KR102013954B1 - Composition for diagnosing hemorrhagic fever with renal syndrome including recombinant nucleocapsid protein derived from seoul virus and puumala virus and recombinent glycoprotein derived from hantaan virus and kit including the same - Google Patents

Abstract

One embodiment of the present invention is a recombinant protein consisting of a nucleocapsid protein (Seoul virus) and Pumala virus (Puumala virus); And a recombinant glycoprotein (Glycoprotein) derived from Hantan virus (Hantaan virus); provides a composition for diagnosing nephrotic syndrome hemorrhagic fever.

Description

COMPOSITION FOR DIAGNOSING HEMORRHAGIC FEVER WITH RENAL SYNDROME INCLUDING RECOMBINANT NUCLEOCAPSID PROTEIN DERIVED FROM SEOUL VIRUS AND PUUMALA VIRUS AND RECOMBINENT GLYCOPROTEIN DERIVED FROM HANTAAN VIRUS AND KIT INCLUDING THE SAME}

The present invention relates to a composition for diagnosing nephrotic syndrome hemorrhagic fever comprising a recombinant nucleocapsid protein derived from Seoul virus and fumala virus and a recombinant glycoprotein derived from Hantan virus, and a diagnostic kit comprising the same.

Hemorrhagic fever with renal syndrome (HFRS) is caused by Hantavirus (Hantavirus), which causes 20 to 300,000 patients worldwide every year, of which less than 5% die. Thousands of patients are classified as group III forensic infections that occur in civilians and soldiers every year.

Nephrotic syndrome hemorrhagic fever, in addition to the typical clinical characteristics, has been shown to be similar to the symptoms of influenza or as a case of intractable infections, and due to the problem of differentiating between febrile infectious diseases such as Tsutsugamushi and leptospirosis, which have high prevalence Therefore, the actual diagnosis is mainly dependent on the serum test results in addition to the clinical findings.

Indirect immunofluorescence assay (IFA) is most commonly used as a serological diagnostic method for hemorrhagic fever of nephrotic syndrome.However, judgment errors may occur due to subjective readings. A long time of 4 hours or more is required.

In addition, enzyme linked immunosorbent assay (ELISA), hemagglutination inhibition (HI), plaque reduction neutralization test (PRNT), western blot analysis (WB), Various tests such as reverse transcriptase polymerase chain reaction (RT-PCR) have been used, but reliable tests that are important for the determination of disease progression, treatment, and prognosis of patients still have problems to be improved.

Recently, there are four methods of manufacturing the most commonly used diagnostic kits: Enzyme Immunoassay (EIA), Aggregation Assay, Dip-stick, and Lateral Flow Assay. The EIA has the advantage of having high sensitivity, but it has a disadvantage in that it takes a long time and requires a special reading device because the specificity is low and a number of pretreatment steps are required before diagnosis. The plate agglomeration method has a disadvantage that the time required for reading is very short, but the sensitivity is low and the result is not easy to read. The tube agglomeration method has a disadvantage that the process is cumbersome, the reading is often ambiguous, and takes more than 2 hours.

Dip-stick (Dip-stick) is applicable to a variety of pathogens and has the advantage of low cost, but has the disadvantage of low sensitivity, specificity. Lateral flow assay method, also called rapid diagnostic kit, is the most recently developed method because it can be diagnosed in a short time. It does not require pretreatment and can be read visually within 15 minutes. There is a limit to securing a specific antigen for a specific pathogen for the application.

The present invention is to solve the above-mentioned problems of the prior art, an object of the present invention is to provide a composition and diagnostic kit capable of diagnosing nephrotic syndrome hemorrhagic fever with excellent diagnostic sensitivity and specificity in a short time.

However, the problem to be solved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

According to one embodiment of the present invention, a recombinant protein consisting of a Nucleocapsid protein derived from Seoul virus and Pumala virus; And a recombinant glycoprotein (Glycoprotein) derived from Hantan virus (Hantaan virus), a composition for diagnosing nephrotic syndrome hemorrhagic fever is provided.

According to one side, the composition can simultaneously detect nephrotic syndrome hemorrhagic fever specific IgM and IgG.

According to one side, the nucleocapsid protein derived from the Seoul virus may be encoded by the nucleotide sequence of SEQ ID NO: 1.

According to one side, the nucleocapsid protein derived from the fumala virus can be encoded by the nucleotide sequence of SEQ ID NO: 2.

According to one side, the recombinant glycoprotein derived from the hantan virus may include the recombination of antigenic determining sites in the glycoprotein of the hantan virus.

According to one side, the recombinant glycoprotein derived from the hantan virus may be encoded by the nucleotide sequence of SEQ ID NO: 3.

According to another embodiment of the present invention, a sample pad (sample pad) is absorbed by the sample; Gold conjugation pads that bind to antibodies in the sample; A test membrane treated with a test line including the composition and a control line including a control composition; And a test strip (absorption pad) in which a residual amount of the sample is absorbed, a nephrotic syndrome hemorrhagic fever diagnostic kit is provided.

According to another embodiment of the present invention, a method of providing information on diagnosing nephrotic syndrome hemorrhagic fever using the diagnostic kit is provided.

According to one aspect of the present invention, by recombining nucleocapsid proteins derived from Seoul virus and fumala virus having excellent antigenicity, and using a recombinant glycoprotein derived from Hantan virus, the diagnosis of nephrotic syndrome bleeding fever in a short time and at the same time diagnosed Sensitivity and specificity can be improved.

It is to be understood that the effects of the present invention are not limited to the above effects, and include all effects deduced from the configuration of the invention described in the detailed description or claims of the present invention.

1 is a result of comparing the nucleocapsid protein amino acid sequence of each of the virus and the conventional virus in accordance with an embodiment of the present invention.
Figure 2 shows the test strip structure in the nephrotic syndrome hemorrhagic fever diagnostic kit according to an embodiment of the present invention.
Figure 3 shows the structure of the nephrotic syndrome hemorrhagic fever diagnostic kit according to an embodiment of the present invention.
Figure 4 confirms the expression and purification of recombinant nucleocapsid protein of Seoul virus and fumala virus in E. coli.
Figure 5 confirms the expression and purification of recombinant nucleocapsid protein of Seoul virus and fumala virus in insect cells.
Figure 6 confirms the recombinant glycoprotein expression and purification of Hantan virus in E. coli.
Figure 7 illustrates the use of nephrotic syndrome hemorrhagic fever diagnostic kit according to an embodiment of the present invention.
Figure 8 shows the detection sensitivity evaluation results of the nephrotic syndrome hemorrhagic fever diagnostic composition applied / unapplied kit according to an embodiment of the present invention.
Figure 9 shows the results of cross-reaction evaluation of the nephrotic syndrome hemorrhagic fever diagnostic composition applied / unapplied kit according to an embodiment of the present invention.

Hereinafter, exemplary embodiments will be described in detail with reference to the accompanying drawings. Like reference numerals in the drawings denote like elements.

Various modifications may be made to the embodiments described below. The examples described below are not intended to be limited to the embodiments and should be understood to include all modifications, equivalents, and substitutes for them.

The terminology used herein is for the purpose of describing particular example embodiments only and is not intended to be limiting of examples. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this specification, terms such as "comprise" or "have" are intended to indicate that there is a feature, number, step, action, component, part, or combination thereof described on the specification, and one or more other features. It is to be understood that the present invention does not exclude the possibility of the presence or the addition of numbers, steps, operations, components, components, or a combination thereof.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art. Terms such as those defined in the commonly used dictionaries should be construed as having meanings consistent with the meanings in the context of the related art and shall not be construed in ideal or excessively formal meanings unless expressly defined in this application. Do not.

In addition, in the description with reference to the accompanying drawings, the same components regardless of reference numerals will be given the same reference numerals and redundant description thereof will be omitted. In the following description of the embodiment, when it is determined that the detailed description of the related known technology may unnecessarily obscure the gist of the embodiment, the detailed description thereof will be omitted.

According to one embodiment of the present invention, a recombinant protein consisting of a Nucleocapsid protein derived from Seoul virus and Pumala virus; And a recombinant glycoprotein (Glycoprotein) derived from Hantan virus (Hantaan virus), a composition for diagnosing nephrotic syndrome hemorrhagic fever is provided.

As used herein, the term "Hemorrhagic Fever with Renal Syndrome (HFRS)" refers to Seoul virus, Pumala virus, Hantaan virus, Dobrava-Belgrade virus ( It refers to a disease caused by Hantavirus virus such as Dobrava-Belgrade virus.

As used herein, the term "diagnose" refers to a series of processes for confirming the presence or characteristics of a pathological condition, through which it is possible to confirm the onset of nephrotic syndrome hemorrhagic fever caused by Hantavirus infection.

The hantavirus is a virus of the genus Bunyaviridae (family) and Hantavirus (genus), a spherical virion having a diameter of 80-120 nm having a coating on its surface. The Hantavirus was a host of rodents in the United States, Brazil, Argentina, Paraguay and Uruguay, and was present in the Americas and is now distributed throughout the world. The total genome of the hantavirus is about 12 kb of single stranded RNA, which is composed of three segments of S (small), M (medium) and L (large).

The hantavirus follows the infection route propagated through the respiratory tract while the feces of rodents are dried, and has a latency period of 2 to 4 weeks in the human body until symptoms of infection appear.

Specifically, the symptom of infection of the Hantavirus includes fever, chills, headaches, and a fever, a respiratory or digestive system problem, a low platelet level, a low blood pressure indicating a rapid pulse and hypoxia, a renal failure and proteinuria, a urinary tract, It can be divided into five stages: diuretic, characterized by large amounts of urine, and recovery, when symptoms improve.

The geographical distribution of the Hantavirus genus causing the nephrotic syndrome hemorrhagic fever is as follows. The Seoul virus is distributed worldwide and the Pumala virus is distributed in Europe, Russia and Korea. The Hantan virus is distributed in Asia, east of Russia, and south of China, while Dobrava-Belgrade virus is distributed in the Balkan region.

Like the geographical distribution of the Hantavirus genus, Hantaviruses causing regional nephrotic syndrome hemorrhagic fever differ in genus. In order to detect an antibody against such a virus and to determine whether nephrotic syndrome hemorrhagic fever is developed, an antigen capable of detecting all antibodies to the genus Hantavirus causing the disease is required. Thus, the present inventors have completed the present invention to prepare a recombinant protein capable of detecting the antibody and use it for diagnosing nephrotic syndrome bleeding.

The present inventors compared the amino acid sequence of each of the viruses with the conventional listening virus for the Hantavirus genus that causes nephrotic bleeding hemorrhagic fever, and the results were obtained as shown in FIG. 1, and the similarities between them were obtained to obtain the results of Table 1 below. .

division Soochong virus Seoul virus Hantaan virus Dobrava-Belgrade virus Puumala virus Soochong virus - - - - - Seoul virus 84.9 - - - - Hantaan virus 95.8 85.7 - - - Dobrava-Belgrade virus 83.2 83.2 83.2 - - Puumala virus 52.1 52.9 54.6 51.3 -

As shown in the results of Fig. 1 and Table 1, it is not possible to detect all the antibodies of the regional nephrotic syndrome hemorrhagic fever with the existing listening virus. Therefore, Seoul virus, which is distributed worldwide, and Nucleocapsid protein of Pumala virus, which is distributed in Europe, Russia, and Korea, are prepared as recombinant proteins and used for diagnosis. sensitivity and specificity can be achieved.

In particular, the nucleocapsid protein of the Seoul virus and the fumala virus and the glycoprotein of the Hantan virus react with the serum of patients with nephrotic syndrome hemorrhagic fever but do not react with normal serum, so hemorrhagic fever with nephrotic syndrome It can act as a specific antigen for diagnosis and exhibit high antigenicity, thereby improving diagnostic sensitivity and specificity.

Recombinant proteins expressed by recombining the coding genes of the nucleocapsid proteins of the Seoul virus and the fumala virus can improve antigenicity as compared to the case of using them alone, and use a mixture of recombinant glycoproteins of the Hantan virus The antigenicity can be further improved. Thus, the composition for diagnosing nephrotic syndrome hemorrhagic fever comprising these proteins can implement excellent diagnostic sensitivity and specificity.

Specifically, the nucleocapsid protein derived from the Seoul virus and the nucleocapsid protein derived from the fumala virus may be encoded by the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

On the other hand, the recombinant glycoprotein derived from the hantan virus may include the recombination of the antigenic determination site in the glycoprotein of the hantan virus. Specifically, the antigenic determining region is a fragment of the glycoprotein 414th amino acid (1,242th base) to 506th amino acid (1,518th base) of the Hantan virus and the 773rd amino acid (2,319th base) to 1,009th amino acid (3,027th). Up to a total of 330aa of the recombinant protein. At this time, the recombinant glycoprotein derived from the hantan virus may be encoded by the nucleotide sequence of SEQ ID NO: 3.

The recombinant nucleocapsid protein may be prepared by recombining the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2 in a microorganism or eukaryotic cell through a genetic recombination method, but is not limited thereto. As a vector for the genetic recombination method, a transfection vector capable of expressing a recombinant protein without infectivity in humans and animals, for example, a baculovirus transition vector, may be used, but is not limited thereto. The base sequence of SEQ ID NO: 3 may also be inserted into a transition vector and then expressed in a microorganism or eukaryotic cell to produce a recombinant glycoprotein.

Since the recombinant nucleocapsid protein and recombinant glycoprotein correspond to proteins derived from Seoul virus, Fumala virus, and Hantan virus, which are mainly distributed in the world, particularly in Asia, the nephrotic syndrome hemorrhagic fever diagnostic composition is distributed worldwide. It can show high sensitivity and specificity for Hantavirus.

In addition, the nephrotic syndrome hemorrhagic fever diagnostic composition can detect antibodies, that is, nephrotic syndrome hemorrhagic fever-specific IgM and IgG produced in patients with nephrotic syndrome hemorrhagic fever, and preferably detect them simultaneously.

According to another embodiment of the present invention, a sample pad (sample pad) is absorbed by the sample; Gold conjugation pads that bind to antibodies in the sample; A test membrane treated with a test line including the composition and a control line including a control composition; And a test strip (absorption pad) in which a residual amount of the sample is absorbed, a nephrotic syndrome hemorrhagic fever diagnostic kit is provided.

Figure 2 shows the test strip structure in the nephrotic syndrome hemorrhagic fever diagnostic kit according to an embodiment of the present invention. Referring to FIG. 2, the test strip may include a sample pad, a gold bonding pad, a reaction film, and an absorption pad.

The sample pad may overlap the gold bond pad to form a first overlap portion, and the gold bond pad may overlap the reaction film to form a second overlap portion. In addition, the reaction film and the absorption pad may form a third overlapping portion.

When a biological sample is deposited on the sample pad, the capillary action causes gold binding pads, such as gold-labeled protein A or gold-labeled goat anti-human IgM antibodies. can pass through a gold binding pad containing a human IgM antibody. The gold particles forming the gold-label may have a diameter of 20 nm to 55 nm, preferably 20 nm to 40 nm, and may act as an indicator dye. As it passes through the gold binding pad, the antibody in the biological sample binds to a gold-labeled protein A or the like to form a complex, and the complex may move along the reaction membrane.

As used herein, the term "biological sample" or "sample" refers to serum, plasma, blood, and the like, of a mammal, including a human suspected of having nephrotic syndrome bleeding. The biological sample may be used by varying the dilution or the dilution concentration according to the diagnostic purpose before dropping onto the sample pad of the diagnostic kit.

When the biological sample has an antibody against Seoul virus, Fumala virus, or Hantan virus, the color change of the reaction line on the reaction membrane is caused by binding to the recombinant nucleocapsid protein and / or the recombinant glycoprotein antigen in the diagnostic kit. As it happens, it can be visually identified.

However, when no antibody to Seoul virus, Fumala virus or Hantan virus is formed in a biological sample, no change can be observed with the naked eye because no binding is made with the antigenic protein.

That is, the antibody against the Seoul virus, fumala virus, or hantan virus may bind to recombinant nucleocapsid protein and / or recombinant glycoprotein antigen immobilized on the reactivity on the reaction membrane.

Formation of such an antigen-antibody complex results in the detection of a reddish violet band as the indicator dye by the gold particles precipitates, thereby positively presenting an antibody against Seoulvirus, Fumalavirus or Hantan virus in the biological sample. Can respond.

The reaction membrane may comprise a suitable material, such as a nitrocellulose membrane (Millipore ^™ XA3J072100) of standard size (10 * 500 nm). The reaction membrane may be arranged in the reaction line and the control line in sequence, these reaction line and the control line may be arranged at intervals of 5mm to 10mm, preferably 6mm to 8mm.

When a reaction line containing the antigenic protein in addition to the control line is developed, it can be determined as a positive response to nephrotic syndrome bleeding fever, whereas when only the control line is developed, it can be determined as a negative response to nephrotic syndrome bleeding. Abnormality of the control line may be confirmed through abnormality of the control line.

The control line is a control composition that binds to the gold-label, eg, rabbit anti-goat IgG polyclonal antibody, goat anti-human IgG polyclonal antibody, rabbit anti-human IgM polyclonal antibody or goat anti-human IgM polyclonal antibody , Anti-chicken IgY polyclonal antibodies, and the like, and preferably include anti-chicken IgY polyclonal antibodies so as not to cause cross-reaction with the sample, but is not limited thereto. That is, the control composition may comprise an antibody protein. In addition, the control line may include the control composition at a concentration of 0.1 mg / ml to 1.0 mg / ml.

The reaction line includes antigenic proteins such as the recombinant nucleocapsid protein and recombinant glycoprotein, and these antigenic proteins may be included in the reaction line at a concentration of 0.5 mg / ml to 1.5 mg / ml.

The gold binding pad may be a suitable marker such as dried colloidal gold labeled portein A, gold-labeled anti-human IgG antibody as a marker or Gold-labeled anti-human IgM antibodies, and the like, and may be located adjacent to the sample pad.

In addition, the gold binding pad may further include a Trehalose solution to optimize the detection conditions. At this time, the concentration of the buffer solution may be 0.1% to 10%.

When the gold-labeled marker is included in the gold binding pad in an excessive amount, the antibody against the hantavirus to be analyzed may increase as a non-specific signal even in a relatively low region, resulting in false positive results. Thus, the gold-labeled marker may be adjusted to dilute the gold-colloidal stock solution so that the optical density (OD) is included at a concentration of 1-6, preferably 1-4.

The absorbent pad may be located at the opposite end of the sample pad and absorb a biological sample, for example, serum, plasma, blood, etc., moved along the reaction membrane by capillary action.

Diagnostic time using the diagnostic kit may be within 15 minutes, specifically 5 minutes to 10 minutes. Diagnostic results can be used to diagnose Hantavirus infection status in patients. For example, biological samples collected from Hantavirus-infected subjects may be positive in order to diagnose nephrotic syndrome hemorrhagic fever, whereas biological samples collected from Hantavirus-infected subjects may be negative. It can be diagnosed that hemorrhagic fever does not develop.

Indirect immunofluorescence antibody (IFA) and the specificity of the sensitivity and specificity for the hemorrhagic fever of nephrotic syndrome of the diagnostic kit comprising a recombinant nucleocapsid protein and recombinant glycoprotein expressed from the nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 3 and As a result of comparative investigation, the diagnostic kit showed very good sensitivity and specificity, and even the initial diagnosis of IgM showed significantly higher sensitivity than the indirect immunofluorescent antibody method. Therefore, the diagnostic kit can effectively diagnose nephrotic syndrome hemorrhagic fever, and in particular, the initial infection can be diagnosed more effectively than the conventional method.

Figure 3 shows the structure of the nephrotic syndrome hemorrhagic fever diagnostic kit according to an embodiment of the present invention. Referring to FIG. 3, the diagnostic kit including the test strip may simultaneously detect renal syndrome hemorrhagic fever specific IgM and IgG by including a plurality of test strips, that is, a region for detecting renal syndrome bleeding specific IgM and IgG, respectively. .

On the other hand, according to another embodiment of the present invention, a method for providing information on the diagnosis of nephrotic syndrome hemorrhagic fever using the nephrotic syndrome hemorrhagic fever diagnostic kit is provided. Specifically, the information providing method comprises the steps of collecting a biological sample from a patient suspected of nephrotic syndrome hemorrhagic fever; And detecting the antibody from the biological sample using the nephrotic syndrome hemorrhagic fever diagnostic kit.

If a biological sample, such as serum, is taken from a patient suspected of having a nephrotic syndrome hemorrhagic fever, and the collected biological sample is dropped onto a sample pad where the sample of the diagnostic kit is absorbed, the biological sample may By reaching the gold conjugation pads so that the antibody in the sample was labeled with a marker in the gold binding pad, e.g., gold-labeled protein A, gold-labeled anti-human IgG antibody. Colloids can be formed by binding to labeled anti-human IgG antibodies and gold-labeled anti-human IgM antibodies.

In this case, the biological sample to be tested may continuously move without being immobilized on the gold binding pad to reach a test line in which the antigenic protein is immobilized.

In the case of biological samples of patients with nephrotic syndrome hemorrhagic fever, an antigen-antibody reaction with the antigen may be caused. That is, antibodies of nephrotic syndrome hemorrhagic fever patients bound to specific markers in the gold binding pads can be visually identified because they bind to the antigenic protein immobilized on the reaction line and form a reddish violet band.

In contrast, markers in the remaining gold binding pads that did not react with the antibody in the biological sample reached the control line and reacted with the control composition to form a reddish violet band, which may indicate the suitability of the test. According to this method, antibodies to nephrotic syndrome hemorrhagic fever can be detected.

Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples are described for the purpose of illustrating the present invention, but the scope of the present invention is not limited thereto.

Example  One: Seoul Virus  And Fumala virus  Derived recombination Nucleocapsid  Protein manufacturing

(1) gene cloning

Nucleocapsid protein coding gene (SEQ ID NO: 357 bp; 119aa) derived from Seoul virus and nucleocapsid protein coding gene (SEQ ID NO: 2; 357 bp; 119aa) derived from fumala virus were linked to the entire nucleocapsid protein gene ( 714bp; 238aa) (Bioneer, Korea).

Nucleocapsid protein gene and pET-32a (+) vector cloned into pBHA vector were digested with restriction enzymes EcoR I and Xho I and electrophoresed in 1.2% agarose gel, followed by QIAGEN gel elution kit (QUAGEN Inc, USA). ) Was recovered using.

The band was cut to the desired size on a UV transilluminator, transferred to a microtube, 100 μl of a salt solution per 0.1 g of agarose gel pure weight was added, and left for 37 to 20 minutes to completely dissolve the gel.

The whole gel lysate was centrifuged at 400 sec intervals for 30 seconds in a tube equipped with a gene binding filter, and 500 µl each of the ethanol solution enclosed in the kit was centrifuged under the same conditions. Centrifugation was performed using a high speed centrifuge (Hanil, 4, 4 min, 13,000 rpm) to completely remove residual ethanol.

Only the ethanol-free filter was removed and transferred to an elution microtube, and 30 μl of elution buffer was added and reacted at room temperature for 5 minutes. The elution was performed using a centrifuge (Hanil, 4, 4 min, 13,000 rpm).

The final result was quantified by Nanodrop (Thermo, Nanodrop 2000), and 100ng of the cut vector and 50ng of the cut gene were mixed and reacted for 4 to 20 minutes. Thereafter, the recombinant nucleocapsid protein gene was cloned by mixing ligase buffer with a 10-fold concentration of 1 μl of ligase and allowing the reaction to stand for 4 to 16 hours.

(2) Preparation and transformation of competent cells

Escherichia coli JM109 incubated for 16 hours in LB medium was inoculated at 1/100 in LB medium and further incubated for 3 to 37 hours, and then temperature-sensitive and divided into 50 ml tubes and centrifuged (eppendorf, 3,000 rpm, 5 min). .

The supernatant was removed, and 5 ml of 0.1 M CaCl _2, which was previously stored cold, was added to each tube to gently release the precipitated Escherichia coli, which was left in ice for 20 minutes and centrifuged (eppendorf, 3,000 rpm, 5 min).

The supernatant was removed from the sterile workbench, a 15% glycerol solution of 0.1M CaCl ₂ was added 2.5 ml per tube, and the precipitate was gently released with a pipette, and then stored in an amount of 100 μl, one of which was used for transformation.

The cloning product and the qualified cells were mixed and left in ice for 20 minutes, and thermal shock was applied at 42, and the mixture was inoculated in LB agar plate medium containing Ampicillin (Ampicillin) and incubated at 37.

One colony colonies were selected, inoculated in 3 ml of ampicillin-added LB medium, and cultured for 18 hours. DNA was extracted using a GeneAll Mini-prep kit (GeneAll., Korea). Restriction enzymes used for cloning were reprocessed on the extracted DNA in the same manner and electrophoresed on 1.2% agarose gel. Electrophoresis resulted in cloning complete DNA was stored in 4.

(3) E. coli-based protein expression and purification

The cloned DNA was transformed into E. coli C43 qualified cells. C43 E. coli colonies cultured in LB agar plate medium was inoculated in LB medium containing ampicillin and incubated for 18 to 18 hours, and then inoculated at 1/100 with fresh LB medium to OD ₆₀₀ = 0.5 to 0.6. IPTG concentration was added to 0.5 mM, incubated for 3 hours, and centrifuged (Hanil, 4, 6,000 rpm). The supernatant was discarded, and the precipitate was suspended in 1X binding buffer (500 mM NaCl, 20 mM Tris-HCl (pH 8.0), 5 mM imidazole), and then crushed 300 times at 10 second intervals by an ultrasonic mill. The supernatant was centrifuged to separate the soluble fraction, and the precipitate was separated into the insoluble fraction.

Fill the column with 5 ml of His-bind resin (Novagen), and then use distilled water, 1X charge buffer (100 mM NiSO4), 1 X binding buffer (500 mM NaCl, 20 mM Tris-HCl (pH 8.0), 5 mM imidazole) Was prepared.

Antigen protein lysate fractions were washed with 1 × washing buffer (500 mM NaCl, 20 mM Tris-HCl (pH 8.0), 40 mM imidazole) and passed through elution buffer (500 mM imidazole, 0.5 M NaCl, 20 mM Tris-Cl pH 8.0). 1 ml each. On the other hand, the insoluble fraction was passed through another column and washed with 1X washing buffer (6M Urea, 500mM NaCl, 20mM Tris-HCl (pH 8.0), 40mM imidazole) and Elution buffer (6M Urea, 500mM imidazole). , 0.5M NaCl, 20mM Tris-Cl pH 8.0) to recover the protein by 1ml.

  Bradford reagent was added to each fraction to recover only those fractions that showed a color change to blue. Dialysis was performed using dialysis buffer (10 mM Sodium phosphate or 10 mM potassium phosphate), and then quantified by BCA and stored at 4. Archived antigens were confirmed for expression using SDS-PAGE (FIG. 4). Based on the expression pattern and yield, for the nucleocapsid protein, only the soluble fraction was used as the injectable antigen.

(4) Insect Cell Based Protein Expression and Purification

The cloned DNA was transformed into E. coli DH10bac. Transformation method was carried out in the same manner as described above. Since the Bacmid gene and the pFast Bac HT A vector in E. coli DH10bac have a site where the internal gene positions can be changed, the genetic position changes occur simultaneously with transformation.

When cultured in LB agar plate medium, it was confirmed that the gene position change was formed by white X-gal and IPTG treatment followed by 37 to 18 hours of incubation. White colonies were inoculated into 3 ml of LB medium containing ampicillin and kanamycin, and cultured for 18 hours, followed by Midi-prep (QIAGENE Inc., USA) using a Midi-prep kit to obtain DNA.

The secured DNA was transduced into insect-derived Sf21 cells. At this time, the Cellfectin® II reagent of Invitrogen (Thermo, USA) was used for transduction. After 2 μg of DNA was reacted with 8 μl of reagent at room temperature for 15 minutes, the reaction was placed in an Sf21 cell culture flask. 72 hours later, the supernatant was recovered and used as a virus. The recovered virus was again infected with Sf21 cells and used for antigen expression.

1/10 of the cell culture volume was infected in the cell culture medium and incubated for 27 to 3.5 days, and then the antigen-expressing cells were recovered and centrifuged (Eppendorf, 4, 1500 rpm) to discard the supernatant and precipitate. After floating with only 1 X binding buffer (500mM NaCl, 20mM Tris-HCl (pH 8.0), 5mM imidazole) it was crushed 36 times at 10 seconds intervals by an ultrasonic grinder. The supernatant was centrifuged to separate the soluble fraction, and the precipitate was separated into the insoluble fraction.

Fill the column with 5 ml of His-bind resin (Novagen), and then use the column with distilled water, 1X charge buffer (100 mM NiSO4), 1X binding buffer (500 mM NaCl, 20 mM Tris-HCl (pH 8.0), 5 mM imidazole). Ready.

Antigen proteins were passed through the column and washed with 1 × washing buffer (6M Urea, 500 mM NaCl, 20 mM Tris-HCl, pH 8.0), 40 mM imidazole. Elution buffer (6M Urea, 500mM imidazole, 0.5M NaCl, 20mM Tris-Cl pH 8.0) was passed through 1ml of protein was recovered. By adding bradford reagent to each fraction, only the fraction showing the color change to blue was recovered. Dialysis was performed using dialysis buffer (10 mM Sodium phosphate or 10 mM potassium phosphate), and then quantified by BCA and stored at 4. Archived antigens were confirmed for expression using SDS-PAGE (FIG. 5).

Example  2: Hantan Virus  Derived Recombinant Glycoprotein

(1) gene cloning

Sites having antigenicity were selected based on the glycoprotein gene nucleotide sequence derived from the Hantan virus, and recombined to prepare a recombinant gene consisting of the nucleotide sequence of SEQ ID NO: 3. Specifically, the total of 330aa (990bp) by combining agarovirus glycoprotein 414aa (1,242bp) to 506aa (1,518bp) 94aa (282bp) and 773aa (2,319bp) to 1,009aa (3,027bp) Recombinant glycoprotein gene consisting of) was synthesized (Bioneer, Korea).

The glycoprotein gene synthesized in the pBHA vector and the pET-22b (+) vector were completely cleaved with restriction enzymes Nde I and Xho I and electrophoresed in 1.2% agarose gel, followed by QIAGEN gel elution kit (QUAGEN Inc, USA). Recovered using.

The band was cut to the desired size on a UV transilluminator, transferred to a microtube, 100 μl of a salt solution per 0.1 g of agarose gel pure weight was added, and left for 37 to 20 minutes to completely dissolve the gel.

The whole gel lysate was centrifuged at 400 sec intervals for 30 seconds in a tube equipped with a gene binding filter, and 500 µl each of the ethanol solution enclosed in the kit was centrifuged under the same conditions. Centrifugation was performed using a high speed centrifuge (Hanil, 4, 4 min, 13,000 rpm) to completely remove residual ethanol.

Only the ethanol-free filter was removed and transferred to an elution microtube, and 30 μl of elution buffer was added and reacted at room temperature for 5 minutes. The elution was performed using a centrifuge (Hanil, 4, 4 min, 13,000 rpm).

The final result was quantified by Nanodrop (Thermo, Nanodrop 2000), and 100ng of the cut vector and 50ng of the cut gene were mixed and reacted for 4 to 20 minutes. Thereafter, the recombinant nucleocapsid protein gene was cloned by mixing ligase buffer with a 10-fold concentration of 1 μl of ligase and allowing the reaction to stand for 4 to 16 hours.

(2) Preparation and transformation of competent cells

E. coli JM109 incubated for 16 hours in LB medium was inoculated at 1/100 in LB medium and further incubated for 3 to 37 hours, and then, the mixture was centrifuged (eppendorf, 3,000 rpm, 5 min) in 4 mL and 50 mL tubes. .

The supernatant was removed, and 5 ml of 0.1 M CaCl _2, which was previously stored cold, was added to each tube to gently release the precipitated Escherichia coli, which was left in ice for 20 minutes and centrifuged (eppendorf, 3,000 rpm, 5 min).

The supernatant was removed from the sterile workbench, a 15% glycerol solution of 0.1M CaCl ₂ was added 2.5 ml per tube, and the precipitate was gently released with a pipette, and then stored in an amount of 100 μl, one of which was used for transformation.

The cloning product and the qualified cells were mixed and left in ice for 20 minutes, and thermal shock was applied at 42, and the mixture was inoculated in LB agar plate medium containing Ampicillin (Ampicillin) and incubated at 37.

One colony colonies were selected, inoculated in 3 ml of ampicillin-added LB medium, and cultured for 18 hours. DNA was extracted using a GeneAll Mini-prep kit (GeneAll., Korea). Restriction enzymes used for cloning were reprocessed on the extracted DNA in the same manner and electrophoresed on 1.2% agarose gel. Electrophoresis resulted in cloning complete DNA was stored in 4.

(3) E. coli-based protein expression and purification

The cloned DNA was transformed into E. coli C43 qualified cells. C43 E. coli colonies cultured in LB agar plate medium was inoculated in LB medium containing ampicillin and incubated for 18 to 18 hours, and then inoculated at 1/100 with fresh LB medium to OD ₆₀₀ = 0.5 to 0.6. IPTG concentration was added to 0.5 mM, incubated for 3 hours, and centrifuged (Hanil, 4, 6,000 rpm). The supernatant was discarded, and the precipitate was suspended in 1X binding buffer (500 mM NaCl, 20 mM Tris-HCl (pH 8.0), 5 mM imidazole), and then crushed 300 times at 10 second intervals by an ultrasonic mill. The supernatant was centrifuged to separate the soluble fraction, and the precipitate was separated into the insoluble fraction.

Fill the column with 5 ml of His-bind resin (Novagen), and then use the column with distilled water, 1X charge buffer (100 mM NiSO4), 1X binding buffer (500 mM NaCl, 20 mM Tris-HCl (pH 8.0), 5 mM imidazole). Ready.

Antigen protein lysate fractions were washed with 1 × washing buffer (500 mM NaCl, 20 mM Tris-HCl (pH 8.0), 40 mM imidazole) and passed through elution buffer (500 mM imidazole, 0.5 M NaCl, 20 mM Tris-Cl pH 8.0). 1 ml each. On the other hand, the insoluble fraction was passed through another column and washed with 1X washing buffer (6M Urea, 500mM NaCl, 20mM Tris-HCl (pH 8.0), 40mM imidazole) and Elution buffer (6M Urea, 500mM imidazole). , 0.5M NaCl, 20mM Tris-Cl pH 8.0) to recover the protein by 1ml.

Bradford reagent was added to each fraction to recover only those fractions that showed a color change to blue. After dialysis of the antigen using dialysis buffer (10mM sodium phosphate or 10mM potassium phosphate) was quantified by BCA method and stored in 4. Archived antigens were confirmed for expression using SDS-PAGE (FIG. 6). Based on the expression pattern and the yield, the glycoprotein was determined to use only the undissolved fraction as the topical antigen.

(4) Insect Cell Based Protein Expression and Purification

The cloned DNA was transformed into E. coli DH10bac. Transformation method was carried out in the same manner as described above. Since the Bacmid gene and the pFast Bac HT A vector in E. coli DH10bac have a site where the internal gene positions can be changed, the genetic position changes occur simultaneously with transformation.

When cultured in LB agar plate medium, it was confirmed that the gene position change was formed by white X-gal and IPTG treatment followed by 37 to 18 hours of incubation. White colonies were inoculated into 3 ml of LB medium containing ampicillin and kanamycin, and cultured for 18 hours, followed by Midi-prep (QIAGENE Inc., USA) using a Midi-prep kit to obtain DNA.

The secured DNA was transduced into insect-derived Sf21 cells. At this time, the Cellfectin® II reagent of Invitrogen (Thermo, USA) was used for transduction. After 2 μg of DNA and 8 μl of the reagent were reacted at room temperature for 15 minutes, the reaction was placed in an Sf21 cell culture flask. 72 hours later, the supernatant was recovered and used as a virus. The recovered virus was again infected with Sf21 cells and used for antigen expression.

1/10 of the cell culture volume was infected in the cell culture medium and incubated for 27 to 3.5 days, and then the antigen-expressing cells were recovered and centrifuged (Eppendorf, 4, 1500 rpm) to discard the supernatant and precipitate. Only 1X binding buffer (500mM NaCl, 20mM Tris-HCl (pH 8.0), 5mM imidazole) was suspended and then crushed 36 times at 10 second intervals by an ultrasonic grinder. The supernatant was centrifuged to separate the soluble fraction, and the precipitate was separated into the insoluble fraction.

Fill the column with 5 ml of His-bind resin (Novagen), and then use the column with distilled water, 1X charge buffer (100 mM NiSO4), 1X binding buffer (500 mM NaCl, 20 mM Tris-HCl (pH 8.0), 5 mM imidazole). Ready.

Antigen proteins were passed through the column and washed with 1 × washing buffer (6M Urea, 500 mM NaCl, 20 mM Tris-HCl, pH 8.0), 40 mM imidazole. Elution buffer (6M Urea, 500mM imidazole, 0.5M NaCl, 20mM Tris-Cl pH 8.0) was passed through 1ml of protein was recovered. By adding bradford reagent to each fraction, only the fraction showing the color change to blue was recovered. Dialysis was performed using dialysis buffer (10 mM Sodium phosphate or 10 mM potassium phosphate), and then quantified by BCA and stored at 4. Archived antigens were confirmed for expression using SDS-PAGE (not shown).

Example  3: Kit  making

In order to confirm the efficacy as a diagnostic agent for nephrotic syndrome hemorrhagic fever for Seoul / Phumala virus nucleocapsid protein and Hantan virus glycoprotein prepared according to Examples 1 and 2 above, a diagnostic using a dispenser (KINEMATIC Automation) Test strip samples were made (FIG. 2).

Specifically, the test strip was composed of a reaction film, a sample pad, an absorption pad, and a gold bonding pad (glass fiber). The reaction membrane used was manufactured by Millipore, and plastic was attached to one surface to prevent damage to the membrane. Three proteins were dipped into the reaction membrane.

One is a protein mixed with antigenic proteins (Seoul / Pumala virus nucleocapsid protein, Hantan virus glycoprotein) expressed in Escherichia coli or insect cells to detect antibodies against nephrotic syndrome hemorrhagic fever virus present in the serum of the patient And dropwise onto the test line.

The other one was spotted on a control line to determine the normal response of the color system, and the other one contained in human serum to remove antibodies that caused antigen-antibody reactions with proteins from E. coli or insect cells. To this end, E. coli or insect cell culture extracts that do not express antigen proteins were added to the pretest line.

Using a dispenser, an appropriate amount of protein was injected into the entire reaction line, the reaction line, and the control line, and then dried at room temperature for 48 hours. 5% trehalose and an appropriate amount of colloidal gold conjugate were absorbed into the glass fiber and dried at room temperature for 48 hours.

The sample pad and absorbent pad are cut to the desired size, and the sample pad is absorbed in a solution made of 1% of Tween 20 in Tris solution or sodium borate solution, and then completely dried at room temperature for 48 hours to prepare serum and plasma. It was easy to absorb.

A gold bonding pad (glass fiber) sprayed with a colloidal gold conjugate was superimposed on the bottom of the reaction film, and a sample pad was superimposed on the bottom of the reaction film. Absorbing pads were overlapped and attached to the reaction membrane.

Two types of final diagnostic strip samples were produced depending on the application. One type was designed to search only IgG for serum nephrotic syndrome hemorrhagic fever antigen protein, and the other type was designed to search only IgM.

Diagnostic strips searching for IgG can detect patients after midterm, and diagnostic strips searching for IgM can be effectively used for initial patient search. In the present invention, two strips were put in one plastic case and manufactured to simultaneously diagnose them (FIG. 3).

Example  4: Kit Performance test

Efficacy tests were performed on patient serum and normal serum using the kit prepared according to Example 3. Specifically, according to the method as shown in Figure 7, the sample was added, the sample diluent was added and the result was read after 15 minutes. At this time, as a control, a kit to which the proteins of Examples 1 and 2 were not applied was used.

As a result of reading, the kit of Example 3 showed relatively strong color development of IgM and IgG. In addition, it was possible to read positive for the negatively read sample in the control group was confirmed a high sensitivity. Accordingly, it can be seen that the kit of Example 3 can quickly, accurately and easily diagnose nephrotic syndrome bleeding fever (FIG. 8).

In addition, as a result of cross-testing with serum of Tsutsugamushi disease patient, which is another acute recessive disease, color reaction appeared in Tsutsugamushi patient sample in the control kit, whereas the cross reaction was confirmed, but the kit of Example 3 was confirmed that no cross-reaction occurred. It was confirmed that the detection specificity was further improved compared to the conventional kit (FIG. 9).

Although the embodiments have been described by the limited embodiments and the drawings as described above, various modifications and variations are possible to those skilled in the art from the above description. For example, the techniques described may be performed in a different order than the described method, and / or the components described may be combined or combined in a different form than the described method, or replaced or substituted by other components or equivalents. Appropriate results can be achieved.

Therefore, other implementations, other embodiments, and equivalents to the claims are within the scope of the following claims.

<110> IMMUNEMED INC. <120> COMPOSITION FOR DIAGNOSING HEMORRHAGIC FEVER WITH RENAL SYNDROME          INCLUDING RECOMBINENT NUCLEOCAPSID PROTEIN DERIVED FROM SEOUL          VIRUS AND PUUMALA VIRUS AND GLYCOPROTEIN DERIVED FROM HANTAAN          VIRUS AND KIT INCLUDING THE SAME <130> APC-2016-0441 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 357 <212> DNA <213> Seoul virus <400> 1 atggcaacta tggaagaaat ccagagagaa atcagtgctc acgaggggca gcttgtgata 60 gcacgccaga aggtcaagga tgcagaaaaa cagtatgaga aggatcctga tgatttaaat 120 aagagggcac tgcatgatcg ggagagtgtc gcagcttcaa tacaatcaaa aattgatgaa 180 ttgaagcgcc aacttgccga caggattgca gcagggaaga acatcgggca agaccgggat 240 cctacagggg tagagccggg cgatcatctc aaggaaagat cagcattaag ctatgggaat 300 acactggacc tgaatagtct tgacattgat gaacctacag gacagacagc tgattgg 357 <210> 2 <211> 360 <212> DNA <213> Puumala virus <400> 2 atgagtgact taaaagatat acaggaggac atagcccgcc atgagcaaca acttgttgtt 60 gccagacaaa agctcaggga tgcagaaaag gcagtggaga tggacccaga tgacgttaac 120 aaaaacacac tgcaagcaag gcaacaaaca gtgtcagcat tggaggataa aattgcagac 180 ttcaagagaa gaatggcaga cattgtgtcc cggaaaaaga tggatactaa gcctgctgac 240 ccgactggga ttgagcctga tgaccacctc aaggagagat caagtcttcg atatggaaat 300 gtccttgatg tgaatgccat tgacattgag gaaccaagtg gtcagacagc agactggtga 360                                                                          360 <210> 3 <211> 993 <212> DNA <213> Hantaan virus <400> 3 atgcggttaa cagaacagca agtgaatttt gtgtgtcagc gagtggacat ggacattgtt 60 gtgtactgca acgggcagag gaaagtaata ttaacaaaaa ctctagttat tggacagtgt 120 atatatacta taacaagctt attctcatta ctacctggag tagcacattc tattgctgtt 180 gaattgtgtg tacctgggtt ccatggttgg gccacagctg ctctgcttgt tacattctgt 240 ttcggatggg ttcttatacc agcaattaca tttatcatac tagtgggcac aggctgtaca 300 gcatgtggtt tatacctaga tcaactgaaa ccagttggta gtgcttataa aattatcaca 360 ataaggtaca gcaggagagt ctgtgttcag tttggggagg aaaacctttg taagataata 420 gacatgaatg attgttttgt atctaggcat gttaaggtct gcataattgg tacagtatct 480 aaattctctc agggtgatac cttattgttt tttggaccgc ttgaaggtgg tggtctaata 540 tttaaacact ggtgtacatc cacatgtcaa tttggtgacc caggagatat catgagtcca 600 agagacaaag gttttttatg ccctgagttt ccaggtagtt tcaggaagaa atgcaacttt 660 gctactaccc ctatttgtga gtatgatgga aatatggtct caggttacaa gaaagtgatg 720 gcgacaattg attccttcca atcttttaat acaagcacta tgcacttcac tgatgaaagg 780 atagagtgga aagaccctga tggaatgcta agggaccata taaacatttt agtaacgaag 840 gacattgact ttgataacct tggtgaaaat ccttgcaaaa ttggcctaca aacatcttct 900 attgaggggg cctggggttc tggtgtgggg ttcacattaa catgtctggt atcactaaca 960 gaatgtccta cctttttgac ctcaataaag tag 993

Claims (8)

A recombinant protein consisting of a Seoul virus encoded by the nucleotide sequence of SEQ ID NO: 1 and a nucleocapsid protein derived from Pumala virus (Puumala virus) encoded by the nucleotide sequence of SEQ ID NO: 2; And
Recombinant glycoprotein derived from Hantan virus (Hantaan virus) encoded by the nucleotide sequence of SEQ ID NO: 3; nephrotic syndrome hemorrhagic fever diagnostic composition comprising a.
The method of claim 1,
The composition is to detect nephrotic syndrome hemorrhagic fever specific IgM and IgG, nephrotic syndrome hemorrhagic fever diagnostic composition.
delete delete The method of claim 1,
The recombinant glycoprotein derived from the hantan virus comprises recombination of antigenic determinants in the glycoprotein of the hantan virus, nephrotic syndrome hemorrhagic fever diagnostic composition.
delete A sample pad on which a sample is absorbed;
Gold conjugation pads that bind to antibodies in the sample;
A test membrane treated with a test line comprising the composition of any one of claims 1, 2 and 5 and a control line comprising a control composition; And
Nephrotic syndrome hemorrhagic fever diagnostic kit comprising a test strip (absorption pad) to absorb the remaining amount of the sample (absorption pad).
A method of providing information for diagnosing nephrotic syndrome hemorrhagic fever using the diagnostic kit of claim 7.

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