WO2006022461A1

WO2006022461A1 - Diagnosis kits for hemorrhagic fever with renal syndrome comprising nucleocapsid protein of hanta virus expressed in transformed plant

Info

Publication number: WO2006022461A1
Application number: PCT/KR2004/002161
Authority: WO
Inventors: Sun Lee; Yong Gyu Ju; Nam Jun Chung; In Hee Lee; Sun Hee Park; Je Geun Yoo; Jim Macpherson; Jako Colette
Original assignee: Nexgen Biotechnologies, Inc.
Priority date: 2004-08-27
Filing date: 2004-08-27
Publication date: 2006-03-02

Abstract

The present invention relates to a novel polynucleotide sequence encoding a nucleocapsid protein of Hanta virus, a plant expression vector comprising the same, a plant transformed with the same, a diagnosis kit for hemorrhagic fever with renal syndrome and a method for detecting an antibody specific to hemorrhagic fever with renal syndrome. The present invention makes it possible to obtain recombinant nucleocapsid proteins with excellent antigenicity of Hanta virus in more cost-effective manner. Furthermore, the recombinant nucleocapsid proteins of this invention permit to diagnose hemorrhagic fever with renal syndrome with excellent specificity and sensitivity.

Description

DIAGNOSIS KITS FOR HEMORRHAGIC FEVER WITH RENAL SYNDROME

COMPRISING NUCLEOCAPSID PROTEIN OF HANTA VIRUS EXPRESSED IN

TRANSFORMED PLANT

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION

The present invention relates to a novel polynucleotide sequence encoding a nucleocapsid protein of Hanta virus, a plant expression vector comprising the same, a plant transformed with the same, a diagnosis kit for hemorrhagic fever with renal syndrome and a method for detecting an antibody specific to hemorrhagic fever with renal syndrome.

DESCRIPTION OF THE RELATED ART Epidemic hemorrhagic fever is an acute fever disease showing symptoms of high fever, kidney failure and hemorrhage and is caused by viruses belonging to the genus, Hanta virus and the family Bunyaviridae. This disease is found around the world and the WHO has called hemorrhagic fever with renal syndrome in 1982 with collecting hemorrhagic nephrosonephritis in the U.S.S.R., nephrophthia epidemica in the Scandinavia and Songo fever in the China.

Hanta virus is well recognized as a pathogen responsible for hemorrhagic fever with renal syndrome in Eurasia and Hantaan virus pulmonary syndrome (HPS) in America. To date, numerous Hanta virions have been isolated from blood and tissues of patients suffering from hemorrhagic fever with renal syndrome and Hantaan virus pulmonary syndrome and pulmonary and intestinal tissues of rodents belonging to the family Cricertidae and Muridae, a host animal of Hanta virus (Chu et al . , Serological relationships among viruses in the Hantavirus genus, family Bunyaviridae. Virology 198: 196-204, 1994) .

Viruses so far known to cause diseases in human include Sin Nombre virus serotype, Hantaan virus serotype, Seoul virus serotype, Belgrade virus serotype, Puumala virus serotype and Black Creek Canal virus serotype. Other viruses such as Prospect Hill virus serotype, Thailand virus serotype and Thottapalayam virus serotype have not been elucidated to cause diseases (Xiao et al . , Phylogenetic analyses of virus isolates in the genus Hantavirus, family Bunyaviridae. Virology 198: 205-217, 1994) . Nine serotypes described above have their specific host animals.

Hantaan virus, Sin Nombre virus, Seoul virus and Puumala virus (belonging to the genus, Hanta virus and the family Bunyaviridae) has a negative-sense RNA genome consisting of large (L) middle (M) and small (S) segments and its size is about 100 nm. Each RNA segment has its nucleocapsid structure that is surrounded by a lipid membrane containing Gl and G2 glycoproteins (Schmaljohn et al. , Characterization of Hantaan virions, the prototype virus of hemorrhagic fever with renal syndrome. J Infect Dis 148: 1005-1011, 1983) . The structural protein involving in an immune reaction is nucleocapsid protein and Gl and G2 membrane proteins. The L segment RNA encodes RNA- dependent RNA polymerase, M segment RNA encodes Gl and G2 glycoproteins and S segment RNA codes for nucleocapsid proteins (Elliott et al. , Bunyaviridae genome structure and gene expression. Curr. Top. Microbiol. Immunol. 169:91-141(1991)) .

Hantaan virus that is the etiologic agent of Korean hemorrhagic fever and hemorrhagic fever with renal syndrome found in the Far East Asia has been isolated from pulmonary and intestinal tissues of Apodemus agrarius and blood and tissues of patients (Lee et al . , Isolation of the epilogic agent of Korean hemorrhagic fever. J^" Infect Dis 137: 298-308, 1978) . Seoul virus responsible for hemorrhagic fever with renal syndrome caused by contact to laboratory rats and urban house rats has been isolated from pulmonary and intestinal tissues of laboratory rats and house 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) . In addition, Belgrade virus that is the etiologic agent of severe hemorrhagic fever with renal syndrome found in the Balkan states has been isolated from blood of Apodemus flavicolis and patient (Avsic-xupanc et al. , Characterization of Dovrava virus: a hantavirus from Slovenia, Yugoslavia. J Med Virol 38: 132-137, 1992) .

Sin Nombre virus has been firstly discovered in the southwestern region of USA and called as Four Corners or Muerto Canyon virus (Nichol et al . , Genetic identification of a hantavirus associated with an outbreak of acute respiratory illness. Science 262:914-917(1993)) . Hemorrhagic fever resulting from Sin Nombre virus has exhibited the lethality more than 60%. Primary animal host of this virus has been elucidated Peromyscus maniculatus (Childs et . al . , Serologic and genetic identification of Peromyscus maniculatus as the primary rodent reservoir for a new hantavirus in southwestern United States. J. Infect. Dis. 169:1271-1280(1994)) .

It has been reported that patients more than two hundred thousands suffering from hemorrhagic fever with renal syndrome occurred every year around the world. The Korea has patients of hemorrhagic fever with renal syndrome around a thousand every year, infected by Hantaan virus and Seoul virus.

Seoul virus has an animal host including house rats and white rats unlike Hantaan virus. Clinical symptoms of patients infected by Seoul virus are similar to those by Hantaan virus. However, Seoul virus exhibits more severe decrease of appetite, nausea, vomiting, gripe and diarrhea than Hantaan virus that are sometimes erroneously diagnosed as intestinal diseases or primary pancreatitis. Furthermore, liver failure is more severe in infection of Seoul virus than Hantaan virus. Puumala virus has been considered to be a primary etiologic agent of hemorrhagic fever with renal syndrome in the Northern Europe and Mediterranean and spread by bank vole {Clethrionomys glareolus) .

Currently, the development of vaccines permits to highly decrease the incidence of patients of hemorrhagic fever with renal syndrome. However, the diseases still threaten the human life around the world. In particular, since the U.S. soldiers stationed in foreign countries are exposed to forest environment, patients of hemorrhagic fever with renal syndrome occur continuously. Therefore, the attempts to prevent the occurrence of hemorrhagic fever with renal syndrome have been made and the researches to develop diagnosis kits and reagents antigens for hemorrhagic fever with renal syndrome have been made. Antigen proteins of virus used in diagnosis kit and vaccine for hemorrhagic fever with renal syndrome have been generally prepared in such a manner that Hantaan virus is infected to brain of mouse aged 1 day for proliferation and Hantaan viruses proliferated then are inactivated with formalin. However, this preparation process is very likely to develop hemorrhagic fever with renal syndrome in persons working for antigen preparation, since a high titer of viruses has been excreted from animal infected during virus proliferation. Furthermore, animal brain- originated proteins remained in Hantaan virus antigen used in diagnosis kits lead to pseudo-positive results and therefore, reliable diagnosis is inhibited.

Drugs to completely treat viral diseases have not been developed yet. Hemorrhagic fever with renal syndrome, one of viral diseases, has not also been completely treated by drugs. Therefore, the most important one in the treatment of hemorrhagic fever with renal syndrome is to diagnose in earlier stage. Accordingly, the diagnosis kits and reagents using antigens for hemorrhagic fever with renal syndrome have been intensively researched.

Throughout this application, several patents and publications are referenced and citations are provided in parentheses . The disclosure of these patents and publications is incorporated into this application in order to more fully describe this invention and the state of the art to which this invention pertains .

SUMMARY OF THE INVENTION

Endeavoring to resolve the problems of such conventional approaches, the inventors have made intensive research to develop a novel plant system for producing nucleocapsid proteins of Hanta virus. As a result, the inventors have discovered that newly designed nucleotide sequences encoding nucleocapsid proteins of Hanta virus have been successfully expressed in plants and recombinant nucleocapsid proteins thus obtained have exhibited excellent antigenicity.

Accordingly, it is an object of this invention to provide a novle polynucleotide sequence encoding a nucleocapsid protein of Hanta virus . It is another object of this invention to provide a plant expression vector comprising a novle polynucleotide sequence encoding a nucleocapsid protein of Hanta virus .

It is still another object of this invention to provide a method for preparing a transformed plant to express a nucleocapsid protein of Hanta virus .

It is further object of this invention to provide a transformed plant to express a nucleocapsid protein of Hanta virus .

It is still further object of this invention to provide a method for preparing a recombinant nucleocapsid protein of Hanta virus .

It is another object of this invention to provide a recombinant nucleocapsid protein of Hanta virus.

It is still another object of this invention to provide a diagnosis kit for hemorrhagic fever with renal syndrome comprising a recombinant nucleocapsid protein of Hanta virus as an antigen.

It is further object of this invention to provide a method for detecting an antibody specific to hemorrhagic fever with renal syndrome.

Other objects and advantages of the present invention will become apparent from the detailed description to follow taken in conjugation with the appended claims and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 represents the expression cassette in the present vector for expressing nucleocapsid proteins of Hanta virus in plants. LB and RB represent left and right border of T-DNA, respectively, PoIyA represents a poly A signal sequence, P35S denotes cauliflower mosaic virus (CaMV) 35S promoter, GUS denotes a gene of β-glucuronidase and npt represents neomycin phosphotransferase II sequence.

Figs. 2a-2d show the existence of the present nucleotide sequences encoding nucleocapsid proteins in cloned pHS737 vector. M denotes 1 kb DNA ladder. Lane 1 corresponds to pHS737 vector digested with Xbal and BamRI, lane 2 corresponds to PCR product of the insert sequence and lane 3 corresponds to pHS737 vector containing the insert sequence digested with Xbal and BamUI .

Figs. 3a-3d represent the results of PCR analysis on the existence of the present nucleotide sequences encoding nucleocapsid proteins in transformed plants. M denotes 1 kb DNA ladder. Lane 1 corresponds to PCR product of positive standard plasmid carrying the nucleotide sequence encoding nucleocapsid protein, lane 2 is PCR product using chromosomal DNA of wild type Nicotiana tabacum, and lanes 3-10 are PCR products using chromosomal DNA of Nicotiana tabacum transformed.

Figs. 4a-4d show the result of the electrophoresis of nucleocapsid proteins purified from plant transformants on SDS- polyacrylamide gel. M denotes a molecular weight marker. Lane 1 corresponds to proteins from wild type Nicotiana tabacum and lane 2 corresponds to nucleocapsid protein of Hanta virus purified from Nicotiana tabacum transformed. Figs. 5a-5d show the results of Western blotting analysis of nucleocapsid proteins of Hanta virus expressed in Nicotiana tabacum transformed. Lanes 1-3 correspond to eluants of fractions 1-3, respectively. Lane 4 corresponds to proteins from wild type Nicotiana tabacum. Figs. 6a-6d represent the antigenicity of nucleocapsid proteins of Hanta virus expressed in plant transformants .

DETAILED DESCRIPTION OF THIS INVENTION

In one aspect of this invention, there is provided a polynucleotide sequence encoding a nucleocapsid protein of Hanta virus, in which said polynucleotide sequence comprises (i) a nucleotide sequence as set forth in SEQ ID N0:l, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7 or (ii) its partial nucleotide sequence. The polynucleotide sequences of this invention code for nucleocapsid proteins of viruses belonging to the genus Hanta virus. In particular, the sequences as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO:7 correspond to those of Hantaan virus, Sin Nombre virus, Seoul virus and Puumala virus, respectively. The nucleocapsid proteins of the genus Hanta virus encoded by the polynucleotide sequences of this invention can serve as an antigen.

The polynucleotide sequences of this invention coding for nucleocapsid proteins of Hanta virus have different sequences from natural-occurring sequences so as to maximize its expression in plants.

The polynucleotide sequences of this invention are designed to: (i) encode natural-occurring nucleocapsid proteins; (ii) have codon usage suitable in the expression in a plant; (iii) have GC content of more than about 50%; and (iv) avoid intron or intron-like sequences in a plant. This novel nucleotide sequences are significantly advantageous in expression in a plant .

The novel polynucleotide of this invention comprises either a full-length sequence or its partial sequence as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7. That is, the partial sequence could be considered as the present polynucleotide sequence only if the amino acid sequence encoded by the partial sequence exhibits antigenicity.

In another aspect of this invention, there is provide a plant expression vector, which comprises (i) the polynucleotide sequence of claim 1; (ii) a promoter that functions in plant cells to cause the production of an RNA molecule operably linked to said nucleotide sequence of (i) ; and (iii) a 3' -non- translated region that functions in plant cells to cause the polyadenylation of the 3' -end of said RNA molecule.

The term "operably linked" refers to functional linkage between a nucleic acid expression control sequence (such as a promoter, signal sequence, or array of transcription factor binding sites) and a second nucleotide sequence, wherein the expression control sequence affects transcription and/or translation of the nucleic acid corresponding to the second sequence. The plant vector system of this invention may be constructed according to the known methods in the art as described in Sambrook et al . , Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001), which is incorporated herein by reference. According to a preferred embodiment, the suitable plant- functional promoters includes the cauliflower mosaic virus

(CaMV) 35S promoter, the nopaline synthetase (nos) promoter, the Figwort mosaic virus 35S promoter, the sugarcane bacilliform virus promoter, the commelina yellow mottle virus promoter, the light-inducible promoter from the small subunit of the ribulose-1, 5-bis-phosphate carboxylase (ssRUBISCO) , the rice cytosolic triosephosphate isomerase (TPI) promoter, the adenine phosphoribosyltransferase (APRT) promoter of Arabidopsis, the rice actin 1 gene promoter, and the mannopine synthase and octopine synthase promoters .

According to a preferred embodiment, the suitable 3'-non- translated region to cause the polyadenylation of the 3'-end of RNA molecules includes that from the nopaline synthase gene of Agrobacterium tumefaciens (nos 3' end) (Bevan et al. , Nucleic Acids Research, 11 (2) :369-385 (1983) ), that from the octopine synthase gene of Agrobacterium tumefaciens, the 3' -end of the protease inhibitor I or II genes from potato or tomato, the CaMV 35S terminator.

It is preferable that the expression vector of this invention carries one or more markers which make it possible to select the transformed host, for example, genes (e.g., neomycin phosphotransferase (πptlJ) and hygromycin phosphotransferase (iipt) ) conferring the resistance to antibiotics such as neomycin, carbenicillin, kanamycin, spectinomycin and hygromycin. Preferably, the expression vector of this invention contains a gene coding a reporter molecule (e.g., luciferase and β-glucuronidase) .

In addition, the expression vector of this invention further comprises a nucleotide sequence to conveniently purify the nucleocapsid protein expressed, which includes but not limited to, glutathione S-transferase (Pharmacia, USA) , maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6X His (hexahistidine; Quiagen, USA) . The most preferable sequence is 6X His because it has not antigenicity and does not interfere desirable folding of the nucleocapsid protein of interest. Due to the additional sequence, the nucleocapsid protein expressed in plants can be purified with affinity chromatography in a rapid and feasible manner.

In still another aspect of this invention, there is provided a method for preparing a transformed plant to express a nucleocapsid protein of Hanta virus, which comprises the steps of: (a) transforming plant cells with the plant expression vector described above; (b) selecting plant cells transformed; and (c) obtaining said transformed plant by regenerating said transformed plant cells.

In further aspect of this invention, there is provided a transformed plant prepared by the method of -this invention.

According to the present method, the transformation of plant cells can be carried out by a wide variety of methods known to one skilled in the art. For example, 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 (U.S. Pat. Nos . 5,004,863, 5,349,124 and 5,416,011) can be performed for transformation. The most preferred is Agrobacterium-mediated transformation because it is possible to bypass the need for regeneration of an intact plant from a protoplast. Agrobacterium-mediated transformation is generally carried out using leaf disc and other tissue such as cotyledons and hypocotyls . This method is the most efficient in dicotyledonous plants .

The selection of transformed cells may be carried out by exposing the transformed cultures to a selective agent such as a metabolic inhibitor, an antibiotic and herbicide. Cells which have been transformed and have stably integrated a marker gene conferring resistance to the selective agent will grow and divide in culture.

The development or regeneration of plants from either plant protoplasts or various explants is well known in the art. The resulting transgenic rooted shoots are planted in an appropriate plant growth medium. The development or regeneration of plants containing the foreign gene of interest introduced by Agrobacterium may be achieved by methods well known in the art (U.S. Pat. Nos . 5,004,863, 5,349,124 and 5,416,011) .

Meanwhile, the present inventors have made attempts to develop novel transformed plants such as Nicotiana tabacum, Cucumis melo, Curcumis sativa, Citrullus vulgaris and Brassica campestris and as a result, have established the most efficient methods for the transformation of certain plant. Such methods have been filed for patent application (PCT/KR02/01461, PCT/KR02/01462 and PCT/KR02/01463) .

According to a preferred embodiment, the plant to be transformed is Nicotiana tabacum, Cucumis melo, Curcumis sativa, Citrullus vulgaris and Brassica campestris.

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

According to a specific embodiment of this invention using Agrobacterium system, the present method comprises: (a') inoculating an explant material from the plant with

Agrobacterium tumefaciens harboring a vector, in which the vector is capable of inserting into a genome of a cell from the plant and contains the following nucleotide sequences: (i) the nucleotide sequence described above encoding the nucleocapsid protein; (ii) a promoter that functions in plant cells to cause the production of an RNA molecule operably linked to the nucleotide sequence of (i) ; and (iii) a 3' -non- translated region that functions in plant cells to cause the polyadenylation of the 3' -end of the RNA molecule; (b' ) regenerating the inoculated explant material on a regeneration medium to obtain regenerated shoots; (c') culturing the regenerated shoots on a rooting medium to obtain a transformed plant, in which the transformed plant is capable of expressing the nucleotide sequence encoding the nucleocapsid protein.

According to the present invention, the preferred explant for transformation includes any tissue derived from seeds germinated. It is preferred to use cotyledon and hypocotyl and the most preferred is cotyledon. Seed germination may be performed under suitable dark/light conditions using an appropriate medium.

Transformation of plant cells is carried out with

Agrobacterium tumefaciens harboring Ti plasmid (Depicker, A. et al . , Plant cell transformation by Agrobacterium plasmids . In Genetic Engineering of Plants, Plenum Press, New York (1983)) . More preferably, binary vector system such as pBinl9, pRD400 and pRD320 is used for transformation (An, G. et al. , Binary vectors" In Plant Gene Res. Manual, Martinus Nijhoff Publisher, New York(1986) ; R. K. Jain, et al . , Biochem. Soc. Trans. 28 : 958 - 961 ( 2000 ) ) .

Inoculation of the explant with Agrobacterium tumefaciens involves procedures known in the art. Most preferably, the inoculation involves immersing the cotyledon in the culture of Agrobacterium tumefaciens to coculture. Agrobacterium tumefaciens is infected into plant cells. Preferably, acetosyringone is employed in the coculturing medium to promote infection of Agrobacterium tumefaciens into explant cell.

The explant transformed with Agrobacterium tumefaciens is regenerated in a regeneration medium, which allows successfully the regeneration of shoots. The regeneration medium used in this invention may contain nutrient basal medium such as MS, B5, LS, N6 and White's, energy source and vitamins, but not limited to. Sugars are useful as energy source and sucrose is the most preferable. In addition, the regeneration medium may further contain MES (2- (N-Morpholino) ethanesulfonic acid Monohydrate) as a buffering agent for pH change and agar as a solid support.

The regeneration medium must contain plant growth regulators. Cytokinin as a plant growth regulator may include but not limited to 6-benzylaminopurine (BAP) , kinetin, zeatin and isopentyladenosine and BAP is the most preferable cytokinin. The auxin (for example, 1-naphthalene acetic acid, indole acetic acid, (2,4-dichlorophenoxy) acetic acid) is contained in the regeneration medium of this invention. The transformed plant is finally produced on a rooting medium by rooting of regenerated shoots .

The transformed plant produced according to the present invention may be confirmed using procedures known in the art. For example, using DNA sample from tissues of the transformed plant, PCR is carried out to elucidate exogenous gene incorporated into a genome of the transformed plant.

Alternatively, Northern or Southern Blotting may be performed for confirming the transformation as described in Maniatis et al . , Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y- (1989) . Where the vector used for the transformation contains a gene coding for β- glucuronidase, it is possible to verify the occurrence of transformation by observing the color development of the regenerated cotyledon immersed in X-gluc (5_rbromo-4-chloro-3- indole-β-D-glucuronic acid) .

In still further aspect of this invention, there is provided a method for preparing a recombinant nucleocapsid protein of Hanta virus, which comprises the steps of: (a) transforming plant cells with the plant expression vector of this invention described above; (b) selecting plant cells transformed; (c) obtaining said transformed plant by regenerating said transformed plant cells,- and (d) preparing said recombinant nucleocapsid protein of Hanta virus from said transformed plant. In still yet further aspect of this invention, there is provided a recombinant nucleocapsid protein of Hanta virus prepared by the present method described above.

The recombinant nucleocapsid proteins of Hanta virus may be provided from the tissues derived from various transformed organs (e.g., stem, leave, root, fruit and seed, etc) and be obtained by purifying the extracts of the tissues.

In this invention, purification methods conventionally used in the art may be employed. For example, various methods including solubility fractionation by use of ammonium sulfate or PEG, size differential filtration and column chromatography (based on size, net surface charge, hydrophobicity or affinity) are available and usually the combination of the methods is used for purification. According to a preferred embodiment of this invention, the recombinant nucleocapsid protein is purified by affinity chromatography. For example, where the nucleocapsid protein contains 6X His at its terminal, Ni-NTA His-binding resin is generally employed to purify the protein of interest in a rapid and feasible manner.

In another aspect of this invention, there is provided a method for detecting an antibody specific to hemorrhagic fever with renal syndrome, which comprises the steps of: (a) preparing a testing fluid from an individual to be tested; (b) allowing for an antigen-antibody reaction between said testing fluid and the recombinant nucleocapsid protein of this invention as an antigen; and (c) detecting the occurrence of said antigen-antibody reaction. The present invention is applicable to a wide variety of biofluid samples (e.g., blood, serum, urine, sweat, etc.) . Preferably, the biofluid sample is blood, more preferably, serum.

For performing the present method, any analysis protocol using antigen-antibody reaction, i.e., immunoassay can be employed. The immunoassay procedures can be found in Enzyme Immunoassay, E. T. Maggio, ed. , CRC Press, Boca Raton, Florida, 1980; and Gaastra, W., Enzyme-linked immunosorbent assay (ELISA), in Methods in Molecular Biology, Vol. 1, Walker, J.M. ed., Humana Press, NJ, 1984. Most preferably, the present method is carried out in accordance with the procedure of ELISA

(enzyme-linked immunosorbent assay) . In particular, the present method executed in accordance with the ELISA method may comprise the steps of: (i) coating wells in a plate with anti- human antibodies; (ii) adding serum to the wells for reaction and washing; (iii) adding the recombinant nucleocapsid protein of Hanta virus to the wells for reaction and washing; (iv) adding to the wells antibodies specific to the nucleocapsid protein for reaction and washing; (v) adding a secondary antibody conjugated to enzyme catalyzing colorimetric reaction or fluorescent material to the wells; and (vi) measuring the binding of the secondary antibody to antibodies specific to the nucleocapsid.

The enzyme catalyzing colorimetric reaction includes, but not limited to, alkaline phosphatase, β-galactosidase and horseradish peroxidase. Where using alkaline phosphatase, bromochloroindolylphosphate (BCIP) , nitro blue tetrazolium (NBT) and ECF may be used as a substrate; in the case of using horseradish peroxidase, chloronaphtol, aminoethylcarbazol, diaminobenzidine, D-luciferin, lucigenin (bis-iV- methylacridinium nitrate) , resorufin benzyl ether, luminol, Amplex Red reagent (lO-acetyl-3, 7-dihydroxyphenoxazine, Pierce) , TMB (3 , 3 , 5, 5-tetramethylbenzidine) and ABTS (2, 2-Azine-di [3- ethylbenzthiazoline sulfonate]) may be used as a substrate.

In still another aspect of this invention, there is provided a diagnosis kit for hemorrhagic fever with renal syndrome, which comprises as an antigen the recombinant nucleocapsid protein of Hanta virus expressed in plants . The recombinant nucleocapsid protein of Hanta virus contained in the present kit is a recombinant protein obtained from transformed plants and shows no infectivity to animal such as human. Furthermore, the recombinant nucleocapsid protein exhibits excellent specificity to antibodies of human infected by Hanta virus and excellent sensitivity. These advantages are demonstrated in Examples described below.

Where the present kit is developed for ELISA, it may comprise plates (coated with nucleocapsid protein of Hanta virus) , buffer, a secondary antibody conjugated to enzyme catalyzing colorimetric reaction or fluorescent material and its substrate.

The present invention makes it possible to obtain recombinant nucleocapsid proteins with excellent antigenicity of Hanta virus in more cost-effective manner. Furthermore, the recombinant nucleocapsid proteins of this invention permit to diagnose hemorrhagic fever with renal syndrome with excellent specificity and sensitivity.

The following specific examples are intended to be illustrative of the invention and should not be construed as limiting the scope of the invention as defined by appended claims .

EXAMPLES

EXAMPLE I: Preparation of Novel Genes Encoding Nucleocapsid Proteins

The present genes coding for nucleocapsid proteins of

Hantaan virus, Sin Nombre virus, Seoul virus and Puumala virus were designed to (i) encode natural-occurring nucleocapsid proteins; (ii) have codon usage suitable in expression in a plant cell; (iii) have GC content of more than about 50%; and (iv) avoid intron or intron-like sequences in a plant. The nucleotide sequences thus newly designed were chemically synthesized in Plant Biotechnology Institute (PBI) , National Research Centre (SK, Canada) . The nucleotide sequences synthesized are indicated in SEQ ID NOs:1 (for Hantaan virus), 3 (for Sin Nombre virus) , 5 (for Seoul virus) and 7 (for Puumala virus) .

EXAMPLE II: Preparation of Plant Expression Vectors Carrying Nucleotide Sequences of Nucleocapsid

The nucleotide sequences encoding nucleocapsid proteins have Xbal and BarriRI restriction sites at their 5'- and 3' -ends, respectively.

The binary vector for plant expression, pHS737 (PBI, National Research Centre, Canada) was digested with Xbal and BarriΑI and purified. The nucleotide sequence encoding nucleocapsid and pHS737 vector were mixed and ligation buffer (KOSCO, Korea) and T4 ligase were added to the mixture to incubate for 1 hr at 16^°C . Thereafter, the resulting vector was transformed into CaCl₂-treated competent E. coli DH5α (Promega, USA) and then the transformed cells with kanamycin resistance were selected by culturing in LB medium containing kanamycin (100 mg/ml) .

From the selected clones, plasmid DNAs were isolated according to the alkaline method and the DNAs were amplified according to the polymerase chain reaction using a primer set: forward primer, 5'-AAT CTA GAA ATG GCT ACT AT-3' and reverse primer, 5'-AAG GAT CCC CGA GCT TGA GA-3' . The PCR was performed using Taq polymerase (Solgent, Korea) under the following condition: pre-denaturation at 96°C for 2 min followed by 35 cycles of annealing at 55^°C for 1 min, extension at 72^"C for 2 min and denaturation at 94"C for 1 min; followed by final extension at 72°C for 10 min.

Amplified products were analyzed by electrophoresis on 1.0% agarose gel to verify the existence of insert sequence in plasmids (see Figs. 2a-2d) .

EXAMPLE III: Transformation of Plant

Example III-l. Preparation of Transformed Agrobacterium tumefaciens GV3101

The recombinant vector, p^'HS737/HTNV, pHS737/SNV, pHS737/SEV or pHS737/PUV prepared in Example II was introduced into Agrobacterium tumefaciens GV3101 (Mp90) {Plant-cell-rep. , 15(11)799-803(1996)) by means of conjugation. Each of the transformed E. coli cells harboring the recombinant vector and Agrobacterium tumefaciens GV3101 (Mp90) was incubated in LB liquid medium to the exponential stage. The two types of cells were mixed at a ratio of 1:1 in eppendorf tube and centrifuged for 30 sec to concentrate, followed by the stationary culture for 1-2 days at 28°C. The eppendorf tubes were mildly shaken to suspend cells and the suspension was spread on LB solid medium containing 50 mg/L of kanamycin and 30 mg/L of gentamicin and incubated for 2 days at 28^°C to select colonies harboring the expression vector. The selected cells transformed with the expression vector were called as GV3101-pHS737/HTNV (for Hataan virus) , GV3101-pHS737/SMV (for Sin Nombre virus) , GV3101- pHS737/SEV (for Seoul virus) and GV3101-pHS737/PUV (for Puumala virus) , respectively. The selected Agrobacterium tumefaciens GV3101 was inoculated into super broth (BHI medium, pH 5.6) and incubated for 2 days at 28^°C. The cultures were used as an infection broth to plants.

Example III-2. Transformation of Plant Cells and Regeneration

The sterilized seeds of Nicotiana tabacum were seeded and cultivated in sterilized condition over 2 weeks for obtaining cotyledons. Agrobacterium tumefaciens GV3101(Mp90) transformed was incubated for 18 hr at 28°C in super broth containing 100 μ M acetosyringone (37 g/1 brain heart infusion broth(Difco) and 0.2% sucrose, pH 5.6) and the cotyledon was then immersed in the culture medium and incubated for 20 min to inoculate with Agrobacterium tumefaciens.

Then, the cotyledon was cocultured under dark culture condition at 25^°C for 2 days in coculturing medium [MSB5 (Murashige & Skoog medium including Gamborg B5 vitamins) containing 3.0% sucrose, 0.5 g/L MES (2- (N-Morpholino) ethanesulfonic acid Monohydrate) , 1.0 mg/L BAP and 0.1 mg/L NAA] . The culture was subjected to clean-up process to remove bacteria and the cotyledon was transferred to a selection medium (MSB5, 3.0% sucrose, 0.5 g/L of MES, 0.6% phytagel, 1.0 mg/L of BAP, 0.1 mg/L of NAA, 100 mg/L of kanamycin and 500 mg/L of carbenicillin, pH 5.6) and cultured at 26±1^°C and 4,000 lux for 2 weeks under 16-hr light condition, resulting in the generation of shoots. The elongated shoots considered to be transformed were selected and cultured on a regeneration medium

(MSB5, 3.0% sucrose, 0.5 g/L of MES, 0.6% agar, 0.01 mg/L of

BAP, 0.1 mg/L of NAA, 500 mg/L of carbenicillin and 100 mg/L of kanamycin) . Then, the regenerated shoots were transferred to a rooting medium (MSB5, 3.0% sucrose, 0.6% agar, 0.5 g/L of MES, 0.1 mg/L of NAA, 100 mg/L of kanamycin and 500 mg/L of carbenicillin) and cultured. The shoots with roots considered to be transformed were selected and analyzed.

EXAMPLE IV: PCR Analysis of Transformed Plants

The transformants prepared in Example III were verified as follows: Using ten mg of the shoots rooted that were considered to be transformed, a genomic DNA for. PCR analysis was obtained according to the method described by Edwards K., et al . {Nucleic Acids Research, 19: 1349(1991)) and then PCR analysis was performed.

The primer sets for PCR analysis of plant transformed with the nucleocapsid sequence are: for Hantaan virus, forward primer, 5'-AAT CTA GAA ATG GCT ACT AT-3' and reverse primer, 5'-AAG GAT CCC CGA GCT TGA GA-3'; for Sin Nombre virus, forward primer, 5'-AAT CTA GAA ATG TCA ACA CT-3' and reverse primer, 5'-AAG GAT CCC CGA GCT TAA GC-3'; for Seoul virus, forward primer, 5'-AAT CTA GAA ATG GCA ACT AT-3' and reverse primer, 5'-AAG GAT CCC TTA TAA CTT CA-3'; and for Puumala virus, forward primer, 5'-AAT CTA GAA ATG AGC GAT TTG-3' and reverse primer, 5'-AAG GAT CCC CAA TTT TGA GTG-3' .

The PCR was performed using Taq polymerase (Solgent, Korea) under the following condition: pre-denaturation at 96^°C for 2 min followed by 35 cycles of annealing at 55°C for 1 min, extension at 72°C for 2 min and denaturation at 94°C for 1 min; followed by final extension at 72^°C for 10 min. The amplified products were analyzed by electrophoresis on 1.0% agarose gel

(see Figs. 3a-3d) . In Figs. 3a-3d, M is molecular size marker, lane 1 is a positive control showing PCR product using wild type nucleotide sequence of each virus, lane 2 is PCR product using chromosomal DNA of wild type Nicotiana tabacum, and lanes 3-10 are PCR products using chromosomal DNA of Nicotiana tabacum transformed. As represented in Figs. 3a-3d, the transformed Nicotiana tabacum shows 1.3 kb DNA bands corresponding to nucleocapsid sequence of each virus.

Therefore, it is recognized that the plants in Example III are transformed with nucleocapsid gene and harbor stably the foreign gene.

EXAMPLE V: Purification of Nucleocapsid Protein from

Transformed Plants

Two g of leaves from the plant transformants were grinded in 2 ml of the homogenization buffer (250 mM sucrose, 1 M Hepes, 1 mM DTT and 1 mM MgCl₂) in the presence of liquid nitrogen and then centrifuged for 30 min at 12,000 rpm and 4^°C, followed by collecting supernatant. The supernatant was loaded on Probond column containing Ni resin (Quiagen GmbH, Germany) . Then, the column was washed twice with the absorption buffer (20 mM sodium phosphate, 500 mM sodium chloride, pH 7.8) to remove plant-derived proteins, after which the column was washed with the washing buffer (20 mM sodium phosphate, 500 mM sodium chloride, pH 6.0) . Thereafter, the column was washed with column volumes of the elution buffer (20 mM sodium phosphate, 500 mM sodium chloride, pH 4.0) to elute nucleocapsid protein of virus.

Since the nucleocapsid protein expressed in plants has 6 his residues at its amino-terminal, it binds to Ni resins having positive charge. After the completion of purification, the nucleocapsid protein was isolated from other plant-derived proteins and purified. The eluted nucleocapsid protein was concentrated using Centricon (Amicon Co., USA) . The eluted nucleocapsid protein was subject to SDS-PAGE and stained with coomassie blue to check its purity. As shown in Figs. 4a-4d, 48 kD band corresponding to nucleocapsid protein, which was not detected in wild type Nicotiana tabacum was observed. In particular, the eluant obtained using Ni resins showed nucleocapsid protein purified without other protein- derived proteins.

Protein quantification of the eluant was performed using Protein assay kit (Bio-Rad) in accordance with Bradford method. The eluant samples with the same concentration were electrophoresed on 8% polyacrylamide gel and were subjected to Western blotting analysis (Peter B. Kaufma et al . , Molecular and Cellular Methods in Biology and Medicine, 108-121, CRC press) . The Western blotting was performed as follows: The band on SDS-PAGE corresponding to nucleocapsid protein was transferred to PVDF membrane using Semi-Dry Transfer Units (Hoefer, USA) and the PVDF membrane was then dried. The PVDF membrane was incubated for 1 hr in 0.5% BSA/TBS (Tris-buffered saline) containing the primary monoclonal antibody specific to nucleocapsid protein (Center of Disease Control, Georgia, USA) and washed three times with TBS. Then, the membrane was incubated with the peroxidase-conjugated secondary antibody (peroxidase-labeled human IgG, KPL, USA) for 1 hr and washed with three times with TBS. The color development was allowed with ABTS developer (KPL, USA) for 30 min. As shown in Pigs. 5a-5d, the 48 kD band corresponding to nucleocapsid protein was observed.

EXAMPLE VI: Diagnosis of Hemorrhagic Fever with Renal Syndrome Using 96-well Plate Containing Nucleocapsid of Hanta Virus

Example VI-I. Measurement of Antibody Titer Specific to

Nucleocapsid of Hanta Virus To verify whether nucleocapsid proteins of Hanta virus purified from plant transformants could be used for diagnosis of hemorrhagic fever with renal syndrome, the antigen-antibody reaction of nucleocapsid proteins was examined using 96-well microtiter plate. The purified nucleocapsid proteins were serially diluted with PBS and placed to each well of 96-well microtiter plate, followed by allowing to stand for immobilization at 4^°C for 8 hr. The plate coated with nucleocapsid was washed three times with the washing buffer (0.05% Tween 20, 0.01 M phosphate, pH 7.6) to remove antigens not absorbed. The primary monoclonal antibodies specific to the nucleocapsid protein (Center of Disease Control, Georgia, USA) were serially diluted with the dilution buffer (0.1% BSA, 0.05% Tween 20, 20 rfl Trisma base, 150 mM NaCl) and their aliquots (100 μl) were inoculated to each well of the plate, followed by incubating to induce antigen-antibody reaction at room temperature for 1 hr.

Thereafter, the plate was washed with the washing buffer, 300 μl of the blocking buffer were added to each well and the plate was then allowed to stand for 1 hr at room temperature, followed by washing with the washing buffer. The peroxidase- conjugated secondary antibodies were added to each well and incubated for 1 hr at room temperature and then washed with the washing buffer. The ABTS developing substrate was added to each well and incubated for 30 min at room temperature, after which the reaction was stopped with 50 μl of the stop buffer. Finally, the absorbance at 405 nm was measured with ELISA reader

(Titertek, USA) . The results are summarized in Tables IA-ID. The abscissa of the Tables represents the dilution fold of Hanta virus nucleocapsid purified (before dilution: 73 ng, 20480-fold dilution: 36 pg) and the ordinate represents the dilution fold in which the initial titer of antibody to the nucleocapsid protein is considered 1. As indicated in Table IA- ID, the nucleocapsid proteins of this invention exhibit excellent antigenicity.

Figs. 6a-6d represent the comparison of antigenicity of the nucleocapsid protein purified from plant transformants and proteins isolated from wild type plant. As represented in Figs. 6a-6d, the nucleocapsid protein purified from plant transformants shows excellent antigenicity; however, the protein isolated from wild type plant shows little or no antigenicity. TABLE IA. Antigenicity of Nucleocapsid protein of Hantaan virus

TABLE IB . Antigenicity of Nucleocapsid protein of Sin Nombre virus

Example VI-2. Diagnosis of Hemorrhagic Fever with Renal Syndrome using Nucleocapsid Proteins Expressed in Plant Transformants

To verify whether nucleocapsid proteins of Hanta virus purified from plant transformants could be used for diagnosis of hemorrhagic fever with renal syndrome, the antibody titer in patient serum suffering from hemorrhagic fever with renal syndrome was measured.

100 μl of anti-human IgM antibody (goat anti-human IgM, KPL, USA) diluted in PBS were aliquoted to each well of 96-well plate and incubated for 8 hr at 4^"C for coating. The plate was then washed three times with the washing buffer (0.05% Tween 20, 0.01 M phosphate, pH 7.6) to remove antibodies not absorbed. Patient serum was diluted in the dilution buffer (0.1% BSA, 0.05% Tween 20, 20 mM Trisma base, 150 tnM NaCl) and its aliquot

(100 μl) was inoculated to each well of the plate, followed by allowing for reaction at room temperature for 1 hr.

100 μl of the diluted nucleocapsid proteins of Hanta virus were plated to each well of the plate and allowed to stand for inducing the secondary antigen-antibody reaction at room temperature for 1 hr. After washing with the washing buffer, 100 μl of monoclonal antibodies specific to the nucleocapsid protein (10 μg/ml, Center of Disease Control, Georgia, USA) were inoculated to each well of the plate and incubated at room temperature for 1 hr, followed by washing.

Thereafter, the peroxidase-conjugated secondary antibodies (peroxidase labeled-anti human IgG, KPL, USA) were added to each well, incubated for 1 hr at room temperature and then washed with the washing buffer. The ABTS developing substrate (KPL, USA) was added to each well and incubated for 30 min at room temperature, after which the reaction was stopped with 50 μl of the stop buffer. Finally, the absorbance at 405 nm was measured with ELISA reader (Titertek, USA) . The results are summarized in Tables 2A-2D. The results are determined as positive when the OD value is more than 1.5. TABLE 2A. Measurement of Antibody Titer Using Nucleoeapsid protein of Hantaan virus

TABLE 2B. Measurement of Antibody Titer Using Nucleocapsid protein of Sin Nombre virus

TABLE 2C. Measurement of Antibody Titer Using Nucleocapsid protein of Seoul virus

TABLE 2D. Measurement of Antibody Titer Using Nucleocapsid protein of Puumala virus

As indicated in Tables 2A-2D, all of normal serums exhibit the antibody titer of less than 100 but serums of patients suffering from hemorrhagic fever with renal syndrome show the antibody titer ranging from 200 to 25600. It could be recognized that the difference in antibody titer is distinctly evident between normal and patient of hemorrhagic fever with renal syndrome. These results demonstrate that the antigen- antibody reaction using Hanta virus nucleocapsid antigen expressed in plant transformants occurs with higher specificity, permitting to diagnose hemorrhagic fever with renal syndrome in more reliable manner.

Having described a preferred embodiment of the present invention, it is to be understood that variants and modifications thereof falling within the spirit of the invention may become apparent to those skilled in this art, and the scope of this invention is to be determined by appended claims and their equivalents.

Claims

What is claimed is:

1. A polynucleotide sequence encoding a nucleocapsid protein of Hanta virus, in which said, polynucleotide sequence comprises (i) a nucleotide sequence as set forth in SEQ ID N0:l, SEQ ID N0:3, SEQ ID NO:5 or SEQ ID NO:7 or (ii) its partial nucleotide sequence.

2. A plant expression vector, which comprises (i) the polynucleotide sequence of claim 1; (ii) a promoter that functions in plant cells to cause the production of an RNA molecule operably linked to said nucleotide sequence of (i) ; and (iii) a 3' -non-translated region that functions in plant cells to cause the polyadenylation of the 3'-end of said RNA molecule.

3. A method for preparing a transformed plant to express a nucleocapsid protein of Hanta virus, which comprises the steps of:

(a) transforming plant cells with the plant expression vector of claim 2;

(b) selecting plant cells transformed; and

(c) obtaining said transformed plant by regenerating said transformed plant cells.

4. A transformed plant prepared by the method of claim 3.

5. A method for preparing a recombinant nucleocapsid protein of Hanta virus, which comprises the steps of:

(a) transforming plant cells with the plant expression vector of claim 2; (b) selecting plant cells transformed;

(c) obtaining said transformed plant by regenerating said transformed plant cells; and

(d) preparing said recombinant nucleocapsid protein of Hanta virus from said transformed plant.

6. A recombinant nucleocapsid protein of Hanta virus prepared by the method of claim 5.

7. A diagnosis kit for hemorrhagic fever with renal syndrome, which comprises the recombinant nucleocapsid protein of Hanta virus of claim 6 as an antigen.

8. A method for detecting an antibody specific to hemorrhagic fever with renal syndrome, which comprises the steps of:

(a) preparing a testing fluid from an individual to be tested;

(b) allowing for an antigen-antibody reaction between said testing fluid and the recombinant nucleocapsid protein of claim 6 as an antigen,- and

(c) detecting the occurrence of said antigen-antibody reaction.