CN112029734A - Recombinant baculovirus and uses thereof - Google Patents

Recombinant baculovirus and uses thereof Download PDF

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Publication number
CN112029734A
CN112029734A CN201910478505.1A CN201910478505A CN112029734A CN 112029734 A CN112029734 A CN 112029734A CN 201910478505 A CN201910478505 A CN 201910478505A CN 112029734 A CN112029734 A CN 112029734A
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vlp
virus
recombinant baculovirus
jev
protein
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郭赐成
林昌棋
高治华
赵德江
徐育麟
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Abstract

The present invention relates to a recombinant baculovirus for producing a viral particle (VLP) comprising a promoter and a foreign gene, wherein the foreign gene is a structural protein encoding the VLP, said structural protein selected from the group consisting of all, a portion of a viral structural protein and combinations thereof. The present disclosure also relates to a method of preparing the VLP by transducing the recombinant baculovirus into a mosquito cell, a use of the VLP for preparing a vaccine, and a method of using the VLP to detect an anti-viral antibody in a biological sample.

Description

Recombinant baculovirus and uses thereof
Technical Field
The present disclosure relates to the field of disease prevention. More specifically, the present disclosure relates to a recombinant baculovirus (baculovir) and its use in the preparation of vaccines for the prevention of infectious diseases.
Background
Many mosquito-borne viruses (e.g., dengue virus (DENV), chikungunya virus (CHIKV), zika virus (ZIKV), Yellow Fever Virus (YFV), West Nile Virus (WNV), or Japanese Encephalitis Virus (JEV)) pose a potentially fatal risk to the health of the public and cause significant economic losses in many countries. In order to maintain the health of the public and avoid economic losses, immunization is generally adopted as a means for avoiding mosquito vector virus infection. In the aspect of vaccine selection, various vaccines prepared by different techniques, such as attenuated vaccines, inactivated vaccines, toxoid vaccines, genetically engineered vaccines, DNA vaccines, etc., can be selected. Among them, with the increasing sophistication of biotechnology, genetic engineering vaccines are also receiving more and more attention. Within the class of genetically engineered vaccines, subunit vaccines are predominant, which use purified viral proteins or glycoproteins as the main component of the vaccine. In addition, in recent years, vaccines containing Virus Like Particles (VLPs) as a main component have been developed, and since the VLPs do not contain gene components of natural arbovirus (arbovirus), the safety of the vaccines can be greatly improved.
For preparing a vaccine of VLP, various expression systems such as bacteria, yeast, insect cells, plant cells, mammalian cells, or cell-free protein expression systems can be generally used. To mimic the cell surface components of the human host and achieve better immune efficacy of the vaccine prepared, mammalian cell expression systems are generally preferred. However, expression vectors for mammalian cells are relatively complex in design and application. In addition, to control the cost of vaccine preparation, the choice of insect cell expression systems is contemplated. However, the expression systems of insect cells that are commonly used at present are mainly insect cells derived from Spodoptera frugiperda (Spodoptera frugiperda), Trichoplusia ni (Trichoplusia ni), Bombyx mori (Bombyx mori), and the like. Because these insects (non-mosquitoes) are not vectors for direct transmission of mosquito vector viruses to humans, VLPs expressed by these cells are used as vaccine components, and their surface components are still different from those of cells that infect human hosts at the earliest stages (mosquito-derived), e.g., the cell membranes have different glycation components and lipid contents, thus limiting the ability of the vaccine to elicit host immune responses.
In view of the above, there is a need in the art to develop a VLP that can more mimic the cell surface components of the earliest infected human host as a vaccine, for example, VLP is directly expressed by mosquito cells to better conform to the infection structure of early human mosquito-borne viruses, thereby improving the immune efficacy of the vaccine.
Disclosure of Invention
The following presents a simplified summary of the invention in order to provide a basic understanding to the reader. This summary is not an extensive overview of the invention, and is intended to neither identify key/critical elements of the invention nor delineate the scope of the invention. Its sole purpose is to present some concepts of the invention in a simplified conceptual form as a prelude to the more detailed description that is presented later.
The present invention relates generally to a recombinant baculovirus and uses thereof. The recombinant virus can express a foreign gene, particularly in mosquito cells, and the foreign gene can be used to encode a structural protein of a VLP, particularly a VLP of an arbovirus, for producing the VLP in mosquito cells.
Furthermore, the use of the above VLPs as a detection reagent is also possible. Because VLPs maintain the appearance and antigenic properties of native viral particles, they retain the sensitivity and specificity of screening for disease, while also increasing the stability of antigen preservation. Furthermore, VLPs do not contain the gene components of the native arbovirus, and therefore, the first line of experiment operators can be protected from arbovirus infection during the process of preparing VLPs.
Accordingly, a first aspect of the present invention provides a recombinant baculovirus, comprising a promoter and a foreign gene, wherein the foreign gene is operably linked to the promoter; wherein the promoter comprises an HzNV-1(Heliothis zea Nudigirus 1) virus early expression gene pag1, an antimicrobial peptide gene b1(cecropin b1), a homeodomain gene 1 (homologus region 1, hr1) or a combination thereof; and wherein the foreign gene is a structural protein encoding a VLP, said structural protein selected from the group consisting of all, a portion of a viral structural protein, and combinations thereof. According to certain embodiments of the present disclosure, the structural protein is selected from the group consisting of capsid protein (C), envelope protein 1(envelope 1, E1), envelope protein 2(envelope 2, E2), envelope protein 3(envelope 3, E3), 6K protein (6K), pre-membrane protein (prM), and envelope protein (envelope, E), and combinations thereof.
Examples of recombinant baculovirus suitable for use in the context of the present invention include, but are not limited to, Autographa californica multinuclear polyhedrosis virus (AcMNPV), Heliothis virescens multinuclear polyhedrosis virus (AfMNPV), Heliothis virescens multinuclear polyhedrosis virus (Antiraphia falcifera MNPV, AfMNPV), Heliothis virescens multinuclear polyhedrosis virus (Anticarsia gemmatalis MNPV, AgMNPV), Bombyx mori multinuclear polyhedrosis virus (Bombyx mori MNPV, BmMNPV), Ectropis gigantes mononuclear polyhedrosis virus (Buzura subsarus single nuclear polyhedrosis virus, BspPV), Heliothis virens mononuclea polyhedrosis virus (BspPV), Heliothis virens mononucleosis virus (SNPV ), Helicoverpa virens mononucleosis virus (Spodopv), Spodoptera multinuclear polyhedrosis virus (Spodoptera PV, Spodoptera virus (Spodoptera PV), SeMNPV) or Trichoplusia ni polyhedrosis virus (Trichoplusia ni MNPV, tnmnnpv). According to a preferred embodiment of the present disclosure, the recombinant baculovirus is a recombinant Autographa californica multinuclear polyhedrosis virus.
According to certain embodiments of the present disclosure, the VLP is selected from the group consisting of Herpesviridae (Herpesviridae), Adenoviridae (Adenoviridae), african swine fever viridae (Asfarviridae), papilloma viridae (Papillomaviridae), polyoviridae (polyoviridae), Poxviridae (Poxviridae), Circoviridae (Circoviridae), small DNA viridae (partoviridae), Reoviridae (Reoviridae), Arteriviridae (artiviridae), Coronaviridae (Coronaviridae), Picornaviridae (Picornaviridae), Filoviridae (cortiviridae), bufoviridae (fuloviridae), Paramyxoviridae (Paramyxoviridae), bombyxoviridae (Rhabdoviridae), Astroviridae (Astroviridae), Caliciviridae (Caliciviridae), and hepatitis (Flaviviridae).
According to a specific embodiment of the present disclosure, the recombinant baculovirus expresses a protein from CHIKV of togaviridae. In one embodiment, the recombinant baculovirus expresses VLP of CHIKV. In a preferred embodiment, the VLP consists of structural proteins C, E1, E2, E3 and 6K. According to another specific embodiment of the present disclosure, the recombinant baculovirus can express a protein from JEV of the family flaviviridae. In one embodiment, the recombinant baculovirus can express VLPs of a JEV. According to yet another specific embodiment of the present disclosure, the recombinant baculovirus can express a protein from DENV of flaviviridae. In one embodiment, the recombinant baculovirus expresses VLPs of a DENV. According to yet another specific embodiment of the present disclosure, the recombinant baculovirus may express a protein from ZIKV of flaviviridae. In one embodiment, the recombinant baculovirus expresses a ZIKV VLP. In a preferred embodiment, the VLP consists of structural proteins prM and E.
A second aspect of the present disclosure relates to a method of producing a VLP using a recombinant baculovirus, particularly in mosquito cells. The method comprises the following steps:
(1) transducing a recombinant baculovirus of the present disclosure into a mosquito cell; and
(2) purifying the mosquito cells or supernatant of step (1) to produce the VLPs.
According to certain embodiments of the present disclosure, mosquito cells that may be used in the methods of the present disclosure include, but are not limited to, the white line mosquito (Aedes albopictus), Aedes pseudobulbifera (Aedes pseudostellatus), Aedes aegypti (Aedes aegypti), Macrophyllus annanensis (Toxorhynchinensis amboinensis), Culex tritaeniorhynchus, Aedes sinensis (Anopheles sinensis), Abies albugineus (Armigers subablatus), or Aedes quinquefasciatus (Culex quinquefasciatus). In a particular embodiment, the mosquito cell is a mosquito cell of a white spotted mosquito, an aegypti mosquito, or an aedes pseudolepidoptera mosquito.
According to one embodiment of the present disclosure, the recombinant baculovirus is a VLP expressing a VLP, particularly a VLP of an arbovirus. In a specific embodiment, the recombinant baculovirus expresses a VLP of CHIKV from togaviridae, wherein the VLP is composed of structural proteins C, E1, E2, E3 and 6K. In yet another specific embodiment, the recombinant baculovirus expresses a VLP of JEV, DENV or ZIKV from the flaviviridae family, wherein the VLP is composed of the structural proteins prM and E.
Based on the foregoing, the present disclosure also encompasses exogenous proteins expressed by recombinant baculoviruses of the present disclosure, such as C, E1, E2, E3, 6K, prM, E, or combinations thereof, as well as VLPs, particularly VLPs of an arbovirus, wherein the VLPs can be produced by a mosquito cell. Thus, the present disclosure also encompasses VLPs produced by the methods of the present disclosure.
It is understood that the use of the VLPs produced by mosquito cells described above for the production of a vaccine is also within the scope of the present invention.
A third aspect of the present disclosure relates to a method of using a VLP to detect an anti-viral antibody in a biological sample, comprising:
(1) mixing the VLP of the present invention with the biological sample; and
(2) detecting a complex formed by the anti-viral antibody and the VLP of step (1) in an immunoassay.
According to one embodiment of the present disclosure, the VLP may be produced from the recombinant baculovirus of the present disclosure, and in particular may be produced in a mosquito cell. In a specific embodiment, the VLP is a CHIKV from togaviridae, wherein the VLP is composed of structural proteins C, E1, E2, E3 and 6K. In yet another specific embodiment, the VLP is a JEV, DENV or ZIKV from the flaviviridae family, wherein the VLP is composed of the structural proteins prM and E.
Based on the above, the present disclosure also encompasses a detection reagent. The detection reagent comprises a VLP of the invention, wherein the VLP can be prepared by a mosquito cell. In addition, the present invention also covers a kit comprising the above detection reagent.
The basic spirit and other objects of the present invention, as well as the technical means and embodiments adopted by the present invention, will be readily understood by those skilled in the art after considering the following embodiments.
Drawings
In order to make the aforementioned and other objects, features, advantages and embodiments of the invention more comprehensible, the following description is given:
FIG. 1 is a schematic diagram of a recombinant baculovirus gene delivery to mosquito cells for the purpose of producing arbovirus VLPs, according to one embodiment of the present disclosure. FIG. 1A depicts a recombinant baculovirus BacMos-JEV prM-E which utilizes the composite promoter of hr1/pag1 to drive the prM-E gene of JEV. The recombinant baculovirus was transduced into mosquito cells to prepare VLPs of JEV. FIG. 1B shows additional recombinant baculoviruses BacMos-DENV prM-E, BacMos-ZIKV prM-E and BacMos-CHIKV 26S (where CHIKV 26S includes C/E1/E2/E3/6K), each using a specific mosquito promoter to drive the prM-E gene of DENV or ZIKV, and the 26S gene of CHIKV, respectively. These recombinant baculoviruses were transduced into mosquito cells to prepare VLPs of DENV, ZIKV and CHIKV.
FIG. 2 is a graph showing the expression of JEV protein in mosquito cells, according to one embodiment of the present disclosure. Fig. 2A presents the results of expression of E glycoprotein of JEV in mosquito cells. Mosquito cells (top to bottom) C6/36 (top), CCL-125 (middle), or AP-61 (bottom), were treated differently, (left to right) transduced with recombinant baculovirus BacMos-JEV prM-E (MOI ═ 5) (left), infected with JEV (MOI ═ 0.5) (middle), or without any treatment (mock) (right). Next, at day 5 post-transduction, or day 2 post-infection, E glycoprotein against JEV was detected by Immunofluorescence (IF) assay. The scale bar is 100 microns. FIG. 2B presents the results of expression of the prM protein of JEV in mosquito cells C6/36. Mosquito cells C6/36 were transduced with recombinant baculovirus BacMos-JEV prM-E (left panel), infected with JEV (middle panel), or not treated at all (mock) (right panel). Subsequently, on day 5 post-transduction, or day 2 post-infection, prM protein of JEV was detected by IF assay. The scale bar is 100 microns. Fig. 2C presents the results of expression of E glycoprotein of JEV in mosquito cells. After mosquito cells C6/36 were transduced with recombinant baculovirus BacMos-JEV prM-E (MOI ═ 5), mosquito cells were collected at specific time points (expressed as days post infection, dpi), and whole cell lysates (total cell lysates) of these cells were analyzed by Western Blot (WB) to detect the expression of the E glycoprotein of JEV. Figure 2D presents the results of expression of JEV protein in the supernatant. Mosquito cells C6/36, CCL-125 or AP-61 were transduced with recombinant baculovirus BacMos-JEV prM-E (MOI 2) or infected with JEV (MOI 0.1), respectively, followed by collection of supernatants at specific time points (indicated as dpi) and analysis with dot blot (dot blot) (left panel) to detect JEV protein and quantification (right panel).
FIG. 3 is a graph showing the expression of proteins ZIKV, DENV type 2 (DENV2) and CHIKV in mosquito cells, according to one embodiment of the present disclosure. Fig. 3A presents the expression of DENV2 and ZIKV E glycoproteins in cells (upper panel) and supernatant (lower panel). Mosquito cells AP-61 were transduced with recombinant baculovirus BacMos-ZIKV prM-E (left panel), with BacMos-DENV2prM-E (middle panel), both MOI ═ 2, or without any treatment (mock) (right panel). On day 4 post-infection, viral E glycoprotein was detected in cells by either IF assay (upper panel) or supernatant by dot blot assay (lower panel). The scale bar is 100 microns. FIG. 3B shows the expression of E2, E1 and C proteins of CHIKV in mosquito cells. Mosquito cells C6/36 were transduced with recombinant baculovirus BacMos-CHIKV 26S (left panel), infected with CHIKV (MOI ═ 1) (right panel), or without any treatment (mock) (middle panel), respectively. Subsequently, on day 5 after transduction or day 2 after infection, the cells were assayed for the E2 protein of CHIKV (upper panel), the E1 protein of CHIKV (middle panel) or the C protein of CHIKV (lower panel) by IF assay. The scale bar is 100 microns. FIG. 3C presents the results of detection of viral glycoproteins secreted by CHIKV and JEV. Mosquito cells AP-61 or C6/36 were transduced with recombinant baculoviruses BacMos-CHIKV 26S or BacMos-JEV prM-E, respectively (both MOI ═ 2). On day 4 post-infection, the supernatants were assayed for E2 for CHIKV (upper panel) or E protein for JEV (lower panel) by dot blot analysis.
Fig. 4 is a graph showing the results of identifying characteristics of mosquito cell-derived VLPs according to an embodiment of the present disclosure. Fig. 4A presents representative results of VLP purification with sucrose density gradient. The JEV VLPs were purified by ultracentrifugation on a sucrose density gradient and 8 fractions (fractions) were taken from the top to the bottom of the sucrose density gradient as samples and the prM and E proteins were detected by dot blot analysis, respectively, the right arrow indicating the location of the VLPs in the sucrose density gradient and the left arrow indicating the dot blot analysis of the corresponding E protein. FIG. 4B presents the results obtained by analysis of the E glycoprotein of JEV by LC-MS/MS. 5 of the samples taken from the sucrose density gradient were subjected to SDS-PAGE and Coomassie blue staining to give a band (band) corresponding to E, and the protein located on this band was taken for mass spectrometer analysis. Bold letters indicate that LC-MS/MS analysis gave a fragment of E glycoprotein corresponding to JEV. Fig. 4C to 4F present the microstructure and size of VLPs. VLP of JEV (C), VLP of DENV2 (D), VLP of ZIKV (E) and VLP of CHIKV (F) were examined by Transmission Electron Microscope (TEM), and enlarged pictures of different magnifications were shown. The area in the upper right picture, which is circled by a frame, is further enlarged to be the lower right picture. The scale bar is 50 nm.
FIG. 5 is a graph illustrating the results obtained using VLPs to detect anti-arbovirus antibodies in patient serum, in accordance with one embodiment of the present disclosure. FIG. 5A presents the results of detection of anti-JEV antibodies by MAC-ELISA. Six JEV-infected patient sera (JE sera), including 58912(JE), 58911(JE), 58833(JE), 58682(JE), 58655(JE) and 58556(JE), and four normal sera (NC sera), including S10700567(NC), S10700568(NC), S10700569(NC) and S10700570(NC), were tested for anti-JEV antibodies in these sera by MAC-ELISA (VLP or active virus (virion), respectively, as antigen). FIG. 5B presents the results of detection of specific antigen versus specific antibody by MAC-ELISA. Seven patient sera (including DENV2 infection (DN pt 159046), JEV infection (JE pt 158941, JE pt 258912), CHIKV infection (CK pt 156851, CK pt 259397), ZIKV infection (ZK pt 156928, ZK pt 256964)) and one normal serum (NC 1S1070568)) were each tested in MAC-ELISA with specific antigens (as shown).
FIG. 6 is a graph illustrating the immunoprotective efficacy of animal vaccination with VLPs inoculated with mosquito cell-derived JEV, in accordance with one embodiment of the present disclosure. FIG. 6A is a schematic of the time course of mice inoculated with three doses of JEV VLPs and blood collected. BALB/c mice (5 per group) were administered three doses of JEV VLPs ( dose 1 or 4 microgram) subcutaneously (with or without adjuvant)Adjuvanted), wherein PBS is the negative control group, and
Figure BDA0002083051570000081
commercially available JEV vaccine served as a reference control. Fig. 6B presents the results of ELISA. Mice 2 weeks after the final immunization were bled and serum-tested for IgG specific to the E glycoprotein of JEV by ELISA. Fig. 6C presents the results of ELISpot analysis. Spleen cells were collected from mice 2 weeks after the final immunization and mixed with VLP antigens. Next, IFN-. gamma.expression was detected by ELISpot. FIG. 6D presents the results of the plaque reduction neutralization assay (focus-reduction micro-neutralization, FR. mu.NT 90). FR μ NT90 was used to test the efficacy of neutralizing antibodies (nabs) against VLPs of GIII against GI and GIII. The experimental data are expressed as mean. + -. standard deviation and are statistically analyzed using Student's t-test (Student's t-test) and P is assigned<0.05 was considered to have significance (. about.P)<0.05,**P<0.01；***P<0.001)。
Detailed Description
In order to make the description of the present invention more complete and complete, the following description is given for illustrative purposes with respect to the embodiments and specific examples of the present invention; it is not intended to be the only form in which the embodiments of the invention may be practiced or utilized. The embodiments are intended to cover the features of the various embodiments as well as the method steps and sequences for constructing and operating the embodiments. However, other embodiments may be utilized to achieve the same or equivalent functions and step sequences.
For convenience of explanation, specific terms described in the specification, examples, and appended claims are collectively described herein. Unless defined otherwise herein, all technical and scientific terms herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The present disclosure relates to recombinant baculoviruses and methods of using the recombinant baculoviruses of the present disclosure to express proteins of an arbovirus, particularly proteins of a VLP of an arbovirus, expressed in a mosquito cell. In addition, the present disclosure also relates to methods of using the VLPs of the present disclosure for detection of arboviruses.
1. Definition of
The term "baculovirus" as used herein refers to an arthropod-specific, double-stranded DNA virus that can be used to control pests (e.g., mosquitoes). Nuclear Polyhedrosis Viruses (NPV) are a subgroup of baculoviruses. A number of baculoviruses are suitable as vectors for expressing foreign proteins in infected hosts, including those that infect Helicoverpa armigera (cotton bollworm, academic name Helicoverpa zea), Helicoverpa tabacum (tobacco budworm, academic name: Heliothis virescens), Helicoverpa fir moth (Douglas-fir month, academic name: Orygia pseudosugata), Lymantria giperda (gypsymo, academic name: Lymantria dispar), Helicoverpa arguta (alfa looper, academic name: Autographa californica), Europaea japonica (European pine sawfly, academic name: Neofilippon seroifera) and apple leafroll moth (codling month, academic name: Cydia pomona). Generally, a baculovirus having a broad host range is preferred, such as Autographa californica multiple nuclear polyhedrosis virus (AcMNPV). Examples of baculovirus suitable for use in the context of the present invention include, but are not limited to, AcMNPV, Spodoptera exigua (Anagrepha falcifera) polynuclear polyhedrosis virus (AfMNPV), Spodoptera glycines (Anticarsia gemmatalis) polynuclear polyhedrosis virus (AgMNPV), Bombyx mori (Bombyxmori) polynuclear polyhedrosis virus (BmMNPV), Ectropis europaea mononuclear polyhedrosis virus (Buzura Suppres mononuclear nucleopolyhedrovirus, BsSNPV), Spodoptera frugiperda (Helicoverpa armigera) mononuclear polyhedrosis virus (HasnpPV), Cotton bollworm (Helicoverpa armigera) mononuclear polyhedrosis virus (HzSNPV), Spodoptera convolvulus virus (Lymanta) polynuclear virus (LdMNPV), Spodoptera frugium virus (Orgypti suta) polynuclear virus (Spodoptera) polynuclear polyhedrosis virus (Spodoptera) and Spodoptera frugipervirus (Spodoptera) polynuclear Spodoptera virus (Spodoptera) polynuclear polyhedrosis virus (Spodoptera) multinuclear polyhedrosophila virus (Spodoptera) multinuclear polyhedrosis virus (Spodoptera) and Spodoptera) multinuclear polyhedrosophila virus (Spodoptera) multinuclear polyhedrosis virus (Spodoptera) multinuclear.
In the context of the present invention, the term "foreign gene" refers to a nucleic acid that is isolated, transferred and linked to a nucleic acid with which it is not associated in nature, and/or that is introduced into and/or expressed in a cell or cell environment in addition to a nucleic acid that is found in the cell or cell environment in nature. The foregoing words encompass nucleic acids originally obtained from a different species or cell type different from the cell type represented by the species, as well as nucleic acids obtained from the same cell line as the cell line represented.
The present disclosure uses "recombinant" to describe a vector or nucleic acid, such as a recombinant viral vector, recombinant polynucleotide, or recombinant polypeptide, to mean that the vector, nucleotide, or polypeptide has been modified (e.g., genetically engineered) in a manner that does not spontaneously occur in nature. Specific examples of recombinant vectors (e.g., recombinant baculovirus vectors) generally refer to recombinant vectors formed by inserting polynucleotides that are not normally present in wild-type viral genomes into the wild-type viral genomes.
The term "operably linked" as used in the present disclosure refers to the linkage of nucleic acid fragments, and when a nucleic acid fragment is linked to other nucleic acid fragments, the function and expression of the nucleic acid fragment can be affected by other nucleic acid fragments. For example, functionally linking a nucleic acid sequence encoding a desired protein to a control sequence (e.g., a promoter) for regulating the expression of the nucleic acid means that the control sequence is placed at an appropriate position relative to the nucleic acid sequence encoding the desired protein such that the control sequence can regulate the expression of the nucleic acid sequence encoding the desired protein. The operable linkage can be achieved by genetic recombination techniques well known in the art, such as site-directed DNA cleavage and ligation using enzymes well known in the art.
The term "transduction" as used in the context of the present invention refers to the introduction of a nucleic acid into a cell or host organism using a vector, such as a recombinant baculovirus vector in the context of the present invention. The introduction of a transgene into a cell by a recombinant baculovirus may thus be referred to as "transduction" of the cell. The transgene may be integrated with the genomic nucleic acid of transduced cells (e.g., 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 passed on to progeny cells or organisms of the recipient cell or host organism. Finally, the introduced transgene may be present extrachromosomally or transiently in the recipient cell or host organism.
"Virus like particle" (VLP) as used in the context of the present invention refers to a self-assembling (self-assembled) gene product obtained by cloning and expressing a viral structural gene (viral structural gene) in a heterologous host expression system (heterologous host system). VLPs are structurally similar to viruses, but are not infectious because they do not contain viral genes and the like. The composition of VLPs of different viruses varies, for example, with reference to Andris Zetins "Construction and Characterization of Virus-Like Particles: A Review" Mol Biotechnol.2013 Jan; 53(1) 92-107.doi 10.1007/s12033-012 and 9598-4, which disclosure is incorporated herein as part of the present disclosure.
As used in this disclosure, the terms "individual" (subject) or "patient" (patient) are used interchangeably and refer to a mammal (including a human) susceptible to infection by an arbovirus. The term "mammal" encompasses all members of the class mammalia, including humans, primates, livestock and farm animals. For example, the livestock or farm animal may be rabbits, pigs, sheep, and cattle. The mammal may also encompass zoo or racing animals, pets, and rodents (e.g., mice and rats). Unless otherwise indicated, "individual" or "patient" generally encompasses both males and females. Further, the term "subject" or "patient" includes animals that benefit from the methods of the present disclosure. For example, the "subject" or "patient" includes, but is not limited to, humans, rats, mice, guinea pigs, monkeys, pigs, sheep, cattle, horses, dogs, cats, birds, and birds. In one example, the subject is a human. In another example, the individual is a mouse.
The term "biological sample" as used in the context of the present invention refers to whole blood samples, plasma samples, serum samples, urine samples, mucus samples, saliva samples and purified or filtered forms thereof taken from mammals suspected to be or infected with arbovirus, including humans. The biological sample may be undiluted or diluted prior to detection by the present recombinant baculovirus, kit and/or method. When antibodies against arboviruses are present in a biological sample, the recombinant baculovirus of the kits and/or methods of the invention is capable of specifically binding to the antibody to form a complex, and which can be detected by immunoassay (e.g., ELISA). Conversely, if an antibody against an arbovirus is not present in the biological sample, the recombinant baculovirus of the kits and/or methods of the invention will not bind to the antibody and no immune complex will form.
Unless otherwise defined in the present specification, in the context of the present invention, the term "antigen" refers to any agent which, after introduction into an immunocompromised human or animal body, can provoke a humoral or cellular immune response. The antigen may be a pure substance, a mixture, a particular material, or a live, attenuated virus. Exemplary suitable antigens include, but are not limited to, proteins, glycoproteins, polypeptides, viruses, or VLPs.
As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Except in the experimental examples, or where otherwise explicitly indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the disclosure are to be understood as being modified by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in this disclosure are approximations that may vary depending upon the desired properties sought to be obtained by the present invention.
2. Recombinant baculovirus
Accordingly, a first aspect of the present invention provides a recombinant baculovirus, comprising a promoter and an exogenous gene, wherein the exogenous gene is operably linked to the promoter; wherein the foreign gene is configured to encode a structural protein of a VLP, particularly a VLP of an arbovirus, said structural protein selected from the group consisting of all, a portion of a viral structural protein and combinations thereof. In some embodiments, the structural protein is selected from the group consisting of C, E1, E2, E3, 6K, prM, and E. In certain embodiments, the exogenous gene may express a structural protein alone, e.g., the exogenous gene may express C alone, E1 alone, E2 alone, E3 alone, 6K alone, prM alone, or E alone. In other embodiments, the exogenous gene can co-express multiple proteins, for example, the exogenous gene can co-express two proteins (e.g., C/E1, C/E2, E1/E2, prM/E, etc.); three proteins (such as C/E1/E2, C/E2/E3, C/E3/6K and the like) are expressed in a combined way; the four proteins are expressed in a combined way (for example, C/E1/E2/E3, C/E2/E3/6K, C/E1/E3/6K, E3/E2/6K/E1, C/E1/6K/prM, C/E1/prM/E and the like); five proteins are expressed in a combined way (such as C/E1/E2/E3/6K, C/E2/E3/6K/prM, C/E1/6K/prM/E and the like); six proteins (such as C/E1/E2/E3/6K/prM, C/E1/E2/E3/6K/E, and the like) are expressed in a combined mode; seven proteins (e.g., C/E1/E2/E3/6K/prM/E, etc.) are expressed in combination. When the foreign gene expresses multiple proteins in combination, the order of the multiple proteins is not essential, and it may be adjusted as necessary. In a specific embodiment, the exogenous gene is a gene that co-expresses both proteins prM/E. In other embodiments, the exogenous gene is a gene that co-expresses five proteins C/E1/E2/E3/6K.
According to an embodiment of the present disclosure, the VLP may be a virus species (species) from the herpesviridae, adenoviridae, african swine fever virus family, papillomaviridae, polyomaviridae, poxviridae, circoviridae, small DNA virus family, reoviridae, arteriviridae, coronaviridae, picornaviridae, celluloviridae, paramyxoviridae, cannonball virus family, astroviridae, caliciviridae, flaviviridae, hepadnaviridae, togaviridae, arenaviridae or the yaviridae families. For example, the virus species of the VLP may be a virus species from a togaviridae family, including, but not limited to, bambushien virus (bamah Forest virus), flexor virus (CHIKV), Eastern equine encephalitis virus (easter equi encephalitis virus), O villus virus (O' nyong-nyong virus), Ross River virus (Ross River virus), german measles virus (Rubella virus), shenglisson Forest virus (Semliki Forest virus), Sindbis virus (Sindbis virus), Western equine encephalitis virus (Western equi encephalitis virus), and venezuelan equine encephalitis virus (venezuelan equi encephalitis virus). In a specific embodiment, the virus species of the Togaviridae family is CHIKV. Alternatively, the virus species of the VLP may be a virus species from a flaviviridae family, including, but not limited to, dengue virus (DENV), Hepatitis C Virus (HCV), Japanese Encephalitis Virus (JEV), hepatotropic virus (pegivirus), West Nile Virus (WNV), Yellow Fever Virus (YFV), and ZIKV. In a particular embodiment, the flaviviridae virus species is JEV. In another specific embodiment, the virus species of the flaviviridae family is DENV. In yet another specific embodiment, the flaviviridae virus species is ZIKV.
According to a particular embodiment of the present disclosure, the recombinant baculovirus expresses a protein of CHIKV, in particular a VLP of CHIKV. In a preferred embodiment, the VLP is composed of structural proteins C, E1, E2, E3 and 6K proteins. According to another particular embodiment of the present disclosure, the recombinant baculovirus can express a protein of JEV, particularly a VLP of JEV. According to yet another specific embodiment of the present disclosure, the recombinant baculovirus can express a protein of DENV, in particular a VLP of DENV. According to yet another specific embodiment of the present disclosure, the recombinant baculovirus may express a ZIKV protein, particularly a ZIKV VLP. In a preferred embodiment, the VLP consists of the structural proteins prM and E.
Promoters suitable for use in recombinant baculoviruses of the invention include, but are not limited to, pag 1: an early expression gene of HzNV-1 virus; p 10: baculovirus late gene; cmv: cytomegalovirus (cytomegalovirus); sv 40: simian virus (simian virus) 40; cir: chimeric Internal Ribosome Entry Sites (IRES) of RhPV virus and EV71 virus; b 1: antibacterial peptide gene b 1; a 4: defensin gene a 4; pub: polyubiquitin (polyubiquitin) gene; heat shock protein 70(hsp70) gene; hhi 1: an HzNV-1 virus early expression gene; hr 1: homeodomain gene 1. The above promoters may be used in combination with each other, i.e., a composite promoter, for example, pag1/sv40 or b1/p10 is used. In one embodiment, the promoter comprises pag 1. In another embodiment, the promoter comprises b 1. In yet another embodiment, the promoter comprises hr 1. In other embodiments, the promoter comprises pag1/b 1. In still other embodiments, the promoter comprises hr1/b 1. In yet other embodiments, the promoter comprises hr1/pag1 (SEQ ID NO: 1). In still other ways, the promoter comprises pag1/hr1/b 1. In the context of the present invention, a recombinant baculovirus vector having the above promoter to express a target foreign gene in a mosquito cell is called BacMos.
Examples of recombinant baculovirus suitable for use in the present disclosure include, but are not limited to, Autographa californica multinuclear polyhedrosis virus (AcMNPV), Heliothis virescens multinuclear polyhedrosis virus (AfMNPV), Heliothis virescens multinuclear polyhedrosis virus (Antiraphica falciparus MNPV), Heliotica polykarya MNPV, Agrimonia hosta multinuclear polyhedrosis virus (Anticarsia gemmatalis MNPV, AgMNPV), Bombyx mori multinuclear polyhedrosis virus (Bombyx mori MNPV, BmPV), Ectropis gigas mononuclear polyhedrosis virus (Bumpera single nuclear polyhedrosis virus, SNPV), Heliothis virens mononuclea polyhedrosis virus (SNPV), Heliothis virens mononuclear polyhedrosis virus (Heliothis virens SNPV, SNPV), Heliothis virens mononucleosis virus (Spodoptera multinuclear Spodoptera Spongopus pv), Spodoptera multinuclear polyhedrosis virus (Spodoptera PV, Spodoptera virus (Spodoptera PV), SeMNPV) or Trichoplusia ni polyhedrosis virus (Trichoplusia ni MNPV, tnmnnpv). According to a preferred embodiment of the present invention, the recombinant baculovirus is a recombinant Autographa californica multinuclear polyhedrosis virus.
According to an optional embodiment of the present disclosure, the recombinant baculovirus may further comprise a reporter gene operably linked to the promoter and the exogenous gene, and encoding a reporter protein that facilitates tracking and/or manipulation of expression. Examples of reporter proteins include, but are not limited to, Green Fluorescent Protein (GFP), Enhanced Green Fluorescent Protein (EGFP), red fluorescent protein of disc anemone (DsRed fluorescent protein, DsRed), Blue Fluorescent Protein (BFP), Enhanced Yellow Fluorescent Protein (EYFP), sea anemone hamno fluorescent protein (anmonia majano fluorescent protein, amFP), buttonhole fluorescent protein (zoxanthus fluorescent protein, zFP), sea anemone fluorescent protein (disco fluorescent protein, dsFP), and coral fluorescent protein (vularia fluorescent protein, clacium fluorescent protein).
Alternatively or additionally, the recombinant baculovirus may further comprise a gene encoding an affinity tag (affinity tag) operably linked to the promoter and the foreign gene to facilitate protein purification by the encoded affinity tag. Examples of affinity tags include, but are not limited to, orotidine 5'-phosphate decarboxylase (orotidine 5' -phosphate decarboxylase), Chitin Binding Protein (CBP), Maltose Binding Protein (MBP), streptococcal tag (Strep-tag), glutathione S-transferase (glutathione-S-transferase, GST), Thioredoxin Reductase (TRX), albumin binding protein (albumin binding protein, ABP), alkaline phosphatase (alkaline phosphatase, AP), biotin carboxyl carrier protein (biotin-carboxyl carrier protein, BCCP), calmodulin binding peptide (calmodulin binding peptide, CBP), peptide 7epitope (bacterial 7epitope, T7-tag), multiple-group tag HA-tag (HA-tag), phage-tag (HA-tag), Mycobacterized-tag (HA-tag), and multiple-group HA tag (HA-tag), and the like, Spot flag (Spot-tag), NE flag (NE-tag), and combinations thereof.
In preparing the recombinant baculovirus of the present disclosure, a gene cassette (cassette) carrying viral proteins of a specific arbovirus is constructed first, and then linked to an appropriate promoter (e.g., pag1, b1, hr1, or a combination thereof) to generate a baculovirus transfer vector; subsequently, the baculovirus DNA is co-transfected (co-transfect) with a transfer vector into a host cell (e.g., insect cell (Sf21)) to produce a recombinant baculovirus of the present disclosure. In a specific embodiment, the baculovirus transfer vector is co-transfected with a flash bacultra baculovirus DNA into an insect host cell. The FlashBACULTRA baculovirus DNA provides the necessary viral backbone, which contains the propagation-essential (replication-essential) genes. Homologous recombination between the recombinant baculovirus transfer vector and the flash bacultra baculovirus DNA in the insect host cell can produce a recombinant baculovirus that can be propagated in the insect host cell and thereby produce the foreign protein of interest encoded by the expressed gene cassette, respectively. The recombinant baculovirus may be further screened and purified, for example by reporting the expression of the polypeptide. Insect host cells suitable for use in the context of the present invention include, but are not limited to: spodoptera frugiperda (S.frugiperda) IPBL-9(Sf9) cells, Sf21 cells, High Five cells and Mimic Sf9 cells. According to certain embodiments of the present disclosure, the insect host cell is an Sf21 cell.
3. Preparation of VLP
A second aspect of the present disclosure relates to a method of producing a VLP using a recombinant baculovirus of the present disclosure, particularly in mosquito cells. The method comprises the following steps:
(1) transducing a recombinant baculovirus of the invention into a mosquito cell; and
(2) purifying the mosquito cells or supernatant of step (1) to produce the VLPs.
According to certain embodiments of the present disclosure, mosquito cells that may be used in the methods of the present disclosure include, but are not limited to, Aedes albopictus (e.g., C6/36 and ATC-15), Aedes pseudobulbifera (e.g., AP-61), Aedes aegypti (e.g., ATC-10 and CCL-125), Aedes manyparis (Toxophynchi amboinensis) (e.g., TRA-171), Aedes trifoliata (Culex tripheliorhynchus), Aedes sinensis (Anopheles sinensis), Aedes albugineus (Armigera subulatus), or Aedes tropicalis (Culex quinquefasciatus). In a particular embodiment, the mosquito cell is a mosquito cell of a white spotted mosquito (e.g., C6/36), an Aedes aegypti (e.g., CCL-125), or an Aedes pseudolepideus (e.g., AP-61).
The recombinant baculovirus of the present invention carries at least one foreign gene of interest encoding a protein of a virus, in particular a VLP, for example comprising C, E1, E2, E3, 6K, prM, E or a combination thereof; and the expression of the foreign gene is driven by a promoter sequence comprising pag1, b1, hr1, or a combination thereof. The VLP protein may be a virus species from the herpesviridae, adenoviridae, african swine fever virus, papillomaviridae, polyomaviridae, poxviridae, circovirus, small DNA virus, reoviridae, arteriviridae, coronavirus, picornavirus, fibiviridae, paramyxoviridae, cannonball virus, astrovirus, caliciviridae, flaviviridae, hepadnavirus, togaviridae, arenaviridae, or bunyavirus families. In a specific embodiment, the recombinant baculovirus expresses a VLP of CHIKV from togaviridae, wherein the VLP is composed of structural proteins C, E1, E2, E3 and 6K. In yet another specific embodiment, the recombinant baculovirus expresses a VLP of JEV, DENV or ZIKV from the flaviviridae family, wherein the VLP is composed of the structural proteins prM and E.
After transduction of a recombinant baculovirus of the present invention (carrying a transfer vector with a gene encoding a VLP) into a mosquito cell, the proteins of the VLP are expressed in the mosquito cell and then self-assembled into mature VLPs. The mature VLPs may be present in the mosquito cells or may be released extracellularly (i.e. in the cell culture supernatant) by various means, such as budding, exocytosis or cytolysis. To determine whether the mosquito cells successfully expressed the viral protein, an immunoassay was used. The immunoassay may be Western Blot (WB) assay, enzyme linked immunosorbent assay (ELISA), Radioimmunoassay (RIA), Immunohistochemistry (IHC) assay, Immunocytochemistry (ICC) assay, Immunofluorescence (IF) assay, dot blot (dot blot) assay, or enzyme-linked immunosorbent assay. In a preferred embodiment, the immunoassay is an IF assay. In another preferred embodiment, the immunoassay is a WB assay. In yet another preferred embodiment, the immunoassay is a dot blot analysis. In yet another preferred embodiment, the immunoassay is an ELISA. In other preferred embodiments, the immunoassay is ELISpot.
After successful confirmation of VLP expression, the mosquito cells or supernatant can be purified to produce the VLPs using any purification method known to those skilled in the art. Such purification methods include, but are not limited to, dialysis (dialysis), ultrafiltration (ultrafiltration), gel filtration chromatography (gel filtration chromatography), density gradient centrifugation (e.g., sucrose density gradient centrifugation), isoelectric focusing (isoelectrofocusing), ammonium sulfate precipitation (ammonium sulfate precipitation), protein a or G columns (protein a or protein G columns), DEAE ion exchange chromatography (DEAE ion exchange chromatography), or affinity chromatography (affinity chromatography).
Based on the foregoing, the present disclosure also encompasses exogenous proteins expressed by recombinant baculoviruses of the present disclosure, such as C, E1, E2, E3, 6K, prM, E, or combinations thereof, and also encompasses a VLP, particularly a VLP of an arbovirus, wherein the VLP is expressed by a mosquito cell. Thus, the present disclosure also encompasses VLPs produced by the methods of the present disclosure. The VLPs of the present invention may be subsequently used for a variety of different purposes, such as vaccine preparation using the VLPs as antigens, or as detection reagents for detecting the presence of antibodies against the virus in a biological sample, and further determining whether the individual from which the biological sample was derived has been immunized or infected with the virus.
4. Detection of antibodies in biological samples using VLPs
A third aspect of the present disclosure relates to a method of detecting an anti-viral antibody in a biological sample using the VLP of the present disclosure, comprising:
(1) mixing the VLP of the present invention with the biological sample; and
(2) detecting a complex formed by the anti-viral antibody and the VLP of step (1) in an immunoassay.
According to an embodiment of the present disclosure, the VLP may be prepared from a recombinant baculovirus of the present disclosure, in particular in a mosquito cell, and may be a virus species from the families herpesviridae, adenoviridae, african swine fever virus, papillomaviridae, polyomaviridae, poxviridae, circoviridae, picornaviridae, reoviridae, arteriviridae, coronaviridae, picornaviridae, celluloviridae, paramyxoviridae, cannonball virus, astroviridae, caliciviridae, flaviviridae, hepadnaviridae, togaviridae, arenaviridae or the yaviridae. In a specific embodiment, the VLP is a VLP of CHIKV from togaviridae, wherein the VLP is composed of structural proteins C, E1, E2, E3 and 6K, which can be used to capture antibodies against CHIKV in a biological sample, which can be serum of an individual who has been vaccinated against CHIKV or who has been infected with CHIKV. In yet another specific embodiment, the VLP is a VLP of JEV, DENV or ZIKV from the flaviviridae family, wherein the VLP is composed of structural proteins prM and E, which can be used to capture antibodies against JEV, DENV or ZIKV in a biological sample, which can be serum of an individual who has been vaccinated with a JEV, DENV or ZIKV vaccine or who has been infected with JEV, DENV or ZIKV.
Biological samples suitable for use in the methods of the present disclosure include, but are not limited to, whole blood samples, plasma samples, serum samples, urine samples, mucus samples, saliva samples, and purified or filtered versions thereof. In a preferred embodiment, the biological sample is a serum sample.
According to a preferred embodiment of the present disclosure, the anti-viral antibody is an IgM or an IgG.
Immunoassays suitable for use in the methods of the present disclosure include, but are not limited to, WB assays, ELISA, RIA, IHC assays, ICC assays, IF assays, dot blot or ELISpot assays. In a preferred embodiment, the immunoassay is an IF assay. In another preferred embodiment, the immunoassay is a WB assay. In yet another preferred embodiment, the immunoassay is a dot blot analysis. In yet another preferred embodiment, the immunoassay is an ELISA. In other preferred embodiments, the immunoassay is ELISpot.
Then, whether the biological sample has anti-virus antibody can be determined according to the result of the immunoassay. When the result of the immunoassay indicates the presence of a complex, it indicates that the biological sample has anti-viral antibodies, indicating that the individual from whom the biological sample was derived has been immunized or infected with the virus; when the result of the immunoassay shows no complex formation, it indicates that the biological sample does not have anti-viral antibodies, indicating that the source individual of the biological sample has not been immunized or infected with the virus.
Based on the above, the present disclosure also encompasses a detection reagent. The detection reagent comprises the VLP of the present invention for detecting an anti-viral antibody present in a biological sample, wherein the VLP is expressed by a mosquito cell. In addition, the present invention also encompasses a detection kit comprising the above detection reagent; a container for containing the detection reagent; and instructions associated with the container and indicating how to use the VLP of the present invention to detect antibodies present in a biological sample.
5. Vaccine composition comprising VLPs
The present disclosure also provides a vaccine composition (vaccine composition) comprising: (i) any of the VLPs of the present invention, and (ii) a pharmaceutically acceptable carrier, wherein the carrier may be an adjuvant (adjuvant). The term "vaccine composition" as used in the context of the present invention may refer to a composition which, when inoculated into a host, has the ability to elicit an immune response in the host which fully or partially protects the host against a disease (e.g. infection by a virus) or reduces its symptoms (e.g. fever, congestion and/or headache). In accordance with certain embodiments of the present disclosure, the present disclosure vaccine composition comprises the present disclosure VLP. The VLP may be a virus species from the herpesviridae, adenoviridae, african swine fever virus, papillomaviridae, polyomaviridae, poxviridae, circovirus, picornaviridae, reoviridae, arteriviridae, coronavirus, picornaviridae, fibrosiviridae, paramyxoviridae, cannonball virus, astrovirus, caliciviridae, flaviviridae, hepadnaviridae, togaviridae, arenaviridae or bunyaviridae families. In one embodiment, the vaccine composition of the present invention comprises a VLP of CHIKV. In another embodiment, the vaccine composition of the present invention comprises VLPs of JEV, DENV or ZIKV. Such vaccine compositions may be used as prophylactic or therapeutic agents to treat particular conditions. The vaccine composition of the present invention may be a monovalent vaccine or a multivalent vaccine. Alternatively, different VLPs of the present invention may be combined, or VLPs of the present invention may be combined with other antigens as a vaccine composition.
VLPs of the present invention may be suspended in a diluent for subsequent use. Exemplary diluents include: water (water), saline (salt), glucose (dextrose), propanol (propanol), ethanol (ethanol), mannitol (mannitol), sorbitol (sorbitol), lactose (lactose), starch (starch), lactitol (lactitol), maltodextrin (maltodextrin), glycerol (glycerol), xylitol (xylitol), trehalose (trehalose), mineral oil (mineral oil), vegetable oil (vegeable oil), sodium chloride (sodium chloride), sodium carbonate (sodium carbonate), sodium bicarbonate (sodium bicarbonate), potassium chloride (potassium chloride), calcium hydrogen phosphate (dicalcium phosphate), calcium carbonate (calcium carbonate), calcium sulfate dihydrate (calcium sulfate), and magnesium carbonate (magnesium carbonate).
Vaccine compositions may be prepared via conventional methods. Exemplary pharmaceutically acceptable carriers include physiological saline, sodium bicarbonate solution and/or adjuvants. The carrier may be selected according to the route of administration of the drug, the normal pharmaceutical process, and the like. Suitable pharmaceutically acceptable carriers and diluents, and the necessity for pharmaceutically acceptable use of the drug, are described in detail in Remington's Pharmaceutical Sciences,18th edition, Mack Publishing co., Easton, Pa (1990). Vaccine compositions also comprise polymers that can deliver drugs in vivo. Reference may be made to Audran R.et al.vaccine 21: 1250-; and Denis-haze et al cell immunol.,225:12-20,2003.
The term "adjuvant" as used in the context of the present invention refers to any substance or mixture of substances used to enhance (enhances), increase (innovations), up-regulate (up-regulates) or diversify (diversities) an immune response (e.g. humoral or cellular) against an antigen. By way of example, the adjuvant may be Freund's complete adjuvant (FA), Freund's incomplete adjuvant (IFA), mineral gel (minor gel), such as aluminum hydroxide (aluminum hydroxide) or aluminum phosphide (aluminum phosphate), surfactant (surface active site, such as lysolecithin), pluronic polyol (pluronic polyol), polyanion (polyanion), peptide (peptide), oil emulsifier (oil emulsifier), hydrocarbon emulsifier (hydro carbo emulsifier), keyhole limpet hemocyanin (keyhole limpet hemocyanin), sulfolipo-cyclodextrin (sulfolipo-cyclodextrin, SL-CD), saponin (such as quini A) cholera toxin (toxin), heat toxin (heat toxin), enterotoxin (immune complex), or enterotoxin (immune complex), lipo-stimulating oligonucleotide (lipoid-stimulating oligonucleotide), lipo-lipoid liposome, SL-CD, saponin (such as quino A) cholera toxin (cholera toxin), heat toxin (heat-stimulating oligonucleotide), or enterotoxin (immune complex, ISS-ODN).
The vaccine composition of the present invention can be administered to a subject in need thereof via an appropriate route. For example, administration can be via transmucosal (transmucosal), parenteral (intravenous), intravenous (intravenous), subcutaneous (subcutaneous) injection or implantation (transplantation), intramuscular (intramuscular), intraspinal (intramural), intraperitoneal (intraepithelial), intradermal (intracutaneous), sternal (intrasternal), intraarticular (intraarticular), intracranial (intraspinal), intralesional (intralesion), intrarectal (intrarectal), intravaginal (intravaginal), intranasal (intragastric), gastrointestinal (intragastric), intratracheal (intratracheal), or intrapulmonary (intrapulmonary) routes.
According to embodiments of the present disclosure, the vaccine composition of the present disclosure is administered to the subject at least 2 times, e.g., 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more times, during an immunization. To produce a particular effect, the vaccine composition may be administered to the individual 2-10 times, with intervals of several days to several years. In a preferred embodiment, the vaccine composition is administered to the subject a total of 3 times, with a time interval of 2 weeks between each administration.
To confirm whether an individual successfully generates an immune response against the corresponding virus following immunization of the individual with the VLP of the present disclosure, the production of individual cytokines (e.g., IL-1, IL-2, IL-6, IL-7, IL-12, IFN- α, IFN- β, IFN- γ, TNF- α, TNF- β) or anti-viral antibodies in a biological sample of the individual may be detected. In a preferred embodiment, the amount of IFN- γ secreted by the subject is measured.
The basic spirit and other objects of the present invention, as well as the technical means and embodiments adopted by the present invention, will be readily understood by those skilled in the art after considering the following embodiments.
Examples
1. Materials and methods
1.1 cells and viruses
At 28 deg.C, 5% CO2In a humidified incubator, C6/36 cell line of the white wire mosquito and CCL-125 cell line of the Aedes aegypti were cultured in RPMI-1640 medium (GIBCO, Invitrogen, CA) supplemented with 10% Fetal Bovine Serum (FBS) and 1 × Antibiotic-antimycin. In addition, 5% CO at 28 ℃2In a humidified incubator, with L-15 medium (GIBCO, Invitrogen, CA) supplemented with 10% FBS and 1 × antibiotic-antimycinCulturing the Aedes pseudolepidopteris AP-61 cell strain. Virus strains JEV/Taiwan/YL0806f/M/2008/KF667317 (genotype I cluster I (Cluster), GI), JEV/Taiwan/YL1106b/M/2011/KF667327 (genotype I cluster II, GI), JEV/Taiwan/TC1006a/M/2010/KF667314 (genotype III cluster I, GIII), JEV Zhongshan (Nakayama) strain, and JaGAr-01 strain were cultured with C6/36 cells, and virus titer was confirmed by spot analysis (plaque assoy) with BHK-12 cells.
1.2 construction of transfer vectors
In part of the preparation of the JEV transfer vector, the NheI-NotI region in pBac-b1-EGFP-Rhir-E was substituted with a synthetic 2.8kb NheI-NotI fragment to construct a transfer vector, wherein the synthetic fragment was adjusted to codons common to insects and contained hr1-pag1-JEV prM-E (accession No. AAB66485, amino acids 106 to 794 thereof) -poly A fragment (SEQ ID NO: 2). In the part for preparing the DENV transfer vector, the SmaI-NotI region in the transfer vector pFastBacT1-Ph-MCS-LIR-hr1pag1-cecrob 1(the construction method is described below) was substituted with a synthetic SmaI-NotI fragment of 2,075bp to construct a transfer vector, wherein the synthetic fragment was adjusted to codons commonly used in insects and contained a JEV prM signal peptide-DENV 2prM-E (accession number ACH61685.1, amino acids at positions 115 to 775 therein) fragment (SEQ ID NO: 3). In the part for preparing the ZIKV transfer vector, the SmaI-NotI region in the transfer vector pFastBacT1-Ph-MCS-LIR-hr1pag1-cecrob1 was substituted with a synthetic 2,307bp SmaI-NotI fragment to construct a transfer vector, wherein the synthetic fragment contains a ZIKV prM-E (accession number AQS26817, amino acids at positions 105 to 794 therein) fragment (SEQ ID NO: 4). In a part for preparing the CHIKV transfer vector, the BglII-PstI region in the above JEV transfer vector was substituted with a synthesized 3,997bp BglII-NotI fragment to construct a transfer vector, wherein the synthesized fragment comprises a fragment (SEQ ID NO: 5) of CHIKV 26S (CHIKV strain LR2006_ OPY1, accession No. KT449801, amino acids at positions 7,567 to 11,310 therein). These transfer vectors were all sequence confirmed. The obtained recombinant baculovirus is named as BacMos-JEV prM-E, BacMos-DENV prM-E, BacMos-ZIKV prM-E and BacMos-CHIKV 26S respectively. According to Kuo, et al, cell-based analysis of Chinese virus membrane fusion using bacterial-expression vectors, journal of viral methods 2011,175, (2),206-15, and Naik, et al, Bacillus as an infection vector for gene delivery into mosquitoes. Sci Rep.2018Dec 12; 8(1) 17778 to prepare recombinant baculovirus. The part of the construction of the transfer vector pFastBacT1-Ph-MCS-LIR-hr1pag1-cecrob1 was a substitution of the SpeI-XhoI region in pFastBac1(Thermo Fisher Scientific) with a synthetic SpeI-XhoI fragment of 1,464bp and a substitution of the SacI-SpeI region with a synthetic SacI-SpeI fragment of 981bp, wherein the synthetic SpeI-XhoI fragment comprises a modified IRES-sRed fragment (SEQ ID NO: 6) and wherein the synthetic SacI-SpeI fragment comprises hr1-pag1-cecropin b1 fragment (SEQ ID NO: 7).
1.3 preparation and purification of mosquito cell-derived VLPs
In 100 ml of culture medium, 1X 108Each recombinant baculovirus was transduced by individual AP-61 cells (MOI ═ 5). After 12 hours of culture, cells were washed 2 times with PBS. Supernatants were collected at day 3 post infection and again at 3 additional days. Next, 200 ml of the supernatant was filtered at 0.45 μm to remove cell debris and concentrated 100X in a dialysis tube (molecular weight cut-off 300,000) (MidiKros Module, Spectrum Repligen, USA). The concentrated VLPs were injected into a 25% sucrose solution and centrifuged at 250,000 × g (rotor model P28S, Hitachi) for 12 hours. The resulting precipitate was then redissolved in NTE buffer and injected into a 15% to 55% sucrose gradient and centrifuged for an additional 18 hours (250,000 Xg rpm, rotor model P28S, Hitachi). Next, 8 fractions from top to bottom were taken as samples for dot blot analysis and TEM analysis. The VLP proteins were analyzed by SDS-PAGE and the band corresponding to E, obtained by staining with Coomassie Brilliant blue (Coomassie blue), was confirmed by capillary LC-MS (model QSTAR XL Q-Tof mass spectrometer, AB Sciex).
1.4 Immunofluorescence (IF) assays
Mosquito cells were transduced with recombinant baculovirus BacMos-JEV prM-E (MOI ═ 5), or infected with a strain of JEV zhongshan (MOI ═ 0.1). On day 3 post-transduction, or day 2 post-infection, cells were fixed and allowed to react for 1 hour with anti-flavivirus monoclonal E antibody (6B6C) (1: 100 dilution) (JEV, DENV and ZIKV detectable E), anti-JEV prM monoclonal antibody (Mybiosource) (1: 80 dilution), anti-CHIKV monoclonal E2 antibody, rabbit anti-CHIKV antibody E1 antibody or anti-CHIKV monoclonal C antibody. Thereafter, the cells were washed 3 times with PBS and then exposed to a fluorescence-labeled secondary antibody (Alexa Fluor 488 or Alexa Fluor 594 labeled) for 1 hour. Finally, the cells were washed with PBS and the cell images were captured with inverted fluorescence microscope.
1.5 dot ink analysis
Mosquito cells were transduced with recombinant baculovirus BacMos-JEV prM-E (MOI ═ 5), or infected with a strain of JEV zhongshan (MOI ═ 0.1). The supernatant was collected and centrifuged at 12,000rpm for 5 minutes, and 100. mu.l of the sample was spotted on a Nitrocellulose (NC) membrane (PROTRAN, Schleicher & Schuell) using a microfiltration device (bio-dot microfiltration apparatus). The NC membranes were blocked (blocked) for 1 hour with Tris Buffer (TBS) containing 5% desfmed milk. Next, the antibody was reacted with E monoclonal antibody against JEV (6B6C) (1: 2000 dilution), rabbit anti-JEV prM serum (Genetex, Irvine, CA) (1: 1000 dilution), or anti-CHIKV E2 monoclonal antibody (in TBS). NC membranes were washed 3 times (15 min each) with TBS containing 0.1% Tween 20 at room temperature to remove unbound antibody. Next, the NC membrane was exposed to a secondary antibody (Chemicon, Billerica, MA, USA) (1: 10,000 dilution) labeled with horseradish peroxidase (HRP) for 1 hour. The NC membranes were washed 3 more times (15 minutes each) with TBS containing 0.1% Tween 20 to remove unbound antibody. Signals were obtained using an HRP-catalyzed Chemiluminescent Substrate (SuperSignal West Pico PLUS Chemiluminescent Substrate) (ThermoFisher Scientific, USA). Thereafter, triplicate blotch images were obtained with a developing machine (Amersham Imager 600photometer) and quantified with image analysis software (ImageJ).
1.6 Transmission Electron Microscopy (TEM)
VLPs were injected onto a carbon film (CF200-CU) (Electron Microcopy Science) with a 200 copper mesh. The mesh was washed 6 times with PBS, then fixed with 4% paraformaldehyde (in PBS) for 15 minutes, washed with PBS and 0.2 vol moore's concentration of sodium cacodylate (sodium cacodylate) buffer, and finally stained with 1% uranyl acetate solution. Inspection was performed by TEM (JEOL JEM-1200EXII, JEOL, Tokyo, Japan).
1.7IgM antibody capture enzyme immunosorbent assay (IgM antibody capture enzyme-linked immunosorbent assay, MAC-ELISA)
Goat anti-human IgM antibody (Jackson, USA) was plated in a 96-well plate (Nunc maxisorp) at 37 ℃ for 1 hour, followed by blocking with blocking buffer (1% BSA in PBS). 100 Xdilution of human serum (including JEV-infected sera: 58912, 58911, 58833, 58682, 58655, 58556 and 58941; DENV 2-infected sera: 59046; ZIKV-infected sera: 56928 and 56964; CHIKV-infected sera: 56851 and 59397; normal sera: S10700567, S10700568, S10700569 and S10700570) was added to the wells and allowed to act at 37 ℃ for 30 minutes. After washing away unbound antibodies, 100 microliters of antigen (including VLPs or supernatants of JEV, DENV, ZIKV, or CHIKV, or the strain JaGAr-01 (1 × 10 per ml)7PFU)) was added to the wells and allowed to act at 37 ℃ for 30 minutes. Followed by another PBS wash, and 4000 Xdilution of AP-labeled monoclonal antibody (D56.3) of mouse anti-flavivirus was added to the wells and allowed to act at 37 ℃ for 30 minutes. The D56.3 monoclonal antibody has cross-reactivity and can be used as a tracking antibody, and has high affinity for epitope (epitope) sensitive to conformational change. Followed by additional washing with PBS and disodium p-nitrophenyl phosphate (p-nitrophenyl-phosphate) (Sigma) was added to the wells and allowed to react for 20 minutes at 37 ℃. Finally, color generation is carried out, and OD405 light absorption values are read.
1.8 immunization of mice
All animal experiments in this study were conducted in compliance with the animal experiment regulations of the farm Commission of the administrative department of Taiwan, and the animal experiment planning of this study was approved by the institutional animal care and use committee of the preventive medicine institute of the State medical institute. At the time of primary vaccination (day 0), groups of 4-week-old BALB master mice (each group n-5) were administered 100 microliters of JEV VLPs (adjuvanted or unadjuvanted)
Figure BDA0002083051570000261
(as a positive control group) and PBS (as a negative control group). Administration of attenuated vaccine to mice
Figure BDA0002083051570000262
Each mouse was given 100 microliters of attenuated vaccine (1/10, a recommended dose for adults) subcutaneously and given boost vaccination (boost) at the same dose over the course of time. Blood was collected on day 35, and after blood coagulation, serum was dispensed by centrifugation and stored at-80 ℃. Meanwhile, on day 35, mice were sacrificed by cervical dislocation (cervical dislocation) and spleens were collected for subsequent cellular immune response experiments.
1.9ELISA assay
Mouse sera were collected before and after immunization. Commercial JEV recombinant E protein (LifeSpan BioSciences, Inc.) was diluted and each well of a 96-well plate was coated with 10 micrograms of concentrated JEV VLP (dissolved in 100 microliters of sodium bicarbonate buffer (NaHCO)318.2 millivol Mole concentration of Na2CO3pH 9.6)) at 4 ℃ overnight. After blocking, 100 microliters of 2 x serial diluted serum samples (from 1: 100) were added to each well and allowed to act overnight at 4 ℃. Next, 100. mu.l of diluted HRP-labeled goat anti-mouse IgG (1: 3000, KPL) was added to each well, followed by color development using o-phenylenediamine (OPD) which is a substrate for HRP. The absorbance at 492 nm was read using a microplate spectrometer.
1.10 lysis plaque reduction neutralization assay (FR. mu. NT90)
To confirm the neutralizing antibody titer of the mice after immunization, FR. mu.NT 90 was performed according to the procedures described in Kojima, A.et al.Stable high-producer cell clone expressing virus-like particles of the Japanese organism viral protein for a second-generation supplement vaccine. journal of virology 2003,77, (16), 8745-55. Briefly, sera were diluted in 2 × sequence (from a concentration of 1: 10) in MEM containing 2% FBS. After heat-inactivated complement was performed, 100 microliters of the dilution of the sequence was exposed to an equal volume of JEV solution containing approximately 100PFU of a virus of JEV genotype 1 or 3. The mixture was added to BHK-21 cells (duplicate, approximately 100PFU per well). After the uptake step, the infected cells were cultured in DMEM containing 5% FBS and 1.4% methylcellulose for 20 hours. Thereafter, the plaques were stained with antibody and quantified. Neutralizing antibody titers were defined as the reciprocal of the highest dilution of serum that caused a 90% reduction in plaque (i.e., PRNT 90).
1.11 detection of cytokines by ELISpot
IFN- γ expression in spleen cells was detected 1 week after the final immunization using ELISpot and following instructions. Briefly, capture antibodies against mouse IFN-. gamma.were plated in 96-well plates and 5X 10 each5Individual spleen cells were added to each well. Thereafter, the samples were exposed to JEV VLP (5. mu.g per well), ConA (as a positive control group) or RPMI 1640 medium (as a negative control group) at 37 ℃ for 48 hours. For IFN-. gamma.detection, a matrix was added after the action of biotin-labeled antibodies and streptavidin-HRP to allow the identification of single cells with a cytokine response. The measurement was performed using an ELISpot reader.
Example 1 expression of viral structural proteins of arbovirus by mosquito cells
This experiment utilized BacMos to efficiently deliver genes into mosquito cells and to prepare VLPs of the jaxivirus. Specifically, recombinant baculovirus BacMos-JEV prM-E contains the hr1-pag1 promoter to drive the prM-E gene of JEV genotype 3 and express proteins in mosquito cells and secrete VLPs (fig. 1A). Similarly, the recombinant baculoviruses BacMos-DENV prM-E, BacMos-ZIKV prM-E and BacMos-CHIKV 26S also contained the hr1-pag1 promoter to drive the prM-E gene of DENV, the prM-E gene of ZIKV and the 26S gene of CHIKV, respectively (FIG. 1B).
Referring to FIG. 2A, in the experimental results of the IF assay, specific mosquito cells transduced with recombinant baculovirus BacMos-JEV prM-E or infected with JEV, except infected CCL-125 cells, all expressed E glycoprotein of JEV in large amounts. Also, the transduced cells expressed more of the E glycoprotein of JEV than the infected cells, which represents the ability of the transduced cells to express the E glycoprotein of JEV, comparable to the infected cells. In addition, IF microscopy can also be used to examine the location of the E glycoprotein of expressed JEV in the cell. As shown in fig. 2A, the E glycoprotein of JEV is expressed in the cytoplasm in both transduced and infected cells. Similarly, prM protein expression of JEV was observed in either transduced C6/36 cells or infected C6/36 cells (FIG. 2B). WB results also showed that the expression level of E glycoprotein of JEV accumulated with increasing time (days post infection) (fig. 2C). After transduction of specific mosquito cells with recombinant infectious virus BacMos-JEV prM-E by dot blot analysis, supernatants collected at specific time points were examined for secretion of E glycoprotein from JEV, while JEV-infected C6/36 cells were used as a reference control (FIG. 2D). As shown in fig. 2D, secreted E glycoprotein of JEV was detectable in both the supernatant of transduced or infected cells. Specifically, transduced AP-61 cells were highly expressed on days 1 to 6 post-infection, whereas transduced C6/36 cells were moderately expressed on days 4 to 6 post-infection, but transduced CCL-125 cells were only slightly expressed even on days 4 to 6 post-infection (FIG. 2D, left panel). The quantitative analysis results of the dot blot analysis are shown in the right column of FIG. 2D.
In addition, the expression of viral proteins of the recombinant baculovirus BacMos-DENV prM-E, BacMos-ZIKV prM-E and BacMos-CHIKV 26S after transduction of mosquito cells was also tested. Referring to fig. 3A, virus expression of E glycoprotein in cells (upper panel) was detected by IF assay or in supernatant (lower panel) by dot blot analysis. Viral E glycoprotein expression was detected in both cells and supernatants at day 4 after DENV and ZIKV transduction of AP-61 cells. Similarly, the expression of the viral structural proteins of CHIKV, including E2, E1 and C, was also detected in CHIKV transduced C6/36 cells or CHIKV infected C6/36 cells (FIG. 3B). To investigate whether there was an antibody-antigen cross-reaction between different virus families (e.g., Togaviridae and Flaviviridae), recombinant baculoviruses BacMos-CHIKV 26S and BacMos-JEV prM-E were transduced with AP-61 and C6/36 cells, respectively. On day 4 post transduction, dot blot analysis supernatants were performed with anti-CHIKV E2 antibody and anti-JEV E antibody. As shown in FIG. 3C, the anti-CHIKV E2 antibody was able to antagonize recombinant baculovirus BacMos-CHIKV 26S transduced AP-61 cells and C6/36 cells, while there was a slight cross-reaction to recombinant baculovirus BacMos-JEV prM-E transduced cells. However, the E antibody against JEV can be specific to AP-61 cells and C6/36 cells transduced by the recombinant baculovirus BacMos-JEV prM-E without generating cross reaction.
Example 2 confirmation of VLP characteristics of mosquito cell-derived JEV
The E glycoprotein profile of JEV was confirmed using a sucrose density gradient experiment, in which the E glycoprotein of JEV was collected from transduced AP-61 cells and concentrated. As shown in FIG. 4A, a distinct band was visible in the middle region of the sucrose density gradient tube. From the results of dot blot analysis, the E glycoprotein of JEV was present in the same fractions (fractions 4 to 8) almost simultaneously with the prM protein. The E glycoprotein of VLPs was further confirmed using a mass spectrometer. Fraction 5 of the sucrose density gradient was subjected to SDS-PAGE followed by staining with Coomassie Brilliant blue and a band corresponding to the E size of JEV was obtained (experimental data not shown) and subjected to LC-MS/MS analysis. The results of the analysis showed that several polypeptides corresponding to the E protein could be identified (coverage 29%) (fig. 4B). VLPs of each arbovirus were observed by microscopy with an electron microscope (fig. 4C to F). As shown in FIG. 4C, the purified fraction enriched in JEV E glycoprotein after negative staining (negatively-stabilized) revealed a VLP of JEV, which is approximately a circular particle with a diameter of 30 nm, in an electrography. Likewise, TEM can be used to observe VLPs of DENV (fig. 4D), ZIKV (fig. 4E) and CHIKV (fig. 4F), all of which are round particles about 30 nm in diameter. Taken together, the above results indicate that mosquito cells transduced with recombinant baculovirus can successfully secrete VLPs.
Example 3 VLPs can present epitopes of active viruses
The structure of VLPs is confirmed by comparing epitope specific antigenicity (epitope specific antigenicity) of both VLPs and active viruses. In this experiment, epitope-specific antigenicity of the JEV VLPs and active viruses was compared by MAC-ELISA, as demonstrated by the analysis of binding of JEV VLPs and active viruses by JEV (JE) -infected human serum or normal human serum (NC) (FIG. 5A). As shown in fig. 5A, both the VLPs and active viruses of JEV exhibited specific binding to JEV-infected sera, while there was only slight binding to normal sera. Both share a similar MAC-ELISA binding pattern (pattern), indicating that VLPs of mosquito cell-derived JEV may exhibit epitopes equivalent to active viruses, demonstrating that VLPs of mosquito cell-derived JEV are truly antigenic. To illustrate the specificity of binding between antigen and antibody, each infection serum (DENV2 infection serum: 59046; JEV infection serum: 58941 and 58912; CHIKV infection serum: 56851 and 59397; ZIKV infection serum: 56928 and 56964; normal serum: S10700568) was analyzed for the binding result of each antigen (VLP or supernatant of DENV; VLP or supernatant of JEV; CHIKV VLP or supernatant; ZIKV VLP or supernatant) by MAC-ELISA (FIG. 5B). As shown in fig. 5B, each infected serum may exhibit varying degrees of binding to each corresponding antigen (e.g., DENV2 infected serum may bind to the VLP or supernatant of DENV), while exhibiting varying degrees (mild to moderate) of cross-reactivity to other non-corresponding antigens.
Example 4 immunization with JEV VLPs elicits both humoral and cellular immune responses
To assess the immunogenicity of JEV VLPs, BALB/c mice were administered three doses (at weeks 4, 6 and 8, respectively) of 1 or 4 micrograms of JEV VLPs (adjuvanted or unadjuvanted) subcutaneously (fig. 6A). At the same time, two additional groups of mice were administered three doses each subcutaneously
Figure BDA0002083051570000301
(commercially available JEV vaccine) or PBS as a positive control or negative control. At week 10, full IgG titers specific for E against JEV were detected by ELISA, and neutralizing antibodies to mice were detected by FR μ NT 90. The results of the experiments show that all VLPs or all VLPs in JEV
Figure BDA0002083051570000302
The immunized mice elicited full IgG antibody titers ranging between 0.2 and 4 of the OD450 reading (fig. 6B), and anti-JEV neutralizing antibodies had titers ranging between 40 and 160 against GI (fig. 6D, left column) and GIII (fig. 6D, right column). Administered to PBSMice with a whole IgG antibody titer ranging between 0.1 and 0.2 of the OD450 reading and a neutralizing antibody titer range against JEV of about only>10. Taken together, the VLPs of JEV can elicit host production of IgG specific to E of JEV, as well as production of neutralizing antibodies to neutralize infection by JEV of GI and GIII. Notably, mice immunized with JEV VLPs had higher whole IgG antibody titers and higher neutralizing antibody titers than mice immunized with JEV VLPs
Figure BDA0002083051570000311
Immunized mice.
This experiment was performed to investigate whether the VLPs of JEV have the ability to elicit a T cell immune response specific to JEV. At 2 weeks after the final immunization, spleen cells from the immunized mice were collected and confirmed for the presence of IFN-. gamma.secreting cells by ELISpot assay. The results of the experiments show that VLPs and
Figure BDA0002083051570000312
the spleen cells of the immunized mice secreted IFN-. gamma.in a significantly higher amount (as measured by the number of spots) than the PBS group (negative control group) (FIG. 6C). In addition, the spleen cells of mice immunized with JEV VLPs also secreted IFN- γ in significantly higher amounts than those immunized with JEV VLPs
Figure BDA0002083051570000313
Immunized mice. From the above experimental results, it was found that the VLPs of JEV can elicit a virus-specific Th1 cellular immune response. The reason why the immunopotency of the inventive arbovirus VLP is presumed to be superior to other VLPs is that VLPs prepared from mosquito cells have a virus envelope structure (e.g., less complicated glycation modification, lipid content, etc.) that is not identical to other VLPs (see: Hafer A. et al. differential interaction of cholesterol by silicon virus growth in major or animal cells. J. Virol 83: 9113-9121.10; He. et al. the structure of the binding virus produced by protein virus and induced by protein virus tissue culture. J. Virol 84: 5270-5276.11; Hsie P and polypeptide PW. Regulation of protein-linked proteins vaccine. J. Virol 84: 5270-5276.11; Hsie P and polypeptideProcessing in A materials infectious cells J Biol Chem 259:2375-2382) so that the mosquito-derived VLP can resemble the infectious structures of human early mosquito vector viruses. Summarizing the above, mice immunized with VLPs of mosquito cell-derived JEV elicited robust humoral and cellular immune responses.
In summary, the recombinant baculovirus provided in the present disclosure can express foreign proteins, particularly VLPs of arboviruses (including JEV, DENV, ZIKV, CHIKV, and the like), in mosquito cells. Second, VLPs derived from mosquito cells can mimic epitopes of active viruses, and are useful as reagents for detecting antibodies against arboviruses. Furthermore, immunization with mosquito cell-derived VLPs allows the host to produce antibodies specific for the arbovirus and to elicit anti-virus-related cytokines with robust humoral and cellular immune responses. Accordingly, the mosquito cell-derived VLPs of the present disclosure are a very potential vaccine candidate.
It should be understood that the foregoing description of the embodiments is given by way of example only and that various modifications may be made by those skilled in the art. The above specification, examples and experimental results provide a complete description of the structure and use of exemplary embodiments of the invention. Although various embodiments of the present invention have been disclosed in the foregoing detailed description, it should be understood that various changes and modifications may be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims. Furthermore, the publications cited in the summary of the invention are considered to be part of the present disclosure.
Sequence listing
<110> Guo give
<120> recombinant baculovirus and use thereof
<130> 19FAP0228CT
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 582
<212> DNA
<213> Artificial sequence (artificial sequence)
<220>
<221> promoter
<223> synthetic sequence
<400> 1
gctagcgtgt tttacaagta gaattctacc cgtaaagcga gtttagtttt gaaaaacaaa 60
tgacatcatt tgtataatga catcatcccc tgattgtgtt ttacaagtag aattctatcc 120
gtaaagcgag ttcagttttg aaaacaaatg agtcatacct aaacacgtta ataatcttct 180
gatatcagct tatgactcaa gttatgagcc gtgtgcaaaa catgagataa gtttatgaca 240
tcatccactg atcgtgcgtt acaagtagaa ttctactcgt aaagccagtt cggttatgag 300
ccgtgtgcaa aacatgacat cagcttatga ctcatacttg attgtgtttt acgcgtagaa 360
ttctactcgt aaagcgagtt cggttatgag ccgtgtgcaa aacatgacat cagcttatga 420
gtcataatta atcgtgcgtt acaagtagaa ttctactcgt aatactcatc gaccaatggc 480
gtcgctcggt tcttatcgca acagagtggg ggccatccgc actataaaaa gccgagactg 540
gtgacgaaca ccatcagtct gattcgagtc gtgttcatac cg 582
<210> 2
<211> 2885
<212> DNA
<213> Artificial sequence (artificial sequence)
<220>
<221> gene
<223> synthetic sequence
<400> 2
gctagcgtgt tttacaagta gaattctacc cgtaaagcga gtttagtttt gaaaaacaaa 60
tgacatcatt tgtataatga catcatcccc tgattgtgtt ttacaagtag aattctatcc 120
gtaaagcgag ttcagttttg aaaacaaatg agtcatacct aaacacgtta ataatcttct 180
gatatcagct tatgactcaa gttatgagcc gtgtgcaaaa catgagataa gtttatgaca 240
tcatccactg atcgtgcgtt acaagtagaa ttctactcgt aaagccagtt cggttatgag 300
ccgtgtgcaa aacatgacat cagcttatga ctcatacttg attgtgtttt acgcgtagaa 360
ttctactcgt aaagcgagtt cggttatgag ccgtgtgcaa aacatgacat cagcttatga 420
gtcataatta atcgtgcgtt acaagtagaa ttctactcgt aatactcatc gaccaatggc 480
gtcgctcggt tcttatcgca acagagtggg ggccatccgc actataaaaa gccgagactg 540
gtgacgaaca ccatcagtct gattcgagtc gtgttcatac cgagatctcc accatgggtg 600
gcaacgaggg ttccatcatg tggctggctt ccctggctgt ggtcatcgct tgcgccggtg 660
ctatgaagct gtctaacttc cagggcaagc tgctgatgac tatcaacaac accgacatcg 720
ctgacgtgat cgtcatccct acttcaaagg gagaaaacag gtgctgggtg cgtgctatcg 780
acgtcggtta cctgtgcgag gacactatca cctacgaatg cccaaagctg accatgggca 840
acgaccctga ggacgtggac tgctggtgcg acaaccagga agtgtacgtc cagtacggaa 900
ggtgcactag gaccagacac tccaagagaa ctcgccgttc agtgtccgtc cagacccacg 960
gtgaatccag cctggtcaac aagaaggaag cttggctgga cagcactaag gccacccgct 1020
acctgatgaa gaccgagaac tggatcatcc gtaaccctgg atacgctttc ctggctgccg 1080
tgctgggatg gatgctgggt tctaacaacg gccagcgcgt ggtcttcact atcctgctgc 1140
tgctggtcgc ccccgcttac tcattcaact gcctgggtat gggcaaccgt gacttcatcg 1200
agggtgcttc cggtgctacc tgggtggacc tggtcctgga aggcgacagc tgcctgacta 1260
tcatggctaa cgacaagcca accctggacg tgcgcatgat caacatcgag gcttctcagc 1320
tggccgaagt ccgttcatac tgctaccacg cttctgtgac tgacatctca accgtcgcca 1380
ggtgccctac cactggagag gctcacaacg aaaagagagc cgactcttca tacgtgtgca 1440
agcagggttt caccgacagg ggatggggta acggctgcgg actgttcggc aagggcagca 1500
tcgacacttg cgctaagttc tcttgcacct caaaggccat cggtagaact atccagcccg 1560
agaacatcaa gtacgaagtg ggtatcttcg tccacggcac cactacctcc gagaaccacg 1620
gaaactactc tgctcaagtg ggtgcctcac aggctgccaa gttcactgtc accccaaacg 1680
ctccttccat cactctgaag ctgggagact acggagaggt gaccctggac tgcgaaccaa 1740
ggagcggcct gaacaccgag gccttctacg tgatgactgt cggaagcaag tctttcctgg 1800
tccacagaga atggttccac gacctggctc tgccctggac ttctccttct tctactgctt 1860
ggaggaacag ggagctgctg atggaattcg aggaagccca cgctaccaag cagtccgtgg 1920
tcgctctggg ttctcaagag ggaggtctcc accaggccct ggctggagct atcgtggtcg 1980
aatactcttc atccgtgaag ctgacttctg gccacctgaa gtgcaggctg aagatggaca 2040
agctggccct gaagggaact acctacggaa tgtgcaccga gaagttctca ttcgctaaga 2100
accccgccga cactggacac ggtaccgtgg tcatcgaact gtcatactcc ggcagcgacg 2160
gaccttgcaa gatccccatc gtgtccgtcg cttccctgaa cgacatgacc cccgtgggac 2220
gcctggtgac tgtcaaccca ttcgtcgcta ccagctcagc caactctaag gtgctggtcg 2280
agatggaacc tcccttcggt gactcataca tcgtgatcgg ccgcggagac aagcagatca 2340
accaccactg gcacaaggct ggctccactc tgggaaaggc cttcagcact accctgaagg 2400
gtgctcaacg tctggctgct ctgggcgaca ctgcttggga cttcggatct atcggcggag 2460
tgttcaactc aatcggcaag gctgtgcacc aggtcttcgg tggcgccttc cgtactctgt 2520
tcggaggtat gtcctggatc acccagggtc tgatgggcgc tctgctgctg tggatgggtg 2580
tgaacgccag ggacagaagc atcgctctgg ccttcctggc tactggcgga gtgctggtct 2640
tcctggctac caacgtccac gcctaactgc agagtagatg ccgaccgaac aagagctgat 2700
ttcgagaacg cctcagccag caactcgcgc gagcctagca agttgtttat tgcagcttat 2760
aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg 2820
cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg gatcggggcg 2880
gccgc 2885
<210> 3
<211> 2075
<212> DNA
<213> Artificial sequence (artificial sequence)
<220>
<221> gene
<223> synthetic sequence
<400> 3
cccgggacta tgggcggtaa cgaaggtagc attatgtggt tggcctcact ggctgtggtc 60
attgcctgtg cgggtgcgat gttccatctc actacccgca acggagagcc acacatgatc 120
gtgtcccgtc aggaaaaggg caagagcctg ctgttcaaga ccggtgacgg cgtcaacatg 180
tgcactctga tggccatgga cctgggagag ctgtgcgaag acaccatcac ttacaagtgc 240
ccactgctga ggcagaacga gcctgaagac atcgactgct ggtgcaactc caccagcact 300
tgggtgactt acggtacttg caccactact ggtgaacaca ggagggaaaa gaggtctgtg 360
gctctggtgc ctcacgtcgg aatgggtctg gaaacccgta ctgaaacctg gatgtcttct 420
gagggtgctt ggaagcacgc tcagcgcatc gaaacctgga tcctgcgtca ccctggcttc 480
actatcatgg ctgccatcct ggcctacacc atcggaacta cccacttcca gcgcgctctg 540
atcttcatcc tgctgaccgc tgtggccccc tccatgacta tgaggtgcat cggtatcagc 600
aacagagact tcgtggaggg cgtctccggt ggcagctggg tggacatcgt cctggaacac 660
ggttcttgcg tcactaccat ggccaagaac aagcccaccc tggacttcga gctgatcaag 720
accgaagcta agcagtcagc cactctgagg aagtactgca tcgaggctaa gctgactaac 780
actactaccg aatcccgctg ccctacccag ggagaaccat cactgaacga ggaacaggac 840
aagaggttcg tgtgcaagca cagcatggtc gacagaggct ggggaaacgg ttgcggcctg 900
ttcggaaagg gaggtatcgt gacctgcgct atgttcactt gcaagaagaa catgaagggc 960
aaggtggtcc agcctgagaa cctggaatac accatcgtga tcactcccca ctctggagag 1020
gaacacgccg tcggtaacga cactggcaag cacggcaagg agatcaagat caccccacag 1080
tcttcaatca ccgaggctga actgactgga tacggtactg tgaccatgga atgctcacct 1140
cgcactggtc tggacttcaa cgagatggtg ctgctgcaga tggaaaacaa ggcctggctg 1200
gtccaccgtc agtggttcct ggacctgcct ctgccatggc tccctggagc tgacacccag 1260
ggatccaact ggatccagaa ggagactctg gtcaccttca agaacccaca cgccaagaag 1320
caggacgtgg tcgtgctggg aagccaggag ggtgctatgc acactgccct gaccggagct 1380
actgaaatcc agatgtccag cggcaacctg ctgttcactg gacacctgaa gtgcaggctg 1440
agaatggaca agctgcagct gaagggcatg tcttactcaa tgtgcaccgg aaagttcaag 1500
gtcgtgaagg agatcgctga aacccagcac ggcactatcg tgatccgcgt ccagtacgag 1560
ggcgacggat ctccctgcaa gatcccattc gagatcatgg acctggaaaa gaggcacgtg 1620
ctgggcagac tgatcaccgt gaaccccatc gtcactgaaa aggactcccc agtgaacatc 1680
gaggccgaac ctcccttcgg agactcttac atcatcatcg gcgtcgagcc tggacagctg 1740
aagctgaact ggttcaagaa gggttcttca atcggccaga tgctggaaac cactatgcgc 1800
ggtgccaagc gtatggctat cctgggtgac actgcttggg acttcggctc tctgggcgga 1860
gtgttcacct caatcggcaa ggccctgcac caggtcttcg gagctatcta cggtgctgcc 1920
ttctctggcg tgtcatggac catgaagatc ctgatcggag tcatcatcac ttggatcggt 1980
atgaactccc gtagcacctc tctgtcagtc tcactggtcc tcgtgggtgt cgttacgctc 2040
tacctcggag ttatggttca agcctagcgg ccgcc 2075
<210> 4
<211> 2307
<212> DNA
<213> Artificial sequence (artificial sequence)
<220>
<221> gene
<223> synthetic sequence
<400> 4
cccgggatgg gcgcagatac cagtgtcgga attgttggcc tcctgctgac cacagctatg 60
gcagcggagg tcactagacg tgggagtgca tactatatgt acttggacag aaacgatgct 120
ggggaggcca tatcttttcc aaccacattg gggatgaata agtgttatat acagatcatg 180
gatcttggac acatgtgtga tgccaccatg agctatgaat gccctatgct ggatgagggg 240
gtggaaccag atgacgtcga ttgttggtgc aacacgacgt caacttgggt tgtgtacgga 300
acctgccatc acaaaaaagg tgaagcacgg agatctagaa gagctgtgac gctcccctcc 360
cattccacta ggaagctgca aacgcggtcg caaacctggt tggaatcaag agaatacaca 420
aagcacttga ttagagtcga aaattggata ttcaggaacc ctggcttcgc gttagcagca 480
gctgccatcg cttggctttt gggaagctca acgagccaaa aagtcatata cttggtcatg 540
atactgctga ttgccccggc atacagcatc aggtgcatag gagtcagcaa tagggacttt 600
gtggaaggta tgtcaggtgg gacttgggtt gatattgtct tggaacatgg aggttgtgtc 660
accgtaatgg cacaggacaa accgactgtc gacatagagc tggttacaac aacagtcagc 720
aacatggcgg aggtaagatc ctactgctat gaggcatcaa tatcagacat ggcttcggac 780
agccgctgcc caacacaagg tgaagcctac cttgacaagc aatcagacac tcaatatgtc 840
tgcaaaagaa cgttagtgga cagaggctgg ggaaatggat gtggactttt tggcaaaggg 900
agtctggtga catgcgctaa gtttgcatgc tccaagaaaa tgaccgggaa gagcatccag 960
ccagagaatc tggagtaccg gataatgctg tcagttcatg gctcccagca cagtgggatg 1020
atcgttaatg acacaggaca tgaaactgat gagaatagag cgaaggttga gataacgccc 1080
aattcaccaa gagccgaagc caccctgggg ggttttggaa gcctaggact tgattgtgaa 1140
ccgaggacag gccttgactt ttcagatttg tattacttga ctatgaataa caagcactgg 1200
ttggttcaca aggagtggtt ccacgacatt ccattacctt ggcacgctgg ggcagacacc 1260
ggaactccac actggaacaa caaagaagca ctggtagagt tcaaggacgc acatgccaaa 1320
aggcaaactg tcgtggttct agggagtcaa gaaggagcag ttcacacggc ccttgctggg 1380
gctctggagg ctgagatgga tggtgcaaag ggaaggctgt cctctggcca cttgaaatgt 1440
cgcctgaaaa tggataaact tagattgaag ggcgtgtcat actccttgtg taccgcagcg 1500
ttcacattca ccaagatccc ggctgaaaca ctgcacggga cagtcacagt ggaggtacag 1560
tacgcaggga cagatggacc ttgcaaggtt ccagctcaga tggcggtgga catgcaaact 1620
ctgaccccag ttgggaggtt gataaccgct aaccccgtaa tcactgaaag cactgagaac 1680
tctaagatga tgctggaact tgatccacca tttggggact cttacattgt cataggagtc 1740
ggggagaaga agatcaccca ccactggcac aggagtggca gcaccattgg aaaagcattt 1800
gaagccactg tgagaggtgc caagagaatg gcagtcttgg gagacacagc ctgggacttt 1860
ggatcagttg gaggcgctct caactcattg ggcaagggca tccatcaaat ttttggagca 1920
gctttcaaat cattgtttgg aggaatgtcc tggttctcac aaattctcat tggaacgttg 1980
ctgatgtggt tgggtctgaa cacaaagaat ggatctattt cccttatgtg cttggcctta 2040
gggggagtgt tgatcttctt atccacagcc gtctctgctt agctgcagct gcagagtaga 2100
tgccgaccga acaagagctg atttcgagaa cgcctcagcc agcaactcgc gcgagcctag 2160
caagttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac 2220
aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc 2280
ttatcatgtc tggatcgggg cggccgc 2307
<210> 5
<211> 3997
<212> DNA
<213> Artificial sequence (artificial sequence)
<220>
<221> gene
<223> synthetic sequence
<400> 5
agatctacca tggagttcat cccaacccaa actttttaca ataggaggta ccagcctcga 60
ccctggactc cgcgccctac tatccaagtc atcaggccca gaccgcgccc tcagaggcaa 120
gctgggcaac ttgcccagct gatctcagca gttaataaac tgacaatgcg cgcggtaccc 180
caacagaagc cacgcaggaa tcggaagaat aagaagcaaa agcaaaaaca acaggcgcca 240
caaaacaaca caaatcaaaa gaagcagcca cctaaaaaga aaccggctca aaagaaaaag 300
aagccgggcc gcagagagag gatgtgcatg aaaatcgaaa atgattgtat tttcgaagtc 360
aagcacgaag gtaaggtaac aggttacgcg tgcctggtgg gggacaaagt aatgaaacca 420
gcacacgtaa aggggaccat cgataacgcg gacctggcca aactggcctt taagcggtca 480
tctaagtatg accttgaatg cgcgcagata cccgtgcaca tgaagtccga cgcttcgaag 540
ttcacccatg agaaaccgga ggggtactac aactggcacc acggagcagt acagtactca 600
ggaggccggt tcaccatccc tacaggtgct ggcaaaccag gggacagcgg cagaccgatc 660
ttcgacaaca agggacgcgt ggtggccata gtcttaggag gagctaatga aggagcccgt 720
acagccctct cggtggtgac ctggaataaa gacattgtca ctaaaatcac ccccgagggg 780
gccgaagagt ggagtcttgc catcccagtt atgtgcctgt tggcaaacac cacgttcccc 840
tgctcccagc ccccttgcac gccctgctgc tacgaaaagg aaccggagga aaccctacgc 900
atgcttgagg acaacgtcat gagacctggg tactatcagc tgctacaagc atccttaaca 960
tgttctcccc accgccagcg acgcagcacc aaggacaact tcaatgtcta taaagccaca 1020
agaccatact tagctcactg tcccgactgt ggagaagggc actcgtgcca tagtcccgta 1080
gcactagaac gcatcagaaa tgaagcgaca gacgggacgc tgaaaatcca ggtctccttg 1140
caaatcggaa taaagacgga tgacagccac gattggacca agctgcgtta tatggacaac 1200
cacatgccag cagacgcaga gagggcgggg ctatttgtaa gaacatcagc accgtgtacg 1260
attactggaa caatgggaca cttcatcctg gcccgatgtc caaaagggga aactctgacg 1320
gtgggattca ctgacagtag gaagattagt cactcatgta cgcacccatt tcaccacgac 1380
cctcctgtga taggtcggga aaaattccat tcccgaccgc agcacggtaa agagctacct 1440
tgcagcacgt acgtgcagag caccgccgca actaccgagg agatagaggt acacatgccc 1500
ccagacaccc ctgatcgcac attaatgtca caacagtccg gcaacgtaaa gatcacagtc 1560
aatggccaga cggtgcggta caagtgtaat tgcggtggct caaatgaagg actaacaact 1620
acagacaaag tgattaataa ctgcaaggtt gatcaatgtc atgccgcggt caccaatcac 1680
aaaaagtggc agtataactc ccctctggtc ccgcgtaatg ctgaacttgg ggaccgaaaa 1740
ggaaaaattc acatcccgtt tccgctggca aatgtaacat gcagggtgcc taaagcaagg 1800
aaccccaccg tgacgtacgg gaaaaaccaa gtcatcatgc tactgtatcc tgaccaccca 1860
acactcctgt cctaccggaa tatgggagaa gaaccaaact atcaagaaga gtgggtgatg 1920
cataagaagg aagtcgtgct aaccgtgccg actgaagggc tcgaggtcac gtggggcaac 1980
aacgagccgt ataagtattg gccgcagtta tctacaaacg gtacagccca tggccacccg 2040
catgagataa ttctgtatta ttatgagctg taccccacta tgactgtagt agttgtgtca 2100
gtggccacgt tcatactcct gtcgatggtg ggtatggcag cggggatgtg catgtgtgca 2160
cgacgcagat gcatcacacc gtatgaactg acaccaggag ctaccgtccc tttcctgctt 2220
agcctaatat gctgcatcag aacagctaaa gcggccacat accaagaggc tgcgatatac 2280
ctgtggaacg agcagcaacc tttgttttgg ctacaagccc ttattccgct ggcagccctg 2340
attgttctat gcaactgtct gagactctta ccatgctgct gtaaaacgtt ggctttttta 2400
gccgtaatga gcgtcggtgc ccacactgtg agcgcgtacg aacacgtaac agtgatcccg 2460
aacacggtgg gagtaccgta taagactcta gtcaatagac ctggctacag ccccatggta 2520
ttggagatgg aactactgtc agtcactttg gagccaacac tatcgcttga ttacatcacg 2580
tgcgagtaca aaaccgtcat cccgtctccg tacgtgaagt gctgcggtac agcagagtgc 2640
aaggacaaaa acctacctga ctacagctgt aaggtcttca ccggcgtcta cccatttatg 2700
tggggcggcg cctactgctt ctgcgacgct gaaaacacgc agttgagcga agcacacgtg 2760
gagaagtccg aatcatgcaa aacagaattt gcatcagcat acagggctca taccgcatct 2820
gcatcagcta agctccgcgt cctttaccaa ggaaataaca tcactgtaac tgcctatgca 2880
aacggcgacc atgccgtcac agttaaggac gccaaattca ttgtggggcc aatgtcttca 2940
gcctggacac ctttcgacaa caaaattgtg gtgtacaaag gtgacgtcta taacatggac 3000
tacccgccct ttggcgcagg aagaccagga caatttggcg atatccaaag tcgcacacct 3060
gagagtaaag acgtctatgc taatacacaa ctggtactgc agagaccggc tgtgggtacg 3120
gtacacgtgc catactctca ggcaccatct ggctttaagt attggctaaa agaacgcggg 3180
gcgtcgctgc agcacacagc accatttggc tgccaaatag caacaaaccc ggtaagagcg 3240
gtgaactgcg ccgtagggaa catgcccatc tccatcgaca taccggaagc ggccttcact 3300
agggtcgtcg acgcgccctc tttaacggac atgtcgtgcg aggtaccagc ctgcacccat 3360
tcctcagact ttgggggcgt cgccattatt aaatatgcag ccagcaagaa aggcaagtgt 3420
gcggtgcatt cgatgactaa cgccgtcact attcgggaag ctgagataga agttgaaggg 3480
aattctcagc tgcaaatctc tttctcgacg gccttagcca gcgccgaatt ccgcgtacaa 3540
gtctgttcta cacaagtaca ctgtgcagcc gagtgccacc ccccgaagga ccacatagtc 3600
aactacccgg cgtcacatac caccctcggg gtccaggaca tctccgctac ggcgatgtca 3660
tgggtgcaga agatcacggg aggtgtggga ctggttgttg ctgttgccgc actgattcta 3720
atcgtggtgc tatgcgtgtc gttcagcagg cactaacttg acaattaagt atgaagggcg 3780
gccgagtaga tgccgaccga acaagagctg atttcgagaa cgcctcagcc agcaactcgc 3840
gcgagcctag caagttgttt attgcagctt ataatggtta caaataaagc aatagcatca 3900
caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca 3960
tcaatgtatc ttatcatgtc tggatcgggg cggccgc 3997
<210> 6
<211> 1464
<212> DNA
<213> Artificial sequence (artificial sequence)
<220>
<221> promoter
<223> synthetic sequence
<400> 6
actagtttaa aacagcctgt gggttgcacc cacccacagg gcccactggg cgctagcact 60
ctggtactga ggtacctttg tgcgcctgtt tttactcccc ttcccccgaa gtaacttaga 120
agctgtaaat caacgatcaa tagcaggtgt ggcacaccag tcataccttg atcaagcact 180
tctgtttccc cggactgagt atcaataggc tgctcgcgcg gctgaaggag aaaacgttcg 240
ttacccgacc aactacttcg agaagcttag taccaccatg aacgaggcag ggtgtttcgc 300
tcagcacaac cccagtgtag atcaggctga tgagtcactg caacccccat gggcgaccat 360
ggcagtggct gcgttggcgg cctgcccatg gagaaatcca tgggacgctc taattctgac 420
atggtgtgaa gagcctattg agctagctgg tagtcctccg gcccctgaat gcggctaatc 480
ctaactgcgg agcacatgct cacaaaccag tgggtggtgt gtcgtaacgg gcaactctgc 540
agcggaaccg actactttgg gtgtccgtgt ttccttttat tcctatattg gctgcttatg 600
gtgacaatca aagagttgtt accatatagc tattggattg gccatccggt gagttaaagc 660
tttataacta taagtaagcc gtgccgaaac gttaatcggt cgctagttgc gtaacaactg 720
ttagtttaat tttccaaaat ttatttttca caatttttag tggatccacc ggtcgccacc 780
atggcctcct ccgagaacgt catcaccgag ttcatgcgct tcaaggtgcg catggagggc 840
accgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc 900
cacaacaccg tgaagctgaa ggtgaccaag ggcggccccc tgcccttcgc ctgggacatc 960
ctgtcccccc agttccagta cggctccaag gtgtacgtga agcaccccgc cgacatcccc 1020
gactacaaga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag 1080
gacggcggcg tggcgaccgt gacccaggac tcctccctgc aggacggctg cttcatctac 1140
aaggtgaagt tcatcggcgt gaacttcccc tccgacggcc ccgtgatgca gaagaagacc 1200
atgggctggg aggcctccac cgagcgcctg tacccccgcg acggcgtgct gaagggcgag 1260
acccacaagg ccctgaagct gaaggacggc ggccactacc tggtggagtt caagtccatc 1320
tacatggcca agaagcccgt gcagctgccc ggctactact acgtggacgc caagctggac 1380
atcacctccc acaacgagga ctacaccatc gtggagcagt acgagcgcac cgagggccgc 1440
caccacctgt tcctgtaact cgag 1464
<210> 7
<211> 981
<212> DNA
<213> Artificial sequence (artificial sequence)
<220>
<221> promoter
<223> synthetic sequence
<400> 7
gagctcgtgt tttacaagta gaattctacc cgtaaagcga gtttagtttt gaaaaacaaa 60
tgacatcatt tgtataatga catcatcccc tgattgtgtt ttacaagtag aattctatcc 120
gtaaagcgag ttcagttttg aaaacaaatg agtcatacct aaacacgtta ataatcttct 180
gatatcagct tatgactcaa gttatgagcc gtgtgcaaaa catgagataa gtttatgaca 240
tcatccactg atcgtgcgtt acaagtagaa ttctactcgt aaagccagtt cggttatgag 300
ccgtgtgcaa aacatgacat cagcttatga ctcatacttg attgtgtttt acgcgtagaa 360
ttctactcgt aaagcgagtt cggttatgag ccgtgtgcaa aacatgacat cagcttatga 420
gtcataatta atcgtgcgtt acaagtagaa ttctactcgt aatactcatc gaccaatggc 480
gtcgctcggt tcttatcgca acagagtggg ggccatccgc actataaaaa gccgagactg 540
gtgacgaaca ccatcagtct gattcgagtc gtgttcatac cgcatatgcc cgggaccatg 600
aacttcagca aagtgtttgc actcgttctg ctcatcgggt tggttttact caccgggcat 660
accgaagccg gtggtctgaa gaagctgggg aagaaattgg aaggagtcgg aaaacgtgta 720
ttcaaagctt ctgagaaagc acttcccgtt ataactggtt acaaagccat aggaaaatag 780
atgcatgcgg ccgcagtaga tgccgaccga acaagagctg atttcgagaa cgcctcagcc 840
ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat 900
aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat 960
catgtctgga tcgggactag t 981

Claims (12)

1. A recombinant baculovirus (baculovir) comprising:
a promoter, and
a foreign gene encoding a structural protein of a Virus Like Particle (VLP), wherein the structural protein is selected from the group consisting of all, a portion of a virus structural protein, and combinations thereof.
2. The recombinant baculovirus of claim 1, wherein said structural protein is selected from the group consisting of capsid protein (capsid, C), envelope protein 1(envelope 1, E1), envelope protein 2(envelope 2, E2), envelope protein 3(envelope 3, E3), 6K protein (6K), pre-membrane protein (prM), and/or envelope protein (envelope, E), and combinations thereof.
3. The recombinant baculovirus of claim 1, wherein the promoter comprises an HzNV-1(Heliothis zea Nudigirus 1) virus early expression gene pag1, an antimicrobial peptide gene b1(cecropin b1), a homeodomain gene 1 (homologus region 1, hr1), or a combination thereof.
4. The recombinant baculovirus of claim 1, wherein the baculovirus is Autographa californica polynuclear polyhedrosis virus.
5. The recombinant baculovirus of claim 1, wherein the VLP is selected from the group consisting of Herpesviridae (Herpesviridae), Adenoviridae (Adenoviridae), african swine fever viridae (Asfarviridae), papilloma viridae (Papillomaviridae), polyoviridae (polyoviridae), Poxviridae (Poxviridae), Circoviridae (Circoviridae), small DNA viridae (partoviridae), Reoviridae (Reoviridae), Arteriviridae (arterividae), Coronaviridae (Coronaviridae), Picornaviridae (Picornaviridae), bulviviridae (Filoviridae), Paramyxoviridae (Paramyxoviridae), Rhabdoviridae (rhaviridae), Astroviridae (Astroviridae), Caliciviridae (Caliciviridae), and Caliciviridae (Flaviviridae), and capsiviridae (Flaviviridae).
6. The recombinant baculovirus of claim 5, wherein said VLP is a Cherovirus (CHIKUngunya virus, CHIKV) from Togaviridae.
7. The recombinant baculovirus of claim 6, wherein said exogenous genes encode structural proteins C, E1, E2, E3 and 6K of said VLP.
8. The recombinant baculovirus of claim 5, wherein said VLP is Japanese Encephalitis Virus (JEV), dengue virus (DENV), or Zika virus (ZIKV) from the Flaviviridae family.
9. The recombinant baculovirus as set forth in claim 8, wherein said exogenous gene encodes structural proteins prM and E of said VLP.
10. A method for making a VLP, comprising:
(1) transducing (transducing) the recombinant baculovirus of any one of claims 1-9 into a mosquito cell; and
(2) purifying the mosquito cells or supernatant of step (1) to produce said VLPs.
11. Use of a VLP for the preparation of a vaccine, wherein said VLP is prepared by the method of claim 10.
12. A VLP produced by the method of claim 10.
CN201910478505.1A 2019-06-03 2019-06-03 Recombinant baculovirus and uses thereof Pending CN112029734A (en)

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Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HIDEKI YAMAJI ET AL.: "Efficient production of Japanese encephalitis virus-like particles by recombinant lepidopteran insect cells", 《AMB》 *
NENAVATH GOPAL NAIK ET AL.: "Baculovirus as an efcient vector for gene delivery into mosquitoes", 《SCIENTIFIC REPORTS》 *
SHIYU DA: "Zika Virus Baculovirus-Expressed Virus-Like Particles Induce Neutralizing Antibodies in Mice", 《VIROLOGICA SINICA》 *

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Application publication date: 20201204