Flavivirus mini-replicon, infectious particle, preparation method and pharmaceutical composition

Abstract

Provided are a flavivirus mini-replicon, a flavivirus single-round infectious particle, a preparation method therefor, and a pharmaceutical composition thereof. The flavivirus mini-replicon comprises a promoter, a 5' untranslated region, a first nucleic acid fragment, an exogenous gene, a second nucleic acid fragment and a 3' untranslated region. The flavivirus single-round infectious particle carries the flavivirus mini-replicon. The pharmaceutical composition contains the flavivirus single-round infectious particle and a pharmaceutically acceptable carrier. The flavivirus mini-replicon can effectively express the exogenous gene and thus can serve as an expression vector for expressing an exogenous gene in mammals and for use in gene therapy.

Description

Flavivirus minireplicons, infectious particles, preparation methods and pharmaceutical compositions Technical Field

[0001] The present invention relates to a replicon, infectious particles, preparation methods and pharmaceutical compositions thereof, in particular to a flavivirus minireplicon, flavivirus single infectious particles, preparation methods and pharmaceutical compositions thereof. Background Art

[0002] A replicon is a self-replicating RNA or DNA sequence. Flavivirus replicons, for example, possess the inherent ability to replicate, enabling them to amplify the original template within host cells, thereby increasing the production of multiple encoded proteins. Therefore, they can be used to express proteins or as gene delivery vectors, for example, to express therapeutic proteins or antigens for vaccine production, or in gene therapy applications.

[0003] Zika virus (ZIKV) and Japanese encephalitis virus (JEV) are both enveloped viruses of the Flaviviridae family. Their genome consists of positive-sense single-strand RNA ((+)ssRNA) approximately 11 kilobases in length. This genome encodes a polyprotein, which is post-translationally modified to form ten mature viral proteins, including three structural proteins and seven non-structural proteins. Flaviviridae virus particles are primarily composed of structural proteins, including capsid proteins, tropism proteins, and envelope proteins. Capsid proteins form an icosahedral capsid that encapsidates the nucleic acid gene; envelope proteins are responsible for forming antigens in Flaviviridae viruses and are associated with their toxicity and pathogenicity; tropism proteins form chaperones with envelope proteins and participate in the maturation process of Flaviviridae viruses. The seven non-structural proteins are involved in Flaviviridae replication.

[0004] Flavivirus replicons are typically designed by removing the structural proteins required for viral assembly while retaining essential elements and nonstructural protein genes for self-replication. These replicons are then embedded in an expression vector with a promoter, enabling self-replication and protein translation in prokaryotic or eukaryotic cells. However, due to the large size of the nonstructural protein genes of Zika and Japanese encephalitis viruses, they must be synthesized in segments during construction. Furthermore, the sequences of Zika and Japanese encephalitis viruses are inherently unstable, leading to large mutations during flavivirus replicon construction. Therefore, improving these challenges in flavivirus replicon construction remains a key challenge.

[0005] Summary of the Invention

[0006] In view of this, an object of the present invention is to provide a flavivirus minireplicon that eliminates redundant genes from Zika virus and Japanese encephalitis virus, retaining only elements relevant to efficient expression of exogenous genes and production of self-replicating RNA. This results in a flavivirus minireplicon with minimized molecular size, effectively reducing the problem of large mutations caused by sequence instability in Zika virus and Japanese encephalitis virus, and enabling efficient expression of exogenous genes. Therefore, it can be used as an expression vector for expressing exogenous genes in mammals and for gene therapy applications.

[0007] Another object of the present invention is to provide a flavivirus infectious particle carrying a flavivirus minireplicon, by transfecting the flavivirus minireplicon into assembly cells to obtain a flavivirus single infectious particle capable of transmitting exogenous genes, and the assembly cells can stably express the structural proteins of Flaviviridae viruses.

[0008] One aspect of the present invention provides a flavivirus minireplicon comprising a promoter, a 5' untranslated region, a first nucleic acid segment, an exogenous gene, a second nucleic acid segment, and a 3' untranslated region, wherein the sequence of the first nucleic acid segment is as shown in SEQ ID NO:1 or SEQ ID NO:2, and when the sequence of the first nucleic acid segment is SEQ ID NO:1, the sequence of the second nucleic acid segment is as shown in SEQ ID NO:3 or SEQ ID NO:4, and when the sequence of the first nucleic acid segment is SEQ ID NO:2, the sequence of the second nucleic acid segment is as shown in SEQ ID NO:5 or SEQ ID NO:6.

[0009] The flavivirus minireplicon may further comprise a third nucleic acid segment connected to the 5' end of the second nucleic acid segment. When the sequence of the second nucleic acid segment is SEQ ID NO:4, the sequence of the third nucleic acid segment is as shown in SEQ ID NO:7. When the sequence of the second nucleic acid segment is SEQ ID NO:6, the sequence of the third nucleic acid segment is as shown in SEQ ID NO:8.

[0010] The flavivirus minireplicon may further comprise a ribozyme and a polyadenylation sequence, wherein the ribozyme and the polyadenylation sequence are linked to the 3' end of the 3' untranslated region.

[0011] The flavivirus minireplicon may further comprise a multiple cloning site constructed between the first nucleic acid fragment and the second nucleic acid fragment.

[0012] The flavivirus minireplicon may further comprise at least one 2A peptide encoding sequence, wherein the at least one 2A peptide encoding sequence is linked to the exogenous gene.

[0013] According to the aforementioned flavivirus minireplicon, the number of the at least one 2A peptide encoding sequence can be two, and each of the at least one 2A peptide encoding sequences is different.

[0014] According to the aforementioned flavivirus minireplicon, the promoter can be a CMV promoter or a SV40 promoter.

[0015] Another aspect of the present invention provides a method for preparing flavivirus single infectious particles, comprising transfecting the flavivirus minireplicon described in the preceding paragraph into an assembly cell to obtain a transfected cell, wherein the assembly cell stably expresses a structural protein of a Flaviviridae virus; and culturing the transfected cell at a culture temperature for an assembly time, and collecting a culture medium after culture, wherein the culture medium after culture contains the flavivirus single infectious particles.

[0016] According to the aforementioned method for preparing flavivirus single infectious particles, the Flaviviridae virus may be Zika virus, Japanese encephalitis virus, dengue virus, yellow fever virus, West Nile virus or tick-borne encephalitis virus.

[0017] According to the aforementioned method for preparing flavivirus single infectious particles, the structural protein may comprise a membrane protein and a set of membrane proteins.

[0018] According to the aforementioned method for preparing flavivirus single infectious particles, the structural protein may further comprise a capsid protein.

[0019] Another aspect of the present invention is to provide a flavivirus single infectious particle, which is prepared by the method for preparing the flavivirus single infectious particle described in the previous paragraph.

[0020] Another aspect of the present invention is to provide a pharmaceutical composition comprising the flavivirus single infectious particles as described in the preceding paragraph and a pharmaceutically acceptable carrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] To make the above and other objects, features, advantages and embodiments of the present invention more apparent, the following descriptions of the accompanying drawings are given:

[0022] FIG1A is a schematic diagram illustrating the construction of the flavivirus minireplicon of the present invention;

[0023] FIG1B is a flow chart illustrating the steps of the method for preparing flavivirus single infectious particles of the present invention;

[0024] FIG2A is a schematic diagram illustrating the construction of flavivirus minireplicons according to Examples 1, 2, and 3 of the present invention;

[0025] FIG2B is a schematic diagram illustrating the construction of the pBR322-Linker vector;

[0026] FIG3A is a schematic diagram showing the construction of ZIKV prM-E expression plasmid, JEV prM-E expression plasmid, and FLAV prM-E expression plasmid;

[0027] FIG3B is a fluorescence micrograph of transfected cells after transfection with the flavivirus minireplicon of Example 1, Example 2, or Example 3 of the present invention;

[0028] Figures 4A, 4B, 4C, and 4D are diagrams showing the quantitative and qualitative analysis results of flavivirus single infectious particles according to Examples 4 and 5 of the present invention;

[0029] FIG5 is a schematic diagram illustrating the construction of flavivirus minireplicons according to Examples 6, 7, and 8 of the present invention;

[0030] 6A and 6B are diagrams showing the quantitative and qualitative analysis results of flavivirus single infectious particles according to Examples 9 and 10 of the present invention;

[0031] Figures 7A, 7B, 7C, 7D, 7E, 7F, 7G, and 7H are graphs showing the analysis results of CFP-YFP expression kinetics in different cells infected with single infectious particles of the flavivirus of Examples 9 and 10 of the present invention at different viral infection doses;

[0032] 8A, 8B, 8C and 8D are graphs showing the analysis results of the survival rates of different cells infected with single infectious particles of the flavivirus of Examples 9 and 10 of the present invention;

[0033] FIG9 is a schematic diagram illustrating the construction of flavivirus minireplicons according to Examples 11 and 12 of the present invention;

[0034] FIG10A and FIG10B are diagrams showing the analysis results of the infectivity and quantitative evaluation of single infectious particles of flaviviruses according to Examples 13 and 14 of the present invention;

[0035] FIG11A and FIG11B are graphs showing analysis results of exogenous protein expression in mice injected with a single dose of flavivirus infectious particles of Example 14 of the present invention;

[0036] FIG12 is a schematic diagram illustrating the construction of flavivirus minireplicons according to Examples 15, 16, and 17 of the present invention;

[0037] FIG13A is a schematic diagram showing the construction of ZIKV C-prM-E expression plasmid, JEV C-prM-E expression plasmid, and FLAV C-prM-E expression plasmid;

[0038] Figure 13B is a fluorescence micrograph of TE671 cells after transfection with the JEV C-prM-E expression plasmid alone, transfection with the ZIKV C-prM-E expression plasmid alone, co-transfection with Example 15 of the present invention and the JEV C-prM-E expression plasmid, or co-transfection with Example 15 of the present invention and the ZIKV C-prM-E expression plasmid;

[0039] FIG14A is a graph showing the results of analyzing the infectivity of single infectious particles of the flavivirus of Example 16; and

[0040] FIG14B is a graph showing the results of analyzing the infectivity of single flavivirus infectious particles of Example 17.

[0041] Wherein, the reference numerals are: 100: flavivirus minireplicon 110: promoter 120: 5' non-translated region 130: first nucleic acid fragment 140: foreign gene 150: second nucleic acid fragment 160: 3' non-translated region 200: method for preparing flavivirus single infectious particles 210, 220: steps DETAILED DESCRIPTION

[0042] The seven nonstructural proteins of Zika virus are related to the replication and translation of viral genes, namely NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5. NS1 is primarily responsible for early viral replication in cells; NS2A participates in viral replication and assembly, and antagonizes the host's innate immune response; NS2B is a cofactor of NS3; NS3, comprising serine protease, nucleoside 5'-triphosphatase (NTPase), and RNA helicase, is the most functional protein; NS4A participates in viral membrane formation and helps the NS3 helicase retain energy during unwinding by regulating adenosine triphosphate (ATPase) activity; NS4B inhibits the host's antiviral mechanisms; NS5, comprising RNA-dependent RNA polymerase (RdRp) and methyltransferase (MTase), is responsible for the composition of viral RNA and the formation of the 5'-end capping structure.

[0043] [Flaviviral minireplicons]

[0044] Please refer to Figure 1A, which is a schematic diagram illustrating the construction of a flavivirus minireplicon 100 of the present invention. The flavivirus minireplicon 100 comprises a promoter 110, a 5' untranslated region 120, a first nucleic acid segment 130, an exogenous gene 140, a second nucleic acid segment 150, and a 3' untranslated region 160. The sequence of the first nucleic acid segment 130 is shown in SEQ ID NO:1 or SEQ ID NO:2. When the sequence of the first nucleic acid segment 130 is SEQ ID NO:1, the sequence of the second nucleic acid segment 150 is shown in SEQ ID NO:3 or SEQ ID NO:4; when the sequence of the first nucleic acid segment 130 is SEQ ID NO:2, the sequence of the second nucleic acid segment 150 is shown in SEQ ID NO:5 or SEQ ID NO:6. Furthermore, the flavivirus minireplicon 100 may further comprise a third nucleic acid segment (not shown) linked to the 5' end of the second nucleic acid segment 150. When the sequence of the second nucleic acid fragment 150 is SEQ ID NO:4, the sequence of the third nucleic acid fragment is shown as SEQ ID NO:7; when the sequence of the second nucleic acid fragment 150 is SEQ ID NO:6, the sequence of the third nucleic acid fragment is shown as SEQ ID NO:8.

[0045] The flavivirus minireplicon 100 is a DNA-derived replicon, and thus the promoter 110 may be a CMV promoter or an SV40 promoter, which have the advantages of being stable in sequence and can be directly transfected into cells in the form of a plasmid for expression.

[0046] Furthermore, the flavivirus minireplicon 100 may further comprise a ribozyme (not shown) and a polyadenine sequence (not shown). The ribozyme and polyadenine sequence are attached to the 3' end of the 3' untranslated region 160. The ribozyme truncates excess RNA after transcription, while the polyadenine sequence drives RNA polyadenylation and termination. The ribozyme may be a hepatitis D virus ribozyme (HDVr), and the polyadenine sequence may be the polyadenylation signal (poly A) of bovine growth hormone (BGH) or the polyadenine sequence of SV40.

[0047] Furthermore, the flavivirus minireplicon 100 may further include a multiple cloning site (MCS) (not shown) constructed between the first nucleic acid segment 130 and the second nucleic acid segment 150, so that the exogenous gene 140 can be inserted into the first nucleic acid segment 130 and the second nucleic acid segment 150 in a variety of possible insertion positions or methods.

[0048] Furthermore, the flavivirus minireplicon 100 may further comprise at least one 2A peptide encoding sequence (not shown). The at least one 2A peptide encoding sequence is linked to the exogenous gene 140. The 2A peptide translated from the 2A peptide encoding sequence has the ability to self-cleave and can cleave the exogenous protein translated from the exogenous gene 140 during a subsequent post-translational modification phase. The number of at least one 2A peptide encoding sequence may be two, and each of the at least one 2A peptide encoding sequence is different. For example, the at least one 2A peptide encoding sequence may be a sequence encoding an F-2A peptide, a sequence encoding a T-2A peptide, a sequence encoding a P-2A peptide, or a sequence encoding an E-2A peptide, which respectively encode an F-2A peptide, a T-2A peptide, a P-2A peptide, and an E-2A peptide. The F-2A peptide is derived from foot-and-mouth disease virus (FMDV), the T-2A peptide is derived from thoseaasigna virus (TaV), the P-2A peptide is derived from porcine teschovirus (PTV), and the E-2A peptide is derived from equine rhinitis A virus (ERAV).

[0049] [Flaviviral single infectious particles]

[0050] 1B , which is a flow chart illustrating the steps of a method 200 for preparing a flavivirus single infectious particle according to the present invention. In FIG1B , the method 200 for preparing a flavivirus single infectious particle comprises steps 210 and 220 .

[0051] Step 210 is to transfect the flavivirus minireplicon into the assembly cells to obtain transfected cells, wherein the assembly cells stably express the structural proteins of the Flaviviridae virus. The Flaviviridae virus can be a member of the Flaviviridae virus family, such as Zika virus (ZIKV), Japanese encephalitis virus (JEV), dengue virus (DV), yellow fever virus (YFV), West Nile virus (WNV), or tick-borne encephalitis virus (TBEV). The structural proteins can include a precursor membrane protein (prM) and an envelope protein (E), and can further include a capsid protein (C).

[0052] Step 220 involves culturing the transfected cells at a temperature for a predetermined time and collecting the culture medium. The culture medium contains flavivirus single infectious particles. The resulting flavivirus single infectious particles are selective for target cells and can be used as a delivery vehicle for exogenous gene expression in vitro and in vivo.

[0053] [Pharmaceutical composition]

[0054] The pharmaceutical composition of the present invention comprises the flavivirus single-shot infectious particles of the present invention and a pharmaceutically acceptable carrier. Due to the ability of the flavivirus single-shot infectious particles to deliver exogenous genes in vitro and in vivo, the pharmaceutical composition of the present invention can be used in targeted gene therapy or cell therapy, such as gene therapy involving gene expression and gene silencing.

[0055] The technical term "pharmaceutically acceptable" refers to an amount sufficient to have a medical effect without causing side effects, and can be easily determined by those skilled in the art based on factors commonly known in the medical field, such as the type of disease, the patient's age, weight, health status, gender, drug sensitivity, route of administration, method of administration, frequency of administration, treatment period, and the planned combination or simultaneous administration of drugs.

[0056] For administration to mammals, the pharmaceutical compositions of the present invention can be prepared by suspending flavivirus single infectious particles in a pharmaceutically acceptable carrier, such as, but not limited to, glycerol, water, buffered saline, ethanol, and other pharmaceutically acceptable salt solutions, such as phosphates and organic acid salts.

[0057] Pharmaceutically acceptable carriers may vary for different routes of administration, including binders, lubricants, disintegrants, excipients, solubilizers, homogenizers, suspending agents, colorants, flavorings, or combinations thereof for oral administration; buffers, preservatives, analgesics, solubilizers, isotonic agents, tranquilizers, or combinations thereof for injections, which may be used in combination; and bases, excipients, lubricants, preservatives, or combinations thereof for topical administration.

[0058] The present invention is further illustrated by the following specific experimental examples, which are intended to facilitate those with common knowledge in the technical field to which the present invention relates, so that they can fully utilize and practice the present invention without excessive interpretation. These experimental examples should not be construed as limiting the scope of the present invention, but are intended to illustrate how to implement the materials and methods of the present invention.

[0059] First embodiment

[0060] In a first embodiment, the 5' untranslated region, first nucleic acid segment, second nucleic acid segment, third nucleic acid segment, and 3' untranslated region used in the flavivirus minireplicon are derived from Zika virus. The sequence of the first nucleic acid segment is shown in SEQ ID NO:1, which comprises the complete capsid gene sequence of Zika virus. The sequence of the second nucleic acid segment is shown in SEQ ID NO:3 or SEQ ID NO:4, wherein the sequence shown in SEQ ID NO:3 comprises the RdRp gene of Zika virus, and the sequence shown in SEQ ID NO:4 comprises the complete NS5 sequence of Zika virus, i.e., the RdRp gene and MTase gene of Zika virus. The sequence of the third nucleic acid segment is shown in SEQ ID NO:7, which comprises a partial NS4A sequence and a complete NS4B sequence of Zika virus.

[0061] 1.1. Construction of a flavivirus minireplicon carrying the GFP gene

[0062] In this experiment, flavivirus minireplicons according to Examples 1, 2, and 3 of the present invention (hereinafter referred to as Examples 1, 2, and 3) were constructed. Please refer to Figures 2A and 2B. Figure 2A schematically illustrates the construction of flavivirus minireplicons according to Examples 1, 2, and 3 of the present invention, while Figure 2B schematically illustrates the construction of the pBR322-Linker vector. The expression vectors used in Examples 1, 2, and 3 were pBR322-Linker vectors, which were modified from the pBR322 vector (New England Biolabs, Inc.). The difference lies in the insertion of the sequence shown in SEQ ID NO: 9 between the restriction enzyme sites EcoRI and BamHI within the multiple cloning site of the pBR322 vector. This sequence comprises the restriction enzyme sites KpnI, NotI, and XhoI, as well as the polyadenylation sequence of SV40 (hereinafter referred to as SV40pA). The promoter included in Examples 1, 2, and 3 is the CMV promoter (represented by "CMV" in FIG. 2A ), the exogenous gene included is the GFP gene (represented by "GFP" in FIG. 2A ), which encodes green fluorescent protein (GFP), and the at least one 2A peptide encoding sequence is two, namely, a T-2A peptide encoding sequence (represented by "T-2A" in FIG. 2A ) and an F-2A peptide encoding sequence (represented by "F-2A" in FIG. 2A ). The ribozyme and polyadenylation sequence included are the HDVr and BGH polyadenylation sequences (hereinafter referred to as "BGHpA"), respectively. The difference between Examples 1 and 2 lies in the second nucleic acid sequence, while the difference between Example 3 and Example 2 lies in the inclusion of a third nucleic acid sequence.

[0063] The insert sequence of Example 1 includes, in order, a CMV promoter as set forth in SEQ ID NO:10, a 5' untranslated region as set forth in SEQ ID NO:11 (represented as 5'UTR in FIG. 2A ), a first nucleic acid sequence as set forth in SEQ ID NO:1 (represented as C in FIG. 2A ), a multiple cloning site as set forth in SEQ ID NO:12, a sequence encoding a T-2A peptide as set forth in SEQ ID NO:13, a GFP gene as set forth in SEQ ID NO:14, a sequence encoding an F-2A peptide as set forth in SEQ ID NO:15, a second nucleic acid sequence as set forth in SEQ ID NO:3 (represented as RdRp in FIG. 2A ), a 3' untranslated region as set forth in SEQ ID NO:16 (represented as 3'UTR in FIG. 2A ), HDVr as set forth in SEQ ID NO:17, and BGHpA as set forth in SEQ ID NO:18.

[0064] The insert sequence of Example 2 of the present invention includes, in order, a CMV promoter as shown in SEQ ID NO:10, a 5' untranslated region as shown in SEQ ID NO:11 (represented as 5'UTR in FIG. 2A ), a first nucleic acid sequence as shown in SEQ ID NO:1 (represented as C in FIG. 2A ), a multiple cloning site as shown in SEQ ID NO:12, a sequence encoding a T-2A peptide as shown in SEQ ID NO:13, a GFP gene as shown in SEQ ID NO:14, a sequence encoding an F-2A peptide as shown in SEQ ID NO:15, a second nucleic acid sequence as shown in SEQ ID NO:4 (represented as NS5 in FIG. 2A ), a 3' untranslated region as shown in SEQ ID NO:16 (represented as 3'UTR in FIG. 2A ), HDVr as shown in SEQ ID NO:17, and BGHpA as shown in SEQ ID NO:18.

[0065] The insert sequence of Example 3 of the present invention comprises, in order, a CMV promoter as set forth in SEQ ID NO:10, a 5' untranslated region as set forth in SEQ ID NO:11 (represented as 5'UTR in FIG. 2A ), a first nucleic acid sequence as set forth in SEQ ID NO:1 (represented as C in FIG. 2A ), a multiple cloning site as set forth in SEQ ID NO:12, a sequence encoding a T-2A peptide as set forth in SEQ ID NO:13, a GFP gene as set forth in SEQ ID NO:14, a sequence encoding an F-2A peptide as set forth in SEQ ID NO:15, a third nucleic acid sequence as set forth in SEQ ID NO:7 (represented as NS4A' and NS4B in FIG. 2A ), a second nucleic acid sequence as set forth in SEQ ID NO:4 (represented as NS5 in FIG. 2A ), a 3' untranslated region as set forth in SEQ ID NO:16 (represented as 3'UTR in FIG. 2A ), HDVr as set forth in SEQ ID NO:17, and BGHpA as set forth in SEQ ID NO:18.

[0066] The constructed Example 1, Example 2 and Example 3 were respectively verified by gel electrophoresis for size and sequence analysis, and were confirmed to contain the above-mentioned insert arrangement.

[0067] 1.2. Evaluation of the role of the Zika virus nonstructural protein NS4B and MTase in RdRp-mediated RNA replication driven by the flavivirus minireplicon using single infectious particles carrying the GFP gene

[0068] In the experiment, assembly cells were first constructed. Please refer to Figure 3A, which is a schematic diagram showing the construction of ZIKV prM-E expression plasmids, JEV prM-E expression plasmids, and FLAV prM-E expression plasmids. The expression vector used was the pcDNA3.1(+ / -) vector (Addgene). The pcDNA3.1(+ / -) vector was cut with EcoRI and XhoI. Then, ZIKV prM-E, which contains the tropism and envelope genes of Zika virus, JEV prM-E, which contains the tropism and envelope genes of Japanese encephalitis virus, and FLAV prM-E, which contains the tropism and envelope genes of Flavivirus (FLAV), were respectively inserted into the pcDNA3.1(+ / -) vector carrying the neomycin (Neo) selection gene to obtain ZIKV prM-E expression plasmids, JEV prM-E expression plasmids, and FLAV prM-E expression plasmids. The sequence of ZIKV prM-E is shown in SEQ ID NO: 19, the sequence of JEV prM-E is shown in SEQ ID NO: 20, and FLAVprM-E represents a nucleotide sequence encoding the membrane tropism protein and envelope protein of a Flaviviridae virus, such as dengue virus, yellow fever virus, West Nile virus, or tick-borne encephalitis virus. The ZIKV prM-E expression plasmid, the JEV prM-E expression plasmid, or the FLAVprM-E expression plasmid were then transfected into HEK293T cells to obtain assembled cells stably expressing the membrane tropism protein and envelope protein of Zika virus (hereinafter referred to as ZIKV prM-E co-expressing cells), assembled cells stably expressing the membrane tropism protein and envelope protein of Japanese encephalitis virus, or assembled cells stably expressing the membrane tropism protein and envelope protein of other Flaviviridae viruses.

[0069] To confirm the RNA self-replication driven by the flavivirus minireplicon, the constructed examples 1, 2, and 3 were transfected into ZIKV prM-E co-expressing cells to obtain transfected cells. The transfected cells were then analyzed for cytopathic effects (CPE) and expression of GFP and the Zika virus RdRp protein.

[0070] FIG3B is a fluorescence micrograph of transfected cells transfected with the flavivirus minireplicons of Examples 1, 2, or 3 of the present invention. Mock represents a control group not transfected with the flavivirus minireplicons. Immunofluorescence staining results showed that after 72 hours, expression of the Zika virus RdRp protein was observed in the transfected cells of Examples 1, 2, and 3. In particular, significant cytopathic effects and strong fluorescent spots were observed in the cells of Examples 2 and 3.

[0071] To evaluate the self-replication of flavivirus minireplicons, single infectious particles of the flavivirus of Example 4 at passage 0 (hereinafter referred to as Example 4, which was transfected with Example 2) and single infectious particles of the flavivirus of Example 5 at passage 0 (hereinafter referred to as Example 5, which was transfected with Example 3) were collected from the culture medium of cells transfected with the ZIKV prM-E co-expressing cells of Example 2 or Example 3. The flavivirus of Example 4 at passage 0 and Example 5 at passage 0 were then used to infect ZIKV prM-E co-expressing cells to obtain flavivirus of Example 4 at passage 1 and Example 5 at passage 1, respectively. The yields of flavivirus single infectious particles of Example 4 and Example 5 at passage 1 were then determined by TCID50 assay and qualitatively analyzed by Western blotting and dot blot.

[0072] Please refer to Figures 4A, 4B, 4C, and 4D, which are graphs showing the quantitative and qualitative analysis results of flavivirus single infectious particles according to Examples 4 and 5 of the present invention. Figure 4B shows the Western blot analysis results of envelope protein (E), Figure 4C shows the Western blot analysis results of tropism protein (prM), and Figure 4D shows the dot blot analysis results of capsid protein (C). In Figures 4B to 4D, the sample in lane 1 is the culture medium of the cells after culture in the control group, the sample in lane 2 is the culture medium of the cells after culture in Example 4, and the sample in lane 3 is the culture medium of the cells after culture in Example 5.

[0073] The results in FIG4A show that the first generation of Example 4 has a potency of 5.5×10 5 TCID50 / ml, while the potency of Example 5 of the first generation was 1.3×10 6 TCID50 / ml. Figures 4B to 4D show Western blotting and dot blot analysis results demonstrating the presence of three Zika virus structural proteins—envelope protein, tropism protein, and capsid protein—in passage 1 of Example 4 and passage 1 of Example 5. These results demonstrate that passage 0 flavivirus single infectious particles have the ability to produce large quantities of passage 1 flavivirus single infectious particles in assembled cells.

[0074] 1.3. Construction of flavivirus minireplicons and flavivirus single infectious particles carrying the CFP / YFP fusion gene

[0075] To provide the flavivirus minireplicons of the present invention with more selective niches and to test the possibility of inserting other exogenous genes, flavivirus minireplicons of Examples 6, 7, and 8 of the present invention (hereinafter referred to as Examples 6, 7, and 8) were experimentally constructed. Please refer to FIG5 , which is a schematic diagram illustrating the construction of the flavivirus minireplicons of Examples 6, 7, and 8 of the present invention. The expression vectors used in Examples 6, 7, and 8 were pBR322-Linker vectors. The promoter contained in Examples 6, 7, and 8 was the CMV promoter (represented as CMV in FIG5 ). The at least one 2A peptide encoding sequence contained two sequences, one encoding a T-2A peptide (represented as T-2A in FIG5 ) and one encoding an F-2A peptide (represented as F-2A in FIG5 ). The ribozyme and polyadenylation sequences contained were HDVr and BGHpA, respectively. The exogenous gene included in Example 6, Example 7 and Example 8 is a CFP / YFP fusion gene (represented by CYP in FIG5 ), which encodes cyan fluorescent protein and yellow fluorescent protein (Cyan fluorescent protein-Yellowfluorescent protein, CFP-YFP).

[0076] The insert sequence of Example 6 of the present invention includes, in order, a CMV promoter as shown in SEQ ID NO:10, a 5' untranslated region as shown in SEQ ID NO:11 (represented as 5'UTR in FIG. 5 ), a first nucleic acid sequence as shown in SEQ ID NO:1 (represented as C in FIG. 5 ), a CFP / YFP fusion gene as shown in SEQ ID NO:21, a first multiple cloning site as shown in SEQ ID NO:22, a sequence encoding a T-2A peptide as shown in SEQ ID NO:13, a sequence encoding an F-2A peptide as shown in SEQ ID NO:15, a second multiple cloning site as shown in SEQ ID NO:23, a second nucleic acid sequence as shown in SEQ ID NO:3 (represented as RdRp in FIG. 5 ), a 3' untranslated region as shown in SEQ ID NO:16 (represented as 3'UTR in FIG. 5 ), HDVr as shown in SEQ ID NO:17, and BGHpA as shown in SEQ ID NO:18.

[0077] The insert sequence of Example 7 of the present invention includes, in order, a CMV promoter as shown in SEQ ID NO:10, a 5' untranslated region as shown in SEQ ID NO:11 (represented as 5'UTR in FIG. 5 ), a first nucleic acid sequence as shown in SEQ ID NO:1 (represented as C in FIG. 5 ), a CFP / YFP fusion gene as shown in SEQ ID NO:21, a first multiple cloning site as shown in SEQ ID NO:22, a sequence encoding a T-2A peptide as shown in SEQ ID NO:13, a sequence encoding an F-2A peptide as shown in SEQ ID NO:15, a second multiple cloning site as shown in SEQ ID NO:23, a second nucleic acid sequence as shown in SEQ ID NO:4 (represented as NS5 in FIG. 5 ), a 3' untranslated region as shown in SEQ ID NO:16 (represented as 3'UTR in FIG. 5 ), HDVr as shown in SEQ ID NO:17, and BGHpA as shown in SEQ ID NO:18.

[0078] The insert sequence of Example 8 of the present invention comprises, in order, a CMV promoter as set forth in SEQ ID NO:10, a 5' untranslated region as set forth in SEQ ID NO:11 (represented as 5'UTR in FIG. 5 ), a first nucleic acid sequence as set forth in SEQ ID NO:1 (represented as C in FIG. 5 ), a CFP / YFP fusion gene as set forth in SEQ ID NO:21, a first multiple cloning site as set forth in SEQ ID NO:22, a sequence encoding a T-2A peptide as set forth in SEQ ID NO:13, a sequence encoding an F-2A peptide as set forth in SEQ ID NO:15, a second multiple cloning site as set forth in SEQ ID NO:23, a third nucleic acid sequence as set forth in SEQ ID NO:7 (represented as NS4A' and NS4B in FIG. 5 ), a second nucleic acid sequence as set forth in SEQ ID NO:4 (represented as NS5 in FIG. 5 ), a 3' untranslated region as set forth in SEQ ID NO:16 (represented as 3'UTR in FIG. 5 ), HDVr as set forth in SEQ ID NO:17, and BGHpA as set forth in SEQ ID NO:18.

[0079] The constructed Example 6, Example 7 and Example 8 were respectively verified by gel electrophoresis for size and sequence analysis, and were confirmed to contain the above-mentioned insert arrangement.

[0080] The constructed Example 7 and Example 8 were then transfected into ZIKV prM-E co-expressing cells, respectively. Passage 0 flavivirus single infectious particles of Example 9 (hereinafter referred to as Example 9, which was transfected with Example 7) and passage 0 flavivirus single infectious particles of Example 10 (hereinafter referred to as Example 10, which was transfected with Example 8) were collected from the cell culture medium of the ZIKV prM-E co-expressing cells transfected with Example 7 or Example 8. Passage 0 Example 9 and passage 0 Example 10 were then used to infect ZIKV prM-E co-expressing cells, respectively, to obtain passage 1 Example 9 and passage 10. The infectivity of passage 0 Example 9 and passage 0 Example 10 was then observed using a fluorescence microscope, and the yield of flavivirus single infectious particles of Example 9 and Example 10 was determined by TCID50 assay.

[0081] Please refer to Figures 6A and 6B for quantitative and qualitative analysis results of flavivirus single infectious particles from Examples 9 and 10 of the present invention, where "Mock" represents a control group uninfected with flavivirus single infectious particles. The results in Figure 6A show that compared to the first-passage Example 9, the 0th-passage Example 10 elicited a more pronounced cytopathic effect, CFP-YFP expression, and Zika virus RdRp protein expression in infected cells, resulting in a higher yield of the first-passage Example 10 compared to the 0th-passage Example 9. The observation results in Figures 6B and 4A demonstrate that the RNA of the flavivirus minireplicons of Examples 3 and 8 possesses excellent self-replication capabilities under the synergistic action of NS4B, MTase, and RdRp, significantly increasing the yield of flavivirus single infectious particles from Examples 5 and 10.

[0082] 1.4. Comparative Analysis of Exogenous Gene Expression in Different Infected Cells by Flavivirus Single Infectious Particles of the Present Invention

[0083] Furthermore, to confirm the expression kinetics of the RNA self-replicating reporter gene driven by the flavivirus minireplicon of the present invention, HEK293T cells, A549 cells, TE671 cells, and SF268 cells were infected with the virus at a multiplicity of infection (MOI) of 0.5, 1, and 2 according to Examples 9 and 10, respectively, and the expression level of CFP-YFP was evaluated by detecting the FRET signal.

[0084] Please refer to Figures 7A to 7H , which show the analysis results of CFP-YFP expression dynamics in different cells infected with single flavivirus infectious particles from Examples 9 and 10 of the present invention at different viral infection doses. Figures 7A and 7B show the analysis results in HEK293T cells, Figures 7C and 7D show the analysis results in A549 cells, Figures 7E and 7F show the analysis results in TE671 cells, and Figures 7G and 7H show the analysis results in SF268 cells. Mock represents a control group not infected with single flavivirus infectious particles. Data in Figures 7A to 7H are presented as mean ± SD, with * indicating p < 0.05, ** indicating p < 0.01, and *** indicating p < 0.001.

[0085] The results in Figures 7A, 7B, 7E, and 7F show that in HEK293T and TE671 cells, the CFP-YFP signal intensity driven by the flavivirus minireplicons of Examples 7 and 8 peaked 72 hours after infection with a single dose of flavivirus infectious particles from Examples 9 and 10. The results in Figures 7C and 7D show that when A549 cells were infected with Examples 9 and 10 at MOIs of 1 and 2, the CFP-YFP signal intensity driven by the flavivirus minireplicons of Examples 7 and 8 decreased 72 hours after infection, but this was not observed in the group infected with an MOI of 0.5. The results in Figures 7G and 7H show that in SF268 cells, the CFP-YFP signal driven by the flavivirus minireplicons of Examples 7 and 8 was lower 48 hours after infection than 24 hours after infection, regardless of the MOI of 0.5, 1, or 2.

[0086] Please also refer to Figures 8A to 8D , which show the analysis results of the survival rate of different cells infected with a single dose of flavivirus infectious particles from Examples 9 and 10 of the present invention. In Figures 8A to 8D , HEK293T cells, A549 cells, TE671 cells, or SF268 cells were infected with Example 9 or Example 10 at MOIs of 0.1, 1, and 2, respectively. Figure 8A shows the analysis results in HEK293T cells, Figure 8B shows the analysis results in A549 cells, Figure 8C shows the analysis results in TE671 cells, and Figure 8D shows the analysis results in SF268 cells. Data in Figures 8A to 8D are presented as mean ± SD, with * indicating p < 0.05, ** indicating p < 0.01, and *** indicating p < 0.001.

[0087] The survival analysis results in Figures 8A to 8D show that infection with Examples 9 and 10 produced cytotoxic effects on HEK293T cells, TE671 cells, A549 cells, and SF268 cells. At the same MOI, cells infected with Example 9 had a higher survival rate than cells infected with Example 10. Furthermore, when cells were infected with Example 10, the survival rate of the infected cells was MOI-dependent. Notably, at an MOI of 2, infection with Example 10 resulted in a greater than 30% decrease in the survival rate of SF268 cells compared to SF268 cells infected with Example 9.

[0088] 1.5. In vivo expression analysis of exogenous genes delivered by flavivirus single infectious particles of the present invention

[0089] To further verify that the flavivirus single infectious particles of the present invention can deliver exogenous genes not only in vitro but also in vivo, flavivirus minireplicons of Examples 11 and 12 of the present invention (hereinafter referred to as Examples 11 and 12) were experimentally constructed. Please refer to FIG9 , which shows a schematic diagram of the construction of the flavivirus minireplicons of Examples 11 and 12 of the present invention. The expression vectors used in Examples 11 and 12 were pBR322-Linker vectors, and the promoters contained in Examples 11 and 12 were CMV promoters (represented as CMV in FIG9 ). The at least one 2A peptide encoding sequence contained in each of the two sequences was two, namely, a T-2A peptide encoding sequence (represented as T-2A in FIG9 ) and an F-2A peptide encoding sequence (represented as F-2A in FIG9 ). The ribozyme and polyadenylation sequences contained in each of the two sequences were HDVr and BGHpA, respectively. The exogenous genes included in Example 11 and Example 12 are the hACE2 gene encoding human angiotensin-converting enzyme 2 (hACE2) (represented as hACE2 in FIG9 ) and the GFP gene encoding GFP (represented as GFP in FIG9 ).

[0090] The insert sequence of Example 11 of the present invention includes, in order, a CMV promoter as shown in SEQ ID NO:10, a 5' untranslated region as shown in SEQ ID NO:11 (represented as 5'UTR in FIG. 9 ), a first nucleic acid sequence as shown in SEQ ID NO:1 (represented as C in FIG. 9 ), an hACE2 gene as shown in SEQ ID NO:24, a sequence encoding a T-2A peptide as shown in SEQ ID NO:13, a GFP gene as shown in SEQ ID NO:14, a sequence encoding an F-2A peptide as shown in SEQ ID NO:15, a second nucleic acid sequence as shown in SEQ ID NO:4 (represented as NS5 in FIG. 9 ), a 3' untranslated region as shown in SEQ ID NO:16 (represented as 3'UTR in FIG. 9 ), HDVr as shown in SEQ ID NO:17, and BGHpA as shown in SEQ ID NO:18.

[0091] The insert sequence of Example 12 of the present invention includes, in order, a CMV promoter as shown in SEQ ID NO:10, a 5' untranslated region as shown in SEQ ID NO:11 (represented as 5'UTR in FIG. 9 ), a first nucleic acid sequence as shown in SEQ ID NO:1 (represented as C in FIG. 9 ), an hACE2 gene as shown in SEQ ID NO:24, a sequence encoding a T-2A peptide as shown in SEQ ID NO:13, a GFP gene as shown in SEQ ID NO:14, a sequence encoding an F-2A peptide as shown in SEQ ID NO:15, a third nucleic acid sequence as shown in SEQ ID NO:7 (represented as NS4A' and NS4B in FIG. 9 ), a second nucleic acid sequence as shown in SEQ ID NO:4 (represented as NS5 in FIG. 9 ), a 3' untranslated region as shown in SEQ ID NO:16 (represented as 3'UTR in FIG. 9 ), HDVr as shown in SEQ ID NO:17, and BGHpA as shown in SEQ ID NO:18.

[0092] The constructed Example 11 and Example 12 were then size-verified by gel electrophoresis and sequence analysis, confirming that they each contained the aforementioned insert arrangement. The constructed Example 11 and Example 12 were then transfected into ZIKV prM-E co-expressing cells. Passage 0 single infectious particles of the flavivirus of Example 13 (hereinafter referred to as Example 13, which was transfected with Example 11) and passage 0 single infectious particles of the flavivirus of Example 14 (hereinafter referred to as Example 14, which was transfected with Example 12) were collected from the culture medium of the ZIKV prM-E co-expressing cells transfected with Example 11 or Example 12. Passage 0 Example 13 and passage 0 Example 14 were then used to infect ZIKV prM-E co-expressing cells, respectively, to obtain passage 1 Example 13 and passage 1 Example 14. The infectivity of Example 13 at passage 0 and Example 14 at passage 0 was observed using a fluorescence microscope, the relative mRNA levels of Example 11 and Example 12 in Example 1 at passage 1 and Example 14 at passage 1 were detected using real-time RT PCR, and the flavivirus single infectious particle yield of Example 13 at passage 1 and Example 14 at passage 1 was determined by TCID50 assay.

[0093] Please refer to Figures 10A and 10B , which show the results of the analysis and quantitative evaluation of the infectivity of flavivirus single infectious particles of Examples 13 and 14 of the present invention. "Mock" represents a control group uninfected with flavivirus single infectious particles. The results show that after infection with passage 0 of Example 13 or passage 0 of Example 14, a mild cytopathic effect was observed in prM-E co-expressing cells, demonstrating that passage 0 of Example 13 and passage 0 of Example 14 are capable of producing large quantities of passage 1 of Example 13 and passage 1 of Example 14 in assembled cells. As shown in Figure 10A , infection with Example 14 resulted in significant hACE2 mRNA expression, resulting in higher production of passage 1 of Example 14 compared to passage 1 of Example 13, as shown in Figure 10B .

[0094] To further evaluate the expression of hACE2 protein in vivo, BALB / c mice were used for the assay. The mice were divided into two groups. The first group received intravenous injection of phosphate buffered saline (PBS) on day 1 and served as the control group. The second group received intravenous injection of PBS on day 1.

[0095] Example 14. On day 6, lung and brain samples collected from each group of mice were comprehensively analyzed. Immunohistochemistry (IHC) staining of lung and brain tissues from different groups was performed using an anti-hACE2 antibody (GeneTex) to examine hACE2 protein expression induced by Example 14.

[0096] Please refer to Figure 11A , which shows the results of IHC staining for hACE2 protein expression in mice injected with the drug from Example 14. The results in Figure 11A show that hACE2 protein expression was observed in the peripheral blood vessels and alveolar regions of the lung tissue of the second group of mice, while no hACE2 protein expression was observed in the first group of mice. Furthermore, hACE2 protein was also observed in the brain capillaries of the second group of mice.

[0097] In the experiment, hematoxylin-eosin (H&E) staining was used to perform histological analysis on the lung and brain tissues of mice to further evaluate the effect of Example 14 on lung and brain inflammation. Please refer to Figure 11B, which shows the H&E staining results of the lung and brain tissues of mice injected with Example 14. The results in Figure 11B show that the second group of mice that received an intravenous injection of Example 14 showed mild inflammation in the lung and brain tissues. In contrast, the lung tissues of the first group of mice that received an intravenous injection of PBS did not show significant changes. The above results show that intravenous injection of Example 14 in mice can lead to the expression of hACE2 protein in both the lungs and brains of mice, indicating that the flavivirus single infectious particles of the present invention have the potential for gene delivery in vivo.

[0098] Second embodiment

[0099] In a second embodiment, the 5' untranslated region, first nucleic acid segment, second nucleic acid segment, third nucleic acid segment, and 3' untranslated region used in the flavivirus minireplicon are derived from Japanese encephalitis virus. The sequence of the first nucleic acid segment is shown in SEQ ID NO:2, which comprises a partial capsid gene sequence of Japanese encephalitis virus. The sequence of the second nucleic acid segment is shown in SEQ ID NO:5 or SEQ ID NO:6, wherein the sequence shown in SEQ ID NO:5 comprises the RdRp gene of Japanese encephalitis virus, and the sequence shown in SEQ ID NO:6 comprises the complete NS5 sequence of Japanese encephalitis virus, i.e., the RdRp gene and MTase gene of Japanese encephalitis virus. The sequence of the third nucleic acid segment is shown in SEQ ID NO:8, which comprises a partial NS4A sequence and a complete NS4B sequence of Japanese encephalitis virus.

[0100] 2.1. Construction of a flavivirus minireplicon carrying the GFP gene

[0101] In this experiment, flavivirus minireplicons according to Examples 15, 16, and 17 of the present invention (hereinafter referred to as Examples 15, 16, and 17) were constructed. Please refer to FIG12 , which is a schematic diagram illustrating the construction of flavivirus minireplicons according to Examples 15, 16, and 17 of the present invention. The expression vectors used in Examples 15, 16, and 17 were pBR322-Linker vectors. The promoters contained in Examples 15, 16, and 17 were CMV promoters (represented as CMV in FIG12 ), the exogenous genes contained were GFP genes (represented as GFP in FIG12 ), the at least one 2A peptide-encoding sequence contained in Examples 16 and 17 was an F-2A peptide-encoding sequence (represented as F-2A in FIG12 ), and the ribozyme and polyadenylation sequences contained in Examples 15, 16, and 17 were HDVr and SV40pA, respectively.

[0102] The insert sequence of Example 15 of the present invention includes, in order, a CMV promoter as shown in SEQ ID NO:10, a 5' untranslated region as shown in SEQ ID NO:25 (represented as 5'UTR in FIG12 ), a first nucleic acid sequence as shown in SEQ ID NO:2 (represented as C in FIG12 ), a GFP gene as shown in SEQ ID NO:14, a second nucleic acid sequence as shown in SEQ ID NO:5 (represented as RdRp in FIG12 ), a 3' untranslated region as shown in SEQ ID NO:26 (represented as 3'UTR in FIG12 ), HDVr as shown in SEQ ID NO:27, and SV40pA as shown in SEQ ID NO:28.

[0103] The insert sequence of Example 16 of the present invention includes, in order, a CMV promoter as shown in SEQ ID NO:10, a 5' untranslated region as shown in SEQ ID NO:25 (represented as 5'UTR in FIG. 12 ), a first nucleic acid sequence as shown in SEQ ID NO:2 (represented as C in FIG. 12 ), a GFP gene as shown in SEQ ID NO:14, a sequence encoding an F-2A peptide as shown in SEQ ID NO:15, a second nucleic acid sequence as shown in SEQ ID NO:6 (represented as NS5 in FIG. 12 ), a 3' untranslated region as shown in SEQ ID NO:26 (represented as 3'UTR in FIG. 12 ), HDVr as shown in SEQ ID NO:27, and SV40pA as shown in SEQ ID NO:28.

[0104] The insert sequence of Example 17 of the present invention includes, in order, a CMV promoter as shown in SEQ ID NO:10, a 5' untranslated region as shown in SEQ ID NO:25 (represented as 5'UTR in FIG. 12 ), a first nucleic acid sequence as shown in SEQ ID NO:2 (represented as C in FIG. 12 ), a GFP gene as shown in SEQ ID NO:14, a sequence encoding an F-2A peptide as shown in SEQ ID NO:15, a third nucleic acid sequence as shown in SEQ ID NO:8 (represented as NS4A' and NS4B in FIG. 12 ), a second nucleic acid sequence as shown in SEQ ID NO:6 (represented as NS5 in FIG. 12 ), a 3' untranslated region as shown in SEQ ID NO:26 (represented as 3'UTR in FIG. 12 ), HDVr as shown in SEQ ID NO:27, and SV40pA as shown in SEQ ID NO:28.

[0105] The constructed Example 15, Example 16 and Example 17 were respectively verified for size by gel electrophoresis and sequence analysis, and were confirmed to contain the above-mentioned insert arrangement.

[0106] 2.2. Evaluation of the self-replication capacity of flavivirus minireplicons using single infectious particles of flavivirus carrying the GFP gene

[0107] Because the flavivirus minireplicon of embodiment 2 lacks the NS2B-NS3 protease of Japanese encephalitis virus, it is unable to cleave the capsid protein at the C-terminal transmembrane domain. Experimentally, assembly cells capable of stably expressing the capsid protein of Japanese encephalitis virus were first constructed. Please refer to FIG13A , which schematically illustrates the construction of ZIKV C-prM-E expression plasmids, JEV C-prM-E expression plasmids, and FLAV C-prM-E expression plasmids. The expression vector used was the pcDNA3.1(+) vector (Addgene). Nucleic acid fragments encoding structural proteins of Flaviviridae viruses were then inserted into the pcDNA3.1(+) vector via multiple cloning sites to produce the ZIKV C-prM-E expression plasmids, JEV C-prM-E expression plasmids, and FLAV C-prM-E expression plasmids. The insert sequence of the ZIKV C-prM-E expression plasmid is the complete capsid gene of Japanese encephalitis virus as shown in SEQ ID NO: 29 (represented as JEV C in FIG. 13A ), the sequence encoding the T-2A peptide as shown in SEQ ID NO: 13 (represented as T-2A in FIG. 13A ), and the ZIKV prM-E as shown in SEQ ID NO: 19. The insert sequence of the JEV C-prM-E expression plasmid is the capsid gene of Japanese encephalitis virus as shown in SEQ ID NO: 29, the sequence encoding the T-2A peptide as shown in SEQ ID NO: 13, and the JEV prM-E as shown in SEQ ID NO: 20. The insert sequence of the FLAV C-prM-E expression plasmid is the capsid gene of Japanese encephalitis virus as shown in SEQ ID NO: 29, the sequence encoding the T-2A peptide as shown in SEQ ID NO: 13, and the FLAV prM-E. prM-E represents a nucleotide sequence encoding the tropism and envelope proteins of a Flaviviridae virus, such as dengue virus, yellow fever virus, West Nile virus, or tick-borne encephalitis virus. The ZIKV C-prM-E expression plasmid, the JEV C-prM-E expression plasmid, and the FLAVC-prM-E expression plasmid were then transfected into TE671 cells, respectively, to generate assembled cells containing ZIKV C-prM-E, JEV C-prM-E, and FLAV C-prM-E co-expressing cells. These assembled cells can stably express the capsid protein of Japanese encephalitis virus, as well as the tropism and envelope proteins of Zika virus, Japanese encephalitis virus, or other Flaviviridae viruses.

[0108] To evaluate the self-replication ability of the RNA driven by Example 15, the constructed ZIKV C-prM-E expression plasmid or JEV C-prM-E expression plasmid was transfected into TE671 cells alone, or Example 15 was co-transfected with the ZIKV C-prM-E expression plasmid or JEV C-prM-E expression plasmid into TE671 cells. Cytopathic effect and GFP expression analyses were performed 72 hours after transfection.

[0109] Please refer to Figure 13B, which shows fluorescence micrographs of TE671 cells transfected with the JEV C-prM-E expression plasmid alone, the ZIKV C-prM-E expression plasmid alone, co-transfected with Example 15 of the present invention and the JEV C-prM-E expression plasmid, or co-transfected with Example 15 of the present invention and the ZIKV C-prM-E expression plasmid. The results in Figure 13B show that TE671 cells co-transfected with Example 15 of the present invention and the JEV C-prM-E expression plasmid, or co-transfected with Example 15 of the present invention and the ZIKV C-prM-E expression plasmid, exhibited significant cytopathic effects and strong fluorescent spots. In contrast, TE671 cells transfected with the JEV C-prM-E expression plasmid alone or the ZIKV C-prM-E expression plasmid alone did not exhibit these effects.

[0110] 2.3. Infectivity of Flavivirus Single Infectious Particles and RNA Self-Replication Driven by Flavivirus Minireplicons

[0111] In order to evaluate the infectivity of the flavivirus single infectious particles of embodiment 2 and the self-replication of RNA driven by the flavivirus minireplicon of embodiment 2, the constructed Example 15 was transfected into JEV C-prM-E co-expressing cells or ZIKV C-prM-E co-expressing cells, respectively. The flavivirus single infectious particles of Example 16 at passage 0 (hereinafter referred to as Example 16, transfected into JEV C-prM-E co-expressing cells) and the flavivirus single infectious particles of Example 17 at passage 0 (hereinafter referred to as Example 17, transfected into ZIKV C-prM-E co-expressing cells) were collected from the culture medium of the JEV C-prM-E co-expressing cells transfected with Example 15 or the ZIKV C-prM-E co-expressing cells transfected with Example 15. C-prM-E co-expressing cells), TE671 cells were infected with Example 16 of passage 0 and Example 17 of passage 0 at an MOI of 1 (hereinafter referred to as high MOI) and an MOI of 0.1 (hereinafter referred to as low MOI), respectively. After 48 hours, the infectivity of Example 16 and Example 17, as well as the cytopathic effect and GFP expression in TE671 cells were observed by fluorescence microscopy.

[0112] Please refer to Figures 14A and 14B , which show the results of analyzing the infectivity of flavivirus single infectious particles according to Embodiment 2. Figure 14A shows the analysis results of Example 16, and Figure 14B shows the analysis results of Example 17. "Mock" represents a control group uninfected with flavivirus single infectious particles. The results show that significant cytopathic effects and strong fluorescent spots were observed in TE671 cells infected with passage 0 of Example 16 and passage 0 of Example 17, but these effects were not observed in the mock-infected control group. This demonstrates that the flavivirus minireplicon of Example 15 has excellent self-replication capacity, enabling the production of large quantities of flavivirus single infectious particles of Examples 16 and 17.

[0113] In summary, the flavivirus minireplicons of the present invention, which eliminate redundant genes from Zika virus and Japanese encephalitis virus, can effectively reduce the problem of large mutations caused by sequence instability in Zika virus and Japanese encephalitis virus. Furthermore, they can effectively express exogenous genes and, therefore, can be used as expression vectors for expressing exogenous genes in mammals and for gene therapy. By transfecting the flavivirus minireplicons of the present invention into assembled cells stably expressing structural proteins of Flaviviridae viruses, the flavivirus single infectious particles of the present invention are obtained. These particles have the ability to serve as delivery vehicles for exogenous gene expression in vitro and in vivo, and can therefore be used as pharmaceutical compositions to deliver exogenous genes or mRNA vaccines, and can be applied in targeted gene therapy or cell therapy. The flavivirus single infectious particles of the present invention can also serve as research platforms to understand the replication mechanisms and pathological phenomena of Zika virus and Japanese encephalitis virus, or for vaccine development and antiviral drug research.

[0114] Although the present invention has been disclosed above in terms of embodiments, they are not intended to limit the present invention. Anyone skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the scope defined by the appended claims.

Claims

1. A flavivirus minireplicon, characterized in that, It comprises a promoter, a 5' untranslated region, a first nucleic acid fragment, a foreign gene, a second nucleic acid fragment, and a 3' untranslated region, wherein the sequence of the first nucleic acid fragment is as shown in SEQ ID NO:1 or SEQ ID NO:2, and when the sequence of the first nucleic acid fragment is SEQ ID NO:1, the sequence of the second nucleic acid fragment is as shown in SEQ ID NO:3 or SEQ ID NO:4, and when the sequence of the first nucleic acid fragment is SEQ ID NO:2, the sequence of the second nucleic acid fragment is as shown in SEQ ID NO:5 or SEQ ID NO:

6.

2. The flavivirus minireplicon according to claim 1, characterized in that, It further comprises a third nucleic acid fragment linked to the 5' end of the second nucleic acid fragment, and when the sequence of the second nucleic acid fragment is SEQ ID NO:4, the sequence of the third nucleic acid fragment is as shown in SEQ ID NO:7, and when the sequence of the second nucleic acid fragment is SEQ ID NO:6, the sequence of the third nucleic acid fragment is as shown in SEQ ID NO:

8.

3. The flavivirus microreplicator according to claim 1, wherein, It further comprises a ribozyme and a polyadenine sequence, and the ribozyme and the polyadenine sequence are linked to the 3' end of the 3' untranslated region.

4. The flavivirus minireplicon according to claim 1, wherein It further comprises a multiple cloning site constructed between the first nucleic acid fragment and the second nucleic acid fragment.

5. The flavivirus microreplicator according to claim 1, characterized in that, It further comprises at least one coding 2A peptide sequence, and the at least one coding 2A peptide sequence is linked to the foreign gene.

6. The flavivirus minireplicon according to claim 5, wherein The number of the at least one coding 2A peptide sequence is two, and each of the at least one coding 2A peptide sequence is different.

7. The flavivirus microreplicator according to claim 1, wherein The promoter is a CMV promoter or an SV40 promoter.

8. A method for preparing single infectious particles of flavivirus, characterized in that, It comprises: Transfecting the flavivirus minireplicon according to any one of claims 1 to 7 into an assembly cell to obtain a transfected cell, wherein the assembly cell stably expresses a structural protein of a flavivirus; and Culturing the transfected cell at a culture temperature for an assembly time and collecting a culture supernatant of the cultured cell, wherein the culture supernatant of the cultured cell contains a flavivirus single infectious particle.

9. The method for preparing a single infectious particle of flavivirus according to claim 8, characterized in that, The flavivirus is Zika virus, Japanese encephalitis virus, dengue virus, yellow fever virus, West Nile virus or tick-borne encephalitis virus.

10. The method for preparing flavivirus single infectious particles according to claim 8, characterized in that, The structural protein comprises a transmembrane protein and an envelope protein.

11. The method for preparing a single infectious particle of flavivirus according to claim 10, characterized in that, The structural protein further comprises a capsid protein.

12. A flavivirus single infectious particle, characterized in that, It is prepared by the method for preparing a flavivirus single infectious particle as described in claim 8.

13. A pharmaceutical composition, characterized in that, It comprises: The flavivirus single infectious particle as described in claim 12; and A pharmaceutically acceptable carrier.

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