A dsRNA, a recombinant vector, a recombinant cell or a recombinant strain thereof and application thereof

Abstract

The application discloses a kind of dsRNA, its recombinant vector, recombinant cell or recombinant strain and application thereof.The application also discloses the construction method of the recombinant vector or the recombinant strain.The recombinant vector constructed by the application can stably express dsRNA in bacillus subtilis, which greatly reduces the synthesis cost of dsRNA, and as important biocontrol bacteria and probiotics, bacillus subtilis has sufficient potential as a carrier to deliver dsRNA, which provides a full-chain solution for solving the production-delivery of dsRNA.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering and relates to a dsRNA, its recombinant vector, recombinant cells or recombinant strains and their applications. Specifically, it relates to a plasmid that can stably express the target dsRNA in Bacillus, its construction method and application. Background Technology

[0002] In RNA interference (RNAi) systems, double-stranded RNA (dsRNA) serves as the core molecule for inducing gene silencing. Through cleavage by the Dicer nuclease, it generates small interfering RNA (siRNA), which then binds to the RNA-induced silencing complex (RISC) to specifically degrade target mRNA, thereby precisely regulating gene expression. Notably, dsRNA not only plays a crucial role in gene silencing but also possesses significant antiviral activity. By inducing a natural immune response within host cells, dsRNA can target RNA intermediates generated during viral replication, effectively inhibiting viral gene expression through the RNAi mechanism, reducing viral load in the host, and achieving antiviral capabilities. Current methods for synthesizing dsRNA, such as chemical synthesis and in vitro transcription, generally suffer from high costs, low yields, and insufficient stability, limiting their application in areas such as agricultural pest and disease 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 Bacillusales, family Bacillusaceae, 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 or secondary metabolites. Furthermore, Bacillus subtilis is also used to express viral membrane proteins and other antigenic sites, serving as a candidate for the preparation of live vector vaccines.

[0004] Bacillus subtilis possesses a mature genetic manipulation platform and excellent industrial fermentation performance, making it an important substrate bacterium in synthetic biology. It is widely used in exogenous protein expression and the production of secondary metabolites, but its application in RNA production is limited. Due to the abundant RNases within its cells, single-stranded RNA (ssRNA) transcribed by traditional single-promoter transcription is unstable and is cleaved and degraded by bacterial RNases, severely restricting the application of Bacillus subtilis in RNA production. Furthermore, its ability to serve as a vector for delivering biologically active RNA to animals remains unproven, which is another important reason for the limited use of Bacillus subtilis in RNA production. Summary of the Invention

[0005] The technical problem solved by the present application is to provide a dsRNA capable of being stably expressed in Bacillus.

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

[0007] The technical problem solved by the present application is to provide a method for constructing a recombinant vector and a recombinant strain.

[0008] The technical problem solved by the present application is to provide the use of the dsRNA, the recombinant vector, the recombinant cell or the recombinant strain in the preparation of an antiviral vaccine or a medicine.

[0009] The technical problem solved by the present application is to provide an antiviral vaccine or a medicine.

[0010] Technical scheme: In order to solve the above technical problems, the dsRNA provided by the present application, the DNA sequence of the dsRNA is shown in SEQ ID NO. 1.

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

[0012] The recombinant vector is an Escherichia coli-Bacillus subtilis shuttle vector.

[0013] The recombinant vector further comprises an Escherichia coli replication origin, a Bacillus subtilis heat-sensitive replication origin, a resistance gene for screening of Escherichia coli, a resistance gene for screening of Bacillus subtilis, a fluorescent protein gene for screening of positive transformation colonies and a xylose-inducible promoter for expression of fluorescent protein.

[0014] The nucleotide sequence of the recombinant vector is shown in SEQ ID NO. 2.

[0015] The method for constructing the recombinant vector comprises the following steps:

[0016] (1) synthesizing the DNA sequence of the dsRNA, amplifying or synthesizing the constitutive promoter Pylb;

[0017] (2) amplifying or synthesizing the gene fragments of the xylose-inducible promoter PxylA and the red fluorescent protein mCherry;

[0018] (3) seamlessly connecting the DNA sequence of the dsRNA of step (1), Pylb and the gene fragments of PxylA and mCherry of step (2) to obtain the recombinant vector.

[0019] The method for constructing the recombinant strain comprises the following steps: after the recombinant vector is transformed into Bacillus by a chemical transformation or electrotransformation method, LB solid culture medium containing xylose and spectinomycin is used for culture, and the positive transformant colonies are picked up by using red fluorescence excitation.

[0020] The application also relates to the application of the dsRNA, the recombinant vector, the recombinant cell or the recombinant strain in preparing a vaccine or a medicine for resisting virus infection.

[0021] The virus comprises avian influenza H9N2 virus.

[0022] The application also relates to a vaccine or a medicine for resisting virus, which comprises the dsRNA or the recombinant vector, the recombinant cell or the recombinant strain.

[0023] Advantages: compared with the prior art, the application has the following remarkable advantages:

[0024] 1. The application adds a pair of bidirectional promoters at both ends of the sequence coding the target RNA, simultaneously starts the transcription of the DNA double chain, obtains the dsRNA, enhances the stability of the target RNA production, and simultaneously the dsRNA can activate the RNAi pathway and play an antiviral role as an important factor of the RNAi pathway.

[0025] 2. The dsRNA containing the bidirectional promoters is constructed into an E. coli-Bacillus shuttle vector, high-copy plasmids can be obtained by using E. coli, and then the plasmids are transformed into Bacillus. Since the plasmid constructed in the application belongs to the integrative plasmid of Bacillus, the plasmid can be integrated into the genome of Bacillus to realize stable expression, the problem that the conventional Bacillus plasmid is easily lost in the subculture process is solved, when the plasmid performs the function, high temperature can be used to eliminate the plasmid in Bacillus, so that the genetically modified Bacillus engineering bacteria no longer contain the exogenous plasmid, and the resistance gene carried by the plasmid can be prevented from drifting, and the environment is friendly.

[0026] 3. The application also constructs the resistance gene and the xylose-induced red fluorescent protein mCherry gene based on the plasmid containing the bidirectional constitutive promoter Pylb, so that when the positive transformant colonies are picked up after transformation, the colonies with good expression effect can be picked up by detecting the red fluorescence intensity of the positive colonies.

[0027] In summary, the plasmid constructed in the application can stably express dsRNA in Bacillus subtilis, greatly reducing the synthesis cost of dsRNA, and also significantly improving the yield and stability of dsRNA. Meanwhile, the delivery of dsRNA by Bacillus subtilis can effectively protect the dsRNA from degradation by a large amount of RNase in the environment and in the animal body, helping to successfully deliver the dsRNA to the animal body, activating the RNAi system of the animal, and achieving the effect of resisting viruses. This provides a full-chain solution for the production and delivery of dsRNA. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 A process diagram for constructing the plasmid pDGds-AAD for stably expressing dsRNA targeting AIV in Bacillus subtilis;

[0029] Figure 2 A plasmid map of the plasmid pDGds-AAD;

[0030] Figure 3 Bacillus subtilis transformed with the plasmid pDGds-AAD Bacillus subtilis 168 coated with LB solid plate containing 1.5% xylose and 100 μg / mL spectinomycin and grown at 37℃ for 10h;

[0031] Figure 4 An agarose gel electrophoresis map of the cDNA amplification product of the recombinant bacteria AAD transformed with the plasmid pDGds-AAD; lane M is Marker; lane 1 is the positive control of in vitro transcribed dsRNA; lane 2 is the cDNA amplification product of the recombinant bacteria AAD; lane 3 is the cDNA amplification product of the wild type Bacillus subtilis 168; Bacillus subtilis

[0032] Figure 5 A graph of the daily weight change of chicks, wherein the PBS group refers to chicks attacked after being dripped with PBS; the RNA group refers to chicks attacked after being dripped with PEI-wrapped dsRNA targeting H9N2; the Bs168 group refers to chicks attacked after being dripped with wild type Bacillus subtilis 168; and the AAD group refers to chicks attacked after being dripped with the recombinant Bacillus subtilis AAD expressing dsRNA targeting H9N2; Bacillus subtilis

[0033] Figure 6 A lung pathological examination graph of chicks, Blank refers to the lung of an untreated and unattacked chick, and the rest of the groups are the same as Figure 5 Description;

[0034] Figure 7 ​​Figure 6 is a lung pathological section HE staining chart of the chick, and the groups are the same as those in figure 5 Figure 6 Description

[0035] Figure 8 Figure 7 is a lung H9N2 virus load detection chart of the chick, β-Actin is used as an internal reference gene, the expression level of H9N2-HA mRNA is detected by SYBR method, 2 -ΔΔct The data is processed, the ordinate data is logarithmically Log2, and Graphpad Prism 9 is used for drawing and difference significance analysis and statistical test;

[0036] Figure 9 Figure 8 is a tracheal H9N2 virus load detection chart of the chick, β-Actin is used as an internal reference gene, the expression level of H9N2-HA mRNA is detected by SYBR method, 2 -ΔΔct The data is processed, the ordinate data is logarithmically Log2, and Graphpad Prism 9 is used for drawing and difference significance analysis and statistical test. DETAILED DESCRIPTION

[0037] The technical solutions of the present application will be further described below in combination with the drawings. The present application is further illustrated by specific examples and drawings. The materials, reagents and the like used in the embodiments of the present application can be obtained from commercial channels unless otherwise specified. The methods used in the following examples are conventional methods unless otherwise specified. The resistance genes and artificially synthesized genes in the present application are all known genes.

[0038] The sources of materials and reagents in the embodiments of the present application are as follows:

[0039] pDG1663 (Shanghai Zeyebio Biotech Co., Ltd., item number: ZY1866), pAX01-mCherry (Shanghai Enzyme Link Biotech Co., Ltd., item number: ml-G13175), Bacillus subtilis Bacillus subtilis 168 (donated by Professor Gao Xuewen, College of Plant Protection, Nanjing Agricultural University, and can also be obtained through commercial channels)

[0040] DMEM medium (Nanjing Sunbioga Biotechnology Co., Ltd.); 10% protein precast gel (Shanghai Yezheng Biomedicine Co., Ltd.); 5×SDS-PAGE (Wuhan Gaoyuntian Biotechnology Co., Ltd.); 4×denatured protein loading buffer (Shanghai Yixing Company); ClonExpress II One Step Cloning Kit (Nanjing Novozyme Biotech Co., Ltd.).

[0041] The reagents used for conversion in this embodiment of the invention are as follows: SP solution (2 mg / mL ammonium sulfate, 14 mg / mL dipotassium hydrogen 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, pH adjusted to 8.0 with NaOH).

[0042] Example 1: Construction and transformation of pDGds plasmid

[0043] like Figure 1 As shown, primers pDG-F and pDG-R were designed to amplify the vector backbone pDG using plasmid pDG1663 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; and primers mCherry-F and mCherry-R were designed to amplify the red fluorescent protein mCherry using plasmid pAX01-mCherry as a template. Bacillus subtilis was used as the amplifying primer. Bacillus subtilis Using the genome of 168 as a template, 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 in Table 2, and the amplification conditions in Table 3. DNA encoding dsRNA targeting AIV was synthesized; the dsRNA DNA sequence is shown in SEQ ID NO. 1. The amplified fragments were ligated into the pDG vector using seamless ligation technology at Universal Biotechnology (Anhui) Co., Ltd., in the order PxylA-mCherry-Pylb (forward)-anti-AIV RNA-Pylb (reverse), to obtain the plasmid vector pDGds-AAD, which expresses the AIV-targeting dsRNA. The plasmid map is shown below. Figure 2 As shown, the nucleotide sequence of the plasmid pDGds-AAD is shown in SEQ ID NO.2.

[0044] Table 1 Primer sequences

[0045]

[0046] Table 2 Amplification System

[0047]

[0048] Table 3 Amplification conditions

[0049]

[0050] Example 2 Transformation of pDGds plasmid vector

[0051] 1. Preparation of Bacillus subtilis competent cells and transformation of pDGds-AAD plasmid

[0052] 1) Pick host bacteria (Bacillus subtilis 168) and inoculate into 20 mL of LB liquid medium, 37 °C, 200 r / min incubate overnight. Bacillus subtilis 168) inoculate into 20 mL of LB liquid medium, 37 °C, 200 r / min incubate overnight.

[0053] 2) Take 800 μL of culture from the overnight culture and inoculate 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), 37 °C, 200 r / min incubate on a shaker for 2 h. Then start to detect OD600, when the culture grows to the end of the logarithmic growth phase (OD600 = 1.0), quickly take 2.5 mL of bacterial solution and inoculate 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), 37 °C, 100 r / min incubate on a shaker for 1.5 h.

[0054] 3) Add 250 μL of solution 6, 37 °C, 100 r / min incubate on a shaker for 10 min, and then divide into 500 μL per tube with a 1.5 mL centrifuge tube.

[0055] 4) Add the constructed plasmid pDGds-AAD (1 μg) to the tube, mix gently, and incubate at 37 °C, 100 r / min on a shaker for 30 min. Then continue to incubate on a 200 r / min shaker for 1.5 h.

[0056] 5) Centrifuge at 4000 r / min to collect the bacterial cells, discard part of the supernatant, leave 100 μL of resuspended bacterial cells, and spread on a 1.5% xylose and 100 μg / mL spectinomycin resistance (Spc + ) LB plate, invert in a 37 °C incubator overnight, and observe under a fluorescence microscope, using a 580 nm wavelength excitation, as shown in Figure 3 , the colonies have a typical Bacillus subtilis morphology and emit bright red fluorescence, and colonies that meet the above characteristics can be determined as positive transformation colonies.

[0057] Example 3 Extraction of bacterial total RNA and obtaining of cDNA

[0058] Single colonies activated by streaking after overnight culture in Example 2 were selected and cultured overnight in resistant liquid LB medium. The bacterial cells were collected by centrifugation at 12000 rpm, resuspended in DEPC water, and then washed twice by centrifugation. Lysozyme at a concentration of 10 U / mL was added, and the mixture was incubated at 37 °C on a shaker for at least 30 min to disrupt the bacterial cell wall. A portion of the bacterial lysate was taken, and whole RNA was extracted using the Trizol method. Recombinant Bacillus subtilis cDNA was obtained by reverse transcription using the Novozymes HiScript IV Q RT SuperMix for qPCR (+gDNAwiper) reverse transcription kit according to the manufacturer's instructions.

[0059] Example 4: Validation of target dsRNA expression

[0060] Primers AIV-F and AIV-R were designed using the DNA sequence encoding the target dsRNA (SEQ ID NO.1) as a template. PCR was performed using the Novizan 2 × Rapid Taq Master Mix kit according to the instructions. The amplification primers, amplification system, and amplification conditions are shown in Tables 4 to 6.

[0061] Table 4 Amplification Primer Sequences

[0062]

[0063] Table 5 Amplification System

[0064]

[0065] Table 6 Amplification Conditions

[0066]

[0067] The amplification products were subjected to 1% agarose gel electrophoresis, and a clear band was observed at 300 bp. Figure 4 ), compared with the blank biological control Bacillus subtilis without transformed plasmid Bacillus subtilis Compared to 168, recombinant bacteria AAD transformed with plasmid pDGds-AAD were able to express the target dsRNA.

[0068] Example 5: Bacillus subtilis stably expressing dsRNA targeting avian influenza H9N2 via nasal drops in chicks resists H9N2 infection.

[0069] Thirty 10-day-old SPF chicks (purchased from Yangzhou Guoji Biotechnology Co., Ltd.) were divided into 5 groups of 6 chicks each. The Blank group received intranasal administration of 0.01 M PBS (pH=7.2) for two consecutive days without challenge; the PBS group received intranasal administration of PBS for two consecutive days with a one-day interval. The viral titer of each chick was 3 × 10⁻⁶. 6H9N2 avian influenza virus (Nanjing Agricultural University Laboratory) 300 μL at 3 x 10 8 CFU / mL of wild-type Bacillus subtilis Bacillus subtilis 168, one day apart, each chick was nasally infected with 3 x 10 6 H9N2 avian influenza virus 300 μL at 3 x 10 8 CFU / mL of recombinant Bacillus subtilis AAD transformed with plasmid pDGds-AAD 200 μL per chick, one day apart, each chick was nasally infected with 3 x 10 6 H9N2 avian influenza virus 300 μL at 3 x 10 6 H9N2 avian influenza virus 300 μL at 3 x 10

[0070] The daily weight of the chicks was subtracted from the previous day to calculate the daily weight gain, as shown in Figure 5 The RNA group, i.e., the preparation of the dsRNA targeting H9N2 packaged with PEI material for nasal administration, the Bs168 group, i.e., wild-type Bacillus subtilis Bacillus subtilis The RNA group, i.e., the preparation of the dsRNA targeting H9N2 packaged with PEI material for nasal administration, the Bs168 group, i.e., wild-type Bacillus subtilis

[0071] The lungs of the chicks were taken and dissected, as shown in Figure 6The results showed that the lungs of chicks challenged with PBS via intranasal instillation exhibited significant lesions, with consolidation of the lung texture, scattered pinpoint to millet-sized hemorrhages on the lung surface, some of which merged into patchy hemorrhages. Some tissues showed necrosis and a grayish tinge, a stark contrast to the unchallenged Blank group. Simultaneously, as... Figure 7 The HE staining images of chick lung sections show that in the unchallenged Blank group, the alveolar structure was intact and uniformly distributed. Numerous alveoli of similar size were arranged in regular polygonal shapes, with open and unobstructed cavities. No localized collapse or adhesions were observed. The alveolar septa were thin and clearly defined, revealing a fine capillary network. No significant thickening or fibrosis was observed. No large aggregations of neutrophils or lymphocytes were seen in the alveolar septa or cavities, indicating that the tissue had not undergone acute or chronic inflammatory stimulation. A small number of scattered blood vessels were visible within the alveolar walls, with unobstructed lumens and continuous endothelium, showing no signs of congestion, hemorrhage, or edema. The interstitial matrix (including elastic fibers and a small amount of connective tissue) of the lung parenchyma was homogeneous, without proliferation or sloughing, and the overall histological appearance was close to that of a healthy group. Following H9N2 avian influenza infection, multiple alveolar cavities showed clusters of deep red erythrocytes (arrows), indicating a significant increase in vascular permeability, accompanied by punctate or patchy hemorrhages. Compared to the control group, the alveolar septa were significantly thickened, no longer thin and clear, but exhibiting an expanded appearance due to cellular infiltration. Numerous neutrophils and monocytes (small round purple-stained cell nuclei clustered) were observed in the alveolar septa and cavities, indicating a strong acute inflammatory response. Driven by inflammatory exudates and cellular infiltration, some alveolar cavities collapsed locally, even blurring the boundaries between adjacent cavities, resulting in fusion and impaired gas exchange. Edema was observed in the lung parenchyma connective tissue, with pale purple gelatinous interstitial tissue visible on sections. Long-term lesions may also be accompanied by fibroblast proliferation, laying the foundation for subsequent fibrosis. Based on the combined results of pathological anatomy and pathological sections, the chicks infected with avian influenza virus showed obvious acute inflammatory response in their lungs, accompanied by hemorrhage and edema caused by increased vascular permeability, as well as alveolar structure destruction and septal thickening. This indicates that intranasal administration of H9N2 avian influenza caused significant lung damage to the chicks, and the H9N2 infection model was established. Meanwhile, chicks challenged with wild-type Bacillus subtilis Bs168 via intranasal instillation also showed obvious lesions in their lungs. Pathological sections showed thickened alveolar septa, with a small number of neutrophils and mononuclear cells scattered in the alveolar septa and cavities. The alveolar septa were slightly thicker than those in the Blank group, with mild edema and inflammatory cell infiltration. This indicates that wild-type strain Bs168 could not alleviate lung damage caused by H9N2 challenge. In contrast, the lungs of chicks challenged with recombinant Bacillus subtilis AAD expressing AIV dsRNA via intranasal instillation were pink, fresh, and soft, with no obvious lesions visible to the naked eye. Pathological sections also showed no significant difference between the tissue and the unchallenged control group Blank, with no obvious pathological changes. This suggests that intranasal instillation of recombinant Bacillus AAD can reduce lung damage in chicks caused by H9N2 infection and help chicks resist H9N2 avian influenza.

[0072] Lungs and tracheas of chicks were ground and RNA was extracted using the Trizol method. cDNA was then obtained by reverse transcription using the Novizan HiScript IV QRT SuperMix for qPCR (+gDNAwiper) reverse transcription kit. qPCR detection was performed using the Novizan ChamQSYBR qPCR Master Mix (Low ROX Premixed) kit according to its instructions. The expression level of H9N2-HA mRNA was detected using β-Actin as an internal reference gene, serving as a standard for viral load in tissues. The primer sequences used in the detection are shown in Table 7.

[0073] Table 7 Primer sequences for qPCR detection

[0074]

[0075] like Figure 8 , 9 As shown, intranasal administration of recombinant Bacillus subtilis significantly reduced the viral load of H9N2, while intranasal administration of PEI-encapsulated dsRNA and Bs168 did not reduce the viral load of H9N2 in the lungs and trachea of ​​chicks.

Claims

1. A recombinant bacterial strain, characterized in that, The recombinant bacteria are obtained by the following steps: transforming the recombinant vector into Bacillus subtilis by a chemical transformation or electrotransformation method, culturing in LB solid medium containing xylose and spectinomycin, and picking up the colonies emitting red fluorescence by fluorescence excitation. The nucleotide sequence of the recombinant vector is shown in SEQ ID NO.

2.

2. The method of constructing a recombinant bacterial strain according to claim 1, wherein, The recombinant bacteria are obtained by the following steps: transforming the recombinant vector into Bacillus subtilis by a chemical transformation or electrotransformation method, culturing in LB solid medium containing xylose and spectinomycin, and picking up the colonies emitting red fluorescence by fluorescence excitation. The nucleotide sequence of the recombinant vector is shown in SEQ ID NO.

2.

3. Use of the recombinant strain of claim 1 in the preparation of a drug for resisting viral infection, wherein the virus is avian influenza H9N2 virus.

4. An antiviral medicament, characterized by comprising the compound of claim 1 or 2. The drug comprises the recombinant strain of claim 1, and the virus is avian influenza H9N2 virus.

Images