Mutant duck tembusu virus strain and construction method and application thereof

Abstract

The application relates to the technical field of molecular biology, and particularly discloses a mutant duck Tembusu virus strain, a construction method and application thereof. A mutant plasmid EDII+III E89G V312A of site-directed mutation E gene is constructed through specific primer combination, and then a mutant duck Tembusu virus strain rDTMUV EDII+III E89G V312A is constructed. The strain can provide a good platform for preparation of subsequent anti-duck Tembusu virus drugs and vaccines.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to a mutant duck Tembusu virus strain, its construction method, and its application. Background Technology

[0002] In April 2010, an unknown duck egg drop disease broke out in southeastern coastal provinces of China, characterized by a sharp decline in egg production and neurological symptoms in infected laying and breeding ducks. Autopsy revealed ovarian congestion, hemorrhage, follicular atresia, and rupture. The morbidity rate was as high as 90%. Duck Tembusu virus (DTMUV) was eventually identified as the pathogen. Currently available commercial vaccines have low protective efficacy and their protective titers are not ideal, requiring repeated vaccinations, thus imposing a heavy economic burden on the duck farming industry.

[0003] DTMUV belongs to the genus Flaviviridae, family Flaviviridae. Its genome contains only one long independent open reading frame, encoding three structural proteins: nucleocapsid (C), membrane (M), and envelope (E); and seven non-structural proteins: NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. For example, the mutation at position 367 of the E protein, from the polar amino acid T to the positively charged polar amino acid K, is a major determinant of cellular adaptation and virulence attenuation in DTMUV. Therefore, constructing duck Tembusu virus mutant strains is of great significance. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide a mutant duck Tembusu virus strain and its construction method, which realizes the mutation of the E gene, thus providing a foundation for subsequent vaccine research.

[0005] Another technical problem that this invention aims to solve is to provide the application of the above-mentioned mutant duck Tembusu virus.

[0006] To address the aforementioned technical problems, this invention provides a primer combination for site-directed mutation of the E gene in duck Tembusu virus, comprising primer one and primer two, wherein primer one is a DNA molecule with a nucleotide sequence as shown in SEQ ID NO: 1, and primer two is a DNA molecule with a nucleotide sequence as shown in SEQ ID NO: 2.

[0007] As an improvement to the above technical solution, primers three and four are also included, wherein primer three is a DNA molecule with a nucleotide sequence as shown in SEQ ID NO: 3, and primer four is a DNA molecule with a nucleotide sequence as shown in SEQ ID NO: 4.

[0008] Accordingly, this invention also discloses a method for constructing a mutant duck Tembusu virus strain, comprising:

[0009] (1) Construct the mutant plasmid EDII+III E89GV312A using the primer combination as described in claim 1 and / or 2;

[0010] (2) The mutant plasmid EDII+III E89G V312A was ligated with pF2 plasmid, pF3 plasmid and pF4 plasmid to obtain the mutant strain cDNA;

[0011] (3) The mutant strain cDNA was transcribed in vitro and then transfected into cells to obtain the mutant duck Tembusu virus strain after rescue.

[0012] As an improvement to the above technical solution, step (1) includes:

[0013] (1.1) The pF1 plasmid was amplified by PCR using the primer combination described above;

[0014] (1.2) Homologous recombination of PCR amplification products and transformation into DH5α competent bacteria;

[0015] (1.3) DH5α competent bacteria were cultured as single-clone bacteria to isolate the mutant plasmid EDII+IIIE89G V312A.

[0016] As an improvement to the above technical solution, in step (1.1), primers 1 and 4 are used to amplify the pF1 plasmid to obtain the E89 segment amplification product; primers 2 and 3 are used to amplify the pF1 plasmid to obtain the V312 segment amplification product.

[0017] As an improvement to the above technical solution, in step (1.2), the amplification products of E89 segment and V312 segment are digested to obtain E89 segment and V312 segment respectively. Then, E89 segment and V312 segment are mixed at a weight ratio of 1:(1-1.5), and after homologous recombination, they are transformed into DH5α competent cells.

[0018] Accordingly, the present invention also discloses a plasmid, characterized in that it is the mutant plasmid EDII+III E89G V312A as described in claim 3.

[0019] Accordingly, the present invention also discloses a mutant duck Tembusu virus strain, which is constructed by the above-described construction method.

[0020] Accordingly, the present invention also discloses the application of the above-mentioned mutant duck Tembusu virus strain in any of the following (1)-(4):

[0021] (1) Prepare a drug for treating duck Tembusu virus;

[0022] (2) Screening for drugs to treat duck Tembusu virus;

[0023] (3) Prepare a vaccine for the prevention of duck Tembusu virus;

[0024] (4) Prepare a kit for detecting duck Tembusu virus.

[0025] Accordingly, the present invention also discloses a vaccine comprising the aforementioned mutant duck Tembusu virus strain.

[0026] Implementing this invention has the following beneficial effects:

[0027] This invention constructs a mutant duck Tembusu virus strain using a specific primer combination, which achieves mutation of the E gene, providing a good platform for the subsequent preparation of drugs and vaccines against duck Tembusu virus. Attached Figure Description

[0028] Figure 1 This is the construction map of the mutant plasmid EDII+III E89G V312A in Example 1;

[0029] Figure 2 This is a sequencing result diagram of the mutant plasmid EDII+III E89G V312A in Example 1;

[0030] Figure 3 This is another sequencing result of the mutant plasmid EDII+III E89G V312A in Example 1;

[0031] Figure 4 This is an electrophoresis diagram of cDNA transcribed into RNA in Example 3; where M is the DNA Marker and 1 is the RNA transcribed from cDNA.

[0032] Figure 5 This is an electrophoresis image of the E gene detected by PCR after virus rescue in Example 4;

[0033] Figure 6 This is a Western blot result image of the virus rescued in Example 4;

[0034] Figure 7 This is a growth curve diagram of the mutant duck Tembusu virus rDTMUV-EDII+III E89G V312A in this invention;

[0035] Figure 8 This is a graph showing the viral content in different parts of ducklings after challenge, as determined by fluorescence quantitative PCR in Example 4. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0037] It should be noted that the pF1, pF2, pF3, and pF4 plasmids used in this invention were all constructed by the Zhaoqing Branch of the Guangdong Provincial Laboratory of Lingnan Modern Agricultural Science and Technology. For specific construction methods, please refer to CN114196683A. All other reagents were commercially available.

[0038] The sequencing and primer synthesis involved in the embodiments of the present invention were completed at Sangon Biotech (Shanghai) Co., Ltd. and Genewiz Biotech Ltd.

[0039] Example 1: Construction of plasmid EDII+III E89G V312A for point mutation of protein E

[0040] (a) Design primers for point mutation E gene and bacterial culture identification PCR;

[0041] Primer 1(+):

[0042] 5'-ggcaacttatgctggatacatatgc-3'.

[0043] Primer 2(-):

[0044] 5'-gtatccagcataagttgccttgggat-3'.

[0045] Primer 1 (SEQ ID NO: 1) and Primer 2 (SEQ ID NO: 2) are used for site-directed mutagenesis of the E89 amino acid (EG) sequence of the target gene.

[0046] Primer 3(+)

[0047] 5'-gcgaagaatcctaccgacactg-3'

[0048] Primer 4(-)

[0049] 5'-gtcggtaggattcttcgctaggg-3'

[0050] Primer 5(+)

[0051] 5'-caaggcaacttatgctgg-3'

[0052] Primer 6(-)

[0053] 5'-gtgtcggtaggattcttcg-3'

[0054] Primer 3 (SEQ ID NO: 3) and primer 4 (SEQ ID NO: 4) were used for site-directed PCR mutation of the target gene at amino acid position 312 (VA). Primer 5 (SEQ ID NO: 5) and primer 6 (SEQ ID NO: 6) were used to identify the mutation effect.

[0055] The primers were sent to a bioengineering company for synthesis.

[0056] (II) PCR amplification of the target gene fragment with the mutation site

[0057] Specifically, the two primer pairs are used interchangeably: Primer 1 + Primer 4 amplifies the E89 segment, and Primer 2 + Primer 3 amplifies the V312 segment.

[0058] Specifically, the PCR system is shown in the table below:

[0059]

[0060] The PCR conditions were as follows: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 55℃ annealing for 15 s, 72℃ extension for 8 min, for a total of 32 cycles; and a final extension at 72℃ for 10 min.

[0061] After the reaction is complete, a small amount of the amplification product is taken for agarose gel electrophoresis. If the target plasmid is correctly amplified, the next step of the experiment can be performed.

[0062] Because the amplification products contain the original template plasmid, digestion is performed to prevent false positive transformants after transformation. Specifically, in this embodiment, Dpn I is used to digest the amplified E89 and V312 segments, respectively. The specific reaction system is shown in the table below:

[0063]

[0064] The specific digestion conditions are as follows: after gently aspirating and mixing the amplification product and Dpn I, briefly centrifuge to collect the product to the bottom of the tube, and then place it at a constant temperature of 37℃ for 1-2 hours.

[0065] (III) Recovery of digestion products

[0066] The digestion products were subjected to gel extraction to recover the 7917bp vector fragment. Specifically, the blade was wiped with alcohol, the electrophoresis gel was placed on a blue light projection gel cutter, the target fragment was identified and cut, and efforts were made to cut only the nucleic acid gel containing the target fragment. The product was transferred to a 2mL EP tube, and XP2 Binding Buffer solution was added to just cover the gel. The tube was heated in a 60℃ constant temperature metal bath, vortexed once every 2-3 minutes until the gel was completely melted. Subsequent operations were performed at room temperature and centrifuged at 12000rpm. The DNA binding column was placed in a 2mL collection tube, and the liquid was added to the binding column. The maximum volume of solution that the binding column can hold at one time is 700μL. Any excess volume can be centrifuged twice for 1 minute each time. The liquid was discarded, 300μL of XP2 Binding Buffer was added, centrifuged for 1 minute, and the filtrate was discarded. Place the binding column back onto the collection tube, add 700 μL of SPW Buffer diluted with anhydrous ethanol to the binding column, centrifuge for 1 min, discard the filtrate, and repeat this operation once. Then centrifuge for 2 min and air dry the ethanol in a clean bench. Add 20-50 μL of ddH2O to the binding column, let stand for 5 min, centrifuge for 1.5 min, and obtain the recovered product.

[0067] (iv) Recombination reaction

[0068] The PCR-amplified mutant fragment was subjected to homologous recombination. The specific recombination system is as follows: The formula is shown in the table below:

[0069]

[0070]

[0071] The specific recombinant reaction conditions are as follows: gently pipette and mix thoroughly, then briefly centrifuge to collect the reaction solution at the bottom of the tube. Incubate at 37°C for 30 minutes; then cool to 4°C or immediately place on ice to cool.

[0072] (V) Transformation of Recombinant Products

[0073] The specific conversion steps are as follows:

[0074] (1) Thaw DH5α competent cells on ice, add 10 μL of recombinant product to 50 μL of DH5α competent cells, gently tap the tube wall to mix (do not shake to mix), and let stand on ice for 30 min.

[0075] (2) After heat shock in a 42℃ water bath for 45 seconds, immediately place it on ice to cool for 2-3 minutes.

[0076] (3) Add 1 mL of SOC medium (without antibiotics) and shake at 37°C for 1 h (200-250 rpm).

[0077] (4) AMP + The LB agar plates were preheated in an incubator at 37°C.

[0078] (5) Take 100 μL of bacterial suspension and gently spread it onto a plate containing AMP using a sterile swab. + On resistant plate solid culture medium.

[0079] (6) Incubate upside down in a 37℃ incubator for 12-16 hours.

[0080] After overnight culture, if the number of clones on the recombinant reaction transformation plate is significantly higher than that of the negative control, several single clones can be selected and inoculated into LB liquid medium containing appropriate antibiotics for overnight culture. Then, plasmids are extracted using the TIAN prep MiniPlasmid Kit, named EDII+III E89G V312A, and subjected to PCR sequencing. The specific sequencing system is shown in the table below:

[0081]

[0082] The PCR conditions were as follows: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 55℃ annealing for 15 s, 72℃ extension for 8 min, for a total of 32 cycles; and a final extension at 72℃ for 10 min.

[0083] Sequencing results as follows Figure 2 and Figure 3 The positive clone sequencing results showed that the EDII+III E89G V312A plasmid was successfully constructed, and it was consistent with the expected target gene sequence.

[0084] Example 2: Obtaining cDNA from the mutant strain

[0085] (I) Plasmid digestion and recovery

[0086] Pre-constructed mutant plasmids were digested with pre-constructed full-length duck Tembusu fragment plasmids pF2, pF3, and pF4. Specifically, Esp3 I (BsmBI) restriction endonuclease was used for digestion. The reaction system was as follows: 4 μL Esp3 I, 5 μL CutSmart buffer, 5 μg fragment, and water added to a final volume of 50 μL. The reaction was carried out at 37°C for 4-5 hours. After purification, fragments F1, F2, F3, and F4 were recovered, with lengths of 3501 bp, 3110 bp, 4006 bp, and 400 bp, respectively. These fragments were then ligated using NEB's high-concentration T4 ligase overnight at 16°C to obtain full-length viral cDNA. The ligation system is shown in the table.

[0087]

[0088] After reacting the above system overnight at 16°C, a small amount of the reaction solution was run on a nucleic acid gel to identify the presence of a long fragment of 11017 bp. If the identification was correct, the reaction solution was purified using the Takara DNA Large Fragment Purification Kit to remove enzyme proteins.

[0089] (II) cDNA Purification

[0090] (1) Add 3 times the amount of Buffer DC to the PCR reaction solution (or other enzyme reaction solution) (if the amount of Buffer DC to be added is less than 100 μL, add 100 μL) and then mix evenly.

[0091] (2) Place the Spin Column from the kit (Omega cycle pure kit (100)-q spin column) onto the Collection Tube.

[0092] (3) Transfer the solution from step (1) above to a Spin Column, centrifuge at 12,000 rpm for 1 minute at room temperature, and discard the filtrate. (Note) Adding the filtrate to the Spin Column and centrifuging again can improve the DNA recovery rate.

[0093] (4) Add 700 μL of Buffer WB to the Spin Column, centrifuge at 12,000 rpm for 30 seconds at room temperature, and discard the filtrate. Please confirm that the specified volume of 100% ethanol has been added to the Buffer WB.

[0094] (5) Repeat step 4.

[0095] (6) Place the Spin Column on the Collection Tube and centrifuge at 12,000 rpm for 1 minute at room temperature.

[0096] (7) Place the Spin Column on a new 1.5 mL centrifuge tube, add 25-30 μL of sterile water or Elution Buffer to the center of the Spin Column membrane, and let it stand at room temperature for 1 minute.

[0097] (8) Elute DNA by centrifuging at 12,000 rpm for 1 minute at room temperature.

[0098] Example 3: In vitro transcription and electroporation, virus rescue

[0099] (I) RNA transcription

[0100] Using the purified full-length cDNA product as a template, mMESSAGEm The T7Kit in vitro transcription kit was used for in vitro transcription. The reaction was performed in PCR at 37°C for 2.5 hours, followed by the addition of 1 μL of GTP and incubation at 37°C for another 0.5 hours until the reaction was complete, yielding infectious RNA. The specific reaction system is shown in the table below:

[0101]

[0102] The transcripts were purified using lithium chloride precipitation. The RNA precipitate was dissolved in 20 μL of nuclease-free water and stored at -80°C.

[0103] (II) Virus Rescue

[0104] The RNA obtained above was transfected into BHK-21 cells using an electroporator, as follows:

[0105] (1) BHK cells were cultured in T75 culture dishes until they were in good growth condition. The culture medium was discarded and 2 μL of PBS was added to wash them twice.

[0106] (2) Prepare the cell culture flasks one day in advance to ensure a cell density of 80% for electroporation the next day. Pre-cool the electroporation flasks after irradiating them with UV light. Place the RNA and PBS on ice. Add 2 mL of 7% FBS DMEM (1% penicillin-dextrose antibody) to three 30 mm culture dishes and preheat them in a 37°C incubator. Digest the cells in the culture flasks, resuspend them in 15 mL of 10% FBS DMEM, transfer them to 50 mL centrifuge tubes, balance the mixture, and centrifuge at 500 g for 5 min at 4°C. Discard the supernatant, resuspend the cells in 15 mL of cold PBS, and centrifuge again (repeat once). Discard the PBS, resuspend the cells in 700 μL of PBS, and aliquot.

[0107] (3) Take 200 μL of the resuspended cells and RNA, mix well, and place on ice for 3 min. Set the electroporator to 105 V, 25 ms. Add the liquid containing the cells and RNA to the electroporation cuvette, place the cuvette in the instrument slot for electroporation, and immediately place the cuvette on ice for 3 min after electroporation. Aspirate the liquid and add it to a preheated culture dish. Gently shake to disperse the cells evenly and incubate at 37℃. After the cells have reached confluence, change the medium (serum-free DMEM + 1% penicillin antibody). After culturing for 3-4 days, freeze and thaw the culture dish three times at -80℃ / 37℃, then centrifuge at 12000 rpm for 5 min. Collect the supernatant and store it. This supernatant is the P0 generation rDTMUV-EDII+III E89G V312A virus solution.

[0108] Example 4: Detection of mutant virus strains and evaluation of immunization effects

[0109] (I) Identification of rDTMUV-EDII+III E89G V312A

[0110] 1. PCR testing to save the virus

[0111] Specifically, viral RNA is extracted, then reverse transcribed into cDNA, and then PCR testing is performed.

[0112] The method for extracting viral RNA is as follows:

[0113] (1) Collect the virus solution after 72 hours, freeze it at -80°C for 3 freeze-thaw cycles, then add 700 μL of Trizol and place it at room temperature for 5 minutes to allow it to fully lyse.

[0114] (2) Centrifuge at 12000 rpm for 5 min and discard the precipitate.

[0115] (3) Add chloroform at a ratio of 200 μL chloroform / μL Trizol, shake to mix, and let stand at room temperature for 15 min.

[0116] (4) Centrifuge at 12000g for 15 min at 4℃.

[0117] (5) Transfer the upper aqueous phase to another centrifuge tube. Do not aspirate the intermediate interface;

[0118] (6) Add isopropanol at a ratio of 0.5 μL isopropanol / μL Trizol, mix well, and let stand at room temperature for 5-10 min.

[0119] (7) Centrifuge at 12000g at 4℃ for 10 min, discard the supernatant, and let the RNA settle at the bottom of the tube.

[0120] (8) Add 75% ethanol at a ratio of 1 μL 75% ethanol / μL Trizol, gently shake the centrifuge tube, and suspend the precipitate.

[0121] (9) Centrifuge at 8000g at 4℃ for 5 minutes and discard the supernatant as much as possible.

[0122] (10) Air dry at room temperature or vacuum dry for 5-10 minutes.

[0123] (11) Dissolve 50 μL of DEPC in water and incubate at 55-60℃ for 5 min.

[0124] The method for reverse transcription of RNA into cDNA is as follows:

[0125] The reaction was performed using TAKARA two-step RT-PCR reverse transcription reagents (as shown in the table below).

[0126]

[0127] The specific reaction conditions are: 37℃ for 15 min; 85℃ for 5 s; and storage at 4℃.

[0128] The PCR assay method is as follows:

[0129] The E gene was amplified by PCR using reverse-transcribed cDNA, with primers shown in the table below. The amplified fragment was then tested by PCR, with primers shown in the table below.

[0130]

[0131] Sequencing results as follows Figure 5 As shown in the figure, it should be noted that 1 is the DF-1 cell blank control; 2 is DTMUV, i.e., the parent virus (DTMUV-QY17 isolated and preserved in our laboratory).

[0132] GenBank Accession No. MT447092), 3 is rDTMUV-QY21, i.e., the recombinant virus constructed using the method of CN114196683A; 4 is P1 generation rDTMUV-EDII+III E89GV312A, 6 is P5 generation rDTMUV-EDII+IIIE89G V312A, and 8 is P10 generation rDTMUV-EDII+III E89G V312A. As can be seen from the figure, the recombinant virus expresses the E gene, indicating successful virus rescue.

[0133] 2. Western Blot Detection for Virus Rescue

[0134] (1) Collect cells, discard cell culture medium, add an appropriate amount of 1×Lysis Buffer according to the cell volume, and lyse the cells at 4℃ for 10-15 min.

[0135] (2) Scrape the cells off with a cell scraper and transfer them to the corresponding labeled 1.5μL EP tubes and incubate on ice for 10-15 min.

[0136] (3) Centrifuge at 12000g for 5 min at 4℃, and slowly aspirate the supernatant into the corresponding labeled 1.5μL EEP tube. Quantify protein using the BCA method and calculate the protein concentration.

[0137] (4) Processing protein samples: Add 5× Loading Buffer, heat at 95℃-100℃ for 5 min, and store at -20℃ for later use.

[0138] (5) Preparation of SDS-PAGE

[0139] 1) Take equal volumes of separating gel buffer and separating gel solution and mix them well, i.e., take 2.0 / 2.7 / 4.0 μL of each solution.

[0140] 2) Add 40 / 60 / 80 μL of modified ammonium persulfate solution to the mixed solution in step 1) and mix well.

[0141] 3) Inject the solution from step 2) into the gel-forming glass plate, and add an appropriate amount of water or ethanol to cover the separating gel.

[0142] 4) After the separating gel solidifies, pour off the upper layer of water or alcohol.

[0143] 5) Take 1.0 / 1.5 / 2.0 μL of stacking gel premix and add 10 / 15 / 20 μL of modified ammonium persulfate solution, then mix well.

[0144] 6) Pour the concentrated gel into the glass plate and insert the comb teeth. After the concentrated gel has solidified, remove the comb teeth and it is ready for electrophoresis.

[0145] (6) Electrophoresis

[0146] 1) Sample loading: Clamp the gel in the electrophoresis tank, add enough 1× electrophoresis solution, and start preparing to load the sample.

[0147] 2) Electrophoresis: two gels are kept at a constant current of 25mA-30mA for about 3 hours.

[0148] 3) Registration: Record information such as the loading order and antibody dilution ratio in the loading record book.

[0149] (7) Immunoblotting (wet transfer)

[0150] Transfer: Place the clamp into the transfer electrophoresis apparatus, add the appropriate 1× transfer buffer, and electrophores at a constant current of 400mA for 120 minutes in an ice bath to perform the transfer.

[0151] (8) Immunochromatography

[0152] Blocking: Prepare a blocking solution (TBST solution containing 5% skim milk). The PVDF membrane should be completely immersed in the blocking solution. Block at room temperature for 2 hours.

[0153] (9) Primary antibody incubation: Incubate overnight at 4 degrees Celsius.

[0154] (10) Washing the membrane: Take out the PVDF membrane, put it in a petri dish, add an appropriate amount of 1×TBST solution, place it on a decolorizing shaker and shake gently. Wash the membrane 3 times, 10 min each time.

[0155] (11) Secondary antibody incubation: Dilute the corresponding secondary antibody with blocking solution and incubate the PVDF membrane at room temperature for 2 hours.

[0156] (12) Washing the membrane: Wash the membrane 3 times with 1×TBST, 10 min each time.

[0157] (13) ECL development: developing color on a gel imaging system.

[0158] The results are shown below. Figure 6 As shown in the figure, E protein expression can be detected, indicating that the virus was successfully rescued.

[0159] (II) Detection of growth characteristics of rDTMUV-EDII+III E89G V312A

[0160] The E protein gene was amplified, identified by PCR, recovered via gel extraction, ligated into a T vector, transformed into competent *E. coli* cells, and after amplification culture, plasmids were extracted and sent to a sequencing company for sequencing. After confirmation that it was the E protein gene, the positive plasmids were divided into 10... -6 -10 -1 Serial dilutions were used as cDNA templates. Following the Takara company's absolute quantitative real-time PCR instructions, a linear equation was obtained on a real-time PCR instrument with copy number logarithm (copy number = plasmid concentration x Aristotle constant / plasmid molecular weight) as the X-axis and CT value as the Y-axis. This resulted in the linear equation for the standard curve. A standard curve was established based on the DTMUV E gene, and the viral copy number of rDTMUV-EDII+III E89G V312A was detected using qPCR absolute quantification. Results are as follows: Figure 7 As shown in the figure, its replication cycle is short.

[0161] (III) Animal pathogenicity experiments

[0162] To avoid the influence of stress on the experimental results, 82 healthy 9-day-old Muscovy ducks were selected and raised to 14 days old, then divided into four groups. The specific groupings are shown in the table below. The infected group ducks received an intramuscular injection of 0.2 mL of virus solution (containing 1000 ELD50) in their legs, while 10 control group ducks received an equal volume of PBS. The infected and control groups were kept separate to ensure no horizontal transmission. Changes in feed intake, defecation, and weight were recorded after infection. On day 14, blood was collected from the subwing vein of the remaining ducks for qualitative testing to determine antibody seroconversion.

[0163]

[0164] After necropsy, liver, spleen, kidneys, brain tissue, pancreas, and trachea of ​​the ducks were collected. RNA was extracted using the Trizol method and reverse transcribed into cDNA, which was then detected by quantitative real-time PCR. The specific reaction system and conditions are as follows:

[0165]

[0166] Reaction conditions: 95℃ for 3 min, 95℃ for 30 s, 60℃ for 15 s, 72℃ for 20 s, 40 cycles.

[0167] After the detection was completed, the linear equation of the standard curve obtained using the DTMUV E gene positive plasmid was y = -3.0777x + 45.74 (R²). 2 =0.9979). Plots were created on the six collected organs, with the logarithm of viral copy number (log10) as the Y-axis and the time after challenge as the X-axis. The results showed that the mutant strain exhibited reduced viral replication ability in the early stages. See [the table below]. Figure 8 The comparison of viral load distribution in various tissues and organs suggests that there is no significant difference in pathogenicity between DTMUV and rDTMUV EDII+III E89G V312A.

[0168] The above description is a preferred embodiment of the invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the invention, and these improvements and modifications are also considered to be within the scope of protection of the invention.

Claims

1. A primer combination for site-directed mutagenesis of E gene in duck Tembusu virus, characterized in that, The primer one is a DNA molecule with a nucleotide sequence as shown in SEQ ID NO: 1, the primer two is a DNA molecule with a nucleotide sequence as shown in SEQ ID NO: 2; the primer three is a DNA molecule with a nucleotide sequence as shown in SEQ ID NO: 3, and the primer four is a DNA molecule with a nucleotide sequence as shown in SEQ ID NO:

4.

2. A method of constructing a mutant strain of duck Tembusu virus, characterized in that, The method comprises the following steps: (1) constructing a mutant plasmid EDII+III E89G V312A by using the primer combination according to claim 1; (2) connecting the mutant plasmid EDII+III E89G V312A with pF2 plasmid, pF3 plasmid and pF4 plasmid to obtain a mutant strain cDNA; (3) transfecting cells after in vitro transcription of the mutant strain cDNA to obtain a mutant duck Tembusu virus strain; In step (1), the method comprises the following steps: (1.1) performing PCR amplification on the pF1 plasmid by using the primer combination according to claim 1; (1.2) homologously recombining and transforming the PCR amplification product into DH5α competent bacteria; (1.3) performing single colony bacterial amplification culture on the DH5α competent bacteria, and isolating to obtain the mutant plasmid EDII+III E89G V312A.

3. The construction method of claim 2, wherein, In step (1.1), the primer one and the primer four are used to amplify the pF1 plasmid to obtain an E89 segment amplification product; and the primer two and the primer three are used to amplify the pF1 plasmid to obtain a V312 segment amplification product.

4. The construction method of claim 3, wherein, In step (1.2), the E89 segment amplification product and the V312 segment amplification product are digested respectively to obtain an E89 segment and a V312 segment, and then the E89 segment and the V312 segment are mixed in a weight ratio of 1: (1-1.5) and transformed into the DH5α competent bacteria after homologous recombination.

5. A plasmid characterized in that, The mutant plasmid EDII+III E89G V312A is as claimed in claim 2.

6. A mutant duck Tembusu virus strain, which is constructed by the construction method according to any one of claims 2-4.

7. The mutant duck Tembusu virus strain according to claim 6 is applied in the following (1) or (2): (1) screening a drug for treating duck Tembusu virus; (2) preparing a vaccine for preventing duck Tembusu virus.

8. A vaccine comprising a polynucleotide of claim 1. The mutant duck Tembusu virus strain according to claim 6.

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