WO2019066389A1 - Nc fusion protein comprising n-terminal domain fragment and c-terminal domain fragment of nucleocapsid protein of mers coronavirus, and kit for diagnosing mers coronavirus infection by using same - Google Patents

Abstract

The present invention provides: an NC fusion protein comprising N-terminal and C-terminal domain fragments of an SRMO coronavirus nucleocapsid protein; and a kit for diagnosing an SRMO coronavirus infection using said NC fusion protein. An NC fusion protein according to the present invention can be mass-produced in E coli much more stably than a full-length nucleocapsid protein. In addition, according to the present invention, when the NC fusion protein represented by SEQ ID NO: 8 is used as the antigen, a specific SRMO coronavirus antibody can be efficiently produced. In addition, since the NC fusion protein reacts with an SRMO coronavirus-induced antibody, the NC fusion protein can be used in an SRMO coronavirus diagnostic kit.

Description

 【Specification】

 Title of the Invention

 An NC fusion protein including an N-terminal domain fragment and a C-terminal domain fragment of a nucleocapsid protein of a mers coronavirus and a kit for diagnosing a mers coronavirus infection using the same

TECHNICAL FIELD

 The present invention relates to an NC fusion protein including an N-terminal domain fragment and a C-terminal domain fragment of a mucin coronavirus nucleocapsid protein, and a kit for diagnosing a mors coronavirus infection using the same.

BACKGROUND ART [0002]

 The middle east respiratory syndrome (MERS) is a serious acute respiratory disease caused by a mers coronavirus infection. MERS coronavirus (MERS coronavius, MERS_CoV) is a newly discovered new beta coronavirus in Saudi Arabia in 2012 (Azhar EI, et al., 2014). The name of the MERS-CoV was named by the International Virus Classification Commission in May 2013 and was formerly called the new coronavirus, SARS-like virus, Middle Eastern SARS, or Saudi SARS. This infection pathway of MERS-CoV is known to be transmitted from camels to humans and camels. In addition, MERS-CoV is transmissible to humans and has a mortality rate of 40%.

 The genome of MERS-CoV is non-segmented and encodes 11 proteins of approximately 30 kb in size. The eleven proteins consist of two replicating polyproteins, four structural proteins, and five non-structural proteins.

 Currently, based on a study of the SARS coronavirus, we are developing a mousse coronavirus vaccine in a primate model. The development of a mors coronavirus vaccine has been shown to increase humoral and cellular immune responses through inoculation with vaccines based on the recombinant receptor binding domain (RBD). A variety of MER virus antivirus are under development.

In addition, products were developed at Antigen Rapid for the rapid diagnosis of MERS-CoV. However, in the case of the product of Antigen Rapid, the nucleocapsid protein Some peptides are expressed in E. coli and purified to make monoclonal antibodies. The Antigen Rapid MERS-CoV Ag Test product is currently in use in Saudi Arabia and the United Arab Emirates for the diagnosis of camel mers. MERS, which has a high mortality rate, is associated with complications such as pneumonia or acute renal failure as well as respiratory infection, so early treatment through rapid diagnosis is very important. However, since there is no MERS rapid diagnostic kit approved for human use, development is necessary.

DETAILED DESCRIPTION OF THE INVENTION

 [Technical Problem]

 It is an object of the present invention to provide an antigen for MERS-CoV diagnosis.

 Another object of the present invention is to provide a kit for the diagnosis of MERS-CoV infection capable of diagnosing MERS-CoV containing the antigen. [Technical Solution]

In order to achieve the above object, the present invention provides a fusion protein comprising an _N- terminal domain fragment and a _c- terminal domain fragment of a nucleocapsid protein of MERS-CoV (hereinafter,

NC fusion protein).

 In addition, the present invention provides a monoclonal antibody that specifically binds to an NC fusion protein of a nucleocapsid protein of MERS-CoV.

 In addition, the present invention provides an expression vector comprising a nucleotide encoding an NC fusion protein of a nucleocapsid protein of MERS-CoV.

 The present invention also provides a host cell transfected with said expression vector.

 The present invention also provides a method for producing an NC fusion protein comprising the step of obtaining an NC fusion protein from said transfected host cell.

 (A) inducing an immune response by injecting an NC fusion protein of a nucleocapsid protein of MERS-CoV as an antigen into an experimental animal; And (b) recovering the antibody from the animal of step (a).

In order to achieve the above other objects, the present invention provides a nucleoside- A kit for the diagnosis of MERS-CoV infection comprising NC fusion protein of protein.

 In addition, the present invention provides a method for providing MERS-CoV infection information comprising the step of adding a sample isolated from a subject to a diagnosis kit for MERS-CoV infection.

【Effects of the Invention】

 The NC fusion protein of nucleoscope protein of MERS-CoV represented by SEQ ID NO: 8 according to the present invention can be mass-produced in E. coli. In addition, the antibody specifically binds to an antibody of MERS-CoV produced in an individual and can be widely used for MERS-CoV diagnosis.

BRIEF DESCRIPTION OF THE DRAWINGS

 Fig. 1 shows an E. coli expression vector pET28a-MERS-CoV-NP_NC capable of expressing an NC fusion protein.

 FIGS. 2A to 2C show the results of confirming the N-terminal domain (a), the C-terminal domain (b) and the NC fusion protein domain (c) using SDS-PAGE. In this case, Sup is a supernatant, ppt is a pellet, FT is a low through, HS-W is a high salt wash, and 7¾) -W is a 7% imidazole wash. .

 Figure 2d shows the results of SDS-PAGE analysis of N-terminal, C-terminal, and NC fusion proteins purified by nickel-affinity chromatography. Here, 5 ug means the amount of each protein loaded.

 FIG. 3 shows the result of ELISA test for confirming the effect of NC fusion protein on the immune antibody formation using a mouse. At this time, 0D means the absorbance (opt i cal dens i ty).

 FIG. 4 is a graph comparing detection sensitivities of a kit using a conventional MERS-CoV diagnostic kit, euroi 隱 flour, and a kit using the NC fusion protein of the present invention. FIG. 5 shows the results of purification by NC fusion protein chromatography of MERS-CoV, SARS-CoV, HCoV-229E and HCoV-0C43 proteins and SDS-PAGE. Here, 5 ug means the amount of each protein loaded.

Figure 6 shows the cross reactivity of MERS-CoV, SARS-CoV, HCoV-229E and HCoV-0C43 proteins with human CoV antibodies. BEST MODE FOR CARRYING OUT THE INVENTION

 In one aspect, the present invention provides a fusion protein of an N-terminal domain fragment and a C-terminal domain fragment of a nucleocapsid protein of a mi ddle east respiratory syndrome (MERS) coronavirus.

 The nucleocapsid of the above-mentioned Mers coronavirus (hereinafter referred to as MERS-CoV) is composed of a total of 11 proteins (two repetitive proteins, four structural proteins, and five nonstructural proteins) encoded by the MERS genome, . These nucleocapsids serve to protect the genetic material when the MERS-CoV is extracellular and to help the gene enter the cell when it penetrates into new host cells. Specifically, the N-terminal domain of the nucleocapsid protein of MERS-CoV may be a polypeptide having the amino acid sequence of SEQ ID NO: 4. Here, the protein may be encoded by the nucleotide sequence of SEQ ID NO: 3. In addition, in the present invention, the fragment of the N-terminal domain has 1 to 134, 10 to 120, 30 to 100, or 50 of the amino acid sequence from amino acid position 36 to 169 of SEQ ID NO: To 80 contiguous amino acid sequences.

 The C-terminal domain of the nucleocapsid protein of MERS-CoV may be a polypeptide having the amino acid sequence of SEQ ID NO: 6. Here, the protein may be encoded by the nucleotide sequence of SEQ ID NO: 5. In the present invention, the fragment of the C-terminal domain has 1 to 117, 10 to 100, 20 to 80, or 30 of the amino acid sequence from amino acid position 246 to 362 of SEQ ID NO: To 50 contiguous amino acid sequences.

 The NC fusion protein of the nucleocapsid protein of MERS-CoV may have the amino acid sequence of SEQ ID NO: 8. In addition, the NC fusion protein can be encoded by the nucleotide sequence of SEQ ID NO: 7.

 In another aspect, the present invention provides a monoclonal antibody that specifically binds to a fusion protein comprising an N-terminal domain fragment and a C-terminal domain fragment of a nucleocapsid protein of MERS-CoV.

The term " antibody ¹ " is a component produced by the stimulation of an antigen in the immune system and specifically binds to a specific antigen to migrate lymph and blood It is a protein that causes antigen-antibody reaction. The antigen-antibody reaction has a high specificity for each antigen. When an antibody is made in the lymphocyte B cells, the antibody produced by the specific antigen does not, in principle, counteract with other antigens. Such high specificity is used in immunoassays, allergy, various diseases, and in the determination of types and types of infectious viruses.

At this time, the vector may be pET28a, and the transformant may be an Escherichia microorganism. In one embodiment, the amino acid residue 36-169 of the N-terminal domain of the MERS-CoV nucleocapsid protein of SEQ ID NO: 4 is subcloned into the NdeI and BamHI sites of the pET28a vector and overexpressed Respectively. Also, amino acid residues 246-362 of the C-terminal domain of the MERS-CoV nucleocapsid protein shown in SEQ ID NO: 6 were subcloned into EcoRI and Xhol sites of pET28a and overexpressed using E. coli strain. Then, each protein was inoculated into E. coli and cultured. ^,

 The term " antigen " is a protein that expresses a virus as a component capable of inducing immunological function among components of the virus. In one embodiment of the present invention, MERS-CoV nucleocapsid protein having the amino acid sequence of SEQ ID NO: Of the NC fusion protein domain acted as an antigen to induce immune antibody formation when administered to an individual.

 The present invention also provides an expression vector comprising a nucleotide encoding said fusion protein, wherein said host cell transfected with said expression vector is provided. The present invention also provides a method for producing an NC fusion protein comprising the step of obtaining an NC fusion protein from said transfected host cell.

 In one embodiment of the present invention, NC fusion proteins were obtained by subcloning the nucleotides encoding the N-terminal and C-terminal domains of the MERS-CoV nucleocapsid proteins in a pET28a vector and inoculating them into E. coli.

(A) inducing an immune response by injecting a fusion protein of an N-terminal domain fragment and a C-terminal domain fragment of an MERS-CoV nucleocapsid protein as an antigen into an experimental animal; And (b) recovering the antibody from the experimental animal of step (a). In addition, the present invention provides a kit for the diagnosis of MERS-CoV infection comprising an NC fusion protein of a nucleocapsid protein of MERS-CoV.

 In one embodiment of the present invention, the NC fusion protein of the MERS-CoV nucleocapsid protein having the amino acid sequence of SEQ ID NO: 8 can specifically bind to the antibody against the mers coronavirus, and thus the MERS-CoV infection diagnostic kit Lt; / RTI > Induced antigen-antibody reaction, MERS-CoV infection could be diagnosed effectively as a low-concentration antigen as compared with the existing MERS-CoV infection diagnostic kit. In particular, the antibody generated from an individual infected with MERS-CoV can bind to the NC fusion protein of the present invention, so that it is possible to easily diagnose whether the individual has been infected with MERS-CoV.

 Also, the present invention provides a method for providing a mers coronavirus infection information, comprising the step of adding a sample isolated from a subject to a diagnosis kit for MERS-CoV infection.

 The term " MERS-CoV infection detection kit " is a kit for rapidly diagnosing MERS-CoV infection and diseases caused therefrom. In one embodiment of the present invention, a strip coated with an antigen on a specimen This kit can be used to detect the test results within 10 to 15 minutes and does not require any equipment or diagnostic equipment to make it easy for anyone to easily detect MERS-CoV infection Which can be diagnosed by the disease.

The above-mentioned " disease caused by Mers-Coronavirus infection " may be a respiratory disease. Severe respiratory symptoms such as a high temperature of 38 ⁰ C or more, coughing or shortness of breath may occur after a latency period of 2 to 14 days after the virus infection . Diarrhea, or constipation. In the case of a chronic disease or immunodeficiency, the complication of pneumonia or acute renal failure may accompany death. DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples. However, the following examples and experimental examples are illustrative of the present invention, and the content of the present invention is not limited by the following examples and experimental examples. Example 1: MERS-€ oV nucleocapsid NC-NC with N-terminal and C- Preparation of fusion protein

 In order to establish the sequence of the MERS-CoV nucleocapsid protein that can be used as an antigen for the development of the diagnostic kit, the structure of the nucleocapsid protein was analyzed. The amino acid sequence of the nucleocapsid protein having a stable form of the secondary structure was determined through the above structural analysis. At this time, the synthesis of the nucleocapsid protein (413 amino acids) of MERS-CoV (Human betacoronavirus 2cEMC / 2012) was carried out in bioneer, and the nucleotide sequence thereof was shown in SEQ ID NO: 1.

 Terminal domain of the MERS-CoV nucleocapside protein shown in SEQ ID NO: 4, in order to prepare a nucleocapsid fusion protein comprising the N-terminal and C-terminal domain fragments of the nucleocapsid protein, 169 were subcloned into the Ndel and BamHI sites of the pET28a vector and overexpressed with nucleotides encoding the 36-169 portion of the N-terminus using the Escherichia coli BL2KDE3) pLysS Rosetta) strain. A nucleotide encoding amino acid residues 246-362 of the C-terminal domain represented by SEQ ID NO: 6 as another site of the nucleocapsid protein was subcloned into the EcoRI and Xhol sites of pET28a and Escherichia coli BL21 (DE3) pLysS Rosetta) was used to overexpress the nucleotides encoding the 246-362 portion of the C-terminus. Alternatively, nucleotides encoding the N-terminal 36-169 and the nucleotides encoding the 246-362 portion of the C-terminal were introduced into the pET28a vector, respectively, Ndel / BamHI and EcoRI / Xh.

 A vector encoding each protein was inoculated into Escherichia coli and incubated at 0 < RTI ID = 0.0 >

IPTG (i sopropyl β-D-1-hi ogalacty anos i de) reagent was added and cultured at 291K for 18 hours. The E. coli expression vector pET28a-MERS-CoV_NP_NC used above is shown in Fig.

 The Hi s-tagged N-terminal, C-terminal and NC fusion proteins were first purified by nickel-affinity chromatography. NC fusion protein was purified once more by HiPrep 16/60 Sephacryl S-200 HR gel-filtration chromatography using phosphate buffered saline (PBS) at 2-fold concentration. Through the above procedure, purified N-terminal, C-terminal and NC fusion protein domains were obtained. The results are shown in Fig.

As shown in Figures 2A-2C, the N-terminal domain (a), the C-terminal domain (b) And NC fusion protein domain (c) were identified on SDS-PAGE. Further, as shown in Fig. 2 (d), each of the domains was purified by chromatography to a purity of 95% or more. At this time, the nucleotide sequence of the NC fusion protein domain is shown in SEQ ID NO: 7, and the 276 amino acid sequences thus prepared are shown in SEQ ID NO: 8. Experimental Example 1. Confirmation of Antibody Production of NC Fusion Protein

 In order to confirm whether or not the NC fusion protein prepared in Example 1 effectively induced antibody production, its antibody production effect was confirmed.

 First, a pre-immune serum was collected and used as a negative control group before injecting the purified NC fusion protein domain antigen into a mouse (Coatech, Korea). For primary immunization (Pr imary i un uni ze), 100 ug of antigen were co-injected with Freund's adjuvant and injected intramuscularly into 4 mice, respectively, and 2 weeks later with Freund's adjuvant, Car boosting was carried out. Two weeks later, primary blood was collected and subjected to ELISA test and secondary boosting. Two weeks later, each mouse was bled and the final serum was separated and subjected to a final ELISA test. At this time, in the ELISA test, 100 ng of antigen was used per well (wel l). In addition, the immunized serum was diluted 1: 100 to 1: 1, 000, 000 by 1XPBS, and anti-mouse IgG-HRP (ABC5001) was diluted at a ratio of 1: 5,000 for secondary confirmation. The color development was detected at an optical density of 405 nm using an ELISA reader, and finally, immunological antibody production was confirmed, which is shown in FIG.

 As shown in Fig. 3, the NC fusion protein showed the effect of forming an immune antibody in all four mice used in the above experiment. This means that the NC fusion protein domain can act as an antigen in the individual and effectively induce the antibody. As a result, it was confirmed that NC fusion protein can be used as an antigen to prepare an antibody for diagnosis of MERS-CoV infection. Experimental Example 2. Comparison of diagnostic performance of MERS ᅳ CoV diagnostic kit and commercial kit developed using NC fusion protein

The superiority of the MRS-CoV diagnostic kit developed using the NC fusion protein prepared in Example 1 was compared with the commercially available MERS-CoV diagnostic kit. ELISA was performed on a plate coated with 400 ng of NC fusion protein antigen on a 40-fold plate of human serum. Secondary antibody HRP-conjugated goat anti-human IgG was diluted 1: 100 ug / ml) and subjected to an indirect ELISA.

 As a commercially available MERS-CoV diagnostic kit, a kit from euroimmun was used. ELISA was performed according to the manufacturer's manual using human serum on the plate coated with the antigen contained in the kit. ELISA was performed by diluting human serum with a sample buffer at a ratio of 1: 100 to the plate contained in the kit according to the manufacturer's protocol Respectively. The results are shown in Fig.

 As shown in FIG. 4, the MERS-CoV diagnostic kit manufactured using the plate coated with the NC fusion protein antigen showed a diagnostic ability four times higher than that of the commercially available MERS-CoV kit . This means that the MERS-CoV diagnostic kit using plates coated with NC fusion protein antigens can be effectively diagnosed at the early stage or at the recovery stage of MERS-CoV infection even in a patient having a low amount of virus in the body. Experimental Example 3. Cross-reactivity determination of human CoV NC fusion protein antibody

 Experimental Example 3.1. Production of human CoV NC fusion protein

MERS-CoV, SARS-CoV, HCoV-229E and HCoV_0C43 nucleocapsid proteins were prepared via an E. coli expression system. Here, the SARS-CoV nucleocapsid protein may be a polypeptide having the amino acid sequence of SEQ ID NO: 9, and the protein may be encoded by the nucleotide sequence of SEQ ID NO: 10. In addition, the HCoV-229E nucleocapsid protein may be a polypeptide having the amino acid sequence of SEQ ID NO: 11, and the protein may be encoded by the nucleotide sequence of SEQ ID NO: In addition, the HCoV-0C43 nucleocapsid protein may be a polypeptide having the amino acid sequence of SEQ ID NO: 13, and the protein may be encoded by the nucleotide sequence of SEQ ID NO: 14. The entire gene of each human CoV nucleocapsid protein was subcloned into the Ndel and BamHI sites of the pET28a vector, and each human CoV nucleocapsid protein was overexpressed using Escherichia coli BL2KDE3 pLysS Rosetta strain. Herein, the method of overexpressing and purifying each of the human CoV nucleocapsid proteins is described in detail in Example 1 The same procedure was followed. Through the above process, purified NC fusion protein domains of SARS-CoV, HCoV-229E and HCoV_0C43 nucleocapsid proteins were obtained, and the results are shown in FIG.

 As shown in FIG. 5, the NC fusion protein domains of MERS-CoV, SARS-CoV, HCoV-229E and HCoV-0C43 nucleocapsid proteins were purified by chromatography to a purity of 95% or more. Experimental Example 3.2. Cross-reactivity measurement with human CoV antibody

It was confirmed that the NC fusion protein of the MERS-CoV, SARS-CoV, HCoV-229E and HCoV-0C43 proteins obtained in Experimental Example 3.1 caused cross-reactivity with the human CoV antibody. Specifically, the monoclonal antibody used in the 96-well plate surface coating was an N-terminal specific antibody of the MERS-CoV nucleocapsid protein and coated at a concentration of 100 ng / well at 4 ^° C and 0 / N coating. Coated 96-well plates were washed once with PBST buffer and then blocked with 2% BSA at 37 ^° C for 1 hour. After blocking, the purified MERS-CoV, SARS-CoV, HCoV-229E and HCoV_0C43 nucleocapsid recombinant proteins were incubated at 100 ng / well to 0.8. ng / well and incubated at 37 [ ^deg.] C for 2 hours. Subsequently, the cells were washed three times for 5 minutes each with PBST. HRP-conjugated MERS-CoV nucleocapsid C-terminal specific mAb was treated at a concentration of 200 ng / well and incubated at 37 ^° C for 1 hour. Then, the cells were washed five times with PBST for 5 minutes each, and then subjected to color reaction with ECL solution. The results are shown in FIG.

 As shown in Figure 6, unlike the NC fusion proteins of the SARS-CoV, HCoV-229E and HCoV-0C43 nucleoside capsid proteins, the MERS-CoV nucleocapside NC fusion protein has cross-reactivity with human CoV antibodies This was done well. This means that the antibody can be used as an antibody to diagnose MERS-CoV.

Claims

Claims:  [Claim 1]  An NC fusion protein comprising an N-terminal domain fragment and a C-terminal domain fragment of a nucleocapsid protein of Mers coronavirus. [Claim 2]  The method according to claim 1,  Wherein the N-terminal domain is a polypeptide having the amino acid sequence of SEQ ID NO: 4. [3]  In Item 1, ^• the N- terminal domain fragment that has an amino acid sequence 50 to 134 consecutive amino acid sequence of from amino acid position number 36 of SEQ ID NO: 4 169-position, characterized in, NC fusion protein. [4]  The method according to claim 1,  Wherein the C-terminal domain is a polypeptide having the amino acid sequence of SEQ ID NO: 6. [Claim 5]  The method of claim 1,  Wherein the C-terminal domain fragment has 50 to 117 contiguous amino acid sequences in the amino acid sequence from amino acid position 246 to 362 of SEQ ID NO: 6. [Claim 6]  The method according to claim 1, Wherein the NC fusion protein has the amino acid sequence of SEQ ID NO: 8 NC fusion protein. [7]  The method according to claim 6,  Wherein the NC fusion protein is encoded by the nucleotide sequence of SEQ ID NO: 7. [8]  A monoclonal antibody that specifically binds to an NC fusion protein according to U. U. [Claim 9]  An expression vector comprising a nucleotide encoding the NC fusion protein of claim 1. Claim 10  Host cells transfected with the expression vector of that paragraph. Claim 11  A method for producing an NC fusion protein, comprising the step of obtaining an NC fusion protein from the transfected host cell of claim 10. Claim 12  (a) injecting a fusion protein according to any one of (1) to (7) into an experimental animal as an antigen to induce an immune response; And  (b) recovering the antibody from the experimental animal of the step). [13] A kit for diagnosing a mars coronavirus infection comprising an NC fusion protein according to claim 1. 14.  13. A method for providing information on a corneal virus infection, comprising the step of adding a sample isolated from a diagnostic object to a diagnostic kit according to claim 13.