TW201610160A - Novel recombinant baculovirus vector and uses thereof - Google Patents

Abstract

Disclosed herein is a recombinant viral construct and its uses thereof. The recombinant viral construct is capable of simultaneously expressing three exogenous proteins, including a classical swine fever virus (CSFV) antigen, a porcine circovirus type 2 (PCV2) antigen, and an immunomodulatory protein. The recombinant viral construct is hence useful as a bio-tool for simultaneously producing multiple antigens of a bi-subunit vaccine.

Description

Novel recombinant baculovirus vector and use thereof

The present disclosure relates to a novel recombinant viral vector. More specifically, the present disclosure relates to a recombinant viral vector and the use of such a recombinant viral vector to prepare a plurality of antigenic proteins of a multi-effect (eg, double-effect) vaccine.

Classical swine fever (CSF) is a common lethal infectious disease in pigs. Its symptoms include fever, bleeding, movement disorders and immunosuppression. The Flaviviridae genus Pestivirus 's classical swine fever virus (CSFV) is the main pathogen causing swine fever. In many countries, cyclical CSFV pandemics often result in huge economic losses. Therefore, it is important to develop a vaccine that is effective against CSFV to reduce the clinical symptoms of CSF and the spread of CSFV in related fields. The vaccine prepared for the structural glycoprotein E2 of CSFV has a protective effect because it can induce a neutralizing antibody reaction in pigs.

Post-weaning multi-systemic wasting syndrome (PMWS) is another global infectious disease of pigs. The disease is often caused by a high mortality rate and a decrease in the efficiency of feed conversion between pigs in the weaned pigs and in the fattening, resulting in considerable economic losses. Circovirus Branch (Circoviridae) ring-like viruses (Circovirus) of porcine circovirus type 2 (porcine circovirus type 2, PCV2) is the major causative agent of PMWS caused. A variety of PCV2 vaccines have been shown to be effective against PMWS and significantly improve aquaculture and mortality rates on inoculated farms. In the known PCV2 vaccine, a vaccine produced against the PCV2 major immunological protein (ie, open reading frame-2, ORF-2) is capable of producing a neutralizing antibody specific for PCV2, And has a good protective effect.

As mentioned above, although there are a variety of vaccines against CSFV or PCV2, most of them are single-acting vaccines, that is, the vaccines contain only a single antigenic protein of CSFV or PCV2. Only against a single virus often limits the scope and efficacy of single-acting vaccines, and must be separately produced and pigs.

Therefore, there is a need in the related art for a multi-effect vaccine containing multiple antigens to protect pigs from infection by different viruses.

SUMMARY OF THE INVENTION The Summary of the Disclosure is intended to provide a basic understanding of the present disclosure. This Summary is not an extensive overview of the disclosure, and is not intended to be an The concept of the present disclosure begins with a simplified form and is described in greater detail below.

The present disclosure relates to a recombinant viral vector capable of simultaneously expressing three exogenous proteins comprising a CSFV antigen or a fragment thereof, a PCV2 antigen or a fragment thereof, and an immunomodulatory protein or a fragment thereof; The recombinant viral vector is used to prepare a double-acting vaccine comprising at least two antigens and a regulatory protein.

One aspect of the present disclosure relates to a recombinant viral vector for use in the preparation of a double-effect vaccine. The recombinant viral vector comprises: (1) a promoter; (2) a first gene expression cassette operably linked to the promoter, wherein the first gene expression group comprises a sequence a first nucleic acid encoding a classical swine fever virus (CSFV) antigen or a fragment thereof, a self-cleaving sequence encoding a self-cleaving peptide, and a code for encoding a type 2 pig ring a second nucleic acid of a virus (porcine circovirus type 2, PCV2) antigen or a fragment thereof; (3) a second gene expression group operably linked to the promoter, wherein the second gene expression group is sequentially included a first ribosomal entry site (IRES) sequence or fragment thereof and a third nucleic acid encoding a reporter polypeptide; and (4) a third gene expression set operably Linked to the promoter, wherein the third gene expression set comprises, in sequence, a second IRES sequence or a fragment thereof and a fourth nucleic acid encoding an immunomodulatory polypeptide or a fragment thereof.

According to some embodiments of the present disclosure, the promoter may be a polyhedrin (polh) promoter, a baculovirus-derived promoter, an actin promoter derived from Bombyx mori, a giant cell. A cytomegalovirus (CMV) promoter or a chicken β-actin promoter (CAG promoter) that binds to a CMV enhancer. In certain embodiments of the present disclosure, the baculovirus-derived promoter may be an immediate-early (IE)1 promoter, an IE2 promoter, a p6.9 promoter, a VP39 promoter, or a p10 promoter. A group of children.

In certain embodiments of the present disclosure, the CSFV antigen is a CSFV-E2 antigen and the amino acid sequence thereof is at least 90% identical to SEQ ID NO: 1.

In certain embodiments of the present disclosure, the PCV2 antigen is the PCV2 open reading frame 2 (PCV2-ORF2) antigen, and the amino acid sequence thereof is at least 90% identical to the sequence number: 2.

According to some embodiments of the present disclosure, the self-cleaving peptide is selected from the group consisting of nuclear inclusion protein a (Nia) protease, P1 protease, 3C protease, L protease, 2A protease, class 3C protease, and class 2A. a group of proteases. In certain embodiments of the present disclosure, the self-cleaving peptide is a class 2A protease comprising a typical gene sequence of Asp-X-Glu-X-Asn-Pro-Gly-Pro, and the self-cleaving point is located in Gan Between the amine acid and the valine acid. In a preferred embodiment of the present disclosure, the self-cleaving peptide is a class 2A protease isolated from Perina nuda virus (PnV) and has an amino acid sequence of SEQ ID NO: 3. In another preferred embodiment of the present disclosure, the self-cleaving peptide is a class 2A protease isolated from the genus Eucalyptus urophylla, and has the amino acid sequence of SEQ ID NO: 4.

According to some embodiments of the present disclosure, the first and second IRES sequences are respectively derived from a 5'-untranslated region (5'-UTR) of a virus, and the virus may be a scorpion venomous moth virus , Rhopalosiphum padi virus (RhPV), Aphid lethal paralysis virus, Black queen cell virus, Cricket paralysis virus, Drosophila C virus Drosophila C virus), Himetobi P virus, Homalodisca coagulata virus-1, Kashmir bee virus, Plautia stali intestine virus, Red fire ant virus A virus having an IRES sequence of 1 ( Solenopsis invicta virus-1), Taura syndrome virus, Encephalomyocarditis virus, or Hepatitis C virus. In certain preferred embodiments of the present disclosure, the first and second IRES sequences are Eucalyptus urophyllin virus IRES having a nucleotide sequence of SEQ ID NO: 5. In other preferred embodiments of the present disclosure, the first and second IRES sequences are rice blast virus IRES having a nucleotide sequence of SEQ ID NO: 6. The first and second IRES sequences need not be the same sequence.

According to some embodiments of the present disclosure, the reporter polypeptide is selected from the group consisting of green fluorescent protein (GFPs), enhanced green fluorescent protein (EGFP), and disc anemone red fluorescent protein ( Discosoma sp. red fluorescent protein (DsRed), blue fluorescence protein (BFP), enhanced yellow fluorescent proteins (EYFP), anemonia majano fluorescent protein (amFP) ), a group of Zoanthus fluorescent protein (zFP), Discosoma fluorescent protein (dsFP), and Clavularia fluorescent protein (cFP).

According to some embodiments of the present disclosure, the immunomodulatory polypeptide may be a fungal immunomodulatory protein Flammunlina velutipes (FIP-FVE), a fungal immunomodulatory protein Ganodera tsugae (FIP-GTS), Fungal immunomodulatory protein ( Volvariella volvacea , FIP-VVO), Ganoderma lucidum Ling-Zhi-8 (LZ-8), fungus immunoglobulin (fungal immunimodulatory protein) Ganoderma microsporum , GMI) or immunomodulatory protein Tremella FuciFormis (TFP). In certain embodiments of the present disclosure, the immunomodulatory polypeptide is an FIP-amino acid sequence at least 90% identical to SEQ ID NO: 7. FVE.

According to some embodiments of the present disclosure, the recombinant virus construct is derived from a virus, wherein the virus is selected from the group consisting of a baculovirus, a gland A group of viruses, adeno-associated viruses, retroviruses, lentiviruses, Sindbis virus, herpes simplex virus, forest virus, and vaccinia virus.

In another aspect, the disclosure provides a double-effect vaccine comprising: a first effective amount of one of a CSFV antigen or a fragment thereof, a second effective amount of one of the PCV2 antigens or a fragment thereof, and a third An effective amount of an immunomodulatory polypeptide or fragment thereof, and a pharmaceutically acceptable adjuvant.

In certain preferred embodiments of the present disclosure, the CSFV antigen is a CSFV-E2 antigen, the PCV2 antigen is a PCV2-ORF2 antigen, and the immunomodulatory polypeptide is a FIP-FVE protein.

The basic spirit and other objects of the present invention, as well as the technical means and implementations of the present invention, will be readily apparent to those skilled in the art of the invention.

The accompanying drawings are a part of this specification for describing various embodiments of the invention, methods, and embodiments. The above and other objects, features, advantages and embodiments of the present invention will become more apparent and understood. The description of the accompanying drawings is as follows: FIG. 1 is a schematic diagram of a recombinant viral vector according to the embodiment 1.1 of the present disclosure; 2 is a specific weight according to embodiment 2.1 of the present disclosure Photograph of group baculovirus infected with Sf21 cells; scale bar represents 50 μm; Figure 3 is a photograph of Western blot analysis of cell proteins after infection of Sf21 cells with specific recombinant baculovirus according to Example 2.2 of the present disclosure; Detection was performed by the following antibodies: (a) an anti-E2 antibody for detecting about 43 kDa (non-glycosylated form) and 55 kDa (glycosylated form) as indicated by the arrows. a CSFV-E2 protein; (b) an anti-PCV2 antibody for detecting a PCV2-ORF2 protein of about 26 kDa as indicated by the arrow; and (c) an anti-FVE antibody for detecting about 12.7 as indicated by the arrow kDa FIP-FVE protein; Figure 4 is a photograph of Sf21 cells infected with the recombinant baculovirus of Example 1.2 according to Example 2.3 of the present disclosure, which was observed by FITC, rhodamine or DAPI filters; Figure 5 is a photograph of the results of an enzyme-linked immunosorbent assay (ELISA) to illustrate (a) anti-CSFV-E2 antibody or (b) anti-PCV2- in mouse serum after injection of a specific immunogen. The content of the ORF2 antibody.

The description of the embodiments of the present invention is intended to be illustrative and not restrictive. The features of various specific embodiments, as well as the method steps and sequences thereof, are constructed and manipulated in the embodiments. However, other specific embodiments may be utilized to achieve the same or equal functionality. With the order of steps.

Unless otherwise stated, the singular "a" or "the" and "the" are used in the plural.

According to the disclosure, terms such as "nucleotide sequence", "polynucleotide" or "nucleic acid" are used interchangeably and mean a double strand of DNA, a single strand. DNA or a transcription product of the DNA (eg, an RNA molecule). It is to be understood that the present disclosure is not related to genetic polynucleotide sequences in nature or in nature. Methods that can be used to isolate or purify (or partially purify) a nucleic acid, polynucleotide or nucleotide sequence of the invention include, but are not limited to, ion-exchange chromatography, molecular screening chromatography (molecular size exclusion chromatography) or genetic engineering techniques such as amplification, subtractive hybridization, cloning, sub-cloning, chemical synthesis, or a combination thereof.

The term "antigen" as used in the specification is understood by the relevant art and includes immunogenic substances (i.e., immunogens), and any immunological unresponsiveness or anergy (ie, the individual defense mechanism is insufficient for external use). Substance of matter). The term "antigen" as used herein refers to a full-length protein and a peptide fragment comprising an epitope.

The term "immunomodulatory" as used herein refers to a humoral or cell that enhances the body, such as a toxic T cell response or a helper T response, when administered to an animal with an immune system. Factors of cellular immune response (for example, Protein or peptide). An immunomodulatory protein can be any protein or active fragment that elicits, enhances or prolongs an immune response in a body.

The term "an effective amount" as used herein refers to a dose sufficient to cause an immune response in a subject. An effective amount can be administered in one or more dosage forms, applications, or dosages.

The term "adjuvant" as used herein refers to a compound or molecule that increases the immune response of an individual when administered together with an antigen to a subject. The use of an adjuvant to enhance and/or prolong the duration of an antigen-priming immune response can be determined in a variety of ways, including, but not limited to, a serum or cellular immune response (eg, a toxic T cell response or a helper T response). ) increase.

The present invention relates to a novel recombinant viral vector capable of simultaneously expressing three exogenous proteins comprising a CSFV antigen or a fragment thereof, a PCV2 antigen or a fragment thereof, and an immunomodulatory protein or a fragment thereof. Therefore, the recombinant viral vector can be used to prepare a double-effect vaccine.

A first aspect of the present disclosure is therefore directed to a recombinant viral vector for use in preparing a double-effect vaccine. The recombinant viral vector comprises: (1) a promoter; (2) a first gene expression group operably linked to the promoter, wherein the first gene expression group comprises a coding sequence The first nucleic acid of the CSFV antigen or a fragment thereof, one for encoding a self-cut a self-cleaving sequence of a peptide, and a second nucleic acid encoding a PCV2 antigen or a fragment thereof; (3) a second gene expression group operably linked to the promoter, wherein the second The gene expression set comprises, in sequence, a first IRES sequence or a fragment thereof and a third nucleic acid encoding a reporter polypeptide; and (4) a third gene expression set operably linked to the promoter, Wherein the third gene expression group comprises a second IRES sequence or a fragment thereof and a fourth nucleic acid encoding an immunomodulatory polypeptide or a fragment thereof.

To prepare a double-effect vaccine comprising multiple antigens of the present disclosure, three gene expression groups are operably linked downstream of the viral vector promoter, thereby preparing a host to simultaneously express at least three exogenous proteins. Viral vectors (eg, two antigens and one immunomodulatory protein).

The first gene expression set is constructed to sequentially comprise a first nucleic acid encoding a CSFV antigen or a fragment thereof, a self-cleaving sequence for encoding a self-cleaving peptide, and a code for encoding a PCV2 antigen or The second nucleic acid of the fragment. In an embodiment of the present disclosure, the CSFV antigen is a CSFV-E2 antigen derived from the structural glycoprotein E2 in the CSFV genome, and the amino acid sequence is at least 90% identical to the sequence number: 1. In an embodiment of the present disclosure, the PCV2 antigen is a PCV2-ORF2 antigen derived from ORF2 in the PCV2 genome, and the amino acid sequence is at least 90% identical to SEQ ID NO: 2. Self-cleaving peptide linked The CSFV-E2 and PCV-ORF-2 antigens were initially expressed as a single protein. Upon self-cleavage by a self-cleaving peptide located between the two antigens and having self-cleaving protease activity, the single protein releases two antigens (ie, CSFV-E2 and PCV2-ORF2).

According to some embodiments of the present disclosure, the self-cleaving peptide may be selected from the group consisting of the Nia protease and the P1 protease of the potato Y virus, the 3C protease of the aphthovirus, the L protease, and the 2A of the enterovirus. Protease and 3C protease, 2A protease and 3C protease of rhinovirus, 2A protease and 3C protease of picornavirus, and 3C of rice tungro spherical virus (RTSV) A group consisting of a protease and a class 2A protease. In certain embodiments of the present disclosure, the self-cleaving peptide is a class 2A protease comprising a typical gene sequence of Asp-X-Glu-X-Asn-Pro-Gly-Pro, and the self-cleaving point is located in glycine Between acid and proline. In a preferred embodiment of the present disclosure, the self-cleaving peptide is a class 2A protease isolated from the genus Eucalyptus urophylla, and has the amino acid sequence of SEQ ID NO: 3. In another preferred embodiment of the present disclosure, the self-cleaving peptide is a class 2A protease isolated from the genus Eucalyptus urophylla, and has the amino acid sequence of SEQ ID NO: 4.

The second gene expression set is constructed to sequentially comprise a first IRES sequence or a fragment thereof and a third nucleic acid encoding a reporter polypeptide. An IRES sequence is a nucleic acid sequence that allows translation to begin in the middle of a message ribonucleic acid sequence and is therefore commonly used for bi-cistronic or multi-effect (multi-cistronic) in the expression system whereby an RNA transcript can simultaneously and independently represent two or more proteins. A variety of IRES sequences found in viruses are used to ensure that viral translation can be continuously activated when host translation is inhibited. The first IRES sequence used to express the reported polypeptide may be derived or engineered from the 5'-UTR region of the virus, wherein the virus may be a scorpion venomous moth virus, a rice blast virus, a locust-killing prion virus, a bee black queen cell virus, IRES sequence of ricin virus, Drosophila C virus, 虱P virus, spider frog virus-1, Kashmir bee virus, soybean locust virus, red fire ant virus-1, peach disease virus, encephalomyocarditis virus or hepatitis C virus Virus. According to some embodiments of the present disclosure, the first IRES sequence is an IRES of the genus Eucalyptus urophylla virus and has a nucleotide sequence of SEQ ID NO: 5. According to other embodiments of the present disclosure, the first IRES sequence is an IRES of rice blast virus and has a nucleotide sequence of SEQ ID NO: 6.

Reported polypeptides are used in the present invention to detect the performance of exogenous proteins and to facilitate subsequent purification steps of the virus. Reportable polypeptides suitable for use in the present disclosure include, but are not limited to, GFP, EGFP, DsRed, BFP, EYFP, amFP, zFP, dsFP, and cFP. In a preferred embodiment of the present disclosure, the reporter polypeptide is EGFP.

The third gene expression set is constructed to sequentially comprise a second IRES sequence or a fragment thereof and a fourth nucleic acid encoding an immunomodulatory polypeptide or a fragment thereof. Similar to the above, the second IRES sequence used to express the immunomodulatory polypeptide may be derived or engineered from the 5'-UTR region of the virus, wherein the virus may be a scorpion venomous moth virus, rice blast virus, aphid lethal poliovirus Bee queen black cell virus, castor disease Virus, Drosophila C virus, 虱P virus, spider frog virus-1, Kashmir bee virus, soybean prion virus, red fire ant virus-1, peach disease virus, encephalomyocarditis virus or hepatitis C virus with IRES sequence . According to some embodiments of the present disclosure, the second IRES sequence is an IRES of the Phyllostachys pubescens virus and has a nucleotide sequence of SEQ ID NO: 5. According to a further embodiment of the present disclosure, the second IRES sequence is an IRES of rice blast virus and has a nucleotide sequence of SEQ ID NO: 6.

It is to be noted that the first IRES sequence in the second gene expression group and the second IRES sequence in the third gene expression group need not be the same sequence. In certain embodiments of the present disclosure, the first IRES sequence is the IRES of the Phyllostachys pubescens virus and the second IRES sequence is the IRES of the rice blast virus.

In view of the fact that immunomodulatory proteins can be used as an adjuvant to enhance the immune response elicited by antigens, this study constructed immunomodulatory proteins in the third gene expression group to enhance the efficacy of vaccines against CSFV and PCV2 by the expression of immunomodulatory proteins. . Immunomodulatory polypeptides suitable for use in the present disclosure include, but are not limited to, FIP-FVE, FIP-GTS, FIP-VVO, LZ-8, GMI, and TFP. In certain embodiments of the present disclosure, the immunomodulatory polypeptide is FIP-FVE having an amino acid sequence at least 90% identical to SEQ ID NO: 7.

The above three sets of gene expression groups are individually ligated downstream of the promoter of a viral vector to prepare a recombinant viral vector of the present disclosure. Viral vectors suitable for use in the present disclosure include, but are not limited to, baculovirus, adenovirus, adeno-associated virus, retrovirus, lentivirus, Sindbis virus, herpes simplex virus, forest virus, and vaccinia virus. In a preferred embodiment of the present disclosure, the recombinant virus construct is derived from a baculovirus. Promoters suitable for use in the present disclosure include, but are not limited to, a polyhedrin promoter, a baculovirus-derived promoter, a silkworm actin-derived promoter, a cytomegalovirus promoter, or a CMV enhancer. Chicken beta -actin promoter (CAG promoter). The baculovirus-derived promoter may be an IE1 promoter, an IE2 promoter, a p6.9 promoter, a VP39 promoter or a p10 promoter, and the like. In a preferred embodiment of the present disclosure, the recombinant virus construction of the present disclosure uses a polyhedrin promoter.

The present disclosure can be co-transfected into an insect host cell as a recombinant baculovirus transfer vector plastid DNA and a Bac-N-Blue viral DNA. The Bac-N-Blue viral DNA provides the necessary viral backbone, which contains the genes required for the propagation of Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The recombinant baculovirus transfer vector and Bac-N-Blue virus DNA of the present disclosure can generate a recombinant virus by homologous recombination in an insect host, and propagate in the insect host, and simultaneously produce three groups of genes. Groups of exogenous proteins encoded separately. The performance of the reported polypeptide is further observed to screen and purify the recombinant virus. Host cells suitable for use in the present disclosure include, but are not limited to, S. furgiperda IPBL-9 (Sf9) cells, Sf21 cells, High Five cells, and Minic Sf9 cells. According to a preferred embodiment of the present disclosure, the insect host cell is a Sf21 cell.

The invention further comprises a double-effect vaccine which is an antigen produced by the recombinant viral vector of the present disclosure, wherein the viral vector Two recombinant antigenic proteins (ie, CSFV-E2 and PCV2-ORF2) and one immunomodulatory polypeptide (ie, FIP-FVE) can be simultaneously and efficiently expressed. The double-acting vaccine comprises: a first effective amount of one CSFV antigen or a fragment thereof, a second effective amount of one of the PCV2 antigens or a fragment thereof, a third effective amount of one of the immunomodulatory polypeptides or fragments thereof, and a pharmaceutically acceptable An acceptable adjuvant.

In certain preferred embodiments of the present disclosure, the CSFV antigen is CSFV-E2, the PCV2 antigen is PCV2-ORF2, and the immunomodulatory polypeptide is FIP-FVE.

The verification of recombinant virus construction of the present disclosure will be described below with reference to a number of examples, as well as the various antigens required for the preparation of a double-effect vaccine using the recombinant virus construction. The disclosed embodiments are only intended to illustrate the disclosure, and the scope of the claims is not limited thereto.

Example

Materials and methods

Cell culture

Sf21 cells were cultured in TNM-FH medium containing 8-10% heat-removed fetal bovine serum (FBS) until the monolayer cells were grown to 80% full.

animal

The animal experiment process was approved by the Central Plains University Animal Center and the Use Committee and complied with the relevant national animal protection regulations.

Male BALB/c mice were purchased from antibody AbKing Biotechnology and housed in a cage with temperature and humidity control at a temperature of about 22 ° C to 24 ° C and a humidity of about 40% to 50%. The cycle is 12 hours. At the beginning of the experiment, each mouse weighed approximately 20 to 25 grams. Drinking water and standard rodent feed are provided for the meal during the test.

Western blot analysis

After infecting the cells with the recombinant virus for 4 days, the proteins in the cell extract were separated by SDS-PAGE and according to the method of Laemmli in the mini Protein III system (Bio-Rad). The protein separated by SDS-PAGE was electrotransferred onto a PVDF (polyvinyldiene difluoride) membrane (Millipore), and Tris-buffered saline (TBS; 100 mM Tris) containing 5% BSA (Sigma). The membrane was soaked (pH 7.4), 100 mM NaCl and 0.1% Tween 20) and gently shaken at room temperature for 1 hour. Next, the membrane was placed in anti-E2 antibody (1:2000) diluted in PBS, anti-PCV2 antibody (1:250) or anti-FVE antibody (1:250), and placed at 4 °C until the next day. . The membrane was washed three times for 5 minutes with shaking in TBS buffer at room temperature to remove unreacted antibodies. The membrane was then placed in horseradish peroxidase (HRP) diluted 1:2000 for 1 hour at room temperature. Finally, the chemluminescence set (Pierce) was used and the HRP on the membrane was detected according to its instructions.

Enzyme immunoassay

The extract isolated from the recombinant baculovirus-infected cell carrying the CSFV-E2 gene or the purified recombinant ubiquitin-PCV2 recombinant protein is phosphate-buffered saline (phosphate-buffered saline, After dilution with PBS), it was added to a 96-well plate and placed at 4 ° C until the next day. The 96-well plate was further immersed in PBS containing 5% by volume of skim milk at room temperature for 1-2 hours. Next, mouse serum was added and allowed to stand at room temperature for 1 hour to detect whether the mouse serum contained an antibody against anti-PCV2-ORF2 antibody or anti-CSFV-E2. The membrane was washed three times for 5 minutes with shaking in a PBS containing 0.1% by volume of Toco 20 to remove unreacted antibodies. HRP-conjugated goat anti-mouse antibody was added to a 96-well plate and allowed to stand at room temperature for 30 minutes. Further, tetramethylbenzidine (TMB) was used as a substrate, and the amount of HRP on the 96-well plate was measured at a wavelength having an optical density (OD) of 490 nm.

Example 1 Construction of recombinant baculovirus

1.1 Construction of pBac-E2-2A-PCV2-PnV339-E-Rhir-FVE

The exogenous CSFV-E2 gene (BamH I and Pst I restriction enzymes), PCV2-cap gene (with Pst I restriction enzyme) and FIP-FVE gene (with Spe I and Not I restriction enzyme) were separately transferred to pBac-mcsI-2A-mcsII-PnV339-EGFP-Rhir-mcsIII multiple cloning site (mcs) I, mcsII and mcsIII. The resulting recombinant plasmid was pBac-E2-2A-PCV2-PnV339-E-Rhir-FVE (Fig. 1).

1.2 Preparation of vAc-E2-2A-PCV2-PnV 339-E-Rhir-FVE

The recombinant plasmid pBac-E2-2A-PCV2-PnV339-E-Rhir-FVE of Example 1.1 was co-transfected into Sf21 cells with Bac-N-Blue virus DNA (purchased from Invitrogen, USA), and then incubated at 27 ° C. After 5 days, homologous recombination between plastid and viral DNA was performed.

The culture medium of the co-transfected cells in which green fluorescence was emitted due to the expression of EGFP was collected, and another batch of Sf21 cells was infected by the terminal dilution method (serious dilution at a ratio of 1:10). The culture broth was collected repeatedly, and the dilution step was performed 3-5 times on another batch of Sf21 cells until a single virus strain was obtained. The resulting recombinant virus is screened from a single plaque.

Example 2 Confirmation of the recombinant baculovirus of Example 1

The infection and performance of the recombinant virus screened in Example 1.2 were further analyzed.

2.1 Use of EGFP expression to detect recombinant baculoviruses that express CSFV-E2, PCV2-ORF2 and/or FIP-FVE proteins

Recombinant baculovirus vAc-E2-PnV339-E-Rhir-mcsII showing only CSFV-E2 protein, (2) only PCV2-ORF2 protein with multiplicity of infection (moi) Recombinant baculovirus vAc-PCV2-PnV339-E-Rhir-mcsII, (3) recombinant baculovirus vAc-E2-PnV339-E-Rhir-FVE showing CSFV-E2 protein and FIP-FVE protein and (4) The same as described in embodiment 1.2 Recombinant baculovirus vAc-E2-2A-PCV2-PnV339-E-Rhir-FVE expressing three antigenic proteins (ie, CSFV-E2, PCV2-ORF2, and FIP-FVE) infected Sf21 cells. A recombinant virus expressing CSFV-E2, PCV2-ORF2, and FIP-FVE was confirmed using a fluorescence microscope (observed under a FITC filter).

The photograph in Figure 2 indicates that a recombinant virus carrying an exogenous gene (ie, vAc-E2-PnV339-E-Rhir-mcsII or vAc-PCV2-PnV339-E-Rhir-mcsII) carries two exogenous properties. a recombinant virus of the gene (ie, vAc-E2-PnV339-E-Rhir-FVE) or a recombinant virus carrying three exogenous genes (ie, vAc-E2-2A-PCV2-PnV339-E-Rhir-FVE), Cells will shed green fluorescence due to the expression of EGFP reporter proteins.

2.2 Western blot analysis to detect CSFV-E2, PCV2-ORF2 and FIP-FVE antigens

To further examine the three exogenous genes carried by the recombinant virus, anti-E2, anti-PCV2 or anti-FVE antibodies were then used in Western blot experiments to confirm the performance of each protein. Briefly, the moi is 1 (1) control group, (2) vAc-E2-PnV339-E-Rhir-mcsII, (3) vAc-PCV2-PnV339-E-Rhir-mcsII, (4) vAc -E2-PnV339-E-Rhir-FVE and (5) vAc-E2-2A-PCV2-PnV339-E-Rhir-FVE infected Sf21 cells. Four days later, proteins from infected cells were collected and analyzed by Western blotting.

The results in Fig. 3 indicate that the Sf21 cells infected with the recombinant virus of Example 1.2 (i.e., vAc-E2-2A-PCV2-PnV339-E-Rhir-FVE) can successfully express CSFV-E2 (Fig. 3a), PCV2-ORF2 (3b) and FIP-FVE protein (Fig. 3c). In addition, different tested recombinant viruses have similar protein expression levels. This result shows that the viral vector constructed by the present invention can simultaneously and efficiently express the characteristics of two antigenic proteins and an immunomodulating protein, making the recombinant virus of Example 1.2 a biological tool which can be used to prepare a multi-effect vaccine.

2.3 Detection of PCV2-ORF2 protein expression by immunofluorescence

After Sf21 cells were seeded in 24-well plates, infection was carried out with a recombinant virus with a moi of 1. Four days after infection, the performance of EGFP was observed by a fluorescence microscope with a FITC filter. The culture solution was discarded, and the 24-well plate was placed at 27 ° C for 1 hour. The cells were fixed with 100 μl of 4% paraformaldehyde and washed 4 times with PBS. After adding 50 μl of methanol, it was washed 4 times with PBS. Then, after shaking for 1 hour with 100 μl of 3% bovine serum albumin (BSA), anti-PCV2 antibody (1:800) was added and allowed to stand for 2 hours. The cells were washed 5 times with PBS, and the cells were labeled with rhodamine-conjugated anti-mouse secondary antibody and allowed to stand for 2 hours. Finally, cells expressing PCV-2 were observed with a conjugated focal microscope with a rhodamine filter.

The photograph of Sf21 cells in Fig. 4 shows that cells infected with the recombinant virus will simultaneously scatter green and red fluorescence due to the expression of EGFP and PCV2. Combined with the results of Western blot analysis, recombinant viruses that simultaneously and efficiently express CSFV-E2, PCV2 and FIP-FVE proteins can be used as a biological carrier for the preparation of multiple antigens in double-effect vaccines.

Example 3 Evaluation of the immunity of a double-acting vaccine of the present disclosure to a living body

In this embodiment, the double-acting vaccine formulation comprises three antigens (ie, CSFV-E2, PCV2-ORF2, and FIP-FVE produced by the recombinant virus of Example 1.2) with or without adjuvant (Buddha) Freund's incomplete adjuvant, FIA); the immunity of this vaccine was evaluated.

Male BALB/c mice PBS (Group A), adjuvant-free 8 μg vaccine composition (Group B), 8 μg of vaccine composition (Group C), 16 μg of vaccine composition (Group D) or 8 μg were administered separately Recombinant sumo-PCV2 protein (group E). Each group contained 4 mice, and each mouse was administered twice. The first and second administrations were performed by intraperitoneal injection at weeks 0 and 2, respectively. To assess the immunogenicity of the treatment, serum samples were collected at weeks 0, 2, 4 and 6 respectively. Figure 5 summarizes the results.

The ELISA results in Figure 5 indicate that anti-CSFV-E2 antibody (Fig. 5a) and anti-- in mouse serum compared to the PBS control group (or group A) after 2 weeks of the first administration. The amount of PCV2-ORF2 antibody (Fig. 5b) will increase. The amount of anti-PCV2-ORF2 antibody and anti-CSFV-E2 antibody eventually reached the maximum amount at week 4 (groups B, C and D). As a control group administered with the vaccine, only anti-PCV2 antibodies were detected in the serum of mice administered with sumo-PCV2 protein (group E), and anti-CSFV-E2 antibodies could not be detected.

Summarizing the above, the results of the various working examples indicate that the novel recombinant virus constructs of the present disclosure (ie, vAc-E2-2A-PCV2-PnV339-E-Rhir-FVE) can simultaneously and efficiently express three exogenous proteins (ie, CSFV-E2, PCV2-ORF2 and FIP-FVE), This property will make this recombinant virus suitable for use as a biological tool for the production of multiple antigens and for the preparation of a double-effect vaccine.

It will be understood that the above-described embodiments and examples are merely illustrative, and that those skilled in the art can align various modifications. The description, examples, and materials set forth above are intended to be illustrative of the present invention and are illustrative of the invention. The present disclosure has been disclosed in the above embodiments, but it is not intended to limit the disclosure, and any person skilled in the art can make various changes and refinements without departing from the spirit and scope of the disclosure. The scope of protection of the disclosure is subject to the definition of the scope of the patent application.

<110> Wu Zongyuan Chen Mingxiang Teng Zhaoyi Chen Yuru

<120> Novel recombinant baculovirus vector and use thereof

<130> P2796-TW

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<170> BiSSAP 1.3

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<211> 363

<212> PRT

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<223> CSFV-E2

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<213> Artificial Sequence

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<223> PCV2-ORF2

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<213> Artificial Sequence

<220>

<223> 2A-like protease-1

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<210> 4

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<212> PRT

<213> Artificial Sequence

<220>

<223> 2A-like sequence-2

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<213> Artificial Sequence

<220>

<223> PnV IRES

<400> 5

<210> 6

<211> 579

<212> DNA

<213> Artificial Sequence

<220>

<223> RhPV IRES

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<223> FIP-FVE

<400> 7

Claims (15)

A recombinant viral vector for preparing a double-effect vaccine comprising: a promoter; a first expression cassette operably linked to the promoter, wherein the first gene expression group is The sequence comprises a first nucleic acid encoding a classical swine fever virus (CSFV) antigen, a self-cleaving sequence encoding a self-cleaving peptide, and a coding for a type 2 porcine circovirus a second nucleic acid of a (porcine circovirus type 2, PCV2) antigen; a second gene expression group operably linked to the promoter, wherein the second gene expression group sequentially comprises a first ribosome in vivo initiation An internal ribosomal entry site (IRES) sequence and a third nucleic acid encoding a reporter polypeptide; and a third gene expression set operably linked to the promoter, wherein the third gene expression group A second IRES sequence and a fourth nucleic acid encoding an immunomodulatory polypeptide are sequentially included. The recombinant viral vector according to claim 1, wherein the promoter is a polyhedrin (polh) promoter, a baculovirus-derived promoter, and a muscle derived from Bombyx mori. A protein promoter, a cytomegalovirus (CMV) promoter or a chicken β -actin promoter (CAG promoter) that binds to a CMV enhancer. The recombinant viral vector of claim 2, wherein the baculovirus-derived promoter is an immediate-early (IE)1 promoter, an IE2 promoter, a p6.9 promoter, and a VP39 Promoter or a p10 promoter. The recombinant viral vector of claim 1, wherein the CSFV antigen is CSFV-E2 and has the amino acid sequence of SEQ ID NO: 1. The recombinant viral vector of claim 1, wherein the PCV2 antigen is PCV2 open reading frame 2 (PCV2-ORF2) and has the amino acid sequence of SEQ ID NO: 2. The recombinant viral vector of claim 1, wherein the self-cleaving peptide is a nuclear inclusion protein a (Nia) protease, a P1 protease, a 3C protease, a L protease, a 2A protease, A class of 3C protease or a class of 2A protease. The recombinant viral vector of claim 6, wherein the self-cleaving peptide is a 2A protease. The recombinant viral vector of claim 1, wherein the first and second IRES sequences are respectively derived from a 5'-untranslated region (5'-UTR) of a virus, which is a eucalyptus Perina nuda virus (PnV), Rhopalosiphum padi virus (RhPV), Aphid lethal paralysis virus, Black queen cell virus, rickett virus (Cricket) Paralysis virus), drosophila C virus, Himetobi P virus, Homalodisca coagulata virus-1, Kashmir bee virus, Soybean toadstool ( Pluutia) Stali intestine virus), Solenopsis invicta virus-1, Taura syndrome virus, Encephalomyocarditis virus or Hepatitis C virus. The recombinant viral vector of claim 8, wherein the first and second IRES sequences are respectively the Eucalyptus urophyllin virus IRES having the nucleotide sequence of SEQ ID NO: 5; and the rice blast virus IRES, A nucleotide sequence having the sequence number: 6. The recombinant viral vector according to claim 1, wherein the reporter polypeptide is green fluorescent protein (GFPs), enhanced green fluorescent protein (EGFP), and disc anemone red fluorescent light. Discosoma sp. red fluorescent protein (DsRed), blue fluorescent protein (BFP), enhanced yellow fluorescent proteins (EYFP), anemonia majano fluorescent protein ( Anemonia majano fluorescent protein) , amFP), Zoanthus fluorescent protein (zFP), Discosoma fluorescent protein (dsFP) or Clavularia fluorescent protein (cFP). The recombinant viral vector according to claim 1, wherein the immunomodulatory polypeptide is fungal immunomodulatory protein Flammunlina velutipes (FIP-FVE), fungal immunomodulatory protein Ganodera tsugae (FIP- GTS), fungal immunomodulatory protein ( Volvariella volvacea , FIP-VVO), Ganoderma lucidum Ling-Zhi-8 (LZ-8), Ganoderma lucidum mycelium fungal immunoregulatory protein (GTS) Fungal immunomodulatory protein Ganoderma microsporum (GMI) or immunomodulatory protein Tremella FuciFormis (TFP). The recombinant viral vector of claim 11, wherein the immunomodulatory polypeptide is FIP-FVE and has the amino acid sequence of SEQ ID NO: 7. The recombinant viral vector of claim 1, wherein the recombinant viral vector is derived from a virus, wherein the virus is selected from the group consisting of a baculovirus, an adenovirus, an adeno-associated virus, a retrovirus, a lentivirus, and a Sindebi. A group of viruses, herpes simplex virus, forest virus, and vaccinia virus. A double-effect vaccine comprising: a first effective amount of one CSFV antigen, a second effective amount of one PCV2 antigen, a third effective amount of one immunomodulatory polypeptide, and a pharmaceutically acceptable adjuvant; The CSFV antigen, the PCV2 antigen, and the immunomodulatory polypeptide are prepared at one time using the recombinant viral vector of claim 1. The double effect vaccine of claim 14, wherein the CSFV antigen is CSFV-E2, the PCV2 antigen is PCV2-ORF2, and the immunomodulatory polypeptide is FIP-FVE.