Annular KANSL1 gene and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to an annular KANSL1 gene and application thereof in regulation and control of replication of type 3 duck hepatitis A virus. The overexpression of the annular KANSL1 gene can significantly promote the replication of the intracellular type 3 duck hepatitis A virus, and the replication of the intracellular type 3 duck hepatitis A virus can be significantly inhibited by inhibiting the expression of the annular KANSL1 gene through small interfering RNA.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the circular KANSL1 gene and its application in regulating the replication of duck hepatitis A virus type 3. Background Technology

[0002] Duck viral hepatitis (DVH) is a major infectious disease threatening the duck farming industry. Its main characteristic is hepatic hemorrhage and necrosis, characterized by rapid onset and high mortality, especially in ducklings under 21 days old, with mortality rates reaching as high as 90% in ducklings aged 7-14 days. The disease is mainly classified into three serotypes, among which genotype 3 duck hepatitis A virus (DHAV-3) is currently the predominant circulating strain in my country, causing severe economic losses to the duck farming industry. However, current prevention and control measures for DHAV-3 infection are significantly inadequate; there are no approved specific vaccines or treatments on the market, posing a significant challenge to clinical prevention and control.

[0003] Circular RNAs (circRNAs) are a class of endogenous non-coding RNAs with closed circular structures. Lacking free ends, they are less susceptible to degradation by exonucleases, exhibiting higher stability and participating extensively in gene expression regulation in eukaryotes. Recent studies have shown that circRNAs play crucial roles in viral infection and host immunity, influencing viral replication through mechanisms such as regulating innate immunity, apoptosis, or acting as competitive endogenous RNA (ceRNA) sponges to adsorb miRNAs. Existing research has confirmed the regulatory effects of specific circRNAs on viral replication. For example, circRNA002006, disclosed in patent CN116622705A, significantly inhibits Coxsackievirus B5 (CVB5) replication, and non-patent literature has reported that circ_0000479 inhibits hantavirus proliferation through the miR-149-5p / RIG-I axis, providing new molecular targets for the prevention and control of viral infections.

[0004] Despite progress in the study of the function of circRNAs in viral infection, the specific role and regulatory mechanism of circRNAs in DHAV-3 infection remain unknown, and the gaps in related research limit the development of DHAV-3 prevention and control strategies. Summary of the Invention

[0005] Based on this, this invention, through functional mining of host circRNAs after infection with duck hepatitis A virus type 3 (DHAV-3), has for the first time discovered that the circular KANSL1 gene (circKANSL1) can significantly promote DHAV-3 replication, while inhibiting its expression can effectively block viral proliferation. This discovery provides a new perspective on revealing the interaction mechanism between DHAV-3 and the host, and lays the foundation for developing DHAV-3 prevention and control strategies targeting circRNAs.

[0006] To achieve the above objectives, the present invention can adopt the following technical solutions:

[0007] In one aspect, the present invention provides a circular KANSL1 gene, the nucleotide sequence of which is shown in SEQ ID NO: 1.

[0008] In another aspect, the present invention provides the application of the above-mentioned overexpression of the circular KANSL1 gene in promoting the replication of duck hepatitis A virus type 3 in vitro.

[0009] Preferably, in the above application, the circular KANSL1 gene is overexpressed by a circular KANSL1 gene overexpression vector, and the expression vector is a pCD2.1-ciR vector containing the circular KANSL1 gene.

[0010] In another aspect, the present invention provides the application of reducing the expression of the above-mentioned circular KANSL1 gene in the in vitro inhibition of duck hepatitis A virus type 3 replication.

[0011] Preferably, in the above applications, the expression of the circular KANSL1 gene is reduced by small interfering RNA, the nucleotide sequence of which is shown in SEQ ID NO: 2 and SEQ ID NO: 3.

[0012] In another aspect, the present invention provides the application of an agent for reducing the expression of the above-mentioned circular KANSL1 gene in the preparation of a formulation for inhibiting the replication of duck hepatitis A virus type 3.

[0013] Preferably, in the above applications, the reagent for reducing the expression of the circular KANSL1 gene is small interfering RNA, and the nucleotide sequence of the small interfering RNA is shown in SEQ ID NO: 2 and SEQ ID NO: 3.

[0014] In another aspect, the present invention provides the application of an agent for reducing the expression of the above-mentioned circular KANSL1 gene in the preparation of a drug for treating duck viral hepatitis.

[0015] Preferably, in the above applications, the reagent for reducing the expression of the circular KANSL1 gene is a small interfering RNA, the nucleotide sequence of which is shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0016] In another aspect, this invention provides the application of a reagent for detecting the expression level of the above-mentioned circular KANSL1 gene in duck hepatitis A virus type 3 resistance breeding.

[0017] The beneficial effects of this invention include: overexpression of the circular KANSL1 gene in this invention can significantly promote the replication of duck hepatitis A virus type 3 in cells, and inhibition of circular KANSL1 gene expression by small interfering RNA can significantly inhibit the replication of duck hepatitis A virus type 3 in cells. Attached Figure Description

[0018] Figure 1 A schematic diagram of the formation of circKANSL1 and the reverse splice site of circKANSL1;

[0019] Figure 2 The results show the tolerance of circKANSL1 to RNase R enzyme digestion;

[0020] Figure 3 To verify the existence of circKANSL1 reverse splicing using forward and reverse primers;

[0021] Figure 4 Map of the circKANSL1 overexpression plasmid vector (PCD2.1-KANSL1);

[0022] Figure 5a The overexpression efficiency of circKANSL1 in duck embryonic primary hepatocytes;

[0023] Figure 5b The results show the changes in DHAV-3 expression after overexpression of circKANSL1 in primary duck embryo hepatocytes;

[0024] Figure 6 This is a schematic diagram of the target sequence of siRNA targeting the circKANSL1 backsplicing site.

[0025] Figure 7a The interference efficiency of circKANSL1 in duck embryo primary hepatocytes;

[0026] Figure 7b The results show the changes in DHAV-3 expression after circKANSL1 was expressed in duck embryo primary hepatocytes. Detailed Implementation

[0027] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0028] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0029] In a first aspect, embodiments of the present invention provide a circular KANSL1 gene, the nucleotide sequence of which is shown in SEQ ID NO: 1.

[0030] Secondly, embodiments of the present invention provide an application of the above-mentioned overexpression of the circular KANSL1 gene in promoting the replication of duck hepatitis A virus type 3 in vitro.

[0031] It should be noted that the present invention found that overexpression of the circular KANSL1 gene in the present invention can significantly promote the replication of duck hepatitis A virus type 3 in cells, so the circular KANSL1 gene can be applied to related studies on duck hepatitis A virus type 3 replication.

[0032] In some specific examples, in the above applications, the circular KANSL1 gene is overexpressed using a circular KANSL1 gene overexpression vector, which is a pCD2.1-ciR vector containing the circular KANSL1 gene.

[0033] It should be noted that the method of overexpressing the circular KANSL1 gene is well known in the field, and an overexpression vector, such as the pCD2.1-ciR vector containing the circular KANSL1 gene, is generally chosen.

[0034] Thirdly, embodiments of the present invention provide an application of reducing the expression of the above-mentioned circular KANSL1 gene in the in vitro inhibition of duck hepatitis A virus type 3 replication.

[0035] In some specific examples, in the above applications, the expression of the circular KANSL1 gene is reduced by small interfering RNA, the nucleotide sequences of which are shown in SEQ ID NO: 2 and SEQ ID NO: 3.

[0036] It should be noted that the methods for reducing the expression of the circular KANSL1 gene are well known in the art. For example, small interfering RNA can be used to reduce the expression of the KANSL1 gene, and the nucleotide sequence of the small interfering RNA can be as shown in SEQ ID NO: 2 and SEQ ID NO: 3.

[0037] Fourthly, embodiments of the present invention provide the application of an agent that reduces the expression of the above-mentioned circular KANSL1 gene in vivo in the preparation of a formulation that inhibits the replication of duck hepatitis A virus type 3.

[0038] In some specific examples, in the above applications, the reagent for reducing the expression of the circular KANSL1 gene is small interfering RNA, the nucleotide sequence of which is shown in SEQ ID NO: 2 and SEQ ID NO: 3.

[0039] It should be noted that reducing the expression of the circular KANSL1 gene in vivo can also inhibit the replication of duck hepatitis A virus type 3 in vivo.

[0040] Fifthly, embodiments of the present invention provide the application of a reagent for reducing the expression of the above-mentioned circular KANSL1 gene in the preparation of a drug for treating duck viral hepatitis.

[0041] In some specific examples, in the above applications, the reagent for reducing the expression of the circular KANSL1 gene is small interfering RNA, the nucleotide sequence of which is shown in SEQ ID NO: 2 and SEQ ID NO: 3.

[0042] Sixthly, embodiments of the present invention provide the application of a reagent for detecting the expression level of the above-mentioned circular KANSL1 gene in duck hepatitis A virus type 3 resistance breeding.

[0043] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0044] In the following examples, the Beijing ducklings and Beijing duck embryos used were provided by the Beijing Duck Germplasm Resource Conservation Farm of the Chinese Academy of Agricultural Sciences; the present invention uses Beijing duck Z7 and Z77 lines for DHAV-3 artificial infection.

[0045] In the following examples, the highly virulent strain DHAV-3112803 was preserved in the key laboratory of animal epidemiology of the relevant department for artificial infection experiments; duck embryonic primary hepatocytes (DEHs) were isolated from 14-day-old specific pathogen-free (SPF) Beijing duck embryos; and human embryonic kidney cells HEK293T were purchased from Beyotime Biotechnology Co., Ltd. and preserved in liquid nitrogen.

[0046] In the following examples, the overexpression plasmid pCD2.1-ciR of CircKANSL1 was purchased from Guangzhou Gise Biotechnology Co., Ltd.

[0047] I. Structural Verification of CircKANSL1

[0048] The CircKANSL1 in this invention is derived from chromosome 28 of the Beijing duck, and its nucleotide sequence is shown in SEQ ID NO.1.

[0049] (I) cDNA Acquisition

[0050] Total RNA was extracted from duck liver samples from each group using the Trizol method, and cDNA was obtained by reverse transcription, as detailed below:

[0051] (1) RNA extraction

[0052] (1-1) Sample Pretreatment

[0053] 1) Adherent cells: Discard the culture medium of duck embryo primary hepatocytes, wash twice with pre-cooled PBS, add 1 mL of Trizol and incubate for 5 minutes, pipette the cells, and collect the liquid into a 1.5 mL centrifuge tube; let stand for 5 minutes to allow the nucleic acid protein complex to be completely separated;

[0054] 2) Tissue: Twenty-four hours after Z7 and Z77 strains of Beijing ducklings were infected with DHAV-3 virus, liver tissue the size of soybeans was taken and placed in 1.5mL centrifuge tubes. 1mL of Trizol was added, and the tissue was ground with a hand grinder until it became liquid. The tissue was then allowed to stand for 5 minutes to allow the nucleic acid-protein complex to be completely separated.

[0055] (1-2) Add 0.2 mL of chloroform (one-fifth of the volume of Trizol) to each centrifuge tube, shake vigorously for 15 seconds, and let stand at room temperature for 5 minutes;

[0056] (1-3) Centrifuge at 12000 r / min for 15 min at 4℃. The solution will separate into layers. Transfer the upper colorless and transparent aqueous phase to a new 1.5 mL centrifuge tube. The volume is about 60% of the volume of Trizol.

[0057] (1-4) Then add an equal volume of pre-cooled isopropanol, mix well, and let stand on ice for 10 minutes to precipitate the RNA in the aqueous phase.

[0058] (1-5) Centrifuge at 4℃, 12000r / min for 10min, and discard the supernatant;

[0059] (1-6) Wash the RNA precipitate twice with pre-cooled 75% ethanol, the volume of which should be the same as the volume of Trizol; centrifuge at 12000 r / min for 5 min at 4℃ and discard the supernatant;

[0060] (1-7) Let it air dry at room temperature for 5-10 minutes;

[0061] (1-8) Add an appropriate amount of DEPC water, and use a pipette tip to agitate several times to fully dissolve the RNA to obtain an RNA solution. Place the solution on ice; after measuring the concentration with a nucleic acid analyzer, store at -80℃.

[0062] (2) Reverse transcription reaction

[0063] 1) Prepare the RNA solution according to the gDNA removal reaction system in Table 1, briefly centrifuge to mix, and incubate at 42°C for 3 min to obtain a pure RNA solution (DNA genome removed); place on dry ice for later use.

[0064] Table 1. DNA Removal Reaction System

[0065] Components volume 5X gDNABuffer 2μL TotalRNA (1ug) <![CDATA[ddH2O]]> Make up to 10 μL

[0066] 2) Add the purified RNA solution from 1) above to the reverse transcription reaction system (Table 2 below) and mix thoroughly; incubate at 42°C for 15 min; incubate at 95°C for 3 min to obtain cDNA; place on ice for subsequent experiments.

[0067] Table 2 Reverse transcription reaction system

[0068] Components volume 10×KingRTBuffer 2μL FastKingRTEnzymeMix 1μL FQ-RT PrimerMix 2μL <![CDATA[ddH2O]]> 5μL

[0069] (II) RT-qPCR reaction

[0070] Using the cDNA prepared above as a template, the expression of each gene in tissues or cells was detected by RT-qPCR; GAPDH was used as an endogenous control for circRNA and mRNA; 2 -ΔΔCT The relative expression levels of each gene were calculated using the method shown in Table 3. All primer sequences were synthesized by Shanghai Sangon Biotech Co., Ltd. In addition, the RT-qPCR reaction system was prepared according to the instructions of ChamQ Blue Universal SYBR qPCR MasterMix from Nanjing Novizan Biotechnology Co., Ltd. Each sample was performed in triplicate, and the reaction volume was 20 μL, as shown in Table 4 below.

[0071] Table 3 Primer pairs for RT-qPCR amplification

[0072]

[0073] Table 4 RT-qPCR reaction system

[0074] Components volume 2×ChamQBlueUniversalSYBRqPCRMasterMix 10μL ForwardPrimer (10μM) 0.6μL ReversePrimer (10μM) 0.6μL TemplatecDNA 1μL <![CDATA[ddH2O]]> 7.8μL

[0075] After the RT-qPCR reaction system is prepared, it is quickly centrifuged and mixed. The eight-tube strip is then placed in a quantitative analyzer for detection. The reaction program is set. The two-step Real-time-qPCR reaction program is: 95℃ for 30s; 95℃ for 10sec, 60℃ for 30sec (40 Cycles); Melt Curve.

[0076] (III) Sanger sequencing

[0077] Based on the sequence information, back-to-back primers (divergent primers) were designed (consistent with the CircKANSL1 primer sequences in Table 3). Using the previously obtained cDNA as a template, PCR amplification was performed. The reaction system and procedure are shown in Tables 5 and 6. Subsequently, agarose gel electrophoresis was performed (specific steps are as follows). The obtained target band was excised and sent to Shanghai Sangon Biotech Co., Ltd. for Sanger sequencing to obtain the reverse splicing site sequence of circKANSL1 (see...). Figure 1 ).

[0078] Table 5 Reaction System

[0079] Components volume Component Volume GreenTaqMix 25μL ForwardPrimer 1.0μL ReversePrimer 1.0μL cDNA or gDNA template 2.0μL <![CDATA[ddH2O]]> 21uL

[0080] The PCR reaction was carried out by uniformly mixing the reagents required for the system in Table 5 and preparing the reaction solution on ice.

[0081] Table 6 Reaction Procedure

[0082]

[0083] The steps for agarose gel electrophoresis are as follows:

[0084] 1) Insert a clean comb into the glue-making tray for later use;

[0085] 2) To prepare 1% agarose gel: Weigh 0.25g of agarose into an Erlenmeyer flask, add 25mL of 1×TAE solution, and heat in a microwave oven for 60s;

[0086] 3) Add 2 μL of nucleic acid dye, mix well, and immediately pour into the tank. Incubate at room temperature for 30 minutes, remove the comb, and place in the electrophoresis tank;

[0087] 4) Add 1×TAE solution to the electrophoresis tank in advance, so that the liquid level is about 5mm higher than the gel surface;

[0088] 5) Load 5 μL of PCR reaction solution and spot the DNA marker next to it;

[0089] 6) 120V voltage for 30 minutes;

[0090] 7) Analyze the electrophoresis results using a gel imaging system;

[0091] (iv) RNase R enzyme resistance test

[0092] The specific steps for digesting total RNA from duck liver tissue using RNase R are as follows:

[0093] (1) Prepare the following reaction system in a sterile microcentrifuge tube, as shown in Table 7.

[0094] Table 7. RNase R enzyme digestion test reaction system

[0095] Components volume component Volume RNA To 1μg 10xRNaseRReactionBuffer 2μL RNase R (20 U / μL) 2U / μgRNA RNase-freeWater Up to 20μL

[0096] (2) Incubate at 37℃ for 30 min;

[0097] (3) Add 1-2 μL LEDTA to terminate the reaction;

[0098] (4) Let it sit at room temperature for 5 minutes;

[0099] (5) Using the digested gene as a template, the expression of related genes was detected by the above RT-qPCR reaction, with linear RNAGAPDH as a positive control.

[0100] The results are as follows Figure 2 As shown in the figure, the results indicate that circKANSL1 has good resistance to RNase R enzyme.

[0101] (V) cDNA / gDNA Validation

[0102] To further verify that the backsplicing site of circKANSL1 is due to backsplicing rather than gene rearrangement, convergence and divergence primers for circKANSL1 were designed (primer sequences are shown in Table 3). Using cDNA and gDNA extracted from duck liver tissue as templates, PCR amplification was performed according to the above-described PCR reaction system and procedure (Tables 5 and 6), and the products were analyzed by agarose gel electrophoresis. The results are as follows: Figure 3 As shown, when cDNA is used as a template, both the convergent and divergent primers of circKANSL1 can amplify the band, and the band sizes are inconsistent; when gDNA is used as a template, only the convergent primer of circPKANSL1 can amplify the band.

[0103] The above results indicate that circKANSL1 forms a stable ring structure in tissues through back splicing.

[0104] II. Functional analysis of circKANSL1 overexpression in DHAV-3 infection

[0105] (I) Construction of circKANSL1 overexpression vector

[0106] Based on the complete sequence of circKANSL1 obtained from the sequencing results, amplification primers were designed. Using the extracted cDNA from duck liver tissue as a template, the circKANSL1 gene sequence was amplified through the PCR reaction system and procedure described above (Tables 5 and 6). The gene sequence was then inserted into the pCD2.1-ciR vector to construct the circKANSL1 overexpression vector, with the restriction enzyme sites being KpnⅠ and BamHI. The primer sequences used to construct the vector are shown in Table 8.

[0107] Table 8 Primers for Vector Construction

[0108]

[0109] In addition, the detailed steps for constructing the vector are as follows:

[0110] (1) PCR amplification of the target gene fragment

[0111] Using the specific primers in Table 8, fragmented PCR amplification was performed using the liver tissue cDNA as a template. First, the circKANSL1 fragment 1 (1-804 bp) was amplified using primers PCD2.1-KANSL1-F1 and PCD2.1-KANSL1-R1. Then, the circKANSL1 fragment 2 (774-1387 bp) was amplified using primers PCD2.1-KANSL1-F2 and PCD2.1-KANSL1-R2. Subsequently, the complete circKANSL1 gene sequence was obtained using overlap PCR. The reaction system and procedure for overlap PCR are shown in Tables 9, 10, 11, and 12.

[0112] Table 9 Overlap PCR Reaction System

[0113] Components volume Component Volume circKANSL1 fragment 1 100g circKANSL1 Fragment 2 100g High-fidelity enzyme (AG) 25μL ddH2O Add water to 45uL

[0114] Table 10 Overlap PCR Reaction Procedure

[0115]

[0116] The reaction solution obtained from the above reaction is added to a new system to continue the reaction.

[0117] Table 11 Overlap PCR Reaction System

[0118] Components volume Component Volume Previous reaction solution 45uL PCD2.1-KANSL1-F1 2.5μL PCD2.1-KANSL1-R2 2.5μL

[0119] Table 12 Overlap PCR Reaction Procedure

[0120]

[0121] The reaction solution obtained from the above reaction was subjected to agarose gel electrophoresis (the specific steps have been described above). The target band was cut off and the DNA was recovered from the agarose gel using the Tiangen Agarose Gel DNA Recovery Kit. The specific method is as follows:

[0122] 1) Place the cut-off gel piece into a clean 1.5mL centrifuge tube and weigh it;

[0123] 2) Add 3 times the volume of sol solution PE to the gel block (if the gel weight is 0.1g, its volume can be regarded as 100μL, then add 300μL of sol solution PE); dissolve the gel at room temperature (25℃) for 10min, and gently turn the centrifuge tube up and down continuously to ensure that the gel block is fully dissolved;

[0124] 3) Add the solution obtained in the previous step to an adsorption column CA5 (place the adsorption column in the collection tube), let it stand at room temperature for 2 min, centrifuge at 12000 rpm for 1 min, discard the waste liquid in the collection tube, and place the adsorption column CA5 into the collection tube.

[0125] 4) Add 600 μL of washing buffer PW to the adsorption column CA5, centrifuge at 12000 rpm for 1 min, discard the waste liquid in the collection tube, put the adsorption column CA5 into the collection tube; let stand for 3 min and then centrifuge again.

[0126] 5) Repeat step 4);

[0127] 6) Place the CA5 adsorption column back into the collection tube, centrifuge at 12000 rpm for 2 min to remove as much rinsing solution as possible; place the CA5 adsorption column at room temperature for several minutes to dry it thoroughly to prevent residual rinsing solution from affecting the next step of the experiment.

[0128] 7) Place the CA5 adsorption column into a clean 1.5 mL centrifuge tube, add an appropriate amount of elution buffer TB to the middle of the adsorption membrane, and incubate at room temperature for 2 min. Centrifuge at 12000 rpm for 2 min and collect the DNA solution, which is the complete circKANSL1 gene sequence with restriction enzyme sites. After measuring the concentration with a nucleic acid analyzer, store at -20℃ for later use.

[0129] (2) Double digestion of plasmid vectors (Takara)

[0130] The pCD2.1-ciR vector was added to the double digestion system (see Table 13), and the mixture was reacted at 37°C for 30 minutes to obtain a mixture containing the double digestion vector.

[0131] Table 13 Double Enzyme Digestion System

[0132] Components volume KpnI enzyme 2μL BamHI enzyme 2μL 10×Buffer 5μL <![CDATA[ddH2O]]> 16μL Total volume 25μL

[0133] The reaction solution obtained from the above reaction was subjected to agarose gel electrophoresis (according to the above method). The target band gel block was cut off and recovered by agarose gel (according to the above method). The collected DNA solution is the double-digested pCD2.1-ciR vector. After the concentration was determined by a nucleic acid analyzer, it was stored at -20℃ for later use.

[0134] (3) Preparation of ligation products

[0135] The complete circKANSL1 gene sequence with restriction sites obtained in (1) and the double-digested pCD2.1-ciR vector obtained in (2) were used to prepare the ligation reaction system in a microcentrifuge tube according to the formula in Table 14 below. The reaction system was reacted at 16℃ for 1 h to obtain the ligation product.

[0136] Table 14 Connection Reaction System

[0137] Components volume 10xT4 DNA Ligase Buffer 1μL T4 DNA Ligase 1μL Target gene fragment 5μL Double enzyme digestion vector 3μL

[0138] (4) Transformation

[0139] 1) In a clean bench, add 10 μL of the ligation product obtained in (4) above to 100 μL of Escherichia coli DH5α competent cells and mix gently.

[0140] 2) Place on ice for 30 minutes;

[0141] 3) Heat shock at 42℃ for 90 seconds;

[0142] 4) Place on ice for 3 minutes;

[0143] 5) Add 900 μL of antibiotic-free LB solution to a centrifuge tube in a clean bench and incubate at 37°C for 1 hour.

[0144] 6) Centrifuge at 6000 rpm for 2 min at room temperature, discard 900 μL of supernatant, and gently pipette the remaining supernatant to mix.

[0145] 7) Spread the mixture onto solid LB medium containing antibiotics and incubate at 37°C for 16 hours;

[0146] 8) Pick a single clone of the strain and transfer it into 2 mL of liquid LB medium (containing antibiotics), and incubate it in a shaker at 37°C for 16 h;

[0147] 9) Extract recombinant plasmid DNA (using Tiangen endotoxin-free plasmid extraction kit) to obtain the circKANSL1 overexpression vector, named PCD2.1-KANSL1 (plasmid vector map shown). Figure 4 (As shown).

[0148] (ii) Overexpression of circKANSL1

[0149] Primary hepatocytes were isolated from 14-day-old Beijing duck embryos. circKANSL1 was overexpressed in these primary hepatocytes (i.e., transfected with the circKANSL1 overexpression vector), and transfected cells were obtained after 24 hours of treatment. In this experiment, GP-transfect-Mate transfection reagent was used for transfection. The specific steps are as follows (using a 24-well plate as an example):

[0150] (1) Cell seeding: Cells are seeded in 24-well plates and cultured until the density reaches 60%-80% before transfection;

[0151] (2) Place the GP-transfect-Mate transfection reagent at room temperature and mix gently before use;

[0152] (3) Add 50 μL of serum-free culture medium to a 1.5 mL sterile centrifuge tube and add an appropriate amount of transfection reagent. Mix gently with a pipette and let stand at room temperature for 5 min.

[0153] (4) At the same time, add 50 μL of serum-free culture medium to another 1.5 mL sterile centrifuge tube, add appropriate RNAoligo / DNA, mix gently with a pipette, and let stand at room temperature for 5 min.

[0154] (5) Add the mixture in (3) to (4), mix gently with a pipette, let stand at room temperature for 20 minutes, and then immediately transfect.

[0155] (6) While the plate is settling, change the medium in the 24-well plate, replacing 0.4 mL of preheated fresh culture medium in each well.

[0156] (7) Add the transfection mixture into the wells. After adding, gently shake the plate to distribute the complex evenly.

[0157] (8) The cells were cultured at 37℃, and after 6 hours the culture medium was replaced with complete medium. The expression of circKANSL1 was detected after 24 hours.

[0158] The overexpression efficiency of the circKANSL1 overexpression vector was detected using RT-qPCR reactions (see Tables 5 and 6). The results are as follows: Figure 5a As shown in the figure, the results indicate that the circKANSL1 overexpression vector (PCD2.1-KANSL1) has a high overexpression efficiency.

[0159] In addition, after infecting transfected cells with DHAV-3 for approximately 20 hours, total RNA was collected, and viral expression levels were detected using RT-qPCR (see Tables 5 and 6). The results are as follows: Figure 5b As shown, the results indicate that overexpression of circKANSL1 promotes DHAV-3 replication.

[0160] III. Functional analysis of circKANSL1 in reducing DHAV-3 infection

[0161] (1) Small interfering RNA design

[0162] Design a small interfering RNA (siRNA) covering the circKANSL1 backsplicing site (see...) Figure 6 )(Remark: Figure 6 The key interfering sequences are the same as those in Table 8, except that the sequence lengths in the schematic diagram are different. Additionally, the interfering RNA sequences are designed with a special sequence (TT) at the end, namely si-circKANSL1-1 (also abbreviated as Si-1) and si-circKANSL1-2 (also abbreviated as Si-2). A negative control siRNA, si-NC, is also included. All of the above small interfering RNAs were synthesized by Shanghai Jima Biotechnology Co., Ltd. The oligonucleotide sequences of the above small interfering RNAs are shown in Table 15 below.

[0163] Table 15 Oligonucleotide sequences of small interfering RNAs

[0164] Small interfering RNA sequence Si-1 5′-CCACGUACCGCUGCUGUCCTT-3′(SEQ ID NO.2) Si-2 5′-GUACCGCUGCUGUCCUCAGTT-3′(SEQ ID NO.3) si-NC 5′-UUCUCCGAACGUGUCACGUTT-3′

[0165] (2) Small interfering RNA interference efficiency test

[0166] To test the interference efficiency of si-circKANSL1-1 (Si-1) and si-circKANSL1-2 (Si-2), Si-1 and Si-2 were transfected in duck embryonic primary hepatocytes using Gemma GP-transfect-Mate transfection reagent. The transfection steps are as follows:

[0167] 1) Cell plating: Duck embryo primary hepatocytes were seeded in 24-well plates and cultured until the density reached 60%-80% before transfection;

[0168] 2) Place the GP-transfect-Mate transfection reagent at room temperature and mix gently before use;

[0169] 3) Add 50 μL of serum-free culture medium to three 1.5 mL sterile centrifuge tubes, add an appropriate amount of transfection reagent, mix gently with a pipette, and let stand at room temperature for 5 min.

[0170] 4) At the same time, add 50 μL of serum-free culture medium to three other 1.5 mL sterile centrifuge tubes, and add appropriate amounts of Si-1, Si-2 and Si-NC respectively. Mix gently with a pipette and let stand at room temperature for 5 min.

[0171] 5) Add the mixture from 3) to 4), mix gently with a pipette, let stand at room temperature for 20 minutes, and then immediately transfect.

[0172] 6) While the plate is settling, change the medium in each well with 0.4 mL of preheated fresh culture medium.

[0173] 7) Add the transfection mixture to the wells, and gently shake the plate after adding to ensure the complex is evenly distributed;

[0174] 8) Incubate cells at 37℃ for 6 hours, then replace with complete culture medium. Collect cell samples 24 hours later and detect circKANSL1 expression level using RT-qPCR (see Tables 5 and 6).

[0175] The results are as follows Figure 7a As shown in the figure, the results indicate that both Si-1 and Si-2 can interfere with the expression of circKANSL, with Si-2 exhibiting higher interference efficiency.

[0176] (3) Test to inhibit DHAV-3 replication

[0177] Si-2, with high interference efficiency, was selected to interfere with the expression of circKANSL1 in duck embryonic primary liver cells. After 24 hours of treatment, DHAV-3 was infected for approximately 20 hours. Total RNA was collected from the cells, and viral expression levels were detected using RT-qPCR (the specific reaction system and procedure have been described above). The results are as follows: Figure 7b As shown, the results indicate that interfering with circKANSL1 inhibits the replication of DHAV-3.

[0178] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. The circular KANSL1 gene, whose nucleotide sequence is shown in SEQ ID NO:

1.

2. The application of overexpression of the circular KANSL1 gene as described in claim 1 in promoting the replication of duck hepatitis A virus type 3 in vitro.

3. The application according to claim 2, characterized in that, The circular KANSL1 gene was overexpressed using a pCD2.1-ciR vector containing the circular KANSL1 gene.

4. The application of the circular KANSL1 gene expression described in requirement 1 in the in vitro inhibition of duck hepatitis A virus type 3 replication.

5. The application according to claim 2, characterized in that, The expression of the circular KANSL1 gene was reduced by small interfering RNA, the nucleotide sequences of which are shown in SEQ ID NO: 2 and SEQ ID NO:

3.

6. The use of the reagent for expressing the circular KANSL1 gene as described in claim 1 in the preparation of a formulation for inhibiting the replication of duck hepatitis A virus type 3.

7. The use of the reagent for reducing the expression of the circular KANSL1 gene as described in claim 1 in the preparation of a medicament for treating duck viral hepatitis.

8. The application according to claim 6 or 7, characterized in that, The reagent used to reduce the expression of the circular KANSL1 gene is small interfering RNA, the nucleotide sequence of which is shown in SEQ ID NO: 2 and SEQ ID NO:

3.

9. The application of the reagent for detecting the expression level of the circular KANSL1 gene as described in claim 1 in the breeding of duck hepatitis A virus type 3.

Images