TWI726212B - Recombinant baculoviruses and uses thereof - Google Patents

Abstract

Disclosed herein are recombinant baculoviruses suitable for introducing an exogenous gene into a pest insect, particularly, disease-transmitting mosquitos. The recombinant baculovirus is characterized in having a promotor that is any of a HzNV-1 viral early expressing genepag 1, a ceropin geneb1 , a defensin genea4, or hsp70 gene; and an exogenous gene operably linked thereto the promoter. Also disclosed herein is a method of introducing an exogenous gene into a pest insect. The method includes transducing the pest insect with a recombinant baculovirus without suppressing the production of microRNAs (miRNAs) in the pest insect, wherein the recombinant baculovirus comprises a promoter ofpa g1, cecropinb1 , defensin genea4, orhsp70.

Description

Recombinant baculovirus and its use

The present disclosure is about modified vectors for gene delivery. More specifically, the present disclosure relates to a recombinant baculovirus, whereby an exogenous gene is delivered to a mosquito (especially a mosquito that transmits diseases).

Aedes aegypti (Aedes aegypti, also known as yellow fever mosquitoes) and A. albopictus (A. albopictus, also known as Asian tiger mosquitoes or forest mosquitoes) can transmit a variety of potentially deadly human viruses (including dengue virus, chick’s disease) (chikungunya) virus, Zika (zika) virus, yellow fever (yellow fever) virus and Mayaro (Mayaro) virus), and also transmit some filarial nematodes (Dirofilaria immitis ) And other diseases. Culex tritaeniorhynchus ( Culex tritaeniorhynchus ) is the main vector of Japanese encephalitis, and Anopheles sinensis (Anopheles sinensis) spreads malaria and lymphatic filariasis. Although those skilled in the art have made unremitting efforts to study mosquito gene regulation and control mosquito-borne diseases, the lack of effective and flexible gene delivery methods still hinders the study of virus/host interaction and mosquito biology. An effective gene delivery system that can span different mosquito species and deliver genes into different organs of cells, larvae and adults can obviously become an indispensable tool for such research, and will have many other important applications in biological research.

In view of this, it is necessary for the prior art in the art to develop a tool and/or method that can transfer genes into mosquitoes, so as to make it easier to study and control mosquitoes to transmit dangerous viruses to humans and/or livestock.

In order to provide readers with a basic understanding, the following provides a brief summary of the present disclosure. This summary of the invention is not an extensive overview of the disclosure, and is not used to identify the key/essential elements of the invention or outline the scope of the invention. Its sole purpose is to present some concepts of this disclosure in a simplified conceptual form as a prelude to the more detailed description presented later.

The present invention as a whole is about modified vectors and their uses. The modified vector is suitable for transferring foreign genes into mosquitoes, especially mosquitoes that can transmit diseases, such as A. aegypti , A. albopictus , and C. tritaeniorhynchus ) And A. sinensis .

Accordingly, the first aspect of the present invention mainly provides a recombinant baculovirus capable of delivering foreign genes into mosquitoes. This recombinant baculovirus is characterized by a HzNV-1 viral early expressing gene (viral early expressing gene) pag1 , an antimicrobial peptide gene (cecropin gene) b1 , a defensin gene (defensin gene) a4 , or a heat shock protein 70 (hsp70) gene promoter, and an exogenous gene operably linked to the promoter.

According to non-essential embodiments of the present disclosure, the recombinant baculovirus may further include a reporter gene, which is operably linked to the promoter and the foreign gene, and is used to encode an expression that is easy to track and/or manipulate The report protein. Examples of reporter proteins include, but are not limited to, green fluorescence protein (green fluorescence protein, GFP), enhanced green fluorescence protein (enhanced green fluorescence protein, EGFP), and discosoma red fluorescent protein (Discosoma sp. red fluorescent protein, DsRed), blue fluorescent protein (blue fluorescence protein, BFP), enhanced yellow fluorescent protein (enhanced yellow fluorescent proteins, EYFP) , anemone Ma Hanuo fluorescent protein (Anemonia majano fluorescent protein, amFP ), Zoanthus fluorescent protein (ZFP), Discosoma fluorescent protein (dsFP), and Clavularia fluorescent protein (CFP). In a preferred embodiment, the reporter protein is EGFP.

Examples of baculoviruses suitable for use in the present invention include, but are not limited to, Autographa californica multiple nucleopolyhedrovirus (AcMNPV), Autographa falclfera MNPV (AfMNPV), Anticarsia gemmatalis MNPV (AgMNPV), Bombyx mori MNPV (BmMNPV), Buzura suppressaria single nucleopolyhedrovirus (BsSNPV), Tomato armyworm Mononuclear Polyhedrosis Virus ( Helicoverpa armigera SNPV, HaSNPV), Helicoverpa zea SNPV (HzSNPV), Leaf Roller Moth Polynuclear Polyhedrosis Virus (Lymantria dispar MNPV, LdMNPV), P. Virus ( Orgyia pseudotsugata MNPV , OpMNPV), Spodoptera frugiperda MNPV (SfMNPV), Spodoptera exigua MNPV (SeMNPV), and Spodoptera exigua MNPV (SeMNPV) Virus ( Trichoplusia ni MNPV, TnMNPV). According to a preferred embodiment of the present disclosure, the recombinant baculovirus is recombinant Autographa californica polynuclear polyhedrosis virus (AcMNPV).

The second aspect of the present disclosure relates to a method for introducing foreign genes into a mosquito body through the recombinant baculovirus of the present invention. The method includes transducing the recombinant baculovirus of the present invention to mosquitoes without inhibiting the production of microRNAs (miRNAs) in the mosquitoes. The recombinant baculovirus of the present disclosure is characterized by having a promoter, which can be a HzNV-1 virus early expression gene pag1 , an antimicrobial peptide gene b1 , a defensin gene a4 , or a heat shock protein 70 (hsp70) Gene; and a foreign gene operably linked to the promoter.

According to the present disclosure, a reagent is used to inhibit the production of microRNA in mosquitoes, and the reagent results in the inhibition of Drosha, Dicer, toll-like receptor 2, STAT1, STAT6, interleukin 7R, or interleukin 1A.

According to an embodiment of the present disclosure, the mosquito is A. aegypti or A. albopictus .

According to embodiments of the present disclosure, mosquitoes may be mosquito cells, larvae, and adults.

Examples of baculoviruses suitable for the present invention include, but are not limited to, Autographa californica polynuclear polyhedrosis virus (AcMNPV), Spodoptera exigua polynuclear polyhedrosis virus (AfMNPV), Spodoptera exigua polynuclear polyhedrosis virus (AcMNPV) AgMNPV), Bombyx mori multinucleated polyhedrosis virus (BmMNPV), Tung-footworm mononuclear polyhedrosis virus (BsSNPV), Tomato armyworm mononuclear polyhedrosis virus (HaSNPV), Helicoverpa armigera mononuclear polyhedrosis virus (HzSNPV), Leaf roller polyhedrosis virus Type Polyhedrosis Virus (LdMNPV), Pseudosciaena chinensis Polynuclear Polyhedrosis Virus (OpMNPV), Spodoptera frugiperda Polynuclear Polyhedrosis Virus (SfMNPV), Beet Armyworm Polynuclear Polyhedrosis Virus (SeMNPV), and Sphagnum sp. Multinuclear polyhedrosis virus (TnMNPV). According to a preferred embodiment of the present disclosure, the recombinant baculovirus is recombinant Autographa californica polynuclear polyhedrosis virus (AcMNPV).

According to an optional embodiment of the present disclosure, the recombinant baculovirus further includes a reporter gene operably linked to the promoter and the foreign gene, and the reporter gene can encode a reporter protein, which can be a green fluorescent protein (GFP), Enhanced Green Fluorescent Protein (EGFP), Disc Anemone Red Fluorescent Protein (DsRed), Blue Fluorescent Protein (BFP), Enhanced Yellow Fluorescent Protein (EYFP), Anemone Mahano Fluorescent protein (amFP), button coral fluorescent protein (zFP), disc sea anemone fluorescent protein (dsFP) or feather coral fluorescent protein (cFP).

According to the embodiments of the present disclosure, the recombinant baculovirus will not replicate in mosquitoes.

According to the embodiments of the present disclosure, foreign genes can be successfully used in the head, proboscis, legs, haltere, midgut malpighian tubule, ovaries, and crops of adult mosquitoes. It is manifested in crop and fat body.

After referring to the following embodiments, those skilled in the art to which the present invention pertains can easily understand the basic spirit and other objectives of the present invention, as well as the technical means and implementation aspects of the present invention.

In order to make the description of the present disclosure more detailed and complete, the following provides an illustrative description for the implementation aspects and specific embodiments of the present invention; this is not the only way to implement or use the specific embodiments of the present invention. The implementation manners cover the features of multiple specific embodiments, and the method steps and sequences used to construct and operate these specific embodiments. However, other specific embodiments can also be used to achieve the same or equal functions and sequence of steps.

For ease of description, the specific terms described in the specification, the embodiments and the appended patent scope are collectively described here. Unless otherwise defined herein, all technical and scientific terms herein have the same meaning as those commonly known to those with ordinary knowledge in the technical field to which the present invention belongs.

The present disclosure is about the recombinant baculovirus and the method for transmitting mosquitoes using the recombinant baculovirus of the present invention.

1. Definition

The term "baculoviruses" as used herein refers to an arthropod-specific double-stranded DNA virus that can be used to control pests and diseases (for example, mosquitoes). Nuclear polyhedrosis viruses (NPV) are a subgroup of baculoviruses. Many baculoviruses are suitable as vectors for expressing foreign proteins in infected hosts, including those infected with cotton bollworm (scientific name Helicoverpa zea ), tobacco budworm (scientific name: Heliothis virescens ), and fir Tussock moth (Douglas-fir tussock moth, scientific name: Orygia pseudotsugata ), gypsy moth (gypsy moth, scientific name: Lymantria dispar ), alfalfa looper (scientific name: Autographa californica ), European pine sawfly , Scientific name: Neodiiprion sertifer ) and apple leaf curling moth (codling moth, scientific name: Cydia pomonella ). Generally speaking, baculoviruses with a wide host range are preferred, such as Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Examples of baculoviruses suitable for the present disclosure include, but are not limited to, AcMNPV, Anagrapha falcifera (Anagrapha falcifera) polynuclear polyhedrosis virus (AfMNPV), Soybean armyworm ( Anticarsia gemmatalis ) polynuclear polyhedrosis virus (AgMNPV), Bombyx mori ( Bombyx mori ) polynuclear polyhedrosis virus (BmMNPV), tung-footworm mononuclear polyhedrovirus ( Buzura suppressaria single nucleopolyhedrovirus, BsSNPV), tomato armyworm ( Helicoverpa armigera ) mononuclear polyhedrosis virus (HaSNPV), cotton bollworm ( Helicoverpa) zea ) Mononuclear Polyhedrosis Virus (HzSNPV), Leaf Roller Moth (Lymantria dispar ) Polynuclear Polyhedrosis Virus (LdMNPV), Orgyia pseudotsugata Polynuclear Polyhedrosis Virus (OpMNPV), Spodoptera frugiperda (Spodoptera frugiperda) Polynuclear polyhedrosis virus (SfMNPV), Spodoptera exigua polynuclear polyhedrosis virus (SeMNPV) and Trichoplusia ni polynuclear polyhedrosis virus (TnMNPV).

"Gene encodes or encoding" refers to a nucleic acid that contains sequence information of structural RNA (such as rRNA, tRNA) or the primary amino acid sequence of a specific protein or peptide. This term specifically includes degenerate codons of the natural sequence (degenerate codons, that is, different codons encoding a single amino acid), or can be introduced into a sequence that conforms to the preferred codons of a specific host cell.

This disclosure uses "an exogenous gene" (an exogenous gene) to generally refer to nucleic acids that are isolated, transfected, and linked to nucleic acids that are not bound to it in their natural state, and/or except for cells or cell environments in their natural state. Other than the discovered nucleic acid or protein, the nucleic acid introduced into and/or expressed in the cell or the cell environment. The aforementioned terms include nucleic acids originally obtained from different species or cell types different from the cell types expressed, and also include nucleic acids obtained from the same cell line as the expressed cell line.

When the “recombinant” used in the present disclosure is used as a modifier of a vector (nucleic acid), such as a recombinant viral vector and sequence modification (such as recombinant polynucleotide and polypeptide), it means The composition has been modified in a way that does not normally occur in nature (e.g., genetically engineered). A specific example of a recombinant vector (such as a recombinant baculovirus vector) may be a recombinant vector in which a polynucleotide that is not normally present in the genome of a wild-type virus is inserted into the genome of the virus.

"Operably linked" as used in the present disclosure refers to the linkage between nucleic acid fragments. Under this functional relationship, when genes are expressed, each gene fragment can operate without functional problems. However, when a nucleic acid fragment is connected to other nucleic acid fragments, its function and performance can be affected by other nucleic acid fragments. For example, the aforementioned term refers to the functional linkage between a nucleic acid sequence encoding a desired protein and a nucleic acid expression control sequence, in such a way that general functions are allowed. The conventional genetic recombination technology in the technical field of the present invention can be used to prepare operable linkages, and at the same time, site-directed DNA cleavage and ligation can be performed by using enzymes known in the art.

The term "transduce" used in the present disclosure refers to the use of a vector (for example, the recombinant baculovirus vector of the present disclosure) to introduce nucleic acid into a cell or host organism. The introduction of transgenes into cells by recombinant baculovirus can be called "transduction" of cells. The transgene may or may not be integrated with the genomic nucleic acid of transduced cells (such as sf12 cells and mosquito C6/36 cells). If the introduced transgene is integrated into the nucleic acid (genomic DNA) of the recipient cell or host organism, it can be stably maintained in the cell or organism and further transmitted, or inherited to the offspring of the recipient cell or host organism Cell or organism. Finally, the introduced transgene may persist outside the chromosomes of the recipient cell or host organism, or temporarily exist in the recipient cell or host organism.

The term "transfection" or "transfection" as used in the present disclosure refers to the process of introducing nucleic acid into host cells without the involvement of viruses or viral particle vectors. In other words, the introduction of nucleic acid into host cells by using an agent (especially when an appropriate amount of nucleic acid and transfection lipid is added) is sufficient to overcome the harmful effects of serum in the medium on transfection. The transfection agent may be a commercially available multivalent cation (for example, polybrene). The effective amount of transfection agent refers to the amount that produces a measurable increase in transfection efficiency for a given host cell cultured with serum.

The "reporter protein" used in the present disclosure refers to a protein that is engineered to behave with the protein of interest and induce visually recognizable characteristics (usually involving fluorescence and luminescent proteins). For example, the gene encoding jellyfish green fluorescent protein (GFP), which causes cells to emit green light under blue light, or luciferase enzyme, which can catalyze the reaction with luciferin to emit light. Examples of reporter proteins include, but are not limited to: GFP, Sea Anemone Red Fluorescent Protein (DsRed), Enhanced Green Fluorescent Protein (EGFP), Blue Fluorescent Protein (BFP), Enhanced Yellow Fluorescent Protein (EYFP) ), Anemone Mahano Fluorescent Protein (amFP), Button Coral Fluorescent Protein (zFP), Disc Anemone Fluorescent Protein (dsFP) and Feather Coral Fluorescent Protein (cFP).

Unless the context clearly indicates otherwise, the singular forms "一" (a, an) and "the" used in this article both include plural forms. Except for the experimental examples, or unless otherwise explicitly stated, it should be understood that all the numerical values used to represent the components and reaction conditions described in the present disclosure are modified by "about". Therefore, unless otherwise stated to the contrary, the numerical parameters disclosed in the present disclosure are approximate values, and can be changed according to the desired properties that the present invention attempts to obtain.

2. Recombinant baculovirus

The present disclosure mainly provides a recombinant baculovirus, which can introduce foreign DNA into a pest insect, especially mosquitoes that can transmit diseases.

In order to identify promoters suitable for the present invention, gene cassettes carrying reporter proteins were constructed and linked to candidate promoter sequences. The candidate promoters include pag1 : an early expression gene of the HzNV-1 virus; p10 : Baculovirus late gene; cmv : cytomegalovirus; sv40 : simian virus 40; lir : RhPV virus and EV71 virus chimeric internal ribosome entry site (IRES) ; B1 : antimicrobial peptide b1 gene; a4 : defensin a4 gene; pub : polyubiquitin (polyubiquitin) gene; and heat shock protein 70 (hsp70) gene; thereby, a baculovirus transfer vector can be generated . The transfection vector can be used together with baculovirus to transfect a host cell (for example, mosquito cell line C6/36), and the performance of the reporter protein is used to evaluate the ability of each candidate promoter sequence. Among all tested candidate promoter sequences, pag1 , antimicrobial peptide b1 gene and hsp70 gene are relatively strong promoters, which significantly drive the performance of operably linked genes, while the remaining promoters show slight performance. Even negligible activity.

Accordingly, the first aspect of the present disclosure relates to a recombinant baculovirus, which is characterized in that it has a HzNV-1 virus early expression gene pag1 , an antimicrobial peptide gene b1 , a defensin a4 gene or a hsp70 gene as A promoter is used to drive the expression of a foreign gene operably linked to the promoter.

In order to construct the recombinant baculovirus of the present disclosure, an expression gene cassette carrying the target foreign gene was constructed and connected to pag1 , antimicrobial peptide b1 , defensin a4 or hsp70 genes to produce a transgenic vector. Then, the transfection vector and baculovirus DNA are co-transfected into host cells to produce recombinant baculovirus.

According to a specific embodiment of the present disclosure, the baculovirus transfer vector is co-transfected with a Bac-N-Blue virus DNA into an insect host cell. Bac-N-Blue viral DNA provides the necessary viral backbone, which contains propagation-essential genes. The homologous recombination between the recombinant baculovirus transfer vector and Bac-N-Blue virus DNA in insect host cells can produce recombinant baculovirus, which can multiply in insect host cells and thereby produce separate genes. Express the target foreign protein encoded by the gene cassette. Recombinant baculovirus can be further screened and purified, for example by reporting the performance of the polypeptide. Insect host cells suitable for the present disclosure include, but are not limited to: S. frugiperda IPBL-9 (Sf9) cells, Sf21 cells, High Five cells, Mimic Sf9 cells, and C6/C36 cells. According to certain embodiments of the present disclosure, the insect host cell is a Sf21 cell. According to certain embodiments of the present disclosure, the insect host cell is a C6/C36 cell. According to embodiments of the present disclosure, transduction does not significantly affect the viability of insect host cells for up to 12 days.

Optionally, the baculovirus vector may include a reporter protein polypeptide. Examples of report polypeptides may include, but are not limited to, blue fluorescent protein (BFP), cyan fluorescent protein (cyan fluorescence protein, CFP), anemone red fluorescent protein (DsRed), green fluorescent protein (GFP) ), enhanced green fluorescent protein (EGFP), enhanced yellow fluorescent protein (EYFP), etc. In certain specific embodiments of the present disclosure, the reporter polypeptide is EGFP. It should be understood that the reported polypeptide (e.g., EGFP) is not an essential technical feature of the technical problem to be solved by the present invention. The technical problem to be solved by the present invention is to introduce the foreign gene of interest into a vector insect (e.g., mosquito).

3. Uses of recombinant baculovirus in this disclosure

The recombinant baculovirus constructed according to the method described in the present disclosure carries at least one target foreign gene, and the expression of the foreign gene will be driven by the promoter sequence of pag1, antimicrobial peptide b1 , defensin a4 or hsp70. The resulting recombinant baculovirus can be used to transduce disease vectors, such as A. aegypti, A. albopictus , C. tritaeniorhynchus , and A. sinensis (A. aegypti). sinensis ) and other mosquitoes.

Accordingly, the present disclosure also includes a method for introducing an exogenous gene into mosquitoes. The aforementioned method includes the following steps: transducing mosquitoes with the recombinant baculovirus of the present invention without inhibiting microRNA in the mosquitoes.

After being infected by a virus, the anti-viral defense mechanism inside the infected host will be activated to resist the aforementioned infected virus. The antiviral defense mechanism includes one or more proteins that activate the immune system, for example, activation of Dosha, Dicer, Exportin-5 (Toll-like receptor-2, TLR- 2), STAT1, STATE, interleukin 7R (interleukin 7R), interleukin 1A and so on. Studies have pointed out that when cells are infected with a baculovirus vector, agents that inhibit protein expression in the antiviral defense pathway can be administered before or together with the baculovirus vector (for example, it can inhibit Dosha, Dicer, and transfusion protein). -5. Toll-like receptor-2 (TLR-2), STAT1, STATE, interleukin 7R and interleukin 1A expression reagents) (please refer to US 2013/0109077A1). Such reagents include, but are not limited to, reagents that can activate the production of microRNAs, which are used to cleave mRNAs in the antiviral defense pathway.

The difference from the content disclosed in US2013/0109077A1 is that the inventors unexpectedly discovered that when the recombinant baculovirus of the present invention is used to infect a vector, no reagent is needed to inhibit the vector's own antiviral defense pathway. According to the embodiments of the present disclosure, the recombinant baculovirus of the present invention can transduce a target exogenous gene to mosquitoes without using Dosha, Dicer, transfusion protein-5, and toll-like receptors (TLR-like receptors) that can inhibit mosquitoes. 2) Reagents for STAT1, STATE, Interleukin 7R and Interleukin 1A.

According to the embodiments of the present disclosure, the recombinant baculovirus of the present invention can be delivered into mosquitoes by GP64 envelope protein.

According to the embodiments of the present disclosure, the recombinant baculovirus is used in mosquitoes of different genera (including but not limited to A. aegypti , A. albopictus , and C. tritaeniorhynchus ). And the larval and adult stages of Anopheles sinensis (A. sinensis) can transduce strong gene expression. The recombinant baculovirus of the present invention is preferably used to transduce A. aegypti or A. albopictus.

According to the embodiments of the present disclosure, the recombinant baculovirus will not replicate in infected mosquitoes.

According to the embodiments of the present disclosure, the foreign gene transduced by the recombinant baculovirus of the present disclosure can be expressed in mosquito tissues. The mosquito tissues include but are not limited to: mosquito's head, snout, legs, balance stick, Markov tubules, ovaries, crops and fat bodies in the midgut.

Various embodiments are provided below to illustrate certain aspects of the present invention, but the present invention is not limited to these aspects.

Example

Materials and Methods

Cells and viruses

_{In a CO 2} humidified incubator (concentration 5%) at a temperature of 28°C, supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 0.1 mM non- Essential amino acid, 1 mM sodium pyruvate and 2% penicillin-streptomycin RPMI-1640 culture medium (supplier: Invitrogen) to cultivate the C6/36 cell line of the white mosquito mosquito and the CCL-125 cell line of the mosquito aegypti . In addition, the Spodoptera frugiperda IPLB-Sf21 (Sf21) cell line was cultured in TC100 insect culture medium containing 10% FBS at 26°C. _{In a CO 2} humidified incubator (5% concentration) at 37°C, Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS and 2% penicillin-streptomycin (DMEM; supplier) : Gibco) Culture HEK-293T and Vero-E6 cells. In this study, the gene body (trade name: flashBAC-ultra) of AcMNPV (AcMNPV) baculovirus (Gene Bank accession number NC_001623.1) was used to prepare recombinant baculovirus. The corresponding frozen virus stocks of the respective media are stored at a temperature of -80°C.

Construction of Transformation Vector and Recombinant Baculovirus

Construct the transformation vector pABspmC to produce vABspmC recombinant baculovirus. In the pTriEx-3 vector (supplier: Novagen), the mammalian sv40 and HzNV-1 pag1 gene promoters were inserted upstream of the TriEX promoter in the opposite direction. The mCherry gene on the pJET-mCherry vector was PCR-amplified and transferred to the downstream of the pag1 promoter to generate the final transfer vector named pABspmC. In this composite vector, the performance of mCherry is driven by the sv40 promoter of the mammalian system and the pag1 promoter of the insect system, respectively. For the promoter analysis, the inventors constructed several transgenic vectors as shown in Figure 3A. In the pAB h EG plastid (supplier: Abvector), the PCR-amplified promoter region was cloned upstream of EGFP to replace the hsp70 promoter on the pAB h EG plastid. pag1 (baculovirus early gene), p10 (baculovirus late gene), cmv (cytomegalovirus), sv40 (simian virus 40), lir (RhPV virus and EV71 virus chimeric internal ribosomal entry region (IRES) )); and the promoter regions of two mosquito genes amplified by PCR from the Aegypti mosquito gene body: antimicrobial peptide b1 and defensin a4 (Gene Bank IDs are HQ285957.1 and HQ285959.1, respectively) ; And commercially available synthetic polyubiquitin gene (Gene Bank ID: GU179018) promoter region ( 565 base pairs upstream of Pub gene) (named respectively: pAB pag1 EG, pAB p10 EG, pAB cmv EG, pAB sv40 EG, pAB lir EG, pAB b1 EG, pAB a4 EG, and pAB pub EG transfer vectors). Follow the standard procedure of the In-Fusion selection kit (supplier: Clontech) to construct the aforementioned transfer vector. The transfer vector pBacb1EG-irEG was constructed according to the conventional breeding method. In short, a NcoI-XbaI EGFP fragment was subcultured between the NcoI and XbaI cleavage sites of pGL3 to generate plastid pGL3-EGFP. The NheI-BglII promoter fragment of the antimicrobial peptide b1 was amplified by PCR and colonized into pGL3-EGFP to generate pGL3-b1EGFP. In addition, the restriction enzyme cleavage region from pGL3-b1-EGFP and NheI-NotI was sub-transplanted to pBac-CHIKV-26S-Rhir-E (Wu YJ, (2008) Acta Pharmacologica Sinica 29(8):965-974 ) To replace the CHIKV-26S region, and insert the NheI-BglII fragment and self-link to generate the pBac-b1-EGFP-Rhir-E vector. Verify all the above transfer vectors through sequencing. Finally, these transfection vectors were co-transfected into Sf21 cells with vAcRP23.Laz (linearized viral DNA of AcMNPV, supplier: Pharmingen) using Cellfectin reagent (supplier: Life Technologies). The recombinant baculovirus was isolated by the end-point dilution method, amplified in a T75 flask, and the virus titer of Sf21 cells was determined _{by the TCID 50 analysis method.}

Baculovirus transduction and infection

On the day before transduction, mosquito C6/36 cells or mammalian cells (unless otherwise specified) were planted in 24-wells at a density of ^{4×10 5} cells per well or 1×10 ^{5 cells.} plate. Wash the cells with 1x DPBS, and add baculovirus with different MOI (as shown below in the respective culture medium without any FBS or antibiotics). Centrifuge the culture plate at 2000 rpm for 32 minutes at room temperature (RT). Culture the transduced mosquito cells or mammalian cells at 28°C or room temperature for 2 hours. Wash the culture plate twice with DPBS containing 0.1% trypsin to remove unadsorbed virus. Add 10%-FBS complete culture solution and culture at different time points. For the baculovirus infection test, the day before the infection, Sf21 cells were ^{planted in a 24-well plate at a density of 2×10 5} cells per well, and different amounts of baculovirus were added to under different MOI conditions Conduct transduction. The culture plate was centrifuged at 2000 rpm at room temperature, and cultured at 26°C in various time frames shown below.

Cell proliferation and cell viability analysis

The recombinant vABspmC baculovirus mosquitoes with MOI=1, 10 and 100 were transduced into C6/36 cells. The harvested cells were trypsinized every 24 hours after transduction, resuspended in 1x DPBS, and then mixed with the same volume of 0.4% trypan blue exclusion dye (supplier: Sigma) to remove dead cells, and measured Proliferation of healthy cells. For cell viability analysis, the relative cell metabolism activity was determined by adding 10% v/v AlamarBlue to these cells and culturing for 4 hours. The decrease in the concentration of AlamarBlue was measured by a fluorescent reader (supplier: EnSpire, PerkinElmer) with an excitation wavelength of 560 nm and an emission wavelength of 590 nm.

Baculovirus neutralization analysis

Take a total volume of 300 µl of 1x DPBS (containing 1 µg of neutralizing monoclonal antibody against GP64 protein (AcV1, supplier: Santa Cruz Biotechnology) or non-neutralizing monoclonal antibody (AcV5, supplier: Santa Cruz Biotechnology) ) Treat vABspmC baculovirus (4×10 ⁵ PFU, MOI=1), and centrifuge at 300 rpm at room temperature. Then the virus and antibody mixture was added to C6/36 cells for 2 hours, and cultured in a CO ₂ humidified incubator (concentration 5%) at 28°C. Then remove the virus and antibody mixture, and wash three times with 1x DPBS (containing 0.1% trypsin) to remove unlinked virus particles. Finally, add 10%-FBS complete culture medium, and culture the cells for two days before the mCherry fluorescence test.

Plastid DNA transfection

Mosquito C6/36 cells were planted in 24-well plates at a density of ^{4×10 5 cells per well.} The next day, mix the recombinant plastid DNA (as shown in Figure 3, 0.5 µg) with the transfection reagent (TransIT-Insect Transfection Reagent; supplier: Mirus) according to the instructions for use, and incubate at room temperature for 30 minutes, then Then add the DNA and transfection reagent mixture to the cells. After 5 hours in a culture environment at 28°C, the cells were removed and washed with 1x DPBS, and then a complete culture solution of 10%-FBS was added. The cells were cultured at a temperature of 28°C for 2 days, and then the cells were collected for flow cytometry analysis.

Prepare cells for flow cytometry analysis

Mosquito C6/36 cells were transduced with baculovirus in a 24-well plate with various designated time ranges. The culture medium was removed from the culture plate, the cells were washed twice with 1x DPBS, and then treated with pre-warmed 100 µl trypsin-EDTA for 5 minutes. Then, 100 µl of MEM medium (modified Eagle’s medium, supplier; Gibco) was added to neutralize the trypsin-EDTA, and the cells were collected in a 1.5 ml microcentrifuge tube (containing 1 ml of 1x DPBS). The cells were centrifuged at 5000 rpm for 5 minutes to remove the MEM medium, and then the cell pellet was washed at least three times with 1x DPBS. Finally, the cells were resuspended in 1 ml of fixation buffer (1% FBS and 1% formaldehyde diluted with 1x DPBS), and analyzed by flow cytometry. By using a flow cytometer to measure mCherry-positive or EGFP-positive cells, and using FlowJo software to measure their mean fluorescence intensities (MFI).

Quantification of baculovirus DNA by real-time qPCR

Sf21 cells infected with baculovirus or C6/36 cells transduced with baculovirus were washed three times with 1x DPBS (containing 0.1% trypsin) to remove free virus particles from the cells. The total cell DNA is extracted through the plastid extraction kit (High Pure PCR Template Preparation Kit, supplier: Roche). A spectrophotometer (model: NanoDrop ND-1000, supplier: Thermo Fisher Scientific) was used for qualitative and quantitative determination of DNA. Perform real-time qPCR on a 96-well plate, place 2 μl DNA (100 ng/μl), 0.6 μl 10 μM specific primer pair (final concentration 300 nM), 6.8 μl H ₂ O and 10 μl per well 2 times staining agent (SYBR Green Master Mix, supplier: ABI). The samples were made in triplicate. Real-time PCR was performed with ABI 7500 FAST system, the number of cycles was set to be greater than 40, and the nucleic acid bonding temperature was 60°C. Evaluate the expression level of each target gene based on relative quantification (RQ) by comparing the critical threshold (CT) method. By normalizing to CT from the endogenous control group genes (actin gene in C6/36 cells, and GAPDH in Sf21 cells), the RQ of the specific gene in each response was evaluated. Three independent transfection experiments were performed, and the data was the result of one independent infection.

Mosquito feeding

Aegypti ( A. aegypti , Kaohsiung strain), Albopictus (A. albopictus , neutralization strain), and Aegypti mosquitoes ( C. tritaeniorhynchus, Peitou strain) and China Anopheles (A. sinensis). The larval stage is fed with crushed fish feed, and the adult stage provides 5% sucrose for free feeding.

Oral mosquito virus infection and intrathoracic vaccination

The frozen virus stock was thawed at 37°C, and serially diluted four or five times in RPMI1640 medium, and then mixed with an equal volume of defibrinated sheep blood. Each virus dilution was presented to female aegypti mosquitoes and white mosquito mosquitoes (starved for 24 hours) 4-5 days after pupa. According to the standard procedure of conventional technology (Thompson and Sarnow ((2003) Virology 315(1): 259-266)), cold-anesthetized mosquitoes were inoculated with virus in the chest cavity.

Example 1: The transduction effect of baculovirus on mosquito C6/36 cells

This example focuses on the use of AcMNPV to deliver gene expression cassettes into host mosquito C6/36 cells. In order to achieve this goal, a recombinant vABspmC baculovirus vector containing sv40 and pag1 promoters was constructed to drive the expression of the mCherry gene (panel A in Figure 1) and transduced with the recombinant vector with MOI=1, 10, and 100, respectively C6/36 cells. The fluorescence of mCherry can be observed at 12 hours (data not shown), and a significant dose-dependent fluorescence intensity can be observed at 48 hours after transduction (Panel B in Figure 1).

Then the transduced cells were collected and analyzed by flow cytometry to determine the transduction efficiency. The circle selection and quantitative results of mCherry-positive cells show that the transduction efficiency of 60% to 65% can be routinely achieved when MOI=1 (Figure 1 D panel), and when MOI=100, the efficiency increases to 95% to 100% (Small C and D in Figure 1).

Next, I want to study whether C6/36 cells will lyse after virus infection to reduce cell proliferation. Expose C6/36 cells to vABspmC virus of various MOIs and test different time ranges, and then stain with 0.4% trypan blue solution. Exclude dead cells to calculate the number of healthy cells. Cell replication is checked every 24 hours until 96 hours after transduction. It was found that normal cell proliferation was performed at all concentrations (MOI) until 48 hours after transduction, and when compared with the control group (pseudo-condition), a slight decrease in cell proliferation was observed at the time point after 48 hours (Especially MOI=100, the result is not shown). Cell viability was analyzed by adding 10% v/v AlamarBlue at four-day intervals to determine cell metabolic activity. In all exposure time ranges, no significant toxicity was found for C6/36 cells with MOI=1 and MOI=10. However, 12 days after transduction, cells with MOI=100 were slightly but significantly reduced ( Results not shown).

At the same time, the inventors also studied whether C6/36 cells support long-lasting gene expression, and monitored the toxicity of baculovirus to C6/36 cells for up to 12 days. As shown in panel E in Figure 1, before reaching saturation, mCherry performance increased as a function of time 8 days after exposure. Therefore, this result indicates that this expression system does not seem to involve the lysis period of baculovirus infection, and C6/36 cells can maintain the baculovirus transduction for a longer period of time.

Since the baculovirus enters insect or mammalian cells through the surface GP64 protein, the GP64-mediated cell entry shall be determined accordingly, where the baculovirus is first neutralized or non-neutralized before being transduced into C6/36 cells The GP64 antibody was processed. It was found that, compared with the negative control group, the baculovirus treated with GP64-neutralizing antibody did not show mCherry fluorescence after 48 hours of transduction (Figure 1, Panel F), which indicates that the baculovirus is indeed Enter the mosquito host through the GP64 surface protein.

In summary, this experimental result shows that the baculovirus can successfully enter mosquito C6/36 cells and consistently drive transgene expression without affecting the proliferation of host cells.

Example 2: Analysis of baculovirus replication in mosquito C6/36 cells

In this example, the replication of baculovirus in mosquito C6/36 cells was analyzed by detecting the amount of viral DNA accumulated in the host cell. For this reason, insect Sf21 cells, the natural host of baculovirus, were used as the control group, and vABspmC and C6/36 cells were infected together.

Panel A in Figure 2 shows the time course of mCherry fluorescence of C6/36 cells (at MOI=10) transduced with vABspmC; and the histogram in Panel B in Figure 2 shows the effect of baculovirus on Sf21 cells And the entry efficiency of C6/36 cells (MOI=10 and MOI=50). It was found that the relative amount of viral DNA in individual MOI cells at 2 hours after infection/transduction was similar in the two hosts, indicating that the baculovirus entered C6/36 and Sf21 cells with similar efficiency (Panel B in Figure 2).

Furthermore, the accumulation of viral DNA in Sf21 cells gradually increased over time (from 12 hours to 48 hours after infection), and reached a plateau at 96 hours after infection. In contrast, from 12 hours to 96 hours after transduction, the virus in C6/36 cells gradually decreased over time, which means that the baculovirus may not replicate in mosquito C6/36 cells (Panel C in Figure 2). Figure).

Example 3: Functional analysis of various baculovirus-incorporated promoters and their efficiency in C6/36 cells

In this experiment, by inserting the DNA sequences of various promoters into the baculovirus genome to test their functionality and efficacy, for this purpose, we construct transgenic vectors with different promoter sequences that drive EGFP expression (as shown in Figure 3A). As shown in the figure).

After each transfection vector is loaded into the baculovirus, C6/36 cells are transduced at MOI=1, so that most cells contain one or less copies of the virus gene body per cell. For control, C6/36 cells were also transfected with 500 ng of the same transfection vector plastid, so that the cells were transfected with an average of 1.15×10 ⁵ copies of plastid DNA. After 48 hours of transduction, the EGFP image was captured (Panel B in Figure 3). The average fluorescence intensity of EGFP-positive cells was quantified by flow cytometry to evaluate the intensity of each promoter in C6/36 cells. Panel C in Figure 3 presents the aforementioned quantitative results.

The results showed that compared with other promoters, the pag1 and b1 promoters are always stronger, and the expression of EGFP driven by b1 is slightly better than that of EGFP driven by pag1. In contrast, the p10 , sv40 , lir , pub, and a4 promoters exhibit less or weaker fluorescence, and the cmv promoter only exhibits minimal activity.

In the plastid transfection system, the pag1 promoter showed better activity, but the activity of other promoters remained at the base value or below the detection limit. In addition, under plastid transfection, the p10 , sv40 , lir, and a4 promoters show similar results as baculovirus transduction (ie, no fluorescence), and the cmv promoter only shows basic activity. Unexpectedly, the pub promoter always exhibits lower fluorescence in the baculovirus transduction system, but maintains its functionality in the plastid transfection system. The inventor believes that this should be due to the baculovirus-mediated cellular response that inhibits the efficiency of the pub promoter.

Example 4: Functional analysis of heat shock protein promoter and its efficacy in C6/36 cells

In this experiment, the transduction vectors containing the b1 promoter and heat shock protein 70 (hsp70) were constructed, and C6/36 cells were transduced according to the similar procedure described in Example 2. After transduction, the flow Promoter activity was measured by cytometer. Figure 4 shows the measurement result.

Compared with the control group, the b1 promoter can drive EGFP expression in C6/36 cells; however, hsp70 exhibits stronger promoter activity, which is useful for baculovirus-mediated gene transfer in C6/36 cells. It is the strongest promoter.

Example 5: Baculovirus-mediated gene expression in larvae (larvae) and adults of various mosquito species

This example is mainly to evaluate the transduction ability of baculovirus to larvae of different mosquito species. Aegypti ( A. aegypti , also known as yellow fever mosquitoes) and A. albopictus (A. albopictus, also known as Asian tiger mosquitoes or forest mosquitoes) can transmit various potentially deadly human viruses (including dengue virus, tragoniasis virus, Zika virus, yellow fever virus and Mayaro (virus) also transmit some filarial nematodes such as canine heartworm and other diseases. The three-spotted house mosquito (C. tritaeniorhynchus ) is the main host of Japanese encephalitis. China Anopheles ( A. sinensis ) spread malaria and lymphatic filariasis.

In order to achieve the aforementioned purpose, a dose of 1×10 ⁵ PFU recombinant vABb1EG-irEG baculovirus was microinjected into the larvae of the aforementioned four mosquito species, and the expression level of EGFP was measured two days after transduction. Figure 5 presents the results. It can be seen that EGFP expression is stronger in Aedes aegypti and Anopheles sinensis. On the contrary, EGFP expression is weak in A. sinensis and A. sinensis (Figure 5A). Small picture).

In order to evaluate the transduction ability of baculovirus in adult mosquitoes, the same recombinant baculovirus (vABb1EG-irEG) was delivered into the larvae of the aforementioned four mosquito species in a dose-dependent manner through intrathoracic microinjection. Interestingly, after 6 days of transduction, as the virus dose increased, the expression of baculovirus-mediated EGFP was higher in adult mosquitoes. At the same time, it was also found that compared with the other three mosquito species, Anopheles sinensis showed a lower EGFP expression level (Panel B in Figure 5). Then, perform RT-qPCR of the total genomic DNA isolated from the transduced adult mosquitoes to detect whether the baculovirus can replicate in vivo. The results showed that the baculovirus did not replicate in adult mosquitoes (data not shown), and this result also confirmed the results of in vitro experiments.

Then, through a predetermined time range, the microinjected mosquitoes are detected at regular time points. It was found that the expression level of EGFP was only slightly expressed on the day after injection, but then gradually increased throughout the experiment until 12 days after injection (Panel C in Figure 5). In order to determine the tissue tropism of baculovirus-mediated exogenous gene expression, the transduced mosquitoes were dissected. Strong EGFP performance can be observed in most tissues (including head, snout, legs, balance stick, midgut Markov tubules, ovary, crop and fat body), but in antennae and wings To a weaker performance (Figure 5, panel D).

In summary, these results indicate that the baculovirus can successfully transduce transgenes in the larvae and adults of the mosquito species tested in this experiment, with no or few tissue barriers.

It should be understood that the foregoing description of the embodiments is only given in the form of examples, and various modifications can be made by those with ordinary knowledge in the technical field to which this field belongs. The above specification, examples and experimental results provide a complete description of the structure and use of the exemplary embodiments of the present invention. Although various specific embodiments of the present invention are disclosed in the above embodiments, they are not intended to limit the present invention. Those with ordinary knowledge in the technical field to which the present invention belongs, without departing from the principle and spirit of the present invention, Various changes and modifications can be made to it, so the protection scope of the present invention should be subject to the scope of the accompanying patent application.

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In order to make the above and other objects, features, advantages and embodiments of the present invention more comprehensible, the description of the accompanying drawings is as follows:

The diagram in Figure 1 shows the result of baculovirus transduction into mosquito C6/36 cells. Panel A shows a composite expression vector used to produce recombinant baculovirus. The composite transfer vector contains sv40 and pag1 promoters, which are designed to express mCherry in mammalian cells and insect cells, respectively. Panel B shows that the baculovirus enters the host cell in a dose-dependent manner, and the recombinant vABspmC baculoviruses of different valences (MOI=1, 10, and 100) are transduced into C6/36 cells. Panel C presents cells that are gating mCherry-positive by flow cytometry (the diagram presents a representative result of multiple biological replicates). Panel D is the quantitative result of mCherry-positive expressing cells to evaluate the transduction efficiency. Panel E shows the results of the remaining baculovirus-mediated gene expression. C6/36 cells were transduced with baculovirus with a concentration of MOI=1, and the expression of fluorescent protein was analyzed for several days. Use a fluorescent microscope to capture mCherry fluorescent images at different time points. Panel F shows GP64-mediated baculovirus entry. C6/36 cells were treated with a baculovirus-antibody (neutralizing or non-neutralizing antibody against GP64 protein) mixture. Panel B and Panel F both show mCherry fluorescence images captured 48 hours after transduction. All experiments were performed in three biological replicates.

The diagram in Figure 2 presents the results of baculovirus replication analysis in C6/36 cells. Panel A shows the mCherry fluorescence image of baculovirus transduction. C6/36 cells were transduced with vABspmC at a concentration of MOI=10, and mCherry fluorescence images were collected at various time points (as shown in the figure). Panel B is a study on the efficiency of baculovirus entry. Baculovirus was transferred into C6/36 cells, or Sf21 cells were infected with MOI=10 and MOI=50 for 2 hours, and then the relative transfer efficiency was quantified by qPCR. Panel C shows the replication efficiency of baculovirus. C6/36 or Sf21 cells were transduced with baculovirus at a concentration of MOI=10, and these cells were collected at various time points, and the intracellular viral DNA was quantified by qPCR. The amount of viral DNA at all time points was normalized based on internal genes (GAPDH in Sf21 cells or actin in C6/36 cells), and the number of viral DNA copies was again normalized 2 hours after transduction. This experiment was carried out three times.

The diagram in Figure 3 presents the efficiency analysis of different baculovirus promoters in C6/36 cells. The small picture A shows the recombinant baculovirus used to produce EGFP, which can pass through various baculovirus, mammalian viruses and mosquito host promoters (from the following genes: pag1 : HzNV-1 virus early expression gene; p10 : baculovirus) Late gene of cytomegalovirus; cmv : cytomegalovirus; sv40 : simian virus 40; lir : chimeric internal ribosome entry site (IRES) of RhPV virus and EV71 virus; b1 : Antimicrobial peptide b1 gene; a4 : defensin a4 gene; pub : driven by polyubiquitin gene. The small picture B shows a fluorescent image. Transfect C6/36 cells with recombinant virus expressing EGFP (concentration MOI=1), or transfect C6/36 cells with 500 ng of recombinant plastid DNA construct used to generate recombinant baculovirus. After 48 hours of transduction, images were taken with a fluorescent microscope. Panel C presents the results of flow cytometry analysis of EGFP fluorescence driven by the target promoter. After 48 hours, C6/36 cells transduced with recombinant baculovirus or transfected with plastid DNA were collected, and the average EGFP fluorescence density was measured by flow cytometry to determine the promoter efficiency. The data presents the average of three biological replicates.

The diagram in Figure 4 shows the effect of the hr1hsp70 promoter on the EGFP expression of mosquito C6/36 cells transduced with baculovirus. C6/36 cells were transduced with recombinant baculovirus vAB hh- EG (concentration MOI=1). After 48 hours, C6/36 cells were collected, and the average fluorescence density of EGFP was measured with a flow cytometer to determine the promoter strength. The experiment was carried out in three repetitions, and only one representative result was presented. The asterisk ( p <0.0005***) indicates the average fluorescence density of EGFP in C6/36 cells transduced by vAB hh -EG and the average fluorescence density of EGFP in the control group and C6/36 cells transduced by vABb1-EG. There are significant differences.

The diagram in Figure 5 presents the results of the analysis of baculovirus transduction in mosquitoes. Panel A shows the results of transducing baculovirus into mosquito larvae. Four different mosquito species (A. aegypti ), A. albopictus , and C. tritaeniorhynchus were microinjected with 1×10 ⁵ PFU of vABb1EG-irEG baculovirus. Anopheles sinensis (A. sinensis )). Two days after transduction, the expression of EGFP was observed under a fluorescence microscope. Panel B shows a dose-dependent performance of baculovirus-mediated transgene in adult mosquitoes. Four different mosquito species were microinjected into the chest cavity with various doses of vABb1EG-irEG baculovirus (1×10 ³ , 10 ⁴ or 10 ^{5 PFU).} Six days after transduction, the expression of EGFP was observed under a fluorescence microscope. Panel C shows the transduction of adult mosquito baculovirus. Four different mosquito species were microinjected into the chest cavity with 1×10 ^{5 PFU of vABb1EG-irEG baculovirus.} After a few days, observe the EGFP performance with a fluorescence microscope (as shown in the figure over time). Panel D shows the tissue tropism of the adult mosquito aegypti. 1×10 ⁵ PFU of vABb1EG-irEG baculovirus was intrathoracically injected into adult Aegypti mosquitoes, and the transduction was observed 15 days later. EGFP fluorescence can be observed in the head, antennae, snout, legs, wings, balance stick, midgut Ma's tubule, ovary, crop and fat body of adult mosquitoes.

Claims (12)

A recombinant baculovirus capable of delivering a foreign gene into a mosquito, comprising a promoter; and the foreign gene is operably linked to the promoter, wherein the promoter is an antimicrobial peptide gene b1 or a Promoter of defensin gene a4. The recombinant baculovirus according to claim 1, further comprising a reporter gene for encoding a reporter protein, wherein the reporter protein is green fluorescent protein (GFP), enhanced green fluorescent protein (nhanced green fluorescence protein (EGFP), discosoma sp.red fluorescent protein (DsRed), blue fluorescence protein (BFP), enhanced yellow fluorescent protein (enhanced yellow fluorescent protein) , EYFP), Anemonia majano fluorescent protein ( Anemonia majano fluorescent protein, amFP), Zoanthus fluorescent protein (ZFP), Discosoma fluorescent protein (dsFP) or Feather Clavularia fluorescent protein (CFP). The recombinant baculovirus according to claim 1, wherein the baculovirus is Autographa californica multiple nucleopolyhedrovirus (Autographa californica multiple nucleopolyhedrovirus, AcMNPV), celeriac californica multiple nucleopolyhedrovirus (Anagraphafalclfera MNPV , AfMNPV) ), Anticarsia gemmatalis MNPV (AgMNPV), Bombyx mori MNPV (BmMNPV), Buzura suppressaria single nucleopolyhedrovirus (BsSNPV), tomato Spodoptera mononuclear polyhedrosis virus ( Helicoverpa armigera SNPV, HaSNPV), cotton bollworm mononuclear polyhedrosis virus ( Helicoverpa zea SNPV, HzSNPV), leaf roller moth polynuclear polyhedrosis virus ( Lymantria dispar MNPV, LdMNPV), P. Polyhedrosis virus ( Orgyia pseudotsugata MNPV , OpMNPV), Spodoptera frugiperda MNPV (SfMNPV), Spodoptera exigua MNPV (SeMNPV) or Spodoptera exigua MNPV (SeMNPV) Polyhedrosis virus ( Trichoplusia ni MNPV, TnMNPV). The recombinant baculovirus according to claim 3, wherein the baculovirus is Autographa californica polynuclear polyhedrosis virus. A method for introducing a foreign gene into a mosquito, comprising transducing a recombinant baculovirus to the mosquito without inhibiting the production of microRNA (miRNA) in the mosquito, wherein the recombinant baculovirus includes a promoter And the exogenous gene is operably linked to the promoter, wherein the promoter is a HzNV-1 virus early expression gene pag1 , an antimicrobial peptide gene b1 , a defensin gene a4 or a hsp70 gene promoter . The method according to claim 5, wherein an agent is used to inhibit the production of microRNA in the mosquito, and the agent results in the inhibition of Drosha, Dicer, toll-like receptor 2, STAT1, STAT6, interleukin 7R, or interleukin 1A performance. The method according to claim 5, wherein the mosquito is Aedes aegypti or A. albopictus . The method according to claim 7, wherein the mosquito is a mosquito cell, a larva or an adult. The method according to claim 5, wherein the baculovirus is Autographa californica polynuclear polyhedrosis virus (AcMNPV), Spodoptera exigua polynuclear polyhedrosis virus (AfMNPV), Spodoptera exigua polynuclear polyhedrosis virus (AgMNPV), Bombyx mori Multinuclear Polyhedrosis Virus (BmMNPV), Tung Geomegularis Mononuclear Polyhedrosis Virus (BsSNPV), Tomato Spodoptera Mononuclear Polyhedrosis Virus (HaSNPV), Helicoverpa armigera Mononuclear Polyhedrosis Virus (HzSNPV), Leaf Roller Moth Polynuclear polyhedrosis virus (LdMNPV), P. chinensis Keratovirus (OpMNPV), Spodoptera frugiperda polynuclear polyhedrosis virus (SfMNPV), Spodoptera frugiperda polynuclear polyhedrosis virus (SeMNPV), or Spodoptera frugiperda polynuclear polyhedrosis virus (TnMNPV). The method according to claim 9, wherein the baculovirus is Autographa californica polynuclear polyhedrosis virus. The method according to claim 5, wherein the baculovirus further comprises a reporter gene for encoding a reporter protein, wherein the reporter protein is green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), Disc Anemone Red Fluorescent Protein (DsRed), Blue Fluorescent Protein (BFP), Enhanced Yellow Fluorescent Protein (EYFP), Anemone Mahano Fluorescent Protein (amFP), Button Coral Fluorescent Protein (zFP) ), Disc Anemone Fluorescent Protein (dsFP) or Feather Coral Fluorescent Protein (cFP). The method according to claim 5, wherein the recombinant baculovirus does not replicate in mosquitoes.

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