KR101995774B1 - A fibronectin Extra Domain B protein scaffold specifically binding to dengue virus - Google Patents

Abstract

The present invention relates to a fibronectin EDB protein scaffold specifically binding to dengue virus. More specifically, the present invention relates to a nucleic acid molecule for coating a fibronectin EDB protein scaffold that specifically binds to the dengue virus NS1 domain, a recombinant vector comprising the nucleic acid molecule, a fibronectin EDB protein scaffold expressed from the recombinant vector and a dengue virus diagnostic composition comprising the same. The fibronectin EDB protein scaffold according to the present invention specifically binds to the NS1 domain, which is a nonstructural glycoprotein of dengue virus, and thus can easily diagnose infection with dengue virus.

Description

A fibronectin Extra Domain B protein scaffold specifically binding to dengue virus that specifically binds to dengue virus.

The present invention relates to a fibronectin EDB protein scaffold specifically binding to a dengue fever virus, and more particularly to a nucleic acid molecule for coating a fibronectin EDB protein scaffold specifically binding to a dengue virus NS1 domain, A fibronectin EDB protein scaffold expressed from the recombinant vector, and a composition for diagnosing dengue virus comprising the recombinant vector.

Dengue fever is an acute fever caused by dengue virus and is a disease mediated by mosquitoes. Dengue virus is a spherical virus with a genetic material of positive single strand RNA belonging to Flaviviridae of the taxonomic Flaviviridae, West Nile virus, , Japanese encephalitis virus, tickborne encephalitis virus and yellow fever virus are the same species. Most are also known as arthropod-borne viruses because they are transmitted by arthropods (mosquitoes or ticks).

Dengue viruses have four serotypes (DENV1, DENV2, DENV3, DENV4), and these four serotypes are known to have about 60-90% sequence homology. The first infection by one serotype has immunity to the serotype, but thereafter the second infection can occur by one serotype of the remaining three. When the second infection occurs, the antigen-antibody Complement activation by conjugates can lead to severe dengue fever, such as dengue hemorrhagic fever or dengue shock syndrome, leading to more severe signs than primary disease.

Dengue virus is a small, cortical structure of about 10.6 Kbp. It has a strand of positive-sense RNA genome consisting of about 11,000 base pairs. This RNA genome contains three structural proteins (capsid protein, membrane protein, envelope protein) And seven nonstructural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5).

The role of these nonstructural proteins is to perform important functions, such as influencing the biological properties such as receptor binding, induction of red blood cell aggregation and neutralizing antibody (Ab) and defense immunity, viral replication and assembly processes essential for virus survival It is known.

In particular, NS1, an unstructured glycoprotein of dengue virus, is a structure containing 352 amino acid residues in the basic polypeptide of ~ 40 KDa and having 12 invariant cysteine residues and 2 N-glycosylation sites. In some studies, intracellular NS1 glycoproteins have been directly or indirectly involved in viral replication, affecting the formation of immunogenic complexes, leading to complement-mediated pathways and antibody-dependent pathways cell cytotoxicity) and is known to cause more severe complications.

Unlike the IgM and IgG antibodies detected after at least 3 to 5 days after onset, NS1 protein binds to the cell surface during viral replication and is released into the circulation 1 day after the onset of the early symptoms, The serum is rapidly increased in concentration and is detected at a high concentration in blood until about 9 days. Therefore, early diagnosis is important for dengue fever infection.

To date, known methods for diagnosing dengue fever include direct detection of viruses in the serum or tissue of a patient, polymerase chain reaction (PCR) for viral nucleic acid detection, or host antibody (IgM, IgG) . In addition, antigen detection methods such as virus culture, immunoassay, fluorescence assay, or mass spectrometry have been used.

However, the methods used are limited in their use due to the nature of the area in which they are developed, as most equipment for diagnosis is expensive and requires skilled personnel. In some sera test, it is difficult to distinguish each serotype due to cross reaction. In case of similar virus, there is a disadvantage that reliability of diagnosis result is low due to duplication detection. In addition, it is known that most of the diagnostic methods are less susceptible to the diagnosis of the second infection than the first infection. In particular, serologic methods may not be able to diagnose dengue virus infection at an early stage of the disease.

Therefore, there is a need to develop a diagnostic test method that can efficiently detect dengue viruses for the diagnosis and treatment of dengue fever virus infection.

An antibody is an immune protein that binds to an antibody from the outside to interfere with the action of an antigen or to remove an antigen. The antibody can bind to a target substance with high affinity and selectivity, And has been widely recognized as a breakthrough therapeutic agent. Antibodies are composed of two immunoglobulins, heavy chain and light chain, which consist of a constant region and a variable region. Several antibody substitution proteins are recombinant proteins, such as antibodies, that can be made to have constant regions and variable regions. A small portion of a small, stable protein is replaced with a random sequence of amino acids to create a library, which is then screened for target substances, And a material with good specificity.

Fibronectin (FN) is a glycoprotein, a major component of the extracellular matrix (ECM), and is known to be an important mediator involved in cell differentiation, adhesion, migration, domain and apoptosis . Among them, Human Fibronectin Type III (FN3) has a β-sandwich structure similar to that of immunoglobulin, and is also applied to the production of antibody replacement proteins. Fibronectin EDB (Extra Domain B) has a structure similar to that of FN3, and has been reported to be expressed in blood vessels and extracellular matrix particularly in cancer, and thus has attracted attention as a target in cancer-specific detection and treatment.

A scaffold is one of the structural features of a protein that has the property of being able to bind to several proteins with different functions.

Accordingly, the present inventors have made intensive efforts to overcome the problems of the prior art. As a result, the inventors of the present invention found that the fibronectin EDB protein scaffold specifically binding to the dengue virus NS1 domain specifically binds to the dengue virus NS1 domain, It was confirmed that the infection can be effectively diagnosed, and the present invention was completed.

KR 10-1763745 B KR 10-1837856 B

Accordingly, it is an object of the present invention to provide a fibronectin EDB protein scaffold capable of specifically diagnosing dengue virus infection by binding specifically to the dengue virus NS1 domain.

The present invention also aims to provide nucleic acid molecules that coat the fibronectin EDB protein scaffold.

The present invention also aims to provide a vector comprising nucleic acid molecules for expressing the fibronectin EDB protein scaffold.

It is another object of the present invention to provide a composition for diagnosing dengue virus comprising a fibronectin EDB protein scaffold specifically binding to the dengue virus NS1 domain.

According to one aspect of the present invention, the present invention provides a method for producing a polypeptide comprising at least one amino acid sequence selected from the group consisting of amino acid sequences represented by the following general formulas 1 to 3:

Provides a fibronectin EDB protein scaffold that specifically binds dengue virus.

[Formula 1] X ¹ X ² X ³ X ⁴ X ⁵ X ⁶ X ⁷

X ¹ is a valine (Val) or arginine (Arg) residue,

X ² is a glutamine (Gln) or methionine (Met) residue,

X ³ is a cysteine (Cys), valine (Val) or threonine (Thr) residue,

X ⁴ is glutamine (Gln), methionine (Met) or cysteine (Cys) residue,

X ⁵ is a cysteine (Cys), arginine (Arg) or leucine (Leu) residue,

X ⁶ is an alanine (Ala), methionine (Met) or leucine (Leu) residue,

X ⁷ stands for glycine (Gly) or leucine (Leu).

[Formula 2] X ⁸ X ⁹ X ¹⁰ X ¹¹

X ⁸ is proline (Pro), histidine (His) or aspartic acid (Asp) residue,

X ⁹ is an alanine (Ala) or proline (Pro) residue,

X ¹⁰ is a cysteine (Cys), isoleucine (Ile) or tryptophan (Trp) residue,

X ¹¹ is valine (Val), arginine (Arg) or threonine (Thr) residue.

[Formula 3] X ¹² X ¹³ X ¹⁴ X ¹⁵ X ¹⁶ X ¹⁷

X ¹² is a histidine (His), proline (Pro) or threonine (Thr) residue,

X ¹³ is an arginine (Arg), tryptophan (Trp) or glutamine (Gln) residue,

X ¹⁴ is isoleucine (Ile), glutamine (Gln) or lysine (Lys) residue,

X ¹⁵ is a histidine (His) or arginine (Arg) residue,

X ¹⁶ is proline (Pro), histidine (His) or valine (Val) residue,

X ¹⁷ is an asparagine (Asn), arginine (Arg) or lysine (Lys) residue.

In the fibronectin EDB protein scaffold of the present invention, the fibronectin EDB protein scaffold is characterized by being represented by the following general formula (4).

[Formula 4]

EVPQLTDLSFVDITDSSIGLRWX ¹ X ² X ³ X ⁴ X ⁵ X ⁶ X ⁷ IIGYRITVVAAGEGIPIFEDFVDX ⁸ X ⁹ X ¹⁰ X ¹¹ YYTVTGLEPGIDYDISVITLIX ¹² X ¹³ X ¹⁴ X ¹⁵ X ¹⁶ X ¹⁷ PTTLTQQT

X ¹ is a valine (Val) or arginine (Arg) residue,

X ² is a glutamine (Gln) or methionine (Met) residue,

X ³ is a cysteine (Cys), valine (Val) or threonine (Thr) residue,

X ⁴ is glutamine (Gln), methionine (Met) or cysteine (Cys) residue,

X ⁵ is a cysteine (Cys), arginine (Arg) or leucine (Leu) residue,

X ⁶ is an alanine (Ala), methionine (Met) or leucine (Leu) residue,

X ⁷ is a glycine (GIy) or leucine (Leu) residue,

X ⁸ is proline (Pro), histidine (His) or aspartic acid (Asp) residue,

X ⁹ is an alanine (Ala) or proline (Pro) residue,

X ¹⁰ is a cysteine (Cys), isoleucine (Ile) or tryptophan (Trp) residue,

X ¹¹ is a valine (Val), arginine (Arg) or threonine (Thr) residue,

X ¹² is a histidine (His), proline (Pro) or threonine (Thr) residue,

X ¹³ is an arginine (Arg), tryptophan (Trp) or glutamine (Gln) residue,

X ¹⁴ is isoleucine (Ile), glutamine (Gln) or lysine (Lys) residue,

X ¹⁵ is a histidine (His) or arginine (Arg) residue,

X ¹⁶ is proline (Pro), histidine (His) or valine (Val) residue,

X ¹⁷ is asparagine (Asn), arginine (Arg) or lysine (Lys) residue,

The other characters are single-character notation of amino acids.

In the fibronectin EDB protein scaffold of the present invention, the fibronectin EDB protein scaffold represented by [Formula 4] includes at least one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOS: .

[SEQ ID NO: 1]

EVPQLTDLSFVDITDSSIGLRWVQCQCAGIIGYRITVVAAGEGIPIFEDFVDPACVYYTVTGLEPGIDYDISVITLIHRIHPNPTTLTQQT

[SEQ ID NO: 2]

≪

[SEQ ID NO: 3]

EVPQLTDLSFVDITDSSIGLRWRMTCLCLIIGYRITVVAAGEGIPIFEDFVDDPWTYYTVTGLEPGIDYDISVITLITQKHVKPTTLTQQT

In the fibronectin EDB protein scaffold of the present invention, the fibronectin EDB protein scaffold is characterized by specific binding to the dengue virus NS1 domain.

The term "domain" of the present invention refers to a region of a protein that can be folded into a stable three-dimensional structure, often irrelevant to the rest of the protein, and can be given specific functions.

NS1 protein, known to be directly or indirectly involved in dengue virus replication, is detected in high concentrations in the blood of infected persons. Various methods for diagnosing dengue fever (serum test, virus culture, etc.) are known, but conventional diagnostic methods are not easy to diagnose dengue fever virus and have low reliability.

Accordingly, the present inventors have invented a fibronectin EDB protein scaffold specifically binding to the dengue virus NS1 domain for easy and reliable diagnosis of dengue virus.

According to an embodiment of the present invention, the phage displaying the EDB library and the NS1 antigen were subjected to bio-panning, and the number of washing was varied during the fourth bio-panning. In this case, the number of phage used at the beginning of each bio-panning is input, and after the washing process, the number of colonies produced by Escherichia coli after being separated by glycine solution is calculated as the number of output phages, I made it.

As a result of the bio-panning as described above, it was confirmed that the ratio of the output phage / input phage increased according to the washing procedure (see Table 1 and FIG. 1). These results indicate that the antibodies capable of binding to the NS1 antigen were selected and sequenced.

According to an embodiment of the present invention, the plasmid DNA of the clones capable of binding to the NS1 domain was isolated and analyzed for the base sequence. As a result, the finally selected EDB amino acid sequence for Dengue virus NS1 diagnosis was identified as SEQ ID NOS: 1 to 3.

[SEQ ID NO: 1]

EVPQLTDLSFVDITDSSIGLRWVQCQCAGIIGYRITVVAAGEGIPIFEDFVDPACVYYTVTGLEPGIDYDISVITLIHRIHPNPTTLTQQT

[SEQ ID NO: 2]

≪

[SEQ ID NO: 3]

EVPQLTDLSFVDITDSSIGLRWRMTCLCLIIGYRITVVAAGEGIPIFEDFVDDPWTYYTVTGLEPGIDYDISVITLITQKHVKPTTLTQQT

According to another aspect of the present invention, there is provided a nucleic acid molecule for coating a fibronectin EDB protein scaffold that specifically binds to a dengue virus NS1 domain comprising an amino acid sequence represented by the following formula [4].

[Formula 4]

EVPQLTDLSFVDITDSSIGLRWX ¹ X ² X ³ X ⁴ X ⁵ X ⁶ X ⁷ IIGYRITVVAAGEGIPIFEDFVDX ⁸ X ⁹ X ¹⁰ X ¹¹ YYTVTGLEPGIDYDISVITLIX ¹² X ¹³ X ¹⁴ X ¹⁵ X ¹⁶ X ¹⁷ PTTLTQQT

X ¹ is a valine (Val) or arginine (Arg) residue,

X ² is a glutamine (Gln) or methionine (Met) residue,

X ³ is a cysteine (Cys), valine (Val) or threonine (Thr) residue,

X ⁴ is glutamine (Gln), methionine (Met) or cysteine (Cys) residue,

X ⁵ is a cysteine (Cys), arginine (Arg) or leucine (Leu) residue,

X ⁶ is an alanine (Ala), methionine (Met) or leucine (Leu) residue,

X ⁷ is a glycine (GIy) or leucine (Leu) residue,

X ⁸ is proline (Pro), histidine (His) or aspartic acid (Asp) residue,

X ⁹ is an alanine (Ala) or proline (Pro) residue,

X ¹⁰ is a cysteine (Cys), isoleucine (Ile) or tryptophan (Trp) residue,

X ¹¹ is a valine (Val), arginine (Arg) or threonine (Thr) residue,

X ¹² is a histidine (His), proline (Pro) or threonine (Thr) residue,

X ¹³ is an arginine (Arg), tryptophan (Trp) or glutamine (Gln) residue,

X ¹⁴ is isoleucine (Ile), glutamine (Gln) or lysine (Lys) residue,

X ¹⁵ is a histidine (His) or arginine (Arg) residue,

X ¹⁶ is proline (Pro), histidine (His) or valine (Val) residue,

X ¹⁷ is asparagine (Asn), arginine (Arg) or lysine (Lys) residue,

The other characters are single-character notation of amino acids.

The term "nucleic acid" of the present invention refers to any two or more covalently bonded nucleotides, or nucleotide analogs or derivatives.

According to another aspect of the present invention, there is provided a vector for expressing a fibronectin EDB protein scaffold that specifically binds to a dengue virus NS1 domain comprising the nucleic acid molecule.

The term "vector" of the present invention refers to a vehicle that is introduced into a host cell to express the desired expression product. As is known to those skilled in the art, such vectors may be readily selected from the group consisting of plasmids, phage, viruses and retroviruses. Generally, vectors compatible with the present invention will include selectable markers, appropriate restriction sites for promoting cloning of the desired nucleic acid, and the ability to enter and / or replicate in eukaryotic or prokaryotic cells.

According to another aspect of the present invention, the present invention provides a fibronectin EDB protein scaffold comprising an amino acid sequence represented by the following formula 4, which can be used for diagnosis of diseases by binding to a specific target related to the disease, The present invention provides a composition capable of specifically diagnosing dengue virus by binding specifically to the dengue virus NS1 domain.

[Formula 4]

EVPQLTDLSFVDITDSSIGLRWX ¹ X ² X ³ X ⁴ X ⁵ X ⁶ X ⁷ IIGYRITVVAAGEGIPIFEDFVDX ⁸ X ⁹ X ¹⁰ X ¹¹ YYTVTGLEPGIDYDISVITLIX ¹² X ¹³ X ¹⁴ X ¹⁵ X ¹⁶ X ¹⁷ PTTLTQQT

X ¹ is a valine (Val) or arginine (Arg) residue,

X ² is a glutamine (Gln) or methionine (Met) residue,

X ³ is a cysteine (Cys), valine (Val) or threonine (Thr) residue,

X ⁴ is glutamine (Gln), methionine (Met) or cysteine (Cys) residue,

X ⁵ is a cysteine (Cys), arginine (Arg) or leucine (Leu) residue,

X ⁶ is an alanine (Ala), methionine (Met) or leucine (Leu) residue,

X ⁷ is a glycine (GIy) or leucine (Leu) residue,

X ⁸ is proline (Pro), histidine (His) or aspartic acid (Asp) residue,

X ⁹ is an alanine (Ala) or proline (Pro) residue,

X ¹⁰ is a cysteine (Cys), isoleucine (Ile) or tryptophan (Trp) residue,

X ¹¹ is a valine (Val), arginine (Arg) or threonine (Thr) residue,

X ¹² is a histidine (His), proline (Pro) or threonine (Thr) residue,

X ¹³ is an arginine (Arg), tryptophan (Trp) or glutamine (Gln) residue,

X ¹⁴ is isoleucine (Ile), glutamine (Gln) or lysine (Lys) residue,

X ¹⁵ is a histidine (His) or arginine (Arg) residue,

X ¹⁶ is proline (Pro), histidine (His) or valine (Val) residue,

X ¹⁷ is asparagine (Asn), arginine (Arg) or lysine (Lys) residue,

The other characters are single-character notation of amino acids.

The fibronectin EDB protein scaffold of the present invention comprises the steps of: obtaining a sample from a subject; Contacting the target with a scaffold of a composition comprising a fibronectin EDB protein scaffold under conditions where an interaction of the antigen-scaffold can be formed; And detecting the target-scaffold complex to detect an antigen in the composition; Can diagnose a disease, i.e., dengue disease, in a subject.

As described above, the fibronectin EDB protein scaffold specifically binds to the NS1 domain, an unstructured glycoprotein of dengue virus, so that it is easy to diagnose dengue virus.

Figure 1 shows the output phage / input phage ratio recovered at each panning step.
Figure 2 shows the ELISA results for NS1 and Streptavidin of 60 recombinant phage recovered in the third step in the NS1 bio-panning process using the fibronectin EDB protein scaffold library.
Figure 3 shows the results of confirming the NS1 binding specificity of EDB amino acids.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

Example 1: Preparation of EDB recombinant phage library

The recombinant phage was produced from the EDB library stored at -80 ° C. 1 ml of the library stored at -80 占 폚 was added to 30 ml SB liquid medium, and the mixture was cultured at a speed of 200 rpm at 37 占 폚 for 20 minutes. Helper phage (1010 pfu) and ampicillin (final concentration 50 μg / ㎖) were added and cultured for the same time for the same time. Then, the cells were transferred to 30 ml of SB liquid medium containing ampicillin (50 μg / ml) and kanamycin (10 μg / ml) and cultured for 16 hours or more under the same conditions to produce recombinant phages.

The culture was centrifuged at a rate of 5,000 rpm for 10 minutes at 4 ° C. The supernatant was mixed with PEG / NaCl at a ratio of 5: 1 and left on ice for 1 hour. The mixture was then centrifuged at 4 ° C for 20 minutes at a rate of 13,000 rpm , The supernatant was carefully removed, and the pellet was resuspended in 1 ml of PBS.

Example 2: Biopanning of EDB-NS1

50 μl of NS1 (10 μg / ml) was added to 8 wells of a 96 well high binding plate, and the plate was allowed to stand at 4 ° C. overnight. The plate was washed once with 200 μl of PBS and then 200 μl of 2% BSA After blocking for 2 hours at room temperature, the solution was discarded and washed three times with 200 μl of PBS.

400 쨉 l of the EDB recombinant phage containing solution prepared in Example 1 and 400 쨉 l of 2% BSA were mixed and added at a rate of 100 쨉 l per well and left at 30 째 C for 1 hour.

After removing all of the 8 wells, the cells were washed three times with 0.05% PBST (tween-20), and then 100 μl of 0.2 M glycine (pH 2.2) was added to each well to elute the phage for 10 minutes. Then, 800 μl of the solution was collected in an Eppendorf tube Was neutralized by adding 200 [mu] l of 1M Tris (pH 9.0).

To measure the number of input phage and output phage for each bio-panning, E. coli cells at OD = 0.7 were mixed and plated on agar medium containing ampicillin. To repeat the panning, 500 μl of the NS1 output phage was mixed with 5 ml of E. coli cells and cultured at 37 ° C. for 30 minutes at a speed of 200 rpm. Then, the cells were cultured in the presence of ampicillin (50 μg / ml) and helper phage ) And incubated for 30 min in the same manner. Then, the cells were transferred to 50 ml SB medium containing ampicillin (50 μg / ml) and kanamycin (10 μg / ml) and cultured in the same manner for one day.

The culture was centrifuged at 5,000 rpm at 4 ° C for 10 minutes, and PEG / NaCl [20% PEG (w / v) and 15% NaCl (w / v)] was added to the supernatant at a ratio of 5: 1 And allowed to stand in ice for 1 hour. After centrifugation at 13,000 rpm at 4 ° C for 20 minutes, the supernatant was completely removed and the phage pellet was suspended in 1 ml of PBS solution and used for secondary bio-panning.

The same method as described above was used for each panning step. As shown in Table 1 below, the washing process was increased 3 times and 5 times (0.05% PBST), respectively, at each step. In Table 1 and FIG. 1 below, the ratio of output phage / input phage increases according to the washing procedure.

Panning step (condition) input phage output phage output / input Condition 1 Binding 30 ° C 1 hr
Washing 3 times
(0.05% PBST)
3.28 x 10 ¹¹
2.6 x 10 ⁷
7.92 x 10 ^-5 Condition 2 Binding 30 ℃ 1hr Washing 3 times
(0.05% PBST)
2.0 x 10 ¹¹
2.1 x 10 ⁶
1.05 x 10 ^-5 Condition 3 Binding 30 ℃ 1hrWashing 5 times
(0.05% PBST)
7.2 x 10 ¹¹
2.2 x 10 ⁷
3.05 x 10 ^-5 Condition 4 Binding 30 ℃ 1hrWashing 5 times
(0.05% PBST)
4.1 x 10 ¹¹
8.39 x 10 ⁷
20.46 x 10 ^-5

Example 3: NS1-specific phage search (colony ELISA)

The E. coli cells were infected with the phage of condition 4 recovered in the bio-panning step in which the output / input phage ratio was the highest in Table 1, and the plaques were smoothed to about 100-200 per plate.

60 plaques were inoculated into 1 ml of SB-Ampicillin (50 μg / ml) culture medium using a sterilized tip, and incubated at 37 ° C for 5 hours with shaking. Then, 30 μl of helper phage was added thereto, For one day. The culture was centrifuged at a speed of 12,000 rpm for 2 minutes, and the supernatant was recovered. Then, 2% BSA was added thereto and used for the phage search. 5 μg / ml of NS1 and Streptavidin were added to 50 wells per well of a 96-well ELISA plate and left at 4 ° C for one day. On the next day, the proteins of all wells were removed and blocked with 2% BSA at room temperature for 2 hours. The solution was discarded and washed three times with 0.1% PBST. 100 쨉 l of phage solution amplified for each clone was dispensed into all wells and allowed to stand at 30 캜 for 1 hour.

After washing three times with 0.1% PBST solution, HRP-conjugated anti-M13 antibody (GE Healthcare) was diluted 1: 2,000, and 100 ㎕ of each was diluted and reacted at 30 캜 for 1 hour. After washing three times with 0.1% PBST, 100 μl of TMB solution was dispensed to induce a color reaction, and 100 μl of 1 M HSO was added to stop the reaction. The results of absorbance measurements at 450 nm are shown in FIG.

In the result of FIG. 2, clones having a higher absorbance than 3 times higher than that of Streptavidin were selected for sequencing (Bioneer, Daejeon, Korea). Sequencing primer was GATTACGCCAAGCTTTGGAGC. The EDB amino acid sequences for the selected dengue virus NS1 diagnostic are shown in Table 2 below.

SEQ ID NO: 1 NS1 @ EDB14 ≪ tb > SEQ ID NO: 2 NS1 @ EDB21 EVPQLTDLSFVDITDSSIGLRW VQVMRMG IIGYRITVVAAGEGIPIFEDFVD HPIR YYTVTGLEPGIDYDISVITLI PWQRHR PTTLTQQT SEQ ID NO: 3 NS1 @ EDB26 EVPQLTDLSFVDITDSSIGLRW RMTCLCL IIGYRITVVAAGEGIPIFEDFVD DPWT YYTVTGLEPGIDYDISVITLI TQKHVK PTTLTQQT

Example 4: Specificity test for NS1

NS1 @ EDB14, NS1 @ EDB21 and NS1 @ EDB26 of SEQ ID NOS: 1 to 3 were used to confirm whether NS1 could be detected by ELISA.

Specifically, 5 ug / ml NS1 was immobilized on a 96-well plate, and then NS1 @ EDB14, NS1 @ EDB21, and NS1 @ EDB26 were added and allowed to stand for 1 hour.

Then, after washing 4 times with 0.05% PBST solution, color development was observed using HRP-conjugated antibody.

As a result, NS1 @ EDB14, NS1 @ EDB21 and NS1 @ EDB26 of NS1 @ EDB14 and NS1 @ EDB21 of SEQ ID NOS: 1 to 3 according to the present invention can detect NS1, , And NS1 @ EDB26 showed specificity for NS1.

<110> Korea Institute of Ceramic Engineering and Technology <120> A fibronectin Extra Domain B protein scaffold specifically          binding to dengue virus <130> DP-2018-0080-KR <160> 3 <170> KoPatentin 3.0 <210> 1 <211> 91 <212> PRT <213> Artificial Sequence <220> <223> NS1 @ EDB14 <400> 1 Glu Val Pro Gln Leu Thr Asp Leu Ser Phe Val Asp Ile Thr Asp Ser   1 5 10 15 Ser Ile Gly Leu Arg Trp Val Gln Cys Gln Cys Ala Gly Ile Ile Gly              20 25 30 Tyr Arg Ile Thr Val Val Ala Ala Gly Glu Gly Ile Pro Ile Phe Glu          35 40 45 Asp Phe Val Asp Pro Ala Cys Val Tyr Tyr Thr Val Thr Gly Leu Glu      50 55 60 Pro Gly Ile Asp Tyr Asp Ile Ser Val Ile Thr Leu Ile His Arg Ile  65 70 75 80 His Pro Asn Pro Thr Thr Leu Thr Gln Gln Thr                  85 90 <210> 2 <211> 91 <212> PRT <213> Artificial Sequence <220> <223> NS1 @ EDB21 <400> 2 Glu Val Pro Gln Leu Thr Asp Leu Ser Phe Val Asp Ile Thr Asp Ser   1 5 10 15 Ser Ile Gly Leu Arg Trp Val Gln Val Met Met Met Gly Ile Ile Gly              20 25 30 Tyr Arg Ile Thr Val Val Ala Ala Gly Glu Gly Ile Pro Ile Phe Glu          35 40 45 Asp Phe Val Asp His Pro Ile Arg Tyr Tyr Thr Val Thr Gly Leu Glu      50 55 60 Pro Gly Ile Asp Tyr Asp Ile Ser Val Ile Thr Leu Ile Pro Trp Gln  65 70 75 80 Arg His Arg Pro Thr Thr Leu Thr Gln Gln Thr                  85 90 <210> 3 <211> 91 <212> PRT <213> Artificial Sequence <220> <223> NS1 @ EDB26 <400> 3 Glu Val Pro Gln Leu Thr Asp Leu Ser Phe Val Asp Ile Thr Asp Ser   1 5 10 15 Ser Ile Gly Leu Arg Trp Arg Met Thr Cys Leu Cys Leu Ile Ile Gly              20 25 30 Tyr Arg Ile Thr Val Val Ala Ala Gly Glu Gly Ile Pro Ile Phe Glu          35 40 45 Asp Phe Val Asp Asp Pro Trp Thr Tyr Tyr Thr Val Thr Gly Leu Glu      50 55 60 Pro Gly Ile Asp Tyr Asp Ile Ser Val Ile Thr Leu Ile Thr Gln Lys  65 70 75 80 His Val Lys Pro Thr Thr Leu Thr Gln Gln Thr                  85 90

Claims (7)

1. A fibronectin EDB protein scaffold specifically binding to dengue virus comprising one or more amino acid sequences selected from the group consisting of the amino acid sequences of SEQ ID NOS:
[SEQ ID NO: 1]
EVPQLTDLSFVDITDSSIGLRWVQCQCAGIIGYRITVVAAGEGIPIFEDFVDPACVYYTVTGLEPGIDYDISVITLIHRIHPNPTTLTQQT
[SEQ ID NO: 2]
&Lt;
[SEQ ID NO: 3]
EVPQLTDLSFVDITDSSIGLRWRMTCLCLIIGYRITVVAAGEGIPIFEDFVDDPWTYYTVTGLEPGIDYDISVITLITQKHVKPTTLTQQT
delete delete The method according to claim 1,
Wherein the fibronectin EDB protein scaffold specifically binds to the dengue virus NS1 domain.
Fibronectin EDB protein scaffold specifically binding to dengue virus.
A nucleic acid molecule that coats a fibronectin EDB protein scaffold specifically binding to the dengue virus of claim 1 or 4.
A vector for expressing a fibronectin EDB protein scaffold specifically binding to dengue virus comprising the nucleic acid molecule of claim 5.
A composition for diagnosing dengue fever virus comprising a fibronectin EDB protein scaffold specifically binding to dengue virus according to claim 1 or 4.

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