WO2017155160A1 - Multi diagnostic kit - Google Patents

Abstract

A multi diagnostic kit according to examples of the present invention comprises a strip, wherein the strip comprises: a tsutsugamushi antigen protein comprising a 56 kDa fusion protein (cr56) of Orientia tsutsugamushi Gilliam, Karp and Kato and a 56 kDa protein (kr56) of Orientia tsutsugamushi Kanwon; a leptospira antigen; and an antigen protein of hemorrhagic fever with renal syndrome.

Description

Multiple diagnostic kit

The present invention relates to multiple diagnostic kits.

In general, a method for diagnosing diseases such as Tsutsugamus disease, leptospirosis, and nephrotic bleeding fever includes a sample, such as blood or urine, of a patient suspected of having the disease on a diagnostic strip to determine a reaction with a substance fixed on the strip. In order to check whether the disease is infected, a lot of methods are used.

However, the prior art as described above can be diagnosed only for a single disease, and in order to check whether a plurality of diseases are infected, tests must be performed separately, and a plurality of samples must be injected.

In order to solve the above problems, the present invention provides a multi-diagnosis kit that can quickly and easily diagnose whether or not Tsutsugamushi disease, leptospirosis, and nephrotic bleeding.

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

Multiple diagnostic kits in accordance with embodiments of the present invention comprise a strip, wherein the strip is 56kDa fusion protein (cr56) of Orientia Tsutsugamus Giliam, Kaf, and Kato and 56kDa protein (kr56) of Orientia Tsutsugamus Kangwon Tsutsugamu antigen protein, leptospira antigen, and nephrotic hemorrhagic antigen protein, including).

The leptospira antigen may comprise a core polysaccharide (cord polysaccharide) prepared from the leptospira lipopolysaccharide (LPS) and the 0-specific side chain (0-specific side chain).

The nephrotic hemorrhagic fever antigen protein may include a nucleocapsid protein prepared from a received virus having an amino acid sequence of SEQ ID NO: 2 or 3 prepared from eukaryotic cells.

According to embodiments of the present invention, the multi-diagnosis kit can diagnose whether or not Tsutsugamushi disease, leptospirosis, and nephrotic bleeding fever. The multiple diagnostic kit can simultaneously diagnose whether a plurality of diseases are infected by a single sample injection. In addition, since the multiple diagnostic kit removes nonspecific antibodies, high accuracy diagnostic results can be obtained.

1 illustrates multiple diagnostic kits in accordance with one embodiment of the present invention.

2 illustrates a strip of multiple diagnostic kits in accordance with one embodiment of the present invention.

Figure 3 shows the kit reaction according to the Tsutsugamushi antigen protein concentration according to an embodiment of the present invention.

Figure 4 shows the kit reaction according to the Tsutsugamushi antigen protein composition according to an embodiment of the present invention.

Figure 5 shows the antigenicity of the purified polysaccharides as a result of SDS-PAGE (A) and Western blotting (B) of the polysaccharides isolated from patoc and lai according to an embodiment of the present invention.

Figure 6 shows the gene structure of nucleocapsid protein, Complete Protein (Complete NP; CNP) and Fragmented Protein (Truncated NP) in the S segment of the hearing virus according to an embodiment of the present invention.

Figure 7 shows the DNA band (about 370bp) of the fragment protein (TNP) and the DNA band (about 1300bp) of the whole protein (CNP) after the PCR reaction according to an embodiment of the present invention.

FIG. 8 shows a fragmentation of fragment protein (TNP) DNA and whole protein (CNP) DNA into a pGem-T-Easy PCR cloning vector according to an embodiment of the present invention.

Figure 9 shows the cloning of the PCR products of pGT-SooV2-TNP and pGT-SooV2-CNP in the baculovirus plasmid vector having His-Taq at the N-terminus according to an embodiment of the present invention.

Figure 10 shows that pBac-SooV2-TNP and pBac-SooV2-CNP according to an embodiment of the present invention using Bac-SooV2-TNP and Bac-SooV2-CNP using a baculovirus expression vector system.

Figure 11 shows the results of Western blot using patients and normal serum for the separation detection of fragment protein (TNP) according to an embodiment of the present invention.

Figure 12 shows the results of Western blot for the purified fractions of fragment protein (TNP) and whole protein (CNP) according to an embodiment of the present invention.

Hereinafter, the present invention will be described in detail through examples. The objects, features and advantages of the present invention will be readily understood through the following examples. The invention is not limited to the embodiments described herein, but may be embodied in other forms. The embodiments introduced herein are provided so that the disclosure may be made thorough and complete, and the spirit of the present invention may be sufficiently delivered to those skilled in the art. Therefore, the present invention should not be limited by the following examples.

Although terms such as first and second are used herein to describe various elements, the elements should not be limited by such terms. These terms are only used to distinguish the elements from one another. Again, where an element is said to be above another element it means that it can be formed directly on another element or a third element can be interposed therebetween.

In the drawings, the size of elements, or the relative sizes between elements, may be somewhat exaggerated for a clearer understanding of the present invention. In addition, the shape of the elements shown in the drawings may be somewhat changed by variations in the manufacturing process. Accordingly, the embodiments disclosed herein are not to be limited to the shapes shown in the drawings unless specifically stated, it should be understood to include some modification.

As used herein, the term 'tsutsugamushi disease' refers to a disease caused by Orientia tsutsugamushi .

As used herein, the term 'leptospirosis' refers to a disease caused by pathogenic spiral bacteria of the genus Leptospira.

As used herein, the term "nephrotic bleeding hemorrhagic fever" refers to a disease caused by Hantan virus and Seoul virus.

As used herein, the term 'diagnosis' refers to identifying the presence or characteristic of a pathological condition, and for the purposes of the present invention, diagnosis refers to identifying Tsutsugamus disease, leptospirosis, and nephrotic bleeding fever.

As used herein, the term "sample" refers to blood, serum, and plasma of mammals, including humans suspected of Tsutsugamus disease, leptospirosis, and nephrotic bleeding.

1 shows a multiple diagnostic kit according to one embodiment of the invention, and FIG. 2 shows a strip of multiple diagnostic kits according to an embodiment of the invention.

1 and 2, the multiple diagnostic kit 10 may include a strip 100, and the strip 100 may include a first strip 100a and a second strip 100b.

The first strip 100a and the second strip 100b may detect Tsutsugamu antibody, leptospira antibody, and nephrotic bleeding antibody in the sample. The first strip 100a can detect the Tsutsugashimu IgM antibody, the leptospira IgM antibody, and the nephrotic hemorrhagic fever IgM antibody, and the second strip 100b is the Tsutsugamus IgG antibody, the leptospira IgG antibody, and the nephrotic hemorrhage IgG antibody. Can be detected.

Each of the first strip 100a and the second strip 100b includes the support member 110, the reaction film 120, the sample pad 130, the gold conjugate pad 140, the prereaction line 150, and the reaction line. 160, a control line 170, and an absorbent pad 180.

The support member 110 is a base layer of the strip 100, and the reaction film 120, the sample pad 130, the gold conjugate pad 140, and the absorbent pad 180 are disposed on the support member 110. Can be.

The reaction film 120 may be a nitrocellulose film. In the reaction film 120, the pre-reaction line 150, the reaction line 160, and the control line 170 may be disposed. The prereaction line 150, the reaction line 160, and the control line 170 may be disposed at an interval of 2.0 to 4.5 mm.

The sample pad 130 may absorb the sample injected through the sample inlet 200. The sample pad 130 of the first strip 100a may be manufactured by soaking in a solution containing 50 mM Tris (pH 8.0), 1% Tween 20, 0.5M NaCl, and 0.1% NaN _3, and then drying it. The sample pad 130 of the second strip 100b may be manufactured by soaking in a solution containing 50 mM Tris (pH 8.0), 1% Tween 20, 0.2% BSA, 0.1% NaN _3, and then drying it.

The gold conjugate pad 140 may be disposed to overlap the sample pad 130. The sample absorbed by the sample pad 130 may move to the gold conjugate pad 140 by capillary action.

Gold conjugate pad 140 may comprise marker antibodies. The marker antibody may comprise anti-human IgM conjugated gold, anti-human IgG conjugated gold, and chicken IgY conjugate gold. The anti-human IgM conjugate gold may be included at a concentration of OD 1 to 3, the anti-human IgG conjugate gold may be included at a concentration of OD 2 to 4, the chicken IgY conjugate gold is OD 0.1 to It may be included at a concentration of 1.

The first strip 100a may comprise the anti-human IgM conjugate gold and the chicken IgY conjugate gold, and the second strip 100b may comprise the anti-human IgG conjugate gold and the chicken IgY conjugate gold It may include.

The marker antibody may be combined with the Tsutsugamu antibody, the leptospira antibody, and the nephrotic bleeding fever antibody to form a complex. The gold conjugate pad 140 may be disposed to overlap with the reaction film 120, such that the composite may move to the reaction film 120. The Tsutsugamu antibody, the leptospira antibody, and the remaining marker antibody not bound to the nephrotic bleeding fever antibody may move to the reaction membrane 120.

The marker antibody may comprise color particles. The colored particles may be colloidal gold particles, colored glass, and plastic beads.

The reaction line 160 may include a first reaction line 161, a second reaction line 162, and a third reaction line 163.

The first reaction line 161 may include leptospira antigen. The leptospira antigen may comprise a core polysaccharide derived from a lipopolysaccharide of leptospira and a 0-specific side chain. The leptospira antigen may be included at a concentration of 2 to 4 mg / ml.

When the leptospira antibody is present in the sample, a complex may be formed by combining the leptospira antibody and the marker antibody in the gold conjugate pad 140. When the complex passes through the first reaction line 161, the leptospira antibody and the leptospira antigen of the first reaction line 161 may be combined. The color particles of the marker antibody are precipitated in the first reaction line 161 by the binding to form a red purple band.

The second reaction line 162 may include a Tsutsugamu antigen protein. The Tsutsugamus antigen protein may be mixed with a 56 kDa gene of Orientia Tsutsugamus Giliam, Kaf, and Kato and a 56 kDa Protein of Orientia Tsutsugamu Kangwon in a volume ratio of 5: 3. The Tsutsugamushi antigen protein may be included at a concentration of 0.4 to 0.8 mg / ml.

When the Tsutsugamu antibody is present in the sample, the Tsutsugamu antibody and the marker antibody may be combined to form a complex in the gold conjugate pad 140. When the complex passes through the second reaction line 162, the Tsutsugamushi antibody and the Tsutsugamushi antigen protein of the second reaction line 162 may be combined. The color particles of the marker antibody are precipitated in the second reaction line 162 by the binding to form a red purple band.

The third response line 163 may include nephrotic bleeding fever antigen protein. The nephrotic hemorrhagic fever antigen protein may include a sensitization virus-derived nucleocapsid protein (CNP) having an amino acid sequence of SEQ ID NO: 2 or a sensitization virus-derived nucleocapsid protein fragment (TNP) having an amino acid sequence of SEQ ID NO: 3 . The nephrotic hemorrhagic fever antigen protein may be included at a concentration of 0.1 to 1.0 mg / ml.

When the nephrotic hemorrhagic fever antibody is present in the sample, a complex may be formed by combining the nephrotic hemorrhagic fever antibody and the marker antibody in the gold conjugate pad 140. When the complex passes through the third reaction line 163, the nephrotic hemorrhagic fever antibody and the nephrotic hemorrhagic fever antigen protein of the third reaction line 163 may be combined. By the binding, the color particles of the marker antibody may form a red violet band while being precipitated in the third reaction line 163.

The Tsutsugamu antibody, the leptospira antibody, and the remaining marker antibody that do not bind to the nephrotic bleeding hemorrhagic antibody may pass through the reaction line 160 to the control line 170.

The prereaction line 150 may include a first prereaction line 151 and a second prereaction line 152.

The first prereaction line 151 may include E. coli extract. The E. coli extract may be combined with a first nonspecific antibody. The first nonspecific antibody may be an antibody bound to a protein derived from E. coli.

The first pre-reaction line 151 may be mistaken as having the E. coli-derived protein contained in the Tsutsugamus antigen protein and the non-specific antibody, so that the first whole reaction line 151 does not actually have an antibody to the Tsutsugamus antigen protein. The possibility of being excluded.

The second prereaction line 152 may include an insect cell Sf (Spodoptera frugiperda) extract. The insect cell Sf extract may be combined with a second nonspecific antibody. The second nonspecific antibody may be an antibody that binds to a protein derived from insect cell Sf.

The second prereaction line 152 is retained even though the protein derived from insect cell Sf21 contained in the nephrotic hemorrhagic fever antigen protein and the non-specific antibody do not actually have an antibody against the nephrotic hemorrhagic fever antigen protein. You can rule out the possibility of being mistaken for doing so.

Control line 170 may comprise a control protein. The control protein may be combined with the marker antibody. The binding of the color particles of the marker antibody to the control line 170 may form a red purple band.

The control protein may be at least one selected from the group consisting of goat anti-chicken IgY polyclonal antibody (Goat anti-chicken IgY), rabbit anti-goat IgG polyclonal antibody, and rabbit anti-goat IgM polyclonal antibody. The control protein may be included at a concentration of 0.1 to 2 mg / ml.

The absorbent pad 180 may absorb the remaining amount of the sample moved along the reaction film 120. The absorption pad 180 may be disposed to overlap the reaction film 120.

Examples of Tsutsugamu antigen protein

Example 1. Antigen Protein Expression and Isolation

1-1. Protein Expression of Chimeric Recombinant 56kDa (cr56)

Orientia Tsutsugamu Giliam, Kaf, Kato tsutsugamushi The chimeric recombinant protein (cr56) comprising the major antigenic sites of Gilliam, Karp, Kato) was expressed and separated and purified in the same manner as described in the examples of Korean Patent Publication No. 2002-0020281.

That is, Gilien, Cap and Kato of Orientia Tsutsugamu were cultured in mouse L-929 cells, harvested, and purified. After enzymatic digestion of the purified strain, DNA was purified by phenol extraction and ethanol precipitation. In addition, based on the nucleotide sequence of the 56kDa protein gene of Orientia, a pair of oligonucleotides were selected in Gilium, Kape, and Kato, respectively, by selecting a protein region showing 30% or more homology between amino acids of the Gilium, Cap and Kato serotypes. Prepare nucleotide primers. With each of these primer pairs, a PCR reaction is carried out using the Orientia Tsutsugamus DNA extracted above as a template to amplify the DNA fragments of Gillium, Cap and Kato. The PCR product is purified and cloned to link to the corresponding restriction enzyme cleavage site of the pTYB12 vector. First, a vector pTG3 was prepared by introducing a DNA fragment of Gilium into a pTYB12 vector, a vector pTGP1 was prepared by introducing a cap DNA fragment into pTG3, and then a pTGPT2 was prepared by introducing a Kato DNA fragment into pTGP1. From pTGPT2, a serial linkage of the DNA fragments of Gillium, Kafe and Kato was cut and introduced into the pET22b (+) vector to prepare a vector pETb7. E. coli is transformed with the produced expression vector, and the transformed E. coli is cultured to induce fusion protein expression. After confirmation by electrophoresis and Western blot, the expressed fusion protein antigen was isolated and purified.

1-2. Expression of Recombinant Kangwon 56kDa Protein (kr56)

Phylogenic analysis results (FEMA Microbiology Letters, 1999, 180: 163-169) show that the global prototypes of Karp, Giliam, Kato and Yeoncheon ( Considering that they can be broadly classified as Yonchon (most similar to Kangwon), it was considered very appropriate to use these four strains as antigens. Karp, Giliam and Kato are shown as cr56. Yeonchon is also presented as kr56 of Kangwon. Therefore, the present inventors decided to use Kangwon 56kDa recombinant protein (kr56) as an auxiliary antigen of chimeric recombinant protein (cr56).

(1) Overview of Kangwon 56kDa protein (kr56) expression

A pair of primers were prepared by modifying the amino acids 84 (250bp) to 445 (1,335bp), known as the major antigenic domain of Kangwon 87-61, to contain Nco I and Sal I recognition sites. Was performed. The PCR product and the expression vector pET30a were digested with Nco I and Sal I restriction enzymes and then ligated with each other. This plasmid was transformed into Escherichia coli BL21 and the proteins were extracted. The primers are shown in Table 1 below.

Primer sequence	Enzyme site	Cloning vector	Polypeptide
F: 5'-CCATGGCGCCAGGATTTAGAGCA-3 '	Nco ISal I	pET30a *	48kDa
R: 5'-GTCGACCAAGACCAGCATAATATGAGAA-3 '

Plasmid pET30a expresses His-Tagged fusion protein.

(2) Cloning and Expression of Genes

DNA amplified by polymerase chain reaction (amino acids 84 (250bp) to 445 (1,335bp) 362 aa (total 1,086bp)) was electrophoresed on 1.2% agarose gel and then QIAGEN gel elution kit (QIAGEN Inc., USA). Bands amplified to the expected size on a UV transilluminator were cut out and transferred to microtubes. Three times the volume of NaI solution of agarose gel was added and then left at 60 ° C. for 5 minutes to completely dissolve the gel. Glass milk was added thereto and stirred for 10 seconds. This was centrifuged at 17,000 g (Hanil, AI.5S-24, 12,000 rpm) for 1 minute at room temperature, and then the supernatant was removed. The microtubes were allowed to stand at room temperature for 5 minutes to dry and then stirred by addition of elution buffer, followed by centrifugation at 17,000 x g (12,000 rpm) to transfer the supernatant to a new microtube. The purified PCR product and the pET30a vector were completely cleaved with restriction enzymes Nco I and Sal I, and then recovered using Geneclean kit II. 100 ng of the cleaved vector and 100 ng of the cleaved PCR product are mixed, and a 10-fold concentration of ligase buffer (250 mM Tris-HCl pH 7.8, 100 mM MgCl 2, 20 mM DTT, 4 mM ATP) is mixed with 18 at 4 ° C. The reaction was carried out for a time.

(3) Preparation and transformation of competent cells

Escherichia coli BL21 incubated in LB medium for 18 hours was diluted 100-fold in LB medium and cultured at 600 nm until the absorbance became 0.3. After standing in ice for 30 minutes, the supernatant was discarded, and E. coli was suspended by adding 0.1 M CaCl 2 to the volume of 1/2 of the medium. It was left for 1 hour in ice, centrifuged at 2,000 g (4,100 rpm) for 10 minutes, and then suspended in 1/10 volume of 0.1 M CaCl 2 of the culture medium, which was used for transformation.

(4) transformation

The ligation product prepared in (3) and the competent cells (competent cells) were mixed and allowed to stand in ice and then subjected to thermal shock at 42 ° C. for 1 minute 30 seconds. After adding LB medium, the cells were incubated at 37 ° C. for 1 hour. Cultures were inoculated in LB agar plates and incubated at 37 ° C.

(5) Isolation of plasmid DNA of transformed Escherichia coli

The transformed E. coli colonies were inoculated in LB medium containing Kanamycin and shaken at 37 ° C. Plasmid DNA extraction was performed using AccuPrep Plasmid Extraction kit. The culture was centrifuged at 750 g (2,500 rpm) for 10 minutes at room temperature, and then only cell precipitates were recovered. Resuspenstion buffer was added thereto, followed by stirring to add lysis buffer and allowed to stand at room temperature for 5 minutes. Neutralization buffer was added and left at room temperature, followed by centrifugation at 17,000xg (12,000 rpm) to transfer the supernatant to a binding column tube. This was centrifuged at 17,000xg (12,000rpm) at room temperature and then the extract was removed. 80% ethanol was aliquoted and centrifuged, and only the binding column was transferred to a new tube. Elution buffer was dispensed and left at room temperature, followed by centrifugation to transfer the supernatant to a new microtube.

(6) Expression and Isolation of Proteins

The culture medium was inoculated with the culture solution of the seed culture step. The inoculation was at 37 ° C. and stirred at 220 rpm for 1 hour 45 minutes after inoculation. When the O.D value was in the range of 0.5 to 0.6, IPTG was inoculated with 0.5 mM and incubated for 3 hours. Then, centrifuged at 3,000 rpm for 30 minutes at 4 ℃, 1xbinding Buffer (6 M Urea, 500 mM NaCl, 20 mM Tris-HCl (pH 8.0), 5 mM Imidazole) into the pellet and using a Sonicator (pulse : 10 sec on, 20 sec off, time: 6 min X 2 times (inverting once in the middle, Amp .: 45%) The cells were disrupted. kr56 was centrifuged at 14,000 rpm for 15 minutes at 4 ° C (rotor 7 in autoclaved ultra centrifuge bottle), the supernatant was refrigerated, and the pellet was resuspended in 1X Binding buffer (50 ml based on 1 L Erlenmeyer flask culture). It was. After leaving for 1 hour in ice (after 30 minutes, inverting), and centrifuged at 14,000 rpm for 30 minutes at 4 ℃ (rotor 7 in autoclaved ultracentrifuge bottle) to obtain a supernatant. The supernatant was diluted 2-fold with 1 × Binding buffer (6M urea), filtered and stored at 4 ° C. (0.45 μm syringe filter).

(7) protein purification

Hisbind resin (Novagen) was charged to the column to 2ml, the column was prepared using distilled water, 1 X charge buffer, 1 X binding buffer. Antigen proteins were passed through the column and washed with 1 X washing buffer. 1 ml of protein was recovered by passing through 1 × Elution buffer (6 M Urea, 500 mM NaCl, 20 mM Tris-HCl, pH 8.0), 500 mM Imidazole. Protein concentration in each fraction was quantified using BCA protein assay, concentrated with Amicon Ultra 30K and then quantified using BCA protein assay.

For the specific amino acid sequence of the Orientia Tsutsugamu Kangwon 56kDa protein included in the Tsutsugamus antigen protein of Example 1, reference may be made to the amino acid sequences disclosed in Korean Patent No. 10-0796772.

Example  2. Depending on antigen concentration Of kit  Reaction (determination of false positives)

Tsutsugamushi antigen proteins prepared according to Example 1 were prepared at concentrations of 0.6 mg / ml, 0.9 mg / ml, 1.2 mg / ml and 1.5 mg / ml, respectively. Four kits containing Tsutsugamu antigen proteins at each concentration were prepared. After evaluating the patient's sample using immunofluorescent antibody, a standard diagnostic test for diagnosing Tsutsugamushi disease, 1 negative serum (DK13) and 3 positive serum (90-202, 00-350, 89) -129) were prepared and applied to each of the diagnostic kits having different antigen concentrations to measure the response.

Figure 3 shows the kit reaction according to the Tsutsugamushi antigen protein concentration according to an embodiment of the present invention.

Table 2 shows the kit response according to the Tsutsugamushi antigen protein concentration.

	Negative serum		Positive serum
	DK13		90-202		00-350		89-129
	IgM	IgG	IgM	IgG	IgM	IgG	IgM	IgG
IFA	-	-	++	+	+	+	++	+
0.6 mg / ml	-	-	++	+	+	+	++	+
0.9 mg / ml	+	+	++	+	+	+	++	+
1.2 mg / ml	+	+	++	+	+	+	++	+
1.5 mg / ml	+	+	++	+	+	+	++	+

Referring to Table 2 and FIG. 3, false positives were shown in the diagnostic kits with Tsutsugamus antigen protein concentrations of 0.9, 1.2, and 1.5 mg / mL, but false positives were not shown in the diagnostic kits with TZTSUGAMUH antigen protein concentrations of 0.6 mg / mL. Did.

Example 3. Kit Reaction According to Mixed Antigen Composition

The Tsutsugamushi antigen protein of Example 1 was prepared at 0.6 mg / ml, and the conventional Tsutsugamushi antigen protein was prepared at the same concentration. The conventional Tsutsugamu antigen protein is a mixture of cr56, kr56, and r21 proteins in a 5: 2: 1 ratio, respectively. A diagnostic kit comprising the Tsutsugamus antigen protein of Example 1 and a diagnostic kit containing the conventional Tsutsugamus antigen protein were prepared, and the reactivity with respect to negative serum and positive serum was measured. As a control, each negative and positive serum was measured for Ab titer according to IFA.

Figure 4 shows the kit reaction according to the Tsutsugamushi antigen protein composition according to an embodiment of the present invention.

Table 3 shows the response sensitivity of the conventional Tsutsugamus antigen protein and the antibodies of each of the Tsutsugamus antigen protein of Example 1 to − = 0, + = 1, ++ = 2, +++ = 3, ++++ = 4 and It is shown together.

division	Negative serum		Positive serum
	Positive Serum 1		Positive serum 2		Positive Serum 3		Positive Serum 4		Positive serum 5
IgM	IgG	IgM	IgG	IgM	IgG	IgM	IgG	IgM	IgG	IgM	IgG
Indirect immunofluorescent antibody test (IFA)	-	-	640	80	640	80	640	1280	-	1280	640	80
Conventional Tsutsugamushi Antigen Protein (cr56: kr56: r21: = 5: 2: 1)	0	0	One	0.5	One	0.5	One	2	0	2	One	0.5
Tsutsugamushi antigen protein of Example 1 (cr56: kr56 = 5: 3)	0	0	1.5	0.75	1.5	0.75	1.5	2.5	0	2.5	1.5	0.75

Referring to Table 3 and Figure 4, when the Tsutsugamu antigen protein does not contain r21 and increased the ratio of kr56, the response sensitivity to the antibody is improved.

Examples of Leptospira Antigens

Example 4. Securing Leptospira Antigen

4-1. Leptospira Strain Culture

After dissolving the EMJH (Leptospira medium base Ellinghausen McCullough Hohnson Harris) to 0.23g / 90ml, sterilization, 10ml of leptospira enrichment EMJH was added to make a medium. The medium was inoculated with leptospira and shaken for 5 days at 30 ° C.

4-2. Leptospira Antigen Preparation

Centrifugation of leptospira bacteria cultured in EMJH medium for 5 days at 4,000xg for 20 minutes, the cells were washed three times with 1X PBS buffer (137mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 2mM KH2PO4) and heated at 100 ° C for 1 hour. It was. Proteasease (Proteinase) K was treated here to 150㎍ / ㎖ and reacted for 16 hours at 37 ℃. Thereafter, EGTA was added to 2 mM and reacted at 70 ° C. for 15 minutes, followed by centrifugation at 4 ° C. and 180,000 × g for 2 hours. Lipid polysaccharide (lipopolysaccharide) was prepared by adding H 2 O to the precipitate (Westpha & Jann, 1965). The purified LPS antigen was treated with acetic acid at 1%, warmed at 100 ° C. for 1 hour, and then centrifuged at 3,000 × g for 20 minutes to remove lipid A (lipid A), thereby preparing a polysaccharide antigen. The amount of the prepared antigen was measured using polysaccharides obtained by treating LPS (Sigma) purified in Escherichia coli in the same manner as the above method as a standard.

Example 5 Antigenicity of Leptospira Polysaccharides

5-1. Western Block Analysis

SDS-PAGE used a Bio-Rad Protean II electrophoresis device to make a 12% gel of 1 mm or 1.5 mm thickness. Transcription of the protein from the electrophoretic gel to the membrane (membrane) was performed for 30 minutes at 80V using a Bio-Rad Transblot cell. The transferred membrane was blocked using 1 × TBS buffer to which 5% skim milk and 0.1% Tween 20 were added. As a primary antibody, rabbit immune serum ( L. interrogans strain WH-19) was used. As a secondary antibody, horse-radish peroxidase conjugated anti-rabbit IgG (Bio-Rad) was diluted 1: 10,000. After completion of the immune response, ECL kit (Amersham Pharmacia Biotech) was developed.

Figure 5 shows the antigenicity of the polysaccharides purified by SDS-PAGE (A) and Western blotting (B) results of the polysaccharides isolated from patoc and lai according to an embodiment of the present invention.

Referring to FIG. 5, the polysaccharides of Leptospira interrogans serovar lai showed similar patterns in the Leptospira interrogans serovar patoc strain Patoc and SDS-PAGE gel profiles, and polysaccharides of 14 kDa or more were commonly present in lai and patoc and acted as antigens. It was estimated.

5-2. Dot blotting

1 ug (1 ug / 1 ul) of the antigen is added to the nitrocellulose membrane and then dried at 37 ° C. for 1 hour. The dried membrane was blocked with 5% skim milk-TBST for 1 hour, and the patient serum, the primary antibody, was diluted 1: 3,000 and reacted for 1 hour. The secondary antibody was reacted by diluting the horse-radish peroxidase conjugate anti-human IgG to 1: 10,000. After the reaction, color development was performed using the ECL kit and compared with the MAT results. It was confirmed that most patients sera showed a positive signal.

Table 4 shows a comparison of MAT and dot-blotting results.

Patient NO.	MAT		Dot-blotting
	lai	canicola
90-34	320	-	-
90-94	640	160	+++
90-227	1280	40	+++
91-23	-	20	-
91-121	-	20	+
91-211	320	-	+++
91-276	20	-	-
94-200	320	-	+
95-2	20	320	-
95-85	-	-	+
96-40	80	80	-
97-39	80	-	+
97-163	1280	80	++
97-212	1280	80	+++
97-330	80	160	+
98-111	640	80	+++
98-156	320	160	+++
98-166	640	-	++

5-3. Enzyme Immunoassay (ELISA)

Polysaccharide ELISA was performed based on the method used by Engvall and Perlmann (1972). After diluting poly-L-lysine (10µg / ml) in 0.01M PBS, the plate was pretreated and reacted at room temperature for 24 hours. After washing twice with PBS buffer containing 0.05% Tween 20, polysaccharides were suspended in PBS and treated with 100 μl per plate. After reacting at 37 ° C. for 1 hour, the plate was washed three times with PBS buffer containing 0.05% Tween 20, and treated with goat serum to block at 37 ° C. The primary antibody consisted of 20 patient sera confirmed with leptospirosis and the serum of 20 normal subjects. The secondary antibody was diluted 1: 10,000 with horse-radish peroxidase conjugated anti-human IgG (Bio-Rad). . Each well was treated with a substrate to observe color development, and the value was measured at 490 nm using an ELISA reader. As a result, the leptospira flagella recombinant protein showed 72-88% sensitivity and 88-90% specificity, whereas the polysaccharide showed 95% sensitivity and 95% specificity, so that the early diagnosis of leptospirosis was possible. Antigen candidates showed better polysaccharides than proteins.

Table 5 shows the sensitivity and specificity of polysaccharides using enzyme immunoassay.

Antigen	Polysaccharide
	patoc	lai
Sensitivity (%) a	95	95
Sensitivity (%) b	95	95

a: Number of sera of patients diagnosed by MAT: 20

b: Number of normal sera: 20

Examples of nephrotic bleeding fever antigen protein

Example 6 Hepatitis Virus and Cultured Cells

Soochong virus strain SooV-2 isolated from domestic Apoddemus peninsulae was passaged in Vero cells. For growth and maintenance of Vero cells, EMEM (Eagel's minimal essential medium) containing 10% fetal bovine serum (FBS) was incubated in a 37 ° C incubator supplied with 5% CO2. About 90% It was used when monolayer culture.

Example 7. Isolation of Viral RNA

After centrifuging the cells in which the virus was propagated at 1,075g (3000rpm) for 3 minutes, 750ul of Trizol LS (Gibco) solution was added and allowed to stand at room temperature for 5 minutes, followed by adding 200ul of chloroform and mixing well. It was left for 10 minutes. The precipitate was removed by centrifugation at 17,000 g (12,000 rpm) at 4 ° C. for 15 minutes, and the same volume of isopropyl alcohol was added to the supernatant and allowed to stand at room temperature for 10 minutes, then again at 17,000 g (12,000 rpm) at 4 ° C. RNA was precipitated by centrifugation for 15 minutes and washed once with 75% ethanol. The RNA precipitate was dried in air for 10 minutes and then dissolved in DEPC treated water.

Example 8. cDNA Synthesis and Chain Enzyme Polymerization

Figure 6 shows the gene structure of the nucleocapsid protein, Complete Protein (Complete NP; CNP) and Fragmented Protein (Truncated NP; TNP) in the S segment of the hearing virus according to an embodiment of the present invention, Figure 7 The DNA band (about 370bp) of the fragment protein (TNP) and the DNA band (about 1300bp) of the whole protein (CNP) after the PCR reaction according to an embodiment of the present invention are shown.

Table 6 shows the PCR primers for the production of the recombinant viral antigen.

Primer	Whole Protein (CNP)	Fragment Protein (TNP)
Fowardprimer	5 ' AGA TCT A ATG GCA ACT ATG GAG GAA TTG 3 '(SEQ ID NO: 4) <Bgl II (AGATCT); AGATCT Added>	5 ' AGA TCT A ATG GCA ACT ATG GAG GAA TTG 3 ′ (SEQ ID NO: 6) <Bgl II; AGATCT Added>
Reverseprimer	5 ′ AAGC TTA TAG TTT TAA AGG TTC TTG GTT TG 3 ′ (SEQ ID NO: 5) <Hind III (AAGCTT); AAGC addition>	5 ' A AGC TTA CCA GTC TGC AGT CTG ACC TGT 3' (SEQ ID NO: 7) <Hind III; Add AAGCTTA>

6 and Table 6, RNA solution 4 isolated in Example 7 to synthesize cDNA from the nucleic acid sequence of SEQ ID NO: 1 (GenBank accession No. AY675350) encoding a nucleocapsid protein (NP) 1 ul of each primer (10 pmole) in Table 6, 5X reverse transcriptase buffer), 10mM dNTPs, 100pmole / ul Oligo (dT), 40units / ul RNasin, 200units / ul MMLV reverse transcriptase mixture was added to ul. After reacting at 30 ° C. for 10 minutes, cDNA was synthesized by reacting at 45 ° C. for 30 minutes, 99 ° C. for 5 minutes, and 5 ° C. for 5 minutes.

PCR was performed by mixing 5 cul of cDNA product, a pair of 10 pmole primer, 10 mM dNTPs, 10X taq buffer, 5 units / ul Taq polymerase, and then reaction once every 95 ° C for 5 minutes, 52 ° C for 2 minutes, and 72 ° C for 2 minutes. The reaction was carried out five times at a cycle of 52 ° C. 1 minute and 72 ° C. 1 minute. 95 ° C 1 minute, 60 ° C 1 minute. The reaction was carried out 20 times at 72 ° C. for 1 minute, and DNA amplification was performed once at 95 ° C. for 1 minute, 60 ° C. for 1 minute, and 72 ° C. for 7 minutes. Amplified DNA was stored at -20 ° C.

Figure 7 shows the DNA band (about 370bp) of the fragment protein (TNP) and the DNA band (about 1300bp) of the whole protein (CNP) after the PCR reaction.

Referring to FIG. 7, PCR of each primer was performed to confirm DNA of the fragment protein (Truncated NP: TNP) at about 370 bp, and DNA of the whole protein (Complete NP: CNP) was also identified as a band at about 1,300 bp.

Example 9 Cloning of Amplification Products

FIG. 8 shows a fragmentation of fragment protein (TNP) DNA and whole protein (CNP) DNA into a pGem-T-Easy PCR cloning vector according to an embodiment of the present invention.

Referring to FIG. 8, DNAs of about 370 bp and about 1,300 bp amplified by a chain enzyme polymerization reaction were electroeluated on an agarose gel to clone into a cloning vector, pGem-T-Easy PCR cloning vector (Invitrogen). It was. DNAs of TNP and CNP were identified by digestion with restriction enzymes Bgl Π and Hind III. This is for the production of CNP (1287bp: 37-1323) and TNP (357bp: 37-393) polypeptides, the amino acid sequence of which is shown in SEQ ID NOs: 2 and 3. The plasmid into which 369bp containing TNP was inserted was named pGT-SooV2-TNP, and the plasmid into which 1299bp including CNP was inserted was named pGT-SooV2-CNP.

Example 10 Cloning into Expression Vectors

Figure 9 shows the cloning of the PCR products of pGT-SooV2-TNP and pGT-SooV2-CNP in the baculovirus plasmid vector having His-Taq at the N-terminus according to an embodiment of the present invention.

9, pBlue-Bac-His2B (Invitrogen), an expression fusion vector containing His-Taq at the N-terminus, was treated with restriction enzymes Bgl Π and Hind III, and pGT-SooV2-TNP PCR products of pGT-SooV2-CNP were also cloned by restriction enzymes Bgl Π and Hind III to obtain pBac-SooV2-TNP and pBac-SooV2-CNP.

Example 11 Construction of Recombinant Baculovirus

Figure 10 shows that pBac-SooV2-TNP and pBac-SooV2-CNP according to an embodiment of the present invention using Bac-SooV2-TNP and Bac-SooV2-CNP using a baculovirus expression vector system.

Referring to FIG. 10, pBac-SooV2-TNP and pBac-SooV2-CNP were converted into Bac-SooV2-TNP and Bac-SooV2-CNP using a Bac-N-Blue baculovirus expression vector system. Made

Sf9 cells ( ⁶ × ² × ¹⁰ ) in the log phase were poured into a 60 mm dish to form a monolayer. To prepare a transfection mixture, 10 ul (0.5 ug) of Bac-N-Blue ^TM DNA was added to a microcentrifuge and the following solution was added.

pBac-SooV2-TNP or pBac-SooV2-CNP (1ug / ul): 4ul

Grace's insect media (without supplements or FBS): 1 ml

Cellfectin reagent (mix well before use and always): 20 ul

After 15 minutes at room temperature, remove the medium from the dish and add 2 ml of Grace's insect media without supplements or FBS, remove the medium from the monolayer, and then prepare the transfection mixture. Put the drops in. After leaving the dish at room temperature for about 4 hours, 1 ml of TNM-FH medium was added, and the dish was sealed and incubated at 27 ° C for 72 hours. The recombinant baculovirus thus cotransfected and replicated is referred to as a virus stock.

Example 12. Recombinant Baculovirus Purification

Virus stock concentrations were measured by an end-point dilution method.

End-point dilution to determine the titer of the virus was performed by diluting the virus inoculation from 10 ^-5 to 10 ^-8 times, and dispensing at a concentration of ¹ × 10 ⁴ cells per well in 96 well plates. Each diluted solution was inoculated to the cultured cells, and the culture was continued at 27 ° C. Virus concentrations were calculated from the ratio of virus infected wells to uninfected wells to determine plaque forming units (PFUs).

For the selection of pure virus, the virus inoculum was diluted 10 ^-2 to 10 ^-8 fold, and each dilution was inoculated into cultured cells dispensed at a concentration of ¹ × 10 ⁴ cells per well in a 96 well plate, and the culture was continued at 27 ° C. . Four days after the inoculation, the cells were observed under the microscope for the formation of intracellular polyhedron and virus infection, and the virus was selected in the wells of the maximum dilution factor.

Example 13. Expression and Isolation of Recombinant Proteins

Insect cells Sf9 cells were seeded about 5 X 10 ⁶ in a 75 cm 2 flask, and then inoculated with 5 MOI of recombinant virus. All cells were harvested after confirming inclusion body formation 5 days after infection. The collected cells were centrifuged at 6,000 g (7,000 rpm) to collect the salivary cells, suspended in 1 X binding buffer (5 mM imidazole, 20 mM Tris-HCl pH7.9, 0.5 M NaCl), and then subjected to ultrasonic grinding at 18 Hz. Stir six times for 15 seconds. The supernatant was stored in soluble form by centrifugation at 23,400 g (14,000 rpm) for 15 minutes. Insoluble form was also suspended in 1 X binding buffer containing 6M Urea and allowed to stand at 4 ° C for 1 hour, followed by centrifugation at 23,400g (14,000rpm) for 30 minutes. lysed pellet) was collected. The harvested pellet was purified through His-bind chromatography. The soluble form, the lysed pellet, and the purified pellet were purified by SDS-PAGE to confirm the fraction of the recombinant protein in the Western blot. As a control, a soluble form of wild-type virus infected cells was used. Western blots were used as primary antibodies for patients with nephrotic bleeding (HFRS) and normal serum.

Figure 11 shows the results of Western blot using patients and normal serum for the separation detection of fragment protein (TNP) according to an embodiment of the present invention. The band of the down arrow appears to be due to partial degradation of the fragment protein. The photo on the left shows the result of nephrotic hemorrhagic fever patient serum as the primary antibody and the serum on the normal subject as the primary antibody. (1 lane: solution fraction of cells infected with wild-type baculovirus, 2 lanes: lysate fraction of cells infected with recombinant baculovirus, 3 lanes: undissolved fraction of cells were treated with buffer containing urea and centrifuged). Supernatant, 4 lanes: 3)

Referring to FIG. 11, it was confirmed that more proteins exist in the soluble form than in the insoluble form of the TNP. In addition to the specific band of 22kDa, which is the TNP fraction, the bands that appear weakly below the western blot by the patient serum are not only soluble forms but also purified pellets. Was estimated to be. It was also demonstrated by the absence of both bands for normal subjects. The lower band was assumed to be due to partial degradation of TNP.

Figure 12 shows the results of Western blot for the purified fractions of fragment protein (TNP) and whole protein (CNP) according to an embodiment of the present invention. (1 lane: cell not infected with virus, 2 lane: cell infected with baculovirus, 3 lane: cell infected with recombinant baculovirus cloned with whole protein (CNP), 4 lane: fragment protein (TNP) Cells infected with the recombinant baculovirus cloned, 5 lanes: cells infected with the recombinant baculovirus cloned with whole protein (CNP), 6 lanes: infected with the recombinant baculovirus cloned with the fragment protein (TNP) cell)

Referring to FIG. 12, it was confirmed that CNP responded only to nephrotic bleeding hemorrhagic fever (HFRS) patients and produced a recombinant protein of about 50 kDa.

Example 14 Purification of Recombinant Proteins by His-bind Affinity Chromatography

After filling his column with 2 ml of His-bind resin (Novagen), the column was prepared using distilled water, 1 X charge buffer, and 1 X binding buffer. The antigen protein was passed through a column, followed by 1 X binding buffer (5 mM imidazole, 20 mM Tris-HCl pH 7.9, 0.5 M NaCl) and 1 X washing buffer (40 mM imidazole, 20 mM Tris-HCl pH7.9, 0.5 M). NaCl). 1 ml of protein was recovered through elution buffer (300 mM imidazole, 0.5 M NaCl, 20 mM Tris-Cl pH 7.9). Protein concentration in each fraction was quantified using the Raleigh method, and the protein was confirmed by SDS-PAGE and Western blot.

Example 15 Recombinant Protein Analysis

15-1. Western-blot

Recombinant proteins purified on 10% gel of 1 mm or 1.5 mm thickness were subjected to electrophoresis at 10 ug per well. Electrophoresis gels were transferred for 40 minutes at 80 volt using a Bio-rad transport machine. The transferred membrane was blocked with 5% skim milk-TBST (Tris-buffered Saline Tween 20) for 1 hour, and the serum of the patient, the primary antibody, was diluted 1: 3000 and reacted for 1 hour. . The secondary antibody was reacted by diluting the horse-radish peroxidase conjugate anti-human IgG to 1: 10000. After the reaction was completed using the ECL kit.

Western blot analysis revealed that recombinant CNP and TNP responded at 50kDa for CNP and 22kDa in sera of nephrotic bleeding hemorrhagic fever but not with normal serum.

15-2. Dot-blot

1 ug of recombinant protein was added dropwise to the nitrocellulose membrane and dried at 37 ° C. for 1 hour. The dried membrane was blocked for 1 hour with 5% skim milk-TBST, and the reaction was performed for 1 hour by diluting the primary antibody patient serum and normal serum 1: 3,000. The secondary antibody, Horse-radish peroxidase conjugate anti-human IgG, was diluted 1: 10,000 and reacted. After the reaction was completed using the ECL kit.

As a result of Dot-blotting with serum of patients with nephrotic bleeding fever infected with Hantan virus or Seoul virus, CNP shows stronger positive signal than TNP as shown in Table 7 below. Appeared to not.

HRFS patientsserum No.	IFA value (HTN)	a Dot-blot Value
	IgG	TNP (IgG)	CNP (IgG)
2001-216	1280	+++	+++
2001-193	80	+	+
2001-36	160	++	+++
2001-42	1280	+++	+++
2000-449	1280	+++	+++
99-445	640	+++	+++
99-485	320	+	++
99-444	640	+++	+++
99-235	320	+	++

a: Data are presented as a strong positive (+++), middle positive (++) or scarcely positive (+) result by Dot-blot.

* Ten normal sera did not react with recombinant NP antigens

15-3. ELISA

The recombinant protein antigen was diluted in 0.05 M carbonate buffer solution (pH 9.6) to 1 ug / ml, coated on a 96 well microplate, and reacted at 4 ° C. for 18 hours. After washing twice with phosphate buffer solution (PBST) containing 0.05% Tween 20 and blocking with 3% BSA for 2 hours at 37 ℃. The primary antibody was used by diluting the serum of rats infected with Hantan, Seoul, Pummala or Prospect Hill virus, and the secondary antibody was horse-radish conjugate anti-rat. IgG (IgM) was used diluted 1: 6000. After color development with a color substrate solution, OD was measured at a wavelength of 490 nm with an ELISA reader.

As shown in Table 8, the recombinant antigen CNP responded to Hantan virus and Seoul virus, but not to Fumala virus and Prospect Hill virus.

Rat Antisera against	Hantaan	Seoul	Pummala	Prospect hill
Reactivity of CNP by ELISA	+++	+++	-	-

So far, specific embodiments of the present invention have been described. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

According to embodiments of the present invention, the multi-diagnosis kit can diagnose whether or not Tsutsugamushi disease, leptospirosis, and nephrotic bleeding fever. The multiple diagnostic kit can simultaneously diagnose whether a plurality of diseases are infected by a single sample injection. In addition, since the multiple diagnostic kit removes nonspecific antibodies, high accuracy diagnostic results can be obtained.

Claims (10)

1. Including a strip,

   The strip,

   A Tsutsugamus antigen protein comprising a 56kDa fusion protein (cr56) of Orientia Tsutsugamus Giliam, a cape, and a Kato and a 56kDa protein (kr56) of Orientia Tsutsugamu River;

   Leptospira antigens; And

   Multiple diagnosis kit, characterized in that it comprises a renal hemorrhagic fever antigen protein.
2. The method of claim 1,

   The strip comprises a first strip and a second strip,

   The first strip detects Tsutsugashimi IgM antibody, Leptospira IgM antibody, and nephrotic bleeding fever IgM antibody,

   The second strip is a multiple diagnostic kit, characterized in that detecting the Tsutsugamu IgG antibody, leptospira IgG antibody, and IgG antibody of hemorrhagic hemorrhagic fever.
3. The method of claim 1,

   The 56kDa fusion protein (cr56) of the Orientia Tsutsugamus Giliam, Kaf, and Kato and the 56kDa protein (kr56) of the Orientia Tsutsugamus Kangwon are included in a volume ratio of 5: 3.
4. The method of claim 1,

   The leptospira antigen is a multiple diagnostic kit, characterized in that it comprises a core polysaccharide (cord polysaccharide) and 0-specific side chain (0-specific side chain) prepared from leptospira lipopolysaccharide (LPS).
5. The method of claim 1,

   The nephrotic hemorrhagic fever antigen protein is a multiple diagnostic kit, characterized in that it comprises a nucleocapsid protein (nucleocapsid protein) prepared from an antivirus having an amino acid sequence of SEQ ID NO: 2 or 3 prepared from eukaryotic cells.
6. The method of claim 1,

   The strip,

   A first reaction line comprising the leptospira antigen;

   A second reaction line comprising the Tsutsugamu antigen protein; And

   Multiple diagnostic kit, characterized in that it comprises a third response line comprising the hemorrhagic fever antigen protein.
7. The method of claim 1,

   The strip,

   A gold conjugate pad comprising a Tsutsugamushi antibody, a leptospira antibody, and a marker antibody bound to a nephrotic bleeding antibody,

   Wherein said marker antibody comprises one or more selected from anti-human IgM conjugate gold, anti-human IgG conjugate gold, and chicken IgY conjugate gold.
8. The method of claim 1,

   The strip,

   A first pre-reaction line comprising an E. coli extract bound to the first nonspecific antibody,

   The first nonspecific antibody is a multiple diagnostic kit, characterized in that the antibody is coupled to E. coli-derived protein.
9. The method of claim 1,

   The strip,

   A second prereaction line comprising an insect cell Sf extract bound to a second nonspecific antibody,

   The second non-specific antibody is a multiple diagnostic kit, characterized in that the antibody is coupled to a protein derived from insect cell Sf.
10. The method of claim 1,

    The strip,

    A control line comprising a control protein bound to the marker antibody,

    Wherein said control protein comprises at least one selected from a goat anti-chicken IgY antibody, a rabbit anti-goat IgG polyclonal antibody, and a rabbit anti-goat IgM polyclonal antibody.

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