KR102243526B1 - Transgenic animal for Nonclinical evaluation of Foot and Mouth Disease Vaccine and producing method thereof - Google Patents

Abstract

The present invention relates to a transgenic animal for a non-clinical evaluation of a hand-foot-and-mouth disease vaccine and relates to a recombinant vector comprising a nucleotide sequence encoding a receptor gene for inducing enterovirus infection and a promoter operably linked to the sequence, a transgenic animal other than humans transformed with the vector, cells or tissues isolated from the animal, and a method for manufacturing the transgenic animal or the cells or tissues isolated therefrom. The recombinant vector, the animal transformed with the recombinant vector, or the cells or tissues isolated therefrom, and the manufacturing method can be usefully used to evaluate the efficacy of an inactivated vaccine of enterovirus type 71 that causes hand-foot-and-mouth disease and its resulting nervous system complications.

Description

Transgenic animal for Nonclinical evaluation of Foot and Mouth Disease Vaccine and producing method thereof

The present invention relates to a transgenic animal capable of evaluating the efficacy of an inactivated vaccine of enterovirus type 71 that causes hand, foot and mouth disease and neurological complications resulting therefrom, and more particularly, encoding a receptor gene that induces enterovirus infection. A recombinant vector comprising a nucleotide sequence and a promoter operably linked to the sequence, a transgenic animal other than a human transformed with the vector, a cell or tissue isolated from the animal, the transgenic animal or a cell isolated therefrom Or it relates to a method of manufacturing the tissue.

Viruses that can cause hand, foot and mouth disease are all enteroviruses, single-stranded RNA viruses without an envelope, and belong to the family Picornaviridae. Enterovirus can be transmitted from person to person in the fecal-oral, respiratory route, or vertically from mother to newborn in the perinatal period. Enteroviruses can survive on the environmental surface and can spread through formites. The incubation period is characteristically 3 to 6 days, and the virus can be excreted from 1 to 3 weeks after infection in a child with or without symptoms, and 7 to 11 weeks through feces. Hand, foot and mouth disease is a disease that is usually accompanied by no fever or mild fever, and is a self-limiting disease in which the rash disappears approximately one week after the onset of the onset, with mild clinical progress. Hand, foot and mouth disease is a clinical syndrome in which a peculiar rash appears, most commonly caused by coxsackie virus A16 (CA16), and can also be caused by enterovirus 71 (EV71), in addition to coxsackie virus A5, A7, A9, It can also be caused by A10, B2, B5, etc. In addition, the Coxsackie virus A16 (CA16) and Enterovirus 71 (EV71) were reported to be infected through common receptors of P-selection glycoprotein ligand-1 and transmembrane proteins of Scavenger receptor Class B member 2 (SCARB2) upon infection.

In the case of hand, foot and mouth disease caused by a relatively new type of enterovirus 71, which is recently prevalent in Southeast Asia, more often than the case caused by Coxsackie virus A16, it has a severe clinical course, and causes nervous system complications at a high rate, especially in young children. It can lead to brain stem encephalitis, neurogenic pulmonary edema, pulmonary hemorrhage, shock and rapid death. Currently, cases of suspected outbreak of hand, foot and mouth disease are being confirmed in Korea, and vaccines are being developed, but a verification system for commercialization of the vaccine has not been developed.

In addition, studies have been reported in connection with the production of animal models related to enterovirus infection in the past, but the existing studies are transgenic models made by specifying only one receptor limited to lysosomes, and there is a limit to the validation of various enteroviruses. .

In this regard, the present inventors have tried to develop a vaccine verification system through animals, and as a result of using the transgenic animal model technology through gene insertion, ANXA2, CD209, CXADR, SCARB2, SELPLG five receptor genes that induce enterovirus infection. Expressed transgenic animals were invented.

Non-Patent Document 1. Seiya Yamayoshi, Ken Fujii, Satoshi Koike., Scavenger receptor B2 as a receptor for hand, foot, and mouth disease and severe neurological diseases. Frontiers in microbiology. 3, 1-6. 2012 Non-patent document 2. Ken Fujii et al., Transgenic mouse model for the study of enterovirus 71 neuropathogenesis. PNAS. 110(36), 14753-14758. 2013

An object of the present invention is to provide a transgenic animal capable of evaluating the efficacy of an inactivated vaccine of Enterovirus type 71 that causes hand, foot and mouth disease and neurological complications resulting therefrom.

In addition, the present invention is to provide a system capable of verifying a vaccine for hand, foot and mouth disease.

In order to achieve the above object, the present invention

It provides a recombinant vector comprising a nucleotide sequence encoding a receptor gene that induces enterovirus infection and a promoter operably linked to the sequence. The receptor gene may include any one or more of ANXA2, CD209, CXADR, SCARB2, and SELPLG. In addition, the promoter may be selected from the group consisting of a thymidine kinase promoter, a beta-actin promoter, and a CMV promoter.

In addition, the recombinant vector may additionally include a Flag tag.

In addition, the present invention provides a transgenic animal other than humans transformed with a recombinant vector comprising a nucleotide sequence encoding a receptor gene that induces enterovirus infection and a promoter operably linked to the sequence.

The transformation is a method selected from the group consisting of a microinjection method, an electroporation method, an incident injection method, a method using sperm, a virus infection method, a direct muscle method, an insulator and a method using a transposon. In addition, the transformation is for the efficacy or safety assay of the hand, foot and mouth vaccine.

In addition, the receptor gene is a virus included in genotype human enterovirus A, human enterovirus B, human enterovirus C, or human enterovirus D. It is possible to induce infection, but it is preferable to induce infection with Enterovirus 71 (Enterovirus, EV71) or Coxsackievirus A 16 (CVA16).

In addition, the present invention provides a cell or tissue isolated from the animal.

In addition, the present invention provides a fertilized egg that has been nuclear-transplanted into cells.

In addition, the present invention

(1) preparing a recombinant vector operably linked by tagging a receptor gene, a CMV promoter and a flag that induces enterovirus infection; And

(2) It provides a method for producing a transgenic animal or cells or tissues isolated therefrom for non-clinical evaluation of a hand, foot or mouth vaccine comprising the step of transforming the vector into a mouse or rat fertilized egg.

In the present invention, the term "hand, foot and mouth vaccine" means an enterovirus vaccine that causes hand, foot and mouth disease. The vaccine includes live vaccines through viral culture, inactivated vaccines and recombinant protein vaccines expressing the envelope protein of enterovirus, enterovirus 71 type inactivated vaccine, C4a genotype virus vaccine line, Coxsackie virus type 16 CVA16 vaccine strains are included, but enterovirus type 71 inactivated vaccines or coxsackie virus type 16 CVA16 vaccine strains are preferred.

In one embodiment, the receptor gene is ANXA2 (Annexin A2), CD209 (DC-SIGN, Cluster of Differentiation 209), CXADR (coxsackie virus and adenovirus receptor), SCARB2 (Scavenger receptor class B member 2), SELPLG (P- Selectin glycoprotein ligand-1 (PSGL-1), CD162) may contain any one or more genes. The receptor gene includes both human or non-human origin, but human origin is preferred.

In the present invention, the term "vector" or "recombinant vector" refers to a plasmid vector as a means for expressing a gene of interest in a host cell; Phagemid vector; Cozmid vector; In addition, viral vectors such as bacteriophage vectors, adenovirus vectors, retroviral vectors, and adeno-associated virus vectors may be included. The vector of the present invention can typically be constructed as a vector for cloning or as a vector for expression. In addition, the vector of the present invention can be constructed using a prokaryotic cell or a eukaryotic cell as a host. When the vector of the present invention is an expression vector and a prokaryotic cell is used as a host, a strong promoter capable of promoting transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, etc.), a ribosome binding site for initiation of translation, and a transcription/translation termination sequence are generally included. When E. coli (eg, HB101, BL21, DH5α, etc.) is used as the host cell, the promoter and operator site of the E. coli tryptophan biosynthetic pathway (Yanofsky, C. (1984), J. Bacteriol., 158:1018- 1024) and a left-facing promoter of phage λ (pLλ promoter, Herskowitz, I. and Hagen, D. (1980), Ann. Rev. Genet., 14:399-445) can be used as a regulatory site.

On the other hand, vectors that can be used in the present invention are plasmids often used in the art (e.g., pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14 , pGEX series, pET series, and pUC19, etc.), phagemid (eg pComb3X), phage (eg λgt4·λB, λ-Charon, λΔz1 and M13, etc.) or viruses (eg SV40, etc.). have.

On the other hand, when the vector of the present invention is an expression vector and a eukaryotic cell is used as a host, a promoter derived from the genome of a mammalian cell (eg, a metallotionine promoter) or a promoter derived from a mammalian virus (eg, adeno Virus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, and private promoter of HSV) can be used, and generally have a polyadenylation sequence as a transcription termination sequence.

In addition, the recombinant vector of the present invention may contain an antibiotic resistance gene commonly used in the art as a selection marker, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin , Neomycin and tetracycline resistance genes.

In the present invention, the term “promoter” refers to a DNA sequence capable of controlling the transcription of a specific nucleotide sequence into mRNA when linked to a specific sequence. In general, promoters do not apply in all cases, but are present at 5'of the desired nucleotide sequence to be transcribed into mRNA (i.e. higher, meaning the sequence located 5'of the reference nucleotide sequence) and RNA polymerase and other transcriptions. It provides a site to which the transcription factor for initiation specifically binds. It also refers to a nucleic acid or nucleic acid sequence having a function of controlling the electrons of one or more genes as described above.

In the present invention, the term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein or RNA of interest to perform a general function. For example, a promoter and a nucleic acid sequence encoding a protein or RNA may be operably linked to affect the expression of the encoding nucleic acid sequence. The operative linkage with the recombinant vector can be prepared using gene recombination techniques well known in the art, and site-specific DNA cleavage and linkage use enzymes generally known in the art.

In the present invention, the term "tag" or "tag sequence" refers to nucleotides, oligonucleotides, polynucleotides or amino acids, peptides, or It refers to the chemical moiety or other chemical substance of a protein.

In the present invention, the term "transformation" means changing the genetic properties of an organism by DNA given from outside. In addition, it means that exogenous DNA is introduced into a host organism (animal in the present invention) such as cells, tissues, and adults, is stably inherited, and exhibits a phenotypic change. Transformation methods include various methods known in the art, such as microinjection, electroporation, particle bombardment, sperm-mediated gene transfer, and viral infection. It can be appropriately selected and applied from techniques using virtual infection, direct muscle injection, insulator, and transposon. Preferably, in the present invention, it can be transformed through microinjection.

The transgenic animal is not limited, but a rodent mouse or rat is preferred.

Recombinant vectors for nonclinical evaluation of hand, foot and mouth vaccines. The animal transformed with the recombinant vector, or cells or tissues isolated therefrom, and the method for producing the transformed animal can be utilized as an effective infection model for serotypes of various enteroviruses in Korea, and accurate enterovirus It can be used very usefully in the development of vaccines as an effective therapeutic agent development and practical use through understanding the infection mechanism, or as an attack vaccination model for additionally developed enterovirus vaccines.

1A shows a cleavage map for a recombinant vector or plasmid containing ANXA2 among receptor genes that induce enterovirus infection, and FIG. 1B shows the result of electrophoresis for the recombinant vector containing ANXA2. 1C shows PCR results confirming that the ANXA2 gene was expressed in an animal model (mouse) transformed with a recombinant vector containing ANXA2 among the receptor genes that induces enterovirus infection. Figure 1d shows information on the primers used in the construction or confirmation of the recombinant vector or plasmid containing the ANXA2 and information on the PCR product size (size).
2A shows a cleavage map for a recombinant vector or plasmid containing CD209 among receptor genes that induce enterovirus infection, and FIG. 2B shows the results of electrophoresis for the recombinant vector containing CD209. 2C shows the PCR results confirming that the CD209 gene was expressed in an animal model (mouse) transformed with a recombinant vector containing CD209 among the receptor genes that induces enterovirus infection. Figure 2d shows information on the primers used to construct or confirm the recombinant vector or plasmid containing the CD209 and information on the PCR product size (size).
Figure 3a shows a cleavage map for a recombinant vector or plasmid containing CXADR among receptor genes that induce enterovirus infection, and Figure 3b shows the results of electrophoresis for the recombinant vector containing CXADR. 3C shows PCR results confirming that the CXADR gene was expressed in an animal model (mouse) transformed with a recombinant vector containing CXADR among the receptor genes that induces enterovirus infection. Figure 3d shows information on the primers used to construct or confirm the recombinant vector or plasmid containing the CXADR and information on the PCR product size (size).
Fig. 4 shows a cleavage map for a recombinant vector or plasmid containing SCARB2 among receptor genes that induce enterovirus infection, and Fig. 4B shows the result of electrophoresis for the recombinant vector containing SCARB2. Figure 4c shows the PCR results confirming that the SCARB2 gene was expressed in an animal model (mouse) transformed with a recombinant vector containing SCARB2 among the receptor genes that induces enterovirus infection. Figure 4d shows information on the primers used to construct or confirm the recombinant vector or plasmid containing SCARB2 and information on the PCR product size (size).
FIG. 5 shows a cleavage map for a recombinant vector or plasmid containing SELPLG among receptor genes inducing enterovirus infection, and FIG. 5B shows the result of electrophoresis for the recombinant vector containing SELPLG. Figure 4c shows the PCR results confirming that the SELPLG gene was expressed in an animal model (mouse) transformed with a recombinant vector containing SELPLG among the receptor genes that induces enterovirus infection. Figure 5d shows information on the primers used to construct or confirm the recombinant vector or plasmid containing the SELPLG and information on the PCR product size (size).
6 shows the results of confirming the incidence of neurological symptoms as a result of infection with hand, foot and mouth disease virus in the animal models of FIGS. 1 to 5.
7 shows the results of confirming the survival rate as a result of infection with the hand, foot and mouth disease virus in the animal models of FIGS. 1 to 5.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are for illustrating the present invention, and the scope of the present invention is not limited to the following examples.

In the present invention, reference sequences of Human ANXA2, CD209, CXADR, SCARB2, and SELPLG used the following known serial number, but are not limited thereto.

Human ANXA2(Genbank accession number): NM_001002857.2, NM_001002858.3, NM_001136015.3, NM_004039.3

Human CD209 (Genbank accession number): NM_001144893.2, NM_001144894.2, NM_001144895.2, NM_001144896.2, NM_001144899.2, NM_021155.4

Human Genbank accession number (CXADR): NM_001207063.2. NM_001207064.2, NM_001207065.2, NM_001207066.1, NM_001338.5

Human SCARB2(Genbank accession number): NM_001204255.2, NM_005506.4

Human SELPLG(Genbank accession number): NM_001206609.2, NM_003006.4

<Example 1.> Construction of Knock in Plasmid for Enterovirus Receptor Candidate

For the production of human ANXA2, CD209, CXADR, SCARB2, SELPLG plasmids, genes were obtained from human cells through cDNA synthesis and PCR, and the five receptor CDS were cloned using a vector that can be fused with the CMV promoter and FLAG tag in the C-term. To make it. Thereafter, the nucleotide sequences of human ANXA2, CD209, CXADR, SCARB2, SELPLG genes and FLAG TAG were analyzed through sequencing (sequence) analysis. After confirming the nucleotide sequence from the CMV promoter to the Poly A tail from the Agarose gel using two restriction enzymes of DrdII / NruI for the prepared plasmid containing the five receptors (Fig. 1b, Fig. 2b, Fig. 3b, Fig. 4b) , Figure 5b), purified and used for mouse embryo microinjection.

<Example 2.> Preparation of TG (Transgeneic mouse) mice expressing enterovirus receptor

2-1. Mouse Embryo Superovulation

PMSG (Pregnant mare's serum Gonadotropin) and HCG (Human Chronic Gonadotropin) were used to induce hyperovulation in mice, and the hormone treatment was performed 48 hours after PMSG 5IU IP injection, followed by HCG 5IU IP injection to induce hyperovulation in mice. .

2-2. Mouse embryo preparation

After performing donor mouse mating and plug check, the Vaginal plug check was executed the morning after mating. Then, female mouse sacrifice and Blastocyst Embryo Collection were performed. After obtaining the Embryo (fertilized egg) of the fertilized mouse through the above process, a recombinant vector or plasmid containing the receptors of 5 types (ANXA2, CD209, CXADR, SCARB2, SELPLG) produced for the production of TG mice was added to the fertilized egg. microinjection was performed.

2-3. Linearized human enterovirus receptors DNA microinjection

Take out the obtained blastocyst embryos, wash them, put them in Petri Dish Drop, and prepare a plasmid containing 5 types of enterovirus receptors (ANXA2, CD209, CXADR, SCARB2, SELPLG) prepared in advance and treated with 2 types of restriction enzymes to form a single line (linearlized). from), and through microinjection, the gene was injected into a fertilized egg fixed with a holding pipet.

2-4. Establishment of transformed cells (Founder) through screening

For F0 born from a surrogate mother implanted with a fertilized egg, two weeks after birth, genomic DNA using a tail cut was used to dissolve the tissue using Proteinase K, and then separated, and the nucleotide sequences of human ANXA2, CD209, CXADR, SCARB2, and SELPLG can be identified. A primer was prepared and tested for gene insertion using PCR. (Fig. 1C, Fig. 2C, Fig. 3C, Fig. 4C, Fig. 5C). The F0 in which the PCR band was detected was designated as a positive transgenic mouse and maintained. Then, human ANXA2, CD209, CXADR, SCARB2, and SELPLG gene-expressing transgenic mice were crossed and propagated.

<Example 3.> Confirmation of enterovirus infection results of transgenic animals

Of the clinical isolates, the incidence of neurological symptoms was as a result of infecting the virus with the iv (intravenous) infection route at a titer of about 106 TCID50 to 5 transgenic mice of 3 weeks of age in the infection model using EV71. The survival rate is as shown in FIG. 7. It was confirmed that the transgenic animals (mouse) prepared above were susceptible to EV71, resulting in neurological symptoms and death after infection.

As described above, the transgenic animal for non-clinical evaluation of the hand, foot and mouth vaccine of the present invention can be usefully utilized as a system capable of verifying the efficacy and safety of the hand, foot, and mouth disease vaccine or the hand, foot, and mouth disease vaccine.

Claims (11)

delete delete delete delete delete delete delete delete delete delete (1) synthesizing cDNA for Genbank accession number NM001207063.2 sequence for the production of human CXADR (coxsackie virus and adenovirus receptor) plasmid;
(2) preparing a recombinant vector operably linked by adding a CMV promoter and a Flag tag;
(3) confirming the nucleotide sequence using DrdII and NruI restriction enzymes;
(4) Inducing mouse hyperovulation by intraperitoneally administering 5 IU of Pregnant mare's serum Gonadotropin (PMSG) and then intraperitoneally administering 5 IU of Human Chronic Gonadotropin (HCG) at 48 hours;
(5) microinjection of the human CXADR recombinant vector into the ovulated fertilized egg;
(6) checking whether the gene is inserted to identify the transgenic mouse;
(7) inoculating a transgenic mouse with a hand, foot and mouth vaccine; And
(8) confirming the incidence and mortality of neurological symptoms including paralysis by infecting the transgenic mice with an EV71 clinical isolate at a titer of 106 TCID50 by intravenous administration; Non-clinical evaluation method of hand, foot and mouth vaccine using human CXADR transgenic mice comprising

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