Construction method and application of Uusu figure virus report replicon

Abstract

The invention discloses a construction method and application of a Uusu figure virus report replicon. According to the invention, a low-copy plasmid pACYC-177B is used as a carrier, all non-structural proteins and non-coding region genes of USUV are retained, 38 amino acids before the N terminal of C protein are retained in the structural protein part, 45 amino acids behind the C terminal of E protein are retained in the structural protein part, and a NanoLuc luciferase tag is inserted between the retained C and E protein genes to obtain a pACYC-TMN-Nlucc-Rep report replicon. The Uu figure virus report replicon pACYC-TMN-Nluc-Rep constructed in the invention can normally work on cells such as BHK21, Vero, DF-1, Hela and the like, and can be effectively translated and replicated. Therefore, the USUV report replicon pACYC-TMN-Nlucc-Rep constructed in the invention can be used as an important research tool to be applied to the research of the USUV.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, and in particular to a construction method and application of a Usutu virus reporter replicon. Background Art

[0002] Usutu Virus (USUV) belongs to the Flaviviridae family ( Flaviviridae ) Orthoflavivirus ( Orthoflavivirus ) Japanese encephalitis virus serotype. USUV is a zoonosis, with mosquitoes as the intermediate host of its transmission and birds as the main host. Clinical manifestations include depression, ataxia, acute death, etc. As one of the hosts of USUV, humans are mostly latently infected after infection, and the main clinical symptoms are high fever, meningitis, jaundice, etc. In addition, USUV can also infect other animals, including poultry, ruminants and mammals. With the increase in human cases infected with USUV, it has gradually attracted the attention of public health departments in some countries in recent years.

[0003] At present, the research on USUV is not in-depth, and the reports mainly focus on the isolation and identification of the virus and the host's immune response. Its pathogenic mechanism and the structural functions of various proteins need further research to determine whether it is consistent with other flaviviruses and whether there are differences. The biosafety assessment of pathogens shows that USUV needs to be tested in biosafety level 3 laboratories (BSL-3) or above. However, due to the high cost of BSL-3 laboratories and the risk of infection and leakage caused by non-standard operations of personnel, it is difficult to study the pathogenic mechanism of USUV and screen antiviral drugs. In order to achieve the purpose of biosafety downgrade, researchers have developed various strategies that are applicable to research in different directions, including replicons, trans-complementation systems, pseudotype viruses, and virus-like particles (VLPs).

[0004] Since the first report of the flavivirus replicon system, multiple types of flavivirus replicons of different species have been successfully constructed and widely used in antiviral drug screening, vaccine development, foreign protein expression, and the study of viral replication and translation molecular mechanisms. It is a powerful tool for studying flaviviruses. Flavivirus replicons can replicate themselves. They contain cis-acting elements at the 5' and 3' ends, as well as non-structural protein genes and some structural protein genes required for RNA replication and translation. Since the replicons lack structural genes, they cannot produce live virus particles. By inserting reporter genes such as NanoLuc luciferase, Renilla luciferase, GFP, etc. at the position where the structural genes are missing, the replication and translation levels of the replicons can be detected in real time by detecting the expression of the reporter genes. The replicon RNA can replicate autonomously but cannot produce live virus particles, thereby eliminating the interference of other life cycles except translation and replication, and can therefore be applied to the study of viral translation and replication mechanisms. At present, there is still no report on the construction of the USUV subgenomic replicon system. Summary of the invention

[0005] The purpose of the present invention is to solve the above technical problems and provide a construction method and application of a Usutu virus reporter replicon.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows: a method for constructing a Usutu virus reporter replicon, comprising the following steps: S1, using the gene fragment P2 of the USUV-TMN strain as a template, amplify the fragment P2, connect it with the double-enzyme-digested (EcoRV, KpnI) vector of the pACYC-TMN-P3 plasmid to obtain a recombinant plasmid named pACYC-TMN-P2-3; S2, using the gene fragment P1 of the USUV-TMN strain as a template, amplify the fragment P1, connect it with the double-enzyme-cut (XhoI, EcoRV) vector of the pACYC-TMN-P2-3 plasmid to obtain a recombinant plasmid named pACYC-TMN-P1-3; S3. Using the duck Tembusu virus (TMUV) NanoLuc reporter replicon as a template, the NanoLuc + FMDV-2A gene sequence was amplified and ligated with the pACYC-TMN-P1-3 plasmid double restriction enzyme digestion (NruI, EcoRV) vector to obtain the replicon pACYC-TMN-Nluc-Rep.

[0007] Preferably, the amplification primers in S1 are TMN-P2-3-F and TMN-P2-3-R, and their sequences are shown in SEQ ID NO.1-2.

[0008] Preferably, the amplification primers in S2 are TMN-P1-3-F and TMN-P1-3-R, and the sequences are shown in SEQ ID NO.3-4.

[0009] Preferably, the amplification primers in S3 are TMN-Nluc-F and TMN-Nluc-R, and the sequences are shown in SEQ ID NO.5-6. The present invention also provides the Usutu virus reporter replicon obtained by the above construction method.

[0010] The present invention also provides the use of the above-mentioned Usutu virus reporter replicon in Usutu virus research. The Usutu virus reporter replicon can be effectively translated and replicated in cells, and the cells include BHK21, Vero, DF-1 and Hela cells.

[0011] Subgenomic reporter replicons were constructed as Figure 1 The USUV replicon gene was synthesized into three fragments, starting with the 5'UTR, and the order from front to back was P1, P2, and P3, each fragment was about 3000bp, and a suitable double restriction site (regardless of the gene sequence) was synthesized at the front end of each fragment to facilitate connection from back to front.

[0012] The USUV strain TMN in the present invention, during gene synthesis, the TMN-P3 fragment is directly synthesized into the low copy plasmid pACYC177B vector, and the vector restriction sites are: SpeI, NotI, named pACYC-TMN-P3. Using the recombination connection method, the pACYC-TMN-P3 plasmid double restriction (EcoRV, KpnI) vector is connected with the TMN-P2 fragment recovered after PCR amplification, and transformed (competent state: E. coli Sure), screened and identified to obtain the plasmid pACYC-TMN-P2-3. Similarly, the pACYC-TMN-P2-3 plasmid is double-restricted (XhoI, EcoRV) and connected with the TMN-P1 fragment to obtain the plasmid pACYC-TMN-P1-3. A double restriction site (NruI, EcoRV) was reserved between the first 38 amino acid gene sequences at the N-terminus of the C protein of the P1 fragment and the last 45 amino acid gene sequences at the C-terminus of the E protein. After cutting, they were connected with the NanoLuc luciferase gene and the FMDV-2A gene to obtain the final TMN strain replicon pACYC-TMN-Nluc-Rep.

[0013] The beneficial effects of the present invention are: The present invention provides a method for constructing a USUV reporter replicon, using a low-copy plasmid pACYC-177B as a vector, retaining all non-structural proteins and non-coding region genes of USUV, retaining the first 38 amino acids of the N-terminus of the C protein in the structural protein part to ensure the formation of the correct RNA secondary structure, and retaining the 45 amino acids after the C-terminus of the E protein to ensure the correct positioning of the NS1 protein. The present invention inserts a NanoLuc luciferase tag between the retained C and E protein genes to obtain the pACYC-TMN-Nluc-Rep reporter replicon.

[0014] At present, the reverse genetics system tools of Usutu virus (USUV) only have infectious clones. The Usutu virus reporter replicon pACYC-TMN-Nluc-Rep constructed in the present invention can work normally on BHK21, Vero, DF-1, Hela and other cells, further improving the USUV reverse genetics platform and reducing the laboratory biosafety requirements for USUV research. Therefore, the Usutu virus (USUV) reporter replicon pACYC-TMN-Nluc-Rep constructed in the present invention can be used as an important research tool in the study of Usutu virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a schematic diagram of the construction of the reporter replicon in the present invention; Figure 2 Schematic diagram of the plasmid map of pACYC-TMN-P3 and pACYC-TMN-P2-3; Figure 3 Schematic diagram of the plasmid map of pACYC-TMN-P1-3; Figure 4 Schematic diagram of the pACYC-TMN-Nluc-Rep plasmid map; Figure 5 The results of TMN replicon replication in BHK21 cells; Figure 6 The working status of TMN replicon in DF-1, Vero and Hela cells.

[0016] Duck Tembusu virus (TMUV) NanoLuc reporter replicon Public literature information: HE Y, WANG X, GUOJ, et al. Replication / Assembly Defective Avian Flavivirus With InternalDeletions in the Capsid Can Be Used as an Approach for Living AttenuatedVaccine [J]. Front Immunol, 2021, 12(694959. DETAILED DESCRIPTION

[0017] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0018] The USUV-TMN strain is Turdus merula Netherlands 2016 (TMN) (GenBank: MN813490.1). The template USUV-TMN strain gene fragment P1 is the base sequence of the 1st to 209th positions plus the 2340th to 5189th positions of the USUV-TMN strain gene sequence; the template USUV-TMN strain gene fragment P2 is the base sequence of the 5190th to 8180th positions of the USUV-TMN strain gene sequence; the template USUV-TMN strain gene fragment P3 is the base sequence of the 8181st to 11064th positions of the USUV-TMN strain gene sequence.

[0019] Example 1 Construction of pACYC-TMN-Nluc-Rep reporter replicon The pACYC-TMN-Nluc-Rep reporter replicon was constructed as Figure 1 The plasmid pACYC in this example is a low copy plasmid pACYC-177B.

[0020] (1) Using the synthetic USUV-TMN gene fragment P2 as a template, primers TMN-P2-3-F / R were used to perform PCR reaction to amplify fragment P2; the fragment P2 was then ligated to the pACYC-TMN-P3 plasmid double-digested (EcoRV, KpnI) vector (see the plasmid map for a schematic diagram). Figure 2 Left, pACYC-TMN-P3 plasmid was directly synthesized by the company and connected to the vector pACYC-177B, the sequence is shown in SEQ ID NO.11), to obtain the recombinant plasmid, named pACYC-TMN-P2-3 (see the schematic diagram of the plasmid map Figure 2 right).

[0021] (2) Using the synthetic USUV-TMN gene fragment P1 as a template, primers TMN-P1-3-F / R were used to perform PCR reaction to amplify fragment P1; fragment P1 was then ligated to the pACYC-TMN-P2-3 plasmid double-digested (XhoI, EcoRV) vector. The plasmid was named pACYC-TMN-P1-3 (see the plasmid map for a schematic diagram). Figure 3 ).

[0022] (3) Using the duck Tembusu virus (TMUV) NanoLuc reporter replicon as a template, amplify the NanoLuc + FMDV-2A gene sequence (fragment Nluc), use primers TMN-Nluc-F / R, perform PCR reaction, amplify the fragment Nluc; connect it with the pACYC-TMN-P1-3 plasmid double restriction enzyme (NruI, EcoRV) vector. The plasmid was named pACYC-TMN-Nluc-Rep (see the schematic diagram of the plasmid map) Figure 4 ).

[0023] The PCR amplification in the above steps used KOD One PCR Master Mix enzyme, and the amplification conditions were 98℃ pre-denaturation for 1min; 98℃ denaturation for 10s, 57℃ annealing for 5s, 68℃ extension for 5s / kb, for a total of 30 cycles; and finally 68℃ extension for 7min. At the end of each round of amplification, gel electrophoresis was performed for identification, and liquid recovery was performed after the bands were confirmed. The primers used in the amplification are shown in Table 1.

[0024] Table 1 List of PCR amplification primers

[0025] Table 2 Single fragment homologous recombination reaction system

[0026] In the recombination ligation reaction between the linearized vector and the target fragment, MonClone TM Hi-Fusion Cloning Mix V2 ligase, the ligation system is shown in Table 2. Then transform into SURE E. coli competent cells, wait for colonies to grow, pick bacteria for colony identification. Then, select PCR-positive colonies for sequencing. Select the single colony with correct sequencing, expand the culture, save and extract the plasmid for later use.

[0027] Example 2 Construction of subgenomic reporter replicon negative control Based on Example 1, a subgenomic reporter replicon negative control was constructed.

[0028] The synthetic USUV-TMN gene fragment P3 was used as a template, and the primers TMN-SfoI-F and TMN-ΔGDD-R were used to amplify the fragment ΔGDD1; the same template was used to amplify the fragment ΔGDD2 using primers TMN-ΔGDD-F and TMN-NdeI-R. The second round of fusion PCR was performed with the primers TMN-SfoI-F and TMN-NdeI-R to fuse the fragment ΔGDD1 / 2 to obtain the fragment ΔGDD; the fragment ΔGDD is the enzyme activity center of the RNA-dependent RNA polymerase (RdRp enzyme) on the missing NS5 protein, that is, the sites G667, D668, and D669. The fragment ΔGDD was connected to the pACYC-TMN-Nluc-Rep plasmid double restriction (SfoI, NdeI) vector, and the recombinant plasmid was named pACYC-TMN-Nluc-ΔGDD-Rep.

[0029] The primer sequences are shown in Table 1. The methods of PCR amplification, ligation, transformation and selection of positive clones were the same as in Example 1.

[0030] Example 3 Application of pACYC-TMN-Nluc-Rep reporter replicon (TMN replicon) 1. Application of replicon in BHK21 cells BHK-21 cells were seeded into 48-well cell plates, and TMN WT and GDD replicons were transfected when the confluence reached 70%-90%. According to the instructions of the full-form gold transfection reagent, 0.3μg plasmid, 0.6μl transfection reagent, and 25μlopti-MEM were prepared for each well, and DMEM maintenance solution containing 2% FBS was replaced after 6 hours of transfection. Cell samples were collected at 12h, 24h, 36h, 48h, 60h, and 72h, and NanoLuc luciferase activity was detected using the Nano-Glo™ Luciferase Assay kit. The specific operation is as follows: (1) Aspirate the cell culture medium, rinse the cells carefully twice with PBS, add 65 μl Glo Lysis buffer, lyse at room temperature for 5 min, scrape the cells into an EP tube, centrifuge at 12000g for 10 min at 4°C, and freeze the supernatant at -80°C for later use; (2) Add 100 μl of Nano-Glo Luciferase Assay Reagent equilibrated to room temperature to a light-proof 96-well plate. Carefully pipette 20 μl of cell lysate into the 96-well plate, mix well, and detect the activity of the NanoLuc reporter gene in a luminometer.

[0031] The results show (see Figure 5), in BHK21 cells, the NanoLuc fluorescence values ​​produced by the TMN replicon WT group and the ΔGDD group had a high expression level 12 hours after transfection, which was caused by the direct translation expression of the transfected plasmid. Subsequently, the NanoLuc luciferase activity in the WT group showed a trend of first rising and then falling, reaching a peak at 48h after transfection, and the luciferase activity was significantly higher than that in the ΔGDD group, which was the proliferation caused by replicon replication. This shows that the TMN replicon can be effectively translated and replicated in BHK21 cells. However, the fluorescence value produced by the ΔGDD group did not fluctuate significantly over time from 12h onwards, and there was no rising curve representing viral replication, indicating that the RdRp enzyme of the NS5 protein of the Usutu virus is the same as other flaviviruses, and the RdRp enzyme activity center is the three GDD amino acids from positions 667 to 669. Mutations in these three amino acids will directly affect viral replication.

[0032] 2. Application of Replicons in Other Cells DF-1, Vero, and Hela cells were seeded into 48-well cell plates and transfected with TMN WT and GDD replicons when the confluence reached 70%-90%. According to the instructions of the full-form gold transfection reagent, 0.3μg of plasmid, 0.6μl of transfection reagent, and 25μl of opti-MEM were prepared for each well. After 6h of transfection, the DMEM maintenance medium containing 2% FBS was replaced. Cell samples were collected at 12h, 24h, 48h, and 72h, and NanoLuc luciferase activity was detected using the Nano-Glo™ Luciferase Assay kit.

[0033] The results show (see Figure 6 ), in DF-1, Vero, and Hela cells, the TMN replicon NanoLuc luciferase activity reached a peak at 48h after transfection, which was about 40 times higher than that at 12h after transfection, and the luciferase activity was significantly higher than that of the negative control ΔGDD group, which was the proliferation caused by the replication of the replicon. This shows that the TMN replicon can be effectively translated and replicated in DF-1, Vero, and Hela cells. Therefore, the USUV reporter replicon pACYC-TMN-Nluc-Rep constructed in the present invention can be used as an important research tool in the study of USUV virus.

[0034] The specification and drawings of the present invention are considered to be illustrative rather than restrictive. On the basis of the present invention, those skilled in the art can make some substitutions and modifications to some of the technical features according to the disclosed technical content without creative labor, all of which are within the protection scope of the present invention.

Claims

1. A method for constructing a Usutu virus reporter replicon, characterized in that: The following steps are involved: S1, using the gene fragment P2 of the USUV-TMN strain as a template, amplify the fragment P2, connect it with the double-enzyme-digested (EcoRV, KpnI) vector of the pACYC-TMN-P3 plasmid to obtain a recombinant plasmid named pACYC-TMN-P2-3; S2, using the gene fragment P1 of the USUV-TMN strain as a template, amplify the fragment P1, connect it with the double-enzyme-cut (XhoI, EcoRV) vector of the pACYC-TMN-P2-3 plasmid to obtain a recombinant plasmid named pACYC-TMN-P1-3; S3. Using the duck Tembusu virus (TMUV) NanoLuc reporter replicon as a template, the NanoLuc+FMDV-2A gene sequence was amplified and ligated with the pACYC-TMN-P1-3 plasmid double restriction enzyme digestion (NruI, EcoRV) vector to obtain the replicon pACYC-TMN-Nluc-Rep.

2. The construction method according to claim 1, characterized in that: The amplification primers in S1 are TMN-P2-3-F and TMN-P2-3-R, and the sequences are shown in SEQ ID NO.1-2.

3. The construction method according to claim 1, characterized in that: The amplification primers in S2 are TMN-P1-3-F and TMN-P1-3-R, and the sequences are shown in SEQ ID NO.3-4.

4. The construction method according to claim 1, characterized in that: The amplification primers in S3 are TMN-Nluc-F and TMN-Nluc-R, and the sequences are shown in SEQ ID NO.5-6.

5. The Usutu virus reporter replicon obtained by the construction method as described in any one of claims 1-4.

6. The use of the Usutu virus reporter replicon in Usutu virus research as claimed in claim 5, characterized in that: The Usutu virus reporter replicon can be efficiently translated and replicated in cells, including BHK21, Vero, DF-1 and Hela cells.

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