution + 250 μL of solution 1 + 250 μL of solution 2 + 250 μL of solution 3 + 250 μL of solution 4 + 250 μL of solution 5). Incubate at 37°C with shaking at 100 rpm for 1.5 h.

[0037] 3) Add 250 μL of solution 6, incubate at 37°C, 100 rpm, and shake for 10 min. Aliquot into 1.5 mL centrifuge tubes, 500 μL each.

[0038] 4) Add the constructed plasmid pDGds-AAD (1 μg) to the tube, mix gently, and incubate at 37°C with a shaker at 100 rpm for 30 min. Then, continue incubation at 200 rpm for 1.5 h.

[0039] 5) Collect the cells by centrifugation at 4000 rpm, discard part of the supernatant, and keep 100 μL to resuspend the cells. Apply 1.5% xylose and 100 μg / mL spectinomycin resistance (Spc + ) LB plate, inverted in a 37 ℃ incubator overnight culture, using a fluorescence microscope, using 580 nm wavelength excitation, as shown in Figure 2. Figure 3 As shown, it can be seen that the colonies have typical Bacillus subtilis morphology and emit striking red fluorescence. Colonies that meet the above characteristics can be determined as positive transformation colonies.

[0040] Example 3 Extraction of bacterial total RNA and acquisition of cDNA Pick the single colony activated by streaking after overnight culture in Example 2 and culture it overnight in resistant liquid LB medium. After the bacterial solution was centrifuged at 12000 rpm to collect the bacteria, resuspend it in DEPC water and then centrifuge and wash the bacteria twice. Add lysozyme at a concentration of 10U / mL and place it in a 37°C shaker for at least 30 minutes to destroy the bacterial cell wall. Take part of the bacterial lysate, extract total RNA using the Trizol method, and use the reverse transcription kit HiScript IV Q RT SuperMix forqPCR (+gDNAwiper) from Novozymes to perform reverse transcription according to the instructions to obtain the cDNA of recombinant Bacillus subtilis.

[0041] Example 4 Verification of expression of target dsRNA Primers AIV-F and AIV-R were designed using the DNA sequence encoding the target dsRNA (shown in SEQ ID NO. 1) as a template. PCR was performed using the 2× Rapid Taq Master Mix kit from Novozymes according to the manufacturer's instructions. The amplification primers, amplification system, and amplification conditions are shown in Tables 4 to 6.

[0042] Table 4 Amplification primer sequences

[0043] Table 5 Amplification system

[0044] Table 6 Amplification conditions

[0045] The amplified product was electrophoresed on 1% agarose gel, and a clear band was observed at 300 bp. Figure 4 ), and the blank biological control Bacillus subtilis without transformed plasmid Bacillus subtilis Compared with 168, the recombinant bacteria AAD transformed with plasmid pDGds-AAD can express the target dsRNA.

[0046] Example 5: Intranasal administration of Bacillus subtilis stably expressing dsRNA targeting avian influenza H9N2 to protect chicks from H9N2 infection Thirty ten-day-old SPF chicks (purchased from Yangzhou Guoji Biotechnology Co., Ltd.) were divided into five groups, with six chicks in each group. The blank group received intranasal PBS (0.01 M, pH = 7.2) for two consecutive days without challenge; the PBS group received intranasal PBS for two consecutive days with one day off. The titer of the virus in each chick was 3 × 10 6 PFU / mL of H9N2 avian influenza virus (laboratory of Nanjing Agricultural University) 300 μL; Bs168 group was intranasally dripped 200 μL / animal with a concentration of 1×108 CFU / mL of wild-type Bacillus subtilis Bacillus subtilis 168, one day after each chick was infected with the virus at a titer of 3 × 10 6 PFU / mL of H9N2 avian influenza virus 300 μL; AAD group for two consecutive days of nasal drops 200 μL / mouse at a concentration of 1×10 8 CFU / mL of recombinant Bacillus subtilis AAD transformed with plasmid pDGds-AAD was added 200 μL / chicken, and the virus titer of each chick was 3×10 6 The RNA group was treated with 300 μL of H9N2 avian influenza virus containing PFU / mL. In the RNA group, polyethylenimine (PEI, Shanghai MacLean Biochemical Technology Co., Ltd., P871834) was mixed with H9N2-targeting dsRNA at a nitrogen-to-phosphorus ratio (N / P) of 6.5:1 (the DNA sequence of dsRNA is shown in SEQ ID NO. 1 and was synthesized by Sangon Biotech (Shanghai) Co., Ltd.). The mixture was administered intranasally at a rate of 200 μL per chick for two consecutive days, with one day between doses. The titer of the virus infecting each chick was 3 × 10 6 300 μL of H9N2 avian influenza virus (PFU / mL) was added. Animals were observed daily and weighed daily after challenge. Animals in the challenge group were sacrificed and samples were collected 6 days after challenge.

[0047] Subtract the chicks’ weight each day from the previous day to calculate the daily weight gain, e.g. Figure 5 As shown, the RNA group is a preparation of dsRNA targeting H9N2 wrapped in PEI material for nasal drops, and the Bs168 group is a wild-type Bacillus subtilis Bacillus subtilis Both the 168 and AAD groups (recombinant Bacillus subtilis AAD expressing dsRNA targeting avian influenza H9N2) were able to reduce weight loss in chicks induced by H9N2 infection. Intranasal administration of AAD was the most significant in restoring weight in chicks. This is because Bacillus subtilis, as a probiotic, improves the intestinal flora and growth performance of animals. Furthermore, AAD's ability to express and deliver dsRNA targeting H9N2 reduces the viral load of H9N2 in animal tissues, minimizing damage to these tissues.

[0048] Take the chick's lungs and perform autopsy. Figure 6 The results showed that the lungs of chicks challenged with PBS intranasally developed obvious lesions, with consolidation of the lungs and pinpoint to millet-sized hemorrhages scattered on the lung surface, some of which merged into sheet-like hemorrhages, and some tissues were necrotic and gray, which was significantly different from the state of the Blank group. Figure 7The HE-stained lung sections of chicks shown in the unchallenged Blank group demonstrate intact and evenly distributed alveoli. Numerous alveoli of similar size formed regular polygons, with wide, unobstructed cavities and no localized collapse or adhesions. The alveolar septa were thin and well-defined, with a discernible network of small capillaries. No significant thickening or fibrosis was observed, and no large numbers of neutrophils or lymphocytes were found within the alveolar septa or cavities, indicating that the tissue had not been subjected to acute or chronic inflammatory stimulation. A small number of scattered blood vessels were visible within the alveolar walls, with unobstructed lumens and continuous endothelial cells, and no signs of congestion, hemorrhage, or edema. The interstitial matrix (including elastic fibers and a small amount of connective tissue) was uniform in structure, with no hyperplasia or desquamation, and the overall histological appearance resembled that of a healthy state. After challenge with H9N2 avian influenza virus, deep red red blood cell aggregates (arrows) were observed in multiple alveolar cavities, indicating a significant increase in vascular wall permeability, accompanied by punctate or patchy hemorrhages. Compared with the control group, the alveolar septa thickened significantly, no longer thin and clear, but instead exhibiting a distended appearance due to cellular infiltration. Numerous neutrophils and monocytes (clusters of small, round, purple-stained nuclei) were observed within the alveolar septa and cavities, indicating a strong acute inflammatory response. Driven by inflammatory exudates and cellular infiltration, some alveolar cavities experienced localized collapse, even blurring the boundaries between adjacent cavities and resulting in fusion, impairing gas exchange. Edema of the lung parenchymal connective tissue was observed, with a pale purple, gelatinous interstitial layer visible on sections. Prolonged lesions may also be accompanied by fibroblast proliferation, laying the foundation for subsequent fibrosis. Based on the results of pathological anatomy and pathological sections, the lungs of chicks infected with avian influenza virus showed obvious acute inflammatory response, accompanied by bleeding and edema caused by increased vascular permeability, as well as destruction of alveolar structure and thickening of the septum. This shows that intranasal administration of H9N2 avian influenza caused obvious lung damage to chicks, and the H9N2 infection model was established. At the same time, the lungs of chicks challenged with wild-type Bacillus subtilis Bs168 after intranasal injection also showed obvious lesions. Pathological sections showed that the alveolar septa were thickened, with a small number of neutrophils and monocytes scattered in the alveolar septa and cavities. The alveolar septa were slightly thicker than those in the Blank group, with slight edema and inflammatory cell infiltration. This indicated that the wild-type strain Bs168 could not alleviate the lung damage caused by H9N2 challenge. The lungs of chicks that were intranasally injected with recombinant Bacillus subtilis AAD expressing AIV dsRNA were pink, fresh and soft, with no obvious lesions. Pathological sections also showed that their tissues were no significantly different from those of the unchallenged control group Blank, and there were no obvious pathological changes in the tissues. This indicates that intranasal administration of recombinant bacteria AAD can reduce lung damage caused by H9N2 infection in chicks and help them resist H9N2 avian influenza.

[0049] Chicken lungs and trachea were ground and RNA was extracted using the Trizol method. cDNA was obtained by reverse transcription using the Novagen HiScript IV QRT SuperMix for qPCR (+gDNAwiper) Reverse Transcription Kit. qPCR was performed using the Novagen ChamQSYBR qPCR Master Mix (Low ROX Premixed) Kit according to the manufacturer's instructions. β-Actin was used as an internal reference gene to measure H9N2-HA mRNA expression, which served as a standard for viral load in tissues. The primer sequences used in the assay are shown in Table 7.

[0050] Table 7 qPCR detection primer sequences

[0051] like Figure 8 、 9 As shown, intranasal drops of recombinant Bacillus subtilis can significantly reduce the viral load of H9N2, while intranasal drops of PEI-encapsulated dsRNA and Bs168 cannot reduce the viral load of H9N2 in the lungs and trachea of chicks.

Claims

1. A dsRNA, characterized in that The DNA sequence of the dsRNA is shown in SEQ ID NO.

1.

2. A recombinant vector, recombinant cell or recombinant strain, characterized in that: It contains the dsRNA according to claim 1.

3. The recombinant vector, recombinant cell or recombinant strain according to claim 2, characterized in that: The recombinant vector is an Escherichia coli-Bacillus shuttle vector.

4. The recombinant vector, recombinant cell or recombinant strain according to claim 3, characterized in that The recombinant vector also includes an Escherichia coli replication origin, a Bacillus subtilis thermosensitive replication origin, a resistance gene for Escherichia coli screening, a resistance gene for Bacillus subtilis screening, a fluorescent protein gene for screening positive transformation colonies, and a xylose-inducible promoter for expressing fluorescent protein.

5. The recombinant vector, recombinant cell or recombinant strain according to claim 2, characterized in that: The nucleotide sequence of the recombinant vector is shown in SEQ ID NO.

2.

6. The method for constructing a recombinant vector according to any one of claims 2 to 5, characterized in that: The following steps are involved: (1) synthesizing the DNA sequence of the dsRNA according to claim 1, and amplifying or synthesizing the constitutive promoter Pylb; (2) Amplify or synthesize the gene fragments of the xylose-inducible promoter PxylA and the red fluorescent protein mCherry; (3) The dsRNA DNA sequence and Pylb from step (1) and the gene fragments of PxylA and mCherry from step (2) are seamlessly connected to obtain the product.

7. The method for constructing the recombinant strain according to claim 2, characterized in that: The method comprises the following steps: transforming the recombinant vector into Bacillus by chemical transformation or electrotransformation, culturing in LB solid culture medium containing xylose and spectinomycin, using fluorescence excitation, and picking colonies emitting red fluorescence.

8. Use of the dsRNA according to claim 1, the recombinant vector, the recombinant cell or the recombinant strain according to claim 2 in the preparation of vaccines or drugs against viral infections.

9. The use according to claim 8, characterized in that The viruses include avian influenza H9N2 viruses.

10. An antiviral vaccine or drug, characterized in that: The vaccine or drug comprises the dsRNA according to claim 1 or the recombinant vector, recombinant cell or recombinant strain according to claim 2.

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