KR102047072B1 - WHOLE PROTEIN GENE OF MERS-CoV NUCLEOPROTEIN AND VACCINE COMPOSITION FOR PREVENTING INFECTION OF MERS-CoV COMPRISING THE SAME - Google Patents

Abstract

The present invention relates to a full-length protein gene of MERS-CoV nuclear protein and a vaccine composition for preventing MERS-CoV infection comprising the same. The full-length protein gene antigen of MERS-CoV nuclear protein represented by SEQ ID NO: 1 according to the present invention can be mass produced in E. coli. In addition, the animal model inoculated with the antigen challenge shows the ability to protect against MERS-CoV, it can be widely used to prevent MERS-CoV.

Description

WHOLE PROTEIN GENE OF MERS-CoV NUCLEOPROTEIN AND VACCINE COMPOSITION FOR PREVENTING INFECTION OF MERS-CoV COMPRISING THE SAME}

The present invention relates to a full-length protein gene of MERS coronavirus nucleoprotein and a vaccine composition for preventing MERS coronavirus infection comprising the same.

Middle east respiratory syndrome (MERS) is a severe acute respiratory disease caused by MERS coronavirus infection. MERS coronavirus (MERS-CoV) is a new beta coronavirus newly discovered in Saudi Arabia in 2012 (Azhar EI, et al., 2014). The MERS-CoV was named by the International Virus Classification Committee in May 2013 and was formerly called the new coronavirus, SARS-like virus, Middle East SAR, or Saudi SARS. The path of MERS-CoV infection is known to spread from bats to camels and humans. In addition, MERS-CoV can spread between humans and has a 40% mortality rate.

The genome of MERS-CoV is non-segmented and is about 30 kb in size, encoding 11 proteins. The eleven proteins consist of two replicating polyproteins, four structural proteins, and five non-structural proteins.

Currently, based on the study of SARS coronavirus, a MERS-CoV vaccine is being developed in a primate model. The MERS coronavirus vaccine under development is a vaccine based on the recombinant receptor binding domain (RBD) and has been shown to increase humoral and cellular immune responses through inoculation. In addition, various MERS virus vaccines are under development, but there are no commercialized vaccines to date, and some pharmaceutical companies are currently developing MERS vaccines, but as a developmental stage, validation through preclinical and clinical trials It is a necessary situation.

In addition, to date, antiviral agents for the treatment of MERS-CoV have not been developed so far, the situation is symptomatic treatment to alleviate the clinical symptoms. Chloroquine, chlorpromazine, loperamide, and lopinavir have been reported to inhibit MERS-CoV proliferation in vitro, but it is not known whether it is effective in real patients.

 Azhar EI, Hashem AM, El-Kafrawy SA, Sohrab SS, Aburizaiza AS, Farraj SA, Hassan AM, Al-Saeed MS, Jamjoom GA, Madani TA. 2014. Detection of the middle east respiratory syndrome coronavirus genome in an air sample originating from a camel barn owned by an infected patient. m Bio 5 (4): e01450-14. doi: 10.1128 / mBio.01450-14.

It is an object of the present invention to provide a vaccine composition for the prevention of MERS-CoV infection.

In order to achieve the above object, the present invention provides an expression vector loaded with a polynucleotide encoding the full-length protein or fragment thereof of the MERS-CoV nucleoprotein.

The present invention also provides a vaccine composition for preventing MERS-CoV infection comprising the expression vector.

In addition, the present invention comprises the steps of (a) introducing the expression vector into the subject as an antigen; And (b) provides a method for producing a MERS-CoV antibody comprising recovering the antibody from the subject.

The present invention also provides a pharmaceutical composition for preventing or treating a disease caused by MERS-CoV infection comprising the antibody.

The present invention also provides a kit for diagnosing MERS-CoV infection comprising an antibody that specifically binds to a DNA gene antigen of MERS-CoV nucleoprotein.

The full-length protein gene antigen of MERS-CoV nuclear protein represented by SEQ ID NO: 1 according to the present invention can be mass produced in E. coli. In addition, the animal model inoculated with the antigen challenge shows the ability to protect against MERS-CoV, it can be widely used to prevent MERS-CoV.

1 is a schematic diagram of a vector used for the production of a DNA gene vaccine of MERS-CoV nucleoprotein.
Figure 2 is a schematic diagram of the expression vector used for the DNA gene vaccine of the MERS-CoV nucleoprotein.
3 shows immunization of nucleoprotein DNA gene vaccines directly to hDPP4 gene-transplanted mice, and shows the effect of antibody formation using purified protein antigens through ELISA experiments.
Figures 4a and 4b is a group of mice not vaccinated with the MERS-CoV nucleoprotein gene vaccine inoculated MERS-CoV, to confirm the weight loss and survival rate according to the hDPP4 gene transplantation to confirm the protection against the virus .
4C and 4D show that the hDPP4 gene-transplanted mouse group inoculated with the MERS-CoV nuclear protein gene vaccine was challenged with MERS-CoV to compare the protection effect against the virus with DNA gene vaccines of the spike protein region. It is shown.

In one aspect, the present invention provides an expression vector loaded with a polynucleotide encoding a full-length protein or fragment thereof of the mers coronavirus nucleoprotein.

The term "mers coronavirus" (hereinafter referred to as MERS-CoV), as used herein, refers to the Middle East Respiratory Syndrome-Corona virus (Middle East Respiratory), which has been identified as the cause of the development of severe acute respiratory diseases first discovered in the Arabian Peninsula. Syndrome-Corona Virus). In addition, MERS-CoV was initially called Human Coronavirus-EMC (Erasmus Medical Center; hCoV-EMC). The MERS-CoV belongs to beta coronavirus lineage 2c and causes severe respiratory disease similar to Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), which appeared in China. MERS-CoV contains four accessory proteins (e.g., accessory proteins 3, 4a, 4b, 5), two open proteins (Open reading frame 1a, and 1b) and four structural proteins (Spike; S, Envelope; E, Nucleocapsid; N, and Membrane; M) and bind to human host cell receptor dipeptidyl peptidase 4 (DPP4) via the Spike (S) protein in structural proteins.

As used herein, the term “nucleoprotein” is a structural protein that wraps a viral gene with a protein membrane. This is one of the major determinants of species specificity.

The MERS-CoV nucleoprotein consists of 413 amino acids represented by SEQ ID NO: 1. It is located at 28,566th to 29,807th nucleotide sequence constituting human beta coronavirus 2c EMC / 2012 (GenBank: JX869059.2). The polynucleotide encoding the MERS-CoV nucleoprotein may have a nucleotide sequence of SEQ ID NO: 2. In addition, the polynucleotide encoding the nuclear protein may have a nucleotide sequence of SEQ ID NO: 3. In this case, the fragment of the MERS-CoV nucleoprotein may be any one selected from the group consisting of the N-terminal domain, C-terminal domain and NC fusion protein of the MERS-CoV nucleoprotein.

The N-terminal domain of the MERS-CoV nucleoprotein may be a polypeptide having an amino acid sequence of positions 10 to 207 of SEQ ID NO: 1. Specifically, the N-terminal domain is 1 to 198, 10 to 180, 20 to 160, 30 to 140 of the amino acid sequence of position 10 to position 207 to position 207 in SEQ ID NO: 1, 50 to 120 or 70 to 100 contiguous amino acid sequences, specifically 50 to 198 contiguous amino acid sequences.

More specifically, the N-terminal domain of the MERS-CoV nucleoprotein may be a polypeptide having an amino acid sequence of SEQ ID NO: 4. The N-terminal domain is a polypeptide identical to SEQ ID NO: 4 as well as a polypeptide whose amino acid is substituted by conservative substitution, 80-99%, 85-99%, preferably 90-99% sequence homology thereof It may include all polypeptides having a.

Further, in the present invention, the N-terminal domain fragment is 1 to 134, 10 to 130, 20 to 110, 30 to 1 of the amino acid sequence of position 1 to position 134 of the amino acid at position 1 of SEQ ID NO: 4 90, 40-70 or 50-60 contiguous amino acid sequences, specifically 50-134 contiguous amino acid sequences.

In addition, the C-terminal domain of the MERS-CoV nucleoprotein may be a polypeptide having an amino acid sequence of positions 208 to 362 of SEQ ID NO: 1. Specifically, the C-terminal domain is 1 to 155, 10 to 140, 20 to 120, 30 to 100 of the amino acid sequence of the amino acid at position 208 to position 362 of SEQ ID NO: 1, 40 to 80 or 50 to 60 contiguous amino acid sequences, specifically 50 to 155 contiguous amino acid sequences.

More specifically, the C-terminal domain of the MERS-CoV nucleoprotein may be a polypeptide having an amino acid sequence of SEQ ID NO: 5. The C-terminal domain is a polypeptide identical to SEQ ID NO: 5 as well as a polypeptide whose amino acid is substituted by conservative substitution, 80-99%, 85-99%, preferably 90-99% sequence homology thereof. It may include all polypeptides having a.

In addition, in the present invention, the C-terminal domain fragment is 1 to 117, 10 to 100, 20 to 80, 30 to 1 of the amino acid sequence of position 1 to position 117 of SEQ ID NO: 5 It may be 60 or 40 to 50 contiguous amino acid sequences, specifically 50 to 117 contiguous amino acid sequences.

The NC fusion protein of the MERS-CoV nucleoprotein means a protein in which an N-terminal domain and a C-terminal domain are bound, and the N-terminal domain and the C-terminal domain are as described above. In addition, the NC fusion protein of the MERS-CoV nucleoprotein can be variously combined into an N-terminal domain fragment and a C-terminal domain fragment.

Specifically, the NC fusion protein of the MERS-CoV nucleoprotein may be a polypeptide having an amino acid sequence of SEQ ID NO: 6. The NC fusion protein has a polypeptide identical to SEQ ID NO: 6 as well as a polypeptide whose amino acid is substituted by conservative substitution, and sequence homology of 80 to 99%, 85 to 99%, preferably 90 to 99% It may include all polypeptides having.

As used herein, the term "conservative substitution" means that one class of amino acids is replaced with another amino acid of the same class. Conservative substitutions do not alter the structure, function or both of the polypeptides. Classes of amino acids for the purpose of conservative substitutions are hydrophobic (eg Met, Ala, Val, Leu), neutral hydrophilic (eg Cys, Ser, Thr), acidic (eg Asp, Glu) , Basic (eg Asn, Gln, His, Lys, Arg), conformational disruptors (eg Gly, Pro) and aromatics (eg Trp, Tyr, Phe) .

As used herein, the term "polynucleotide-loaded expression vector" refers to a vector obtained by PCR digesting a fragment obtained by PCR amplifying the full-length protein or fragment thereof of the MERS-CoV nuclear protein represented by SEQ ID NO: 1. It means that the insertion is made to express the genetic material in the cell.

The vector can be a viral or nonviral vector. The viral vector may be any one selected from the group consisting of adenovirus, adeno-associated virus, helper-dependent adenovirus and retroviral vector. In addition, the nonviral vector may be a plasmid, liposomes, or the like.

In one embodiment of the present invention, the pGX-10 vector prepared by modifying the pMT3 vector was cut using EcoRI and XbaI restriction enzymes, and then a fragment obtained by PCR amplifying the MERS-CoV nuclear protein represented by SEQ ID NO: 1 was inserted. Thus, an expression vector loaded with a polynucleotide encoding the MERS-CoV nucleoprotein was prepared.

In another aspect, the present invention provides a vaccine composition for preventing MERS-CoV infection comprising an expression vector loaded with a polynucleotide encoding a full-length protein or fragment thereof of the MERS-CoV nucleoprotein.

As used herein, the term "vaccine composition" means a composition containing at least one immunologically active component that induces an immunological response in an animal.

The vaccine composition may be in any form known in the art, such as, but not limited to, solid forms suitable for liquid or injectable forms or suspensions. Such formulations may also be emulsified or encapsulated in liposomes or soluble glass, or prepared in the form of aerosols or sprays. They can also be included in transdermal patches. In the case of solutions or injections, propylene glycol and, if necessary, may contain an amount of sodium chloride sufficient to prevent hemolysis (eg about 1%).

The vaccine composition of the present invention may comprise a pharmaceutically acceptable carrier or diluent. Suitable carriers for the vaccine are known to those skilled in the art and may include, but are not limited to, proteins, sugars, and the like. Such carriers can be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous carriers may be propylene glycol, polyethylene glycol, edible oils such as olive oil, and injectable organic esters such as ethyl oleate.

Aqueous carriers may comprise water, alcohol / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral carriers may include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactically treated Ringer's or fixed oils. Intravenous carriers may include electrolyte supplements such as those based on Ringer's dextrose, liquids and nutritional supplements, and the like. Preservatives and other additives such as antimicrobial agents, antioxidants, chelating agents, inert gases and the like may further be present. Preferred preservatives may include formalin, thimerosal, neomycin, polymyxin B and amphotericin B.

In addition, the vaccine composition may further comprise an adjuvant (adjuvant, immunoadjuvant). The adjuvant may refer to a compound or mixture that enhances the immune response and / or promotes the rate of absorption after inoculation and may include any absorption-promoting agent. Acceptable adjuvants include Freund's complete adjuvant, Freund's incomplete adjuvant, saponins, mineral gels such as aluminum hydroxide, surfactants such as lysocithin, pluron polyols, polyanions, peptides, oils or hydrocarbon emulsions, keyhole rimpets It may include, but is not limited to, mocyanine, dinitrophenol, and the like.

The vaccine composition of the present invention can be administered via any one of the routes of administration selected from the group consisting of oral, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous or nasal, preferably administered by injection. Do.

The vaccine composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term “pharmaceutically effective amount” means an amount sufficient to exhibit a vaccine effect, and an amount that does not cause side effects or serious or excessive immune responses, and the level of an effective dose is a disorder to be treated. The severity of the disorder, the activity of the specific compound, the route of administration, the rate of removal of the protein, the duration of treatment, the drug used in combination with or concurrently with the protein, the age, weight, sex, diet, general health status and medical conditions of the individual It can depend on various factors, including factors.

In one embodiment of the present invention, in the efficacy test of MERS-CoV nucleoprotein gene DNA vaccine, DNA gene antigen of MERS-CoV nucleoprotein acts as a vaccine to effectively induce cellular immune responses and immune antibodies in the individual and , MERS-CoV was effectively defended. Through this, it was confirmed that the gene antigen of MERS-CoV nuclear protein can be used as a vaccine for preventing MERS-CoV.

In another aspect, the present invention provides a method for preparing a human body, comprising the steps of: (a) introducing into an individual an expression vector loaded with a polynucleotide encoding a full-length protein or fragment thereof of the MERS-CoV nucleoprotein; And (b) recovering the antibody from the individual, thereby providing an antibody manufacturing method that specifically binds to the MERS-CoV nucleoprotein.

The term “antigen” refers to a protein expressed by a virus as a component capable of generating immune function among the components of the virus.

In addition, the introduction of the expression vector may be carried out by injection into intradermal, subcutaneous or muscle tissue.

The term "antibody" is a protein produced by stimulation of an antigen in the immune system, which is a protein that specifically binds to a specific antigen and floats lymph and blood to generate an antigen-antibody reaction. Antigen-antibody responses have high specificity for each antigen. This is because when antibodies are made in B cells of lymphocytes, antibodies produced by specific antigens do not, in principle, react with other antigens. Such high specificity is used for the examination of immunity, allergy, various diseases and types and types of infections.

As used herein, the term “an antibody that specifically binds to a MERS-CoV nucleoprotein” refers to an N-terminal domain, a C-terminal domain, an N-terminal domain, and a C-terminal domain to which the NERS-CoV nucleoprotein binds. An antibody that specifically binds to a fusion protein or full-length protein.

In one embodiment of the present invention, the gene of MERS-CoV nucleoprotein acted as an antigen that induces immune antibody formation when administered to a subject.

The present invention also provides a pharmaceutical composition for preventing or treating a disease caused by MERS-CoV infection comprising the antibody.

The disease caused by MERS-CoV infection may be a respiratory disease, and severe respiratory symptoms such as high fever, cough or difficulty breathing may occur after 38 days after the virus infection. Digestive symptoms, such as diarrhea or constipation in some cases, chronic diseases or immunocompromised patients, such as pneumonia or acute renal failure may be accompanied by death.

The present invention provides a kit for diagnosing MERS-CoV infection comprising an antibody that specifically binds to a MERS-CoV nucleoprotein.

The antibody that specifically binds to the MERS-CoV nucleoprotein is as described above, and in one embodiment, the MERS-CoV nucleoprotein having the amino acid sequence of SEQ ID NO: 1 may specifically bind to the antibody thereto. Can be applied to MERS-CoV infection diagnostic kits.

Hereinafter, the present invention will be described in detail by Examples and Experimental Examples. However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.

Example  One. MERS - CoV  Nuclear Protein Gene DNA Vaccine Preparation

The nuclear protein of MERS-CoV used in the following experimental example was prepared by obtaining a nucleotide sequence of MERS-CoV (Human betacoronavirus 2c EMC / 2012) through USA NIH NCBI, and synthesized through Bioneer Corporation. The nucleotide sequence is represented by SEQ ID NO: 1.

The MERS-CoV nucleoprotein represented by SEQ ID NO: 1 is a vector pMT3 (Sambrook, et al., Molecular cloning 2nd Ed., Vol 3: 16.20; Kaufman RJ, et al., Mol. Cell Biol. 9,946-958, (1989 PGX-10 vector prepared by modifying)) was cut using EcoRI and XbaI restriction enzymes (FIG. 1), and then prepared by inserting PCR amplified fragments of the nuclear protein full-length gene (FIG. 2).

Experimental Example  One. MERS - CoV  Confirmation of antibody production of nuclear protein DNA gene

In order to confirm whether the DNA gene antigen of the MERS-CoV nucleoprotein prepared in Example 1 effectively induces antibody production, the effect of antibody production was confirmed.

First, the purified pGX-MCoV-NC gene antigen of the nuclear protein was immunized with electroporation (50V) at 10 ug per horse to the thigh muscles of 6 to 8 week old mice (hDPP4 transgenic Mouse C57bl / 6N). . Three weeks after the primary immunization, a second boost was performed. Three weeks later, mice were bled and final serum isolated to perform ELISA test experiments. At this time, in the ELISA test, the nuclear protein antigen was used 100 ng per well. In addition, the immune sera were diluted in 1 × PBS at a ratio of 1: 100, 1: 500, 1: 1,000, 1: 5,000, 1: 10,000, 1: 50,000, 1: 100,000 and 1: 500,000, and the antibody was purified for secondary confirmation. Mouse IgG-HRP (ABC5001) was used at a 1: 5,000 dilution. Color development was detected at an optical density of 405 nm to confirm whether an immune antibody was generated first, as shown in FIG. 3.

As shown in FIG. 3, all of the nucleoprotein genes showed an immune antibody formation effect in the mice used in the experiment. This means that the genetic vaccine of the nuclear protein can act as an antigen in the individual to effectively induce the antibody.

Experimental Example  2. MERS - CoV  Confirmation of the efficacy of the nuclear protein gene vaccine

In order to confirm the protective ability after challenge inoculation of the gene antigen of MERS-CoV nucleoprotein, virus challenge inoculation efficacy evaluation was performed. As a control group, a group not vaccinated was used. This is shown in Figures 4a and 4b. After challenge inoculation with the mouse nasal cavity of the MERS-CoV nucleoprotein gene of 5 LD50, body weight and survival rate were observed for 2 weeks. For comparison, the receptor binding domain (RRD), S1, and NTD (N-terminal domain) domains of the MERS-CoV spike protein were prepared using the same vector of Example 1, followed by analysis of the gene antigen of MERS-CoV nuclear protein. The defense ability was confirmed. This is shown in Figures 4c and 4d.

As shown in Figures 4c and 4d, the DNA gene antigen of the MERS-CoV nucleoprotein acted as a vaccine to effectively induce cellular immune responses and immune antibodies in the individual, and effectively defended against MERS-CoV. Through this, it was confirmed that the gene antigen of MERS-CoV nuclear protein can be used as a vaccine for preventing MERS-CoV.

<110> Korea Research Institute of Bioscience and Biotechnology <120> WHOLE PROTEIN GENE OF MERS-CoV NUCLEOPROTEIN AND VACCINE          COMPOSITION FOR PREVENTING INFECTION OF MERS-CoV COMPRISING THE          SAME <130> FPD / 201711-0087 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 413 <212> PRT <213> Artificial Sequence <220> <223> MERS CoV nucleoprotein <400> 1 Met Ala Ser Pro Ala Ala Pro Arg Ala Val Ser Phe Ala Asp Asn Asn   1 5 10 15 Asp Ile Thr Asn Thr Asn Leu Ser Arg Gly Arg Gly Arg Asn Pro Lys              20 25 30 Pro Arg Ala Ala Pro Asn Asn Thr Val Ser Trp Tyr Thr Gly Leu Thr          35 40 45 Gln His Gly Lys Val Pro Leu Thr Phe Pro Pro Gly Gln Gly Val Pro      50 55 60 Leu Asn Ala Asn Ser Thr Pro Ala Gln Asn Ala Gly Tyr Trp Arg Arg  65 70 75 80 Gln Asp Arg Lys Ile Asn Thr Gly Asn Gly Ile Lys Gln Leu Ala Pro                  85 90 95 Arg Trp Tyr Phe Tyr Tyr Thr Gly Thr Gly Pro Glu Ala Ala Leu Pro             100 105 110 Phe Arg Ala Val Lys Asp Gly Ile Val Trp Val His Glu Asp Gly Ala         115 120 125 Thr Asp Ala Pro Ser Thr Phe Gly Thr Arg Asn Pro Asn Asn Asp Ser     130 135 140 Ala Ile Val Thr Gln Phe Ala Pro Gly Thr Lys Leu Pro Lys Asn Phe 145 150 155 160 His Ile Glu Gly Thr Gly Gly Asn Ser Gln Ser Ser Ser Arg Ala Ser                 165 170 175 Ser Val Ser Arg Asn Ser Ser Arg Ser Ser Ser Gln Gly Ser Arg Ser             180 185 190 Gly Asn Ser Thr Arg Gly Thr Ser Pro Gly Pro Ser Gly Ile Gly Ala         195 200 205 Val Gly Gly Asp Leu Leu Tyr Leu Asp Leu Leu Asn Arg Leu Gln Ala     210 215 220 Leu Glu Ser Gly Lys Val Lys Gln Ser Gln Pro Lys Val Ile Thr Lys 225 230 235 240 Lys Asp Ala Ala Ala Ala Lys Asn Lys Met Arg His Lys Arg Thr Ser                 245 250 255 Thr Lys Ser Phe Asn Met Val Gln Ala Phe Gly Leu Arg Gly Pro Gly             260 265 270 Asp Leu Gln Gly Asn Phe Gly Asp Leu Gln Leu Asn Lys Leu Gly Thr         275 280 285 Glu Asp Pro Arg Trp Pro Gln Ile Ala Glu Leu Ala Pro Thr Ala Ser     290 295 300 Ala Phe Met Gly Met Ser Gln Phe Lys Leu Thr His Gln Asn Asn Asp 305 310 315 320 Asp His Gly Asn Pro Val Tyr Phe Leu Arg Tyr Ser Gly Ala Ile Lys                 325 330 335 Leu Asp Pro Lys Asn Pro Asn Tyr Asn Lys Trp Leu Glu Leu Leu Glu             340 345 350 Gln Asn Ile Asp Ala Tyr Lys Thr Phe Pro Lys Lys Glu Lys Lys Gln         355 360 365 Lys Ala Pro Lys Glu Glu Ser Thr Asp Gln Met Ser Glu Pro Pro Lys     370 375 380 Glu Gln Arg Val Gln Gly Ser Ile Thr Gln Arg Thr Arg Thr Arg Pro 385 390 395 400 Ser Val Gln Pro Gly Pro Met Ile Asp Val Asn Thr Asp                 405 410 <210> 2 <211> 1242 <212> DNA <213> Artificial Sequence <220> <223> MERS CoV nucleoprotein <400> 2 atggcatccc ctgctgcacc tcgtgctgtt tcctttgccg ataacaatga tataacaaat 60 acaaacctat ctcgaggtag aggacgtaat ccaaaaccac gagctgcacc aaataacact 120 gtctcttggt acactgggct tacccaacac gggaaagtcc ctcttacctt tccacctggg 180 cagggtgtac ctcttaatgc caattctacc cctgcgcaaa atgctgggta ttggcggaga 240 caggacagaa aaattaatac cgggaatgga attaagcaac tggctcccag gtggtacttc 300 tactacactg gaactggacc cgaagcagca ctcccattcc gggctgttaa ggatggcatc 360 gtttgggtcc atgaagatgg cgccactgat gctccttcaa cttttgggac gcggaaccct 420 aacaatgatt cagctattgt tacacaattc gcgcccggta ctaagcttcc taaaaacttc 480 cacattgagg ggactggagg caatagtcaa tcatcttcaa gagcctctag cttaagcaga 540 aactcttcca gatctagttc acaaggttca agatcaggaa actctacccg cggcacttct 600 ccaggtccat ctggaatcgg agcagtagga ggtgatctac tttaccttga tcttctgaac 660 agactacaag cccttgagtc tggcaaagta aagcaatcgc agccaaaagt aatcactaag 720 aaagatgctg ctgctgctaa aaataagatg cgccacaagc gcacttccac caaaagtttc 780 aacatggtgc aagcttttgg tcttcgcgga ccaggagacc tccagggaaa ctttggtgat 840 cttcaattga ataaactcgg cactgaggac ccacgttggc cccaaattgc tgagcttgct 900 cctacagcca gtgcttttat gggtatgtcg caatttaaac ttacccatca gaacaatgat 960 gatcatggca accctgtgta cttccttcgg tacagtggag ccattaaact tgacccaaag 1020 aatcccaact acaataagtg gttggagctt cttgagcaaa atattgatgc ctacaaaacc 1080 ttccctaaga aggaaaagaa acaaaaggca ccaaaagaag aatcaacaga ccaaatgtct 1140 gaacctccaa aggagcagcg tgtgcaaggt agcatcactc agcgcactcg cacccgtcca 1200 agtgttcagc ctggtccaat gattgatgtt aacactgatt ag 1242 <210> 3 <211> 1242 <212> DNA <213> Artificial Sequence <220> <223> MERS-CoV DNA vaccine sequence <400> 3 atggcttctc ctgccgctcc tagagccgtg tctttcgccg acaacaacga catcaccaac 60 accaacctgt ccagaggcag gggcagaaac cccaagccta gagctgcccc taacaacacc 120 gtgtcctggt acaccggcct gacacagcat ggaaaggtgc ccctgacctt tccacctgga 180 cagggcgttc cactgaacgc caattctacc cctgctcaga acgccggcta ttggcggaga 240 caggaccgga agatcaacac cggcaacggc atcaagcagc tggcccctag atggtacttc 300 tactacaccg gcaccggacc tgaggccgct ctgcctttta gagctgtgaa ggacggcatc 360 gtgtgggtgc acgaagatgg cgctaccgac gctccttcta ccttcggcac ccggaatcct 420 aacaacgact ccgccatcgt gacccagttt gcccctggaa caaagctgcc caagaacttc 480 cacatcgaag gcaccggcgg caactcccag agttcctcta gagcctcttc tctgtcccgg 540 aactcctctc ggtctagctc ccagggctct agatccggca acagcaccag aggcacaagc 600 cctggaccat ctggaatcgg agctgtcggc ggcgatctgc tgtatctgga cctgctgaat 660 agactgcagg ccctggaatc cggcaaagtg aagcagtccc agcctaaagt gatcaccaag 720 aaggatgccg ccgctgccaa gaacaagatg cggcacaagc ggacctccac caagtccttc 780 aatatggtgc aggccttcgg cctgagaggc cctggggatc tgcagggcaa tttcggagat 840 ctgcagctga acaagctggg caccgaggat cctagatggc cccagattgc tgagctggct 900 cctaccgcct ctgccttcat gggcatgtcc cagttcaagc tgacccacca gaacaacgac 960 gaccacggca accccgtgta cttcctgaga tactccggcg ccatcaagct ggaccctaag 1020 aaccccaact acaacaagtg gctggaactg ctggaacaga acatcgacgc ctacaagaca 1080 ttccccaaga aagagaagaa gcagaaggcc cctaaagagg aatccaccga ccagatgtcc 1140 gagcctccta aagaacagag agtgcagggc agcatcaccc agcggacaag aaccagacct 1200 tccgtgcagc ccggacctat gatcgacgtg aacaccgact ga 1242 <210> 4 <211> 134 <212> PRT <213> Artificial Sequence <220> <223> N-terminal domain of MERS CoV nucleoprotein <400> 4 Ala Pro Asn Asn Thr Val Ser Trp Tyr Thr Gly Leu Thr Gln His Gly   1 5 10 15 Lys Val Pro Leu Thr Phe Pro Pro Gly Gln Gly Val Pro Leu Asn Ala              20 25 30 Asn Ser Thr Pro Ala Gln Asn Ala Gly Tyr Trp Arg Arg Gln Asp Arg          35 40 45 Lys Ile Asn Thr Gly Asn Gly Ile Lys Gln Leu Ala Pro Arg Trp Tyr      50 55 60 Phe Tyr Tyr Thr Gly Thr Gly Pro Glu Ala Ala Leu Pro Phe Arg Ala  65 70 75 80 Val Lys Asp Gly Ile Val Trp Val His Glu Asp Gly Ala Thr Asp Ala                  85 90 95 Pro Ser Thr Phe Gly Thr Arg Asn Pro Asn Asn Asp Ser Ala Ile Val             100 105 110 Thr Gln Phe Ala Pro Gly Thr Lys Leu Pro Lys Asn Phe His Ile Glu         115 120 125 Gly Thr Gly Gly Asn Ser     130 <210> 5 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> C-terminal domain of MERS CoV nucleoprotein <400> 5 Ala Lys Asn Lys Met Arg His Lys Arg Thr Ser Thr Lys Ser Phe Asn   1 5 10 15 Met Val Gln Ala Phe Gly Leu Arg Gly Pro Gly Asp Leu Gln Gly Asn              20 25 30 Phe Gly Asp Leu Gln Leu Asn Lys Leu Gly Thr Glu Asp Pro Arg Trp          35 40 45 Pro Gln Ile Ala Glu Leu Ala Pro Thr Ala Ser Ala Phe Met Gly Met      50 55 60 Ser Gln Phe Lys Leu Thr His Gln Asn Asn Asp Asp His Gly Asn Pro  65 70 75 80 Val Tyr Phe Leu Arg Tyr Ser Gly Ala Ile Lys Leu Asp Pro Lys Asn                  85 90 95 Pro Asn Tyr Asn Lys Trp Leu Glu Leu Leu Glu Gln Asn Ile Asp Ala             100 105 110 Tyr Lys Thr Phe Pro         115 <210> 6 <211> 251 <212> PRT <213> Artificial Sequence <220> <223> NC fusion protein of MERS CoV nucleoprotein <400> 6 Ala Pro Asn Asn Thr Val Ser Trp Tyr Thr Gly Leu Thr Gln His Gly   1 5 10 15 Lys Val Pro Leu Thr Phe Pro Pro Gly Gln Gly Val Pro Leu Asn Ala              20 25 30 Asn Ser Thr Pro Ala Gln Asn Ala Gly Tyr Trp Arg Arg Gln Asp Arg          35 40 45 Lys Ile Asn Thr Gly Asn Gly Ile Lys Gln Leu Ala Pro Arg Trp Tyr      50 55 60 Phe Tyr Tyr Thr Gly Thr Gly Pro Glu Ala Ala Leu Pro Phe Arg Ala  65 70 75 80 Val Lys Asp Gly Ile Val Trp Val His Glu Asp Gly Ala Thr Asp Ala                  85 90 95 Pro Ser Thr Phe Gly Thr Arg Asn Pro Asn Asn Asp Ser Ala Ile Val             100 105 110 Thr Gln Phe Ala Pro Gly Thr Lys Leu Pro Lys Asn Phe His Ile Glu         115 120 125 Gly Thr Gly Gly Asn Ser Ala Lys Asn Lys Met Arg His Lys Arg Thr     130 135 140 Ser Thr Lys Ser Phe Asn Met Val Gln Ala Phe Gly Leu Arg Gly Pro 145 150 155 160 Gly Asp Leu Gln Gly Asn Phe Gly Asp Leu Gln Leu Asn Lys Leu Gly                 165 170 175 Thr Glu Asp Pro Arg Trp Pro Gln Ile Ala Glu Leu Ala Pro Thr Ala             180 185 190 Ser Ala Phe Met Gly Met Ser Gln Phe Lys Leu Thr His Gln Asn Asn         195 200 205 Asp Asp His Gly Asn Pro Val Tyr Phe Leu Arg Tyr Ser Gly Ala Ile     210 215 220 Lys Leu Asp Pro Lys Asn Pro Asn Tyr Asn Lys Trp Leu Glu Leu Leu 225 230 235 240 Glu Gln Asn Ile Asp Ala Tyr Lys Thr Phe Pro                 245 250

Claims (13)

A DNA vaccine composition for preventing MERS coronavirus infection comprising an expression vector loaded with a polynucleotide consisting of a nucleotide sequence encoding a full length protein of MERS coronavirus nucleoprotein. The method of claim 1,
The full-length protein of the mers coronavirus nucleoprotein is a polypeptide having an amino acid sequence of SEQ ID NO: 1, characterized in that the DNA vaccine composition for preventing mers coronavirus infection. The method of claim 2,
The polypeptide is characterized in that the encoded by the nucleotide sequence of SEQ ID NO: 2, 3, mers coronavirus infection prevention DNA vaccine composition. delete delete delete delete delete delete delete delete delete delete

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