In-vivo and in-vitro double-visual recombinant mouse poxvirus genome, recombinant mouse poxvirus strain and application of recombinant mouse poxvirus genome and recombinant mouse poxvirus strain

Abstract

The invention provides an in-vivo and in-vitro double visual recombinant mouse poxvirus genome, a recombinant mouse poxvirus strain and application, and belongs to the technical field of gene engineering. The invention provides a recombinant mouse poxvirus genome. The recombinant mouse poxvirus genome comprises a mouse poxvirus genome, a red fluorescent protein gene mKate and a Gaussian luciferase protein gene Gluc, wherein the red fluorescent protein gene mKate and the Gaussian luciferase protein gene Gluc are inserted between the mouse poxvirus genome A11R and the mouse poxvirus genome A12L. According to the invention, the area A of the mouse poxvirus is modified, and the double-reporter gene is inserted between A11R and A12L for the first time, so that the mouse poxvirus-resistant high-throughput drug screening method can be used for anti-orthopoxvirus high-throughput drug screening, the in-vivo effect of the drug can be more intuitively displayed, and the operation is simple and convenient. The recombinant virus obtained by the invention has good hereditary stability, has similar biological characteristics and virulence to a wild type virus, can visualize infection and distribution of the virus in a mouse body, and can be used for in-vitro high-throughput screening of drugs and development of corresponding vaccines.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to a recombinant vaccinia virus genome and a recombinant vaccinia virus strain with in-vivo and in-vitro dual visualization and application. BACKGROUND

[0002] The vaccinia virus belongs to the family of poxviridae and the genus of orthopoxvirus. The vaccinia virus has high genetic similarity with the smallpox virus and the monkeypox virus, and the disease caused by the vaccinia virus has many commonalities with the human orthopoxvirus infection. In addition, the mouse is convenient as an experimental animal, so the mouse becomes an ideal model for studying the pathogenic mechanism of the orthopoxvirus and developing treatment / prevention measures.

[0003] The orthopoxvirus A11R encodes a non-structural protein, which is necessary for the formation of the virus membrane; the A12L encodes a core protein, which is crucial for the replication of the virus. The orthopoxvirus A11R and A12L are highly conserved. The insertion of the exogenous gene in the region between the two essential genes promotes the stability of the exogenous gene transfer with the parent virus.

[0004] The biofluorescent tracing technology is a brand-new analysis and detection technology for molecular and gene expression developed in recent years. In the field of molecular biology research, the technology combined with the fluorescent tracing can be used to monitor and detect the labeled molecules at the cell and animal levels. Gluc is derived from the marine copepod Gossiplea perpusilla, and is the smallest naturally secreted luciferase known in vivo. Gluc has extremely high sensitivity and can detect extremely low concentrations of substrates, with a sensitivity of 2000 times. In the detection process, the high luminescence efficiency and secretion characteristics of Gluc make the detection signal have high signal-to-noise ratio, which helps to reduce the interference of background noise and improve the accuracy of data; Gluc shows good stability under different temperature and pH conditions and is not easy to degrade, thereby ensuring the consistency and reliability of the experimental results; Gluc can be continuously secreted into the culture medium, so that researchers can perform real-time detection during the experiment and track the dynamic changes of cell activity. This is particularly useful for studying time-dependent effects and drug responses; Gluc also has a wide range of applications. In proteomics research, Gluc helps to improve the identification efficiency of phosphorylated peptides and can be used to identify proteins lacking basic amino acids. Combined with other proteases, it can effectively improve the sequence coverage of protein identification and improve the reliability of detection. In summary, Gluc has shown significant advantages in multiple fields with its high sensitivity, high signal-to-noise ratio, stability, real-time detection capability and wide range of applications.

[0005] Meanwhile, because the larger the gene inserted into the genome, the greater the impact on the genome, even if it can be successfully expressed, it is difficult to be stably passaged, and based on the requirement of biological fluorescence tracing, double fluorescence is a necessary choice, but at present, there is a lack of double fluorescence marker recombinant vaccinia virus technology which can be stably passaged. SUMMARY

[0006] The application provides a recombinant vaccinia virus genome and a recombinant vaccinia virus strain which can be double visualized in vivo and in vitro, wherein a double reporter gene is inserted into a region A of a vaccinia virus genome, and the double reporter gene can maintain similar replication dynamic characteristics and plaque size as the wild type virus, can maintain similar virulence as the wild type virus in vivo, and can be stably passaged.

[0007] The application provides a recombinant vaccinia virus genome, comprising a vaccinia virus genome and a red fluorescent protein gene mKate and a Gaussian luciferase protein gene Gluc inserted between A11R and A12L of the vaccinia virus genome.

[0008] In a preferred mode of the application, the red fluorescent protein gene mKate is expressed under the control of a p11 promoter, and the nucleotide sequence of the p11 promoter is shown in SEQ ID No. 2.

[0009] In a preferred mode of the application, the Gaussian luciferase protein gene Gluc is expressed under the control of a p7.5 promoter, and the nucleotide sequence of the p7.5 promoter is shown in SEQ ID No. 3.

[0010] In a preferred mode of the application, the nucleotide sequence between A11R and A12L of the recombinant vaccinia virus genome is shown in SEQ ID No. 10.

[0011] The application further provides a homologous recombination vector for cloning the above-mentioned recombinant vaccinia virus genome, wherein the homologous recombination vector comprises an upstream homologous arm with a nucleotide sequence shown in SEQ ID No. 1 and a downstream homologous arm shown in SEQ ID No. 4.

[0012] The application further provides a recombinant vector for expressing the above-mentioned recombinant vaccinia virus genome.

[0013] The application further provides a recombinant vaccinia virus strain comprising the above-mentioned recombinant vaccinia virus genome.

[0014] The application further provides a construction method of the above-mentioned recombinant vaccinia virus strain, comprising transforming a wild strain of vaccinia virus with the above-mentioned homologous recombination vector or the above-mentioned recombinant vector to obtain the recombinant vaccinia virus strain.

[0015] The application also provides a primer pair for detecting the recombinant vaccinia virus strain, which comprises an upstream primer with a nucleotide sequence as shown in SEQ ID No. 8 and a downstream primer with a nucleotide sequence as shown in SEQ ID No. 9.

[0016] The application also provides application of the recombinant vaccinia virus genome or the homologous recombinant vector or the recombinant vector or the recombinant vaccinia virus strain in screening of anti-vaccinia virus drugs and / or preparation of vaccines.

[0017] Beneficial effects: the application provides a recombinant vaccinia virus genome, which comprises a vaccinia virus genome and a red fluorescent protein gene mKate and a Gaussian luciferase protein gene Gluc inserted between A11R and A12L of the vaccinia virus genome. The application is modified in the A region of the vaccinia virus, and the double reporter genes are inserted between A11R and A12L for the first time, which can be used for in vitro rapid and efficient anti-vaccinia virus high-throughput drug screening, can more intuitively display the in vivo effect of the drug, and is simple to operate and easy to popularize.

[0018] The application inserts the double reporter genes between A11R and A12L of the vaccinia virus genome by the method of homologous recombination, and the obtained virus has good genetic stability, which can be maintained for at least 7 generations, and the recombinant vaccinia virus has similar replication kinetics characteristics and plaque size to the wild-type virus in vitro, and also has similar virulence to the wild-type virus in vivo. The recombinant vaccinia virus can visualize the infection and distribution of the virus in mice, and can be used for in vitro high-throughput drug screening and development of corresponding vaccines. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 FIG. 1 is a diagram of construction and verification results of ECTVA carrying mKate and Gluc double reporter genes in Example 1, wherein A is the cell pathogenesis and fluorescent protein expression of Vero cells infected by ECTVA and the parent virus ECTV-GD01; B is a schematic diagram of F4 primer design for identification of ECTVA; C is a nucleic acid gel electrophoresis diagram for PCR identification of ECTVA; D is a report gene expression of ECTVA detected by indirect immunofluorescence method (Bar = 100 μm);

[0020] Figure 2Figure for identification results of genetic stability of ECTVA in Example 2, wherein A is the mKate fluorescent protein expression of ECTVA P1-P7 passage strains after infecting Vero cells (Bar = 500 μm); B is the Gluc protein expression of ECTVA P1-P7 passage strains after infecting Vero cells; C is the PCR identification nucleic acid gel electrophoresis map of ECTVA P1-P7 passage strains; D is the luciferase expression of ECTVA after P1-P7 passage virus infecting Vero cells; E is the protein expression of ECTVA after P1-P7 passage virus infecting Vero cells detected by Western blot;

[0021] Figure 3 Figure for research results of comparing in vitro replication kinetics of recombinant virus ECTVA and ECTV-GD01 in four cell lines in Example 3, wherein A is the fluorescent photos of ECTVA and ECTV-GD01 infecting 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 the Gluc replication kinetics curves of ECTVA and ECTV-GD01 infecting the above four cell lines; C is the copy number replication kinetics curves of ECTVA and ECTV-GD01 infecting the above four cell lines;

[0022] Figure 4 Figure for comparing plaque morphology and size of recombinant virus ECTVA and ECTV-GD01 in Example 4, wherein A is the comparison of plaque size and morphology of ECTVA and ECTV-GD01 formed on Vero cells; B is the comparison of immune plaque size and morphology of ECTVA and ECTV-GD01 formed on CEF cells (Bar = 200 μm); C is the comparison of immune plaque size and morphology of ECTVA and ECTV-GD01 formed on BHK cells (Bar = 200 μm); D is the comparison of immune plaque size and morphology of ECTVA and ECTV-GD01 formed on Vero cells (Bar = 200 μm); E is the comparison of immune plaque size and morphology of ECTVA and ECTV-GD01 formed on HeLa cells (Bar = 200 μm);

[0023] Figure 5Figure for the application results of recombinant virus ECTVA in the anti-positive orthopoxvirus drug ST-246 and CDV pharmacodynamic evaluation in Example 5, wherein A is the fluorescence expression diagram of mKate of ECTVA 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 ECTVA quantitatively determined according to luciferase experiment; C is the plaque picture of ECTVA formed 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 mKate of ECTVA under the action of different concentrations of CDV (Bar = 200 μm); F is the inhibitor dose response curve of CDV to recombinant virus ECTVA quantitatively determined according to luciferase experiment; G is the plaque picture of ECTVA formed 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;

[0024] Figure 6 Figure for the application results of ECTVA in vivo biological characteristics in Example 6, wherein A is the body weight change of four-week-old Balb / c mice after intranasal challenge with different doses of ECTVA; B is the survival curve of 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 ECTVA; D is the light intensity change of in vivo live imaging of mice in C. DETAILED DESCRIPTION

[0025] The application provides a recombinant ectromelia virus genome, comprising an ectromelia virus genome and a red fluorescent protein gene mKate and a Gaussian luciferase protein gene Gluc inserted between A11R and A12L of the ectromelia virus genome.

[0026] In an embodiment of the ectromelia virus ECTV genome, the genome of the ectromelia virus ECTV-China-C-Tan-GD01 (ECTV-GD01) strain is used as the basis, wherein the ECTV-GD01 strain has been disclosed in the 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.).

[0027] The application inserts a double fluorescent marker gene between A11R and A12L of the genome of the ECTV-GD01 strain, wherein the red fluorescent protein gene mKate is expressed by a p11 promoter, the nucleotide sequence of the p11 promoter is shown as SEQ ID No. 2; the Gaussian luciferase protein gene Gluc is expressed by a p7.5 promoter, the nucleotide sequence of the p7.5 promoter is shown as SEQ ID No. 3. Therefore, in the embodiment of the application, the recombinant genome comprises mKate-p11-p7.5-Gluc connected in sequence, and after the insertion, the nucleotide sequence between A11R and A12L of the recombinant ectromelia virus genome is shown as SEQ ID No. 10.

[0028] The application also provides a homologous recombination vector for cloning the above-mentioned recombinant ectromelia virus genome, wherein the homologous recombination vector comprises an upstream homologous arm with the nucleotide sequence shown as SEQ ID No. 1 and a downstream homologous arm shown as SEQ ID No. 4.

[0029] In addition to the upstream homologous arm and the downstream homologous arm, the homologous recombination vector of the application also comprises a promoter sequence, such as the p11 promoter sequence shown as SEQ ID No. 2 and the p7.5 promoter sequence shown as SEQ ID No. 3.

[0030] The base vector of the homologous recombination vector of the application can be a plasmid vector, such as a PUC57 vector as the base vector in an embodiment, and then the sequences shown as SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 4 are connected in sequence and inserted into the PUC57 vector to obtain a PUC57-A-Gluc-mKate recombinant plasmid.

[0031] The application also provides a primer set for detecting the PUC57-A-Gluc-mKate recombinant plasmid, comprising a primer F1 with the nucleotide sequence shown as SEQ ID No. 5, a primer F2 with the nucleotide sequence shown as SEQ ID No. 6 and a primer F3 with the nucleotide sequence shown as SEQ ID No. 7.

[0032] The application also provides a set of PCR detection system and procedures, each PCR reaction system is 25 μL, comprising: 12.5 μL 2x Phanta Max MasterMix, 1 μL of upstream primer (10 μM), 1 μL of downstream primer (10 μM), 5 μL of template DNA and 5.5 μL of ddH2O. The PCR program is set as: pre-denaturation 95℃ for 3 min; amplification cycle 95℃ for 15 s, 60℃ for 15 s, 72℃ for 60 s, cycle 35 times; extension 72℃ for 5 min.

[0033] The application also provides a recombinant vector for expressing the recombinant vaccinia virus genome.

[0034] The recombinant strain according to the application comprises a basic strain and a double reporter gene sequence introduced into the basic strain. As an embodiment, the basic strain according to the application is a vaccinia virus; as another embodiment, the vaccinia virus according to the application is vaccinia virus ECTV-GD01, and the recombinant vector used in the examples is PUC57-A-Gluc-mKate.

[0035] The application also provides a recombinant vaccinia virus strain comprising the recombinant vaccinia virus genome.

[0036] The recombinant vaccinia virus strain according to the application can obtain the recombinant vaccinia virus ECTVA which efficiently expresses red fluorescent protein and Gaussian luciferase, and the recombinant virus is obtained for at least 7 generations in succession, has good genetic stability, and is verified by the examples, and has similar replication kinetics characteristics and plaque size to wild-type virus in vitro, and also has similar virulence to wild-type virus in vivo.

[0037] The application also provides a construction method of the recombinant vaccinia virus strain, comprising transforming a wild-type vaccinia virus with the homologous recombination vector or the recombinant vector to obtain the recombinant vaccinia virus strain.

[0038] The application synthesizes a DNA fragment in series of an upstream homologous arm sequence, an mKate red fluorescent protein gene sequence, a p11 promoter sequence, a p7.5 promoter sequence, a Gluc Gaussian luciferase gene sequence, and a downstream homologous arm sequence; clones the synthesized DNA fragment into a linearized vector obtained by cutting a basic vector PUC57 with EcoRV to obtain a PUC57-A-mKate-Gluc vector;

[0039] The virus is inoculated on Vero cells at an MOI of 0.05, and then the homologous recombination vector PUC57-A-mKate-Gluc is transfected, and the virus-infected supernatant and cells are harvested after 48 hours;

[0040] After repeated freeze-thawing for 3 times, the recombinant liquid is inoculated in BHK-21 cells, and 3-5 independent red fluorescent single plaques with large areas are picked up after 48 hours;

[0041] After repeated freeze-thawing for 3 times, one of the red fluorescent single plaques is inoculated in BHK-21 cells again, and 3-5 independent bright red single plaques are picked up again;

[0042] Repeat this step, and after 4 single spot purifications, use primer pair F4 for PCR identification; the monoclonal identified successfully is the recombinant mousepox virus ECTVA.

[0043] The primer pair F4 of the present application comprises an upstream primer with the nucleotide sequence shown in SEQ ID No. 8 and a downstream primer with the nucleotide sequence shown in SEQ ID No. 9. The system for PCR identification of the present application comprises, in 25 μL, 2x Phanta Max Master Mix 12.5 μL, 1 μL of each of the upstream primer and the downstream primer, 100 ng of template cDNA, and 25 μL of ddH2O. The PCR amplification procedure of the present application is as follows: pre-denaturation at 95℃ for 30 s; denaturation at 95℃ for 15 s, annealing at 60℃ for 15 s, extension at 72℃ for 60 s, for 35 cycles; and further extension at 72℃ for 5 min.

[0044] The present application also provides a set of primer pairs for detecting the recombinant mousepox virus strain described above, comprising an upstream primer with the nucleotide sequence shown in SEQ ID No. 8 and a downstream primer with the nucleotide sequence shown in SEQ ID No. 9.

[0045] The present application also provides the use of the recombinant mousepox virus genome described above, or the homologous recombinant vector described above, or the recombinant vector described above, or the recombinant mousepox virus strain described above, in screening anti-mousepox virus drugs and / or preparing vaccines.

[0046] The antiviral drug of the present application can be an anti-orthopoxvirus drug, such as an anti-mousepox virus drug, and in particular an anti-mousepox virus ECTV-GD01 drug. The vaccine of the present application can be an anti-orthopoxvirus vaccine, such as an anti-mousepox virus vaccine, and in particular an anti-mousepox virus vaccine.

[0047] In order to further illustrate the present application, the recombinant mousepox virus genome, the recombinant mousepox virus strain, and the use thereof provided by the present application are described in detail below in conjunction with examples, but they should not be understood as limiting the scope of protection of the present application.

[0048] Example 1

[0049] The recombinant mousepox virus carrying mKate and Gluc is constructed and expression identified as follows:

[0050] 1.1 Construction of homologous recombinant plasmid

[0051] The upstream homologous arm sequence (SEQ ID No. 1), p11 promoter sequence (SEQ ID No. 2), p7.5 promoter sequence (SEQ ID No. 3), and downstream homologous arm sequence (SEQ ID No. 4) were synthesized in series; the synthesized gene fragment was cloned into the base vector PUC57 to become the PUC57-A-mKate-Gluc vector (the gene synthesis and fragment ligation were completed by GenScript Biotech Co., Ltd.); the recombinant vector was transformed into E. coli DH5a competent cells, uniformly coated on an LB plate containing ampicillin, and single colonies were picked and cultured in 5 mL LB liquid containing ampicillin for 12-16 h to obtain plasmids, which were sequenced using forward primers F1 (SEQ ID No. 5), F2 (SEQ ID No. 6), and F3 (SEQ ID No. 7). The colonies with correct sequencing were expanded for culture.

[0052] 1.2 Obtaining and identifying recombinant viruses

[0053] Vero cells were plated in a 12-well plate at 2x10 5 cells per well one night in advance and cultured in a 5% CO2, 37°C incubator for 16-20 h. When the Vero cells in the 12-well plate grew to 90%, 500 μL of ECTV-GD01 virus diluent with an MOI of 0.05 was added to each well, and the cells were cultured 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. Each well was transfected with 1 μg of PUC57-A-mKate-Gluc plasmid (plasmid: HP transfection reagent = 1:2), and 1 mL of 2% FBS DMEM was added to each well. The cell culture plate was placed in a 5% CO2, 37°C incubator for 48 h, then frozen in a -80°C refrigerator, and after repeated freeze-thawing three times, BHK-21 cells were inoculated and plaque-picked.

[0054] After 48 h of inoculation of BHK cells, low-melting-point agarose was used for fixation, and single, large, bright red fluorescent plaques were observed under a fluorescence microscope and placed in culture medium and frozen in a -80°C refrigerator. After repeated freeze-thawing three times, the next generation was inoculated. According to this cycle, after four times of continuous single-plaque purification, one generation was expanded on Vero cells for PCR identification (primers F4: SEQ ID No. 8 and SEQ ID No. 9) and immunofluorescence identification. The successfully identified recombinant vaccinia virus was named ECTVA. The nucleotide sequence inserted between A11R and A12L of ECTV-GD01 is shown in SEQ ID No. 10.

[0055] The recombinant vaccinia virus ECTVA emitted bright red fluorescence after 48 h of infection of Vero cells, while the parent virus and the cell control did not emit fluorescence Figure 1(A); Primer pairs designed on the two arms of homologous recombination, such as F4 Figure 1 As shown in Figure B, the recombinant virus band size is 2001 bp, while the parent virus band size is 339 bp. The recombinant virus shows no obvious band at 339 bp, indicating that the recombinant virus has been purified completely, and the insertion position and size of the target band are correct. Figure 1 (C)

[0056] One day in advance, Vero cells were seeded into poly-L-lysine-treated eight-chamber cells. When the cell density reached 90%, the cells were inoculated with virus at an MOI of 0.05 and cultured at 37°C with 5% CO2 for 72 h. The culture medium was discarded, and the cells were fixed with 4% paraformaldehyde for 30 min and washed three times with PBS. The cells were then incubated with 0.5% Triton X-100 at room temperature for 20 min and washed three times with PBS. The cells were blocked with 10% goat serum at 37°C for 2 h. Rabbit-derived Gluc polyclonal antibody (Nanolight, 401P) was diluted 1:500 with 10% goat serum, and mouse-derived A27L monoclonal antibody (Santa Cruz, 8115) was diluted 1:10 with 10% goat serum. The cells were incubated at 37°C for 1 h and washed three times with PBS. FITC-labeled goat anti-rabbit IgG antibody (Invitrogen, F-2765) was diluted 1:1000 with 10% goat serum, and Alexa Fluor was diluted 1:1000 with 10% goat serum. TM Donkey anti-mouse IgG antibody labeled with 647 (Invitrogen, A-31571) was incubated with DAPI diluted 1:1000 in 10% goat serum at 37°C for 2 hours, followed by washing three times with PBS. Mounting tablets were added, and the mixture was observed using a Leica laser scanning confocal microscope. Images were taken and stored under the microscope. Recombinant mousepox virus expressed Gluc protein in the cytoplasm, while the parent virus and blank cells did not. Figure 1 (D); Recombinant mousepox virus-infected cells showed obvious expression of mKate red fluorescent protein, while the parent virus and blank cells did not express it. Figure 1 (D); A27L, as a viral membrane protein control, was expressed on the cell membranes of cells infected with recombinant virus and cells infected with maternal virus, while it was not expressed in blank cells. Figure 1 (D); DAPI staining showed all cell nuclei to be blue; the above experiments demonstrate that the recombinant mousepox virus was successfully constructed and the target protein was correctly expressed.

[0057] Example 2

[0058] Genetic stability determination of recombinant mousepox virus ECTVA

[0059] 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.

[0060] Vero cells were plated into 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);

[0061] The culture medium was discarded, 1 mL of PBS was added for washing, the cells were scraped off with a scraper, centrifuged at 1200 rpm for 10 min, the PBS was discarded, 200 μL of cell lysis solution containing PMSF was added to each sample, and after incubation on ice for 30 min, 6x protein loading was boiled for 15 min, 15 μL was loaded onto each well for SDS-PAGE gel electrophoresis, and electrotransferred to a nitrocellulose membrane (NC membrane). Subsequently, the NC membrane was blocked with 5% skim milk-TBS buffer at room temperature for 2 h. The membrane was incubated with 1:500 diluted primary antibody Gluc polyclonal antibody (NanoLight, 401P) and 1:500 diluted primary antibody F13L at 4°C overnight. After incubation, the membrane was washed with TBST (Tris salt buffer containing Tween-20) for 3 times, 5 min each time. Then, 1:1000 diluted secondary antibody (Goat anti-Rabbit IgG (H+L) Secondary Anticody HRP, purchased from Zhongsu Jinqiao, ZB-2301) was incubated at 37°C for 2 h in the dark. After incubation, the membrane was washed with TBST for 3 times, 5 min each time. The membrane was scanned and imaged using a multifunctional imaging system (Wuhan Saiver Biological Technology Co., Ltd., product number SCG-W5000) instrument 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 A), while the parent virus could only detect a band at 41 kDa, indicating that each generation of virus could effectively express Gluc protein.

[0062] Vero cells were plated into 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, with 6 replicate wells for each generation of virus. After 48 h of infection, 20 μL of cell supernatant from 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 after gentle shaking, the reading was taken using a chemiluminescence detection instrument (Promega Glomax, USA). The Gluc luminescence signal value of P1-P7 generations of recombinant viruses was 10 5.23 ~10 6.01 , and the Gluc luminescence signal value of the maternal virus-infected cells and the blank cells was the background value 10 1.3 , which showed that the recombinant virus could stably express Gluc Figure 2 in B).

[0063] Example 3

[0064] Comparison of cell line replication kinetics of recombinant mousepox virus ECTVA and maternal virus ECTV-GD01

[0065] 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 cells / well, the inoculation density of BHK cells was 1 x 10 6 cells / well, the inoculation density of Vero cells was 2 x 10 5 cells / well, and the inoculation density of HeLa cells was 5 x 10 5 cells / well. The inoculation density of cells in the 96-well plates was 1.5 x 10 4 cells / well. The cells were cultured in a 37°C 5% CO2 incubator for 12-16 h. The cells in the 12-well plates were used for cytopathic effect and fluorescence photography, and the cells in the 96-well plates were used for subsequent determination of virus replication kinetics curves; when the cell growth density reached 90%, ECTVA and ECTV-GD01 were inoculated into the above four cell lines at an infection dose of 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. The cytopathic effect and fluorescence production in the 12-well plates were photographed and recorded at the corresponding time points.

[0066] The harvested cell suspension was frozen at -80℃. For testing, 20 μL of the suspension was used to determine the Gluc expression level and plot the Gluc replication kinetics curve. Viral nucleic acid was extracted using a viral DNA extraction kit (Vazyme, China) according to the manufacturer's instructions, using 100 μL of suspension. Quantification was performed using Probe qPCR Mix (QN213, Vazyme, China) on a LightCycler 96 real-time PCR instrument (Roche Diagnostics Ltd., Switzerland) according to the established procedure, and replication kinetics curves were plotted.

[0067] The results are as follows Figure 3 As shown, both recombinant mousepox virus and the parent virus can infect these four cell lines. Figure 3 The values ​​in A, B, and C represent the broad host range that mousepox virus can infect, from primary chicken embryo cells to mouse and human cell lines.

[0068] For CEF cells: such as Figure 3 As shown in Figure A, cells infected with the recombinant virus ECTVA began to show sporadic fluorescence from 36 hours onwards. The intensity of the red fluorescence gradually increased over time, reaching its maximum at 84 hours. The diseased cells began to detach from the cells between 72 and 84 hours later, and completely detached from the cells at 120 hours. Therefore, the fluorescence intensity decreased after 96 hours. Figure 3 As shown in Figure B, luciferase activity assays revealed that the luciferase value of ECTVA peaked at 84 hours, subsequently decreasing due to cell shedding. In contrast, the luciferase value of the parent virus remained extremely low and unchanged. Figure 3 As shown in Figure C, viral copy number detection revealed that the two viruses exhibited similar replication kinetics curves on CEF cells, rapidly amplifying from 12 to 36 hours, reaching a replication plateau at 84 hours, and then remaining unchanged thereafter.

[0069] For BHK cells: such as Figure 3 As shown in Figure A, cells infected with the recombinant virus ECTVA began to show sporadic fluorescence after 36 hours, and the fluorescence intensity gradually increased over time, reaching its maximum after 84 hours. The diseased cells began to detach after 84 hours and completely detached after 120 hours; therefore, the fluorescence intensity decreased after 96 hours. Figure 3 As shown in Figure B, luciferase activity assays revealed that the luciferase value of ECTVA peaked between 96 and 120 h, while the luciferase value of the parent virus remained extremely low and unchanged. Figure 3 As shown in Figure C, viral copy number detection revealed that the two viruses exhibited similar replication kinetics curves on BHK cells, rapidly amplifying from 12 to 60 hours, reaching a replication plateau at 96 hours, and then remaining unchanged thereafter.

[0070] For Vero cells: such as Figure 3As shown in Fig. 1A, the recombinant virus ECTVA-infected cells began to show sporadic fluorescence from 24 h, and the fluorescence intensity gradually increased with time. The fluorescence intensity reached the maximum at 60-72 h, and the diseased cells began to fall off after 60 h. Large pieces of cells fell off at 72-84 h, and the cells completely fell off at 96 h. Therefore, the fluorescence intensity decreased after 60-72 h. Figure 3 As shown in Fig. 1B, luciferase activity detection found that the Gluc value of ECTVA reached a peak at 48-60 h, and then decreased due to cell shedding. The luciferase reading value of the parent virus was very low and did not change with time. Figure 3 As shown in Fig. 1C, virus copy number detection found that the two viruses had similar replication kinetics curves on Vero cells. They rapidly amplified at 12-36 h, reached a replication plateau at 60 h, and then remained unchanged.

[0071] For HeLa cells: as Figure 3 As shown in Fig. 2A, ECTVA-infected cells began to show sporadic fluorescence from 24 h, and the fluorescence gradually increased with time. The fluorescence intensity reached the maximum at 60-72 h, and then decreased with cell shedding. Large pieces of cells fell off at 72-84 h, and the cells completely fell off at 96 h, and the cells were fragmented at 96 h. The parent virus did not fluoresce. Figure 4 As shown in Fig. 2B, luciferase activity detection found that the Gluc value of ECTVA reached a peak at 60 h, and then decreased due to cell shedding. The luciferase reading value of the parent virus was very low and almost unchanged. Figure 4 As shown in Fig. 2C, virus copy number detection found that the two viruses had similar replication kinetics curves on HeLa cells. They rapidly amplified at 12-36 h, reached a replication plateau at 60 h, and then remained unchanged.

[0072] The above experiments show that the recombinant virus ECTVA maintains the same in vitro replication kinetics as the parent virus ECTV-GD01. The time at which the viral load reaches a plateau is related to the type of cell line.

[0073] Example 4

[0074] Comparison of plaque and immune plaque size and morphology of recombinant vaccinia virus ECTVA and parent virus ECTV-GD01

[0075] To further define the biological characteristics of recombinant ectromelia virus ECTVA in vitro, the present application compared the plaque morphology and size of recombinant ectromelia virus ECTVA and the parental virus ECTV-GD01 on Vero cells. Vero cells were seeded in 12-well plates and when the cells reached 90% confluence, ECTVA and ECTV-GD01 were inoculated at MOI = 0.0001 and MOI = 0.0005. The cell plates were placed in a 37°C incubator and after 2 hours of adsorption, the medium was discarded and replaced with 2x DMEM containing 2% FBS and 2.4% Avicel, and the plates were incubated at 37°C for another 120 hours. Then the medium was discarded and 1 mL of 0.1% crystal violet-4% paraformaldehyde fixing solution was added to each well, and the cells were fixed and stained for 30 minutes. After the staining-fixing solution was removed and the cells were washed with running water, the plates were air-dried and photographed for comparison. It was observed that both viruses formed plaques with central empty spaces after 120 hours. As shown in Figure 4 Figure 1A, there was no significant difference in the size and shape of the plaques formed by ECTVA and the parental virus.

[0076] To further investigate the spreading ability of recombinant ectromelia virus ECTVA and the parental virus ECTV-GD01 on different cell lines, ECTVA and ECTV-GD01 were inoculated at MOI = 0.0001 on CEF, BHK, Vero and HeLa cells, respectively, and at 24 hours and 48 hours after infection, the cells in the 6-well plates were fixed with 4% paraformaldehyde for 20-30 minutes, and then washed twice with PBS. After the cells were treated with 3% hydrogen peroxide in the dark for 10 minutes, they were blocked with PBS containing 3% bovine serum albumin (BSA) for 30 minutes. The cells were incubated with 1:2000 diluted rabbit anti-poxvirus polyclonal antibody (Bio-Rad, 9503-2057) at room temperature for 1 hour, and then washed three times with PBS. Then, 1:2000 diluted horseradish peroxidase-labeled goat anti-rabbit IgG H+L (Zhongshanjinqiao, ZB-2301) was added and incubated for 30 minutes. After washing three times with PBS, 800 μL of TRUEBLUE ready-to-use color developing solution was added to each well, and color development was performed at room temperature in the dark for 5-8 minutes. After the reaction was terminated with distilled water, the blue-purple spots in each well were counted. As shown in Figure 4 Figure 1B, the present application found that the immune plaques of both viruses at 48 hours were larger than those formed at 24 hours. For CEF cells Figure 4 Figure 1B), the immune plaques formed by ECTVA at 48 hours were smaller than those formed by ECTV-GD01; while for BHK Figure 5 Figure 1C), Vero and HeLa cells, the immune plaques formed by both viruses at 48 hours were similar in size and shape Figure 5 Figure 1D and E). In summary, recombinant ectromelia virus ECTVA and ECTV-GD01 maintained similar plaque and immune plaque morphology and size.

[0077] Example 5

[0078] Application analysis of recombinant vaccinia virus ECTVA in ST-246 and CDV pharmacodynamics evaluation

[0079] In order to evaluate whether recombinant vaccinia virus ECTVA can be used for pharmacodynamics evaluation of antiviral drugs, the present application selects two positive drugs ST-246 (tecovirimat, purchased from Shanghai Tao Shu Biological Technology Co., Ltd.) and CDV (cidofovir, purchased from Beijing Wakai Biological Technology Co., Ltd.) for experiments. Vero cells were inoculated in 96-well plates at a density of 1.5×10 4 cells per well, and the cells were grown to about 90%, then gradient-diluted drugs were added. ST-246 was serially diluted by 5 times from 2 μM for 6 concentrations, and CDV was serially diluted by 2 times from 100 μM for 6 concentrations, 100 μL was added to each well, and three replicate wells were set for each concentration, and cell control wells and virus control wells were set to add cell maintenance fluid, and then incubated in a constant temperature incubator at 37°C, 5% CO2 for 2 h. Then 100 μL containing 100 PFU ECTVA was added to each well, and 100 μL of cell maintenance fluid was added to the cell control wells and virus control wells. Continue to incubate in a 37°C incubator for 72 h. Then observe the expression of red fluorescent protein under a fluorescence microscope. 20 μL of virus supernatant was aspirated and added to a black plate, and 70 μL of renilla luciferase assay buffer was added to each well, and the GloMax chemiluminescence instrument was read.

[0080] The Gluc inhibition rate of each well = (Gluc average in virus control wells - Gluc value in this well) / (Gluc average in virus control wells - Gluc average in cell control wells) * 100%.

[0081] Finally, the 50% inhibition concentration (EC 50 ) of the drug on the virus was calculated by Graphpad, and the virus inhibition rate curve was drawn. Similarly, Vero cells were inoculated in 12-well plates at a density of 2×10 5 cells per well, and the cells were grown to about 90%, then 500 μL of the above gradient-diluted drugs were added to each well, and 500 μL of 100 PFU ECTVA was added to each well after 2 h, and then incubated for 72 h, then fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, counted the plaques, counted, and calculated the EC 50 .

[0082] As Figure 5As shown in Figure A, as the concentration of ST-246 decreases, the viral fluorescence intensity in the cell pores 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 7.56 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 10.83 nM ( Figure 5 (D). The inhibition effects of Gluc inhibition and plaque reduction experiments were very similar.

[0083] 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 12.31 μM ( Figure 6 (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 ECTVA infection of cells can provide a high-throughput and rapid evaluation of the drug's anti-opioid effect.

[0084] Example 6: Evaluation of the in vivo biological characteristics of ECTVA

[0085] To evaluate the in vivo virulence of recombinant mousepox virus ECTVA in mice, 4-week-old female BALB / c mice purchased from Beijing Vital River Biotechnology Co., Ltd. were randomly divided into groups and allowed to acclimatize to the environment for one day. One day later, mice were anesthetized by intraperitoneal injection of an anesthetic and then injected intranasally with 40 μL of a viral dilution of ECTVA. The control group received 40 μL of PBS. The 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 body weight was recorded for 12 consecutive days. The results showed that recombinant mousepox virus ECTVA caused death in Balb / c mice; compared with the parent virus ECTV-GD01, ECTVA significantly reduced the median lethal dose (LD50). 50 ), but still less than 25 PFU ( ​Fig. 2A, B).

[0086] 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 10000 PFU, 100 PFU, 50 PFU and 25 PFU of virus were respectively injected into each mouse by nose drop, 3 mice in each group, and the bioluminescence in the mice was observed every day.

[0087] 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, inject for 6 min, then put it into the anesthesia box, and after the mouse is completely anesthetized, put it into the dark warehouse of the instrument, with the mouse's abdomen facing up, close the warehouse door and start imaging. The bioluminescence signal collection time is in auto mode; save the photo data; finally take out the mouse and put it back into the mouse cage for normal feeding. It is found that the bioluminescence in the mouse body gradually increases with the increase of the infection days ​ Fig. 2C, D). Although it is nose drop infection, no obvious bioluminescence is observed in the lung and respiratory tract, and the recombinant mousepox virus ECTVA is mainly distributed in the abdominal cavity of the mouse ​ Fig. 2C), which is consistent with the characteristics of mousepox virus targeting mouse liver and spleen.

[0088] In summary, the recombinant mousepox virus carrying the red fluorescent protein gene and the Gaussian luciferase gene can efficiently express the red fluorescent protein and the Gaussian luciferase protein. The recombinant virus has genetic stability on the cell line. Compared with the wild virus, the recombinant virus has similar replication kinetics and plaque size and morphology. 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 the mouse body.

[0089] Although the above embodiment describes the present application in detail, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained according to the present embodiment without creativity, which all belong to the protection scope of the present application.

Claims

1. A recombinant vaccinia virus genome, characterized in that, The recombinant vaccinia virus genome comprises a vaccinia virus genome and a red fluorescent protein gene mKate and a Gaussia luciferase protein gene Gluc inserted between A11R and A12L of the vaccinia virus genome.

2. The recombinant vaccinia virus genome of claim 1, wherein, The red fluorescent protein gene mKate is expressed under the control of a p11 promoter, and the nucleotide sequence of the p11 promoter is shown as SEQ ID No.

2.

3. The recombinant vaccinia virus genome of claim 1, wherein, The Gaussia luciferase protein gene Gluc is expressed under the control of a p7.5 promoter, and the nucleotide sequence of the p7.5 promoter is shown as SEQ ID No.

3.

4. The recombinant vaccinia virus genome according to any one of claims 1 to 3, wherein, The nucleotide sequence between A11R and A12L of the recombinant vaccinia virus genome is shown as SEQ ID No.

10.

5. A homologous recombination vector for cloning the genome of the recombinant mousepox virus according to any one of claims 1 to 4, characterized in that, The homologous recombination vector comprises an upstream homologous arm with the nucleotide sequence shown as SEQ ID No. 1 and a downstream homologous arm shown as SEQ ID No.

4.

6. A recombinant vector expressing the recombinant vaccinia virus genome according to any one of claims 1 to 4.

7. A recombinant vaccinia virus strain comprising the recombinant vaccinia virus genome according to any one of claims 1 to 4.

8. The method of constructing a recombinant vaccinia virus strain according to claim 7, wherein, The recombinant vaccinia virus strain is obtained by transforming a wild-type vaccinia virus strain with the homologous recombination vector according to claim 5 or the recombinant vector according to claim 6.

9. A set of primer pairs for detecting the recombinant strain of the vaccinia virus of claim 7, characterized in that, The upstream primer has the nucleotide sequence shown as SEQ ID No. 8 and the downstream primer has the nucleotide sequence shown as SEQ ID No.

9.

10. Use of the recombinant vaccinia virus genome according to any one of claims 1 to 4, the homologous recombination vector according to claim 5, the recombinant vector according to claim 6, or the recombinant vaccinia virus strain according to claim 7 in screening anti-vaccinia virus drugs and / or preparing vaccines.

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