KR20170104260A - Multiple Diagnostic kit - Google Patents

Abstract

A multiple diagnostic kit in accordance with embodiments of the present invention includes a strip, wherein the strip contains antigen protein of Orientia tsutsugamushi gilliam, and leptospira, and hemorrhagic fever with renal syndrome which contain 56 kDa fusion protein (cr56) of Orientia tsutsugamushi gilliam, Karp, Kato, and 56kDa fusion protein (kr56) of Orientia tsutsugamushi Kangwon.

Description

Multiple Diagnostic kit

The present invention relates to multiple diagnostic kits.

Generally, as a method for diagnosing diseases such as Tsutsugamushi disease, leptospirosis, and hemorrhagic fever after hemorrhagic fever, a sample of blood or urine of a patient suspected of the disease is put on a diagnostic strip to judge a reaction with a substance immobilized on the strip A method for confirming whether or not the disease is infected is widely used.

However, the above-described conventional techniques can only diagnose a single disease, and in order to check whether a plurality of diseases are infected, it is necessary to individually inspect and inject a plurality of samples.

In order to solve the above problems, the present invention provides a multiple diagnostic kit capable of quickly and easily diagnosing the infection of Tsutsugamushi disease, leptospirosis, and hemorrhagic fever after nephrotic syndrome.

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

The multiple diagnostic kits according to embodiments of the present invention comprise a strip comprising a 56 kDa fusion protein (cr56) of Orientia tsutsugamushi gillianam, cap, and kato, and a 56 kDa protein (kr56) of Orientia tsutsugamushi ), Leptospira antigens, and nephrotic hemorrhagic fever antigen proteins.

The leptospira antigen may comprise a core polysaccharide and a 0-specific side chain made from lipopolysaccharide (LPS) of Leptospira.

The renal hemorrhagic fever antigen protein may comprise a nucleocapsid protein prepared from Suwon virus having the amino acid sequence of SEQ ID NO: 2 or 3 prepared from eukaryotic cells.

According to embodiments of the present invention, multiple diagnostic kits can diagnose the infection of Tsutsugamushi disease, leptospirosis, and hemorrhagic fever after nephrotic syndrome. The multiple diagnostic kits can simultaneously diagnose multiple infectious diseases by injecting a single sample. In addition, since the multiple diagnostic kits eliminate nonspecific antibodies, highly accurate diagnosis results can be obtained.

1 shows a multiple diagnostic kit according to an embodiment of the present invention.
2 shows a strip of a multiple diagnostic kit according to an embodiment of the invention.
FIG. 3 shows the kit reaction according to the tsutsugamushi antigen protein concentration according to an embodiment of the present invention.
FIG. 4 shows a kit reaction according to the composition of tsutsugamushi antigen protein according to an embodiment of the present invention.
FIG. 5 shows the antigens of polysaccharides purified by SDS-PAGE (A) and Western blotting (B) of polysaccharides isolated from patoc and lai according to an embodiment of the present invention.
Figure 6 shows the gene structure of nucleocapsid protein, complete NP (CNP) and truncated NP (TNP) in S segment of Succulent virus according to one embodiment of the present invention.
FIG. 7 shows the DNA band (about 370 bp) and the DNA band (about 1300 bp) of the whole protein (CNP) of the fragment protein (TNP) after the PCR reaction according to an embodiment of the present invention.
FIG. 8 shows the cloning of the fragment protein (TNP) DNA and the whole protein (CNP) DNA into the pGem-T-Easy PCR cloning vector according to an embodiment of the present invention.
FIG. 9 shows a PCR product of pGT-SooV2-TNP and pGT-SooV2-CNP cloned into a baculovirus plasmid vector having His-Taq at the N-terminus according to an embodiment of the present invention.
FIG. 10 shows pBac-SooV2-TNP and pBac-SooV2-CNP according to an embodiment of the present invention prepared using Bac-SooV2-TNP and Bac-SooV2-CNP using a baculovirus expression vector system.
Figure 11 shows the results of western blotting using patient and normal human serum for isolation detection of fragment protein (TNP) according to one embodiment of the present invention.
Figure 12 shows the results of western blotting of purified fractions of fractional protein (TNP) and total protein (CNP) according to one embodiment of the present invention.

Hereinafter, the present invention will be described in detail with reference to examples. The objects, features and advantages of the present invention will be easily understood by the following embodiments. The present invention is not limited to the embodiments described herein, but may be embodied in other forms. The embodiments disclosed herein are provided so that the disclosure may be thorough and complete, and that those skilled in the art will be able to convey the spirit of the invention to those skilled in the art. Therefore, the present invention should not be limited by the following examples.

Although the terms first, second, etc. 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 each other. In addition, when an element is referred to as being on another element, it may be directly formed on the other element, or a third element may be interposed therebetween.

The sizes of the elements in the figures, or the relative sizes between the elements, may be exaggerated somewhat for a clearer understanding of the present invention. In addition, the shape of the elements shown in the drawings may be somewhat modified by variations in the manufacturing process or the like. Accordingly, the embodiments disclosed herein should not be construed as limited to the shapes shown in the drawings unless specifically stated, and should be understood to include some modifications.

As used herein, the term " Tsutsugamushi disease " refers to a disease in which Orientia tsutsugamushi causes pathogenicity.

As used herein, the term " leptospirosis " refers to diseases caused by pathogenic strains of the genus Leptospira.

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

As used herein, the term " diagnosis " refers to identifying the presence or characteristic of a pathologic condition, and for the purpose of the present invention, diagnosis refers to identifying tsutsugamushi disease, leptospirosis, and hemorrhagic fever after nephritis.

As used herein, the term " sample " refers to blood, serum, plasma, and the like of mammals including humans with suspected tsutsugamushi disease, leptospirosis, and febrile hemorrhagic fever.

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

Referring to Figures 1 and 2, multiple diagnostic kits 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 can detect the tsutsugamushi antibody, the leptospira antibody, and the nephrotic range hemorrhagic fever antibody in the sample. The first strip 100a can detect the tsutsugamushi IgM antibody, the leptospira IgM antibody and the nephropathy hemorrhagic fever IgM antibody and the second strip 100b can detect the tsutsugamushi IgG antibody, the leptospira IgG antibody, and the IgG antibody of the nephrotic hemorrhagic fever Can be detected.

Each of the first strips 100a and the second strips 100b includes a support member 110, a reaction film 120, a sample pad 130, a gold conjugate pad 140, an entire reaction line 150, A cleaning line 160, a cleaning 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 absorption pad 180 are disposed on the support member 110 .

The reaction film 120 may be a nitrocellulose film. The reaction line 120, the reaction line 150, the reaction line 160, and the cleaner 170 may be disposed. The entire reaction line 150, the reaction line 160, and the reference line 170 may be disposed at an interval of 2.0 to 4.5 mm.

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

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

Gold conjugate pad 140 may comprise a marker antibody. The marker antibody may include anti-human IgM conjugated gold, anti-human IgG conjugated gold, and chicken IgY conjugated gold. The anti-human IgM conjugate gold may be contained at a concentration of OD 1 to 3, and the anti-human IgG conjugate gold may be contained at a concentration of OD 2 to 4, 1 < / RTI >

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 conjugated gold . ≪ / RTI >

The marker antibody may be combined with the tsutsugamushi antibody, the leptospira antibody, and the nephrotic hemoglobin antibody to form a complex. The gold conjugate pad 140 may be disposed to overlap with the reaction film 120 so that the complex can move to the reaction film 120. The tsutsugamushi antibody, the leptospira antibody, and the other marker antibody that is not bound to the nephrotic range hemorrhagic fever antibody may migrate to the reaction membrane 120.

The marker antibody may comprise color particles. The color particles may be colloidal gold particles, color 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 contain a Leptospira antigen. The leptospira antigen may include core polysaccharides derived from lipopolysaccharide of Leptospira and O-specific side chains. The leptospira antigen may be contained at a concentration of 2 to 4 mg / ml.

When the leptospira antibody is present in the sample, the leptospira antibody and the marker antibody bind to each other at the gold conjugate pad 140 to form a complex. 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. By the binding, the color particles of the marker antibody can be precipitated on the first reaction line 161 to form a red purple band.

The second reaction line 162 may comprise a tsutsugamushi antigen protein. The tsutsugamushi antigen protein can be obtained by mixing the 56 kDa protein of Orientia tsutsugamushi radix, 56 kDa protein of Kanto and 56 kDa protein of Orient tsutsugamushi radish with 5: 3 volume ratio. The tsutsugamushi antigen protein may be contained at a concentration of 0.4 to 0.8 mg / ml.

When the tsutsugamushi antibody is present in the sample, the tsutsugamushi antibody and the tag antibody can be combined with each other in the gold conjugate pad 140 to form a complex. When the complex passes through the second reaction line 162, the tsutsugamushi antigen and the tsutsugamushi antigen protein of the second reaction line 162 may be combined. By the binding, the color particles of the marker antibody can be precipitated on the second reaction line 162 to form a red purple band.

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

When the nephrotic hemorrhagic heat antibody is present in the sample, the nephrotic hemoglobin heat antibody and the marker antibody bind to each other in the gold conjugate pad 140 to form a complex. When the complex passes through the third reaction line 163, the nephrotic range hemorrhagic heat antibody and the nephrotic range hemorrhagic fever antigen protein of the third reaction line 163 may be combined. By the binding, the color particles of the marker antibody can be precipitated on the third reaction line 163 to form a red purple band.

The marker antibody that does not bind to the tsutsugamushi antibody, the leptospira antibody, and the nephrotic range hemorrhagic fever antibody can be transferred to the control line 170 through the reaction line 160.

The entire reaction line 150 may include a first pre-reaction line 151 and a second pre-reaction line 152.

The first total reaction line 151 may include an E. coli extract. The E. coli extract may be combined with the first non-specific antibody. The first non-specific antibody may be an antibody that binds to a protein derived from Escherichia coli.

The first total reaction line 151 may be misjudged to be possessed even though the E. coli -based protein contained in the tsutsugamushi antigen protein binds with the nonspecific antibody and does not actually have an antibody against the tsutsugamushi antigen protein It is possible to exclude the possibility of

The second total reaction line 152 may comprise an insect cell Sf (Spodoptera frugiperda) extract. The insect cell Sf extract may be combined with a second nonspecific antibody. The second non-specific antibody may be an antibody that binds to a protein derived from an insect cell Sf.

Although the second pre-reaction line 152 binds to the nonspecific antibody and the protein derived from the insect cell Sf21 contained in the nephrotic range hemorrhagic fever antigen protein does not actually possess the antibody against the nephrotic hemorrhagic fever antigen protein, It is possible to exclude the possibility that it may be misjudged.

Cannulation 170 may comprise a control protein. The control protein may be associated with the marker antibody. By the binding, the color particles of the marker antibody can be precipitated on the control line 170 to 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, 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 absorption 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 with the reaction film 120.

Tsutsugamushi  Antigenic protein Example

Example  1. Antigen Protein Expression and Isolation

1-1. Kai Meric  Recombination Of 56 kDa (cr56)  Protein expression

Orientia Tsutsugamushi Giliam, Cap, Kato ( Orientia tsutsugamushi Gillam, Karp, Kato) was expressed and purified by the same method as described in the example of Korean Patent Publication No. 2002-0020281.

 Namely, Gilliam, Cap, and Kato of Orientia tsutsugamushi are cultured in mouse L-929 cells, harvested and purified. After purification of the purified strain, the DNA is purified by phenol extraction and ethanol precipitation. On the basis of the nucleotide sequence of Orientia tsutsugamushi 56 kDa protein gene, a protein region having a homology of 30% or more in amino acid sequence between Gyllium, Cap, and Kato serotype was selected, and a pair of oligosaccharides To prepare a nucleotide primer. DNA fragments of Gilliam, Kapp and Kato are amplified by PCR using Orientia tsutsugamushi DNA extracted from the above primer pair as a template. The PCR product is purified and cloned into the restriction enzyme cleavage site of the pTYB12 vector. First, a vector pTG3 is prepared by introducing a DNA fragment of a glial into a pTYB12 vector. A vector pTGP1 is prepared by introducing a caffe DNA fragment into pTG3, and then a DNA fragment of pTGP1 is introduced into pTGP1 to prepare pTGPT2. From pTGPT2, a serial link of the DNA fragment of Gilliam, CAP and Kato is cut out and introduced into the pET22b (+) vector to construct vector pETb7. E. coli is transformed with the produced expression vector, and the transformed E. coli is cultured to induce expression of the fusion protein. After confirming by electrophoresis and Western blotting, expressed fusion protein antigen was isolated and purified.

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

References to phylogenic analysis results (FEMA Microbiology Letters, 1999, 180: 163-169) show that the major global serotype prototypes Karp, Gilliam, Kato, Yonchon (most similar to Kangwon), it was considered very appropriate to use these four strains as antigens. Karp, Gilliam, and Kato are presented as cr56. Yonchon is also presented as a similar kr56 in Kangwon. Therefore, the present inventors decided to use the Gangwon 56 kDa recombinant protein (kr56) as a secondary antigen of chimeric recombinant protein (cr56).

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

A pair of primers was prepared by modifying NcoI and SalI recognition sites from a partial amino acid 84 (250 bp) to 445 (1,335 bp), which is known as a major antigenic domain of Kangwon 87-61, Respectively. The PCR product and the expression vector pET30a were digested with NcoI and SalI restriction enzymes, and ligated to each other. The plasmid was transformed into Escherichia coli BL21 and protein was extracted. The primers are shown in Table 1 below.

Primer sequence
Enzyme site
Cloning vector
Polypeptide
F: 5'-CCATGGCGCCAGGATTTAGAGCA-3 '
Nco I

Sal I
pET30a *

48 kDa

R: 5'-GTCGACCAAGACCAGCATAATATGAGAA-3 '

Plasmid pET30a expresses a His-Tagged fusion protein.

(2) Cloning and Expression of Gene

The amplified DNA (amino acid 84 (250bp) to 445 (1,335bp)) was amplified by PCR using a 1.2% agarose gel, followed by a QIAGEN gel elution kit (QIAGEN Inc., USA). The amplified bands were cut out on a transilluminator to the expected size and transferred to a microtube. Three times as much NaI solution as the volume of the agarose gel was added, and the mixture was allowed to stand at 60 DEG 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 microtube was allowed to stand at room temperature for 5 minutes, dried, added with elution buffer, stirred, and centrifuged at 17,000 x g (12,000 rpm) to transfer the supernatant to a new microtube. The thus purified PCR product and pET30a vector were completely digested with restriction enzymes Nco I and Sal I and recovered in the same manner using Geneclean kit II. 100 ng of the digested vector and 100 ng of the digested PCR product were mixed and ligated with 10-fold concentration of ligase buffer (250 mM Tris-HCl pH 7.8, 100 mM MgCl 2, 20 mM DTT, 4 mM ATP) Lt; / RTI >

(3) Production and transformation of competent cells

Escherichia coli BL21 cultured in LB medium for 18 hours was diluted 100-fold in LB medium and re-cultured at 600 nm until the absorbance became 0.3. After allowing to stand 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 volume of the medium. The mixture was allowed to stand in ice for 1 hour, centrifuged at 2,000 g (4,100 rpm) for 10 minutes, and then suspended in 1/10 volume of 0.1M CaCl2 in the culture medium and used for transformation.

(4) Transformation

The ligated product prepared in (3) above was mixed with competent cells, left in ice, and heat shocked at 42 ° C for 1 minute and 30 seconds. LB medium was added, followed by incubation at 37 ° C for 1 hour. The culture was inoculated on LB agar plate medium and cultured at 37 ° C.

(5) Plasmid DNA isolation of transformed E. coli

The transformed E. coli single colonies were inoculated on LB medium containing kanamycin and cultured at 37 ° C with shaking. For plasmid DNA extraction, AccuPrep Plasmid Extraction kit was used. The culture was centrifuged at room temperature for 10 minutes at 750 g (2,500 rpm), and only the cell precipitate was collected. After adding resuspenstion buffer, it was added with lysis buffer, and left at room temperature for 5 minutes. Neutralization buffer was added and left at room temperature. The supernatant was transferred to a binding column tube by centrifugation at 17,000 × g (12,000 rpm). It was centrifuged at 17,000xg (12,000 rpm) at room temperature and then the extract was removed. Then, 80% ethanol was dispensed and centrifuged, and only the binding column was transferred to a new tube. Then, the elution buffer was dispensed and left at room temperature, and centrifuged to transfer the supernatant to a new microtube.

(6) Protein Expression and Isolation

The culture medium of the seed culture process was inoculated to the culture medium. The inoculation was carried out at 37 ° C and the mixture was stirred at 220 rpm for 1 hour and 45 minutes after inoculation. When the value of O.D showed a value of 0.5 to 0.6, IPTG was inoculated at 0.5 mM and cultured for 3 hours. After centrifugation at 3,000 rpm for 30 min at 4 ° C, 1x binding buffer (6 M Urea, 500 mM NaCl, 20 mM Tris-HCl (pH 8.0), 5 mM Imidazole) was added to the pellet : 10 sec on, 20 sec off, time: 6 min X 2 (inverting once in the middle), Amp .: 45%) cells were disrupted. kr56 was centrifuged at 14,000 rpm for 15 minutes at 4 ° C (rotor 7 in autoclaved ultracentrifuge bottle), the supernatant was stored in the refrigerator, and the pellet was resuspended in 1X Binding buffer (50 ml based on 1 L Erlenmeyer flask culture) Respectively. After incubation for 1 hour in ice (inverting after 30 min.), The mixture was centrifuged at 14,000 rpm for 30 min at 4 ° C (rotor 7 in autoclaved ultracentrifuge bottle), and the supernatant was obtained. The supernatant was diluted 2X with 1X Binding buffer (6M urea), filtered (0.45 μm syringe filter) and stored at 4 ° C.

(7) Protein purification

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

The specific amino acid sequence of Orientia tsutsugamushi Gangwon 56 kDa protein contained in the tsutsugamushi antigen protein of the present Example 1 can be referred to the amino acid sequence disclosed in Patent No. 10-0796772.

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

The tsutsugamushi antigen protein prepared according to Example 1 was prepared at a concentration of 0.6 mg / ml, 0.9 mg / ml, 1.2 mg / ml and 1.5 mg / ml, respectively. Four kits containing each concentration of Tsutsugamushi antigen protein were prepared. The patient's samples were evaluated using immunofluorescent antibody method, a standard diagnostic test for the diagnosis of tsutsugamushi disease, and one negative serum (DK13) and three positive sera (90-202, 00-350, 89 -129) were prepared and applied to each of the diagnostic kits having different antigen concentrations to measure the response.

FIG. 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 reaction according to the concentration of tsutsugamushi antigen protein.

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-positive results were obtained in the diagnostic kit in which the concentration of tsutsugamushi antigen protein was 0.9, 1.2 and 1.5 mg / mL, but false-positive in the diagnostic kit in which the tsutsugamushi antigen protein concentration was 0.6 mg / mL I did.

Example  3. Depending on mixed antigen composition Kit  reaction

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 tsutsugamushi antigen protein is a mixture of cr56, kr56, and r21 proteins at a ratio of 5: 2: 1, respectively. A diagnostic kit comprising the tsutsugamushi antigen protein of Example 1 and a diagnostic kit comprising the conventional tsutsugamushi antigen protein were prepared and the response to negative and positive sera was measured. As a control, Ab titer according to IFA was measured for each negative serum and positive serum.

FIG. 4 shows a kit reaction according to the composition of tsutsugamushi antigen protein according to an embodiment of the present invention.

Table 3 shows the response sensitivities of the conventional tsutsugamushi antigen protein and the tsutsugamushi antigen protein of Example 1 to - = 0, + = 1, ++ = 2, +++ = 3, ++++ = 4 As shown in Fig.

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 The tsutsugamushi of Example 1
Antigen protein
(cr 56: kr 56 = 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 FIG. 4, when rs21 was not included in the tsutsugamushi antigen protein and the ratio of kr56 was increased, the sensitivity to the antibody was improved.

Of leptospira antigen Example

Example  4. Securing Leptospira antigen

4-1. Leptospira culture

EMJH (Leptospira medium base Ellinghausen McCullough Hohnson Harris) was dissolved in 0.23 g / 90 ml and sterilized. Ten ml of leptospira enrichment EMJH was added to the culture medium. This medium was inoculated with leptospirosis and cultured at 30 DEG C for 5 days with shaking.

4-2. Preparation of leptospira antigen

Leptospira cultured for 5 days in EMJH medium was centrifuged at 4,000 × g for 20 minutes. The cells were washed three times with 1 × PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4) and heated at 100 ° C. for 1 hour Respectively. Proteinase K (150 / / ml) was added thereto, followed by reaction at 37 캜 for 16 hours. Then, EGTA was added to make 2 mM, followed by reaction at 70 ° C for 15 minutes, followed by centrifugation at 4 ° C and 180,000 × g for 2 hours. The precipitate was suspended by adding H2O to produce a lipopolysaccharide (Westpha & Jann, 1965). The thus purified LPS antigen was treated with 1% acetic acid, heated at 100 ° C for 1 hour, centrifuged at 3,000 xg for 20 minutes, and lipid A was removed to prepare a polysaccharide antigen. The amount of the prepared antigen was measured using a polysaccharide obtained by treating LPS (Sigma) purified from Escherichia coli in the same manner as above as a control.

Example  5. Antigenicity of Leptospira polysaccharide

5-1. Western  Block analysis

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

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

5, the polysaccharide of Leptospira interrogans serovar lai showed similar pattern in SDS-PAGE gel profile with Leptospira interrogans serovar patoc strain Patoc, and the polysaccharide component of 14 kDa or more existed commonly in lai and patoc, .

5-2. Dot blotting

One ug of antigen (1 ug / 1 ul) was applied to the nitrocellulose membrane and then dried at 37 캜 for 1 hour. The dried membrane was blocked with 5% skim milk-TBST for 1 hour. The primary antibody, patient serum, was diluted 1: 3,000 and reacted for 1 hour. The secondary antibody was diluted 1: 10,000 with horse-radish peroxidase conjugate anti-human IgG. After the reaction, ECL kit was used for color development and compared with MAT. Positive signals were seen in most patient sera.

Table 4 compares the results of MAT and Dot-blotting.

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)

The polysaccharide ELISA was performed based on the method used by Engvall and Perlmann (1972). The plate was pre-treated with 0.01 M PBS diluted with poly-L-lysine (10 μg / ml) and reacted at room temperature for 24 hours. After washing twice with PBS buffer containing 0.05% Tween 20, the polysaccharide was suspended in PBS and treated with 100 μl per well. After reacting at 37 ° C for 1 hour, the plate was washed three times with PBS buffer containing 0.05% Tween 20, treated with chlorine serum, and blocked at 37 ° C. Twenty patients with leptospirosis and 20 normal controls were used. The secondary antibody was diluted 1: 10,000 with horse-radish peroxidase-conjugated anti-human IgG (Bio-Rad) . The substrate was treated with each well to observe the color development, and the value was measured at 490 nm using an ELISA reader. As a result, Leptospira flagella recombinant protein showed sensitivity of 72-88% and specificity of 88-90%, compared with 95% sensitivity and 95% specificity of this polysaccharide. As a result, the early diagnosis of leptospirosis The candidates for the antigen showed better polysaccharide than protein.

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

Hemorrhagic fever  Antigenic protein Example

Example  6. Washing virus  And cultured cells

The Soochong virus strain SooV-2 isolated from domestic white thrips (Apodemus peninsulae) was subcultured in Vero cells. Vero cells were grown and maintained in EMEM (Eagel's minimal essential medium) containing 10% fetal bovine serum (FBS) at 37 ° C in a 5% It was used when monolayer culture was performed.

Example  7. Isolation of viral RNA

Cells grown with the virus were centrifuged at 1,075 g (3000 rpm) for 3 minutes, and 750 T of Trizol LS (Gibco) solution was added. After incubation at room temperature for 5 minutes, 200 클로 of chloroform was added and mixed well. And allowed to stand for 10 minutes. The precipitate was removed by centrifugation at 17,000 g (12,000 rpm) at 4 ° C for 15 minutes, the same volume of isopropyl alcohol was added to the supernatant, the mixture was allowed to stand at room temperature for 10 minutes and then again at 17,000 g (12,000 rpm) For 15 minutes to precipitate RNA and wash once with 75% ethanol. RNA precipitates were 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 nucleocapsid protein, complete NP (CN) and truncated NP (TNP) in S segment of Succulent virus according to an embodiment of the present invention, (About 370 bp) and a DNA band (about 1300 bp) of the whole protein (CNP) of the fragment protein (TNP) after the PCR reaction according to an embodiment of the present invention.

Table 6 shows PCR primers for the production of recombinant virus recombinant antigens.

Primer Total protein (CNP) Fragment protein (TNP) Foward
primer 5 ' AGA TCT ATG GCA ACT ATG GAG GAA TTG 3 '(SEQ ID NO: 4)
≪ BglII (AGATCT); AGATCT addition> 5 ' AGA TCT A ATG GCA ACT ATG GAG GAA TTG 3 '(SEQ ID NO: 6)
<Bgl II; AGATCT addition> Reverse
primer 5 ' AAGC TTA TAG TTT TAA AGG TTC TTG GTT TG 3' (SEQ ID NO: 5)
&Lt; Hind III (AAGCTT); AAGC addition> 5 ' A AGC TTA CCA GTC TGC AGT CTG ACC TGT 3' (SEQ ID NO: 7)
<Hind III; AAGCTTA addition>

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

PCR was performed by mixing 5 μl of cDNA product, 10 pmole primer pair, 10 mM dNTPs, 10 × taq buffer and 5 units / μl Taq polymerase, followed by one cycle of reaction at 95 ° C for 5 min, 52 ° C for 2 min and 72 ° C for 2 min, , 52 [deg.] C for 1 minute, and 72 [deg.] C for 1 minute. 95 ° C for 1 minute, 60 ° C for 1 minute. The reaction was performed 20 times at 72 ° C for 1 minute, and DNA was amplified by reacting once at 95 ° C for 1 minute, 60 ° C for 1 minute, and 72 ° C for 7 minutes. The amplified DNA was stored at -20 ° C.

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

Referring to FIG. 7, the DNA of the truncated NP (TNP) was confirmed at about 370 bp by performing PCR with each primer, and the DNA of the complete protein (Complete NP: CNP) was also confirmed as a band at about 1,300 bp.

Example  9. Amplification products Cloning

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

8, about 370 bp and about 1,300 bp of DNA amplified by the sequential enzymatic polymerization were electroeluted in an agarose gel and cloned into a pGem-T-Easy PCR cloning vector (Invitrogen) as a cloning vector. Respectively. DNAs of TNP and CNP were confirmed by digesting 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 369 bp inserted plasmid containing TNP was named pGT-SooV2-TNP and the 1299 bp inserted plasmid containing CNP was named pGT-SooV2-CNP.

Example  10. Expression vector Cloning

FIG. 9 shows a PCR product of pGT-SooV2-TNP and pGT-SooV2-CNP cloned into a 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 And pGT-SooV2-CNP were cloned by treatment with restriction enzymes Bgl Π and Hind Ⅲ to obtain pBac-SooV2-TNP and pBac-SooV2-CNP.

Example  11. Recombination Baculovirus  making

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

10, pBac-SooV2-TNP and pBac-SooV2-CNP were transformed into Bac-SooV2-TNP and Bac-SooV2-CNP using a Bac-N-Blue baculovirus expression vector system Manufactured

Sf9 cells (2X10 ⁶ ) in the log phase were poured into a 60 mm dish to become a monolayer. To prepare a transfection mixture, 10 ul (0.5 ug) of Bac-N-Blue ^™ DNA was placed in a microcentrifuge and the following solution was added.

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

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

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

After allowing to stand at room temperature for 15 minutes, the dish medium was removed and 2 ml of Grace's insect medium without supplement or FBS was added. After removing the medium from the monolayer, the prepared transfection mixture was transferred to a 60 mm dish And put the drop. The dish was allowed to stand 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 is used as a virus stock.

Example  12. Recombination Baculovirus  refine

The concentration of virus stock was measured by the end-point dilution method.

The end point (end-point) was diluted to determine the concentration (titer) of the diluted virus to virus inoculation amount of 10 ^-5 to 10 ^-8 times the step, and in a 96-well plate, each dispensed with 1X10 ⁴ cells per well of a concentration Cultured cells were inoculated with each dilution and cultured at 27 ° C. The virus concentration was determined from the ratio of the virus-infected well to the non-infected well to determine the plaque forming unit (PFU).

For the selection of the pure virus, the virus inoculum was diluted to 10 &lt; ^-2 &gt; to 10 &lt; ^-8 &gt; times, and each diluted solution was inoculated into cultured cells at a concentration of ¹ x 10 ⁴ cells per well in a 96 well plate, . From day 4 after inoculation, the presence of intracellular polyhedra and virus infection were observed under a microscope to select viruses in the wells of the maximum dilution ratio, and pure viruses were selected through three or more purification steps in the same manner.

Example  13. Expression and isolation of recombinant proteins

Insect cells Sf9 cells were seeded in a 75 cm 2 flask at about 5 × 10 ⁶ , and then inoculated with 5 MOI of recombinant virus. At 5 days after infection, inclusion body formation was confirmed and all the cells were collected. The collected cells were centrifuged at 6,000 g (7,000 rpm), and the precipitates were collected and suspended in 1 X binding buffer (5 mM imidazole, 20 mM Tris-HCl pH 7.9, 0.5 M NaCl) The mixture was stirred six times for 15 seconds. After centrifugation at 23,400 g (14,000 rpm) for 15 minutes, the supernatant was stored in soluble form. The insoluble form, which is a needle, was floated in a 1 × binding buffer containing 6M Urea, allowed to stand at 4 ° C for 1 hour, and centrifuged at 23,400g (14,000rpm) for 30 minutes to remove supernatant lysed pellet. The collected lysed pellet was purified through His-bind chromatography (purified pellet). The soluble form, lysed pellet, and purified pellet were subjected to SDS-PAGE to determine the presence of recombinant protein in each fraction by Western blotting. As a control, a soluble form of wild-type virus-infected cells was used. Western blot analysis was performed using HFRS and normal human serum as primary antibodies.

Figure 11 shows the results of western blotting using patient and normal human serum for isolation detection of fragment protein (TNP) according to one embodiment of the present invention. The band on the down arrow appears to be due to partial degradation of the fragment protein. The left image shows the serum of the hemorrhagic fever after the primary antibody and the serum of the normal person as the primary antibody. (1 lane: solution fraction of cells infected with wild-type baculovirus, 2 lane: lysis fraction of cells infected with recombinant baculovirus, 3 lane: cell lysate was treated with buffer containing urea and centrifuged And then the supernatant and the fraction of 4 lane: 3 purified)

Referring to FIG. 11, it was confirmed that TNP had more proteins in a soluble form than in an insoluble form. In addition to the specific band of 22 kDa of the TNP fraction, a weakly bands beneath the specific band were strongly expressed in the Western blot by the patient serum in the soluble form as well as in the purified pellet, Respectively. In addition, it was proved that normal bands do not show both bands. The band below was estimated to be due to partial degradation of TNP.

Figure 12 shows the results of western blotting of purified fractions of fractional protein (TNP) and total protein (CNP) according to one embodiment of the present invention. (1 lane: cells not infected with virus, 2 lane: cells infected with baculovirus, 3 lane: cells infected with recombinant baculovirus cloned with total protein (CNP), 4 lane: , 5 lane: cells infected with recombinant baculoviruses cloned with whole protein (CNP), 6 lane: recombinant baculoviruses cloned with fragment protein (TNP), and cells infected with recombinant baculovirus cell)

Referring to FIG. 12, it was confirmed that CNP reacts only with patients with post-hemorrhagic fever (HFRS) and produces recombinant proteins of about 50 kDa.

Example  14. His-bind affinity By chromatography  Purification of recombinant proteins

His-bind resin (Novagen) was added to the column to 2 ml, and the column was prepared with distilled water, 1 X charge buffer and 1 X binding buffer. After the antigen protein was passed through the column, 1 × binding buffer (5 mM imidazole, 20 mM Tris-HCl pH 7.9, 0.5 M NaCl) and 1 × washing buffer (40 mM imidazole, 20 mM Tris- NaCl). The protein was eluted with 1 ml of elution buffer (300 mM imidazole, 0.5 M NaCl, 20 mM Tris-Cl pH 7.9). Protein concentration in each fraction was quantitated using the Raleigh method, and proteins were identified by SDS-PAGE and Western blotting.

Example  15. Recombinant protein analysis

15-1. Western-blot

The recombinant proteins purified at 10% gel at 1 mm or 1.5 mm thickness were subjected to electrophoresis at 10 ug / well. The electrophoresed gel was transferred at 80 volts for 40 minutes 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 primary antibody, patient serum, was diluted 1: 3000 and reacted for 1 hour . The secondary antibody was reacted with horse-radish peroxidase conjugate anti-human IgG diluted 1: 10000. After the reaction, the cells were developed using an ECL kit.

Western blot analysis showed that recombinant CNP and TNP did not react with normal normal serum, although they were reacted with 50 kDa of CNP and 22 kDa of TNP in serum of patients with nephrotic hemorrhagic fever.

15-2. Dot-blot

One μg of recombinant protein was loaded onto a nitrocellulose membrane and allowed to dry for 1 hour at 37 ° C. The dried membrane was blocked with 5% skim milk-TBST for 1 hour, and the primary antibody, patient serum and normal serum were diluted to 1: 3,000 and reacted for 1 hour. The secondary antibody, Horse-radish peroxidase conjugate, was diluted 1: 10,000 with anti-human IgG. After the reaction, the cells were developed using an ECL kit.

Dot-blotting was performed with sera of patients with hemorrhagic fever after infection with Hantan virus or Seoul virus. As shown in Table 7 below, CNP showed a stronger positive signal than TNP, .

HRFS patients
serum 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 (pH 9.6) to 1 ug / ml, coated on a 96-well microplate, and reacted at 4 캜 for 18 hours. After washing twice with phosphate buffer (PBST) containing 0.05% Tween 20, the cells were blocked with 3% BSA at 37 ° C for 2 hours. Primary antibodies were diluted with serum from rats infected with Hantaan, Seoul, Pummala or Prospect Hill viruses. Secondary antibodies were obtained from Horse-radish conjugate anti-rat IgG (IgM) was diluted to 1: 6000 and used. After coloring with a chromogenic substrate solution, the OD was measured at 490 nm with an ELISA reader.

As shown in Table 8, the recombinant antigen CNP reacts with hanthan virus and Seoul virus, but does not react with fumarovirus and prospective heil virus.

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

Hereinafter, specific embodiments of the present invention have been described. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

10: Multiple diagnosis kit 100: Strip
100a: first strip 100b: second strip
110: support member 120: reaction membrane
130: Sample pad 140: Gold conjugate pad
150: total reaction line 151: first total reaction line
152: second overall reaction line 160: reaction line
161: first reaction line 162: second reaction line
163: Third reaction line 170: T /
180: Absorption pad 200: Sample inlet

Claims (10)

Comprising a strip,
Wherein the strip comprises:
A tsutsugamushi antigen protein comprising a 56 kDa fusion protein (cr56) of Oriental tiatsugamushi gilliam, capy, and kato; and a 56 kDa protein (kr56) of Orientia tsutsugamushi gangwon;
Leptospira antigen; And
Lt; RTI ID = 0.0 &gt; a &lt; / RTI &gt; renal hemorrhagic fever antigen protein. The method according to claim 1,
Wherein the strip comprises a first strip and a second strip,
Said first strip detecting a tsutsugamushi IgM antibody, a leptospira IgM antibody, and a nephrotic hemoglobin IgM antibody,
Wherein said second strip detects a tsutsugamushi IgG antibody, a leptospira IgG antibody, and an IgG antibody of nephrotic hemorrhagic fever. The method according to claim 1,
Wherein the 56 kDa fusion protein (cr56) of Orientia tsutsugamusi giliam, cap, and kato and the 56 kDa protein (kr56) of the Orient tiatsugamushi gangwon are contained in a volume ratio of 5: 3. The method according to claim 1,
Wherein said leptospira antigen comprises a core polysaccharide and a 0-specific side chain made from lipopolysaccharide (LPS) of Leptospira. The method according to claim 1,
Wherein said nephrotic hemorrhagic fever antigen protein comprises a nucleocapsid protein prepared from Suwon virus having an amino acid sequence of SEQ ID NO: 2 or 3, which is prepared from eukaryotic cells. The method according to claim 1,
Wherein the strip comprises:
A first reaction line containing the leptospira antigen;
A second reaction line comprising the tsutsugamushi antigen protein; And
And a third reaction line containing the nephrotic range hemorrhagic fever antigen protein. The method according to claim 1,
Wherein the strip comprises:
A gold conjugate pad comprising a tsutsugamushi antibody, a leptospira antibody, and a marker antibody that binds to a nephrotic range hemorrhagic fever antibody,
Wherein the marker antibody comprises at least one selected from anti-human IgM conjugated gold, anti-human IgG conjugated gold, and chicken IgY conjugated gold. The method according to claim 1,
Wherein the strip comprises:
A first total reaction line comprising an E. coli extract which is bound to a first nonspecific antibody,
Wherein the first non-specific antibody is an antibody that binds to a protein derived from Escherichia coli. The method according to claim 1,
Wherein the strip comprises:
And a second pre-reaction line comprising an insect cell Sf extract coupled with a second nonspecific antibody,
Wherein the second non-specific antibody is an antibody that binds to a protein derived from an insect cell Sf. The method according to claim 1,
Wherein the strip comprises:
And a control line comprising a control protein that binds to said marker antibody,
Wherein the control protein comprises at least one selected from the group consisting of 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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