KR100454727B1 - Diagnostic kit of hemorrhagic fever with renal syndrome using Baculovirus expressed nucleocapsid protein derived from Hantann virus 91011 - Google Patents

Abstract

The present invention relates to a diagnostic kit for hemorrhagic fever with renal syndrome (HFRS) using a hancleovirus-derived nucleocapsid protein. Baculovirus transfer vector containing the S geneome segment encoding, a recombinant baculovirus transfected with the baculovirus transfer vector, a nucleus expressed from a recombinant baculovirus in insect cells The present invention relates to an antibody measuring method and diagnostic kit for hemorrhagic fever of nephrotic syndrome using a cleocapsid protein and a nucleocapsid antigen protein purified therefrom. The diagnostic kit of nephrotic hemorrhagic fever of the present invention is more convenient than the conventional fluorescence assay using the Hantan virus nucleocapsid protein having the highest antigenicity, and is excellent in sensitivity and specificity, which is useful for diagnosis and analysis of hemorrhagic fever of nephrotic syndrome. Can be used.

Description

Diagnostic kit of hemorrhagic fever with renal syndrome using Baculovirus expressed nucleocapsid protein derived from Hantann virus 91011}

The present invention relates to a diagnostic kit for renal hemorrhagic hemorrhagic fever using a Hantan virus-derived nucleocapsid protein, and specifically, a small gene segment encoding a nucleocapsid protein of Hantan virus 91011 strain. ), A baculovirus transfer vector, a recombinant baculovirus transformed from the baculovirus transfer vector, a nucleocapsid protein expressed in an insect cell infected with the recombinant baculovirus, The present invention relates to an antibody measurement method and diagnostic kit for renal syndrome hemorrhagic fever using the purified nucleocapsid antigen protein.

Hemorrhagic fever with renal syndrome (HFRS) is an acute infectious disease caused by viruses belonging to the genus Hantavirus of the family Bunyaviridae . It was widely known as a fever, and it was called by various names in various parts of the world.However, in 1982, the World Health Organization decided to call this disease the name of a different disease in the country.

Patients with nephrotic hemorrhagic fever that are prevalent in Korea are mainly caused by infection with Hantan virus and Seoul virus, and patients with nephrotic hemorrhagic fever caused by Hantan virus and Seoul virus have severe clinical symptoms. Mortality of 4-7%. Patients with nephrotic hemorrhagic fever infected with these viruses have symptoms of acute febrile disease with chills, headache, vomiting and abdominal pain after incubation for 1 to 4 weeks after the virus infection. It causes bleeding in the intestines. After that, severe back pain, vomiting and conjunctival hemorrhage are observed. After about one week, hypotension and shock symptoms occur, and the urinary tract caused by nephritis progresses, and in some cases, kidney failure occurs. Otherwise, after the urinary period is progressed through the urinary system to reach the recovery phase, it normally takes about 1 to 2 months to recover, and even after recovery, kidney function may remain as a sequelae.

The host animal of the hantan virus is Apodemus agrarius, which is a wild field mouse, and the host animal of the Seoul virus is known to be a house mouse and an experimental white mouse. The host animal of the virus does not cause the same symptoms as when infected with a human virus, but grows while being present in the natural ecosystem in an infected state. Nephrotic pathogenesis of hemorrhagic fever is present in the air after the virus excreted in saliva, urine, and stool from host animals, rats, mice and experimental rats infected with Hantan virus and Seoul virus. It is transmitted to susceptible animals and humans.

On the other hand, Hantan virus is a small virus of about 80 to 100 nm in size, including the negative ribonucleic acid (RNA) gene, which is composed of three large, medium and small fragments, like the virus belonging to other fields. . The ribonucleic acid of hantan virus forms a complex by combining with nucleocapsid protein and polymerase ptorein capable of copying the virus ribonucleic acid gene, and the Golgi cell membrane into which the virus-derived glycoprotein is inserted. Derived capsular proteins surround these complexes. The small gene segment, which is a three-segment viral gene like the Hantan virus, contains the genetic information of the nucleocapsid protein that binds to ribonucleic acid, and the medium gene segment is present in the membrane. Contains the genetic information of the viral glycoprotein, the glycoprotein is responsible for recognizing the receptor (receptor) of the cell (virus) during virus infection and produces a neutralizing antibody of the virus. In addition, a large gene segment contains genetic information of a polymerase protein capable of replicating a viral ribonucleic acid gene. Among these virus-derived proteins, antibodies to nucleocapsid proteins encoded by small genes are first produced in large quantities, and viruses belonging to the hantavirus show high cross-reaction between antibodies to the nucleocapsid proteins. In particular, the cross-reaction between the Hantan virus and Seoul virus is very high, it is almost impossible to distinguish between the two viruses by the fluorescent antibody method and immunoenzyme antibody method commonly used for virus detection.

About 10 to 150,000 cases of nephrotic hemorrhagic fever caused by the infection of Hantan virus and Seoul virus occur in Asia and about 1,000 cases occur in Korea every year. Since the formalin inactivation vaccine developed in Green Cross has been used, the number of patients with nephrotic hemorrhagic fever has decreased rapidly. In addition, studies on the development of diagnostic kits and diagnostic reagents for diagnosing hemorrhagic fever patients with nephrotic syndrome using antigens produced during vaccine development are being actively conducted. Currently, viral antigen proteins used in the development of diagnostic kits and vaccines for the diagnosis of hemorrhagic fever with nephrotic syndrome are called Hanmalan virus, which is obtained by injecting 84/105 weeks of Hantan virus into the brain of a 1-day-old cow, and multiplying it with formalin. Antigen obtained by activation treatment. However, in the case of the diagnostic kit and vaccine using antigens propagated using animal brain tissue, a large amount of virus is excreted from infected animals in the process of propagating viruses using animal brain tissue, resulting in animal breeders and viruses. Infecting people who are involved in the process of purifying antigens may cause hemorrhagic fever with nephropathy, and incompletely inactivated in the process of inactivating a large amount of proliferated virus with formalin, which has been a problem in the manufacture of polio vaccines in the past. Viruses can infect people who are performing diagnostic tasks. In addition, animal brain tissue-derived protein components remaining in the Hantan virus antigen used in the diagnostic kit may be misdiagnosed as pseudo-positive in the test serum, which has raised the problem of inhibiting accurate diagnosis.

In order to solve the problems of the conventional nephrotic hemorrhagic fever diagnostic kit, the present inventors use a baculovirus transfer vector that grows only in insect cells and is not infectious to humans and mammals. By using the Hantanvirus nucleocapsid antigen protein obtained by expressing a bovine gene, the gene of Hantanvirus nucleocapsid protein, which has the highest antigenicity, the nucleocapsid protein of Hantan virus shows the same immunological antigenicity as that of the original virus. The present invention has been completed by providing a diagnostic method and a diagnostic kit for detecting hemorrhagic fever caused by Hantan virus and Seoul virus by using it as an antigen.

It is an object of the present invention to provide a diagnostic kit for nephrotic hemorrhagic fever using Hantan virus-derived nucleocapsid protein for accurate and convenient diagnosis of nephrotic hemorrhagic fever.

Figure 1a is a comparison of the small gene (SEQ ID NO: s) of the small gene (SEQ ID NO: 91011 strain) and the Hantan virus 76/118 strain,

Figure 1b is the nucleotide sequence of Figure 1a is continued,

2 is a comparison of amino acid sequences derived from bovine gene nucleotide sequences of Hantan virus 91011 strain and Hantan virus 76/118 strain,

Figure 3 is a schematic diagram showing the manufacturing process of baculovirus transfer vector (Baculovirus transfer vector) containing a bovine gene of Hantan virus 91011,

Figure 4 shows the results of electrophoresis by cutting the pCR-HTNs vector cloned bovine gene of the Hantan virus 91011 strain with restriction enzymes Xho I and EcoR I,

FIG. 5 is a schematic diagram illustrating a process for producing a recombinant HTN-s-Baculovirus transfected with a pCR-HTNs vector cloned with a bovine gene of Hantan virus 91011.

FIG. 6 shows the results of confirming by the polymerase chain reaction that the bovine gene of the Hantan virus 91011 strain was inserted into the recombinant recombinant baculovirus (HTN-s-Baculovirus).

Lane 1: 1 kb DNA ladder

Lane 2: HTN-s-Baculovirus

Lane 3: wild type baculovirus

7 is a schematic diagram illustrating a process for purifying the Hantan virus 91011 strain nucleocapsid protein expressed in Sf9 insect cells transfected with the recombinant HTN-s-baculovirus,

FIG. 8 shows the results obtained by staining the Hantan virus nucleocapsid protein obtained by each purification step of FIG. 7 with Coassie blue after electrophoresis on an SDS-polyacrylamide gel.

M: Prestained Molecular Weight Marker

Lane 1: Sf9 cell lysate infected with recombinant baculovirus

Lane 2: Sf9 cell lysate not infected with the recombinant baculovirus

Lane 3: Eluate after binding Sf9 cell lysate infected with recombinant baculovirus to nickel resin column

Lanes 4 and 5: the effluent washed with column wash buffer

Lanes 6, 7 and 8: protein eluted with column elution buffer

FIG. 9 shows Western blot analysis of the Hantanvirus 91011 strain nucleocapsid protein obtained by purification from Sf9 insect cells transfected with the recombinant HTN-s-baculovirus, followed by electrophoresis on an SDS-PAGE gel. blotanalysis),

M: Prestained Molecular Weight Marker

Lane 1: Sf9 cell lysate infected with recombinant baculovirus

Lane 2: Eluate after binding Sf9 cell lysate infected with recombinant baculovirus to nickel resin column

Lanes 3 and 4: drains washed with column wash buffer

Lanes 5 and 6: protein eluted with column elution buffer

FIG. 10 shows the results of analysis of serum of patients with renal hemorrhagic fever or normal persons using a nitrocellulose membrane adsorbed with a nucleocapsid protein purified from Sf9 insect cells transfected with the recombinant HTN-s-baculovirus.

M: Prestained Molecular Weight Marker

Lane 1: Serum of Patient with Renal Syndrome Hemorrhagic Fever 1

Lane 2: Serum of Nephrotic Hemorrhagic Fever Patient 2

Lane 3: Serum of Nephrotic Hemorrhagic Fever Patient 3

Lane 4: Serum of Nephrotic Hemorrhagic Fever Patient 4

Lane 5: serum of normal person 1

Lane 6: serum of normal 2

In order to achieve the above object, the present invention is a baculovirus transfer vector comprising a bovine gene encoding a Hantan virus nucleocapsid protein; A recombinant baculovirus transfected with the baculovirus transfer vector; It provides a Hantan virus nucleocapsid protein expressed from the recombinant recombinant baculovirus in insect cells.

In addition, the present invention provides a method for measuring the antibody of hemorrhagic fever with nephrotic syndrome using the Hantanvirus nucleocapsid protein expressed from the recombinant baculovirus.

In addition, the present invention provides a nephrotic hemorrhagic fever diagnostic kit comprising a nucleocapsid antigen protein derived from a Hantan virus small gene.

Finally, the present invention provides a biological microchip coated on the surface of the hantan virus nucleocapsid antigen protein for the diagnosis of nephrotic bleeding fever.

Hereinafter, the present invention will be described in detail.

The present invention provides an expression vector comprising a bovine gene encoding a Hantan virus nucleocapsid protein; A baculovirus transfer vector comprising a hantan virus nucleocapsid antigen protein gene obtained from the expression vector; A recombinant baculovirus transfected with the baculovirus transfer vector; It provides a Hantan virus nucleocapsid protein expressed from the recombinant recombinant baculovirus in insect cells.

We used Hantan virus 91011 strain isolated from spleen tissue of a patient who died of nephrotic hemorrhagic fever. As a result of analyzing the gene sequencing, the Hantan virus 91011 strain is composed of 1696 nucleotide sequences as shown in SEQ ID NO: 1 and shows about 7% difference from the nucleotide sequence of 76/118 strain, which is the prototype of the Hantan virus. (See FIGS . 1A and 1B ), the amino acid sequences derived from the respective base sequences showed a difference of about 3% (see FIG. 2 ).

In order to diagnose nephrotic hemorrhagic fever using a nucleocapsid protein having the best antigenicity among all of the genes of the Hantan virus, the present inventors first provide an expression vector comprising a Hantan virus small gene region encoding a nucleocapsid protein ( 3 ). The expression vector was confirmed that the target gene was correctly inserted by the restriction enzyme treatment (see Figure 4 ).

In addition, the present inventors cloned the nucleocapsid protein gene fragment obtained from the expression vector to provide a baculovirus transfer vector that is infected and expressed only in insect cells without being infected with humans and animals. The Hantanvirus nucleocapsid protein gene inserted into the baculovirus transfer vector was made to be expressed under the control of the polyhedrin promotor of baculovirus.

It was confirmed that the baculovirus transfer vector containing the Hantan virus nucleocapsid gene was correctly inserted into the E. coli transformant by restriction enzyme treatment, and the nucleotide sequence of the Hantan virus nucleocapsid gene inserted into the baculovirus transfer vector was inserted. The analysis confirmed that the Hantanvirus nucleocapsid protein gene described in SEQ ID NO: 5 was inserted in the correct direction.

In addition, the present invention provides a recombinant baculovirus expressing the Hantan virus nucleocapsid protein in Sf9 insect cells.

The recombinant baculovirus was cotransfected into insect cell lines using a baculovirus transfer vector with a baculovirus DNA using a cationic liposome mediation method, and finally, in a medium containing X-gal. Screened (see FIG. 5 ).

Polymerase chain reaction (PCR) and Western blot analysis were performed to confirm that the recombinant baculovirus contains a Hantan virus nucleocapsid protein gene and actually expresses the protein in insect cells ( 6, 8 and 9 ), which was deposited on August 18, 2000 to the Biotechnology Research Institute Gene Bank (Accession Number: KCTC 0854BP).

In addition, the present invention provides a Hantan virus nucleocapsid protein expressed in insect cells from a recombinant baculovirus.

Hantanvirus nucleocapsid protein

1) infecting the insect cell with the recombinant baculovirus;

2) inducing the expression of a Hantavirus nucleocapsid protein in insect cells;

And 3) separating and purifying the protein from insect cells.

According to the expression vector used in the step 2, such as isopropyl-β-D-thiogalactopyranoid (IPTG) or X-gal to induce the expression of the protein of interest from the recombinant baculovirus in insect cells It may be added to the medium as an inducer.

In the step 3, the method for purifying the Hantan virus nucleocapsid protein is obtained by crushing insect cells using lysis buffer and ultrasonic crushing to obtain crushed precipitates, and then crushing the bacteria by the appropriate chromatography according to the expression vector used. It consists of purifying. In the present invention, six histidine residues present at the amino terminus of the Hantanvirus nucleocapsid protein expressed from the recombinant baculovirus are eluted with a cation-containing nickel resin column to make the cell-derived protein or nonspecific. Pure Hantanvirus nucleocapsid protein from which bound baculovirus-derived protein was removed was isolated and purified (see FIG. 7 ).

The separated and purified proteins were electrophoresed on a SDS-PAGE gel for each purification step, followed by staining with Comassie blue. The SDS-PAGE gel was transferred to a nitrocellulose membrane and subjected to Western blot analysis. It was confirmed that the hantan virus nucleocapsid protein was purely isolated and purified (see FIGS . 8 and 9 ).

In addition, the present invention provides a method for measuring the antibody of hemorrhagic fever with nephrotic syndrome using the Hantan virus nucleocapsid protein expressed in Sf9 insect cells using the recombinant baculovirus.

The antibody measurement method of the hemorrhagic fever with nephrotic syndrome is based on the Hantan virus and Seoul virus.

Collecting a sample from a patient with nephrotic bleeding hemorrhagic fever (Step 1) and detecting the antibody against the Hantan virus bovine gene-derived nucleocapsid antigen protein (Step 2).

The sampling step of step 1 may be preferably performed through a general serum separation method.

As a specific antibody detection method used in step 2 of the antibody measurement method of hemorrhagic fever with nephrotic syndrome, any assay method based on antigen-antibody binding may be used. Among them, the ELISA (enzyme-linked immunosorbent assay) method and the immunoblot method which are commonly used for the analysis of antigen-antibody binding are preferable.

In a preferred embodiment, the present inventors provide immunoblot and ELISA methods for measuring antibodies by adsorbing a Hantanvirus nucleocapsid antigen protein on a substrate to measure antibodies of nephrotic hemorrhagic fever.

The method of measuring the antibody by adsorbing the antigenic protein of the hemorrhagic fever of nephrotic syndrome to a substrate

1) adsorbing a Hantanvirus nucleocapsid antigen protein to a substrate;

2) washing by adding serum of a sample to the membrane to which the antigen protein is adsorbed, and then washing it;

3) reacting by adding an antibody having a chromophore or a fluorescent substance bound thereto; And

4) Induce color development by adding a coloring agent to the membrane and then observing the specificity of the antibody reaction.

The substrate of step 1 may be a nitrocellulose membrane, a 96 well plate synthesized with a polyvinyl resin, a 96 well plate synthesized with a polystyrene resin, a glass slide glass, and the like. In the present invention, the anti-neoplastic hemorrhagic fever antibody was measured by adsorbing a Hantan virus nucleocapsid antigen protein on the nitrocellulose membrane.

Peroxidase, alkaline phosphatase (Alkaline Phosphatase) may be used as the chromophore bound to the antibody in step 3, and fluorescent materials may be used such as FITC, RITC, and the like. Antibodies were used.

In Step 4, 4CN (4-chloro-1-naphtol), DAB (Diaminobenzidine), and AEC (Aminoethyl carbazole) may be used. In the present invention, 4CN decomposed and developed by the peroxidases was used.

The inventors of the present invention diagnosed the serum of patients with nephrotic hemorrhagic fever and normal persons by adsorbing Hantan virus nucleocapsid protein to the nitrocellulose membrane, and found that only Hantan virus nucleocapsid protein was detected only in the serum of human confirmed patients with fluorescence antibody method. Confirmation (see FIG. 9 ).

In addition, the method of measuring the antibody using the ELISA method

1) adsorbing an anti-human antibody to the well plate;

2) reacting and washing the sample by adding serum of the sample to the well plate;

3) reacting with the addition of a Hantan virus nucleocapsid antigen protein;

4) adding and reacting a monoclonal antibody against said nucleocapsid antigen protein;

5) washing the well plate and reacting by adding a secondary antibody bound to a chromophore or a fluorescent substance; And

6) Color development is performed by adding a color substrate and measuring absorbance with an ELISA reader.

The anti-human antibodies of step 1 may be IgG, IgM, Ig A, Ig D and Ig E, and in the present invention, their antibody titers were measured using antibodies against IgG and IgM.

Peroxidase, alkaline phosphatase (Alkaline phosphatase) may be used as the chromophore bound to the antibody in step 5, fluorescent material may be used FITC, RITC, etc., in the present invention peroxidase is bound Antibodies were used.

In step 6, the colorant substrate solution may be ABTS [2, 2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)], OPD (o-Phenylenediamine), TMB (Tetramethyl Benzidine) can be used, in the present invention ABTS was used that was degraded by peroxidase and developed.

The ELISA method was used to diagnose nephrotic hemorrhagic fever in patients with hemorrhagic fever with nephrotic syndrome and in normal and uninfected animals with Hantan virus and Seoul virus. Was detected in the serum of patients with nephrotic hemorrhagic fever and infected animals, the antibody titer was very high. ELISA diagnostic method using the Hantan virus nucleocapsid protein of the present invention was more effective than the fluorescent antibody method. It was confirmed that the specificity and sensitivity for the very good (see Table 1 and Table 2 ).

In the antibody measurement method of hemorrhagic fever of nephrotic syndrome of the present invention, the antibody to the antigenic protein can be detected by immobilizing the antigenic protein on a biological microchip. For example, samples can be analyzed in large quantities using known biological microchips and automated microarray systems in addition to ELISA methods.

In addition, the present invention provides a nephrotic hemorrhagic fever diagnostic kit comprising a nucleocapsid antigen protein derived from the Hantan virus small gene to diagnose nephrotic hemorrhagic fever.

The Hantan virus-derived nucleocapsid antigen protein included in the nephrotic hemorrhagic fever diagnostic kit may use a recombinant protein expressed by a recombinant virus from insect cells. As a vector for producing a recombinant virus, it is preferable to use a transfer vector capable of expressing a recombinant protein in insect cells without infecting humans and animals. In the present invention, a baculovirus transfer vector is used as a preferred embodiment. It was.

The diagnostic kit may diagnose hemorrhagic fever with nephrotic syndrome by quantitatively or qualitatively analyzing the antibody of the antigenic protein, and may use an ELISA method for analysis of the antibody. For example, the diagnostic kit may be provided to perform an ELISA using a 96 well microtiter plate coated with the recombinant antigen protein on a surface thereof. Such diagnostic kits may include antigenic proteins, appropriate buffers, standard antibodies, secondary antibodies labeled with chromogenic enzymes or fluorescent materials, chromogenic substrates, and the like.

In addition, the diagnostic kit may use an automated analysis method using a biological microchip for diagnosing hemorrhagic fever with nephrotic syndrome. For example, the diagnostic kit may be configured to perform ELISA using a chip such as a glass slide coated on the surface of the recombinant antigen protein. Such diagnostic kits include biological microchips with antigenic proteins immobilized on the surface, suitable buffers, standard antibodies, secondary antibodies, and the like.

Finally, the present invention provides a biological microchip coated on the surface of the Hantan virus nucleocapsid antigen protein for diagnosing nephrotic hemorrhagic fever.

Microchips are manufactured by combining molecular biology knowledge with mechanical and electronic engineering techniques, with hundreds to hundreds of thousands of DNA or proteins densely packed in very small spaces. This allows microchips to search for at least several hundred genes or proteins at the same time in a short time, making it easier and faster to discover and diagnose new genes than with conventional biotechnologies. Microchips can be divided into oligonucleotide chips and cDNA chips according to the size of the genetic material and can be selected and used according to the genes and experimental purposes to be found. Therefore, the microchip using the Hantan virus nucleocapsid antigen protein can search the serum of numerous patients suspected of nephrotic bleeding fever simultaneously and quickly.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Cloning of the Hantan Virus 91011 Address Gene Encoding Nucleocapsid Protein

In order to diagnose nephrotic hemorrhagic fever using the nucleocapsid protein having the highest antigenicity among all the Hantan virus genes, the present inventors first cloned the hantanvirus subgene region encoding the nucleocapsid protein.

To obtain the Hantanvirus bovine gene DNA, VeroE6 (Eagles minimal essential medium, EMEM) in 10% fetal bovine serum, 100 units / ml penicillin and 100 μg / ml streptomycin Vero C1008, ATCC CRL 1586) cells were extracted from the RNA of 91011 strain of Hantan virus cultured for 7 days. Specifically, 1000 μl of Trizol solution and 200 μl of chloroform were added to 100 μl of the culture of Hantan virus 91011 strain, and allowed to stand in an ice bath for 20 minutes, followed by centrifugation at 4 ° C. and 15,000 rpm for 20 minutes. To recover only the supernatant. The same amount of isopropanol (isopropanol) was added to the supernatant, followed by mixing for 1 hour at -20 ° C. The supernatant was centrifuged at −4 ° C. and 15,000 rpm for 30 minutes to discard the supernatant and recover only the sinking RNA precipitate. 1 ml of 70% ethanol (Ethanol) was added to the RNA precipitate, followed by light washing, and then DEPC-treated distilled water was added to dissolve viral RNA of Hantan virus 91011 strain.

In order to synthesize complementary DNA of viral RNA of the isolated Hantan virus 91011 strain, RT-PCR was performed using the 3 ′ terminal primer as described in SEQ ID NO: 2 . In order to obtain a gene fragment of the ORF (Open Reading Frame) region encoding the nucleocapsid protein site of the Hantan virus 91001 strain, the complementary DNA of the Hantan virus 91011 strain synthesized as described above and a restriction enzyme XhoI recognition site were included. A polymerase chain reaction (PCR) was performed using the 3 'terminal downstream primer described in SEQ ID NO: 3 and the upstream primer of the 5' terminal depicted in SEQ ID NO: 4 . DNA polymerase (DNA polymerase) used in PCR is a mixture of Vent DNA polymerase (Invitrogen, Netherland) and Taq DNA polymerase (Invitrogen, Netherland) in a certain amount to minimize the error occurrence rate due to non-specific binding during gene amplification. Electrophoresis was performed on the amplified PCR product on an agarose gel to confirm that a DNA band having a size of about 1.7 kb was detected, and then the DNA band at the position was cut from the gel, and a GinClean kit (BIO 101, USA) was used. Purification by Taq DNA polymerase was added to the purified DNA and reacted with dATP at 72 ° C. for 5 minutes to generate an A base overhang at the 3 ′ end. As such, the nucleocapsid protein gene having the A base overhang generated at the 3 ′ end was inserted into the T base overhang of the pCR II vector (Invitrogen, Netherland) to prepare a cloning vector (pCR II-HTN S) . ). The cloning vector pCR II-HTN S was transformed into Escherichia coli ( E. coli INVαF ') to select an E. coli transformant comprising a Hantan virus bovine gene fragment.

In order to confirm that the bovine gene encoding Hantan virus 91001 strain nucleocapsid protein was correctly inserted into the vector, the plasmid DNA of the expression vector pCR II-HTN S which was amplified in a large amount from the E. coli transformant was transformed into restriction enzymes EcoR I and Xho I. After cutting, electrophoresis was performed to examine band shape. As a result, the Hantan virus 91011 element gene fragment of about 1.7 kb size and the vector gene fragment of about 3.9 kb size were detected, indicating that the Hantan virus 91011 element gene was correctly inserted into the expression vector pCR II-HTN S of the present invention. It was confirmed ( FIG. 4 ).

Example 2 Preparation of Baculovirus Transfer Vector pBlueBacHis-HTN011S

To prepare a baculovirus transfer vector containing a bovine gene of Hantan virus 91011 strain, 100 μg / ml of the E. coli transformant transformed with the expression vector pCR-HTN S prepared in Example 1 above. Amplified pCR-HTN S clone was obtained by culturing in LB culture medium containing Ampicillin. PCR-HTN S plasmid DNA isolated from this was digested with restriction enzymes XhoI and EcoRⅠ, electrophoresis was isolated from 1.2% agarose gel, stained with ethidium bromide, and irradiated with UV light to about 1.7 kb in size. It was confirmed that nucleocapsid protein gene band was detected. The gene bands were cleaved from the gels and purified by JinClean kit. The pBlueBacHis plasmid (Invitrogen, Netherland) DNA treated with restriction enzymes XhoI and EcoR I was purified in the same manner as described above, mixed with the nucleocapsid protein gene at the same molar concentration, and T4 DNA Ligase (GIBBCO-BRL). , USA) was used for 12 hours in a 4 ℃ water bath to prepare a baculovirus transfer vector pBlueBacHis-HTN S containing a Hantan virus nucleocapsid protein gene. The Hantanvirus nucleocapsid protein gene inserted into the baculovirus transfer vector pBlueBacHis-HTN S was prepared to be expressed under the control of the polyhedrin promotor of baculovirus. The baculovirus transfer vector pBlueBacHis-HTN S was transformed into Escherichia coli ( E. coli JM 109), and the E. coli transformants expressing the Hantan virus nucleocapsid protein gene were selected.

In order to confirm that the baculovirus transfer vector containing the Hantan virus nucleocapsid gene is inserted into the E. coli transformant, the E. coli transformant is cultured in an LB culture medium containing 100 µg / ml ampicillin, and then grown in large quantities. The transfer vector DNA was extracted from a small amount of E. coli recovered from the digested enzyme and digested with restriction enzymes, followed by electrophoresis on 1.2% agarose gel to confirm the cleaved band. As a result, as shown in Example 1, the Hantan virus 91011 element gene fragment having a size of about 1.7 kb and the transition vector gene fragment having a size of about 4.9 kb were detected, and the hantan virus 91011 was transferred to the baculovirus transition vector pBlueBacHis-HTN of the present invention. It was confirmed that the address gene was inserted.

Further, as a result of analyzing the nucleotide sequence of the Hantanvirus nucleocapsid gene inserted into the baculovirus transition vector pBlueBacHis-HTN S, the Hantanvirus nucleocapsid protein gene described in SEQ ID NO: 5 in the baculovirus transition vector of the present invention. It is confirmed that is inserted in the correct direction.

Example 3 Preparation of Recombinant HTN S-Beculovirus

In order to prepare a baculovirus in which the hantan virus nucleocapsid protein gene was recombined, the baculovirus transfer vector pBlueBacHis-HTN S clone propagated from the E. coli transformant of Example 2 was transferred to Bac-N-Blue AcNPV DNA (Invitrogen). , Co-fection with the insect cell line Sf9 cells (Invitrogen, Netherland) using liposome mediation method of cation together with Netherland. After 4 h co-transfection at room temperature with TNM-FH medium without fetal bovine serum, infected Sf9 cells were transferred to complete TNM-FH medium with 10% fetal bovine serum and cultured at 27 ° C. for 3 days. It was. The supernatant was recovered by centrifugation of the culture broth, and the agar containing 10% fetal bovine serum and 150 μg / ml of X-gal was superimposed on Sf9 cells cultured in monolayer to separate the recombinant baculovirus from the supernatant. After adding TNM-FH, the cells were incubated at 27 ° C. for several days. Recombinant baculovirus co-infected with baculovirus transfer vector is a plaque colored blue because X-gal is decomposed by the β-galactosidase gene included in the transfer vector. After separation, the recombinant baculovirus was purely inoculated by inoculating Sf9 cells cultured in a monolayer ( FIG. 5 ).

In order to confirm that the isolated recombinant recombinant baculovirus contains the Hantanvirus nucleocapsid protein gene and actually expresses it, the Hantanvirus nucleocapsid protein gene was confirmed by amplification by PCR from the recombinant baculovirus DNA. Western blot analysis was performed to detect and confirm the antigen of the Hantanvirus nucleocapsid protein expressed in recombinant baculovirus.

First, in order to confirm whether the recombinant baculovirus contains the Hantanvirus nucleocapsid protein gene by PCR, the DNA of baculovirus extracted from the infected cells by culturing for 3 days at 27 ° C. is described as SEQ ID NO: 6 . PCR was performed using a forward primer and a reverse primer of SEQ ID NO . As a result of confirming the PCR product obtained by electrophoresis, it was confirmed that the Hantan virus nucleocapsid protein gene of about 1.5 kb size was detected ( FIG. 6 ).

In addition, in order to confirm that the Hantanvirus nucleocapsid protein is actually expressed in the recombinant baculovirus, cells infected with the recombinant baculovirus were pulverized and subjected to electrophoresis on an SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gel. The expressed proteins were isolated and transferred to nitrocellulose membranes and subjected to western blot analysis with monoclonal antibodies that specifically reacted with the Hantanvirus nucleocapsid protein. As a result, it was confirmed that the Hantanvirus nucleocapsid protein having a molecular weight of 49.5 kD was expressed from the recombinant baculovirus (lane 1 of FIG. 9 ).

The recombinant baculovirus identified in this manner was separated twice by pure plaque separation method using agar overlapping medium to obtain the recombinant HTN011S-baculovirus expressing the Hantan virus nucleocapsid protein, which was obtained from the Biotechnology Research Institute. It was deposited with the Gene Bank on August 18, 2000 (Accession No .: KCTC 0854BP).

Example 4 Isolation and Purification of Hantan Virus Nucleocapsid Protein

The following experiment was performed to isolate and purify the expressed Hantanvirus nucleocapsid protein from the recombinant baculovirus comprising the nucleocapsid protein gene of Hantanvirus 91011 strain.

Sf9 cells cultured in a spinner flask using 150 ml complete TNM-FH medium containing 10% fetal bovine serum were infected with the recombinant gene baculovirus of Example 4 and 3-4 at 27 ° C. After culturing for one day, centrifugation was performed using a Sorvall centrifuge at 3000 rpm. 150 ml of pH 7.8 phosphate buffer (20 mM Sodium Phosphate, 500 mM Sodium Chloride) refrigerated in an ice bath was added to the precipitated infected cells, and the cells were washed twice. The supernatant was removed by centrifugation again, and the precipitated cells obtained therefrom were stored in a pH 7.8 cytolysis buffer containing a protease inhibitor (20 mM Sodium Phosphate, 500 mM Sodium Chloride, 5 mM EDTA). 15 ml of 1% Triton X-100, 200 μM PMSF, 10 μg / ml Leupeptin, 5 μg / ml Aprotinin was added and suspended for 10 minutes, and then dissolved in ice solution three times for 15 seconds using an ultrasonic mill. . The lysed cell solution was centrifuged at 8000 rpm for 30 minutes to obtain a supernatant, which was then added to a Probond column containing nickel resin and gently shaken for 10 minutes at room temperature while the Hantan virus in the infected cell supernatant. The nucleocapsid protein was allowed to adsorb well to the nickel resin. The resin with the nucleocapsid protein 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 cell-derived proteins or non-specifically bound baculovirus-derived proteins. It was. The same amount of pH 6.0 wash buffer solution (20 mM Sodium Phosphate, 500 mM Sodium Chloride) was added to the washed resin, followed by washing with gentle shaking for 2 minutes at room temperature, followed by the same amount of pH 4.0 elution buffer solution (20 mM Sodium Phosphate). , 500 mM Sodium Chloride) was added to elute the Hantanvirus nucleocapsid protein. Hantanvirus nucleocapsid proteins expressed from recombinant baculovirus have six histidine residues at the amino terminus and are easily isolated by binding to a cation bearing nickel resin. Through this process, only the Hantan virus nucleocapsid protein from which all of the cell-derived or baculovirus-derived proteins were removed was purified. The eluted protein was concentrated to a desired concentration using a centricon manufactured by Amicon, and the proteins obtained in each purification step were electrophoresed on a 12% SDS-PAGE gel and then transferred to a nitrocellulose membrane. Western blot analysis was performed using monoclonal antibodies that specifically respond to protein antigens.

As shown in FIG . 8 , the Hantanvirus nucleocapsid protein expressed from the recombinant baculovirus was electrophoresed for each purification step on an SDS-PAGE gel and stained with Comassie blue. 49.5 kD of the Hantanvirus nucleocapsid protein, which was not detected in the uninfected Sf9 cell lysate (lane 2), was detected in the Sf9 cell lysate infected with the recombinant baculovirus and the subsequent eluate (lane 1, 4, 5, 6, 7 and 8), especially when eluted with a column filled with nickel resin (lane 8), pure Hantanvirus nucleocapsid protein with all cell-derived or baculovirus-derived proteins removed was detected. It was.

In addition, the SDS-PAGE gel was transferred to a nitrocellulose membrane and subjected to Western blot analysis. As a result, 49.5 kD of Hantanvirus nucleocapsid protein was infected with Sf9 cell lysate infected with recombinant baculovirus and then purified. It was confirmed that the eluent was detected in ( FIG. 9 ).

As a result of analyzing the amino acid sequence of the recombinant protein recovered therefrom, it was confirmed that the hantanvirus nucleocapsid protein was a polypeptide consisting of the amino acids set forth in SEQ ID NO: 8 .

Example 5 Diagnosis of Hemorrhagic Fever Nephrotic Syndrome Using Nitrocellulose Membrane Adsorbed Nucleocapsid Antigen Protein

To confirm that the Hantanvirus nucleocapsid protein expressed from the recombinant baculovirus can be used for diagnosis of nephrotic hemorrhagic fever, 10 μg / ml of the Hantanvirus nucleocapsid protein was electrophoresed on an SDS-PAGE gel followed by nitro. The reaction pattern of the adsorbed antigens adsorbed on the cellulose membrane with serum of normal hemorrhagic fever or normal serum was investigated in comparison with the fluorescent antibody method.

Specifically, 5% skim milk was blocked by blocking the Hantanvirus nucleocapsid antigen protein adsorbed on the nitrocellulose membrane, and then serum of nephrotic hemorrhagic fever patients or normal persons was added to the nitrocellulose membrane having a width of 4 to 5 mm, respectively, at room temperature. The reaction was carried out for one hour. The nitrocellulose membrane was washed three times at room temperature with phosphate buffer containing 0.02% Tween 20 for 3 minutes and added with anti-human IgG Goat serum (KPL, USA) labeled with Peroxidase. Reaction and washing were performed in the same manner as above. Then 4CN (4-chloro-1-naphtol) and hydrogen peroxide solution were added to the nitrocellulose membrane and reacted for 30 minutes at room temperature to induce color development. After completion of the color reaction, the nitrocellulose membrane was washed with tap water to remove the color reagent, and the specificity of the antibody reaction was observed by observing the pigment deposited on the membrane surface.

As a result, in the nitrocellulose membrane which reacted with the serum of a person suspected of bleeding fever that was suspected of nephrotic syndrome, a clear band of 49.5 kD of the Hantanvirus nucleocapsid protein was identified, but the nitrocellulose reacted with normal serum. No reaction could be observed at all in the membrane ( FIG. 10 ). Therefore, it was confirmed that nephrotic hemorrhagic fever can be diagnosed by using a Hantanvirus nucleocapsid antigen protein expressed from a recombinant baculovirus adsorbed on a nitrocellulose membrane.

Example 6 ELISA Diagnosis of Hemorrhagic Fever with Nephrotic Syndrome Using 96-well Plate Adsorbed Nucleocapsid Antigen Protein

Differential diagnosis of serum from patients with renal hemorrhagic fever and laboratory animals infected with Hantan virus or Seoul virus through ELISA (Enzyme Linked Immuno-Sorbent Assay) method using nucleocapsid antigen protein expressed from recombinant baculovirus in Sf9 insect cells We investigated whether we could. Serum collected from a month after infection from 20 patients sera suspected of nephrotic bleeding fever and confirmed by laboratory diagnosis by fluorescent antibody method and 4 rats infected with 1000 pfu of Hantan virus and Seoul virus were used as experimental groups. Serum from 10 normal human serum and 4 experimental animal rats not infected with the virus was used as a control.

<6-1> IgM antibody titers in serum of patients with renal hemorrhagic fever

First, in order to measure the IgM antibody titer in serum of patients with renal hemorrhagic fever, anti-human IgM antibody (0.01 M Phosphate, pH 7.6) diluted in a certain amount in a 96-well plastic plate made of polyvinyl (Polyvinyl) anti-human IgM antibody) was dispensed at 100 μl per well and reacted at 4 ° C. for 12 hours. The antibody-adsorbed 96 well plate was washed three times with a wash solution (phosphate buffer solution containing 0.1% Tween 20 [0.01 M Phosphate, pH 7.6]) to remove the antibody that was not adsorbed onto the plate, and then diluted with a 5% skim milk. Serum samples diluted in several dilutions with 0.1% Tween 20 phosphate buffer solution [0.01 M Phosphate, pH 7.6] were dispensed at 100 μl per well and reacted at 37 ° C. or room temperature for 1 hour. Wash three times. Subsequently, 100 ml (10 ng of protein per well) of the nucleocapsid protein dissolved in the diluent at 10 μg / ml was dispensed into the 96 well plate, reacted at 37 ° C. or room temperature for 1 hour, and washed three times with a washing solution. It was. Here, 100 μl of a monoclonal antibody to a nucleocapsid antigen protein diluted at a constant dilution rate was dispensed per well for reaction at 37 ° C. or room temperature for 1 hour, washed three times with a washing solution, and peroxy diluted at a constant dilution rate in a dilution solution. 100 μl of Days labeled secondary antibody was dispensed and reacted and washed in the same manner as above. Finally, 100 μl of ABTS color substrate was added to each well, followed by reaction for 30 minutes at 37 ° C. or room temperature, and the results were read at a wavelength of 405 nm. The results were positively read by diluting fold with an absorbance value of 2.0 or more using an ELISA reader (ELISA Reader, Titertek, USA) of 405 nm and the results are shown in Table 1 below.

<6-1> IgG antibody titers in serum of patients with renal hemorrhagic fever

In addition, in order to determine the IgG antibody titer in the serum of patients with renal hemorrhagic fever, 10 μg / ml in a coating buffer (Coating buffer, 67 mM Na ₂ Co ₃ , 29 mM NaHCo ₃ , NaN ₃ 0.3 M; pH 9.6) Nucleocapsid antigen protein dissolved at a concentration of 100 μl (10 ng of protein per well) was added to each well of a 96 well plastic plate, and reacted at 4 ° C. for 12 hours for adsorption. The 96 well plate was washed three times with a wash solution (phosphate buffer solution containing 0.1% Tween 20 [0.01 M Phosphate, pH 7.6]) to remove unadsorbed protein, followed by dilution (5% skim milk and 0.1% Tween 20). 100 μl of the samples diluted in multiple dilutions with the phosphate buffer solution [0.01 M Phosphate, pH 7.6]) was dispensed and reacted at 37 ° C. or room temperature for 1 hour. This was washed again three times with the washing solution, and 100 μl of the peroxides labeled secondary antibody diluted with a certain dilution factor in the diluent was dispensed and reacted and washed in the same manner as above. Finally, 100 μl of ABTS color substrate was added to each well of the plate and reacted at 37 ° C. or room temperature for 30 minutes, and the results were read at a wavelength of 405 nm. The results were read in the same manner as in <6-1>, and the results are shown in Table 1 below.

number serum ELISA diagnosis method Fluorescent Antibody Method Purified Gene Recombinant Nucleocapsid Protein Cell Lysis Solution Infected with HTNV 91001 Cell Lysis Solution Infected with HTNV 91001 IgM IgG IgM IgG One Patient 1 3200 800 3200 800 256 2 Patient 2 3200 800 3200 1600 256 3 Patient 3 12800 400 12800 1600 1024 4 Patient 4 25600 400 25600 1600 1024 5 Patient 5 1600 200 3200 400 256 6 Patient 6 6400 800 3200 800 1024 7 Patient 7 25600 400 25600 400 2048 8 Patient 8 6400 100 6400 200 512 9 Patient 9 800 1600 800 1600 128 10 Patient 10 1600 6400 3200 6400 256 11 Patient 11 800 6400 800 12800 256 12 Patient 12 25600 12800 25600 25600 1024 13 Patient 13 100 12800 100 12800 128 14 Patient 14 <100 12800 <100 25600 256 15 Patient 15 25600 25600 25600 25600 4096 16 Patient 16 25600 25600 25600 25600 1024 17 Patient 17 6400 1600 3200 1600 128 18 Patient 18 1600 <100 1600 <100 128 19 Patient 19 3200 6400 6400 6400 256 20 Patient 20 3200 25600 3200 25600 512 21 Normal 1 <100 <100 <100 <100 <32 22 Normal 2 <100 <100 <100 <100 <32 23 Normal 3 <100 <100 <100 <100 <32 24 Normal 4 <100 <100 <100 <100 <32 25 Normal 5 <100 <100 <100 <100 <32 26 Normal 6 <100 <100 <100 <100 <32 27 Normal 7 <100 <100 <100 <100 <32 28 Normal 8 <100 <100 <100 <100 <32 29 Normal 9 <100 <100 <100 <100 <32 30 Normal 10 <100 <100 <100 <100 <32

As shown in Table 1 , the serum of normal persons showed antibody titers of 100 or less in both IgM and IgG, whereas the serum titers of patients with nephrotic hemorrhagic fever showed antibody titers of 800 to 25600 when IgM was measured (patient 14 was 100 or less), and IgG was measured to show antibody titers ranging from 200 to 25600 (patient 18 was 100 or less). Patients with nephrotic hemorrhagic fever were distinguished from normal individuals, and showed high specificity for the Hantan virus nucleocapsid protein antigen. It was.

<6-3> Antibody titer of laboratory animals infected with Hantan virus and Seoul virus

Example <6-1> was measured and IgG antibody titers for the Hantaan virus nucleocapsid antigen protein in each experiment was collected from the animal serum rats infected with Hantaan virus and Seoul virus in the same manner and the results in Table 2 Shown in

number Infection virus IgG ELISA Diagnosis Method Fluorescent Antibody Method Purified Gene Recombinant Nucleocapsid Protein Cell Lysis Solution Infected with HTNV 91001 Cell Lysis Solution Infected with HTNV 91001 H-1 HTNV ^a 91011 25600 25600 4096 H-2 12800 25600 4096 H-3 6400 6400 512 H-4 25600 12800 4096 S-1 SEOV ^b 80/39 12800 25600 4096 S-2 25600 25600 4096 S-3 25600 25600 4096 S-4 6400 6400 1024 N-1 Uninfected <100 <100 <32 N-2 <100 <100 <32 N-3 <100 <100 <32 N-4 <100 <100 <32

a: Hantan Virus b: Seoul Virus

As shown in Table 2 , IgG antibody titers were less than or equal to 100 in the serum of rats uninfected with Hantan virus and Seoul virus, whereas nucleocapsid antigen proteins were detected in serum of experimental animals infected with Hantan virus and collected one month after. IgG antibody titer was 6400-25600, and IgG antibody titer of nucleocapsid antigen protein in serum of experimental animals collected one month after Seoul virus infection was also 6400-25600 in Hantan virus. It was confirmed that the sensitivity to the antibody was very excellent.

As described above, the present invention reveals that the Hantanvirus nucleocapsid protein expressed from the recombinant baculovirus in insect cells exhibits very excellent antigenicity, and provides a diagnostic method and a diagnostic kit using the same. Diagnosis method and diagnostic kit of renal syndrome hemorrhagic fever of the present invention is more convenient than the conventional fluorescence analysis method and excellent in sensitivity and specificity can be usefully used for the diagnosis and analysis of renal syndrome hemorrhagic fever.

Claims (14)

Baculovirus transfer vector comprising a small gene segment as described in SEQ ID NO: 5 . The recombinant baculovirus transfected with the baculovirus transfer vector of claim 1 (Accession Number: KCTC 0854BP). A hantanvirus nucleocapsid protein expressed from the recombinant baculovirus of claim 2 and having an amino acid sequence as set forth in SEQ ID NO: 8 . 1) taking a sample from a patient suffering from nephrotic hemorrhagic fever; And 2) A method for diagnosing nephrotic hemorrhagic fever in a mammal, except for humans, comprising detecting the antibody from the sample using the Hantan virus nucleocapsid protein of claim 3 as an antigen. A method for measuring the antibody of hemorrhagic fever with nephrotic syndrome using the hantan virus nucleocapsid protein of claim 3 as an antigen. The method of claim 5, 1) adsorbing a Hantanvirus nucleocapsid antigen protein to a substrate; 2) washing by adding serum of a sample to the membrane to which the antigen protein is adsorbed, and then washing it; 3) reacting the substrate by adding an antibody having a chromophore or fluorescent substance bound thereto; And 4) The method of measuring the antibody of hemorrhagic fever with nephrotic syndrome comprising the step of measuring the absorbance after inducing color development by adding a coloring agent to the substrate. The glass of claim 6, wherein the substrate of step 1 is a nitrocellulose membrane, a 96 well plate synthesized with a polyvinyl resin, a 96 well plate synthesized with a polystyrene resin, and a glass slide glass. Antibody measurement method of hemorrhagic fever with nephrotic syndrome, characterized in that selected from the group consisting of. The method of claim 6, wherein the chromophore bound to the antibody in step 3 is selected from the group consisting of peroxidase and alkaline phosphatase. 7. The method of claim 6, wherein the fluorescent material bound to the antibody in step 3 is selected from the group consisting of FITC and TRITC. The method of claim 6, wherein the coloring agent in step 4 is 4CN (4-chloro-1-naphtol), DAB (Diaminobenzidine), AEC (Aminoethyl carbazole), ABTS [2, 2'-Azino-bis (3-ethylbenzothiazoline-6- sulfonic acid)], OPD (o-Phenylenediamine), TMB (Tetramethyl Benzidine) is an antibody measuring method of hemorrhagic fever of nephrotic syndrome characterized in that it is selected from the group consisting of. Claim 3 antibody measurement kit of hemorrhagic fever of nephrotic syndrome using the hantan virus nucleocapsid protein of claim 3. 12. The diagnostic kit for hemorrhagic fever of nephrotic syndrome according to claim 11, further comprising a microtiter plate, an assay buffer, a standard antibody, a secondary antibody, and a coloring substrate. The diagnostic kit for hemorrhagic fever of nephrotic syndrome according to claim 12, wherein the secondary antibody is bound to a chromophore or a fluorescent substance. 12. The diagnostic kit of nephrotic hemorrhagic fever according to claim 11, wherein the nucleocapsid antigen protein is immobilized on a biological microchip.