KR100514438B1 - A recombinant protein useful for the detection of tsutsugamushi infection, a gene encoding the same, and a diagnostic kit using the same - Google Patents

Abstract

The present invention provides a recombinant protein having the surface antigen activity of Orientia tsutsugamushi Pajoo useful for the detection of Tsutsugamushi infection, a gene encoding the same, an expression vector comprising the gene, a host cell transformed with the expression vector, Provided are a method for producing the recombinant protein from the host cell, and a diagnostic kit for detecting infection of Tsutsugamu containing the recombinant protein.

The recombinant protein according to the present invention not only has high antigenicity, but also can be easily cloned into an expression vector, thereby enabling mass production of antigen, thereby effectively preparing a diagnostic reagent or a diagnostic kit.

Description

A recombinant protein useful for the detection of tsutsugamushi infection, a gene encoding the same, and a diagnostic kit using the same}

The present invention provides a recombinant protein having the surface antigen activity of Orientia tsutsugamushi Pajoo useful for the detection of Tsutsugamushi infection, a gene encoding the same, an expression vector comprising the gene, a host cell transformed with the expression vector, The present invention relates to a method for producing the recombinant protein from the host cell, and a diagnostic kit for detecting an infection of Tsutsugamu containing the recombinant protein.

Tsutsugamus disease is an acute pyrogenic disease caused by the infection of Orientia Tsutsugamushi , which is called in Asia in Japan, China, Malaysia, Thailand, Vietnam, etc. (Tsutsugamushi disease in Korea. -27,1994), most prevalent in October, most often between September and early December (Kim et al. 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, high fever, chills, headache, molecular papule 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. 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. In addition, acute febrile diseases such as leptospirosis, rash and bleeding hemorrhagic fever are called at the same time.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 (Park et al., 45: 497). -509,1993, Bae, Hyun-Ju Institute of Infection Studies, p79-87, 2001).

The bacteriological diagnosis that isolates the causative organism Orientia Tsutsugamushi from the patient's blood requires susceptible animal inoculation, cell culture and biosafety facilities (Biosafty level 3) to isolate the causative organism, and the identification time is 3 weeks or longer. As a result, it is impossible to use the test results in actual patient treatment.

Therefore, the diagnosis of Tsutsugamus is usually made by clinical symptoms and serological diagnosis (Jang et al. Korean Journal of Microbiology 24: 281-289, 1989). Traditionally, Tsutsugamu bacteria are classified as Gillam, Karp, Kato serotypes (Derrick, EH, et al., Lancet. 13: 833-838, 1964, Bennet, BL, et al., J. Immunol. 62: 453- 461, 1949, Shishido, A., et al., Jpn. J. Med. Sci. Biol. 11: 383-399, 1958), and serologically different strains have been isolated from Korea, Japan, and Thailand. According to Boryong, Yeoncheon, Korea, isolated strains (Chang, WH, et al., J. Clin.Microbiol. 28: (4) 685-688, 1990, Seong, SY, et al., Microbiol Immunol. 41 (6): 437-443, 1997) and Shimokoshi, Kawasaki, kuroki, TA716, TA763, TH1817 (Tamura. A., et al., Microbiol. Immunol. 28 (8): 873-882, 1984. , Yamamoto.S., Et al., Microbiol.Imunmun. 30 (7): 611-620, 1986, Ohashi.N., Et al., J. Clin.Microbiol. 28 (9): 2111-2113, 1990 Serological classifications, etc., are becoming more sophisticated.

Serological methods include nonspecific Weil-Felix reactions, complement binding tests, indirect immunofluorescent antibodies, enzyme immunoassays, indirect immune peroxidase tests, and passive hemagglutination (Wolf, JW, 32). : 352-360, 1931, Yamamoto, S., et al., J. Clin.Microbiol. 15: 1128-1132, 1982, Suto T., Clin.Virol. 84: 425-429, 1980, Kim et al. Korean Journal of Microbiology 24: 281-289, 1989; The Korean Medical Association Journal 31: 608-611, 1988; Kim et al. Korean Society of Internal Medicine 50: 77-86, 1996) Micro Indirect Immunofluroscence Assay (MIFA) is widely used, but TSUSUGAMU bacteria used for diagnosis are passaged in cell culture and the culture period is over two weeks. Many problems are known due to objective standardization, such as being expensive and requiring skill in reading the diagnosis (Robi nson et al., Am. J. Trop. Med. 25: 900-905, 1976).

Recently, the method of diagnosing Tsutsugamushi disease using PCR (Sugita Y., et al., J. Med. Microbiol. 37: 357-360, 1992) has been studied. Passive hamagglutination assay (PHA) (Kim IS, et al., J. Clin. Microbiol. 31: 2057-2060) in which a recombinant antigen of a surface antigen protein (56kDa) of Boryeong isolates is attached to sheep red blood cells in Korea. , 1993) and diagnostic kits using the same and diagnostic methods using ELISA (Kim IS, et al., J. Clin. Microbiol. 31: 598-605, 1993). However, the passive hemagglutination method, which 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 (Ching WM, et al., Clin. Diagn. Lab. Immunol. 5 (4): 519-526, 1998), which improved the recombinant 56kDa antigenic site of Karp strains abroad, rapid immunochromatography Rapid immunochromatographic flow assay (RAF) (Ching WM, et al., Clin. Diagn. Lab. Immunol. 8 (2): 409-414, 2001) has 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 Orientia tsutsugamushi Pajoo in Korea, and found the gene of the coat protein (56kDa) and the coat protein expressed from the gene from this strain, NCNC I gene bank authorization number (NCBI) genbank accession number (AF430142) was obtained.

On the other hand, in order to develop a diagnostic kit for detecting Tsutsugamu infection using the surface antigen protein, the surface antigen protein should be cloned into an expression vector effectively and expressed in a host cell to be prepared in large quantities. However, when an expression vector including a gene encoding a foreign protein is prepared using a common general vector, the recovery rate is inefficient for industrial use in the expression and purification of the recombinant recombinant protein and host E. coli cells. It is frequently produced as a result, there is a problem that mass production is difficult. Furthermore, when the N- and C-terminus of the protein (antigen) is changed for cloning, the three-dimensional structure of the antigen is changed, resulting in a problem of low antigenicity, ie sensitivity and specificity.

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

Accordingly, an object of the present invention is to provide a novel recombinant protein having a surface antigen activity of Orientia tsutsugamushi Pajoo, which is useful for the detection of Tsutsuga mucin infection, and a gene encoding the same.

It is also an object of the present invention to provide an expression vector comprising the gene and a host cell transformed with the expression vector.

It is also an object of the present invention to provide a method for producing the recombinant protein from the host cell.

It is also an object of the present invention to provide a diagnostic kit for detecting infection of Tsutsuga mucin bacteria comprising the recombinant protein.

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 Provided variants having surface antigenic activity of Orientia tsutsugamushi Pajoo with homology over 90% with sequence.

Recombinant protein represented by the amino acid sequence of SEQ ID NO: 1 is an amino acid sequence is additionally introduced at both ends of the 56kDa protein encoding the specific surface antigen of the various antigenic determinants of Orientia tsutsugamushi Pajoo. That is, 22 amino acids of GSRISLEWLPLKNKFNSFKEIR 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 can be effectively used in diagnostic kits for the detection of Tsutsugamu infection by exhibiting high sensitivity and specificity as can be seen in the following test examples.

In addition, the variant refers to a protein having a modification such as substitution, deletion, or insertion in the amino acid sequence or 56 kDa surface antigen sequence added to the sock end, and despite such modification, the amino acid of SEQ ID NO: 1 And a protein having a surface antigenic activity of Orientia tsutsugamushi Pajoo with homology greater than or equal to 90% with the sequence.

The present invention provides a gene encoding the recombinant protein or variant. The gene according to the present invention includes a pre- and post-sequence together with the gene encoding the surface antigen of Orientia tsutsugamushi Pajoo, and is preferably a gene represented by the nucleotide sequence of SEQ ID NO: 2. The gene according to the present invention contains a restriction enzyme action site that co-operates with the inserted vector, so that it can be cloned effectively into the expression vector, thereby enabling mass production of antigens essential for diagnosing Tsutsugamus.

The present invention provides an expression vector comprising the gene. Expression vectors of the present invention may include a conventional promoter sequence, translation initiation sequence and terminator sequence to express the recombinant protein. The expression vector of the present invention is preferably designed using a vector in which histidine is fused to the N-terminal portion of the protein so as to facilitate purification of the recombinant protein. As shown in FIG. 1, the gene according to the present invention is expressed in pET-15b (Novagen). It is more preferable that it is p56kDa manufactured by inserting in USA.

The present invention also provides a host cell transformed with the expression vector. As host cells usable in the present invention, mammalian cells such as prokaryotic cells such as bacteria, eukaryotic cells such as yeast, or multicellular organism cells may be used, but E. coli is economically preferable for mass production through expression. More preferably, the host cell of the present invention is Escherichia coli NIH / BL / 56kDa-ET15B of Accession No. KCCM 10489.

In addition, the present invention provides a method for producing the recombinant protein or variant comprising the step of culturing and isolating the host cell. Preferably, the production method of the present invention may further comprise a purification step by chromatography using His-binding resin.

The present invention also provides a diagnostic kit for detecting infection of Tsutsuga mucin in humans and mammals, comprising the recombinant protein or variant. In the diagnostic kit of the present invention, the recombinant protein or variant is preferably prepared by the production method according to the present invention. In addition, the detection method used in the diagnostic kit of the present invention is preferably an ELISA method or a rapid immunochromatographic flow assay (RFA).

In the case of the ELISA kit, a sample of the primary antibody is reacted on a plate coated with the recombinant protein antigen of the present invention, and then a secondary antibody labeled with peroxidase is bound, followed by OPD (orange), ABTS (green) or TMB (pink). It can be configured to react with the substrate of). In the case of a rapid immunochromatographic flow assay (RFA) kit, the recombinant protein antigen of the present invention is adsorbed and immobilized on gold, and then dipped into a membrane on a glass pad, and a horseradish peroxidase or alkaline phosphatase is added thereto. The bound secondary antibody may be instilled and then reacted by dropping the sample serum onto a glass pad.

The diagnostic kit of the present invention may further include one or more recombinant antigens for diagnosing other pathogens other than Tsutsugashimi.

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 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, in order to amplify the gene for cloning into the vector from the DNA fragment amplified by 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 oligonucleotides were confirmed by UV light 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 Distilled water was added to a solution containing 0.5 µl of LA Taq DNA polymerase (Takara, Japan), and 1 µl each of the primer (the oligonucleotide synthesized in Example 2), so that the total amount was 100 µl. For the gene sequence of interest, the template DNA is the chromosomal DNA of Orientia Tsutsugashimu isolated from Example 1 and the primer is an oligonucleotide of SEQ ID NO: 3 and SEQ ID NO: 4; For gene amplification for cloning of 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.

For the PCR reaction, a temperature cycler (Thermal cycler, Perkin-Elmer, USA) was used, denaturation (94 ℃, 30 seconds), annealing, (50 ℃, 30 seconds), extension ( 72 ° C., 1 minute) was performed 30 times, and finally, 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 and the expression vector pET-15b (Novagen, USA) were cut into Xho I (NEB, USA) and BamH I (NEB, USA), respectively. 0.1 μg of the expression vector and 30 ng of the recombinant protein gene were mixed, and 10 μl of conjugate concentrate (10 mM DTT and 100 mM MgCl ₂ , 600 mM Tris-HCl containing 10 mM ATP, pH7.5) 1 μl and T4 DNA ligase (NEB) US, USA) 10 units (Unit) was added to adjust the total volume to 10 μl and reacted for 18 hours at 16 ℃ to prepare a cyclic plasmid in which the gene is inserted into the expression vector pET-15b (Takara, Japan), This 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, treated with 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 broken at 15,000 psi and kept cold in ice, and then centrifuged at 8,000 rpm for 10 minutes at 4 ° C. 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 procedure 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 at room temperature. Bound for -3 hours. Carefully fill the column with a supernatant-filler mixture to avoid foaming and elute the mixture, then wash buffer (10 mM IMDZ, 20 mM Na ₂ HPO ₄ , 0.5 M NaCl, 8 M Urea (pH 6.0)) 10 The column was washed four times with mL. The washed protein was passed through 3 ml of elute 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 change of protein during the purification process was performed by electrophoresis with 10% SDS-PAGE to confirm that the purification process proceeded normally. (See FIG. 3) In FIG. 3, lane 1 shows cell lysate, lane 2 effluent, lanes 3-5 for washed solution, and lanes 6-9 for 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

ELISA IgG, IgM was performed using the recombinant protein purified in Example 7 as an antigen, 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 with a predetermined amount of purified recombinant protein in a coating solution (0.159 g Na ₂ CO ₃ , 0.293 g NaHCO ₃ per 100ml) and put in 100 μL in 96 well plate (18-24 hours). Completely remove the recombinant protein solution and buffer (PBS) with 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 resistant solution, wash 5 times with Buffer (PBS-T) containing 0.01% Tween-20, and dilute the patient's serum to be used as the primary antibody in a 1: 100 dilution buffer. 100 μl of sugar was added and reacted at 37 ° C. for 1 hour. Each groove was washed five times with 200 μl buffer and the secondary antibody was diluted 1: 25,000 in PBS with antiserum IgG (Sigma, USA) labeled with peroxidase and placed in 100 μl in each groove. 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 groove was washed five 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 96-well plate which was sufficiently washed was completely removed, and then 100 μl of the coloring reagent was added to the grooves. 20 minutes after the start of color development, 50 µl of 2 normal (N) hydrogen peroxide (H ₂ O ₂ ) was added to each groove to stop the reaction, and 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 a MIFA titer of 128 or higher or the presence of clinical symptoms, 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) serum. The number of times divided by the number of each serum tested.

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 from Table 1, ELISA (IgG) using recombinant antigen protein using sera determined to be 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. In particular, IgM levels are important indicators for early infection, and the results of ELISA (IgM) using recombinant antigenic proteins are significant.

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. Anti-human-IgG (alkaline phosphatase, AP) bound (Anti-human-IgG, Sigma, USA) was diluted 1: 3,000 to 5% skim milk added TBS and reacted at room temperature for 2 hours Washed three times for 5 minutes with TTBS. 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 Tsutsugamus positive sera, lane 6 is negative Tsutsugamus negative sera, lanes 7 are leptospirosis negative serum, lanes 8-11 are leptospirosis positive serum, lanes 12-13 nephrotic syndrome hemorrhagic fever Positive serum, lanes 14-15 for nephrotic syndrome bleeding negative serum, lanes 16-17 for hepatitis B viral sera, 18-19 lanes for viral hepatitis C positive serum, lanes 20-22 for malaria positive serum, 23 Lane -24 shows the results of cross-reaction testing on malaria negative sera. Western blot using recombinant protein as antigen showed specific bands in all patients with Tsutsugamus positive patients and did not cross-react with similar diseases such as leptospirosis, nephrotic syndrome hemorrhagic fever serum, 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 this recombinant antigen can be effectively used for differential diagnosis.

As described in detail above, the recombinant protein or variant having the surface antigen activity of Orientia tsutsugamushi Pajoo according to the present invention shows excellent reactivity with respect to the antibody in the serum of the Tsutsugamushi infected or patient, The gene according to the present invention encoding the variant is not only artificially designed to facilitate its expression and genetic manipulation, but is also cultured easily by being inserted into and expressed in E. coli transformants compared to the culture of Tsutsugamus bacteria, which is difficult and time consuming. To maximize industrial utility. Therefore, the protein or variant according to the present invention can be used as a useful ingredient in the production of diagnostic reagents or diagnostic kits for Tsutsugamushi, or vaccines for the prevention or treatment of Tsutsugamushi, and can also be used for the production of antibodies to Orientia Tsutsugamushi. have.

FIG. 1 schematically shows a process of constructing an expression vector p56kDa comprising a gene encoding an 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 p56kDa,

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 from recombinant proteins,

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

<110> Republic of Korea (National Institute of Health) <120> A recombinant protein useful for the detection of tsutsugamushi infection, a gene encoding the same, and a diagnostic kit using the same <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 560 <212> PRT <213> Escherichia coli <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> Escherichia coli <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

Claims (10)

A recombinant protein having surface antigen activity of Orientia tsutsugamushi Pajoo having the amino acid sequence of SEQ ID NO: 1. Gene encoding the recombinant protein according to claim 1. The gene of claim 2, wherein the gene is represented by the nucleotide sequence of SEQ ID NO: 2. 4. An expression vector comprising the gene according to claim 2. The expression vector according to claim 4, wherein the expression vector is p56kDa. A host cell transformed with the expression vector according to claim 4. The host cell according to claim 6, which is Escherichia coli NIH / BL / 56kDa-ET15B of Accession No. KCCM 10489. A method for producing a recombinant protein according to claim 1 comprising culturing and isolating the host cell according to claim 6. A diagnostic kit for detecting infection of Tsutsugamu bacteria in humans and mammals, comprising the recombinant protein according to claim 1. 10. The diagnostic kit of claim 9, wherein the detection method is an ELISA method or an immunochromatographic flow assay (RFA).