Poultry flavivirus NS1 trans-complementary pseudovirus system and application

Abstract

The invention discloses an avian flavivirus NS1 trans-complementary pseudovirus system and application thereof. According to the pseudovirus system disclosed by the invention, a BHK21 cell line for stably expressing USUV-NS1 protein carrying a V5 label, namely a BHK21-NS1 stably transfected cell line, is constructed by utilizing a lentivirus packaging method; meanwhile, deletion mutation is introduced into the NS1 protein of USUV, infectious clone plasmids are constructed, and the 157th-251st amino acid of the NS1 protein is deleted by the deletion mutation. The infectious clone plasmid is transfected with a BHK21-NS1 stably transfected cell line, so that the trans-complementary virus USUV-delta NS1 which can be rescued and proliferated only in the BHK21-NS1 stably transfected cell line is obtained. The USUV-deltaNS1 trans-complementary virus is subjected to continuous passage on a BHK21-NS1 cell for 10 generations, so that the TMN-deltaNS1 virus with high yield is obtained, and complete live virus is not generated by recombination in the passage process. Therefore, the avian flavivirus NS1 trans-complementary pseudovirus system disclosed by the invention can be used as a tool to be applied to USUV vaccine preparation and related antibody evaluation.

Description

Technical Field

[0001] The invention relates to the field of molecular biotechnology, and in particular to a pseudovirus system and application of avian yellow virus NS1 trans-complementation. Background Art

[0002] Usutu Virus (USUV) belongs to the Orthoflavivirus genus of the Flaviviridae family and the Japanese encephalitis virus serotype. Current research reports on USUV mainly include trans-complementation system, pseudotype virus, and virus-like particles (VLP). In comparison, the single-round infectious USUV produced by the trans-complementation system contains a complete viral skeleton, with the highest integrity, closer to the real virus, and can be studied in all aspects of the virus under BSL-2. The trans-complementation system is an important technology for studying basic biology and genetics in molecular virology. The currently reported method mainly introduces the defective viral genome into a stable cell line containing the corresponding complementary gene. The virus obtained in this way can only produce progeny viruses in the stable cell line and cannot be expanded to other cells, thereby achieving the purpose of safety degradation.

[0003] In flaviviruses, the trans-complementation system has been constructed and widely used as a tool for basic research. The flavivirus nonstructural protein NS1 is a multifunctional glycoprotein of approximately 46-55 kDa, which has membrane-associated and soluble secreted forms, and is important for RNA replication, the production of infectious virus particles, and regulates the host immune response. Flaviviruses lacking NS1 cannot proliferate, but can be rescued by trans-complementation of the full-length NS1 protein. Pseudoviral particles produced using the trans-complementation system can be used in vaccine development and neutralizing antibody evaluation. Since the pseudovirus produced can only replicate and produce toxins in cells expressing specific proteins, vaccines based on pseudoviral particles can stimulate the body to produce higher immune protection in a safe manner compared to traditional vaccines. In general, the pseudoviral system of flaviviruses has been effectively used as an effective tool in multiple research fields. At present, there are still no reports on the construction of the USUV trans-complementation system. Summary of the invention

[0004] The purpose of the present invention is to solve the above problems existing in the prior art and to provide a pseudovirus system and application of NS1 trans-complementation of avian yellow virus.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows: a pseudovirus system for trans-complementation of avian yellow virus NS1, in which the infectious clone plasmid of USUV-TMN strain lacking part of the NS1 gene is transfected into the BHK21 stable cell line to rescue the trans-complementation virus USUV-ΔNS1; the BHK21 stable cell line can stably express USUV-NS1 protein; the trans-complementation virus USUV-ΔNS1 is a mutant virus after the 157-251 amino acids are missing in the NS1 protein sequence of USUV.

[0006] Preferably, the method for preparing the BHK21 stable cell line comprises the following steps:

[0007] A1. Using pUC57-TMN-P1 plasmid as template, PCR amplification was performed to obtain the target fragment, which was then connected to the pLJM1 plasmid double-digested (NheI, EcoRI) vector to construct a recombinant plasmid named pLJM1-TMN-NS1-V5;

[0008] A2, transfecting HEK293T cells with the recombinant plasmid pLJM1-TMN-NS1-V5, filtering and purifying to obtain a virus suspension;

[0009] A3. Transfect BHK21 cells with the virus suspension in A2, and obtain positive cells BHK21-NS1 through puromycin resistance screening, which are stable transfected cells.

[0010] Preferably, the primers for PCR amplification in A1 are pLJM1-TMN-E45-F and pLJM1-TMN-NS1-V5-R, and the nucleotide sequence is shown in SEQ ID N0.1-2.

[0011] Preferably, the method for preparing the infectious clone plasmid of the USUV-TMN strain lacking a portion of the NS1 gene comprises the following steps:

[0012] B1, using plasmid pUC57-TMN-P1 as a template, PCR amplification was performed to obtain fragment a and fragment b; a and b were subjected to fusion PCR to amplify the final fragment c; fragment c was connected with the double restriction enzyme digestion (XhoI, EcoRV) vector of pACYC-TMN-P2-3 plasmid to obtain a recombinant plasmid, which was named as pACYC-TMN-ΔNS1 intermediate;

[0013] B2. Using plasmid pACYC-TMN-CprME as a template, the target fragment was amplified and ligated with the pACYC-TMN-ΔNS1 intermediate plasmid double-digested (NruI, EcoRV) vector in B1 to obtain an infectious clone plasmid of the USUV-TMN strain lacking part of the NS1 gene, named pACYC-TMN-ΔNS1; the sequence of the plasmid pACYC-TMN-CprME is shown in SEQ ID N0.13.

[0014] Preferably, the amplification primers for the fragment a are TMN-P1-3-F and TMN-NS1-N156-R; the amplification primers for the fragment b are TMN-NS1-C253-F and TMN-P1-3-R; the amplification primers for the fragment c are TMN-P1-3-F and TMN-P1-3-R; the amplification primers for the target fragment are TMN-C-1F and TMN-E-1R; the primer nucleotide sequences are shown in SEQ ID N0.3-8.

[0015] Further preferably, the preparation method of the plasmid pUC57-TMN-P1 is: connecting the artificially synthesized TMN-P1 fragment to the vector pUC57, and the sequence of the TMN-P1 fragment is shown in SEQ ID N0.11.

[0016] Further preferably, the preparation method of the pACYC-TMN-P2-3 plasmid is: using the synthesized USUV-TMN strain gene fragment P2 as a template, using primers TMN-P2-3-F / R, performing a PCR reaction, and amplifying to obtain fragment P2; the sequence of the primers TMN-P2-3-F / R is shown in SEQ ID N0.9-10; the fragment P2 is connected to the pACYC-TMN-P3 plasmid double enzyme digestion (EcoRV, KpnI) vector to obtain a recombinant plasmid, named pACYC-TMN-P2-3; the sequence of the pACYC-TMN-P3 plasmid is shown in SEQ ID N0.12.

[0017] The present invention also provides the use of the above-mentioned pseudovirus system of NS1 trans-complementation of avian yellow virus in the preparation of Usutu virus vaccine.

[0018] The beneficial effects of the present invention are:

[0019] 1) The present invention provides a pseudovirus system for trans-complementation of NS1 of avian yellow virus. The pseudovirus system uses a lentiviral packaging method to construct a BHK21 cell line that stably expresses USUV-NS1 protein carrying a V5 tag, namely, a BHK21-NS1 stable cell line; in order to achieve the purpose of trans-complementation, the present invention introduces a deletion mutation into the NS1 protein of USUV to construct an infectious clone plasmid, and the deletion mutation causes the NS1 protein to lack amino acids 157-251. The infectious clone plasmid is transfected into the BHK21-NS1 stable cell line to obtain a trans-complementation virus USUV-ΔNS1 that can be rescued and proliferated only in the BHK21-NS1 stable cell line.

[0020] 2) The USUV-ΔNS1 trans-complemented virus prepared by the pseudovirus system was continuously passaged on BHK21-NS1 cells for 10 generations, and a high yield of TMN-ΔNS1 virus was obtained, and no complete live virus was recombined during the passage process. Therefore, the avian yellow virus NS1 trans-complemented pseudovirus system of the present invention can be used as a tool in the preparation of USUV vaccines and related antibody evaluation. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the pLJM1-TMN-NS1-V5 plasmid map.

[0022] Figure 2 Verification of three monoclonal TMN-NS1-V5 BHK21 stable cell lines (WB results are shown in the left figure, and RT-PCR results are shown in the right figure).

[0023] Figure 3 This is a verification of the stability of the TMN-NS1-V5 BHK21 stable cell line (WB results are shown in the upper figure, and RT-PCR results are shown in the lower figure).

[0024] Figure 4 Schematic diagram of the pACYC-TMN-ΔNS1 intermediate plasmid (left) and pACYC-TMN-ΔNS1 plasmid (right).

[0025] Figure 5 Schematic diagram for the construction of pACYC-TMN-ΔNS1.

[0026] Figure 6 WB (left) and IFA (right) results for TMN-ΔNS1 trans-complementation virus validation.

[0027] Figure 7 RT-qPCR (upper) and IFA (lower) of TMN-ΔNS1 trans-complemented virus proliferation.

[0028] Figure 8This is the WB verification result of the stability of TMN-ΔNS1 trans-complemented virus.

[0029] Fig. 9 Schematic diagram of the plasmid map of pACYC-TMN-P3 (left) and the plasmid map of pACYC-TMN-P2-3 (right). DETAILED DESCRIPTION

[0030] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0031] Example 1 Construction of BHK21 stable cell line expressing USUV-NS1 protein

[0032] 1. Construction of pLJM1-TMN-NS1-V5 lentiviral packaging plasmid

[0033] Using pUC57-TMN-P1 plasmid as template, primers pLJM1-TMN-E45-F and pLJM1-TMN-NS1-V5-R were used to amplify the fragment. The PCR amplification used KOD One PCR Master Mix enzyme, and the amplification conditions were 98°C pre-denaturation for 1 min; 98°C denaturation for 10 s, 57°C annealing for 5 s, 68°C extension for 5 s / kb, for a total of 30 cycles; and finally 68°C extension for 7 min. At the end of the amplification, gel electrophoresis was performed for identification, and the liquid was recovered after the bands were confirmed. The primers used in the amplification are shown in Table 1. The recovered fragment was connected to the pLJM1 plasmid double enzyme digestion (NheI, EcoRI) vector. Use MonClone TM Hi-Fusion Cloning Mix V2 ligase will linearize the vector and target fragment for recombination ligation reaction. The ligation system is shown in Table 2. Then transform it into SURE E. coli competent cells, wait for colonies to grow, and pick bacteria for colony identification. Then, select PCR-positive colonies for sequencing. Select the single colony with correct sequencing, expand the culture, save it, and extract the plasmid for use. The successfully constructed plasmid is named pLJM1-TMN-NS1-V5 (see the schematic diagram of the plasmid map) Figure 1 ).

[0034] Table 1 PCR amplification primer list

[0035]

[0036] Table 2 Single fragment homologous recombination reaction system

[0037]

[0038] 2. Rescue, Filtration and Purification of Lentivirus

[0039] HEK293T cells were inoculated into a 5cm cell culture dish, and when the confluence reached more than 90%, the lentiviral packaging plasmids were co-transfected: pLJM1-TMN-NS1-V5 4μg, pCMV-VSV-G 2μg, psPAX2 3μg. According to the instructions of the full-form gold transfection reagent, 500μl opti-MEM, 18μl transfection reagent, and replaced with 1640 medium containing 20% ​​FBS after transfection for 6h. After transfection for 48h, the cell supernatant was collected and the cell debris was filtered out with a 0.22μm filter. In the 5ml filtered virus initial solution, 1.25ml of 5X PEG-8000NaCl mother solution was added, and mixed every 20-30min. After 3-5 times, it was placed at 4℃ overnight. Centrifuge at 4℃, 4000g for 20min, aspirate the supernatant, let the tube stand for 1-2min, and aspirate the residual liquid. Add appropriate amount of DMEM to dissolve the lentiviral precipitate. The concentrated virus suspension is packaged and stored in finished tubes and stored at -80℃.

[0040] 3. Construction and monocloning of TMN-NS1-V5 BHK21 stable cell line

[0041] BHK21 cells were inoculated into 6-well plates. When the confluence reached 50%, the supernatant was discarded, 500 μL of lentiviral concentrate was mixed with 500 μL of fresh DMEM medium, and after infecting the cells for 6 hours, the maintenance medium was replaced with DMEM medium containing 2% FBS. After 48 hours, the cells were screened according to the puromycin resistance (puromycin, puro) carried by the pLJM1-TMN-NS1-V5 plasmid. The remaining positive cells after screening were stable cells. BHK21-NS1 positive cells were diluted into 96-well culture plates, the cells were diluted in equal gradients, and a single cell well was selected for expansion culture.

[0042] 4. BHK21-NS1 cell line verification and cell line stability

[0043] Three cell lines with better cell status were selected for WB and RT-PCR verification. Figure 2 As shown in the figure, the three monoclonal cell lines of BHK21-NS1 can express NS1 protein and have the corresponding gene, and the Mock group has no target protein band and PCR band. Comparison found that monoclonal cell line No. 3 expressed more NS1 protein, so cell line No. 3 was selected for further expansion and the next experiment. BHK21-NS1 cells were passaged daily at a ratio of 1:3 until the F11 generation, and the expression of NS1 protein and gene was verified by WB and RT-PCR methods. Figure 3 As shown, no gene loss occurred in BHK21-NS1 cells during the passaging process to the F11 generation and the protein expression was normal.

[0044] Example 2 Construction of infectious clone plasmid of USUV-TMN strain lacking part of NS1 gene

[0045] 1. Construction of pACYC-TMN-ΔNS1 intermediate plasmid

[0046] Plasmid pUC57-TMN-P1 was used as a PCR amplification template, and primers TMN-P1-3-F and TMN-NS1-N156-R were used to amplify fragment a, and primers TMN-NS1-C253-F and TMN-P1-3-R were used to amplify fragment b; a and b were subjected to fusion PCR, and primers TMN-P1-3-F / R were used to amplify the final fragment c (primers are shown in Table 3). Fragment c was connected with the pACYC-TMN-P2-3 plasmid double restriction digest (XhoI, EcoRV) vector to obtain a recombinant plasmid named pACYC-TMN-ΔNS1 intermediate (see the schematic diagram of the plasmid map) Figure 4 ).

[0047] Plasmid pUC57-TMN-P1 is an artificially synthesized plasmid. The TMN-P1 fragment was directly synthesized by the company and connected to the vector pUC57. The sequence of the TMN-P1 fragment is shown in SEQ ID N0.11.

[0048] The preparation method of pACYC-TMN-P2-3 plasmid is as follows: using the synthesized USUV-TMN strain gene fragment P2 as a template, using primers TMN-P2-3-F / R, PCR reaction, amplifying fragment P2; connecting the amplified fragment P2 with the pACYC-TMN-P3 plasmid double-enzyme digested (EcoRV, KpnI) vector (pACYC-TMN-P3 plasmid is directly synthesized by the company and connected to the vector pACYC-177B, the plasmid map schematic diagram is shown in Fig. 9 Left, the sequence is shown in SEQ ID NO.12), and the recombinant plasmid was obtained and named pACYC-TMN-P2-3 (the schematic diagram of the plasmid map is shown in Fig. 9 right).

[0049] The gene fragment P2 of the USUV-TMN strain is the base sequence of the gene sequence of the USUV-TMN strain from base 5190 to base 8180. The USUV-TMN strain is Turdus merula Netherlands 2016 (TMN) (GenBank: MN813490.1).

[0050] 2. Construction of pACYC-TMN-ΔNS1 plasmid

[0051] like Figure 5As shown, plasmid pACYC-TMN-CprME was used as a PCR amplification template, and primers TMN-C-1F and TMN-E-1R were used to amplify the fragment by PCR; then the fragment was connected with the pACYC-TMN-ΔNS1 intermediate plasmid double restriction enzyme digestion (NruI, EcoRV). The plasmid was named pACYC-TMN-ΔNS1 (see the schematic diagram of the plasmid map for details). Figure 4 ).

[0052] The sequence of plasmid pACYC-TMN-CprME is shown in SEQ ID NO.13. The fragment was directly synthesized by the company and connected to the vector pACYC-177B.

[0053] The PCR amplification described in this example uses KOD One PCR MasterMix enzyme, and the amplification conditions are 98°C pre-denaturation for 1 min; 98°C denaturation for 10 s, 57°C annealing for 5 s, 68°C extension for 5 s / kb, for a total of 30 cycles; and finally 68°C extension for 7 min. At the end of each round of amplification, gel electrophoresis was performed for identification, and liquid recovery was performed after the bands were confirmed. Using MonClone TM Hi-FusionCloning MixV2 ligase performs recombination ligation reaction between the linearized vector and the target fragment. The ligation system is shown in Table 2. Then transform it into SURE E. coli competent cells, wait for colonies to grow, and pick bacteria for colony identification. Then, select PCR-positive colonies for sequencing. Select the single colony with correct sequencing, expand the culture, save it, and extract the plasmid for use.

[0054] Table 3 PCR amplification primer list

[0055]

[0056] Example 3 Rescue and Characterization of Transcomplemented Virus TMN-ΔNS1

[0057] 1. Rescue verification of trans-complementation virus TMN-ΔNS1

[0058] The BHK21-NS1 cells prepared in Example 1 were inoculated into a 6-well cell plate, and the recombinant plasmid pACYC-TMN-ΔNS1 prepared in Example 2 was transfected when the confluence reached 70%-90%. According to the instructions of the full-form gold transfection reagent, 2 μg of plasmid, 4 μl of transfection reagent, and 200 μl of opti-MEM were prepared for transfection in each well, and the DMEM maintenance solution containing 2% FBS was replaced after 6 hours of transfection. 72 hours after transfection, WB samples were collected, and the viral supernatant was continuously passaged to the F2 generation, and WB samples were collected 72 hours after infection. WB sample collection was lysed with RIPA lysis buffer at 4°C for 30 minutes, centrifuged at 4°C 12000g for 10 minutes, and the supernatant protein sample was collected. The reverse complementation virus TMN-ΔNS1 was verified using a homemade USUV-E protein polyclonal antibody (rabbit). The negative control was the same operation on BHK21 cells. The protein expression sample of TMN-CprME packaging plasmid was used as a positive control to verify the working condition of USUV-E protein polyclonal antibody.

[0059] like Figure 6 As shown, the trans-complemented virus TMN-ΔNS1 can be rescued on BHK21-NS1 cells and the supernatant contains effective trans-complemented virus TMN-ΔNS1, which can be passaged normally. No trans-complemented virus was produced in the negative control. Accordingly, the corresponding indirect immunofluorescence experiment (IFA) was performed in a 12-well cell plate, and the cell slides were collected 72 hours after transfection. Fix with 4% paraformaldehyde, wash with PBST, permeabilize with 0.25% Triton-PBS at 4°C for 1 hour, and wash with PBST; add 5% BSA-PBS to block at 37°C for 1 hour and wash with PBST; incubate with homemade USUV-E protein polyclonal antibody (mouse) as the primary antibody overnight and wash with PBST; incubate with FITC fluorescently labeled goat anti-mouse IgG as the secondary antibody and wash with PBST, and the cell nuclei are stained with DAPI; finally, add glycerol to seal the slide and observe under a fluorescence microscope. The results are shown in Figure 6 As shown, only BHK21-NS1 cells transfected / treated with TMN-ΔNS1 had fluorescence, ie, the trans-complemented virus was present, while the negative control group did not have the trans-complemented virus.

[0060] 2. Study on the characteristics of the trans-complemented virus TMN-ΔNS1

[0061] BHK21-NS1 cells were inoculated into 5 cm cell plates. When the cells reached 70%-90% confluence, plasmid pACYC-TMN-ΔNS1 was transfected. The supernatant virus solution was collected 96 hours after transfection. The supernatant virus solution was diluted 10 times and infected with BHK21-NS1 cells or BHK21 cells (negative control) plated on 12-well plates according to the standard dose. The supernatant and cell samples were collected 1d, 2d, 4d, and 6d after infection and placed at -80°C. Total RNA was extracted from the supernatant and cell samples using the Trizol method and then subjected to RT-qPCR experiments. The results are shown in Figure 7 As shown, in BHK21-NS1 cells infected with TMN-ΔNS1, the amount of TMN subgenomic RNA was large and significantly proliferated, especially on the 4th and 6th days after infection. The intracellular level reached the highest level on the 4th day after infection, and the extracellular trans-complemented viral RNA continued to proliferate 6 days after infection, possibly reaching a higher level. The intracellular and extracellular trans-complemented viral TMN-ΔNS1 subgenomic RNA levels increased by about 100 times compared to the detection level 1 day after infection, while the TMN subgenomic RNA in the control group BHK21 cells did not change or proliferate significantly. The matching IFA experimental results are shown in Figure 7 The above indicated that TMN-ΔNS1 trans-complemented virus had excellent translation and replication capabilities in BHK21-NS1 cells.

[0062] 3. Stability of trans-complemented virus TMN-ΔNS1

[0063] In order to ensure the safety of TMN-ΔNS1, that is, the inability to recombinantly produce wild-type live virus, in this example, TMN-ΔNS1 was continuously passaged on BHK21-NS1 cells for 10 generations, and each generation of trans-complemented virus was used to infect BHK21 cells, and cell WB samples were collected 72 hours after infection. The WB results are shown in Figure 2. Figure 8 As shown, in BHK21-NS1 cells, a large amount of USUV-E protein was expressed in F0-F10 cell samples, while no USUV-E protein could be detected in BHK21 cells after infection, indicating that a high yield of TMN-ΔNS1 virus was obtained in BHK21-NS1 cells, and no recombination or reversion to replication-competent wild-type virus occurred during the passaging process.

[0064] The specification and drawings of the present invention are considered to be illustrative rather than restrictive. On the basis of the present invention, those skilled in the art can make some substitutions and modifications to some of the technical features according to the disclosed technical content without creative labor, all of which are within the protection scope of the present invention.

Claims

1. A pseudovirus system for trans-complementation of NS1 of avian yellow virus, characterized in that: The infectious clone plasmid of the USUV-TMN strain lacking part of the NS1 gene was transfected into the BHK21 stable cell line to rescue the trans-complemented virus USUV-ΔNS1; the BHK21 stable cell line can stably express the USUV-NS1 protein; the trans-complemented virus USUV-ΔNS1 is a mutant virus after the 157-251 amino acids are missing in the NS1 protein sequence of USUV.

2. The pseudovirus system according to claim 1, characterized in that: The method for preparing the BHK21 stable cell line comprises the following steps: A1. Using pUC57-TMN-P1 plasmid as template, PCR amplification was performed to obtain the target fragment, which was then connected to the pLJM1 plasmid double-digested (NheI, EcoRI) vector to construct a recombinant plasmid named pLJM1-TMN-NS1-V5; A2, transfecting HEK293T cells with the recombinant plasmid pLJM1-TMN-NS1-V5, filtering and purifying to obtain a virus suspension; A3. Transfect BHK21 cells with the virus suspension in A2, and obtain positive cells BHK21-NS1 through puromycin resistance screening, which are stable transfected cells.

3. The pseudovirus system according to claim 2, characterized in that: The primers for PCR amplification in A1 are pLJM1-TMN-E45-F and pLJM1-TMN-NS1-V5-R, and the nucleotide sequence is shown in SEQ ID NO.1-2.

4. The pseudovirus system according to claim 1, characterized in that: The method for preparing the infectious clone plasmid of the USUV-TMN strain lacking a portion of the NS1 gene comprises the following steps: B1, using plasmid pUC57-TMN-P1 as a template, PCR amplification was performed to obtain fragment a and fragment b; a and b were subjected to fusion PCR to amplify the final fragment c; fragment c was connected with the double restriction enzyme digestion (XhoI, EcoRV) vector of pACYC-TMN-P2-3 plasmid to obtain a recombinant plasmid, which was named as pACYC-TMN-ΔNS1 intermediate; B2. Using plasmid pACYC-TMN-CprME as a template, the target fragment was amplified and ligated with the pACYC-TMN-ΔNS1 intermediate plasmid double-digested (NruI, EcoRV) vector in B1 to obtain an infectious clone plasmid of the USUV-TMN strain lacking part of the NS1 gene, named pACYC-TMN-ΔNS1; the sequence of the plasmid pACYC-TMN-CprME is shown in SEQ ID N0.

13.

5. The pseudovirus system according to claim 4, characterized in that: The amplification primers for the fragment a are TMN-P1-3-F and TMN-NS1-N156-R; the amplification primers for the fragment b are TMN-NS1-C253-F and TMN-P1-3-R; the amplification primers for the fragment c are TMN-P1-3-F and TMN-P1-3-R; the amplification primers for the target fragment are TMN-C-1F and TMN-E-1R; the primer nucleotide sequences are shown in SEQ ID N0.3-8.

6. The pseudovirus system according to claim 2 or 4, characterized in that: The preparation method of the plasmid pUC57-TMN-P1 is as follows: connecting the artificially synthesized TMN-P1 fragment to the vector pUC57, and the sequence of the TMN-P1 fragment is shown in SEQ ID NO.

11.

7. The pseudovirus system according to claim 4, characterized in that: The preparation method of the pACYC-TMN-P2-3 plasmid is as follows: using the synthesized USUV-TMN strain gene fragment P2 as a template, using primers TMN-P2-3-F / R, performing a PCR reaction, and amplifying to obtain fragment P2; the sequence of the primers TMN-P2-3-F / R is shown in SEQ ID N0.9-10; the fragment P2 is connected to the pACYC-TMN-P3 plasmid double enzyme digestion (EcoRV, KpnI) vector to obtain a recombinant plasmid, named pACYC-TMN-P2-3; the sequence of the pACYC-TMN-P3 plasmid is shown in SEQ ID N0.

12.

8. Use of the avian yellow virus NS1 trans-complemented pseudovirus system as claimed in claim 1 in the preparation of Usutu virus vaccine.

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