KR100508499B1 - Diagnosis Kits for Hemorrhagic Fever with Renal Syndrome Comprising Nucleocapsid Protein from Hantann virus Expressed in Plants - Google Patents

Abstract

The present invention provides a novel polynucleotide encoding a nucleocapsid protein of a hantan virus and a plant expression vector comprising the same. The present invention also provides a method for producing a transgenic plant expressing a nucleocapsid protein of a hantan virus and a plant transformant. The present invention provides a method for producing a nucleocapsid protein of hantan virus and a nucleocapsid protein of hantan virus. On the other hand, the present invention provides a kit for antibody diagnosis of nephrotic bleeding fever and a method for measuring the antibody of nephrotic bleeding fever. According to the present invention, a recombinant nucleocapsid protein of Hantan virus exhibiting excellent antigenicity at low cost can be obtained, and when using this, nephrotic bleeding fever can be diagnosed with excellent sensitivity and specificity.

Description

Diagnosis Kits for Hemorrhagic Fever with Renal Syndrome Comprising Nucleocapsid Protein from Hantann virus Expressed in Plants}

The present invention relates to a nephrotic hemorrhagic fever diagnostic kit comprising a nucleocapsid protein of a peat virus expressed in a plant, and more particularly, to a novel polynucleotide encoding a nucleocapsid protein of a peat virus, for plant expression comprising the same. The present invention relates to a vector, a plant transformed with the vector, a kit for antibody diagnosis of nephrotic bleeding fever, and a method for measuring antibodies of nephrotic bleeding fever.

The gene of the Hantan virus belonging to the Hantavirus genus of the field of Illegals consists of a three-segment single-line structure consisting of large (L) large, medium (M) middle, and small (S) small segments. It is a small negative-sense RNA virus about 100 nm in size. Each of these RNAs has its own nucleocapsid structure and is wrapped in a lipid membrane made of glycoproteins called G1 and G2 (Schmaljohn et al., Characterization of Hantaan virions, the prototype virus of hemorrhagic fever with renal syndrome.J Infect Dis 148: 1005-1011, 1983). Among structural proteins of Hantan virus, nucleocapsid proteins and G1 and G2 membrane proteins are involved in the immune response. L segment RNA encodes RNA dependent RNA polymerase, M segment RNA encodes G1 and G2 glycoproteins, and S segment RNA encodes nucleocapsid protein.

Hantavirus is well known as a pathogen causing nephrotic bleeding fever on the Eurasian continent and Hantan virus pulmonary syndrome in the Americas. To date, a large number of Hantavirus strains have been found in the blood and tissues of patients with nephrotic hemorrhagic fever and Hantavirus pulmonary syndrome, as well as in many species belonging to the family's natural host animals, the Family Cricertidae and the Family Muridae. It has been isolated from lung tissue of rodents (Chu et al., Serological relationships among viruses in the Hantavirus genus, family Bunyaviridae. Virology 198: 196-204, 1994).

Viruses that have been identified to cause disease in humans to date include the Sin Nombre virus serotype, the Hantaan virus serotype, the Seoul virus serotype, and the Belgrade virus. ) Serotypes, Puumala virus serotypes and Black Creek Canal virus serotypes. Prospect Hill virus serotypes, which are not yet known pathogenic, Thailand serotype and Thottapalayam virus serotype (Xiao et al., Phylogenetic analyses of virus isolates in the genus Hantavirus, family Bunyaviridae. Virology 198: 205-217, 1994) . These nine viral serotypes vary in host animal depending on the virus belonging to each serotype.

Hantan virus, a pathogen of Korean hemorrhagic fever and nephrotic bleeding fever that occurs in Far East Asia, has been isolated from the lung tissue of Apodemus agrarius and the blood and tissue of patients (Lee at al., Isolation of the epilogic agent of Korean hemorrhagic fever. J Infect Dis 137: 298-308, 1978), Seoulvirus, a pathogen of nephrotic hemorrhagic fever that occurs in contact with animal labs and rats in urban areas around the world, was isolated from the lung tissue of mice and rats (Lee et al. , Isolation of Hantaan virus, the etiologic agent of Korean hemorrhagic fever from wild urban rats.J Infect Dis 146: 638-644, 1982), Bell, a pathogen of severe nephrotic bleeding fever in countries of the Balkans basin in Europe. Grade virus was isolated from Apodemus flavicolis and blood of patients (Avsic-xupanc et al., Characterization of Dovrava virus: a hantavirus from Slovenia, Yugoslavi a.Med Virol 38: 132-137, 1992).

It is known that more than 200,000 patients with nephrotic bleeding fever occur every year in the world. In Korea, about 1,000 patients with nephrotic bleeding fever are reported every year. Known.

Currently, the number of patients with nephrotic bleeding fever has been reduced by the development of vaccines, but it is still occurring worldwide. Especially, in the United States, patients with nephrotic bleeding fever continue due to the presence of US troops stationed overseas. We are constantly trying to prevent this from happening. Accordingly, studies on the development of diagnostic kits and diagnostic reagents for diagnosing patients with nephrotic bleeding hemorrhagic fever using antigens are being actively conducted.

Currently, the virus antigen protein used in the development of diagnostic kits and vaccines for the diagnosis of nephrotic bleeding hemorrhagic fever is an antigen obtained by inactivating formalin with a hantan virus obtained by inoculating the brain of a one-day-old mouse in large quantities. The use of such antigens causes the development of nephrotic hemorrhagic fever by infecting animal breeders and people involved in the process of purifying viral antigens by excreting large amounts of virus from infected animals in the process of propagating viruses using animal brain tissue. There is a risk of making. In addition, animal serum tissue-derived protein components remaining in the Hantan virus antigen used in the diagnostic kit may cause the test serum to be misrepresented as pseudo-positive, which causes a problem of inhibiting accurate diagnosis.

Throughout this specification, numerous citations and patent documents are referenced and their citations are indicated. The disclosures of cited documents and patents are incorporated herein by reference in their entirety, so that the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The present inventors obtained a plant transformant by transforming a plant with a nucleocapsid protein gene having the highest antigenicity among all genes of the Hantan virus, and then isolated and purified Hantan virus nucleocapsid antigen protein is identical to the virus. The present invention has been completed by confirming that it shows immunological antigenicity, and confirming that it can be used to diagnose nephrotic bleeding hemorrhagic fever caused by Hantan virus.

Accordingly, it is an object of the present invention to provide novel polynucleotides encoding the nucleocapsid proteins of the Hantan virus.

Another object of the present invention is to provide a plant expression vector comprising a novel polynucleotide encoding a nucleocapsid protein of a hantan virus.

Still another object of the present invention is to provide a method for producing a transgenic plant expressing a nucleocapsid protein of a hantan virus.

Another object of the present invention is to provide a plant transformant that expresses the nucleocapsid protein of the Hantan virus.

Another object of the present invention to provide a method for producing a nucleocapsid protein of a hantan virus.

Another object of the present invention is to provide a nucleocapsid protein of hantan virus.

It is another object of the present invention to provide a kit for diagnosing antibodies of nephrotic bleeding hemorrhagic fever comprising nucleocapsid protein of hantan virus as an antigen.

Another object of the present invention to provide a method for measuring the antibody of nephrotic bleeding hemorrhagic fever.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to one aspect of the present invention, the present invention provides a polynucleotide encoding a nucleocapsid protein of a peat virus comprising a nucleotide sequence described in SEQ ID NO: 1 or a portion thereof.

The novel polynucleotide of the present invention encodes a nucleocapsid protein of a hantan virus that can act as an antigen, and has a modified sequence to optimize expression in plants.

The design of the polynucleotides of the invention is carried out by the following criteria: (i) to make the GC content at least 50%, (ii) to have the plant's preferred codons, and (i) to remove intron-like sequences. will be.

The novel polynucleotides of the invention include some sequences as well as the nucleotide sequences set forth in SEQ ID NO: 1. That is, when the amino acid sequence encoded by the above partial sequence shows antigenicity, it is included in the polynucleotide of the present invention.

According to another aspect of the present invention, the present invention provides an antibody comprising (i) the nucleotide sequence described in SEQ ID NO: 1 or a portion thereof; (Ii) a promoter operably linked to the polynucleotide of (iii) and acting on plant cells to form RNA molecules; And (iii) a 3'-non-detoxification site that acts on plant cells to cause polyadenylation of the 3'-end of the RNA molecule.

According to a preferred embodiment of the present invention, a promoter suitable for the present invention can be used any conventionally used in the art for the transformation of plants, for example, the cauliflower mosaic virus (CaMV) 35S promoter , Nopaline synthase (nos) promoter, pigwarm mosaic virus 35S promoter, sugacran basilisform virus promoter, commelina yellow mottle virus promoter, ribulose-1,5-bis-phosphate carboxylase small sub Photoinduced promoter of ssRUBISCO, rice cytosol triosphosphate isomerase (TPI) promoter, adenine phosphoribosyltransferase (APRT) promoter of arabidopsis and octopine synthase promoter.

According to a preferred embodiment of the present invention, the 3'-non-detoxification site causing the polyadenylation suitable for the present invention is derived from the nopalin synthase gene of Agrobacterium turmeration (nos 3 'end) ( Bevan et al., Nucleic Acids Research , 11 (2): 369-385 (1983)), derived from the Octopine synthase gene of Agrobacterium tuberculosis, of the protease inhibitor I or II gene of tomato or potato 3 'terminal portion and CaMV 35S terminator.

Optionally, the vector additionally carries a gene encoding a reporter molecule (eg, luciferase and β-glucuronidase). In addition, the vector of the present invention may be selected as an indicator for antibiotics (e.g. neomycin, carbenicillin, kanamycin, spectinomycin, hygromycin, and the like) and resistance genes (e.g. neomycin phosphotransferase ( nptII ), hygro Mycin phosphotransferase ( hpt ), and the like).

According to another aspect of the present invention, the present invention comprises the steps of (a) transforming a plant cell with the plant expression vector of claim 2; (b) selecting the transformed plant cells; And (c) regenerating the plant from the transformed plant cells to obtain a transformed plant. The method provides a method for producing a transformed plant expressing a nucleocapsid protein of a hantan virus.

According to another aspect of the present invention, the present invention provides a plant transformant produced by the method of the present invention described above.

In the method of the present invention, the transformation of plant cells can be carried out according to a conventional method known in the art, which is electroporation (Neumann, E. et al., EMBO J. , 1: 841 (1982) ), Particle bombardment (Yang et al., Proc. Natl. Acad. Sci. , 87: 9568-9572 (1990)) and Agrobacterium-mediated transformation (US Pat. Nos. 5,004,863, 5,349,124 and 5,416,011) Include. Of these, Agrobacterium-mediated transformation is most preferred. Agrobacterium-mediated transformation is generally carried out with leaf discs and other tissues (cotyledon and hypocotyls). Agrobacterium-mediated transformation is most efficient in dicotyledonous plants.

Selection of transformed plant cells can be carried out by exposing the transformed culture to a selection agent (eg metabolic inhibitors, antibiotics and herbicides). Plant cells that are transformed and stably contain marker genes that confer selective resistance are grown and divided in the cultures described above.

Methods of developing or regenerating plants from plant protoplasts or various implants are well known in the art. The transformed root shoots formed are imbedded in a suitable plant growth medium. Development or regeneration of plants comprising foreign genes introduced by Agrobacterium can be accomplished according to methods known in the art (US Pat. Nos. 5,004,863, 5,349,124 and 5,416,011).

On the other hand, the present inventors have made extensive research efforts to develop novel transgenic plants (eg, tobacco, melons, cucumbers, watermelons and rapeseeds), and as a result, have established the most efficient method for the transformation of specific plants. Such a method is disclosed in PCT / KR02 / 01461, PCT / KR02 / 01462 and PCT / KR02 / 01463, which patent documents are incorporated herein by reference.

The method of the invention is applied to a variety of plants, but preferably to tobacco, melons, cucumbers, watermelons and rapeseeds.

In the present invention, the preferred transformation method is carried out using the Agrobacterium system, more preferably using the Agrobacterium tumefaciens -binary vector system.

In a method using the Agrobacterium system, one specific embodiment includes the following steps: (a ') an Agrobacterium tumer embedded in a genome DNA of a plant cell and having a vector having the sequence Infecting an plant plant with Agrobacterium tumefaciens : the nucleotide sequence described in SEQ ID NO: 1 or a portion thereof; (Ii) a promoter operably linked to the polynucleotide of (iii) and acting on plant cells to form RNA molecules; And (iii) a 3'-non-detoxification site that acts in plant cells to cause 3'-end polyadenylation of the RNA molecule; (b ') re-differentiating the infected implant in a regeneration medium to obtain a redifferentiated shoot; And (c ') culturing the re-differentiated shoots in a rooting medium to obtain a transformed plant.

In one specific embodiment described above, suitable implants for transformation include any tissue derived from the germinated seed, preferably cotyledon and hypocotyl, most preferably cotyledon. Seed germination is carried out under suitable dark / light conditions using a suitable medium. Transformation of plant cells is carried out with Agrobacterium trimers comprising Ti plasmids (Depicker, A. et al., Plant cell transformation by Agrobacterium plasmids.In Genetic Engineering of Plants, Plenum Press, New York (1983) ).

More preferably, binary vector systems such as pBin19, pRD400, pRD320 and pHS737 are used (An, G. et al., “Binary vectors” In Plant Gene Res. Manual, Martinus Nijhoff Publisher, New York (1986); RK Jain, et al., Biochem. Soc. Trans. 28: 958-961 (2000).

Infection of implants with Agrobacterium trimmers includes methods known in the art. Most preferably, the infection process comprises coculture by impregnating cotyledon in a culture of Agrobacterium tumerification. As a result, Agrobacterium trimmers are infected into plants. Co-cultures preferably include acetosyringone, which is intended to help transform Agrobacterium into plants.

Explants transformed with Agrobacterium trimerization are re-differentiated in regeneration medium, which successfully leads to regeneration of shoots. According to the present invention, the regeneration medium includes, but is not limited to, nutrient basal mediums, energy sources and plant growth regulators such as MS, B5, LS, N6 and White's. The energy source is more preferably a saccharide, most preferably sucrose. On the other hand, the re-differentiation medium of the present invention may further include MES to serve as a pH buffer, and may further include agar as a solid support.

The regeneration medium of the present invention includes plant growth regulators. Cytokinins contained in plant growth regulators include, but are not limited to, 6-benzylaminopurine (BAP), kinetin, zeatin, isopentyl adenosine, and the like, most preferably 6-benzylaminopurine. On the other hand, auxin-based growth regulators such as 1-naphthalene acetic acid, indole acetic acid, and (2,4-dichlorophenoxy) acetic acid may also be included in the regeneration medium.

Re-differentiated tissue is rooted in the rooting medium to produce a transgenic plant.

Plants transformed according to the present invention is confirmed whether or not transformed by methods known in the art. For example, PCR can be performed using DNA samples obtained from tissues of transformed plants to identify foreign genes inserted into the genome of the transformed plants. Alternatively, Northern or Southern blotting can be performed to confirm transformation (Maniatis et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989)). On the other hand, if the vector used for transformation contains the β-glucuronidase gene, a part of the re-differentiated cotyledon tissues may be treated with X-gluc (5-Bromo-4-Chloro-3-Indole-β-D-Glucuronic Acid By visually observing the color development by immersion in a substrate solution, such as) can be easily confirmed whether the transformation.

According to another aspect of the present invention, the present invention comprises the steps of (a) transforming a plant cell with the above-described vector of the present invention; (b) selecting the transformed plant cells; (c) regenerating the plant from the transformed plant cells to obtain a transformed plant; And (d) obtaining a nucleocapsid protein of the hantan virus from the transgenic plant.

According to another aspect of the present invention, the present invention provides a nucleocapsid protein of hantan virus produced by the above-described method.

Nucleocapsid proteins of the Hantaan virus can be obtained from tissues derived from various organs (e.g. stems, leaves, roots, fruits, seeds, etc.) of the plant transformants of the present invention, and the extracts obtained from the tissues are conventionally purified. You can get it through the process. In the present invention, the purification step may be carried out by employing a purification method commonly used in the art. For example, solubility fractionation using ammonium sulfate or PEG, ultrafiltration separation according to molecular weight, separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity), etc. Various methods may be used and are usually purified using a combination of the above methods.

In a specific embodiment of the present invention, when there is 6 x His at the N-terminus of the nucleocapsid protein of the hantan virus of the present invention, the nucleocapsid of the desired hantan virus using a Ni-NTA His-binding resin column Proteins can be isolated and purified quickly and easily.

According to the method of the present invention, it is possible to mass-produce a hantan virus-derived nucleocapsid protein at low cost by using a plant transformant.

According to another aspect of the present invention, the present invention provides a method for producing a test fluid, comprising the steps of: (a) obtaining a testing fluid from a subject under test; (b) inducing an antigen-antibody reaction with the test solution using the nucleocapsid protein of the hantan virus of the present invention as an antigen; And (c) provides a method for measuring the antibody of nephrotic bleeding fever comprising measuring the occurrence (occurrence) of the antigen-antibody response in step (b).

In the present invention, it can be applied to various biological fluids (eg, blood, serum, urine, sweat, etc.), but is preferably blood, and more preferably serum.

The method of the present invention can be used for all assays based on antigen-antibody binding. Most preferably, it is performed according to an enzyme-linked immunosorbent assy (ELISA) method. More specifically, (i) adsorbing an anti-human antibody to the well plate; (Ii) reacting and washing the sample by adding serum of the sample to the well plate; (Iii) adding and reacting the hantan virus nucleocapsid protein; (Iii) adding and reacting a monoclonal antibody against the hantan virus nucleocapsid protein; (Iii) washing the well plate and reacting by adding a secondary antibody bound to a chromophore or a fluorescent substance; And (iii) determining whether or not the secondary antibody is bound.

The color development enzymes include, but are not limited to, alkaline phosphatase, β-galactosidase, horse radish peroxidase, and the like. In addition, when using alkaline phosphatase, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), ECF substrate, etc. are used as a substrate, and when using a horse radish peroxidase, Chloronaphthol, aminoethylcarbazole, diaminobenzidine, luminol, ABTS (2,2'-Azine-di [3-ethylbenzthiazoline sulfonate]) and the like are used as the substrate.

According to another aspect of the present invention, the present invention provides a kit for antibody diagnosis of nephrotic bleeding fever comprising the nucleocapsid protein of the hantan virus of the present invention described above as an antigen.

The hantan virus nucleocapsid protein included in the diagnostic kit of the present invention is a recombinant protein expressed in plants, which is highly infectious in humans and animals, and has excellent specificity and sensitivity to the hantan virus antibody. It is proven.

When the diagnostic kit of the present invention is basically developed for ELISA, a plate (or a plate coated with a peat virus nucleocapsid protein), a buffer, a secondary antibody labeled with a chromophore or a fluorescent substance, a chromogenic substrate, and the like It may include.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention more specifically, and the scope of the present invention is not limited to these examples according to the gist of the present invention.

Example 1 Synthesis of Novel Genes of Hantan Virus Nucleocapsid Protein

Unlike the nucleotide sequence encoding the existing Hantan virus nucleocapsid protein, while encoding the 429 amino acids (see SEQ ID NO: 2 sequence) identically to the native Hantan virus nucleocapsid protein gene, the plant's preferred optimal The nucleotide sequence of was designed. The design criteria for the new gene is first, to make the GC content above 50%, to have the plant's preferred codons, and to remove intron-like sequences. The nucleotide sequences thus designed were chemically synthesized at the Plant Biotechnology Institute (hereinafter referred to as "PBI") and the National Research Center (SK, Canada). DNA encoding the synthesized hantan virus nucleocapsid protein is described in SEQ ID NO: 1.

Example 2: Construction of Plant Expression Vector of Hantan Virus Nucleocapsid Protein Gene

The synthetic gene encoding the nucleocapsid of the Hantan virus is composed of 1287 bp and synthesized to have a recognition site of Xba I at the 5'-end and Bam HI at the 3'-end.

PHS737 (PBI, National Research Center, Canada), a plant expression binary vector, was digested with restriction enzymes Xba I and Bam HI enzymes, digested, and isolated and purified. The 1284 bp synthetic gene and the cleaved pHS737 vector were mixed and added to the ligation buffer (KOSCO, Korea) and T4 ligase (KOSCO, Korea) and reacted at 16 ° C. for at least 1 hour. The reactants were then transformed into competent cells treated with CaCl ₂ ( E. coli strain DH5α (Promega, USA) and then antibiotic resistant strains were selected in LB medium containing the antibiotic kanamycin (100 mg / ml).

Plasmid DNA was isolated from the selected clones using an alkaline method and polymerized using forward primer 5'-AAT CTA GAA ATG GCT ACT AT-3 'and reverse primer 5'-AAG GAT CCC CGA GCT TGA GA-3'. Enzyme chain reaction (PCR) was performed. The enzyme used in the PCR amplification was Taq polymerase (Solgent, Korea), and the temperature conditions were set as follows: predenature for 2 minutes at 96 ° C, denature for 1 minute at 94 ° C, and connect for 1 minute at 55 ° C. The reaction was repeated 35 times at 72 ° C. for 2 minutes, and further extended at 72 ° C. for 10 minutes.

The amplification product was then analyzed on a 1.0% agarose gel to confirm that the desired insert sequence was in the plasmid (see Figure 2).

Example 3: Transformation of Plants

Example 3-1 Preparation of Agrobacterium tumefaciens GV3101 Infected Broth

The recombinant pHS737 / SNV vector prepared in Example 2 was introduced into Agrobacterium turmeration GV3101 (Mp90) by conjugation. Escherichia coli S17-1 with recombinant pHS737 / HTNV vector and Agrobacterium Tumperfections GV3101 (Mp90) were each cultured in liquid LB medium to log phase. Each of these cells were mixed together 1: 1 in an Eppendorf tube and then centrifuged for 30 seconds to concentrate the cells, followed by stationary culture at 1-2C for 1-2 days. The Eppendorf tube containing the cells over time was gently shaken to float the concentrated cells, and then plated in LB solid medium to which 50 mg / L kanamycin and 30 mg / L gentamicin were added. Colonies were selected after incubation for days. The strain into which the vector was introduced was named Agrobacterium turmeration GV3101-pHS737 / HTNV (Alt-Morbe et al., J. Bacteriol. 178: 4248-4257 (1996)).

Agrobacterium trimmers GV3101-pHS737 / HTNV with pHS737 / HTNV were inoculated in Superbros (BHI medium, pH 5.6) and incubated at 28 ° C. for 2 days, and the culture was used for plant infection.

Example 3-2 Transformation and Regeneration of Plant Cells

First, the seeds of the sterilized tobacco were sown, and fresh young leaves which were sterilely cultured for two weeks or longer were collected. Super broth: 37 g / l Brain heart infusion broth (Difco) containing 100 μM acetosyringone was added to Agrobacterium turmeration GV3101 (Mp90) with pHS737 vector with the Hantan virus nucleocapsid gene. , 0.2% sucrose, pH 5.6) for 18 hours, and then the cotyledon sections were immersed in the culture and mixed for 20 minutes.

Then, coculture medium [3.0% sucrose, 0.5 g / L MES (2- (N-Morpholino) ethanesulfonic acid Monohydrate), 1.0 mg / L BAP, 0.1 mg / L NAA under cancer culture conditions at 25 ° C. Co-cultured with MSB5 (Murashige & Skoog medium including Gamborg B5 vitamins) for 2 days. Then, through a clean up step to remove bacteria, leaf sections were removed in a medium based on selection medium (MSB5, 0.5 g / L MES, 3% sucrose and 0.6% pitagel 1.0 mg / L, NAA 0.1 mg / L, carbenicillin 500 mg / L and kanamycin 100 mg / L) were cultured for 2 weeks and rooted shoots presumed to be transformed were selected and re-differentiated medium (MSB5, 500 incubated in mg / L of MES, BAP 0.01 mg / L, NAA 0.1 mg / L, carbenicillin 500 mg / L, kanamycin 100 mg / L, 3% sucrose, 0.6% agar). The regenerated shoots were transferred to rooting medium (MSB5, 500 mg / L of MES, NAA 0.1 mg / L, carbenicillin 500 mg / L, kanamycin 100 mg / L, 3% sucrose, 0.6% agar) for rooting. One individual was estimated as a transformant and analyzed using the method of Example 4 below.

Example 4 PCR Analysis of Transgenic Plants

Confirmation of the transformed plants obtained in Example 3 was carried out as follows: 10 mg of shoots selected as transformants in selection medium using the Edward method ( Nucleic Acids Research , 19: 1349 (1991)) After the plant genome DNA was isolated, PCR was carried out using this as template DNA.

The primers used for PCR analysis of the transgenic plant of the Hantan virus nucleocapsid protein gene correspond to the nucleotide sequence of the nucleocapsid protein gene, and the forward primer is 5'-AAT CTA GAA ATG GCT ACT AT-3 ', The reverse primer is 5'-AAG GAT CCC CGA GCT TGA GA-3 '. PCR reaction was performed by Taq polymerase (Solgent, Korea), total denaturation at 96 ° C for 2 minutes, denaturation at 94 ° C for 1 minute, connection at 55 ° C for 1 minute, and polymerization at 72 ° C for 2 minutes. Repeated a further time, after further extension for 10 minutes at 72 ℃ reaction product was analyzed on 1.0% agarose gel (see Figure 3). In Figure 3, column M is a molecular weight marker, column 1 is a PCR product of a positive standard plasmid containing a Hantan virus nucleocapsid protein gene, column 2 is a PCR product based on chromosomal DNA of wild tobacco, columns 3 to 10 PCR products based on the DNA of the selected tobacco transformants are shown. As can be seen in Figure 3, in the plant transformant of the present invention was observed a 1.3 kb DNA band corresponding to the Hantan virus nucleocapsid protein gene was confirmed that the transformation.

Example 6: Isolation and Purification of Hantan Virus Nucleocapsid Protein in Transgenic Plants

In a mortar containing an appropriate amount of liquid nitrogen, 1 g of the leaves of the transformed plant were cut and pulverized. 2 ml of extract buffer (250 mM Sucrose, 1 M Hepes, 1 mM MgCl ₂ , 1 mM DTT) containing protease remover per 0.5 g of plant weight was finely ground. The extract was centrifuged at 12,000 rpm and 4 ° C. for at least 30 minutes, then the supernatant was removed and transferred to a new tube. The lysed cell solution was added to a Probond column (Quiagen GmbH, Germany) containing nickel resin, and gently shaken for 10 minutes at room temperature to allow the Hantan virus nucleocapsid protein in the cell solution to adsorb well to the nickel resin. .

The resin to which the nucleocapsid protein was adsorbed was washed twice with the same amount of pH 7.8 adsorption buffer solution (20 mM Sodium Phosphate, 500 mM Sodium Chloride) to remove plant cell-derived protein adsorbed on the resin. The same amount of pH 6.0 washing buffer (20 mM Sodium Phosphate, 500 mM Sodium Chloride) was added to the washed resin, and the mixture was gently shaken at room temperature to wash the resin again, and then the same amount of pH 4.0 elution buffer (20 mM Sodium) was added. Phosphate, 500 mM Sodium Chloride) was added to elute the Hantan virus nucleocapsid protein.

Hantan virus nucleocapsid proteins expressed in plant transformants have six histidine residues at the amino terminus and are easily isolated by binding to a cationically nickel nickel resin. Through this process, only the Hantan virus nucleocapsid protein from which all plant cell proteins were removed was purified. The eluted protein was concentrated using Centricon (Amicon Co., U.S.A.).

As shown in FIG. 4, 48 kD of agar virus nucleocapsid protein, which was not detected in wild-type tobacco, was detected in cell eluate as a result of electrophoresis of the hantan virus nucleocapsid protein on an SDS-PAGE gel. In particular, in the case of eluting with a nickel resin column, it was confirmed that pure Hantan virus nucleocapsid protein from which all plant cell proteins were removed was detected.

Hantan virus nucleocapsid protein purified by the above method was added to the stain assay (Bradford) method (Protein assay kit, Bio-Rad) and measured the absorbance at 595 nm with a UV-spectrophotometer and BSA (Bovine Serum Albumin ) Protein quantification was performed in comparison with the standard. The extracted protein was adjusted to the same amount and each sample was subjected to 8% polyacrylamide gel electrophoresis, and Western blot analysis was performed (see Peter B. Kaufma et al., Molecular and Cellular Methods in Biology and Medicine , 108-121, CRC press). Western blot analysis was performed as follows. The protein in the SDS-PAGE gel was transferred to PVDF membrane using Semi-Dry Transfer Units (Hoefer, USA). After confirming that all proteins in the gel had migrated to the PVDF membrane, the PVDF membrane was dried. This PVDF membrane was reacted for 1 hour in a 0.5% BSA / TBS (Tris-Buffered Saline) solution to which a monoclonal antibody (Center of disease control, Georgia, USA), which specifically reacts with a peat virus nucleocapsid protein antigen, was added. The PVDF membrane was then washed three times for 5 minutes with TBS solution. Then, after reacting with peroxidase labeled secondary antibody (peroxidase labeled-anti human IgG, KPL, USA) for 1 hour, the PVDF membrane was washed three times for 10 minutes with TBS solution. Finally, the PVDF membrane was added to the left buffer solution to which the ABTS coloring substrate solution (KPL, USA) was added and reacted for 30 minutes at room temperature, and the result was read (FIG. 5). As can be seen in Figure 5, Western blot analysis was carried out as a result of the 48 kD of the hantan virus nucleocapsid protein was confirmed.

Example 7: Diagnosis of Renal Syndrome Hemorrhagic Fever Using 96-well Plate Adsorbed with Hantan Virus Nucleocapsid Protein

Example 7-1 Determination of Antibody Titers against Hantan Virus Nucleocapsid Protein

In order to confirm that the Hantan virus nucleocapsid protein isolated and purified from plant transformants can be used for diagnosis of nephrotic bleeding hemorrhage, the nucleocapsid protein was subjected to antigen-antibody reaction using 96-well microtiter plates. Measured.

The purified nucleocapsid protein in a 96-well microtiter plate was serially diluted in phosphate buffer, dispensed 100 μl per well, and left at 4 ° C. for at least 8 hours to fix the antigen in the well. The plate containing the protein antigen adsorbed was washed three times with washing buffer (0.05% Tween 20, 0.01 M Phosphate, pH 7.6) to remove the non-adsorbed antigen from the plate, and the single protein against the new Nombrevirus nucleocapsid protein antigen. Clone antibodies (Centers of disease control, Georgia, USA) were diluted in diluted dilution (0.1% BSA, 0.05% Tween 20, 20 mM Trisma base, 150 mM NaCl) in aliquots of 100 μl per well and then at room temperature. The reaction was timed longer to induce antigen-antibody reaction. Then, each well was washed three times with washing buffer, and then the blocking buffer was dispensed into each well, and reacted at room temperature for 1 hour, followed by washing in the same manner as above. Peroxidase labeled secondary antibody (peroxidase labelled-anti human IgG, KPL, U.S.A.) was aliquoted and reacted and washed in the same manner as above. Finally, the color was reacted for 30 minutes at room temperature by adding ABTS color substrate (KPL, USA) to each well, and the result was read at 405 nm using an ELISA reader (ELISA Reader, Titertek, USA). Table 1 shows. The horizontal axis of the table represents the dilution fold of the nucleocapsid protein of the purified Hantan virus (amount before dilution: 73 ng, 20480 fold dilution: 36 pg), and the vertical axis shows the dilution of the initial titer of the antibody against the protein as 1. It represents a multiple. As can be seen in Table 1, the nucleocapsid proteins of the present invention exhibit excellent antigenicity.

Figure 6 is a schematic of the antibody titer group reacting with 36 pg of purified hantan virus compared to the protein isolated from wild-type plants. As shown in the figure, the antigenicity of the nucleocapsid protein isolated and purified from recombinant plants was excellent, whereas the protein isolated from wild-type plants showed no antigenicity at all.

Dilution multiples of nucleocapsid protein Antibody Dilution Factor 10 20 40 80 160 320 640 1280 2560 5120 10240 20480 100 <3.0 <3.0 <3.0 <3.0 2.33 2.36 2.18 2.15 2.10 1.97 1.71 1.55 200 <3.0 <3.0 <3.0 <3.0 2.24 2.26 2.09 2.03 2.03 1.89 1.60 1.45 400 <3.0 <3.0 <3.0 2.83 2.21 2.22 2.00 2.01 1.98 1.69 1.55 1.34 800 <3.0 <3.0 2.75 2.61 2.09 2.01 2.00 2.01 1.94 1.58 1.50 1.21 1600 <3.0 2.63 2.51 2.33 1.99 1.87 1.82 1.78 1.87 1.43 1.3 1.14 3200 2.58 2.40 2.31 2.23 1.95 1.79 1.75 1.45 1.76 1.24 1.13 0.87 6400 2.40 2.32 2.19 2.11 1.68 1.56 1.43 1.19 1.64 1.15 0.83 0.68 1280 2.05 1.9 1.74 1.68 1.25 1.20 0.9 0.9 0.65 0.66 0.68 0.53

Example 7-2 Diagnosis of Renal Syndrome Hemorrhagic Fever

In order to confirm that the Hantan virus nucleocapsid protein isolated and purified from plant transformants can be used for the diagnosis of nephrotic bleeding fever, antibody titers in serum of patients with nephrotic bleeding fever were measured.

100 μl of anti-human IgM antibody (goat anti-human IgM, KPL, USA) diluted in phosphate buffer in 96-well microtiter plates was dispensed per well, and the antibody was left at 4 ° C. for at least 8 hours. Fixed to wells. The antibody-adsorbed plate was washed three times with washing buffer (0.05% Tween 20, 0.01 M Phosphate, pH 7.6) to remove the antibody that did not adsorb on the plate, and the patient serum was diluted (0.1% BSA, 0.05% Tween 20). , 20 mM Trisma base, 150 mM NaCl) was diluted with a constant dilution factor and dispensed 100 μl per well, followed by reaction at room temperature for at least 1 hour, and washed three times with the same washing solution.

100 μl of Hantan virus nucleocapsid protein dissolved in the dilutions by concentration was dispensed into each well and left at room temperature for 1 hour or more to induce a second antigen-antibody reaction. After washing each well again with washing buffer, 100 μl of each monoclonal antibody (Center of disease control, Georgia, USA) against the nucleocapsid antigen protein dissolved in the diluent at a concentration of 10 ug / ml After reacting for 1 hour at room temperature, the solution was washed three times with a washing solution.

Then peroxidase labeled secondary antibody (peroxidase labeled-anti human IgG, KPL, U.S.A.) was dispensed and reacted and washed in the same manner as above. Finally, the color was reacted for 30 minutes at room temperature by adding ABTS color substrate (KPL, USA) to each well, and the result was read at 405 nm using an ELISA reader (ELISA Reader, Titertek, USA). Table 2 shows. The result was positive reading of dilution factor with absorbance value of 1.5 or more.

number serum Antibody titers One Patient 1 800 2 Patient 2 800 3 Patient 3 6400 4 Patient 4 400 5 Patient 5 6400 6 Patient 6 200 7 Patient 7 200 8 Patient 8 800 9 Patient 9 6400 10 Patient 10 6400 11 Patient 11 12800 12 Patient 12 400 13 Patient 13 25600 14 Patient 14 12800 15 Patient 15 25600 16 Patient 16 12800 17 Patient 17 800 18 Patient 18 1600 19 Patient 19 200 20 Patient 20 25600 21 Normal 1 <100 22 Normal 2 <100 23 Normal 3 <100 24 Normal 4 <100 25 Normal 5 <100 26 Normal 6 <100 27 Normal 7 <100 28 Normal 8 <100 29 Normal 9 <100 30 Normal 10 <100

As shown in Table 2, the serum of normal subjects showed all antibody titers of 100 or less, whereas the serum titers of patients with nephrotic hemorrhagic fever showed 200-25600, which clearly distinguished patients from nephrotic hemorrhagic fever. This shows that the antigen-antibody reaction using the Hantan virus nucleocapsid protein antigen isolated from the recombinant plant occurs with a high specificity, and it was confirmed that this can discriminate patients with nephrotic hemorrhagic fever.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

As described in detail above, the present invention provides a novel polynucleotide encoding a nucleocapsid protein of a hantan virus and a plant expression vector comprising the same. The present invention also provides a method for producing a transgenic plant expressing a nucleocapsid protein of a hantan virus and a plant transformant. The present invention provides a method for producing a nucleocapsid protein of hantan virus and a nucleocapsid protein of hantan virus. On the other hand, the present invention provides a kit for antibody diagnosis of nephrotic bleeding fever and a method for measuring the antibody of nephrotic bleeding fever.

According to the present invention, a recombinant nucleocapsid protein of Hantan virus exhibiting excellent antigenicity at low cost can be obtained, and when using this, nephrotic bleeding fever can be diagnosed with excellent sensitivity and specificity.

1 is a schematic diagram showing the manufacturing process and gene map of a plant transformation vector comprising a nucleocapsid protein gene of the Hantan virus.

Figure 2 is a photograph showing the results of electrophoresis by cleavage of the pHS737 vector cloned with nucleocapsid protein gene of Hantan virus with restriction enzymes Xba I and Bam HI. M, 1 kb DNA ladder; Lane 1, pHS737 vector digested with Xba I and Bam HI; Lane 2, PCR product of the insertion sequence; And lane 3, pHS737 vector (pHS737 / HTNV) comprising a nucleocapsid gene digested with Xba I and Bam HI.

Figure 3 is a photograph showing the results of PCR analysis of the Hantan virus nucleocapsid protein gene in a transgenic plant. M, 1 kb DNA ladder; Lane 1, PCR product of a positive standard plasmid containing the nucleocapsid protein gene of Hantan virus; Lane 2, PCR product based on the chromosomal DNA of wild tobacco; And lanes 3-10, PCR products based on DNA of selected tobacco transformants.

Figure 4 is a photograph of the results of electrophoresis of the Hantan virus nucleocapsid protein expressed in the transgenic plant on SDS-polyacrylamide gel. M, molecular weight marker; Lane 1, protein of wild tobacco; And lane 2, nucleocapsid protein of Hantan virus expressed in transgenic plants.

Figure 5 is a photograph showing the results of Western blot analysis of nucleocapsid protein of Hantan virus expressed in the transgenic plant. M, molecular weight marker; Lane 1, section 1 eluate; Lane 2, section 2 eluate; Lane 3, section 3 eluate; And lane 4, wild type tobacco

6 is a graph showing the antigenicity of nucleocapsid proteins of Hantan virus expressed in transgenic plants.

<110> NEXGEN BIOTECHNOLOGIES, INC. <120> Diagnosis Kits for Hemorrhagic Fever with Renal Syndrome Comprising Nucleocapsid Protein from Hantann Virus Expressed in Plants <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 1287 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence encoding nucleocapsid of hantann virus <220> <221> CDS (222) (1) ... (1287) <400> 1 atg gct act atg gag gaa ctc caa cgc gaa atc aac gcc cat gaa ggg 48 Met Ala Thr Met Glu Glu Leu Gln Arg Glu Ile Asn Ala His Glu Gly 1 5 10 15 caa ttg gtg ata gcc cgc cag aaa gtg aga gac gcc gag aaa cag tat 96 Gln Leu Val Ile Ala Arg Gln Lys Val Arg Asp Ala Glu Lys Gln Tyr 20 25 30 gag aaa gat cca gat gaa ctt aac aag agg act ctc acc gat aga gaa 144 Glu Lys Asp Pro Asp Glu Leu Asn Lys Arg Thr Leu Thr Asp Arg Glu 35 40 45 gga gtc gca gtt tca att cag gct aag ata gat gag ctt aag cga cag 192 Gly Val Ala Val Ser Ile Gln Ala Lys Ile Asp Glu Leu Lys Arg Gln 50 55 60 ctc gcc gac cgg atc gct acc gga aaa aat cta ggg aag gag caa gat 240 Leu Ala Asp Arg Ile Ala Thr Gly Lys Asn Leu Gly Lys Glu Gln Asp 65 70 75 80 ccc aca ggt gtc gaa cca ggt gac cat tta aaa gag agg tcg atg ctt 288 Pro Thr Gly Val Glu Pro Gly Asp His Leu Lys Glu Arg Ser Met Leu 85 90 95 agc tac ggt aac gtc ctc gat ctt aat cac ctg gat att gac gag ccc 336 Ser Tyr Gly Asn Val Leu Asp Leu Asn His Leu Asp Ile Asp Glu Pro 100 105 110 aca gga cag acg gct gat tgg ctg tct atc att gtg tat ctt act tct 384 Thr Gly Gln Thr Ala Asp Trp Leu Ser Ile Ile Val Tyr Leu Thr Ser 115 120 125 ttc gtt gtt cca atc ctt ttg aaa gct cta tac atg ctc acc acg aga 432 Phe Val Val Pro Ile Leu Leu Lys Ala Leu Tyr Met Leu Thr Thr Arg 130 135 140 gga agg cag act acc aag gat aat aaa gga acc cgc att aga ttt aag 480 Gly Arg Gln Thr Thr Lys Asp Asn Lys Gly Thr Arg Ile Arg Phe Lys 145 150 155 160 gat gac agt agt ttc gaa gac gtg aac ggt ata cgt aag cca aag cat 528 Asp Asp Ser Ser Phe Glu Asp Val Asn Gly Ile Arg Lys Pro Lys His 165 170 175 ctg tac gtc agc ttg cct aac gcc caa tct agc atg aag gca gag gag 576 Leu Tyr Val Ser Leu Pro Asn Ala Gln Ser Ser Met Lys Ala Glu Glu 180 185 190 att acc cca ggg agg tat cgt aca gct gtt tgt ggc ttg tac ccc gct 624 Ile Thr Pro Gly Arg Tyr Arg Thr Ala Val Cys Gly Leu Tyr Pro Ala 195 200 205 caa atc aag gcc aga caa atg atc tcg ccg gtt atg tct gtt att gga 672 Gln Ile Lys Ala Arg Gln Met Ile Ser Pro Val Met Ser Val Ile Gly 210 215 220 ttc ctt gcg ctc gct aag gat tgg tct gac agg atc gaa caa tgg ctt 720 Phe Leu Ala Leu Ala Lys Asp Trp Ser Asp Arg Ile Glu Gln Trp Leu 225 230 235 240 att gaa cca tgc aaa ctt ttg cct gac acc gct gca gtg agc ttg ctg 768 Ile Glu Pro Cys Lys Leu Leu Pro Asp Thr Ala Ala Val Ser Leu Leu 245 250 255 ggt ggt cct gcc act aat aga gac tac ctt agg cag cga cag gtg gca 816 Gly Gly Pro Ala Thr Asn Arg Asp Tyr Leu Arg Gln Arg Gln Val Ala 260 265 270 ctg gga aac atg gaa act aag gag agc aag gct att cgc cag cac gct 864 Leu Gly Asn Met Glu Thr Lys Glu Ser Lys Ala Ile Arg Gln His Ala 275 280 285 gaa gcc gca ggg tgt tcc atg atc gaa gat att gag tcc cca tca tca 912 Glu Ala Ala Gly Cys Ser Met Ile Glu Asp Ile Glu Ser Pro Ser Ser 290 295 300 atc tgg gtt ttt gcc ggt gca cca gat cgt tgc cct ccg aca tgc ttg 960 Ile Trp Val Phe Ala Gly Ala Pro Asp Arg Cys Pro Pro Thr Cys Leu 305 310 315 320 ttc ata gca gga att gcc gag ctg ggc gct ttt ttc tca att ttg caa 1008 Phe Ile Ala Gly Ile Ala Glu Leu Gly Ala Phe Phe Ser Ile Leu Gln 325 330 335 gac atg aga aat aca ata atg gcg tcc aaa act gtc ggc aca tca gaa 1056 Asp Met Arg Asn Thr Ile Met Ala Ser Lys Thr Val Gly Thr Ser Glu 340 345 350 gag aaa ttg cgg aag aag agt agc ttt tac caa tcc tat ttg cgt agg 1104 Glu Lys Leu Arg Lys Lys Ser Ser Phe Tyr Gln Ser Tyr Leu Arg Arg 355 360 365 acc caa tct atg ggt atc caa cta ggc cag cgt atc att gta ctc ttc 1152 Thr Gln Ser Met Gly Ile Gln Leu Gly Gln Arg Ile Val Leu Phe 370 375 380 atg gtg gcg tgg ggc aaa gag gct gta gat aac ttt cac ctt gga gac 1200 Met Val Ala Trp Gly Lys Glu Ala Val Asp Asn Phe His Leu Gly Asp 385 390 395 400 gat atg gac cct gag ctt cgt act ctc gca caa tcc ttg atc gat gtt 1248 Asp Met Asp Pro Glu Leu Arg Thr Leu Ala Gln Ser Leu Ile Asp Val 405 410 415 aaa gtt aag gag att tcc aat caa gaa cct ctc aag ctc 1287 Lys Val Lys Glu Ile Ser Asn Gln Glu Pro Leu Lys Leu 420 425 <210> 2 <211> 429 <212> PRT <213> Artificial Sequence <400> 2 Met Ala Thr Met Glu Glu Leu Gln Arg Glu Ile Asn Ala His Glu Gly 1 5 10 15 Gln Leu Val Ile Ala Arg Gln Lys Val Arg Asp Ala Glu Lys Gln Tyr 20 25 30 Glu Lys Asp Pro Asp Glu Leu Asn Lys Arg Thr Leu Thr Asp Arg Glu 35 40 45 Gly Val Ala Val Ser Ile Gln Ala Lys Ile Asp Glu Leu Lys Arg Gln 50 55 60 Leu Ala Asp Arg Ile Ala Thr Gly Lys Asn Leu Gly Lys Glu Gln Asp 65 70 75 80 Pro Thr Gly Val Glu Pro Gly Asp His Leu Lys Glu Arg Ser Met Leu 85 90 95 Ser Tyr Gly Asn Val Leu Asp Leu Asn His Leu Asp Ile Asp Glu Pro 100 105 110 Thr Gly Gln Thr Ala Asp Trp Leu Ser Ile Ile Val Tyr Leu Thr Ser 115 120 125 Phe Val Val Pro Ile Leu Leu Lys Ala Leu Tyr Met Leu Thr Thr Arg 130 135 140 Gly Arg Gln Thr Thr Lys Asp Asn Lys Gly Thr Arg Ile Arg Phe Lys 145 150 155 160 Asp Asp Ser Ser Phe Glu Asp Val Asn Gly Ile Arg Lys Pro Lys His 165 170 175 Leu Tyr Val Ser Leu Pro Asn Ala Gln Ser Ser Met Lys Ala Glu Glu 180 185 190 Ile Thr Pro Gly Arg Tyr Arg Thr Ala Val Cys Gly Leu Tyr Pro Ala 195 200 205 Gln Ile Lys Ala Arg Gln Met Ile Ser Pro Val Met Ser Val Ile Gly 210 215 220 Phe Leu Ala Leu Ala Lys Asp Trp Ser Asp Arg Ile Glu Gln Trp Leu 225 230 235 240 Ile Glu Pro Cys Lys Leu Leu Pro Asp Thr Ala Ala Val Ser Leu Leu 245 250 255 Gly Gly Pro Ala Thr Asn Arg Asp Tyr Leu Arg Gln Arg Gln Val Ala 260 265 270 Leu Gly Asn Met Glu Thr Lys Glu Ser Lys Ala Ile Arg Gln His Ala 275 280 285 Glu Ala Ala Gly Cys Ser Met Ile Glu Asp Ile Glu Ser Pro Ser Ser 290 295 300 Ile Trp Val Phe Ala Gly Ala Pro Asp Arg Cys Pro Pro Thr Cys Leu 305 310 315 320 Phe Ile Ala Gly Ile Ala Glu Leu Gly Ala Phe Phe Ser Ile Leu Gln 325 330 335 Asp Met Arg Asn Thr Ile Met Ala Ser Lys Thr Val Gly Thr Ser Glu 340 345 350 Glu Lys Leu Arg Lys Lys Ser Ser Phe Tyr Gln Ser Tyr Leu Arg Arg 355 360 365 Thr Gln Ser Met Gly Ile Gln Leu Gly Gln Arg Ile Val Leu Phe 370 375 380 Met Val Ala Trp Gly Lys Glu Ala Val Asp Asn Phe His Leu Gly Asp 385 390 395 400 Asp Met Asp Pro Glu Leu Arg Thr Leu Ala Gln Ser Leu Ile Asp Val 405 410 415 Lys Val Lys Glu Ile Ser Asn Gln Glu Pro Leu Lys Leu 420 425

Claims (8)

A polynucleotide encoding a nucleocapsid protein of a hantan virus consisting of the nucleotide sequence set forth in SEQ ID NO: 1. (Iii) the polynucleotide of claim 1; (Ii) a promoter operably linked to the polynucleotide of (iii) and acting on plant cells to form RNA molecules; And (iii) a 3'-non-detoxification site that acts on plant cells to cause 3'-end polyadenylation of the RNA molecule. Method for producing a transgenic plant expressing the nucleocapsid protein of the Hantan virus comprising the following steps: (a) transforming a plant cell with the plant expression vector of claim 2; (b) selecting the transformed plant cells; And (c) regenerating the plant from the transformed plant cells to obtain a transformed plant. Plant transformant prepared by the method of claim 3. Method for producing a nucleocapsid protein of the hantan virus comprising the following steps: (a) transforming a plant cell with the plant expression vector of claim 2; (b) selecting the transformed plant cells; (c) regenerating the plant from the transformed plant cells to obtain a transformed plant; And (d) obtaining a nucleocapsid protein of hantan virus from the transgenic plant. Nucleocapsid protein of the Hantan virus prepared by the method of claim 5. The diagnostic kit for antibody of nephrotic bleeding hemorrhage comprising the nucleocapsid protein of the hantan virus of claim 6 as an antigen. A method for measuring antibody in nephrotic bleeding fever comprising the following steps: (a) obtaining a testing fluid from the subject under test; (b) inducing an antigen-antibody reaction with the test solution using the nucleocapsid protein of the Hantan virus of claim 6 as an antigen; And (c) measuring the occurrence (occurrence) of the antigen-antibody response in step (b).