Recombinant African swine fever virus strain co-expressing Gaussian luciferase and green fluorescent protein and application of recombinant African swine fever virus strain

Abstract

The invention discloses a recombinant African swine fever virus strain co-expressing Gaussian luciferase and green fluorescent protein and application of the recombinant African swine fever virus strain. According to the invention, a Gluc and EGFP reporter gene transfer vector started by a p72 promoter is constructed; and inserting into a termination codon of an ASFV MGF300-4L gene by using homologous recombination to obtain a recombinant African swine fever virus strain co-expressing Gluc and EGFP (Enhanced Green Fluorescent Protein). As the first recombinant ASFV which expresses fluorescent protein and luciferase and does not lack any viral gene, the inserted reporter genes EGFP and Gluc can be stably expressed, and the biological characteristics of the reporter virus have no significant difference from that of a parent strain; compared with the existing recombinant ASFV virus strain without the virus gene, the recombinant African swine fever virus strain constructed by the invention has more representativeness when being used for replacing a parent virus strain to research infection and pathogenic mechanisms of the virus or used for screening antiviral drugs.

Description

Technical Field

[0001] The present invention relates to a recombinant African swine fever virus strain, in particular to a recombinant African swine fever virus strain co-expressing Gaussia luciferase and green fluorescent protein and an application thereof, belonging to the field of a recombinant African swine fever virus strain expressing a reporter gene and an application thereof. Background Art

[0002] African swine fever (ASF) is an acute, severe, and highly contagious disease caused by African swine fever virus (ASFV) infecting pigs (Galindo I, Alonso C. African swine fever virus: A review. Viruses. 2017; 9(5): 103.). The ASFV genome is 170 to 194 kb in length and can encode 68 structural proteins and more than 100 non-structural proteins (Wang Y, Kang W, Yang W, Zhang J, Li D, Zheng H. Structure of African swine fever virus and associated molecular mechanisms underlying infection and immunosuppression: a review. Front Immunol. 2021; 12: 715582.). Since it was introduced into China in 2018, it has gradually evolved from the initial strong strain of genotype II to weak strains of genotype I and recombinant strong strains of genotype II and genotype I, making the prevention, control and eradication of ASF extremely difficult. Since there is no commercial vaccine or drug yet, the development of safe and effective drugs is very important for the prevention and control of ASF. The vaccine development cycle of ASFV is long, and a large number of clinical trials are required during the development process to evaluate its effectiveness. At present, there is no safe and effective vaccine in China, so screening drugs that can inhibit ASFV is one of the important measures to control the spread of ASF.

[0003] At present, there are two main methods for quantitative analysis of ASFV infection. The first is to detect the virus titer of ASFV; the second is to detect the genome copy number of ASFV by fluorescence quantitative PCR (Wang Tao, Han Yu, Pan Li, et al. Establishment of TaqMan fluorescence quantitative PCR for African swine fever virus MGF360-13L gene. Chinese Agricultural Science, 2021, 54(5): 1073-1080.). The former requires fresh porcine red blood cells during the experiment, and the virus must be diluted into different dilutions, and 6 to 8 replicates are required for each dilution; while the latter requires extraction of viral genomic DNA for fluorescence quantitative PCR. The above two methods are cumbersome, time-consuming, laborious, and low-throughput. Therefore, it is necessary to construct a high-throughput method that can be used to quickly detect ASFV infection.

[0004] Thanks to the development of molecular biology, great progress has been made in the development of ASFV reporter viruses in recent years. Various types of reporter genes, such as EGFP and mCheery fluorescent protein genes (Petrini S, Righi C, Mészáros I, D'Errico F, Tamás V, Pela M, Olasz F, Gallardo C, Fernandez-Pinero J, E, Magyar T, Feliziani F, Zádori Z. The production of recombinant African swine fever virus Lv17 / WB / Rie1 strains and their in vitro and in vivo characterizations. Vaccines. 2023; 11(12): 1860.) and luciferase gene. The fluorescent protein gene is mainly used to track virus infection in vitro, or to replace the target gene to construct a gene-deficient strain, while the luciferase gene is mostly used to quantify the level of virus replication in cells in vitro (Kang Li, Zhou Shijun, Song Jie, et al. Construction of recombinant African swine fever virus expressing luciferase and green fluorescent protein dual reporter genes. Journal of Virology, 2021, 37(5): 1128-1134; CN116515772A).

[0005] Among the recombinant ASFVs expressing dual reporter genes, there are reporter viruses that express both EGFP and Gluc (rASFV-Gluc-EGFP) and reporter viruses that express both EGFP and Fluc (rASFV-Fluc-EGFP), but these two are constructed by replacing the K145R or MGF505-1R gene in the parental strain with the reporter gene (Kang Li, Zhou Shijun, Song Jie, et al. Construction of recombinant African swine fever virus expressing dual reporter genes of luciferase and green fluorescent protein. Journal of Virology, 2021, 37(5): 1128-1134). The loss of these genes may affect the biological characteristics of ASFV, and there is a possibility of interaction between the proteins encoded by these viral genes and drugs, which may lead to the omission of some drugs that may have an inhibitory effect. Summary of the invention

[0006] One of the purposes of the present invention is to provide a recombinant African swine fever virus strain that co-expresses Gluc and EGFP reporter genes without lacking viral genes;

[0007] The second purpose of the present invention is to provide a recombinant African swine fever virus strain for constructing the co-expression of Gluc and EGFP reporter genes.

[0008] The third purpose of the present invention is to apply the recombinant African swine fever virus strain co-expressing Gluc and EGFP reporter genes to anti-African swine fever virus drug screening, neutralizing antibody determination, disinfectant evaluation or research on African swine fever virus infection and pathogenic mechanism.

[0009] To achieve the above purpose, the main technical solutions adopted by the present invention include:

[0010] One aspect of the present invention is to provide a recombinant African swine fever virus strain rASFV-Gluc / EGFP that co-expresses Gluc and EGFP reporter genes without lacking viral genes, and its microbial preservation number is: CCTCC NO: V202469; its classification name is: African swine fever virus rASFV-Gluc / EGFP; its preservation time is August 13, 2024; the preservation unit is: China Center for Type Culture Collection; the preservation address is: Wuhan University, Wuhan, China.

[0011] Another aspect of the present invention is to provide a method for constructing the recombinant African swine fever virus strain rASFV-Gluc / EGFP that co-expresses Gluc and EGFP reporter genes, comprising:

[0012] (1) The present invention constructs a Gluc and EGFP reporter gene transfer vector driven by the p72 promoter; by using homologous recombination, it is inserted after the stop codon of the ASFV MGF300-4L gene to obtain a recombinant African swine fever virus strain that co-expresses Gluc and EGFP

[0013] In a preferred embodiment of the present invention, in step (1), Gluc and EGFP reporter genes driven by p72 promoter are inserted after the stop codon of ASFV MGF300-4L gene by overlapping PCR and homologous recombination.

[0014] In a preferred specific embodiment of the present invention, in step (2), the recombinant African swine fever virus strain rASFV-Gluc / EGFP expressing Gluc and EGFP reporter genes is rescued using ASFV-WT as the parent strain through homologous recombination.

[0015] In a preferred embodiment of the present invention, the ASFV-WT parent strain described in step (2) is ASFVHLJ / 18 strain (GenBank accession number: MK333180.1).

[0016] The present invention inoculates constructed rASFV-Gluc / EGFP and ASFV-WT into primary porcine alveolar macrophages (PAMs) respectively, and observes fluorescence expression under an inverted fluorescence microscope 24 hours after infection; at the same time, cell supernatant is aspirated to detect Gluc activity in the culture medium, and the results show that Gluc and EGFP genes of rASFV-Gluc / EGFP can be expressed normally.

[0017] In order to verify whether the insertion of Gluc and EGFP genes affects the ability of ASFV to adsorb red blood cells, the present invention inoculated PAMs with rASFV-Gluc / EGFP and ASFV-WT respectively, took cells not infected with the virus as blank controls, added 1% porcine red blood cell suspension 48 hours after infection, and observed red blood cell adsorption 12 hours later; the test results showed that the insertion of Gluc and EGFP genes did not affect the red blood cell adsorption of ASFV.

[0018] In order to verify whether the insertion of Gluc and EGFP genes affects the virus particle morphology of ASFV, the present invention infected PAMs with rASFV-Gluc / EGFP and ASFV-WT respectively, and observed the particle morphology of the virus in the cells by transmission electron microscopy after 24 hours; the observation results showed that the insertion of Gluc and EGFP genes did not affect the virus particle morphology of ASFV and the formation of virus factories.

[0019] The results of the one-step growth curve and the multi-step growth curve showed that the replication levels of rASFV-Gluc / EGFP and ASFV-WT in PAMs were basically similar, with no significant difference. The experimental results showed that the insertion of Gluc and EGFP reporter genes did not affect the replication level of ASFV

[0020] In order to evaluate the genetic stability of Gluc and EGFP genes in rASFV-Gluc / EGFP, the present invention continuously propagated rASFV-Gluc / EGFP on PAMs for 20 generations, took ASFV-WT as a control, observed the expression of green fluorescence during the propagation process, detected Gluc in the cell supernatant of the 1st, 10th and 20th generations, and extracted genomic DNA of the 1st and 20th generations for PCR identification and whole genome sequencing; the test results showed that rASFV-Gluc / EGFP had good genetic stability during the propagation of PAMs.

[0021] For the dual-reporter virus rASFV-Gluc / EGFP, viral infection can be qualitatively determined or the virus can be traced by observing EGFP expression under a fluorescence microscope, and viral titer can be quantified by measuring the Gluc activity in the supernatant of infected cells. Based on these two characteristics, it can be used to determine neutralizing antibodies or evaluate the effectiveness of antiviral drugs and disinfectants.

[0022] In addition, the present invention has developed a high-throughput screening (High Throughput Screening, HTS) method based on luciferase readings based on rASFV-Gluc / EGFP, and the applicability of the method has been proved by evaluating the ratio of signal to background (Signal-to-background ratio S / B), coefficient of variation (Coefficient of variation, CV) and Z factor (Z factor), and by using genistein as a positive drug for testing, it is found that the inhibitory effect of the drug on the virus can be obviously detected, proving the feasibility of screening antiviral drugs using rASFV-Gluc / EGFP. In actual use, it is possible to first observe whether the drug has an inhibitory effect on the virus by a fluorescent microscope, and then select a sample with an inhibitory effect to detect luciferase activity, which can greatly save the experimental cost and achieve the purpose of HTS drug screening.

[0023] The rASFV-Gluc / EGFP constructed by the present invention is the first recombinant ASFV expressing green fluorescent protein and luciferase without deleting any viral genes. The inserted exogenous reporter genes EGFP and Gluc can be stably expressed, and the biological characteristics are not significantly different from those of ASFV-WT. Compared with the existing recombinant reporter ASFV virus lacking viral genes, rASFV-Gluc / EGFP is more representative when replacing the parent strain to study viral infection, pathogenic mechanism or for drug screening.

[0024] In summary, the present invention constructed a recombinant ASFV (rASFV-Gluc / EGFP) that co-expressed Gluc and EGFP reporter genes without lacking viral genes through homologous recombination, and confirmed that the recombinant virus was genetically stable and its biological properties were similar to those of the parent strain. Statistical analysis also proved that the dual-reporter virus was feasible for HTS screening of antiviral drugs. The rASFV-Gluc / EGFP constructed by the present invention can be used for high-throughput screening of anti-ASFV drugs, evaluation of neutralizing antibody activity and disinfectants, and as a tool strain for studying the infection and pathogenic mechanism of ASFV.

[0025] Definitions of terms used in this invention

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods, devices and materials are now described.

[0027] The term "host cell" or "recombinant host cell" means a cell comprising a polynucleotide of the present invention, regardless of the method used for insertion to produce a recombinant host cell, such as direct uptake, transduction, f-mating, or other methods known in the art. The exogenous polynucleotide may be maintained as a non-integrating vector such as a plasmid or may be integrated into the host genome.

[0028] The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also means oligonucleotide analogs, which include PNA (peptide nucleic acid), DNA analogs used in antisense technology (phosphorothioate, phosphoramidate, etc.). Unless otherwise specified, a specific nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including but not limited to degenerate codon substitutions) and complementary sequences as well as explicitly specified sequences. In particular, degenerate codon substitutions can be achieved by generating a sequence in which the 3rd position of one or more selected (or all) codons is substituted with mixed bases and / or deoxyinosine residues.

[0029] The term "recombinant" refers to a host cell or organism, such as a strain, a bacterium or a plant cell (such as a plant), into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the host's genome, or the nucleic acid molecule can also exist in the form of an extrachromosomal molecule. Such an extrachromosomal molecule can be self-replicating. Transformed cells, tissues or plants are understood to include not only the end product of the transformation process, but also its transgenic progeny. "Untransformed", "non-transgenic" or "non-recombinant" hosts refer to wild-type organisms such as bacteria or plants, which do not contain heterologous nucleic acid molecules.

[0030] The term "promoter" refers to any nucleic acid sequence (such as a DNA sequence) that is recognized and bound (directly or indirectly) by a DNA-dependent RNA polymerase during the initiation of transcription, resulting in the generation of an RNA molecule complementary to the transcribed DNA; this region may also be referred to as a "5' regulatory region". A promoter is typically located upstream of the 5' untranslated region (UTR) present in front of the coding sequence to be transcribed and has multiple regions that serve as binding sites for RNA polymerase II and other proteins such as transcription factors to initiate transcription of operably linked genes. The promoter itself may contain subelements (i.e., promoter motifs) such as cis elements or enhancer domains that regulate the transcription of operably linked genes. The promoter and the connected 5'UTR are also referred to as "promoter regions". BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 .Construction and identification of rASFV-Gluc / EGFP; A: Construction diagram of rASFV-Gluc / EGFP; B: PCR identification of rASFV-Gluc / EGFP.

[0032] Figure 2 .Identification of EGFP and Gluc expression in PAMs infected with rASFV-Gluc / EGFP; A: Fluorescence expression of PAMs after rASFV-Gluc / EGFP and ASFV-WT infection (Scale bar = 400 μm); B: Gluc activity detection of PAMs after rASFV-Gluc / EGFP and ASFV-WT infection.

[0033] Figure 3 .Identification of red blood cell adsorption characteristics after rASFV-Gluc / EGFP infected PAMs (Scale bar = 400 μm).

[0034] Figure 4 .Analysis of virus particle morphology of rASFV-Gluc / EGFP; A: Virus particle morphology of rASFV-Gluc / EGFP and ASFV-WT (scale bar = 500 nm); B: Virus factory composition of rASFV-Gluc / EGFP and ASFV-WT (scale bar = 2 μm).

[0035] Figure 5 .Growth kinetic curves of rASFV-Gluc / EGFP and ASFV-WT on PAMs; A: One-step growth curve of rASFV-Gluc / EGFP and ASFV-WT on PAMs; B: Multi-step growth curves of rASFV-Gluc / EGFP and ASFV-WT on PAMs.

[0036] Figure 6 .Genetic stability analysis of rASFV-Gluc / EGFP; A: Fluorescence expression of rASFV-Gluc / EGFP on PAMs of the 1st, 10th and 20th generations (Scale bar = 400 μm); B: Luciferase activity of rASFV-Gluc / EGFP and ASFV-WT on PAMs; C: Schematic diagram of sequence alignment of rASFV-Gluc / EGFP-F20 and rASFV-Gluc / EGFP.

[0037] Figure 7 Establishment of HTS method based on rASFV-Gluc / EGFP; A. Dynamic range of Gluc signal expression of rASFV-Gluc / EGFP in PAMs; B. Quality evaluation of HTS method based on rASFV-Gluc / EGFP; C. HTS drug inhibition experiment based on rASFV-Gluc / EGFP. DETAILED DESCRIPTION

[0038] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, it should be understood that the embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0039] 1. Biological materials, reagents and biosafety

[0040] 1.1 Cells, viruses, strains and plasmids

[0041] Primary porcine alveolar macrophages (PAMs), porcine erythrocytes, and Escherichia coli DH5α were isolated and preserved by the Swine Severe Infectious Disease Innovation Team of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences (hereinafter referred to as the inventor's team); ASFV HLJ / 18 strain (GenBank accession number: MK333180.1) (hereinafter referred to as ASFV-WT), pOK12 plasmid, and transfer vector pOK12-MGF300-2R-p72-EGFP containing the EGFP gene controlled by the p72 promoter were preserved by the inventor's team (Wang T, Luo R, Zhang J, Lu Z, Li LF, Zheng YH, Pan L, Lan J, Zhai H, Huang S, Sun Y, Qiu HJ. The MGF300-2R protein of African swine fever virus is associated with viral pathogenicity by promoting the autophagic degradation of IKKαand IKKβ through the recruitment of TOLLIP.PLoS Pathog.2023;19(8):e1011580.).

[0042] 1.2 Main materials and reagents

[0043] RPMI 1640 medium containing L-glutamine (Cat. No. C11875500BT), 0.25% EDTA trypsin (Cat. No. 25200056), fetal bovine serum (Cat. No. 10099141C) and penicillin-streptomycin solution (Cat. No. 15140122) were purchased from Gibco; DL5000 DNA Marker (Cat. No. 3428Q) and 2× PrimeSTAR HS Premix (Cat. No. R040Q) were purchased from TaKaRa; restriction endonuclease EcoRI (Cat. No. FD0274) and Pierce Gaussia luciferase glow detection kit (Cat. No. 16161) were purchased from Thermo Fisher scientific company; DNA gel recovery kit (catalog number: D2500-01) was purchased from Omega; endotoxin-free plasmid extraction kit (catalog number: TP117) and nucleic acid extraction or purification kit (catalog number: Y1814) were purchased from Tiangen Biochemical Technology; QIAamp blood DNA extraction kit (catalog number: 51104) was purchased from Qiagen; X-treme GENE HP DNA transfection reagent (catalog number: 6366236001) was purchased from Roche; homologous recombination enzyme (catalog number: C112-01) was purchased from Novozymes; genistein (catalog number: HY-14596) was purchased from MCE; CCK-8 cell proliferation-toxicity detection kit (catalog number: K1018) was purchased from Apbai Company; primer synthesis and sequencing identification were completed by Beijing Ruibo Xingke Biological.

[0044] 1.3. Biosafety

[0045] All live virus experiments involving ASFV in the following experiments were conducted in the National Animal Disease Prevention and Control High-Level Biosafety Laboratory (ABSL-3) of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The waste generated during the experiment was subjected to standardized disinfection and sterilization and other harmless treatments, meeting biosafety requirements.

[0046] Example 1 Construction and identification of dual reporter virus rASFV-Gluc / EGFP

[0047] 1 Test method

[0048] 1.1 Construction and identification of pOK12-EGFP / Gluc transfer vector

[0049] The inventors' team found in previous studies that inserting the EGFP gene sequence controlled by the p72 promoter after the MGF300-4L gene stop codon did not affect viral replication, and the EGFP gene could be expressed normally and stably. Therefore, this experiment chose to insert the Gluc and EGFP reporter genes after the MGF300-4L gene stop codon. The construction strategy of the transfer vector is as follows Figure 1 As shown in A. The p72 promoter-controlled Gluc (p72-Gluc) reporter gene expression cassette was synthesized by Ruibo Company and inserted into the pOK12 vector through the EcoRI restriction site, named pOK12-p72-Gluc. Design amplification primers p72-Gluc-F / R (Table 1), use pOK12-p72-Gluc as a template, and obtain the p72-Gluc fragment by PCR amplification; At the same time, use pOK12-MGF300-2R-p72-EGFP as a template and design primers p72-EGFP-F / R (Table 1) for PCR amplification to obtain the p72-EGFP gene fragment; select the ASFV-WT strain MGF300-4L gene termination codon about 1kb before and after as the homology arm, use nucleic acid extraction or purification kit to extract ASFV genomic DNA as a template, and use primers 4L-LA-F / R and 4L-RA-F / R (Table 1) to PCR amplify the left and right homology arms. The overlapping PCR method was used to connect the four fragments in the order of left homology arm, p72-Gluc, p72-EGFP, and right homology arm, and PCR amplified by primer pair 4L-LA-F / R. Overlapping PCR reaction system (50 μL): upstream primer (10 μmol / L) 2 μL; downstream primer (10 μmol / L) 2 μL; template 2 μL; 2× PrimeSTAR HS Premix 25 μL; ddH 2 O was added to 50 μL. Overlap PCR amplification was divided into two steps. The first PCR amplification: 95℃5min, 95℃20s, 55℃20s, 72℃1min, 15 cycles; 72℃ extension for 10min; the second PCR amplification (adding primer pair 4L-LA-F / R): 95℃5min, 95℃20s, 55℃20s, 72℃4min, 35 cycles; 72℃ extension for 10min. The purified PCR product was connected to the pOK12 vector after EcoRI digestion, transformed in DH5α, and finally the positive clone was picked, the plasmid was extracted, and the transfer vector with correct sequencing was named pOK12-Gluc-EGFP and stored at -20℃.

[0050] 1.2 Construction of dual reporter virus rASFV-Gluc / EGFP

[0051] The transfer vector pOK12-p72-Gluc-p72-EGFP was transfected into PAMs using X-treme GENE HP DNA transfection reagent. ASFV-WT was inoculated with a multiplicity of infection (MOI) of 1 24 h after transfection and incubated at 37°C with 5% CO 2 The cells were cultured in an incubator for 3 to 5 days, and the virus was collected after fluorescence appeared in the cells and named rASFV-Gluc / EGFP-P1. The rASFV-Gluc / EGFP was purified by limiting dilution. First, rASFV-Gluc / EGFP-P1 was mixed with PAMs and plated in a 96-well plate (10 5 cells / well) at 37°C with 5% CO 2 Culture in an incubator for 3 to 5 days, collect the cells with more fluorescence expression in the wells and the culture medium supernatant, and use the collected virus liquid for the next round of purification in a similar way. After that, as the fluorescence expression increases, the dilution ratio of the virus is continuously increased during the purification process for purification. After 7 to 8 rounds of continuous purification, the proportion of green fluorescence in cells infected with the virus is more than 70%. Use a nucleic acid extraction or purification kit to extract the genomic DNA of the virus liquid as a template, and use the primer pair D-JD-F / R (Table 1) for PCR amplification to identify whether the recombinant virus is completely purified. After the virus is confirmed to be purified in the first PCR identification, two more rounds of purification are performed based on the virus liquid, and PCR verification is performed to ensure its complete purification. The final purified virus is named rASFV-Gluc / EGFP.

[0052] 1.3 PCR identification of dual reporter virus rASFV-Gluc / EGFP

[0053] Using rASFV-Gluc / EGFP or ASFV-WT as template, genomic DNA was extracted by nucleic acid extraction or purification kit, and a pair of primers (D-JD-F / D-JD-R) were designed upstream and downstream of the MGF300-4L stop codon for amplification (Table 1). PCR reaction system (50 μL): upstream primer (10 μmol / L) 1 μL; downstream primer (10 μmol / L) 1 μL; template 5 μL; 2× PrimeSTARHS Premix 25 μL; ddH 2 The volume was filled to 50 μL with 0.05 μL. PCR reaction program: 95°C for 5 min, 95°C for 20 s, 55°C for 20 s, 72°C for 30 s, 30 cycles; 72°C for 10 min extension.

[0054] Table 1 PCR primers used in this experiment

[0055]

[0056] 2 Test results

[0057] By overlapping PCR, according to the map constructed by double reporter virus ( Figure 1 A) A transfer vector pOK12-Gluc / EGFP expressing Gluc and EGFP genes was constructed, and the correctness of the inserted fragment was verified by sequencing. Then, the recombinant virus expressing Gluc and EGFP reporter genes was rescued by homologous recombination using ASFV-WT as the parent strain. The virus was purified by limiting dilution method, and after 11 rounds of purification and screening, the purified virus was obtained and named rASFV-Gluc / EGFP.

[0058] The purified rASFV-Gluc / EGFP genomic DNA was extracted and PCR was performed using primers D-JD-F / R. The results showed that the rASFV-Gluc / EGFP group had a single band of approximately 2277 bp in size, and no bands similar to those in the ASFV-WT group appeared ( Figure 1 B). This indicates that rASFV-Gluc / EGFP has been completely purified. To further prove the correct insertion of Gluc and EGFP genes, whole genome high-throughput sequencing of rASFV-Gluc / EGFP was performed (Table 2).

[0059] The results showed that compared with ASFV-WT, rASFV-Gluc / EGFP had additional Gluc and EGFP genes controlled by the p72 promoter, and no deletion or mutation occurred in other sites. The above results showed that rASFV-Gluc / EGFP successfully inserted the gene sequences of Gluc and EGFP, and rASFV-Gluc / EGFP had been purified.

[0060] Table 2 Whole genome high-throughput sequencing results

[0061]

[0062] Experimental Example 1: Dual reporter virus rASFV-Gluc / EGFP expression of EGFP and Gluc activity detection test

[0063] 1 Test method

[0064] Using Pierce TMGluc activity was detected by Gluc luciferase glow detection kit. PAMs were infected with rASFV-Gluc / EGFP, and the cell culture supernatant was aspirated 24 hours after infection. After inactivation at 70°C for 10 minutes, the inactivated culture supernatant was added to a light-proof 96-well plate (20 μL / well), and 30 μL of working solution was added to each well. After the reaction lasted for 10 minutes and the fluorescence signal expression was stable, Gluc activity was detected in a focused multifunctional fluorescence analyzer (Cat. No.: 2105-0010, PerkinElmer).

[0065] 2 Test results

[0066] rASFV-Gluc / EGFP and ASFV-WT were inoculated into PAMs, and the fluorescence expression was observed under an inverted fluorescence microscope 24 hours after infection. At the same time, the cell supernatant was aspirated and the Gluc activity in the culture medium was detected, with cells not infected with the virus as blank controls. No green fluorescence expression was found in cells infected with ASFV-WT and uninfected cells, while obvious green fluorescence was observed in cells infected with rASFV-Gluc / EGFP ( Figure 2 A). In the Gluc activity test, it was found that the Gluc activity detected in the culture supernatant of cells infected with rASFV-Gluc / EGFP reached 10 6 The relative light units (RLU) of the cells in the ASFV-WT group and the blank control group were similar, and no obvious Gluc activity was detected ( Figure 2 B). The above results indicate that the Gluc and EGFP genes of rASFV-Gluc / EGFP can be expressed normally.

[0067] Experimental Example 2 Experiment on the adsorption effect of dual reporter virus rASFV-Gluc / EGFP on red blood cells

[0068] 1 Test method

[0069] To verify whether the insertion of Gluc and EGFP genes affects the ability of ASFV to adsorb red blood cells, rASFV-Gluc / EGFP and ASFV-WT were inoculated into PAMs, and cells not infected with the virus were used as blank controls. 1% porcine red blood cell suspension was added 48 hours after infection, and red blood cell adsorption was observed 12 hours later. The specific method is as follows:

[0070] PAMs were plated in 96-well plates (10 5After the cells adhered to the wall, 5 MOI of rASFV-Gluc / EGFP was inoculated. At the same time, ASFV-WT (5 MOI) infected PAMs was used as a positive control, and cells not infected with ASFV were used as a negative control. After 48 hours of virus infection, 10 μL / well of 1% fresh porcine red blood cell suspension was added and the cells were returned to 37°C with 5% CO 2 The cells were cultured in an incubator and the adsorption of the virus to red blood cells was observed under an inverted fluorescence microscope (Product No.: TS100, Nikon) after 12 h.

[0071] 2 Test results

[0072] The results showed that typical erythrocyte adsorption occurred in cells infected with rASFV-Gluc / EGFP and ASFV-WT, with no significant difference between the two, while no erythrocyte adsorption occurred in uninfected cells ( Figure 3 ). The experimental results showed that the insertion of Gluc and EGFP genes did not affect the red blood cell adsorption of ASFV.

[0073] Experimental Example 3 Particle morphology observation test of dual reporter virus rASFV-Gluc / EGFP

[0074] 1 Test method

[0075] To verify whether the insertion of Gluc and EGFP genes affects the virion morphology of ASFV, rASFV-Gluc / EGFP and ASFV-WT were infected with PAMs, and the particle morphology of the virus in the cells was observed by transmission electron microscopy 24 hours later. The specific experimental method is as follows:

[0076] PAMs were plated in a 6-well plate. After 24 hours, rASFV-Gluc / EGFP and ASFV-WT were inoculated into different wells at 5 MOI. After 18 hours of infection, the cells and culture medium were collected, centrifuged at 3000r / min for 5 minutes, and fixed with 2% glutaraldehyde for 12 hours. The samples were then sent to the electron microscopy group of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences for detection. The detection method is to add 4% paraformaldehyde and 1% glutaraldehyde in 0.1M HEPES buffer (pH=7.2) to the sample and fix it at 4°C for 2 hours. After removing the fixative, 50%, 70%, 90% and 100% dimethylformamide were used for gradient dehydration at 4°C for 15 minutes. After dehydration, the cells were embedded in LR white resin and polymerized under ultraviolet irradiation at -20°C for 10 days. Finally, the sections were cut according to the method described in the literature (Andrés G, Charro D, Matamoros T, Dillard RS, Abrescia NGA. The cryo-EM structure of African swine fever virus unravels a unique architecture comprising two icosahedral protein capsids and two lipoprotein membranes. J Biol Chem. 2020; 295(1): 1-12.), and the sections were observed and photographed using a transmission electron microscope.

[0077] 2 Test results

[0078] The results showed that the virus factories in PAMs infected with rASFV-Gluc / EGFP or ASFV-WT were composed of abundant virus precursors, immature viruses, mature viruses, etc. There was no significant difference in the composition of virus factories and the morphology of virus particles between the two groups ( Figure 4 A, B). The above test results show that the insertion of Gluc and EGFP genes does not affect the virion morphology of ASFV. Test Example 4 One-step growth curve and multi-step growth curve determination test of dual reporter virus rASFV-Gluc / EGFP

[0079] 1 Test method

[0080] To evaluate whether the proliferation ability of rASFV-Gluc / EGFP in PAMs is similar to that of the parental strain, rASFV-Gluc / EGFP and ASFV-WT were inoculated into PAMs (10 5 The virus titer was measured at 12, 24, 48, 72 and 120 h after infection in 4% paraformaldehyde (PNA) cells / well. The specific test method is as follows:

[0081] PAMs were plated in 24-well plates (10 5 After the cells adhered to the wall, rASFV-Gluc / EGFP or ASFV-WT were inoculated at 0.01 and 5 MOI, respectively, and the culture medium was replaced 2 hours after infection. The cells and culture supernatant were collected 12, 24, 48, 72, and 120 hours after virus infection, and the virus liquid collected at different time points was diluted with PAMs at 10 -1 ~10 -7 Each dilution was repeated 8 times and plated in a 96-well plate (10 5 10 μL / well of 1% porcine erythrocyte suspension was added 4 days after infection. The results of erythrocyte adsorption in PAMs were observed and recorded daily. The half erythrocyte adsorption (HAD) at each time point was calculated using the Reed & Muench method. 50 ), and finally use Graphpad Prism

[0082] The growth curve was drawn using 8.0.2 software.

[0083] 2 Test results

[0084] The results of one-step growth curve and multi-step growth curve showed that the replication levels of rASFV-Gluc / EGFP and ASFV-WT in PAMs were basically similar, with no significant difference ( Figure 5 A, B). This indicates that the insertion of Gluc and EGFP genes does not affect the replication level of ASFV.

[0085] Experimental Example 5 High-throughput sequencing and genetic stability analysis of the whole genome of the dual reporter virus rASFV-Gluc / EGFP

[0086] 1 Test method

[0087] To evaluate whether the Gluc and EGFP genes of rASFV-Gluc / EGFP can be stably expressed after multiple passages, rASFV-Gluc / EGFP was continuously passaged on PAMs for 20 generations, with ASFV-WT as the control. The expression of fluorescence was observed during the passage process, and the Gluc in the cell supernatant of the 1st, 10th and 20th generations was detected. At the same time, the genomic DNA of the 1st and 20th generations was extracted for PCR identification and whole genome sequencing. The specific experimental methods are as follows:

[0088] PAMs were plated in 24-well plates (10 5 50 μL of rASFV-Gluc / EGFP was added to each well and incubated at 37°C with 5% CO 2Culture in an incubator for 72 hours, observe the fluorescence expression and detect Gluc activity, and collect and store the virus solution for the next round of passage until the virus is passed to the 20th generation. Use a nucleic acid extraction or purification kit to extract the genomic DNA of the 1st, 10th, and 20th generation virus solution, and use it as a template for PCR identification. At the same time, use the QIAamp blood DNA extraction kit to extract the genomic DNA of the 1st and 20th generations, respectively, and perform whole genome sequencing, splicing, and assembly. SnapGene software was used to compare the sequence of the assembled virus genome.

[0089] 2 Test results

[0090] The results showed that when rASFV-Gluc / EGFP was propagated to the 20th generation, both EGFP and Gluc reporter genes were stably expressed, green fluorescence was observed in PAMs infected at the 20th generation, and Gluc activity was detected in the culture supernatant ( Figure 6 A, B). The whole genome of the 20th generation (F20) was sequenced (Table 2). The experimental results also showed that the average sequencing depth of rASFV-Gluc / EGFP at the 20th generation was not significantly different from that of the first generation, and no base mutations or deletions were found in the comparison of the whole genomes of the two ( Figure 6 C, D). The above test results show that rASFV-Gluc / EGFP has good genetic stability during the passage of PAMs.

[0091] Experimental Example 6 Infection dose and infection time test of dual reporter virus rASFV-Gluc / EGFP in HTS

[0092] 1 Test method

[0093] In order to select a suitable infection dose and infection time in HTS, the dynamic range of the Gluc signal (RLU) secreted by rASFV-Gluc / EGFP in PAMs was detected. PAMs were infected with rASFV-Gluc / EGFP at different MOIs, and the Gluc activity in the culture supernatant was detected at 24, 36, 48, and 72 hpi; the specific test method is as follows:

[0094] PAMs were inoculated with rASFV-Gluc / EGFP at an MOI of 0.2, and the culture medium was collected 36 hours after infection for Gluc activity detection. The signal-to-background ratio (S / B), coefficient of variation (CV), and Z factor were used to evaluate the HTS effect based on Gluc activity.

[0095] The calculation method is as follows: (1) S / B = average signal of positive control / average signal of negative control; (2) CV = standard deviation of negative control / average of negative control; (3) (σ = standard deviation, p = positive control, n = negative control, μ = mean value). S / B represents the ratio of signal to background value, CV reflects the deviation of signal in HTS, and the effect is best when it is below 20%, while the Z factor between 0.5 and 1 is considered to be of good quality in HTS (Zhao X, Wang Y, Cui Q, Li P, Wang L, Chen Z, Rong L, Du RA parallel phenotypic versus target-based screening strategy for RNA-dependent RNA polymerase inhibitors of the influenza a virus. Viruses. 2019; 11(9): 826.).

[0096] 2 Test results

[0097] according to Figure 7 The results shown in A show that when the inoculation dose is 0.2MOI or higher, an obvious Gluc signal can be detected in the culture supernatant 24h after infection, and the intensity of the fluorescent signal increases with the increase of infection time. Based on this result, the virus infection dose and infection time were set to 0.2MOI and 36h. In order to determine whether this scheme is suitable for HTS, S / B, CV and Z factor are used to evaluate its suitability in HTS. S / B reflects the ratio between the positive control signal and the background signal; CV reflects the signal deviation in HTS, and it is generally believed that CV≤20% has the best effect; Z factor is a combination of the signal range and the coefficient of variation, and is an indicator of the feasibility of quantitative screening conditions and measurement methods in HTS. When 0.5≤Z factor≤1, the method is considered to be suitable for HTS. Figure 7As shown in B, S / B, CV and Z factors are 74, 14% and 0.65, respectively, which indicates that rASFV-Gluc / EGFP is very suitable for HTS when the infection dose is 0.2 MOI and the infection time is 36h. Genistein has been shown to inhibit the replication of ASFV in vitro (Arabyan E, Hakobyan A, Kotsinyan A, Karalyan Z, Arakelov V, Arakelov G, Nazaryan K, Simonyan A, Aroutiounian R, Ferreira F, Zakaryan H. Genistein inhibits African swine fever virus replication in vitro by disrupting viral DNA synthesis. Antiviral Res. 2018; 156: 128-137.).

[0098] To further verify the feasibility of this method, HTS was performed using genistein as a positive control. The results are shown in Figure 7B. The Gluc signal intensity of the negative control group (DMSO) was 2.6 times that of the group with genistein added. This inhibitory effect is consistent with the effects reported in other articles (Zhang Zhenjiang. Screening of small molecule compounds that inhibit the replication of African swine fever virus and research on their mechanism of action [D]. Northeast Agricultural University, 2023). This result shows that it is feasible to use this condition for HTS screening of antiviral drugs.

Claims

1. A recombinant African swine fever virus strain co-expressing Gluc and EGFP reporter genes, It is characterized in that Its microbiological deposit number is: CCTCC NO:V202469.

2. A method for constructing the recombinant African swine fever virus strain according to claim 1, It is characterized in that include: (1) Inserting Gluc and EGFP reporter genes driven by p72 promoter after the stop codon of ASFV MGF300-4L gene to construct a transfer vector expressing Gluc and EGFP reporter genes; (2) Recombining the constructed transfer vector expressing Gluc and EGFP reporter genes by homologous recombination to obtain a recombinant African swine fever virus strain rASFV-Gluc / EGFP that co-expresses Gluc and EGFP reporter genes.

3. The method according to claim 2, It is characterized in that In step (1), the Gluc and EGFP reporter genes driven by the p72 promoter are inserted after the stop codon of the ASFV MGF300-4L gene by overlapping PCR and homologous recombination to construct a transfer vector expressing the Gluc and EGFP reporter genes.

4. The method according to claim 2, It is characterized in that In step (2), the recombinant African swine fever virus strain rASFV-Gluc / EGFP expressing Gluc and EGFP reporter genes is rescued using ASFV-WT as the parent strain through homologous recombination.

5. The method according to claim 2, It is characterized in that The ASFV-WT parent strain described in step (2) is the ASFVHLJ / 18 strain, and its GenBank accession number is: MK333180.

1.

6. Use of the recombinant African swine fever virus strain according to claim 1 in screening anti-ASFV drugs.

7. Use of the recombinant African swine fever virus strain according to claim 1 in evaluating the activity of neutralizing antibodies against African swine fever virus.

8. Use of the recombinant African swine fever virus strain according to claim 1 in evaluating the inhibitory activity of African swine fever virus disinfectants.

9. Use of the recombinant African swine fever virus strain according to claim 1 in the preparation and study of ASFV infection or antiviral drug screening.

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