In-vivo and in-vitro double visual recombinant mouse pox virus ECTVC genome, recombinant virus, construction method and application

Abstract

The invention provides an in-vivo and in-vitro double visual recombinant mouse pox virus ECTVC genome, a recombinant virus, a construction method and application, and belongs to the technical field of gene engineering. The method comprises the following steps: improving a wild type rat poxvirus genome, namely knocking out a non-coding region between C12R and C13R, and inserting a red fluorescent protein gene mKate and a Gaussian luciferase gene Gluc by using a homologous recombination method. The invention also provides a recombinant mouse poxvirus ECTVC containing the recombinant mouse poxvirus ECTVC genome, the obtained recombinant virus has good hereditary stability, and the stability of the recombinant virus can be continuously maintained for at least seven generations. The recombinant virus provided by the invention can be used for in-vitro high-throughput screening of drugs, and can realize visualization of replication and distribution of the virus in a mouse body.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to a recombinant ectromelia virus CTCV genome and recombinant virus which can be visualized in vivo and in vitro, and a construction method and application. BACKGROUND

[0002] The ectromelia virus belongs to the poxviridae family and orthopoxvirus genus, and is a double-stranded DNA virus. It can cause the ectromelia disease characterized by ulcerative necrosis of the tail and foot and toe shedding. Only a trace amount of virus can cause upper and lower respiratory tract infection in mice, accompanied by characteristic skin rash. In the past century, ectromelia virus strains have been isolated from different periods and different laboratories around the world, involving the United States, Asia and Europe and other regions. The ectromelia virus is similar to human smallpox virus infection in terms of high mortality rate, low infection dose and respiratory disease progression. Therefore, it is urgent to develop improved vaccines or small molecule compounds against orthopoxvirus infection, and the construction of recombinant ectromelia virus with a reporter gene can be used for high-throughput screening of broad-spectrum anti-orthopoxvirus drugs.

[0003] Gaussia luciferase (Gluc) is a secreted luciferase, and the expressed enzyme can be secreted to the extracellular space through the endoplasmic reticulum. This allows the enzyme activity to be detected without lysing the cells during the experiment, thereby simplifying the operation steps and reducing the interference to the cells. And it can be detected in real time during the experiment. In addition to the simple operation, Gluc also has the advantages of high sensitivity, high signal-to-noise ratio, stability and specificity.

[0004] Currently, only one study has reported the insertion of firefly luciferase gene between C12R and C13R of the ectromelia virus, and the virus maintains similar virulence to the parent virus. There is no report on the insertion of double reporter genes, and there is no report on the use of recombinant ectromelia virus with reporter genes for drug screening against orthopoxvirus. SUMMARY

[0005] The present application provides a recombinant ectromelia virus CTCV genome and recombinant virus which can be visualized in vivo and in vitro, and a construction method and application. The present application inserts the red fluorescent protein gene mKate and the luciferase gene Gluc into the C region of the ectromelia virus by homologous recombination technology, establishes an in vitro anti-orthopoxvirus drug screening platform, and realizes the visualization of the replication and infection of the ectromelia virus in mice in vivo.

[0006] The present application provides a recombinant ectromelia virus CTCV genome, which comprises replacing the non-coding region between C12R and C13R of the wild-type ectromelia virus genome, and the replacement comprises replacing the red fluorescent protein gene mKate and the Gaussia luciferase gene Gluc.

[0007] In a preferred mode of the present application, the replacement comprises replacing the nucleotide sequence at positions 733-1065 in the non-coding region between C12R and C13R of the vaccinia virus genome.

[0008] In a preferred mode of the present application, the red fluorescent protein gene mKate is expressed by the promoter p11.

[0009] The Gaussian luciferase gene Gluc is expressed by the promoter p7.5.

[0010] In a preferred mode of the present application, the nucleotide sequence at positions 733-1065 between C12R and C13R after the replacement is shown in SEQ ID No. 19.

[0011] The present application also provides a construction method of the recombinant vaccinia virus ECTVC genome, comprising the following steps: knocking out the non-coding region between C12R and C13R of the wild-type vaccinia virus genome by using a gene editing method, and then inserting a tandem gene by using a homologous recombination method to construct the recombinant vaccinia virus ECTVC genome.

[0012] The tandem gene comprises a red fluorescent protein gene mKate, a promoter p11, a Gaussian luciferase gene Gluc, and a promoter p7.5.

[0013] In a preferred mode of the present application, when the CRISPR-Cas9 method is used for knocking out, the designed sgRNA sequence is shown in SEQ ID No. 1-SEQ ID No. 4.

[0014] The present application also provides a homologous recombination vector comprising an upstream homologous arm comprising a red fluorescent protein gene mKate, a promoter p11, a promoter p7.5, and a downstream homologous arm comprising a Gaussian luciferase gene Gluc.

[0015] The nucleotide sequence of the upstream homologous arm is shown in SEQ ID No. 6, the nucleotide sequence of the promoter p11 is shown in SEQ ID No. 7, the nucleotide sequence of the promoter p7.5 is shown in SEQ ID No. 8, and the nucleotide sequence of the downstream homologous arm is shown in SEQ ID No. 9.

[0016] The present application also provides a recombinant vaccinia virus comprising the recombinant vaccinia virus ECTVC genome.

[0017] The application also provides two pairs of primer pairs for detecting the recombinant mousepox virus, including primer pair F6 and primer pair F7, wherein the nucleotide sequence of the upstream primer of primer pair F6 is shown as SEQ ID No. 15, the nucleotide sequence of the downstream primer of primer pair F6 is shown as SEQ ID No. 16; the nucleotide sequence of the upstream primer of primer pair F7 is shown as SEQ ID No. 17, and the nucleotide sequence of the downstream primer of primer pair F7 is shown as SEQ ID No. 18.

[0018] The application also provides application of the recombinant mousepox virus ECTVC genome or the recombinant mousepox virus ECTVC genome constructed by the construction method or the homologous recombination vector or the recombinant mousepox virus in antiviral drug screening and / or vaccine preparation.

[0019] Beneficial effects: The application improves the wild-type mousepox virus genome, including knocking out the non-coding region between C12R and C13R, and then inserting the red fluorescent protein gene mKate and the Gaussian luciferase gene Gluc by the method of homologous recombination. The application also provides a recombinant mousepox virus ECTVC containing the recombinant mousepox virus ECTVC genome, and the obtained recombinant virus has good genetic stability, which can be continuously maintained for at least 7 generations. The application first constructs the recombinant mousepox virus ECTVC carrying the mKate and Gluc double reporter genes based on the CRISPR-Cas9 combined with the homologous recombination technology, and it is proved by the examples that the recombinant virus has similar replication kinetics and plaque size and morphology compared with the wild virus. The recombinant virus can be used for in vitro high-throughput screening of drugs. The recombinant virus can visualize the distribution and spread of the virus in mice. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 FIG. 1 is a diagram of the construction and verification results of the ECTVC carrying mKate and Gluc in Example 1, wherein A is the cell lesion and fluorescent protein expression after the ECTVC and the parent virus ECTV-GD01 infect Vero cells; B is a schematic diagram of primer design for identifying ECTVC; C is a nucleic acid gel electrophoresis map of single plaque PCR identification of ECTVC; D is the expression of the reporter gene of ECTVC detected by indirect immunofluorescence method (scale Bar = 100 μm);

[0021] Figure 2Figure for genetic stability identification results of ECTVC in Example 2, wherein A is mKate protein expression after Vero cells were infected with ECTVC P1-P7 passage strains (Bar = 200 μm); B is Gluc protein expression after Vero cells were infected with ECTVC P1-P7 passage strains; C is nucleic acid gel electrophoresis map of PCR identification of ECTVC P1-P7 passage strains; D is Gluc protein expression after Vero cells were infected with ECTVC P1-P7 passage viruses;

[0022] Figure 3 Figure for in vitro replication kinetics results of recombinant virus ECTVC compared with ECTV-GD01 in Example 3, wherein A is fluorescence photos of ECTVC and ECTV-GD01 infected cells CEF, BHK, Vero and HeLa cells observed by fluorescence microscope, from top to bottom are CEF, BHK, Vero and HeLa cells (Bar = 100 μm); B is Gluc replication kinetics curve of ECTVC and ECTV-GD01 infected above four cell lines; C is copy number replication kinetics curve of ECTVC and ECTV-GD01 infected above four cell lines;

[0023] Figure 4 Figure for plaque morphology and size of recombinant virus ECTVC compared with ECTV-GD01 in Example 4, wherein A is comparison of plaque size and morphology of ECTVC and ECTV-GD01 formed on Vero cells; B is comparison of immune plaque size and morphology of ECTVC and ECTV-GD01 formed on CEF cells (Bar = 200 μm); C is comparison of immune plaque size and morphology of ECTVC and ECTV-GD01 formed on BHK cells (Bar = 200 μm); D is comparison of immune plaque size and morphology of ECTVC and ECTV-GD01 formed on Vero cells (Bar = 200 μm); E is comparison of immune plaque size and morphology of ECTVC and ECTV-GD01 formed on HeLa cells (Bar = 200 μm);

[0024] Figure 5Figure for the application results of recombinant virus ECTVC in the anti-positive orthopoxvirus drug ST-246 and CDV pharmacodynamics evaluation in Example 5, wherein A is the fluorescence expression diagram of ECTVC mKate under the action of different concentrations of ST-246 (Bar = 200 μm); B is the inhibitor dose response curve of ST-246 to recombinant virus ECTVC quantitatively determined according to luciferase experiment; C is the plaque picture formed by ECTVC on Vero cells under the action of different concentrations of ST-246; D is the dose response curve fitted by plaque reduction experiment in figure C; E is the fluorescence expression diagram of ECTVC mKate under the action of different concentrations of CDV (Bar = 200 μm); F is the inhibitor dose response curve of CDV to recombinant virus ECTVC quantitatively determined according to luciferase experiment; G is the plaque picture formed by ECTVC on Vero cells under the action of different concentrations of CDV; H is the dose response curve fitted by plaque reduction experiment in figure C;

[0025] Figure 6 Figure for the in vivo biological characteristics of ECTVC in Example 6, wherein A is the body weight change of Balb / c mice after intranasal challenge with different doses of ECTVC; B is the survival curve of Balb / c mice after challenge with corresponding dose in A; C is the in vivo live imaging of Balb / c mice after intranasal challenge with different doses of ECTVC; D is the light intensity change of in vivo live imaging of mice in C;

[0026] Figure 7 Figure for the in vivo virulence change of mice after infection with ECTVC in Example 7, wherein A is the body weight curve of mice and control group mice after administration; B is the survival curve of mice and control group mice after administration; C is the in vivo bioluminescence imaging diagram of mice compared with control group mice after administration; D is the light intensity change of in vivo live imaging of mice in C; E is the viral load in blood, liver, spleen, lung and kidney of mice compared with control group mice after administration. DETAILED DESCRIPTION

[0027] The application provides a recombinant ectromelia virus ECTVC genome, which comprises replacing a non-coding region between C12R and C13R of a wild type ectromelia virus genome, and the replacement comprises replacing a red fluorescent protein gene mKate and a Gaussian luciferase gene Gluc.

[0028] In an embodiment of the present application, the wild-type ectromelia virus ECTV-China-C-Tan-GD01 (ECTV-GD01) genome is taken as an example for illustration, and the ECTV-GD01 has been disclosed in an article (Huo S, Wu C, Qi Z, et al. Identification of a novel ectromelia virus from rodent: Implications for use as an in vivo infection model for vaccine and antiviral research [J]. Virologica Sinica, 2025.).

[0029] The ECTV-GD01 genome is taken as a basic genome, and the basic genome is improved, including replacing the nucleotide sequence at positions 733-1065 in the non-coding region between C12R and C13R of the basic genome with a red fluorescent protein gene mKate and a Gaussian luciferase gene Gluc, and the red fluorescent protein gene mKate is expressed by a promoter p11, and the nucleotide sequence of the promoter p11 is shown in SEQ ID No. 7; the Gaussian luciferase gene Gluc is expressed by a promoter p7.5, and the nucleotide sequence of the promoter p7.5 is shown in SEQ ID No. 8. In an embodiment of the present application, the nucleotide sequence at positions 733-1065 between C12R and C13R after the replacement is shown in SEQ ID No. 19.

[0030] The present application also provides a construction method of the recombinant ectromelia virus ECTVC genome, including the following steps: using a gene editing method to knock out the non-coding region between C12R and C13R of the wild-type ectromelia virus genome, and then using a homologous recombination method to insert a tandem gene to construct the recombinant ectromelia virus ECTVC genome.

[0031] The tandem gene includes a red fluorescent protein gene mKate, a promoter p11, a Gaussian luciferase gene Gluc, and a promoter p7.5.

[0032] The gene editing method of the application comprises CRISPR-Cas9, and the sgRNA for knocking out the region is designed according to the application, the nucleotide sequences of the sgRNA and the complementary strand are respectively shown as SEQ ID No. 1 and SEQ ID No. 2, and AAAC is added in front of the 5' end of SEQ ID No. 1, and C is added at the 3' end to become SEQ ID No. 3, and CACCG is added in front of the 5' end of SEQ ID No. 2 to become SEQ ID No. 4, and the sgRNA shown as SEQ ID No. 3 and SEQ ID No. 4 is annealed and connected to the basic vector to construct a gene editing vector. In an embodiment of the application, PX458del is used as the basic vector of the gene editing vector, and the PX458del has been disclosed in an article (Wu YB, Zhao L, Ren J, et al. Establishment of a highly efficient recombinant vaccinia virus system based on CRISPR-Cas9 [J]. Chinese Journal of Experimental and Clinical Virology, 2021, 35(2): 199-204. DOI: 10.3760 / cma.j.cn112866-20210111-00008.). In the specific operation, the annealed sgRNA is connected to the basic vector PX458del linearized by BbsI; and the CRISPR-Cas9 recombinant vector is transfected into Vero cells to express Cas9 protein.

[0033] The application further provides a primer F1 for detecting the CRISPR-Cas9 homologous recombination vector, and the nucleotide sequence of the primer F1 is shown as SEQ ID No. 5.

[0034] The application further provides a homologous recombination vector comprising an upstream homologous arm containing a red fluorescent protein gene mKate, a promoter p11, a promoter p7.5 and a downstream homologous arm containing a Gaussia luciferase gene Gluc.

[0035] The nucleotide sequence of the upstream homologous arm is shown as SEQ ID No. 6, the nucleotide sequence of the promoter p11 is shown as SEQ ID No. 7, the nucleotide sequence of the promoter p7.5 is shown as SEQ ID No. 8, and the nucleotide sequence of the downstream homologous arm is shown as SEQ ID No. 9.

[0036] In one embodiment of the present application, the upstream homologous arm sequence, the SacI restriction endonuclease sequence, the PstI restriction endonuclease sequence, the p11 promoter sequence, the p7.5 promoter sequence, the BglII restriction endonuclease sequence, the Smal restriction endonuclease sequence, and the downstream homologous arm sequence are connected in series by gene synthesis, taking PUC57 as the basic vector of the homologous recombination vector; the synthesized DNA fragment is cloned into the basic vector PUC57 to become the PUC57-C vector; the PUC57-C vector is linearized by using BglII and Smal restriction endonucleases; T4 DNA ligase is used to ligate the linearized PUC57-C and the Gluc fragment with the same enzyme cutting end to become the PUC57-C-Gluc vector; then the PUC57-C-Gluc vector is linearized by using the SacI restriction endonuclease enzyme cutting site and the PstI restriction endonuclease enzyme cutting site; the mKate gene fragment is cloned by using the primer pair F2; the mKate gene fragment and the PUC57-C-Gluc vector are connected by seamless cloning to become the final recombinant vector PUC57-C-Gluc-mKate, and the recombinant vector PUC57-C-Gluc-mKate comprises the upstream homologous arm shown in SEQ ID No. 6, the promoter p11 shown in SEQ ID No. 7, the promoter p7.5 shown in SEQ ID No. 8, and the downstream homologous arm shown in SEQ ID No. 9.

[0037] In the present application, a pair of primer pair F2 for cloning the mKate gene in the homologous recombination vector is also involved; the nucleotide sequences of the upstream primer F2-F and the downstream primer F2-R of the primer pair F2 are shown in SEQ ID No. 10 and SEQ ID No. 11, respectively. The present application also provides three primer sequences F3, F4, and F5 for detecting the homologous recombination vector, and the nucleotide sequences thereof are shown in SEQ ID No. 12, SEQ ID No. 13, and SEQ ID No. 14, respectively.

[0038] The present application also provides a recombinant vaccinia virus comprising the recombinant vaccinia virus ECTVC genome.

[0039] In one embodiment of the present application, the ectromelia virus ECTV-GD01 is used as the original strain, CRISPR-Cas9 plasmid is first transfected, 24 hours later, the virus is inoculated, and then the homologous recombination vector PUC57-C-Gluc-mKate is transfected to obtain a recombination liquid; the recombination liquid is purified by single spot for 14 rounds, and the single clone is identified and amplified to obtain the recombinant virus. In the present application, the CRISPR-Cas9 recombination vector is transfected into Vero cells one day in advance, the homologous recombination vector is transfected after the ectromelia virus ECTV-GD01 is inoculated, and the virus-infected cell supernatant and cells are harvested after 48 hours. After repeated freeze-thawing, the recombinant ectromelia virus ECTVC which efficiently expresses red fluorescent protein and Gaussian luciferase is obtained by 14 rounds of single spot purification on BHK cells and PCR amplification identification by the above F2 and F3 primers. The present application does not have special limitations on the specific process of transfection, and the steps of conventional cell transfection in the art can be used. As an embodiment, the system of PCR amplification in the present application is 25 μL, including: 2×Phanta Max MasterMix 12.5 μL, upstream primer and downstream primer each 1 μL, template cDNA 100 ng and the rest of ddH2O. The program of PCR amplification in the present application is: 95℃ pre-denaturation for 30s; 95℃ denaturation for 15s, 60℃ annealing for 15s, 72℃ extension for 60s, 35 cycles; 72℃ extension for 5min.

[0040] The recombinant ectromelia virus ECTVC constructed by the present application can not only successfully obtain efficient expression of red fluorescent protein and Gaussian luciferase, but also obtain the recombinant virus for at least 7 consecutive generations, which has good genetic stability.

[0041] The present application also provides two pairs of primer pairs for detecting the above-mentioned recombinant ectromelia virus, including primer pair F6 and primer pair F7, wherein the nucleotide sequence of the upstream primer of primer pair F6 is shown in SEQ ID No. 15, the nucleotide sequence of the downstream primer of primer pair F6 is shown in SEQ ID No. 16; the nucleotide sequence of the upstream primer of primer pair F7 is shown in SEQ ID No. 17, and the nucleotide sequence of the downstream primer of primer pair F7 is shown in SEQ ID No. 18.

[0042] The present application also provides a set of PCR detection system and program, each PCR reaction system is 25 μL, including: 12.5 μL 2×PhantaMax MasterMix, 1 μL upstream primer (10 μM), 1 μL downstream primer (10 μM), 5 μL template DNA and 5.5 μL ddH2O. The PCR program is set as: pre-denaturation at 95℃ for 3min; amplification cycle at 95℃ for 15s, 60℃ for 15s, 72℃ for 60s, 35 cycles; extension at 72℃ for 5min.

[0043] The application also provides the use of the recombinant ectromelia virus ECTVC genome or the recombinant ectromelia virus ECTVC genome constructed by the construction method or the homologous recombination vector or the recombinant ectromelia virus in antiviral drug screening and / or vaccine preparation.

[0044] The antiviral drug can be an anti-orthopoxvirus drug, such as an anti-ectromelia virus drug, especially an anti-ectromelia virus ECTV-GD01 drug. The vaccine can be an anti-orthopoxvirus vaccine, such as an anti-ectromelia virus vaccine, especially an anti-ectromelia virus ECTV-GD01 vaccine.

[0045] In order to further illustrate the application, the recombinant ectromelia virus ECTVC genome and the recombinant virus, the construction method and the application provided by the application are described in detail in combination with the examples below, but they cannot be understood as limiting the protection scope of the application.

[0046] Example 1

[0047] The recombinant ectromelia virus carrying mKate and Gluc is constructed and expressed and identified, and the steps are as follows:

[0048] 1.1 Construction of CRISPR-Cas9 recombinant plasmid

[0049] The sgRNA is designed by using the CRISPOR website online. The sgRNA is designed according to the highest Doench'16 score of the nucleotide sequence in Predicted Efficiency. The nucleotide sequence of the sgRNA and the nucleotide sequence of the complementary strand are shown in SEQ ID No. 1 and SEQ ID No. 2, respectively. The 5' end of SEQ ID No. 1 is added with AAAC, and the 3' end is added with C to become SEQ ID No. 3. The 5' end of SEQ ID No. 2 is added with CACCG to become SEQ ID No. 4, which is synthesized by a company, and the sgRNA is resuspended with distilled water to 100 μM.

[0050] The two sgRNA single strands are phosphorylated and annealed; the annealing step is 37℃ for 30 min, 95℃ for 5 min, and then reduced to 25℃ at a speed of 5℃ / min.

[0051] The reagent ratio for sgRNA phosphorylation and annealing: oligo 1 (100 μM) 1 μL, oligo 2 (100 μM) 1 μL, 10×T4 Ligation Buffer (NEB) 1 μL, ddH2O 6.5 μL and T4 PNK (NEB) 0.5 μL.

[0052] PX458 plasmid contains Cas9 protein gene and gRNA gene sequence broadcast position, when the plasmid transfection cells, Cas9 protein can be under the guidance of gRNA cutting specific position. In order to make the poxvirus infection better in the cytoplasm, the team has removed the nuclear localization signal at both ends of the Cas9 protein gene in the previous study, the rest of the elements remain unchanged, the modified plasmid is named PX458del. Linearization of PX458del vector with BbsI, digestion with BbsI restriction endonuclease at 37℃ for 2h.

[0053] After the phosphorylation and annealing of sgRNA, the linearized vector PX458del is connected, and the connection product is transformed into E. coli DH5α competent cells, uniformly coated on LB plate containing ampicillin, and single colonies are picked and cultured in 5mL LB liquid containing ampicillin for 12-16h to obtain plasmid, and forward primer F1 (SEQ ID No. 5) is used for sequencing, and the correct colony is expanded to obtain CRISPR-Cas9 recombination vector.

[0054] 1.2 Construction of homologous recombination plasmid

[0055] The upstream homology arm sequence (SEQ ID No. 6), Sac I restriction endonuclease sequence, Pst I restriction endonuclease sequence, p11 promoter sequence (SEQ ID No. 7), p7.5 promoter sequence (SEQ ID No. 8), Bgl II restriction endonuclease sequence, Smal restriction endonuclease sequence, downstream homology arm sequence (SEQ ID No. 9) are connected in series for gene synthesis; the above synthesized gene fragment is cloned into the basic vector PUC57 to become the PUC57-C vector (the gene synthesis and fragment connection are completed by Nanjing Kingsriver Biotechnology Co., Ltd.); the PUC57-C vector is linearized using Bgl II and Smal restriction endonucleases; T4 DNA ligase is used to connect the above linearized PUC57-C and Gluc fragment with the same enzyme cutting end to become the PUC57-C-Gluc vector; then the PUC57-C-Gluc vector is linearized using the Sac I restriction endonuclease enzyme cutting site and the Pst I restriction endonuclease enzyme cutting site; the mKate gene fragment is cloned using the primer pair F2 (SEQ ID No. 10 and SEQ ID No. 11); the mKate gene fragment and the PUC57-C-Gluc vector are connected using seamless cloning to become the final recombinant vector PUC57-C-Gluc-mKate; the recombinant vector is transformed into the E. coli DH5α competent cells, uniformly coated on the LB plate containing ampicillin, and single colonies are picked and cultured in 5 mL LB liquid containing ampicillin for 12-16 h to extract plasmids, and the forward primers F3 (SEQ ID No. 12), F4 (SEQ ID No. 13) and F5 (SEQ ID No. 14) are used for sequencing, and the colonies with correct sequencing are expanded and cultured.

[0056] 1.3 Obtaining and identifying the recombinant virus

[0057] The Vero cells are prepared one night in advance at 2x10 5Vero cells were inoculated in 12-well plates and placed in a 5% CO2, 37°C incubator for 16-20 h. When the Vero cells in the 12-well plates reached 90%, 1 μg of PX458del-gRNA plasmid (plasmid: HP transfection reagent 1:2) was transfected per well, and the plates were placed in a 5% CO2, 37°C incubator for 24 h; the liquid was discarded, and each well was washed once with 1 mL of 2% FBS DMEM; 500 μL of ECTV-GD01 virus dilution solution with MOI = 0.05 was added to each well, and the plates were placed in a 5% CO2, 37°C incubator for 2 h; the liquid was discarded, and each well was washed twice with 1 mL of 2% FBS DMEM; 1 μg of PUC57-C-Gluc-mKate plasmid (plasmid: HP transfection reagent 1:2) was transfected per well, and 1 mL of 2% FBS DMEM was added to each well, and the cell culture plates were placed in a 5% CO2, 37°C incubator for 48 h, and then frozen at -80°C. After repeated freezing and thawing three times, BHK-21 cells were inoculated and plaque picking was performed.

[0058] After 48 h of inoculation of BHK cells, low-melting-point agarose was used for fixation, and the plates were observed under a fluorescence microscope. Single, relatively large, bright red fluorescent plaques were picked and frozen at -80°C. After repeated freezing and thawing three times, the next generation was inoculated. According to this cycle, after 14 times of single-plaque purification, one generation was expanded on Vero cells for PCR identification and immunofluorescence identification. The recombinant mousepox virus identified successfully was named ECTVC. The nucleotide sequence of the recombinant mousepox virus after replacement between ECTV-GD01 C12R-C13R 766-1065 is shown in SEQ ID No. 19.

[0059] The primer sequences are as follows:

[0060] F6-F (SEQ ID No. 15): 5'-CGTATCAGATGTAGATGACGCGG-3';

[0061] F6-R (SEQ ID No. 16): 5'-CCGGCGTTCATCAATAGTTGTAT-3';

[0062] F7-F (SEQ ID No. 17): 5'-GTAGTATATGGAAGAGAGTCTCACG-3';

[0063] F7-R (SEQ ID No. 18): 5'-CGCCAATATATGTAGCGGTGT-3'.

[0064] The recombinant mousepox virus emits bright red fluorescence after 48 h of infection of Vero cells, while the parent virus and the cell control do not emit fluorescence Figure 1 The schematic diagram of the design of primer pairs F6 and F7 is shown inFigure 1 The recombinant virus had no band, and the size of the band of the parent virus was 287 bp; the size of the band of the recombinant virus was 1903 bp, and the size of the band of the parent virus was 574 bp when primer pair F7 was used for PCR detection, indicating that the insertion position and size of the target band of the recombinant virus were correct. Figure 1 Middle C);

[0065] Vero cells were inoculated into polylysine-treated eight-chambered cells one day in advance, and when the cell density reached 90%, the cells were inoculated with the virus at an MOI of 0.05, and cultured at 37°C, 5% CO2 for 72 h; the culture medium was discarded, and the cells were fixed with 4% polyformaldehyde for 30 min, and washed with PBS for 3 times; 0.5% Triton X-100 was used for incubation at room temperature for 20 min, and washed with PBS for 3 times; 10% goat serum was used for blocking at 37°C for 2 h; rabbit-derived Gluc polyclonal antibody (Nanolight, 401P) was diluted with 10% goat serum at 1:500, mouse-derived A27L monoclonal antibody (Santa Cruz, 8115) was diluted with 10% goat serum at 1:10, and incubated at 37°C for 1 h, and washed with PBS for 3 times; FITC-labeled goat anti-rabbit IgG antibody (Invitrogen, F-2765) was diluted with 10% goat serum at 1:1000, Alexa Fluor 647-labeled donkey anti-mouse IgG antibody (Invitrogen, A-31571) was diluted with 10% goat serum at 1:1000, and DAPI diluted with 10% goat serum at 1:1000 was incubated at 37°C for 2 h, and washed with PBS for 3 times; mounting medium was added, and a Leica laser scanning confocal microscope was used for observation, and photographs were taken under the microscope for preservation. The recombinant vaccinia virus can express Gluc protein in the cytoplasm, and the parent virus and blank cells do not express it. TM Figure 1 Middle D); recombinant vaccinia virus-infected cells can express obvious mKate red fluorescent protein, and the parent virus and blank cells do not express it (Middle D); A27L as a viral membrane protein control is expressed on the membranes of recombinant virus-infected cells and parent virus-infected cells, and blank cells do not express it (Middle D); DAPI stains all cell nuclei blue; the above experiments show that the recombinant vaccinia virus is successfully constructed, and the target protein is correctly expressed. Figure 1 Figure 1

[0066] Example 2

[0067] Identification of genetic stability of recombinant vaccinia virus ECTVC

[0068] ​​​The virus amplified from a single plaque to a T25 flask was considered as passage 1 (P1), and the P1 virus was serially passaged on Vero cells for 7 passages. The genetic stability of the recombinant virus was evaluated by the expression of mKate fluorescent protein, Gluc luciferase quantification, PCR detection of the size and location of the target band, and Western blot analysis of Gluc protein.

[0069] Vero cells were plated in 6-well plates one day in advance, and the next day when the cell density reached 90%, P1-P7 viruses and the parent virus were inoculated at 0.1 MOI. After 48 hours of infection, the expression of mKate fluorescent protein was observed under a fluorescence microscope, and the results showed that P1-P7 recombinant viruses could stably express mKate protein, and obvious red fluorescence could be seen, while the parent virus and uninfected virus cells did not fluoresce. Figure 2 A); discard the culture medium, wash once with 1 mL PBS per well, scrape the cells with a spatula, centrifuge at 1200 rpm for 10 min, discard the PBS, and add 200 μL of cell lysis solution containing PMSF to each sample, incubate on ice for 30 min. Add 6x protein loading, boil for 10 min, and load for SDS-PAGE gel electrophoresis, and electrotransfer to nitrocellulose membrane (NC membrane). Then, use 5% skim milk-TBS buffer to block the NC membrane at room temperature for 2 h. Incubate the membrane with 1:500 diluted Gluc polyclonal antibody (NanoLight, 401P) and 1:500 diluted F13L primary antibody at 4°C overnight. After incubation, wash the membrane with TBST (Tris salt buffer containing Tween-20) 3 times for 5 min each time. Then incubate with 1:1000 diluted secondary antibody (Goat anti-Rabbit IgG (H+L) Secondary Antibody HRP, purchased from Zhongsu Jinqiao, ZB-2301) at 37°C for 2 h in the dark. After incubation, wash the membrane with TBST again 3 times for 5 min each time. Scan and image the membrane using a multifunctional imaging system (Wuhan Saiver Biological Technology Co., Ltd., product number SCG-W5000) to detect the expression levels of the exogenous target protein Gluc and the viral protein F13L. In the cells infected with each generation of recombinant virus, specific bands were detected at 19 kDa (Gluc) and 41 kDa (F13L) ( Figure 2 D), while the parent virus could only detect a band at 41 kDa, indicating that each generation of recombinant virus could effectively express Gluc protein.

[0070] Vero cells were plated in 96-well plates one day in advance, and the next day when the cell density reached 90%, P1-P7 generations of viruses were inoculated at 0.1 MOI, 6 replicates for each generation of virus. After 48 h of infection, 20 μL of cell supernatant in each well was taken into a black flat-bottom reaction plate (CORNING, 3925), 70 μL of renilla luciferase assay buffer (Promega, E2810) was added to each well, and then the chemical luminescence detection instrument (Promega Glomax, USA) was used for reading. The Gluc luminescence signal value of P1-P7 generations of recombinant viruses was 10 6.31 ~10 7.11 , and the Gluc luminescence signal value of the maternal virus-infected cells and the blank cells was the background value 10 1.5 , which showed that the recombinant virus could stably express Gluc Figure 2 in the B) part.

[0071] The cells in the 96-well plates were taken for nucleic acid extraction, and PCR detection was performed using the F6 and F7 primer pairs described in Example 1. It was found that no band was detected for the 7 generations of recombinant viruses using the F6 primer pair, while the maternal virus had a band size of 287 bp, indicating that the recombinant virus had been purified. It was found that the band size of the 7 generations of recombinant viruses was 1903 bp using the F7 primer pair, while the band size of the maternal virus was 574 bp, indicating that the recombinant virus did not lose fragments during the passage process, and the insertion position and size were correct.

[0072] Example 3

[0073] Comparison of cell line replication kinetics of recombinant vaccinia virus ECTVC and maternal virus ECTV-GD01

[0074] CEF, BHK, Vero and HeLa cells were inoculated in 12-well plates and 96-well plates one day in advance. The inoculation density of CEF in the 12-well plates was 8 x 10 6 / well, the inoculation density of BHK cells was 1 x 10 6 / well, the inoculation density of Vero cells was 2 x 10 5 / well, and the inoculation density of HeLa cells was 5 x 10 5 / well. The inoculation density of cells in the 96-well plates was 1.5 x 10 4 / well plates were cultured in 37 °C 5% CO2 incubator for 12-16 h. Cells in 12-well plates were used for cytopathic effect and fluorescence photography, and cells in 96-well plates were used for subsequent virus collection, Gluc replication kinetics and virus growth curve determination; when the cell growth density reached 90%, the ECTVC and ECTV-C-Tan-GD01 were respectively infected into the above four cell lines according to the infection dose MOI = 0.05; from 2 h after virus infection, the cells and supernatant in the 96-well plates were collected every 12 h. A total of 120 h were collected, and the cytopathic effect and fluorescence production in the 12-well plates were photographed and recorded at the corresponding time points; the harvested cell suspension was frozen at -80 °C, and the virus nucleic acid in 100 μL of the suspension was extracted using a virus DNA extraction kit (Vazyme, China) according to the instructions, and the quantitative PCR was performed on a LightCycler 96 fluorescence quantitative PCR instrument (Roche Diagnostics Ltd., Switzerland) using Probe qPCR Mix (QN213, Vazyme, China) according to the established procedure to draw the replication kinetics curve. 20 μL of the suspension was taken to determine the Gluc expression and draw the Gluc replication kinetics curve.

[0075] For CEF cells: as shown in FIG. 1A, the recombinant virus ECTVC-infected cells began to appear sporadic fluorescence from 36 h, and the red fluorescence intensity gradually increased with time, reaching a maximum at 84 h. The cytopathic cells began to fall off after 72-84 h, and completely fell off at 120 h; therefore, the fluorescence intensity decreased after 96 h. Figure 3 As shown in FIG. 1B, luciferase activity detection found that the Gluc value of ECTVC reached a peak at 84 h, and then decreased due to cell shedding. The luciferase reading of the parent virus was very low and unchanged. Figure 3 As shown in FIG. 1C, virus copy number detection found that the two viruses had similar replication kinetics curves on CEF cells, rapidly amplified at 12-36 h, reached a replication plateau at 84 h, and then remained unchanged. Figure 3

[0076] For BHK cells: as shown in FIG. 2A, the recombinant virus ECTVC-infected cells began to appear sporadic fluorescence from 36 h, and the fluorescence intensity gradually increased with time, reaching a maximum at 84 h. The cytopathic cells began to fall off after 84 h, and completely fell off at 120 h; therefore, the fluorescence intensity decreased after 96 h. Figure 3 As shown in FIG. 2B, luciferase activity detection found that the Gluc value of ECTVC reached a peak at 96-120 h, and the luciferase reading of the parent virus was very low and unchanged. Figure 3 Figure 3 ​​As shown in Fig. 2A, virus copy number detection found that the two viruses had similar replication kinetic curves on BHK cells, rapidly expanded from 12 to 60 h, reached a replication plateau at 96 h, and then remained unchanged.

[0077] For Vero cells: as Figure 3 As shown in Fig. 2A, the recombinant virus ECTVC-infected cells began to appear sporadic fluorescence from 24 h, and the fluorescence intensity gradually increased with time, reaching a maximum at 60-72 h. The diseased cells began to fall off after 60 h, and large pieces fell off at 72-84 h, and completely fell off at 96 h; therefore, the fluorescence intensity decreased after 60-72 h. Figure 3 As shown in Fig. 2B, luciferase activity detection found that the Gluc value of ECTVC reached a peak at 48-60 h, and then decreased due to cell shedding. The maternal virus had very low luciferase reading, and did not change with time. Figure 3 As shown in Fig. 2C, virus copy number detection found that the two viruses had similar replication kinetic curves on Vero cells, rapidly expanded from 12 to 36 h, reached a replication plateau at 60 h, and then remained unchanged.

[0078] For HeLa cells: as Figure 3 As shown in Fig. 2A, the recombinant virus ECTVC-infected cells began to appear sporadic fluorescence from 24 h, and the fluorescence intensity gradually increased with time, reaching a maximum at 60-72 h. The diseased cells began to fall off after 60 h, and large pieces fell off at 72-84 h, and completely fell off at 96 h; therefore, the fluorescence intensity decreased after 60-72 h. Figure 3 As shown in Fig. 2B, luciferase activity detection found that the Gluc value of ECTVC reached a peak at 60 h, and then decreased due to cell shedding. The maternal virus had very low luciferase reading, and did not change with time. Figure 4 As shown in Fig. 2C, virus copy number detection found that the two viruses had similar replication kinetic curves on Vero cells, rapidly expanded from 12 to 36 h, reached a replication plateau at 60 h, and then remained unchanged.

[0079] The above experiments show that the recombinant virus ECTVC maintains the same in vitro replication kinetics as the maternal virus ECTV-GD01, and the time to reach the plateau of viral load growth is related to the type of cell line.

[0080] Example 4

[0081] Comparison of plaque and immune plaque size and morphology of recombinant vaccinia virus ECTVC and maternal virus ECTV-GD01

[0082] To further clarify the in vitro biological characteristics of recombinant ectromelia virus ECTVC, the present application compared the plaque morphology and size of recombinant ectromelia virus ECTVC and the parent virus ECTV-GD01 on Vero cells. Vero cells were seeded in 12-well plates, and when the cell confluence was 90%, ECTVC and ECTV-GD01 were inoculated at MOI = 0.0001 and MOI = 0.0005. The cell plate was placed in a 37°C incubator, adsorbed for 2 h, and then the medium was discarded and replaced with 2% FBS, 2.4% Avicel, 2x DMEM, and incubated in a 37°C incubator for 120 h. Then the medium was aspirated, 1 mL of 0.1% crystal violet-4% paraformaldehyde fixing solution was added to each well, and fixed and stained for 30 min. The staining-fixing solution was aspirated and washed with running water until clean, then air-dried and photographed for comparison. Observation found that both viruses formed a central empty plaque at 120 h. As shown in Figure 4 ECTVC and the parent virus had no significant difference in plaque size and shape.

[0083] To further investigate the spreading ability of recombinant ectromelia virus ECTVC and the parent virus ECTV-GD01 on different cell lines, ECTVC and ECTV-GD01 were inoculated at MOI = 0.0001 on CEF, BHK, Vero and HeLa cells, respectively, and at 24 h and 48 h after infection, the cells in the 6-well plate were fixed with 4% paraformaldehyde for 20-30 min, and then washed with PBS twice. After the cells were treated with 3% hydrogen peroxide in the dark for 10 min, they were blocked with PBS containing 3% bovine serum albumin (BSA) for 30 min. After incubation at room temperature for 1 h with 1:2000 diluted rabbit anti-poxvirus polyclonal antibody (Bio-Rad, 9503-2057), the cells were washed with PBS three times, and then 1:2000 diluted horseradish peroxidase-labeled goat anti-rabbit IgG H+L (Zhongshanjingqiao, ZB-2301) was added for incubation for 30 min. After washing with PBS three times, 800 μL of TRUEBLUE ready-to-use color developing solution was added to each well, and color development was carried out at room temperature in the dark for 5-8 min. After the reaction was terminated with distilled water, the blue-purple spots in the wells were counted. As shown in Figure 5 As shown in Fig. B, the immune plaques of both viruses at 48 h were larger than the immune plaques they formed at 24 h. The immune plaques formed by ECTVC on the four cell lines were all smaller than the plaques formed by the parent virus ECTV-GD01 on the four cell lines, which reflected that ECTVC weakened the spreading ability among cells.

[0084] Example 5

[0085] Application analysis of recombinant ectromelia virus ECTVC in ST-246 and CDV pharmacodynamic evaluation

[0086] To evaluate whether recombinant mousepox virus ECTVC can be used for pharmacodynamic evaluation of antiviral drugs, this invention selected two positive control agents, ST-246 (tecovir, purchased from Shanghai Taoshu Biotechnology Co., Ltd.) and CDV (cidofovir, purchased from Beijing Wokai Biotechnology Co., Ltd.), for the experiment. Vero cells were cultured at 1.5 × 10⁶ cells per well. 4 Cells were seeded at a density of [number] cells per well in 96-well plates. When the cells reached approximately 90% saturation, serially diluted drugs were added. ST-246 was serially diluted 5-fold (6 concentrations) starting at 2 μM, and CDV was serially diluted 2-fold (6 concentrations) starting at 100 μM. 100 μL was added to each well, with three replicates for each concentration. Cell control and virus control wells were also prepared, with cell maintenance medium added to each well. The plates were incubated at 37°C with 5% CO2 for 2 hours. Then, 100 μL of ECTVC containing 100 PFU was added to each well, and 100 μL of cell maintenance medium was added to each cell control and virus control well. The plates were incubated at 37°C for another 72 hours. The expression of red fluorescent protein was then observed under a fluorescence microscope. 20 μL of viral supernatant was added to a black plate, and 70 μL of renilla luciferase assay buffer was added to each well. Readings were performed using a GloMax chemiluminescence analyzer.

[0087] The inhibition rate of Gluc in each well = (average Gluc value in virus control wells - Gluc value in this well) / (average Gluc value in virus control wells - average Gluc value in cell control wells) * 100%.

[0088] Finally, Graphpad was used to calculate the 50% inhibitory concentration (EC50) of the drug against the virus. 50 The viral inhibition rate curve was plotted. Similarly, Vero cells were used at a density of 2 × 10⁶ cells per well. 5 Cells were seeded at a density of 100 μL in 12-well plates. When the cells reached approximately 90% cellularity, 500 μL of the serially diluted drug was added to each well. After 2 hours, 500 μL of 100 PFU of ECTVC was added to each well. Cells were cultured for another 72 hours, then fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and the cells were counted and EC50 was calculated. 50 .

[0089] like Figure 5 As shown in Figure A, as the concentration of ST-246 decreases, the viral fluorescence intensity in the cell wells gradually increases. Viral fluorescence is almost invisible at 1000 nM, 200 nM, and 40 nM, but begins to appear below 8 nM, with the intensity gradually increasing. The calculated EC... 50 The value is 12.93 nM ( Figure 5 (B). This is consistent with literature reports that ST-246 has an inhibitory effect at the nanomolar level. As the compound concentration decreased, the viral plaques gradually increased (Figure 5 EC calculated by the plaque experiment (C) 50 The value is 11.94 nM ( Figure 5 (D). The inhibition effects of Gluc inhibition and plaque reduction experiments were very similar.

[0090] Similarly, such as Figure 5 As shown in Figure E, the intensity of viral fluorescent protein in cell pores gradually increased with decreasing CDV drug concentration. Viral fluorescence was almost invisible at 100 μM, 75 μM, and 50 μM, but began to appear below 25 μM, with the intensity gradually increasing. This invention detected Gluc expression at 72 h and calculated EC... 50 The value is 16.2 μM ( Figure 5 (F). Compared to the classic plaque reduction experiment, the number of viral plaques in the wells gradually increased as the drug concentration gradually decreased. Figure 6 EC obtained from the plaque experiment (G) 50 The value is 10.92 μM ( Figure 6 (H). The above experiments demonstrate that detecting the Gluc inhibition rate after ECTVC infection of cells can provide a high-throughput and rapid evaluation of the drug's anti-opioid effect.

[0091] Example 6

[0092] Evaluation of the in vivo biological characteristics of recombinant mousepox virus ECTVC

[0093] To evaluate the in vivo virulence of recombinant mucinous mousepox virus ECTVC in mice, 4-week-old female BALB / c mice purchased from Beijing Vital River Biotechnology Co., Ltd. were randomly assigned to groups and allowed to acclimatize to the environment for one day. One day later, mice were anesthetized by intraperitoneal injection and then injected intranasally with 40 μL of a viral dilution of ECTVC. The control group received 40 μL of PBS. Viral doses were 1000 PFU / mouse, 100 PFU / mouse, 50 PFU / mouse, 25 PFU / mouse, 10 PFU / mouse, and 1 PFU / mouse. Mice were weighed and their weight recorded for 12 consecutive days. The results showed that recombinant mucinous mousepox virus ECTVC caused death in Balb / c mice; compared with the parent virus ECTV-GD01, ECTVC significantly reduced the median lethal dose (LD50). 50 ), but still less than 25 PFU ( Figure 7 (A and B in the middle).

[0094] In order to evaluate whether the recombinant mousepox virus reporter gene Gluc can be used for in vivo experiments, the present application uses live imaging technology to observe the imaging of Gluc in mice. 4-week-old female Balb / c mice were randomly divided and then respectively injected with 10000 PFU, 100 PFU, 50 PFU and 25 PFU of virus per mouse by nasal drops, 3 mice in each group, and the bioluminescence in the mice was observed every day.

[0095] The live imaging operation steps are as follows: turn on the live imaging instrument (Guangzhou Bolvteng Biotechnology Co., Ltd.), and turn on the anesthesia system; inject 130 μg of substrate coelenterazine solution into each mouse for imaging, and place the mouse in the anesthesia box after 6 minutes of injection, and then place the mouse in the instrument dark chamber after the mouse is completely anesthetized, so that the abdomen of the mouse faces upwards, close the chamber door and start imaging. The bioluminescence signal collection time is in auto mode; save the photo data; and finally take out the mouse and place it back in the mouse cage for normal feeding. It is found that the bioluminescence in the mouse gradually increases with the increase of the infection days. Although it is nasal infection, no obvious bioluminescence is observed in the lungs and respiratory tract, and the recombinant mousepox virus ECTVC is mainly distributed in the abdominal cavity of the mouse, which is consistent with the characteristics of mousepox virus targeting the liver and spleen of the mouse Figure 7

[0096] Example 7

[0097] In vivo efficacy evaluation of recombinant mousepox virus ECTVC

[0098] In order to evaluate whether ECTVC can be used for in vivo efficacy evaluation of antiviral drugs, the present application uses the method of infection on the 0th day, and each mouse is injected with 50 PFU of ECTVC by nasal drops, and each mouse is given CDV 10 mg / kg per day from the 1st to the 6th day, and the control group is only infected without drug administration. The bioluminescence in the mouse is observed every day. On the 8th day, the mouse is dissected, and the blood, liver, spleen, lung and kidney of the mouse are taken. There is no significant difference in the body weight of the two groups of mice Figure 7 Figure 7 Figure 7 ​ ​

[0099] Although the above examples make a detailed description of the present application, it is only a part of the examples of the present application, but not all the examples, and other examples can be obtained according to the present examples without creativity, which all belong to the protection scope of the present application.​​​​​​

Claims

1. A recombinant vaccinia virus ECTVC genome, characterized in that, The replacement comprises replacing the nucleotide sequence at positions 733-1065 of the non-coding region between C12R and C13R of the wild-type vaccinia virus genome.

2. The recombinant vaccinia virus ECTVC genome of claim 1, wherein, The replacement comprises replacing the nucleotide sequence at positions 733-1065 of the non-coding region between C12R and C13R of the vaccinia virus genome.

3. The recombinant vaccinia virus ECTVC genome according to claim 1 or 2, wherein, The red fluorescent protein gene mKate is expressed by the promoter p11; and the Gaussia luciferase gene Gluc is expressed by the promoter p7.

5.

4. The recombinant vaccinia virus ECTVC genome of claim 3, wherein, The nucleotide sequence at positions 733-1065 between C12R and C13R after the replacement is shown in SEQ ID No.

19.

5. A method of constructing the ECTV genome of recombinant vaccinia virus according to any one of claims 1 to 4, characterized in that, The method comprises the following steps: The recombinant vaccinia virus ECTVC genome is constructed by knocking out the non-coding region between C12R and C13R of the wild-type vaccinia virus genome using a gene editing method, and then inserting a tandem gene using a homologous recombination method. The tandem gene comprises a red fluorescent protein gene mKate, a promoter p11, a Gaussia luciferase gene Gluc, and a promoter p7.

5.

6. The method of construction according to claim 5, wherein, When knocked out using a CRISPR-Cas9 method, the designed sgRNA sequence is shown in SEQ ID No. 1-SEQ ID No.

4.

7. A homologous recombination vector, characterized in that, The upstream homologous arm comprises a red fluorescent protein gene mKate, a promoter p11, a promoter p7.5, and a downstream homologous arm comprising a Gaussia luciferase gene Gluc. The nucleotide sequence of the upstream homologous arm is shown in SEQ ID No. 6, the nucleotide sequence of the promoter p11 is shown in SEQ ID No. 7, the nucleotide sequence of the promoter p7.5 is shown in SEQ ID No. 8, and the nucleotide sequence of the downstream homologous arm is shown in SEQ ID No.

9.

8. A recombinant vaccinia virus comprising the recombinant vaccinia virus ECTVC genome according to any one of claims 1-4.

9. Two pairs of primer pairs for detecting the recombinant mousepox virus according to claim 8, characterized in that, The primer pair F6 comprises an upstream primer with a nucleotide sequence shown in SEQ ID No. 15 and a downstream primer with a nucleotide sequence shown in SEQ ID No. 16; and the primer pair F7 comprises an upstream primer with a nucleotide sequence shown in SEQ ID No. 17 and a downstream primer with a nucleotide sequence shown in SEQ ID No.

18.

10. Use of the recombinant vaccinia virus ECTVC genome according to any one of claims 1-4, or the recombinant vaccinia virus ECTVC genome constructed using the construction method according to claim 5 or 6, or the homologous recombination vector according to claim 7, or the recombinant vaccinia virus according to claim 8, in antiviral drug screening and / or vaccine preparation.

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