KR20050083241A - Nucleotide sequence for preventing diseases from coxsackievirus infection for manufacturing vaccine and vector containing the same - Google Patents

Abstract

In the present invention, an immune response by injecting a vector containing a nucleic acid sequence capable of preventing heart disease and other diseases, including myocarditis and dilated cardiomyopathy caused by the infection of Coxsackievirus B, is injected into the body. It is suggested how to cause this.

The nucleic acid sequence inserted according to the present invention contains only the minimum sequence capable of causing an immune response in the coxsackievirus genome, thereby solving the problem that may be caused by the injection of a large amount of foreign genes. .

Description

Nucleotide Sequence for Preventing Diseases from Coxsackievirus Infection for Manufacturing Vaccine and Vector Containing the Same}

The present invention relates to a DNA vaccine sequence and a vector comprising the same, and more particularly, to a nucleic acid sequence for preparing a DNA vaccine comprising a specific sequence in the coxsackievirus genome and a vector into which the nucleic acid sequence is inserted.

Coxsackievirus (Coxsackievirus, CV) is a human enterovirus belonging to the Picornavirus (Picornavirus) family, and is largely divided into A type and B type. Coxsackievirus B (CVB) is known to six genotypes (CVB1 to CVB6) to date, and the genome consists of a single-stranded positive-sense RNA.

Genomic analysis of CVB revealed that all CVB viruses have a 5 'untranslating region (5' UTR), a protein coding region with an open reading frame (ORF), 3 '. Consists of UTR and terminal poly-A tail. The protein coding region of CVB can be largely divided into regions of P1, P2 and P3, and the P1 region encodes four capsid proteins of CVB, and P2 and P3 are non-structures required for the growth of CVB. It encodes a protein. The P1 region encoding the four capsid proteins can be subdivided into VP4 (1A), VP2 (1B), VP3 (1C) and VP1 (1D), depending on the portion encoding each capsid protein, and the P2 and P3 regions Are classified into 2A (protease), 2B, 2C and 3A, 3B (Vpg), 3C (protease) and 3D (polymerase), respectively. In particular, the four capsid proteins encoded in the P1 region have about 60 copies in each virion. All CVBs consist of approximately 7,400 nucleotide sequences and are found to differ in particular in the structural protein coding regions VP1, VP2 and 5 'UTR, 3' UTR.

On the other hand, since the smallpox vaccine was manufactured by Edward Jenner more than 200 years ago, it has been used in the clinical practice of various vaccines. In general, a vaccine may be defined as an antigen used to immunize an uninfected person by using an infectious disease, a pathogen of an infectious disease itself or a part thereof, and may be classified into a live vaccine and a killed vaccine. . However, in the case of conventional vaccines, live vaccines may be converted into toxic strains, which may cause side effects, particularly in children. In the case of vaccinated vaccines, the duration of immunity is short, so booster injections are often performed. I have a problem that I need.

Due to the recent rapid development of molecular biology, it is possible to use a subunit vaccine as a protein obtained by expressing and isolating and in vitro expressing a nucleic acid fragment containing a specific gene of a pathogen through genetic recombination. It was. Such vaccination increases the humoral immune response in vivo, but in most cases it has been found to be ineffective for cell mediated immune response (CMI).

Due to this problem, a new type of vaccine is being developed that can activate CMI. A representative vaccine is a recombinant vaccine method that injects genes of other pathogens into an attenuated vaccine strain. The recombinant bacteria or viruses thus produced are expressed in cells when a heterologous gene is expressed in a cell when infected in vivo, thereby presenting antigenic peptides in MHC class I as well as humoral immunity. It is reported to be effective in inducing reactions.

Regarding CVB3, US Pat. Nos. 6,071,742 and 6,323,024 (Steven M. Tracy et al.) Replace 5 'UTR, a non-translated region of the CVB3 genome, with a UTR of PV1, another enterovirus, or a specific base that forms a UTR. Disclosed is a viral vector in which a mouse IL4 (murine IL4, mIL4), which is a kind of cytokine, is inserted into a genome of CVB3, which is mutated with a base to attenuate the activity of CVB3. In the vicinity of the inserted mIL4, there is a sequence recognized by CVB3 protease 2A, and the mIL4 foreign gene is inserted between capsid protein D and protease 2A (P2-A), which is expressed together with the genome of CVB3 when injected in vivo. As a result, the disease caused by the infection of CVB3 can be controlled early. However, in this case, in addition to the foreign gene, other genes of the virus or bacteria used as a vector are also expressed, and the original virulent type is passed down from generation to generation, especially by replacing some bases with other bases. By reversion, there is a problem that can cause substantial disease.

The present invention has been proposed to solve the above problems, and an object of the present invention is to provide a nucleic acid sequence for vaccine production that can prevent the disease that can be caused by coxsackie virus in advance.

Another object of the present invention is to inoculate only a specific sequence of coxsackieviruses and inoculate it in vivo, thereby artificially causing an immune response, thereby preventing diseases such as cardiac diseases such as cardiomyopathy caused by infection of coxsackieviruses. An object of the present invention is to provide a plasmid recombinant expression vector for preparing a vaccine.

For the above purpose, in one aspect of the present invention provides a nucleic acid sequence for vaccine production for the prevention of diseases caused by coxsackievirus infection consisting of SEQ ID NO: 2 to SEQ ID NO: 6. The nucleic acid sequence of SEQ ID NO: 1 to SEQ ID NO: 6 is preferably characterized in that the cDNA sequence derived from CVB3 H3 strain.

On the other hand, in another aspect of the present invention provides a recombinant plasmid expression vector in which a sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 6 is inserted therein. In particular, the plasmid expression vector into which the SEQ ID NO: 1 to SEQ ID NO: 6 of the present invention can be inserted is basically inserted in addition to the cloning site that can be recognized by one or more restriction enzymes. A transcriptional regulatory site that regulates expression, a poly (A) adenylation signal site (pA) and ColEl origin of replication that induces the inserted sequence to be stably transcribed in eukaryotes, and may include an inserted sequence It is preferable to further include an antibiotic resistance gene that can determine whether the expression. In particular, plasmid vectors that can be used as parent vectors in the context of the present invention are pCK expression vectors or variants thereof that contain all such sequences. Hereinafter, with reference to the accompanying drawings of the present invention will be described in more detail.

Coxsackievirus is an intestinal virus of the family of piconaviruses. In particular, coxsackievirus type B (CVB) is known to be the causative agent of aseptic meningitis (CVB1-5) and insulin-dependent diabetes mellitus (CVB4). In particular, Coxsackievirus B3 (hereinafter referred to as CVB3) has been found to be closely associated with heart diseases such as myocarditis and dilated cardimyopathy (DCM). In other words, CVB3 is thought to be the cause of the cardiac disease, as chronic myocarditis or CVB3 is found in about 5 to 50% of DCM patients.

To date, the immune mechanisms of myocarditis and dilated cardiomyopathy caused by CVB3 are not clear, but when CVB3 infects heart cells, myosin is released and antibodies against the released myosin attack normal cardiac cells. Autoimmune myocarditis by research reports that the protein expressed by the infection of CVB3 mimics the antigens of normal heart cells, and normal heart tissues are attacked by antibodies designed to attack the viral proteins and destroy the cardiac tissues. (autoimmune myocarditis) has been studied to cause. In other words, the immune response caused by infection with CVB3 may proceed in a direction that adversely affects host cells in addition to the mechanism of protecting host cells, thereby controlling the protective mechanisms and harmful effects caused by the infection of CVB3. How is it required?

On the other hand, since recent reports of plasmid DNA injection directly into animal muscle have been reported (Wolff JA et al, Direct gene transfer into mouse muscle in vivo, 1990, Science 247: 1465-1468). Attempts have been made to develop so-called 'DNA vaccines' using the method. In a DNA vaccine, since there is a gene for a target antigen to be cloned into a bacterial plasmid, the metabolic mechanism of the host cell is used to direct the synthesis of the antibody injected into the plasmid. Such attempts to synthesize antigens in cells express genes in cells when injected into a living body of an animal, thus creating a condition similar to that in which pathogens are infected, compared with conventional immunization using extracellular recombinant proteins or dead pathogens. It has many advantages. In other words, the foreign gene transfected into the plasmid has a strong promoter or enhancer inserted into the plasmid to efficiently express the foreign gene in the cell. Thus expressed proteins of the pathogen are loaded into MHC Class I and MHC Class II through antigen processing and antigen presentation pathways to induce antigen-specific humoral and cell mediated immune responses. have. Furthermore, plasmid vectors contain nucleic acid sequences that stimulate immunity, such as cPG motifs, which can greatly induce cell-mediated immunity in animals. In particular, it is possible to express a large amount of protein by plasmid purification and injection relatively simply, and to omit the process of purely separating and purifying the protein expressed in the conventional vaccine manufacturing process, and by simultaneously injecting genes of various pathogens, It has the advantage of inducing a translation reaction against.

Therefore, the present inventors inserted only a specific sequence of the region expressing the capsid protein of the genome of CVB into the plasmid, and then transfected into an animal, especially a mouse, and artificially challenged the immune response. It has been found that the present invention can be completed to complete the present invention.

The present inventors first of all the region expressing the capsid protein of CVB3, VP1 (SEQ ID NO: 1), VP2 (SEQ ID NO: 2), VP3 (SEQ ID NO: 3) and VP1-1 (SEQ ID NO: 4) artificially separated from the VP1, The nucleic acid sequences of VP1-2 (SEQ ID NO: 5) and VP-1-3 (SEQ ID NO: 6) were inserted into the recombinant plasmid vector, and the recombinant plasmid expression vector containing the specific nucleic acid sequence was transfected into the mouse. As a result of experimenting with the immune response caused by the expression of a specific nucleic acid sequence, it has been found that the immune response can be induced only by inserting a specific nucleic acid sequence rather than the entire genome of CVB3.

The entire CVB genome sequence used in the present invention is a cDNA (Knowlton, KU et al., A mutation in the puff region of VP2 attenuates the myocarditisc phenotype of an infectious cDNA) of the already published Woodruff variant of CVB3. of the Woddruff variant of coxsackievirus B3, Journal of Virology, 70 (11), 7811-7818, 1996). The 5 'primers used to amplify SEQ ID NO: 1 to SEQ ID NO: 6 include the Kozak sequence (CACC) and Transcription initiation codons (ATG) were included to enhance translation efficiency, and 3 'primers contained transcription termination codons (TAA). In addition, a 5 'primer and a 3' primer were respectively inserted into sequences recognizable by restriction enzymes so that the amplified SEQ ID NO: 1 to SEQ ID NO: 6 could be cut by the same restriction enzyme.

In particular, the VP1-1, VP1-2, and VP1-3 sequences of SEQ ID NO: 1 to SEQ ID NO: 6 artificially separated in the present invention are hydrophobic (hydorphobicity) to the VP1 region of SEQ ID NO. It is classified according to surface probability.

On the other hand, recombinant plasmid vectors that can be used in the context of the present invention, like conventional plasmid vectors, contain a sequence that can be recognized by various restriction enzymes so that foreign genes can be inserted between the sequences cut by the restriction enzymes. Multi-cloning site (MCS), antibiotic resistance genes such as ampicillin and neomycin ( Amp ^r , Neo ^r, etc.) to confirm expression in E. coli, and ColE1 origin of replication to enable replication in E. coli Should be

In particular, in order to stably express heterologous genes inserted therein in eukaryotic cells such as mice, the recombinant plasmid expression vector is a promoter capable of inducing transcription in eukaryotic cells in addition to the genes in the expression vector. Poly (A) adenylation signal sites which may be present.

In this case, as a promoter capable of inducing transcription in eukaryotic cells, a conventionally used SV40 promoter (pSV40) may be used, and preferably, IE (HCMV) of human cytomegalovirus (HCMV) capable of inducing strong and stable transcription in eukaryotic cells. Immediate early) A vector into which a gene promoter (pCMV) is inserted can be used. More preferably, the upstream of the polycloning region includes the pCMV, and is designed to include all sequences immediately preceding the ATG of exon 1, intron A, and exon 2 in the 5 'UTR of the HCMV IE gene. Can be maximized. In addition, the poly (A) adenylation signal site preferably includes a poly (A) adenylation signal site of bovine growth hormone (BGH). For example, the pCK vector disclosed in Korean Patent Application Laid-Open No. 10-2002-0013486 as a plasmid vector into which the nucleic acid sequence of SEQ ID NO: 1 to SEQ ID NO: 6 can be inserted according to the present invention (Virome, Korea) or the pCA vectors modified with pCK vectors can be used in particular.

1 shows a schematic cleavage map of a pCA vector used in the present invention. As shown, the pCA vector used as the parent vector of the present invention is composed of approximately 4,346 bp, and a transcriptional regulatory region including a promoter and enhancer of the HCMV IE gene to induce strong and stable transcription in eukaryotic cells. Downstream, the poly (A) adenylation signal site of BGH is included. Between the transcriptional regulatory site and the poly (A) adenylation signal site, a multicloning that can be recognized by various restriction enzymes such as Kpn I, BamH I, EcoR I, EcoR V, Not I, Xho I, Xba I and Pme I can be recognized. The site (MCS) is located so that the foreign gene can be inserted. On the other hand, as a factor for confirming the insertion of foreign genes include ampicillin resistance gene (Ampicillin) and the origin of the ColE1 replication to enable replication.

Figure 2 schematically shows the position in the CVB3 genome of SEQ ID NO: 1 to SEQ ID NO: 6 and the position inserted into pCA, a plasmid expression vector of the nucleic acid sequence consisting of SEQ ID NO: 1 to SEQ ID NO: 6 according to an embodiment of the present invention It is shown as.

As described above, all coxsackieviruses, including CVB3, consist of regions that are not translated at the 5 'and 3' ends (5 'UTR, 3' UTR), and the portion encoding substantially the specific protein is a capsid protein ( It is divided into a P1 region encoding VP1 to VP4) and a P2 and P3 region encoding nonstructural proteins (2A to 2C, 3A to 3D). In the present invention, particularly, the VP1 (SEQ ID NO: 1), the VP2 (SEQ ID NO: 2), the VP3 (SEQ ID NO: 3) region of the capsid protein coding region of the P1 region, and the VP1-1 (SEQ ID NO: 4), which is a partial region of the VP1 region, The nucleic acid sequences of VP1-2 (SEQ ID NO: 5) and VP1-3 (SEQ ID NO: 6) are amplified by PCR amplification, and the above amplified products cut by the restriction enzyme are cut with the same restriction enzyme, for example, pCA of FIG. Inserted into the polycloning site (MCS) of the vector, the pCA-VP1 recombinant vector, pCA-VP2 recombinant vector, pCA-VP3 recombinant vector, pCA-VP1-1 recombinant vector, pCA-VP1-2 recombinant vector and pCA-VP1- 3 recombinant vectors were prepared.

Each modified pCA expression vector thus prepared was injected into BALB / c mice, which are model mice, to measure the degree of antibody production following injection, and then lethal CVB3 at a time after a predetermined time after injection of the expression vector. H strain was injected to induce an artificial immune response (Challenge), and the survival rate, weight and heart tissue damage of the mice injected with each pCA expression vector was measured.

The present inventors through the above process, the expression vector is inserted into the nucleic acid sequence of SEQ ID NO: 1 to SEQ ID NO: 6 humoral immune response to produce antibodies in the mouse, as well as cellular immune response involving T lymphocytes We confirmed that it proceeded.

In addition, the inventors of the present invention prepared SEQ ID NO: 1 (VP1), SEQ ID NO: 2 (VP2), SEQ ID NO: 3 (VP3), SEQ ID NO: 4 (SEQ ID NO: VP1-1), SEQ ID NO: 5 (VP1-) 2), pCA modified expression vector in which SEQ ID NO: 6 (VP1-3) is inserted is' pCA-VP1 ',' pCA-VP2 ',' pCA-VP3 ',' pCA-VP1-1 'and' pCA- VP1-2 'and' pCA-VP1-3 'were named and deposited on February 9, 2004 at the Korea Agricultural Microbiological Conservation Center (KACC, 225 Seodun-dong, Suwon-si, Gyeonggi-do, Korea). Accession numbers KACC 95201 (pCA-VP1), KACC 95202 (pCA-VP2), KACC 95203 (pCA-VP3), KACC 95204 (pCA-VP1-1) and KACC 95205 (pCA-VP1-), respectively. 2), and KACC 95206 (pCA-VP1-3).

Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples are merely illustrated to illustrate the present invention in detail, but the present invention is not limited thereto.

Example 1 Preparation of Vector

In this example, PCR amplification was performed for a part of the cDNA sequence of the coxsackie virus CVB3 (CVB3 H3 strain). The entire sequence of CVB3 was based on the cDNA of the Woodruff variant of CVB3, which has already been published. In this example, the amplified sequence is VP1 (SEQ ID NO: 1), which is the region encoding the capsid protein in the CVB3 genomic region. ), VP2 (SEQ ID NO: 2) and VP3 (SEQ ID NO: 3) and VP1-1 (SEQ ID NO: 4), VP1-2 (SEQ ID NO: 5), and VP1-3 (SEQ ID NO: 6) regions that are part of the VP1 region. Sequence.

First, primers complementarily binding to the 5 'end and 3' end of each nucleic acid sequence were used to amplify the nucleic acid sequence consisting of SEQ ID NO: 1 to SEQ ID NO: 6. The 5 'sense primers used in the present examples were 5'-cagcaaagcttgaccatgggccccgtgtgagacgcgcgataac-3' (SEQ ID NO: 7, VP1 region), 5'-tcactaagcttgaccatgtcccccacagtagaggagtgc-3 '(SEQ ID NO: 9, VP2 region), and 5'-cgtttaactact-3ga '(SEQ ID NO: 11, VP3 region), 5'-cagcaggatccgaccatgggccccgtggaagacgcgataac-3' (SEQ ID NO: 13, VP1-1 region), 5'-acgttggatccgaccatgactcaacagccctcaaccacac-3 '(SEQ ID NO: 15, VP1-2 region), and 5'-aaaaaaaagtcccaaaccat -3 '(SEQ ID NO: 17, VP1-3 region), and the 3' antisense primer was 5'-cctgactcgagttaaaatgcgcccgtatttgtcattg-3 '(SEQ ID NO: 8, VP1 region, VP1-3 region), 5'-atggtctcgagttactggtgcccggccaaacgtagtc-3' ( SEQ ID NO: 10, VP2 region), 5'-tcttcctcgagttactggaaaaagttttgctgcgaaat-3 '(SEQ ID NO: 12, VP3 region), 5'-gttgactcgagttaagtacttgttatgacaaacgtc-3' (SEQ ID NO: 14, VP1-1 region), and 5'-tgcttctcgagttagtagattctaatgg-3 ' SEQ ID NO: 16, VP1-2 region).

PCR amplification was performed in accordance with the usual procedures, mass amplification of SEQ ID NO: 1 to SEQ ID NO: The nucleic acid sequence of the 6 BamHⅠ and pCA vector digested with XhoⅠ restriction enzymes, and the cloning site is cleaved by the same restriction enzyme (viromed, Korea) and ligation. PCA-VP1, pCA-VP2, pCA-VP3, pCA-VP1-1, pCA-VP1-2, pCA-VP1-3 plasmid expression vectors having the nucleic acid sequences of SEQ ID NOS: 1 to 6 inserted therein were prepared. Each prepared plasmid was secured in large quantities using the "EndoFree ™ -Maxi prep kit (Qiagen, Hilden, Germany).

Example 2: Electroporation and Immunogenesis in Vivo

In this embodiment, pCA-VP1, pCA-VP2, pCA-VP3, pCA-VP1-1, pCA-VP1-2 and pCA-VP1-3 recombinant plasmid expression vectors prepared in Example 1 were injected into mice ( After injection, an immune response by artificial immune challenge was conducted.

3 is a view schematically showing an immune response according to the present embodiment. Im in the figure shows that the recombinant plasmid expression vector in which the nucleic acid sequence of SEQ ID NO: 1 to SEQ ID NO: 6 is inserted into the mouse to be immunized (immunization), Bl means blood sampling (bleeding), Ch Is an indication of challenge.

First, ketamine (100 mg / kg) and xylazine (5 mg / kg) were injected into the peritoneum of BALB / c strain mice purchased from Daehan Biolink Co., Ltd., at 8 weeks old mice bred at the animal breeding center of the Korea Food and Drug Administration. Kg), the mixture was injected, anesthetized the mice, and then, at intervals of 2 weeks (day 0, day 14), 15 μg of the modified pCA recombinant expression vector prepared in Example 1 and the pCA vector without any foreign genes were inserted. Each was injected into the right quadriceps muscle of BALB / c mice (30 μl / leg). Injection of the strain vector by electroporation into the mouse was performed according to conventional methods using an electroshock generator (Electro Square Porator, ECM 830, BTX, San Diego, USA).

Four weeks after the last immunization by injection of the modified pCA expression vector, the mice were immunized by injecting a lethal dose (1 × 10 ³ plaque forming unit [pfu] / mouse) of CVB3 (H3). Mice were observed over 46 days after immunization.

Examples 3 to 5: Antibody production detection of modified vector injected into mouse

In Examples 3 and 4 it was confirmed through Example 2 whether the antibody is produced in the serum of the mouse injected with a modified pCA expression vector inserted with the nucleic acid sequence of SEQ ID NO: 1 to SEQ ID NO: 6.

Example 3: Immune Response Detection of Injected Vectors-Western Blotting

In the present embodiment, when the pCA modified expression vector and the pCA vector were inoculated 7 days after the first inoculation (first western blotting) and after 14 days after the second inoculation (second western) Blotting) and serum from each mouse was subjected to Western blotting.

First the cell lysate of HeLa Cell (ATCC CCL-2) injected with the modified pCA expression vector and pCA vector was separated on 12% sodium dodecyl sulfate-polyacrylamide gel and then transferred onto nitrocellulose. The modified pCA vector and the serum obtained from the immunized mice immunized with the pCA vector according to Example 2 were used as primary antibodies to detect the presence of specific immunoglobulins against the target protein of the CVB3 H3 strain. Serum from mice used for the first western blotting could not detect the presence of specific proteins, but among the sera used for the second western blotting, pCA-VP1, pCA-VP3, pCA-VP1- 1, pCA-VP1-2 was confirmed to induce specific immunoglobulin from the serum obtained from the mice injected.

Example 4 Immune Response Detection of Recombinant Expression Vectors: Immunofluorescence Staining

In this example, immunofluorescent staining was performed on the serum of mice used in the second western blotting of the serum of Example 3. First, serum from immunized mice was injected into the same recombinant expression vector and provided to immobilized HeLa cells (ATCC-CCL2) to be used as primary antibodies. It was then incubated with a secondary antibody (Santa Cruz Biotechnology, USA) conjugated with anti-mouse fluorescent isothiocyanate (FITC). Slides were washed, mounted and examined using a fluorescence microscope (Carl Zeiss, Gottingen, Germany).

5 is a photograph showing the results of immunofluorescence staining according to the present embodiment, wherein the modified pCA vector into which the nucleic acid sequences of pCA-VP1, pCA-VP3, pCA-VP1-1 and pCA-VP1-2 are inserted is inserted. It was confirmed that the primary antibody bound to the secondary antibody was formed from the serum obtained after injection into.

Through this example, it was confirmed that a specific polyclonal antibody can be induced when a specific sequence of CVB3, particularly the nucleic acid sequences of the VP1-1 and VP1-2 regions, is injected into a mouse and then expressed. The relative amounts of antibodies identified in Examples 3 and 4 are shown in Table 1.

Table 1. Amount of Antibodies Produced in Serum of Mice by Injection of Expression Vectors

Injected vector (immunogen) Induction level of antibody pCA-VP1 + pCA-VP2 - pCA-VP3 + pCA-VP1-1 +++ pCA-VP1-2 +++ pCA-VP1-3 -

Example 5: Neutralizing Antibodies of Recombinant Expression Vector

In this example, a plaque reduction neutralizing test (PRNT) was performed on the serum of mice obtained after injecting the pCA modified expression vectors identified in Examples 3 and 4.

First, the serum of the mice obtained in Example 3 was heat-inactivated at about 56 ° C. for 30 minutes, initially diluted 10-fold in Dulbecco's Modified Eagle serun-free medium (DMEM) on ice, and then serially diluted 2-fold serially. I was. Each diluted medium was mixed with the same amount of CVB3 H3 containing about 100 pfu of CVB3 and incubated at 37 ° C. for 60 minutes. HeLa cells (ATCC CCL-2) were infected with a mixed medium of serum and virus to detect the extent of excess infection with the respective mixed medium of serum and virus, and the non-infected (pCA vector injection) serum and negative control. A mixed medium of virus was used and CVB3 antiserum (ATCC, VR-1034 / HO) was used as positive control.

Infected HeLa cells were analyzed on DMEM (Seakem GTG; FMC; Fockland, USA) containing 5% and 1.5% agarose of fetal bovine serum (FBS) on plates with six wells formed. . After 2-3 days of incubation at 37 ° C., crystal violet was used to stain the plaques. Table 2 below shows the neutralizing antibody titers by injection of the recombinant expression vector used in this example. Neutralizing antibody titre according to the present example means the reciprocal of the highest dilution concentration with a 50% reduction in the degree of virus infection in the obtained serum. It can be seen that the neutralizing antibody was not induced in the serum obtained after the injection of the modified pCA expression vector into which the nucleic acid sequence of SEQ ID NO: 1 to SEQ ID NO: 6 was inserted.

Table 2. Neutralizing Results by Injection of Expression Vectors

Injection Vector (Immune Source) Chinese Antibody Reverse negative control (pCA) <1:10 pCA-VP1 <1:10 pCA-VP2 <1:10 pCA-VP1-1 <1:10 pCA-VP1-2 <1:10 pCA-VP1-3 <1:10 CVB antiserum > 1:80

Example 6 Efficacy of Recombinant Expression Vector Following CVB3 Immunogenesis

As shown in FIG. 3, after 4 weeks of immunization with mice injected with the second recombinant expression vector, lethal dose of CVB3 (H3 strain, 10 ³ pfu / mouse) was introduced into the peritoneum of the mouse. Artificially challenged (challenge). As a control, a pCA vector without a foreign gene was inserted, and recombinant plasmid expression vectors were administered to 8 to 10 mice, respectively, and survival rate until 46 days after immunization had no change in mortality. Was compared.

6A and 6B are graphs showing survival rates of mice inserted with a specific sequence up to 46 days after artificially inducing a recombinant plasmid vector into which a specific nucleic acid sequence was inserted and artificially induced, and 18 days after immunization. And survival rates for day 46. As shown, all mice injected with the pCA expression vector with no foreign gene inserted and artificially induced immunization died 18 days after immunization. On the contrary, according to the present invention, mice injected with the modified pCA expression vector into which the nucleic acid sequences of SEQ ID NO: 1 to SEQ ID NO: 6 were inserted and immunized showed a survival rate of about 40 to 75%. That is, 46 days after the artificial immunization, the survival rate of the mouse for each nucleic acid sequence was 42.8% of the pCA-VP1 vector into which the nucleic acid sequence of SEQ ID NO: 1 was inserted and pCA- into which the nucleic acid sequence of SEQ ID NO: 2 was inserted. 60% VP2 vector, 75% pCA-VP3 vector with the nucleic acid sequence of SEQ ID NO: 3, 12.5% pCA-VP1-1 vector with the nucleic acid sequence of SEQ ID NO: 4, pCA- with nucleic acid sequence of SEQ ID NO: 5 50% of the VP1-2 vector and 37.5% of the pCA-VP1-3 vector into which the nucleic acid sequence of SEQ ID NO: 6 was inserted.

When the pCA-VP1 vector into which the nucleic acid sequence (VP1 region) of SEQ ID NO: 1 was inserted through these results was injected, the nucleic acid sequence of pCA-VP2 vector and SEQ ID NO: 3 into which the nucleic acid sequence of SEQ ID NO: 2 (VP2 region) was inserted It was confirmed that the survival rate was lower than when the pCA-VP3 vector into which the (VP3 region) was inserted was injected. In particular, in the partial sequence of the nucleic acid sequence of the VP1 region of CVB3, the VP-1-2 and VP1-3 regions, which are C-terminal regions, rather than the VP1-1 region, which is the N-terminal coding region of the expressed protein, are more efficient. there was.

Example 7: Comparison of body weight of mice after immunization and artificial immunization

In this example, a modified pCA expression vector in which the nucleic acid sequence of SEQ ID NO: 1 to SEQ ID NO: 6 is inserted according to Example 6 is injected, and the state of health of the mouse is determined at 46 days after artificial immunization. Body weights were measured for each group of mice to see. As a control, the body weights of mice injected with the peritoneum with expression vectors of pCA, pCA-VP1, pCA-VP2, and pCA-VP3 were measured without artificial immunization.

Figure 7 shows the average body weight measured for each group of mice measured by this example. The average body weight of mice immunized with the injection of expression vectors without artificial immunization was 27.8-30.4 g. On the other hand, the average body weights of mice that induced immunity and an artificial immune response by injection of pCA-VP1, pCA-VP2, and pCA-VP3 expression vectors ranged from 19.7 to 21.9 g and expressed pCA-VP1-2 and pCA-VP1-3. 24.6 ~ 26.8 g were used to induce the immune and human immune responses by the vector. After injecting the pCA-VP1-1 vector and artificially immunizing, the 46 mouse group only survived 46 days after immunization, so the result was omitted.

Mice injected only with the expression vector without artificial immunogenicity had a higher body weight than mice that were immunized, and compared with the skin and activity, mice that were not immunogenic were found to be healthy.

Example 8: Histological Comparison of Immune Induced Mouse Hearts

In Example 6, 10 μl of EVANS Blue dye (PBS 10 mg / ml) to identify CVB3 infection or cardiac disease induced by immunity in each group of mice surviving 46 days after immunization was induced. 12 hours after injection into the peritoneum of the model mouse, the heart of the mouse was captured according to a conventional method. On the other hand, a heart captured from a mouse that did not inject any expression vector into the peritoneum and was not artificially immunized was used as a negative control, and a heart obtained from a mouse 7 days after infection with CVB3 without injection of the expression vector Was used as positive control. In addition, the heart was captured according to the same procedure for the cardiac tissues obtained from mice immunized without immunization and injected with expression vectors containing the nucleic acid sequences of SEQ ID NOS: 1 to 3, and which did not induce artificial immunization.

The frozen portion of each group of mouse heart tissue captured was checked for histological disease. Infected heart cells in heart tissue are stained red by the Evans Blud dye penetrated into the tissue.

Figure 8a is a photograph of mouse heart tissue injected with negative control, positive control and pCA vector, Figure 8b is immunized by injecting pCA-VP1, pCA-VP2 and pCA-VP3 vectors, and then induced an artificial immune response The cardiac tissue of the mouse was not taken, and FIG. 8C is a photograph of the cardiac tissue of the mouse subjected to vector injection and artificial immunization.

As can be seen in the photo, positive control with CVB3 infection (center of FIG. 8A) was significantly damaged in heart tissue, but the pCA vector (FIG. 8A right) without the negative control (left of FIG. 8A) and immunization, pCA-VP1 In the case of the vector (left of FIG. 8B), the pCA-VP2 vector (in FIG. 8B), and the pCA-VP3 vector (right of FIG. 8B), it can be seen that no damage to the heart tissue occurred. In addition, as shown in FIG. 8C, the cardiac tissue of the mouse that has undergone the immunization and the artificial immunization by the injection of the expression vector is also significantly reduced when compared with the positive control. This fact means that even if the recombinant vector used in the present invention is introduced into the mouse, autoimmunity that destroys heart tissue does not proceed.

In the above description of the preferred embodiment of the present invention, but this is only an example, and various modifications and changes to the present invention are possible. In addition, the fact that all such modifications and changes belong to the scope of the present invention will become more apparent through the appended claims.

The plasmid vector into which the specific sequence of CVB3 identified by the present invention is inserted, particularly because it inserts only a minimal nucleic acid sequence capable of inducing an immune response, can be produced in large quantities with excellent expression efficiency and low cost.

In addition, by injecting some nucleic acid sequences in place of the entire genome of CVB3 that can cause autoimmunity as well as the actual disease when introduced in vivo, it was confirmed that it does not involve an autoimmune reaction that can be caused by in vivo inflow.

1 is a schematic cleavage map of a pCA plasmid vector used to insert a nucleic acid sequence of the invention;

Figure 2 is a schematic diagram showing the genomic composition of coxsackievirus (CVB3 H strain) cDNA used in the present invention and the positional relationship of specific sequences inserted into recombinant vectors;

3 is a schematic diagram showing a process for inducing an immune response using the recombinant plasmid vector used in the present invention;

Figure 4 is a photograph showing the analysis results of Western blotting performed to determine the expression in vivo of the recombinant plasmid vector used in the present invention;

Figure 5 is a photograph of the heart tissue in the immunity-induced state after injection of the recombinant plasmid vector used in the present invention;

6A and 6B are graphs illustrating the survival rate over time of mice injecting an expression vector including a specific nucleic acid sequence in vivo and causing an immune response according to one embodiment of the present invention;

7 is a graph showing the weight of each group of mice measured according to whether or not inducing an immune response and injecting an expression vector containing a specific nucleic acid sequence in vivo according to an embodiment of the present invention;

8A to 8C are histological examinations of the damage state of the mouse heart tissue according to whether injection of a vector having a specific nucleic acid sequence inserted therein and whether an artificial immune response is induced.

<110> National Institute of Health in Korea <120> NUCLEOTIDE SEQUENCE FOR PREVENTING DISEASES FROM COXSACKIEVIRUS INFECTION FOR MANUFACTURING VACCINE AND VECTOR CONTAINING THE SAME <160> 17 <170> KopatentIn 1.71 <210> 1 <211> 852 <212> DNA <213> Artificial Sequence <220> <223> cDNA of Coxsackievirus B VP1 region <400> 1 ggccccgtgg aagacgcgat aacagccgcc atagggagag ttgcggacac cgtgggtaca 60 gggccaacca actcagaggc tataccagca ctcactgctg ctgagacagg tcacacgtcg 120 caagtagtgc cgagtgacac catgcagaca cgccacgtta agaactacca ttcaaggtct 180 gagtcgacca tagagaactt cctatgtagg tcagcatgcg tgtactttac agagtatgaa 240 aactcaggcg ccaagcggta tgctgaatgg gtaataacac cacgacaagc ggcacaactt 300 aggagaaagc tagaattctt tacctacgtc cggtttgacc tggagctgac gtttgtcata 360 acaagtactc aacagccctc aaccacacag aaccaggatg cacagatcct aacacaccaa 420 attatgtatg taccaccagg tgggcctgtg ccagacaaag tcgattctta cgtgtggcaa 480 acatctacga atcccagtgt gttttggacc gagggaaacg ccccgccgcg tatgtccgta 540 ccgtttttga gcattggcaa cgcttattca aatttctatg atgggtggtc tgaattttcc 600 aggaacgggg tttacggtat caacacacta aacaacatgg gcacgctata tgcaagacat 660 gtcaatgctg gaagcacggg accaataaaa agcaccatta gaatctactt caaacctaag 720 catgtcaaag cgtggatacc tagaccacct agactctgcc aatacgagaa ggcaaagaac 780 gtgaacttcc aacccagcgg agttaccact actaggcaaa gcatcactac aatgacaaat 840 acgggcgcat tt 852 <210> 2 <211> 789 <212> DNA <213> Artificial Sequence <220> <223> cDNA of Coxsackievirus B VP2 region <400> 2 tcccccacag tagaggagtg cggatacagt gacagggtga gatcaatcac actaggtaac 60 tccaccataa cgactcagga atgcgctaac gtggtggtag gctatggagt gtggccagat 120 tatctgaagg atagcgaggc tacagcagag gaccaaccga cccaaccaga cgttgccaca 180 tgtaggttct atacccttga ctctgtacaa tggcagaaaa cctcaccagg atggtggtgg 240 aagctgcctg atgctttgtc gaacttagga ctgtttgggc agaacatgca gtaccactac 300 ttgggccgaa ctgggtatac catacatgtg cagtgcaatg catccaagtt ccaccaagga 360 tgcttgctag tagtgtgtgt accggaagct gagatgggtt gcgcaacgct aaacaacacc 420 ccatccagtg cagaattgct ggggggcgat agcgccaaag agtttgcgga caaaccggtt 480 gcatccgggt ccaacaagtt ggtacagagg gtggtgtata atgcaggcat gggggtgggt 540 gttggaaacc ttaccatttt ccctcaccag tggatcaatc tacgcaccaa caatagtgct 600 acaattgtga tgccatacac caacagcgta cctatggata acatgtttag gcataacaac 660 gtcaccctaa tggttatccc atttgtaccg ctagattact gccctgggtc taccacgtac 720 gtcccaatca cgatcacgat agccccaatg tgtgccgagt acaatggact acgtttggcc 780 gggcaccag 789 <210> 3 <211> 714 <212> DNA <213> Artificial Sequence <220> <223> cDNA of Coxsackievirus B VP3 region <400> 3 ggcttaccaa ccatgaacac tccggggagc tgtcaatttc tgacatcaga cgacttccaa 60 tcaccatctg ccatgccgca atacgacgtc acgccagaga tgaggatacc tggtgaggtg 120 aagaacttga tggaaatagc tgaggttgac tcagttgtcc cggtccaaaa tgttggagag 180 aaggtcaact ccatggaagc gtaccagata cctgtgagat ccaatgaagg atctggaacg 240 caagtattcg gcttcccact gcaaccaggg tattcgagtg ttttcagtcg gacgctccta 300 ggagagatct tgaactatta cacccattgg tcgggcagca taaagctcac gtttatgttc 360 tgtggttcgg ccatggccac tggaaaattc cttttggcat actcaccacc aggcgctggg 420 gctcccacaa aaagggttga tgctatgctt ggcactcatg tagtttggga tgtggggcta 480 caatcaagtt gcgtgctgtg cataccctgg ataagccaaa cacactaccg gtatgttgct 540 tcagatgagt ataccgcagg gggttttatt acgtgctggt atcaaacaaa catagtcgtc 600 ccagcagatg cccaaagctc ctgttacatc atgtgtttcg tatcagcatg caatgatttc 660 tctgtcaggc tattgaagga cactcctttt atttcgcagc aaaacttttt ccag 714 <210> 4 <211> 369 <212> DNA <213> Artificial Sequence <220> <223> cDNA of Coxsackievirus B partial VP1 region <400> 4 ggccccgtgg aagacgcgat aacagccgcc atagggagag ttgcggacac cgtgggtaca 60 gggccaacca actcagaggc tataccagca ctcactgctg ctgagacagg tcacacgtcg 120 caagtagtgc cgagtgacac catgcagaca cgccacgtta agaactacca ttcaaggtct 180 gagtcgacca tagagaactt cctatgtagg tcagcatgcg tgtactttac agagtatgaa 240 aactcaggcg ccaagcggta tgctgaatgg gtaataacac cacgacaagc ggcacaactt 300 aggagaaagc tagaattctt tacctacgtc cggtttgacc tggagctgac gtttgtcata 360 acaagtact 369 <210> 5 <211> 342 <212> DNA <213> Artificial Sequence <220> <223> cDNA of Coxsackievirus B partial VP1 region <400> 5 actcaacagc cctcaaccac acagaaccag gatgcacaga tcctaacaca ccaaattatg 60 tatgtaccac caggtgggcc tgtgccagac aaagtcgatt cttacgtgtg gcaaacatct 120 acgaatccca gtgtgttttg gaccgaggga aacgccccgc cgcgtatgtc cgtaccgttt 180 ttgagcattg gcaacgctta ttcaaatttc tatgatgggt ggtctgaatt ttccaggaac 240 ggggtttacg gtatcaacac actaaacaac atgggcacgc tatatgcaag acatgtcaat 300 gctggaagca cgggaccaat aaaaagcacc attagaatct ac 342 <210> 6 <211> 147 <212> DNA <213> Artificial Sequence <220> <223> cDNA of Coxsackievirus B partial VP1 region <400> 6 tacttcaaac ctaagcatgt caaagcgtgg atacctagac cacctagact ctgccaatac 60 gagaaggcaa agaacgtgaa cttccaaccc agcggagtta ccactactag gcaaagcatc 120 actacaatga caaatacggg cgcattt 147 <210> 7 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Primer for Amplifying Coxsackievirus B VP1 region <400> 7 cagcaaagct tgaccatggg ccccgtggaa gacgcgataa c 41 <210> 8 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Primer for Amplifying Coxsackievirus B VP1 region <400> 8 cctgactcga gttaaaatgc gcccgtattt gtcattg 37 <210> 9 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Primer for Amplifying Coxsackievirus B VP2 region <400> 9 tcactaagct tgaccatgtc ccccacagta gaggagtgc 39 <210> 10 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Primer for Amplifying Coxsackievirus B VP2 region <400> 10 atggtctcga gttactggtg cccggccaaa cgtagtc 37 <210> 11 <211> 40 <212> DNA <213> Artificial Sequence <220> Primer for Amplifying Coxsackievirus B VP3 region <400> 11 cgtttaagct tgaccatggg cttaccaacc atgaacactc 40 <210> 12 <211> 38 <212> DNA <213> Artificial Sequence <220> Primer for Amplifying Coxsackievirus B VP3 region <400> 12 tcttcctcga gttactggaa aaagttttgc tgcgaaat 38 <210> 13 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Primer for Amplifying Coxsackievirus B partial VP1 region <400> 13 cagcaggatc cgaccatggg ccccgtggaa gacgcgataa c 41 <210> 14 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Primer for Amplifying Coxsackievirus B partial VP1 region <400> 14 gttgactcga gttaagtact tgttatgaca aacgtc 36 <210> 15 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Primer for Amplifying Coxsackievirus B partial VP1 region <400> 15 acgttggatc cgaccatgac tcaacagccc tcaaccacac 40 <210> 16 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Primer for Amplifying Coxsackievirus B partial VP1 region <400> 16 tgcttctcga gttagtagat tctaatggtg cattttattg 40 <210> 17 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Primer for Amplifying Coxsackievirus B partial VP1 region <400> 17 aaaagggatc cgaccatgta cttcaaacct aagcatgtc 39

Claims (10)

A nucleic acid sequence for preparing a vaccine for preventing a disease due to infection with coxsackievirus B selected from the group consisting of SEQ ID NOs: 1 to 6. The method of claim 1, SEQ ID NO: 1 to SEQ ID NO: 6 nucleic acid sequence for vaccine production, characterized in that obtained from c-DNA of CVB3 H strain. Recombinant plasmid expression vector for the preparation of a vaccine for coxsackievirus B, wherein a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6 in the sequences constituting the Coxsackievirus B genome is inserted into at least one cloning site. . The method of claim 3, wherein The recombinant plasmid expression vector includes a poly (A) adenylated signal site (BA) of bovine growth hormone (BGH), an antibiotic resistance gene and ColE1 origin of replication and a heterologous transcriptional regulatory region in addition to the at least one or more cloning sites. Recombinant plasmid expression vector for vaccine preparation of virus B. The method of claim 3, wherein The recombinant plasmid expression vector is a recombinant plasmid expression vector for vaccine preparation of coxsackievirus B having accession number KACC 95021. The method of claim 3, wherein The recombinant plasmid expression vector is a recombinant plasmid expression vector for vaccine preparation of coxsackievirus B having accession number KACC 95022. The method of claim 3, wherein The recombinant plasmid expression vector is a recombinant plasmid expression vector for producing a vaccine of coxsackievirus B having accession number KACC 95023. The method of claim 3, wherein The recombinant plasmid expression vector is a recombinant plasmid expression vector for vaccine preparation of coxsackievirus B having accession number KACC 95024. The method of claim 3, wherein The recombinant plasmid expression vector is a recombinant plasmid expression vector for vaccine preparation of coxsackievirus B having accession number KACC 95025. The method of claim 3, wherein The recombinant plasmid expression vector is a recombinant plasmid expression vector for vaccine preparation of coxsackievirus B having accession number KACC 95026.