KR100561687B1 - A kit capable of detecting tsutsugamushi, Leptospira and Hantaan virus infection simultaneously - Google Patents

Abstract

The present invention is a recombinant protein represented by the amino acid sequence of SEQ ID NO: 1 or amino acid of SEQ ID NO: 1 A variant having a surface antigen activity of Orientia tsutsugamushi Pajoo with at least 90% homology with the sequence, a recombinant protein represented by the amino acid sequence of SEQ ID NO: 7 or an amino acid of SEQ ID NO: 7 A variant having a homologous activity of Leptospira interrogans serovar lai HY10 having at least 90% homology with the sequence, and a recombinant protein represented by the amino acid sequence of SEQ ID NO: 10 or the amino acid of SEQ ID NO: 10 A diagnostic kit capable of simultaneously detecting infections caused by Tsutsugashimi, Leptospira interrogans, and Hantan virus, including variants having a nuclear protein antigen activity of Hantaan virus 76-118 with at least 90% homology with the sequence. to provide.

Tsutsugamu City, Leptospira, Hantan Virus

Description

A kit capable of detecting tsutsugamushi, Leptospira and Hantaan virus infection simultaneously}

Figure 1 schematically shows the process of constructing an expression vector p56 kDa comprising a gene encoding Orientia tsutsugamushi Pajoo recombinant surface antigen.

Figure 2 shows the results of 12% SDS-PAGE of the cell lysate of E. coli transformed with the expression vector p56 kDa.

Figure 3 shows the 10% SDS-PAGE results to confirm the purification of the expressed recombinant protein.

4A and 4B are scattergrams of IgG and IgM ELISA reactivity and corresponding microindirect immunofluorescence antibody (MIFA) titers obtained with recombinant proteins.

Figure 5 shows the results of the cross-reaction test (Western blotting) with Tsutsugamusosis and similar diseases.

Figure 6 schematically shows the arrangement of a high-speed immunochromatography kit for diagnosing three types of pyrogenic diseases of the present invention.

Figure 7 schematically shows the process of constructing an expression vector pBAD-HTN comprising a gene encoding a recombinant nuclear protein antigen N protein of Hantaan virus 76-118.

The present invention relates to Orientia tsutsugamushi Pajoo, Leptospira interrogans serovar lai HY10 and Hantaan virus 76-118, each of which comprises recombinant protein antigens specific for Orientia tsutsugamushi Pajoo. The present invention relates to a diagnostic kit capable of simultaneously detecting the presence or absence of an antibody against leptospira and a hantan virus.

Tsutsugamus disease is an acute pyrogenic disease caused by the infection of Orientia Tsutsugamushi , which is called in Asia, including Japan, China, Malaysia, Thailand, and Vietnam (Chang, Korea's Tsutsugashi disease, Seoheung Publishing Co., Ltd.). p21-27,1994), most prevalent in October, most often between September and early December (Kim et al., Infections 20: 105-116, 1988). Most people are infected by ticks that have Tsutsugamu bacteria while working outdoors in the fields or camping. Clinical symptoms are characterized by the appearance of ulcers with eschar on the aspirated area of infected ticks, with high fever, chills, headache, spotty rash, and lymphadenopathy.

The diagnosis of Tsutsuga's disease is largely made by clinical diagnosis based on clinical symptoms and physicochemical findings, bacteriological diagnosis by isolates of causative bacteria, and serological diagnosis measuring the presence of antibodies in serum (Kim et al., Infection 20: 105-116, 1988). , Jang et al., Korean Journal of Microbiology 24: 281-289, 1989). Clinical diagnosis is made by proving the presence of epidermis, rash, and lymphadenopathy in patients with acute fever, but early clinical manifestations are often nonspecific and are found without careful attention depending on the location of the eschar. It is difficult to do, and when re-infection, there is a problem in the diagnosis of the skin may not appear. Also, at the same time, acute febrile diseases such as leptospirosis, rash and bleeding hemorrhagic fever are called.In the early stage of the onset, the clinical findings of these diseases are similar and there are many problems in differential diagnosis by only clinical symptoms. 497-509,1993, Hyun-Ju Bae, Int. Of Intology, p79-87, 2001). The acute febrile illness of autumn mentioned above has been on the rise since 1998 due to active climate warming, economic and leisure activities. In 2001, 2,638 cases of Tsutsugamushi's cases were reported to the statutory epidemic computer network, and an estimated 50,000 similar patients were reported each year (2002 Infectious Diseases Annual Report 2003, National Institutes of Health).

The bacteriological diagnosis that isolates the causative organism Orientia tsutsugamushi from the patient's blood should be equipped with susceptible animal inoculation, cell culture and biosafety (Biosafty level 3) facilities to isolate the causative organism, and the identification time is three weeks. Because of this, it is impossible to use test results in actual patient treatment. Therefore, the diagnosis of Tsutsuga's disease is usually made by clinical symptoms and serological diagnosis (Intestinal et al., Korean Journal of Microbiology 24: 281-289, 1989). Traditionally, Tsutsugamu bacteria have been classified into Gillam, Karp and Kato serotypes, and as the serologically different strains are isolated from Korea, Japan, and Thailand, the isolates of Boryong, Yeonchon, Serological classifications such as Shimokoshi, Kawasaki, Kuroki, TA716, TA763, and TH1817 are becoming more sophisticated. Serological methods include nonspecific Weil-Felix reaction, complement binding test, indirect immunofluorescent antibody, enzyme immunoassay, indirect immune peroxidase test, passive hemagglutination. Micro Indirect Immunofluroscence Assay (MIFA) using double cell cultures is widely used.However, Tsutsugamu bacteria used for diagnosis are passaged as cell culture and culture period is longer than 2 weeks. Many problems are known due to objective standardization, such as the high cost of manufacture and the high cost of the preparation and the need for skill in reading the diagnosis (Robinson et al., Am. J. Trop. Med. 25: 900-905, 1976).

On the other hand, PCR diagnostics, a gene detection method for clarifying regional and clinical characteristics according to the phenotype of Tsutsuga mucin bacteria, was studied (Sugita Y. et al., J. Med. Microbiol. 37: 357-360, 1992). In Korea, passive hamagglutination assay (PHA), which attaches a recombinant antigen of surface antigen protein (56 kDa) of Boryeong isolate, to sheep red blood cells (Kim IS et al., J. Clin.Microbiol. 31: 2057-2060, 1993) And a diagnostic kit using the same and an ELISA method (Kim IS et al., J. Clin. Microbiol. 31: 598-605, 1993) have been developed. However, the passive agglutination method that is widely used has a low positive predictive value compared to the indirect immunofluorescent antibody method, which is not suitable for early diagnosis.

In addition, ELISA method for improving the recombinant 56 kDa antigen site of Karp strain in foreign countries (Ching WM et al., Clin. Diagn. Lab. Immunol. 5 (4): 519-526, 1998), rapid immunochromatography (Rapid) immunochromatographic flow assays (RAF) (Ching WM et al., Clin. Diagn. Lab. Immunol. 8 (2): 409-414, 2001) have been developed. However, in order to diagnose imported goods in frontline medical institutions and clinical centers, the price of the kit is expensive and the reality is low, and there is a need to replace the product with domestic isolates. The present inventors have purely isolated and identified Orientia tsutsugamushi Pajoo in Korea, and discovered the gene of the envelope protein (56 kDa) and the envelope protein expressed from the gene from this strain. NCBI genbank accession number AF430142 was obtained.

Leptospirosis is a common infectious disease that is widely distributed around the world and is caused by Leptospira interrogans . About 240 pathogenic serotypes are known in Leptospira interrogans, and 90% of leptospirosis occurring in Korea has been found to belong to serotype lai (Oh Hee-bok et al., National Institute of Health, 24: 64-). 76 (1990); Oh Hee-bok et al., Korean Journal of Microbiology, 26: 253-262 (1991). Currently, the diagnosis of leptospirosis is recognized by microscopic agglomeration (MAT) using live bacteria as a standard diagnostic method (Faine S. Guidelines for the control of Leptospirosis, Offset publication No. 67, WHO (1982)). However, it is difficult to objectively standardize such that leptospira used for diagnosis should be kept passaged in a viable state, culture takes 5-7 days, the medium used is expensive, and the skill of reading the diagnosis is required. there was.

In order to solve this problem, various diagnostic methods, for example, ELISA method, dipstick method (using immunochromatography) and PCR method have been studied. However, both the conventional ELISA method and the dipstick method use the leptospira cells as antigens, so there is a drawback of culturing the leptospira bacteria. In addition, the PCR method is required to vary the primer according to the type of specimen, there is a disadvantage that the price is expensive. In order to overcome this drawback, the inventors have found that a recombinant FlaB truncation, which cleaves some amino acids at the N- and C-terminus of FlaB, the flagella antigen of Leptospira interrogans serovar lai HY10, serves as an antigen. It has been revealed that the sensitivity and specificity is high (see Patent Publication No. 10-2002-0078629). The disclosure of the above Patent Publication No. 10-2002-0078629 is incorporated in the specification of the present invention by reference.

In the diagnosis of hemorrhagic fever with nephrotic syndrome, immunofluorescent antibodies have been the most common method used to date, but the number of samples to be processed is limited during mass examination, and special equipment such as tissue culture facility and fluorescence microscope is required. The disadvantage is that a skilled inspector is required for accurate judgment. In addition, ELISA, hemagglutination inhibition, and hemagglutination have been used for the purpose of serological diagnosis and research of hemorrhagic fever with nephrotic syndrome.

On the other hand, the renal syndrome hemorrhagic fever diagnostic kit developed in Korea includes an insoluble carrier particle aggregation method and a diagnostic kit using the ELISA method, but when using any of the methods, the inspector prepares the sample dilution and the test reagent and fills it with a plate. There is inconvenience.

In addition, a method of simultaneously detecting these three types of exothermic diseases has been disclosed. Patent Publication No. 10-1998-0077901 discloses a step of blotting the bacterial syndrome of nephrotic syndrome hemorrhagic fever, Tsutsugashimi and leptospira in a predetermined position on the nitrocellulose membrane, respectively; Reacting the sample and the control serum with the nitrocellulose membrane blotted with the antigens, respectively, for reacting; Detecting the antigen-antibody reactants produced by the reaction using alkaline phosphatase-labeled goat anti human immunoglobulin antibody and BCIP / NBT substrate solution, respectively; And a simultaneous differential diagnosis method of nephrotic syndrome hemorrhagic fever, Tsutsuga's syndrome, and leptospirosis, comprising comparing the detection result in the sample and control serum. However, these diagnostic kits also use Leptospira and Tsutsugamus bacteria or Hantan virus itself as antigens, which requires time, money and effort to cultivate, isolate and purify each bacterium and virus. There was a disadvantage.

Accordingly, the present inventors have repeatedly studied to develop a recombinant protein that can be mass-produced by a genetic engineering method and a gene encoding the same, while maintaining high surface antigen activity of Orientia tsutsugamushi Pajoo, a pure domestic isolate. As a result, when a specific amino acid sequence was introduced at both ends of the surface antigen protein of 56 kDa, it was found that not only can an expression vector be effectively produced, but also the sensitivity and specificity essential for diagnosis can be maintained. It was completed.

Accordingly, the present invention includes a novel recombinant protein having surface antigenic activity of Orientia tsutsugamushi Pajoo, which is useful for the detection of Tsutsugamushi bacteria infection, Orientia tsutsugamushi , Leptospira interrogans It is an object of the present invention to provide a kit that can simultaneously detect the presence or absence of Hantaan virus .

In order to achieve the above object, the present invention is a recombinant protein represented by the amino acid sequence of SEQ ID NO: 1 or amino acid of SEQ ID NO: 1 A variant having a surface antigen activity of Orientia tsutsugamushi Pajoo with at least 90% homology with the sequence, a recombinant protein represented by the amino acid sequence of SEQ ID NO: 7 or an amino acid of SEQ ID NO: 7 A variant having a homologous activity of Leptospira interrogans serovar lai with at least 90% homology with the sequence, and a recombinant protein represented by the amino acid sequence of SEQ ID NO: 10 or the amino acid of SEQ ID NO: 10 Diagnosis capable of simultaneous detection of antibodies against infection of Tsutsugashimi, Leptospira interrogans, and Hantan virus, including variants having a nuclear protein antigen activity of Hantaan virus 76-118 with a sequence homology of at least 90% Provide the kit.

In the present invention, the recombinant protein represented by the amino acid sequence of SEQ ID NO: 1 has an amino acid sequence additionally at both ends of the 56 kDa protein encoding specific surface antigens in various antigenic determinants of Orientia tsutsugamushi Pajoo It is introduced. That is, 22 amino acids of SEQ ID NO: 9 are further linked to the N-terminus, and 3 amino acids of GSF are further linked to the C-terminus. Recombinant protein having the amino acid sequence of SEQ ID NO: 1 exhibits high sensitivity and specificity, as can be seen in the following test examples, can be effectively used in diagnostic kits for the detection of Tsutsugamu infection.

In addition, the variant having the surface antigen activity of Orientia tsutsugamushi Pajoo refers to a protein having a modification such as substitution, deletion, or insertion in the amino acid sequence added to the sock end or the surface antigen sequence of 56 kDa, Amino acid of SEQ ID 1 And a protein having a surface antigenic activity of Orientia tsutsugamushi Pajoo with homology greater than or equal to 90% with the sequence.

In the present invention, the recombinant protein represented by the amino acid sequence of SEQ ID NO: 7 removes the sock end of the flag FlaB, a flagellar antigen, among the various antigens of Leptospira interrogans serovar lai HY10. Sensitivity and specificity have been increased so that it can be applied to diagnosis.

Said variant with flagellar antigenic activity of Leptospira interrogans serovar lai HY10 refers to a protein with modifications such as substitutions, deletions, or insertions in the amino acid sequence of SEQ ID NO. And the amino acid of SEQ ID NO: 7 And a protein having a homologous activity of Leptospira interrogans serovar lai HY10 with at least 90% homology with the sequence.

In the present invention, the recombinant protein represented by the amino acid sequence of SEQ ID NO: 10 is a sequence representing the N protein, which is a nuclear protein antigen of Hantaan virus 76-118, and has high sensitivity and specificity for the virus. It is known to be applicable.

The variant having the nuclear protein antigen activity of Hantaan virus 76-118 refers to a protein having a modification such as substitution, deletion, or insertion in the amino acid sequence of SEQ ID NO: 10, and despite such modification, amino acid It contains a protein having a nuclear protein antigen activity of Hantaan virus 76-118 having a homology of 90% or more with the sequence.

In the present invention, the diagnostic kit may use any type of kit as long as it can be used in an analysis method for detecting the presence or absence of the three types of antigens. Such diagnostic kits are well known to those skilled in the art, and provided that the three kinds of antigens can be readily reconstituted by those skilled in the art. Such a diagnostic kit may include, for example, an ELISA kit, and a kit using an immunochromatography method, but is not limited thereto. The kit using the immunochromatography may be, for example, in the form of a sample pad, a conjugate pad, a debris virus antigen line, a leptospirosis antigen line, a Tsutsugamushi antigen line, a control line and an absorption pad. The sample pad is a pad for loading a sample such as serum. The conjugate pad comprises a conjugate of a substance (eg, a gold particle) that contains a detectable moiety and a substance that binds to a target substance (eg, an antibody that specifically binds to each antigen) in the sample pad. It is a pad. The three antigen lines are immobilized with the respective antigens in a porous membrane (eg, nitrocellulose (NC) membrane), and each antigen line is separated at regular intervals so that the detection signal can be well resolved. . The control line is for confirming that the sample is well developed, and includes a substance (eg, goat anti-human IgG) that always binds to the conjugate included in the conjugate pad. The absorbent pad serves to allow the sample to move well along the porous membrane. Therefore, in the immunochromatography kit, when a sample is added to a sample pad, it is developed through three kinds of antigen lines and a control line via a conjugate pad. At this time, the target substance present in the sample passes through the conjugate pad, binds to the conjugate substance, and becomes a detectable form, and then, when specific antibodies are present, passes through three antigen lines to specifically detect each antigen. By binding, the presence of the three pyrogenic disease pathogens is detected. However, the three types of pyrogenic disease diagnostic kits of the present invention are not limited to the above examples, and other forms of immunochromatography kits and ELISA kits may be included. The kit of the present invention may further include a reagent required for an immunochromatographic reaction, a detection device, and the like.

The three kinds of protein antigens used in the present invention may be amplified or prepared by a method known in the art, for example, recombinant DNA technology, and then inserted into a suitable vector, and then used in a suitable host cell. It can be produced by transforming, culturing the cells and then separating the protein from the culture, but is not limited to the above examples.

The present invention is characterized by including three kinds of recombinant protein antigens having high sensitivity and specificity to these microorganisms, respectively, so that the presence of Orientia Tsutsugamushi, Leptospira interogans or Hantan virus can be detected.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited by these examples.

Example

Example 1 Pure Isolation and Chromosome DNA Isolation of Orientia Tsutsugamu

Orientia tsutsugamushi Pajoo was inoculated into L ₉₂₉ cell line (Korea Cell Line Bank) incubated in DMEM medium supplemented with 2% fetal bovine serum, followed by CO ₂ incubator (34%). Incubated). After confirming the growth of microorganisms by MIFA, microorganisms were purified by using 40% Percoll and ultra centrifuge (Bekman Sw 55Ti). Chromosome DNA of Orientia tsutsugamushi Pajoo was isolated using a QIAamp blood DNA kit (Qiagen).

Example 2. Oligonucleotide Synthesis, Purification and Analysis

A gene encoding the Orientia Tsutsugamu surface antigen protein was amplified to synthesize two primers (SEQ ID NO: 3 and SEQ ID NO: 4) of 27mers to identify the nucleotide sequence. In addition, to amplify the gene for cloning into the vector from the DNA fragment amplified with the oligonucleotide, two primers of 30mers (SEQ ID NO: 5 and SEQ ID NO: 6) having a restriction enzyme recognition site inserted therein were synthesized.

In the above, oligonucleotides were synthesized using an automated nucleotide synthesizer (ABI-Pharmacia, USA). Each oligonucleotide thus synthesized was electrophoresed on 15% polyacrylamide gel (TE-boric acid, pH8.3) to separate synthetic oligonucleotides. After electrophoresis, the purity and concentration of the oligonucleotide were confirmed by ultraviolet rays having a wavelength of 260 nm and 280 nm. The oligonucleotide solution thus separated was lyophilized under reduced pressure at -70 ° C.

Example 3 Polymerase Chain Reaction (PCR)

Template DNA (50 ng), 10 μl of 10 × LA Taq polymerase buffer (500 mM KCl, 15 mM MgCl ₂ and 10 mM Tris-HCl, pH8.3), 10 μl of dNTP's mixture (dGTP, dATP, dTTP, dCTP each 1.25 mM Per liter) and primer (1 oligonucleotide synthesized in Example 2), and distilled water was added to a solution containing 0.5 µl of LA Taq DNA polymerase (Takara, Japan) to make 50 or 100 µl in total. It was. For the target gene base sequence, the template DNA is the genomic DNA of Orientia Tsutsumashi isolated in Example 1 and the primer is an oligonucleotide of SEQ ID NO: 3 and SEQ ID NO: 4; In the case of gene amplification for cloning recombinant proteins, the template DNA is a DNA fragment resulting from PCR for sequencing and the primers are oligonucleotides of SEQ ID NO: 5 and SEQ ID NO: 6.

A thermocycler (Thermal cycler, Perkin-Elmer, USA) was used for the PCR reaction and 30 times of denaturation (94 ° C, 30 seconds), annealing (50 ° C, 30 seconds), extension (72 ° C, 1 minute) was performed. Finally, the mixture was further reacted at 72 ° C. for 5 minutes. Then, a PCR purification kit (Promega, USA) was used to remove the polymerase, and the purified PCR product was used for later experiments.

Example 4 Preparation of Expression Vectors

In order to express the gene, the second amplified gene in Example 3 and the expression vector pET-15b (Novagen, USA) were cut with Xho I and BamH I, respectively, and 0.1 μg of the expression vector and 30ng of the recombinant protein gene were mixed. 1 μl of 10 mM conjugated concentrate (10 mM DTT and 100 mM MgCl ₂ , 600 mM Tris-HCl containing 10 mM ATP, pH7.5) and 10 units of T4 DNA ligase (NEB, USA) The volume was adjusted to 10 μl and reacted at 16 ° C. for 18 hours to prepare a cyclic plasmid in which the gene was inserted into the expression vector pET-15b (Novagen, USA), which was named p56kDa. The plasmid is 7308 bp (see Figure 1).

Figure 1 schematically shows the construction of the expression vector according to the present invention. The expression vector p56kDa was inserted into the transformant Escherichia coli BL21 (Novagen, USA), and re-extracted the expression vector p56kDa from the transformed Escherichia coli BL21 to process restriction enzymes and subjected to 1% agarose gel electrophoresis. By confirming that the double restriction enzyme site was present in the electric expression vector p56kDa, it was confirmed that the recombinant protein gene of the present invention was correctly inserted. In addition, as a result of determining the nucleotide sequence of the DNA fragment prepared from the plasmid (expression vector), it was confirmed to have the nucleotide sequence of SEQ ID NO: 2.

Example 5. Preparation of Transformant

10 ng of the expression vector p56kDa prepared in Example 4 was added to 20 μl of E. coli NovaBlue (Novagen, USA) already prepared for transformation, and reacted for about 30 minutes in ice and at 40 ° C. for 40 seconds. Heat shock was applied to allow the recombinant vector to enter the transformant Escherichia coli. After standing in ice for 2 minutes, 80 μl of SOC liquid medium (GIBCO BRL, USA) was added and shaken at 37 ° C. for 1 hour in solid LB medium containing 100 μg / ml of empicillin (Sigma, USA). The transformants were selected by culturing.

At this time, the transformant containing the expression vector p56kDa was named Escherichia coli NIH / NV / 56kDa-GEMT (Esherichia coli ), and the transformant containing the p56kDa vector was transformed into Escherichia coli BL21 (Novagen, USA). It was named Escherichia coli NIH / BL / 56kDa-ET15B (Esherichia coli ), and was deposited with the Korea Microorganism Conservation Center on April 30, 2003 (Accession No. KCCM 10489).

Example 6 Expression of Recombinant Proteins

The transformant E. coli NIH / BL / 56kDa-ET15B was inoculated into LB solid medium containing 100 μg / ml of empicillin, incubated at 37 ° C. for 24 hours, and the cultured cells were suspended in 5 ml of LB liquid medium. After adjusting the absorbance to 0.6 at 600 nm, 5 ml of this culture was inoculated into 500 ml of LB liquid medium containing 100 µg / ml of empicillin and shaken at 200 rpm at 37 ° C. to obtain 0.7-1.0 at 600 nm absorbance. . Subsequently, IPTG (isopropyl-β-D-thiogalactopylanoside, Bioneer, Korea) was added so that the final concentration was 1 mM, and the expression was induced by shaking culture (200 rpm) for 4-5 hours at 37 ° C. and 200 rpm at 37 ° C. After incubation, the cells were collected by centrifugation, suspended in 1 / 15M phosphate buffered saline (pH7.4), and the cells were crushed to crush the 10% SDS-PAGE (see FIG. 2). In FIG. 2, lane M is a molecular weight size marker, the first lane is the result of electrophoresis of cell lysate before IPTG induction, and the second lane is the result of electrophoresis of the lysate of bacteria cultured for 4 hours after IPTG induction. Indicates the position of the expressed recombinant protein. As shown in Figure 2, it can be seen that the recombinant protein is expressed in the transformant E. coli NIH / BL / 56kDa-ET15B.

Example 7. Purification of Recombinant Proteins

Incubate E. coli BL21 with recombinant protein, add IPTG to a final concentration of 1 mM, induce induction by shaking culture (200 rpm) for 1.5 hours at 37 ° C, and centrifuge at 5000 rpm for 10 minutes at 4 ° C. Was recovered and washed twice with PBS. After the prepared cells were suspended in 10 ml of PBS, the cells were cooled in ice, crushed at 15,000 psi, and kept in ice, followed by centrifugation at 4 ° C. at 8,000 rpm for 10 minutes. The insoluble precipitate was washed twice with 10 ml of PBS, then suspended in 7-8 ml of lysis buffer (20 mM Na ₂ HPO ₄ , 0.5 M NaCl, 8 M Urea (pH 7.8)) and overnight at 4 ° C. Dissolved. Repeat this process for 10 minutes at 12,000rpm, centrifuge for 10 minutes, take the supernatant, wash 4 ml of 50% Ni-NTA filler (Qiagen) 2-3 times with a solution and mix with the supernatant 2 at room temperature Bound for -3 hours. Fill the column with a supernatant-filler mixture carefully to avoid foaming, elute the mixture, and then wash 10 ml (10 mM IMDZ, 20 mM Na ₂ HPO ₄ , 0.5 M NaCl, 8 M Urea, pH 6.0). The column was washed four times. The washed protein was passed through 3 ml of elution buffer (300 mM IMDZ, 20 mM Na ₂ HPO ₄ , 0.5 M NaCl, 8 M Urea, pH 6.0) to recover the recombinant protein. The protein change in the purification process was subjected to electrophoresis with 10% SDS-PAGE to confirm whether the purification process was normally performed (see Fig. 3). In FIG. 3, lane 1 is a cell lysate, lane 2 is an effluent, lanes 3-5 are washed, and lanes 6-9 are the results for the eluate. It was observed that the antigenic protein with histidine residues was purified by a column containing nickel filler. The expression level of the purified recombinant antigen protein was 765 [mu] g (17 [mu] g / ml) on average.

Example 8. ELISA Reaction Using Purified Recombinant Protein Antigen

Using the recombinant protein purified in Example 7 as an antigen, ELISA IgG, IgM was performed, and the antibody titer measured by MIFA was compared with sensitivity and specificity.

Samples were selected according to the antibody titer measured by MIFA in the serum stored in this laboratory, and serum that did not cross-react with leptospirosis and nephrotic syndrome hemorrhagic fever was used. ELISA was diluted to a certain amount of purified recombinant protein with a coating solution (0.159 g Na ₂ CO ₃ per 100ml, 0.293 g NaHCO ₃ ) and put 100 ㎕ in 96 well plate and left for 18-24 hours at 4 ℃. Completely remove the recombinant protein solution and buffer (PBS) containing 2% fetal bovine serum (8 g NaCl, 0.2 g KH ₂ PO ₄ , 2.17 g Na ₂ HPO ₄ .7H ₂ O, 0.2 g / L KCl, blocking solution) 200 μl was added and reacted at 37 ° C. for 2 hours. Completely remove the blocking solution, wash 5 times with Buffer (PBS-T) containing 0.01% Tween-20, and dilute the patient serum to be used as the primary antibody 1: 100 with buffer and 100 μl per well. Each reaction was carried out at 37 ℃ for 1 hour. Each well was washed 5 times with 200 μl buffer and the secondary antibody was diluted antiserum IgG (Sigma, USA) labeled with peroxidase 1: 25,000 in PBS, 100 μl in each well and 1 at 37 ° C. Reacted for hours. Secondary antibodies for detecting IgM were also used diluted 1: 25000 with antiserum IgM (Sigma, USA) labeled with peroxidase. After the reaction, each well was washed 5 times with buffer. 20 ml of 1 phosphate buffer containing urea hydrogen peroxide (268 mg) and 1 phenylenediamine dihydrochloride (8 mg) After dissolving in distilled water for 10 minutes or more, the water of the well-washed 96-well plate was completely removed, and 100 µl of the color reagent was added to the well. 20 minutes after the start of color development, the reaction was stopped by adding 50 µl of 2 normal (N) hydrogen peroxide (H ₂ O ₂ ) to each well, and the absorbance was measured at 450 nm. Fig. 4A is a scattergram of titer by MIFA corresponding to IgG ELISA reactivity of serum of Tsutsuga's patient obtained with recombinant protein. Scattergram of titer by method (IFA), dotted line represents mean plus 2D response of control healthy persons. Table 1 shows the sensitivity and specificity of the recombinant protein ELISA. Active infection was defined as either MIFA titer 1: 128 or higher, or the presence of clinical symptoms, and the cutoff used was determined by the mean plus 2SD response of control healthy persons, with the numbers in parentheses being either ELISA positive (sensitivity) or negative (specificity). ) The number of sera divided by the number of sera tested.

Table 1.

Measure responsiveness(%) Specificity (%) Recombinant Protein IgM 83 (25/30) 97 (29/30) Recombinant Protein IgG 83.8 (62/74) 96.6 (84/87)

As can be seen in Table 1, ELISA (IgG) results using recombinant antigenic protein using sera determined as positive or negative for Tsutsugamus showed 83.8% sensitivity to MIFA IgG and 83% sensitivity to IgM. In comparison of specificity, IgG showed 96.6% and IgM 97% for MIFA. Especially in the diagnosis of diseases, IgM levels

 The result of ELISA (IgM) using recombinant antigenic protein is significant because it is an important indicator used for early infection.

Example 9. Efficacy Measurements and Cross Reactions on Purified Recombinant Antigens

SDS-PAGE electrophoresis was performed by taking a certain amount of the recombinant protein purified solution prepared in Example 6 and then using a semi-dry transfer (Hoefer, USA) to a nitrocellulose (NC) membrane (Bio-Rad, USA). Was transferred. The finished membrane was washed three times for 5 minutes with 1 × Tris-buffered saline (TBS; 50 mM Tris-HCl, 200 mM NaCl, pH 7.4) and then 1 × containing 5% skim milk (Difco Co., USA). Blocking was performed for 24 hours with TBS-0.05% Tween 20 (TTBS). The NC membrane treated with skim milk was washed three times with TTBS for 5 minutes each time, and the control and Tsutsugamushi positive and negative patient serum were diluted 1: 100 with TTBS added with 5% skim milk to the NC membrane and stirred at room temperature for 18 hours. After washing 3 times with TTBS 5 minutes each. Alkaline phosphatase (AP) conjugated anti-human-IgG (Sigma, USA) was diluted with TBS added with 5% skim milk at 1: 3,000, and reacted at room temperature for 2 hours, and washed three times with TTBS for 5 minutes. Color development was performed using 5% NBT (nitro blue tetrazolium chloride in 70% dimethylformamide, Sigma, USA) and 5% BCIP (5-bromo-4-chloro-3-indolyl phosphate in 100% dimethylformamide, Sigma, USA). NC membranes were developed.

Cross-reaction test with other similar diseases was performed by Western blotting test in the same way as above for serum of patients with similar diseases such as leptospirosis, nephrotic syndrome hemorrhagic fever, viral hepatitis and malaria (see FIG. 5). ). Lane 1 of Figure 5 is negative control group lanes 2-5 is Tsutsumamosis positive serum, lane 6 is Tsutsugamus negative sera, lanes 7 are leptospirosis negative serum, lanes 8-11 are leptospirosis positive serum, lanes 12-13 nephrotic syndrome hemorrhagic fever Positive sera, lanes 14-15 are nephrotic syndrome hemorrhagic fever negative sera, lanes 16-17 are positive hepatitis B virus, lanes 18-19 are positive hepatitis C virus, lanes 20-22 are malaria positive serum, 23 Lane -24 shows the results of cross-reaction testing on malaria negative sera. Western blot using recombinant protein as an antigen showed specific bands in all Tsutsugamus positive patients serum and did not cross-react with similar diseases such as leptospirosis, nephrotic syndrome hemorrhagic fever serum and viral hepatitis positive and malaria positive serum. However, the response of 8 lanes of leptospira-positive serum is considered to be a double infection with Tsutsugamus. On the other hand, Tsutsugamosis, Leptospirosis, Nephrotic Syndrome Hemorrhagic Fever Negative Patients all showed negative results, indicating that the recombinant antigenic protein has high antibody specificity, and such recombinant antigen can be effectively used for differential diagnosis.

Example 10 Preparation and Testing of Three Fever Disease Rapid Test Kits

(1) Determination of titrant antigen and gold-conjugate concentrations

Among the antigens used in this Example, the purified Tsutsugamus recombinant 56K antigen was prepared according to the above example, and the Leptospira recombinant FlaB antigen (having the amino acid sequence of SEQ ID NO: 7 and the nucleotide sequence of SEQ ID NO: 8) was disclosed in the patent. Prepared according to the method disclosed in No. 10-2002-0078629, the purified Hantan virus recombinant N antigen (having the amino acid sequence of SEQ ID NO: 10) was subjected to the nucleotide sequence of SEQ ID NO: 11 from Hantaan virus 76-118 (National Institutes of Health). amplification, and it is also through the process as shown in Preparation 7, the pBAD / Thio TOPO a recombinant vector in a recombinant (Invitrogen) vector and the N gene, by transforming it to the E.coli culture of the transformant E.coli obtained And purified from the culture were used.

Purified Tsutsugamushi r56K antigen, purified Hantan virus recombinant N antigen, and leptospira recombinant FlaB antigen were injected and analyzed by concentration, and 500 µg / ml of three recombinant antigens were determined as appropriate antigen concentrations.

Protein A was conjugated using a 20 nm gold suspension prepared according to the method of Greg et al. And O.D. It was suspended in 10 mM Borax buffer at a concentration of 10. The protein A-gold conjugate was sprayed at 30 μl / cm onto a conjugate pad with 20 mM Tris-HCl (pH 7.4) containing bovine serum, sucrose, and Tween-20 and dried, and analyzed by O.D. 5.0 to 8.0 were determined at the proper concentration.

(2) Manufacture of high speed inspection kit

Antigen was diluted in 10 mM phosphate buffer (pH 7.2) and sprayed onto the NC membrane (Satorius) at a concentration of 1.0 μl / cm using a divider (CAMAG or BIODOT) as shown in FIG. 6. Three simultaneous diagnostic kits sprayed each antigen onto the same NC membrane at 3 mm intervals.

As a control, goat anti-human IgG (Sigma) was diluted to 1.0 mg / ml and sprayed in the same manner as the antigen. After drying the injected NC membrane, the conjugate pad, the sample pad, and the absorbent pad sprayed with the protein A-gold conjugate were attached to a laminator card, and cut at 0.4 cm intervals. Was produced.

The test dropped 5 μl of serum onto the strip, added 100 μl of assay buffer (3% BSA, 10 mM Tris-HCl, pH 8.0) and read the results after 5-10 minutes. The prepared kit evaluated the adequacy of the reaction with positive serum and negative serum. Serum used in the experiment was lepttospirosis using MAT according to the standard diagnostic method, Tsutsugamusosis and nephrotic syndrome hemorrhagic fever using IFA was used to determine the positive and negative. The results are shown in Table 2. The results are shown in Table 2.

Table 2. Sensitivity and Specificity of the Simultaneous Diagnosis Rapid Test Kit for Three Fever Diseases

disease % Sensitivity (number of serums) % Specificity (number of serums) Leptospirosis 95.0 (20) 99.3 (151) Tsutsugamusosis 97.1 (71) 98.9 (91) HFRS 84.6 (26) 91.8 (170)

In addition, commercial diagnostic kits, Dipstick (Immunochromatography) kit and passive hemagglutination kit, were tested according to standard diagnostic methods to determine sensitivity and specificity. The results are shown in Table 3.

Table 3. Sensitivity and Specificity of Commercial Diagnostic Kits

Commercial kit % Sensitivity (number of serums) % Specificity (number of serums) Leptospirosis (Dipstick Kit) 85.7 (14) 81.8 (33) Tsutsugamosis (Dipstick Kit) 87 (87) 81 (59) HFRS (PHA Kit) 83.3 (6) 57.1 (7)

Example 9 Preparation and Test of ELISA Kit for Diagnosing Three Fever Diseases

(1) antigen

The three antigens used were the same as those used in Example 8. Each antigen was used after purifying histidine-tagged protein using a Ni-NTA column (Qiagen) and confirming the presence of the antigen using SDS-PAGE. Antigenicity was tested by Western blotting using an anti-His monoclonal antibody (Qiagen) and serum or rabbit immuno-immune serum (antiserum obtained by inoculating the antigen into rabbits) of each patient and used as antigen. Protein quantification of the antigen was quantified using BCA protein analysis (Pierce, Inc.) and stored at −70 ° C. and used for kit preparation.

(2) secondary antibodies

HRP anti-human IgG and HRP anti-human IgM (Sigma) were diluted and used at appropriate concentrations.

(3) substrate and suspension solution

TMB (Moss Inc) was used as the substrate for HRP and 0.5 M sulfuric acid solution was used as reaction stopper.

(4) diluent and washing liquid

Phosphate buffer (pH 7.2) containing 0.005% Tween 20 (Sigma) was used as the diluent and left wing solution.

(5) Proper antigen concentration and concentration of conjugate

As a result of the check board titration, the titer antigen titration was titrated at 1 μg / ml, and HRP anti-human IgG and IgM (Sigma) were titrated at 0.2 μg / ml. Since the reaction was shown, an ELISA kit was prepared at the above concentration.

Sample serum was tested using the prepared ELISA kit. Serum used for the test was tested in the same manner as used in Example 8 and divided into positive and negative serum. The results are shown in Table 4.

Table 4. Sensitivity and Specificity of ELISA Kits

antigen % Sensitivity (serum count) % Specificity (serum count) IgG IgM IgG IgM rFlaB 90.0 (20) 100 (20) 91.4 (151) 80.4 (151) r56K 97.2 (71) 85.7 (91) 85.7 (91) 94.5 (91) N 96.2 (26) 80.1 (26) 79.4 (170) 83.5 (170)

As described in detail above, the recombinant protein or variant of SEQ ID NO: 1 having the surface antigen activity of Orientia tsutsugamushi Pajoo according to the present invention showed excellent reactivity to the antibody in the serum of Tsutsugamushi infected or patient .

In addition, the three pyrogenic disease kits according to the present invention use the recombinant 56KDa protein, the recombinant FlaB protein, and the recombinant N protein of the present invention, and thus have very high sensitivity and specificity in diagnosing leptospirosis, Tsutsugamusosis, and nephrotic syndrome hemorrhagic fever. In addition, these three types of fever disease can be diagnosed effectively.

<110> Shin Jin Medics Inc.          Republic of Korean (National Institute of Health)          Genobiotech Co., Ltd. <120> A kit for detecting tsutsugamushi pajoo, Leptospira interrogans          serova lai and Hantaan virus infection simultaneously <160> 11 <170> KopatentIn 1.71 <210> 1 <211> 560 <212> PRT <213> Artificial Sequence <220> <223> modified 56 kDa protein <400> 1 Gly Ser Arg Ile Ser Leu Glu Trp Leu Pro Leu Lys Asn Lys Phe Asn   1 5 10 15 Ser Phe Lys Glu Ile Arg Met Lys Lys Ile Met Leu Ile Ala Ser Ala              20 25 30 Met Ser Ala Leu Ser Leu Pro Phe Ser Ala Ser Ala Ile Glu Leu Gly          35 40 45 Asp Glu Gly Gly Leu Glu Cys Gly Pro Tyr Ala Lys Val Gly Val Val      50 55 60 Gly Gly Met Ile Thr Gly Ala Glu Ser Ala Arg Leu Asp Gln Ala Asp  65 70 75 80 Thr Thr Gly Lys Lys His Leu Pro Leu Thr Thr Ser Met Pro Phe Gly                  85 90 95 Gly Thr Leu Asn Ala Gly Met Thr Ile Thr Pro Trp Leu Arg Ala Glu             100 105 110 Leu Gly Val Met Tyr Leu Arg Asn Ile Ser Ala Glu Val Glu Val Gly         115 120 125 Lys Gly Lys Val Glu Val Gly Lys Gly Lys Ala Asp Ser Gly Gly Gly     130 135 140 Thr Asp Ala Pro Ile Arg Lys Arg Phe Lys Leu Thr Pro Pro Gln Pro 145 150 155 160 Thr Ile Met Pro Ile Ser Ile Ala Asp Arg Asp Phe Gly Ile Asp Ile                 165 170 175 Arg Asn Ile Pro Gln Ala Gln Ala Gln Ala Ala Gln Pro Gln Leu Asn             180 185 190 Asp Glu Gln Arg Ala Ala Ala Arg Val Ala Trp Leu Lys Asn Tyr Ala         195 200 205 Gly Ile Asp Tyr Arg Val Lys Asp Pro Asn Asn Pro Asn Gly Pro Met     210 215 220 Val Ile Asn Pro Val Leu Leu Asn Ile Pro Gln Gly Asn Pro Asn Pro 225 230 235 240 Val Gly Asn Pro Pro Gln Arg Ala Asn Gln Pro Ala Asp Phe Ala Ile                 245 250 255 His Asn His Glu Gln Trp Arg Tyr Met Val Leu Asp Leu Leu His Tyr             260 265 270 Gln Met Leu Ile Asn Leu Ala Ile Leu Leu Ser Lys Tyr Leu Ser Asp         275 280 285 Lys Ile Thr Gln Ile Tyr Asn Asp Ile Arg Pro Phe Ala Asp Ile Val     290 295 300 Gly Ile Asp Val Pro Asn Gly Ala Leu Pro Asn Ser Ala Ser Val Glu 305 310 315 320 Gln Ile Gln Asn Lys Met Gln Glu Leu Asn Glu Leu Leu Glu Glu Val                 325 330 335 Arg Asp Ser Phe Glu Gly Tyr Ile Gly Gly Asn Ala Phe Ala Asn Gln             340 345 350 Ile Gln Leu Asn Phe Val Ile Pro Gln Gln Ala Gln Gln Gln Gln Gln         355 360 365 Gln Gly Gln Gly Gln Gln Gln Gln Ala Gln Ala Thr Ala Gln Glu Ala     370 375 380 Ala Ala Ala Ala Ala Val Arg Leu Leu Asn Gly Asn Asp Gln Ile Val 385 390 395 400 Gln Leu Tyr Lys Asp Leu Val Lys Leu Lys Arg His Ala Gly Phe Lys                 405 410 415 Lys Ser Met Asp Lys Leu Ala Ala Gln Gln Glu Glu Asp Ala Lys Gln             420 425 430 Lys Glu Glu Asp Ala Lys Gln Lys Glu Gly Ser Cys Lys Lys Asp Asp         435 440 445 Lys Glu Gly Val Ser Lys Glu Thr Glu Phe Asp Leu Ser Met Ile Val     450 455 460 Gly Gln Val Lys Leu Tyr Ala Asp Leu Val Ala Thr Glu Ser Phe Ser 465 470 475 480 Ile Tyr Ala Gly Val Gly Ala Gly Leu Ala His Thr Tyr Gly Lys Ile                 485 490 495 Asp Asp Lys Asp Ile Lys Gly His Thr Gly Met Val Ala Ser Gly Ala             500 505 510 Leu Gly Val Ala Ile His Ala Ala Glu Gly Val Tyr Val Asp Ile Glu         515 520 525 Gly Ser Tyr Met Tyr Ser Phe Ser Lys Ile Glu Glu Arg Tyr Ser Ile     530 535 540 Asn Pro Leu Met Ala Ser Ile Ala Val Arg Tyr Asn Phe Gly Ser Phe 545 550 555 560 <210> 2 <211> 1683 <212> DNA <213> Artificial Sequence <220> <223> gene for modified 56 kDa <400> 2 ggctcgagaa ttagtttaga atggttacca ctaaaaaata aatttaattc ttttaaggag 60 attagaatga aaaaaattat gttaattgct agtgcaatgt ctgcgttgtc gttgccgttt 120 tctgctagtg cgatagaatt gggggatgaa ggaggattag agtgtggtcc ttatgctaaa 180 gttggagttg tcggaggaat gattactggc gcagaatctg ctcgcttgga tcaagctgat 240 actactggca aaaaacactt gccattaaca acctcgatgc catttggtgg tacattaaat 300 gcaggtatga caattactcc atggcttaga gcagagctag gggttatgta ccttagaaat 360 ataagcgctg aggttgaagt aggtaaaggc aaggttgaag taggtaaagg caaggcagat 420 tctggaggtg ggacagatgc tcctatacgt aagcggttta aacttacacc tcctcagcct 480 actataatgc ctataagtat agctgatcgt gactttggga ttgatattcg taacatacct 540 caggcgcaag cgcaagctgc gcagcctcag cttaatgatg agcaacgtgc tgcagctagg 600 gtcgcttggt taaagaatta tgctggtatt gactataggg taaaagatcc taataatcct 660 aatgggccta tggttataaa tccggtgttg ttaaatattc cacagggtaa ccctaatcct 720 gttggaaatc caccgcagcg agcaaatcag cctgcagatt ttgcgataca taaccatgag 780 caatggaggt atatggtatt ggacttgctg cattatcaaa tgctaataaa cctagcgatc 840 ctcctgtcaa agtatctaag tgataaaatt actcagatat ataatgatat aaggccattt 900 gctgatatag ttggtattga tgttcctaat ggtgctttgc ctaatagtgc atctgtcgaa 960 cagatacaga ataaaatgca agaattaaac gagctattag aagaggtcag agattctttt 1020 gaggggtata ttggtggtaa tgcttttgct aatcagatac agttgaattt tgtcataccg 1080 cagcaagcac agcagcagca gcagcagggg caagggcagc aacagcaagc tcaagctaca 1140 gcgcaagaag cagcagcagc agcagctgtt aggcttttaa atggcaatga tcagattgtg 1200 cagttatata aagatcttgt taaattgaag cgtcatgcag gatttaagaa atctatggat 1260 aaattagctg ctcaacaaga agaagatgca aagcaaaaag aagaagatgc aaagcaaaaa 1320 gaaggtagct gcaagaagga cgataaagaa ggagtaagca aagaaacaga gtttgatctg 1380 agtatgattg ttggtcaagt taaactctat gctgatctag ttgcaactga atcattctca 1440 atatatgctg gtgttggtgc agggttagct catacttatg gaaaaataga tgataaggat 1500 attaaagggc atacaggcat ggttgcatca ggggcgcttg gtgtagcaat tcatgctgct 1560 gagggtgtgt atgtggacat agaaggtagt tatatgtact cattcagtaa aatagaagag 1620 aggtattcaa taaatcctct tatggcaagt attgctgtac gctataactt ctagggatcc 1680 ttt 1683 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> synthetic primer <400> 3 tagtgtggct aaataattag tttagaa 27 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> synthetic primer <400> 4 tttaaagaaa aactagaagt tatagcg 27 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> synthetic primer <400> 5 ggctcgagaa ttagtttaga atggttacca 30 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> synthetic primer <400> 6 aaaggatccc tagaagttat agcgtacagc 30 <210> 7 <211> 205 <212> PRT <213> Artificial Sequence <220> <223> modified FlaB protein <400> 7 Leu Arg Gln Ala Glu Arg Asn Thr Glu Asp Gly Met Ser Phe Ile Gln   1 5 10 15 Thr Ala Glu Gly Phe Leu Glu Gln Thr Ser Asn Ile Gln Arg Ile              20 25 30 Arg Val Leu Ala Ile Gln Thr Ser Asn Gly Ile Tyr Ser Asn Glu Asp          35 40 45 Arg Gln Leu Val Gln Val Glu Val Ser Ala Leu Val Asp Glu Val Asp      50 55 60 Arg Ile Ala Ser Gln Ala Glu Phe Asn Lys Phe Lys Leu Phe Glu Gly  65 70 75 80 Gln Phe Ala Arg Gly Ser Arg Val Ala Ser Met Trp Phe His Met Gly                  85 90 95 Pro Asn Gln Gln Arg Glu Arg Phe Tyr Ile Gly Thr Met Thr Ser Lys             100 105 110 Ala Leu Lys Leu Val Lys Ala Asp Gly Arg Pro Ile Ala Ile Ser Ser         115 120 125 Arg Gly Glu Ala Asn Asp Val Ile Gly Leu Ala Asp Ala Ala Leu Thr     130 135 140 Lys Ile Met Lys Gln Arg Ala Asp Met Gly Ala Tyr Tyr Tyr Leu Glu 145 150 155 160 Tyr Thr Ala Lys Gly Leu Met Gly Ala Tyr Glu Asn Met Gln Ala Ser                 165 170 175 Glu Ser Arg Ile Arg Asp Ala Asp Met Ala Glu Glu Val Val Ser Leu             180 185 190 Thr Thr Lys Gln Ile Leu Val Gln Ser Gly Thr Ala Met         195 200 205 <210> 8 <211> 771 <212> DNA <213> Artificial Sequence <220> <223> gene for modified FlaB <400> 8 ctcaccgttc tctaaagttc aacgaacttg ctgtggacaa gacgatgaaa gctgtgtctt 60 ccggtatgcg gattaattcc gctgcggacg acgcttccgg acttgcggtt tctgaaaagc 120 ttagaacgca gatcaacggt ctgcgtcagg ccgaaagaaa caccgaagac ggaatgagtt 180 tcattcaaac tgccgaggga tttctggagc agacgtctaa catcattcaa agaattcggg 240 tgctcgccat ccagacttcg aatggtatct acagcaacga agataggcag cttgtgcagg 300 tggaagtatc tgcgctggtg gatgaagtcg atcgaattgc ttctcaggct gaatttaata 360 agttcaaact ttttgaaggc caattcgcta gaggttccag ggttgcatcc atgtggtttc 420 atatgggtcc aaaccaaaat cagcgtgaaa gattttacat aggcacgatg acttcaaagg 480 ctctgaagct tgtaaaagcg gacgggaggc cgatcgcgat ctcttctcgg gagaggctaa 540 cgacgtgatc ggtttggcag atgctgccct tacgaagatc atgaagcaga gagcggatat 600 gggagcttat tataataggc ttgaatatac cgcaaagggt ctgatgggtg cgtatgaaaa 660 tatgcaggca tctgaatcta gaattcgaga cgccgatatg gcggaggaag ttgtctcgct 720 gaccacaaaa caaatacttg tacagagtgg tacggcaatg ttggatcccc g 771 <210> 9 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequences added to the N-terminal of 56 kDa protein <400> 9 Gly Ser Arg Ile Ser Leu Glu Trp Leu Pro Leu Lys Asn Lys Phe Asn   1 5 10 15 Ser Phe Lys Glu Ile Arg              20 <210> 10 <211> 429 <212> PRT <213> Hantaan virus 76-118 <400> 10 Met Ala Thr Met Glu Glu Leu Gln Arg Glu Ile Asn Ala His Glu Gly   1 5 10 15 Gln Leu Val Ile Ala Arg Gln Lys Val Arg Asp Ala Glu Lys Gln Tyr              20 25 30 Glu Lys Asp Pro Asp Glu Leu Asn Lys Arg Thr Leu Thr Asp Arg Glu          35 40 45 Gly Val Ala Val Ser Ile Gln Ala Lys Ile Asp Glu Leu Lys Arg Gln      50 55 60 Leu Ala Asp Arg Ile Ala Thr Gly Lys Asn Leu Gly Lys Glu Gln Asp  65 70 75 80 Pro Thr Gly Val Glu Pro Gly Asp His Leu Lys Glu Arg Ser Met Leu                  85 90 95 Ser Tyr Gly Asn Val Leu Asp Leu Asn His Leu Asp Ile Asp Glu Pro             100 105 110 Thr Gly Gln Thr Ala Asp Trp Leu Ser Ile Ile Val Tyr Leu Thr Ser         115 120 125 Phe Val Val Pro Ile Leu Leu Lys Ala Leu Tyr Met Leu Thr Thr Arg     130 135 140 Gly Arg Gln Thr Thr Lys Asp Asn Lys Gly Thr Arg Ile Arg Phe Lys 145 150 155 160 Asp Asp Ser Ser Phe Glu Asp Val Asn Gly Ile Arg Lys Pro Lys His                 165 170 175 Leu Tyr Val Ser Leu Pro Asn Ala Gln Ser Ser Met Lys Ala Glu Glu             180 185 190 Ile Thr Pro Gly Arg Tyr Arg Thr Ala Val Cys Gly Leu Tyr Pro Ala         195 200 205 Gln Ile Lys Ala Arg Gln Met Ile Ser Pro Val Met Ser Val Ile Gly     210 215 220 Phe Leu Ala Leu Ala Lys Asp Trp Ser Asp Arg Ile Glu Gln Trp Leu 225 230 235 240 Ile Glu Pro Cys Lys Leu Leu Pro Asp Thr Ala Ala Val Ser Leu Leu                 245 250 255 Gly Gly Pro Ala Thr Asn Arg Asp Tyr Leu Arg Gln Arg Gln Val Ala             260 265 270 Leu Gly Asn Met Glu Thr Lys Glu Ser Lys Ala Ile Arg Gln His Ala         275 280 285 Glu Ala Ala Gly Cys Ser Met Ile Glu Asp Ile Glu Ser Pro Ser Ser     290 295 300 Ile Trp Val Phe Ala Gly Ala Pro Asp Arg Cys Pro Pro Thr Cys Leu 305 310 315 320 Phe Ile Ala Gly Ile Ala Glu Leu Gly Ala Phe Phe Ser Ile Leu Gln                 325 330 335 Asp Met Arg Asn Thr Ile Met Ala Ser Lys Thr Val Gly Thr Ser Glu             340 345 350 Glu Lys Leu Arg Lys Lys Ser Ser Phe Tyr Gln Ser Tyr Leu Arg Arg         355 360 365 Thr Gln Ser Met Gly Ile Gln Leu Gly Gln Arg Ile Val Leu Phe     370 375 380 Met Val Ala Trp Gly Lys Glu Ala Val Asp Asn Phe His Leu Gly Asp 385 390 395 400 Asp Met Asp Pro Glu Leu Arg Thr Leu Ala Gln Ser Leu Ile Asp Val                 405 410 415 Lys Val Lys Glu Ile Ser Asn Gln Glu Pro Leu Lys Leu             420 425 <210> 11 <211> 1290 <212> DNA <213> Hantaan virus 76-118 <400> 11 atggcaacta tggaggaatt acagagggaa atcaatgccc atgagggtca attagtgata 60 gccaggcaga aggtgaggga tgcagaaaaa cagtatgaaa aggatccaga tgagttgaac 120 aagagaacat taactgaccg agagggcgtt gcagtatcta tccaggcaaa aattgatgag 180 ttaaaaaggc aactggcaga taggattgca actgggaaaa accttgggaa ggaacaagat 240 ccaacagggg tggagcctgg agaccatctg aaagagaggt caatgctcag ttatggtaat 300 gtgctggatt taaaccattt ggatattgat gaacctacag gacagacagc agactggctg 360 agcatcatcg tctatcttac atcctttgtc gtcccgatac ttctgaaagc tctgtatatg 420 ttgacaacaa gggggaggca aactaccaag gataataaag ggacccggat tcgatttaag 480 gatgatagct cgttcgagga tgttaacggt atccggaaac caaaacatct ttacgtgtcc 540 ttgccaaatg cacagtcaag catgaaggca gaagagatta cacctggtag atatagaaca 600 gcagtctgtg ggctctaccc tgcacagatt aaggcacggc agatgatcag tccagttatg 660 agtgtaattg gttttctagc attagcaaag gactggagtg atcgtatcga acaatggtta 720 attgaacctt gcaagcttct tccagataca gcagcagtta gcctccttgg tggtcctgca 780 acaaacaggg actacttacg gcagcggcaa gtggcattag gcaatatgga gacaaaggag 840 tcaaaggcta tacgccagca tgcagaagca gctggctgta gcatgattga agatattgag 900 tcaccatcat caatatgggt ttttgctgga gcaccagacc gttgtccacc aacatgtttg 960 tttatagcag gtattgctga gcttggggca tttttttcca tcctgcagga catgcgaaat 1020 acaatcatgg catctaagac agttggaaca tctgaggaga agctacggaa gaaatcatca 1080 ttttatcagt cctacctcag aaggacacaa tcaatgggga tacaactagg ccagagaatt 1140 attgtgctct tcatggttgc ctggggaaag gaggctgtgg acaacttcca cttaggggat 1200 gatatggatc ctgagctaag gacactggca cagagcttga ttgatgtcaa agtgaaggaa 1260 atctccaacc aagagccttt gaaactctaa 1290

Claims (4)

Orientia tsutsugamushi , Leptospirintero, comprising a recombinant protein represented by the amino acid sequence of SEQ ID NO: 1, a recombinant protein represented by the amino acid sequence of SEQ ID NO: 7, and a recombinant protein represented by the amino acid sequence of SEQ ID NO: 10 A diagnostic kit that can simultaneously detect antibodies against infection of Leptospira interrogans and Hantaan virus . The diagnostic kit of claim 1, wherein the kit is an immunochromatography or ELISA kit. The diagnostic kit according to claim 2, wherein the three kinds of protein antigens are immobilized on the nitrocellulose membrane. The diagnostic kit according to claim 2, wherein the three kinds of protein antigens are immobilized on a polymer support.

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