KR102297546B1 - Composition comprising antibody of IPNH protein for diagnosing Acanthamoeba keratitis and diagnosis kit of Acanthamoeba keratitis using the same - Google Patents

Abstract

The present invention relates to a composition for diagnosing the differentiation of aciamoebic keratitis comprising an inosineuridine preferring nucleoside hydrolase (IPNH) antibody, and to a diagnostic kit for acanthamoebaic keratitis using the same. A composition comprising a specific antigen-antibody reaction occurs only in IPNH secreted from Acanthamoeba, enabling rapid and accurate diagnosis and discrimination of the cause of keratitis compared to the existing Acanthamoebaic keratitis diagnosis method, and using the composition It is expected to have high utilization in the manufacture of amoebic keratitis diagnostic kits, therapeutic agents, and contact lens disinfectants.

Description

Composition comprising antibody of IPNH protein for diagnosing Acanthamoeba keratitis and diagnosis kit of Acanthamoeba keratitis using the same

The present invention relates to a composition for differential diagnosis of aciamoebic keratitis, comprising an inosineuridine preferring nucleoside hydrolase (IPNH) antibody, and a kit for diagnosing aciamoebic keratitis using the same.

The cornea is the outermost surface of the eyeball, which occupies about 1/6 of the outer membrane of the eye and refracts light. Corneal epithelium consisting of 5-6 cell layers, Bowman's layer consisting of cell-free collagen fibers, Corneal stroma occupying 90% of the thickness of the cornea, Dua's layer, Descemet's membrane, which consists of 3 to 4 layers of cells, and 6 layers of the corneal endothelium with a fixed number of cells and extremely limited regeneration.

Because the cornea is located at the front of the eye, it can be damaged by trauma, infection, and other immune responses. The most common corneal diseases include corneal abrasions, corneal dystrophy, corneal ulcers, corneal neovascularization, fuchitsushi muscular dystrophy, keratitis, and keratoconus. .

Human keratitis is a disease in which bacteria or viruses invade the cornea and cause inflammation. It can be caused by Staphylococcus sp. , Pseudomonas sp. , Fusarium and Acanthamoeba sp. If not properly treated after the onset of inflammation, it can cause painful ulcers, and in severe cases, eye perforation or even blindness. To prevent these symptoms, it is important to find the cause of keratitis as soon as possible. Among keratitis, keratitis caused by Acanthamoeba infection shows symptoms such as redness, excessive tear production, foreign body sensation, and pain when exposed to light. not.

Amoebic keratitis is diagnosed by collecting a tissue sample from the cornea of a patient with keratitis, and the current diagnostic method is through microscopic observation and PCR. Diagnosis time of about 1 week is required. As a result, early diagnosis is difficult and the treatment time is longer. Therefore, there is an urgent need for a novel method for differential diagnosis of aciamoebic keratitis for rapid and early treatment.

Meanwhile, a primer set for detecting Acanthamoeba and a method for detecting keratitis-inducing Acanthamoeba using the same (Korean Patent No. 10-1308516) are being studied, but this is only a method for detecting Acanthamoeba using a primer set. There is no technology for differentially diagnosing Acanthamoebaic keratitis using an antibody against a specific secreted protein.

In order to solve the above problems, an object of the present invention is to provide a composition for differential diagnosis of Acanthomoebic keratitis, comprising an antibody that specifically binds to Inosineuridine preferring nucleoside hydrolase (IPNH) protein.

Another object of the present invention is to provide a kit for differential diagnosis of Acanthamoeba keratitis comprising the composition.

In addition, the present invention provides information for differential diagnosis of keratitis in patients with Acanthamoebaic keratitis, comprising measuring an antigen-antibody reaction of an antibody specific to Inosineuridine preferring nucleoside hydrolase (IPNH) protein with respect to a biological sample derived from a subject It is another object to provide a method of providing.

Accordingly, there is provided a composition for differential diagnosis of Acanthamoeba keratitis, comprising an antibody that specifically binds to Inosineuridine preferring nucleoside hydrolase (IPNH) protein.

In one embodiment of the present invention, the IPNH (Inosineuridine preferring nucleoside hydrolase) protein may be the amino acid sequence of SEQ ID NO: 1.

In another embodiment of the present invention, the IPNH (Inosineuridine preferring nucleoside hydrolase) protein may be secreted from the pathogenic Acanthomoebae.

In addition, the present invention provides a kit for differential diagnosis of Acanthamoeba keratitis comprising the composition.

In addition, the present invention provides information for differential diagnosis of keratitis in patients with Acanthamoebaic keratitis, comprising measuring an antigen-antibody reaction of an antibody specific to Inosineuridine preferring nucleoside hydrolase (IPNH) protein with respect to a biological sample derived from a subject provide a method for providing

The composition containing the IPNH (Inosineuridine preferring nucleoside hydrolase) protein antibody of the present invention specifically causes an antigen-antibody reaction only in IPNH secreted from Acanthamoeba, so that it is faster and more accurate than the conventional diagnosis method of Acanthamoebaic keratitis. It is possible to identify the cause, and it is expected that the composition will have high utility in the manufacture of a diagnosis kit for Acanthamoeba keratitis, a therapeutic agent, and a contact lens disinfectant using the composition.

Figure 1 shows the ORF of IPNH (Inosineuridine preferring nucleoside hydrolase) in Acanthamoeba secreted protein, Figure 1a shows the nucleotides of IPNH (Inosineuridine preferring nucleoside hydrolase), Figure 1b shows the amino acids of IPNH (Inosineuridine preferring nucleoside hydrolase) it has been shown
Figure 2 shows the results of confirming the protein expression by culturing after securing the IPNH (Inosineuridine preferring nucleoside hydrolase) antibody of the present invention.
Figure 3 shows a schematic diagram of the preparation of the IPNH (Inosineuridine preferring nucleoside hydrolase) antibody of the present invention.
Figure 4 shows the results of comparing the similarity with IPNH (Inosineuridine preferring nucleoside hydrolase) of keratitis causative bacteria with respect to the high antigenic part of the protein structure of IPNH (Inosineuridine preferring nucleoside hydrolase).
Figure 5 shows Acanthamoeba and IPNH (Inosineuridine preferring nucleoside hydrolase) detected in Acanthamoeba culture medium.
Figure 6 shows the specific detection results of the IPNH (Inosineuridine preferring nucleoside hydrolase) antibody against the type of Acanthamoeba.

The present inventors intensively studied IPNH (Inosineuridine preferring nucleoside hydrolase) among proteins secreted by pathogenic Acanthomoebae to prepare antibodies corresponding thereto, and Staphylococcus aureus, Pseudomonas aeruginosa, which can cause human corneal cells and keratitis. , Fusarium solani, Acanthamoeba castellanii and clinical isolate Acanthamoeba spp. The present invention was completed by confirming that the antigen-antibody reaction was specifically caused only to Acanthamoeba among the cell lysates and cultures of

Accordingly, the present invention provides a composition for differential diagnosis of Acanthamoebaic keratitis, comprising an antibody that specifically binds to Inosineuridine preferring nucleoside hydrolase (IPNH) protein.

As used herein, the term “antibody” includes immunoglobulin molecules having immunological reactivity with a specific antigen, and includes both monoclonal and polyclonal antibodies. The antibody also includes forms produced by genetic engineering such as chimeric antibodies (eg, humanized murine antibodies) and heterologous antibodies (eg, bispecific antibodies).

The term "Inosineuridine preferring nucleoside hydrolase (IPNH)" used in the present invention is an enzyme that hydrolyzes all commonly occurring purine and pyrimidine nucleosides into ribose and related bases. IPNH of the present invention is secreted by various microorganisms. Although not limited to IPNH, it is preferably IPNH secreted from Fusarium solani, Staphylococcus aureus, Pseudomonas aeruginosa and Acanthamoeba castellanii , and more preferably IPNH (Inosineuridine preferring nucleoside hydrolase) secreted from the pathogenic Acanthamoeba castellanii.

IPNH (Inosineuridine preferring nucleoside hydrolase) according to the present invention may consist of the amino acid sequence of SEQ ID NO: 1, and 70% or more, preferably 80% or more, more preferably 90% or more, most preferably from SEQ ID NO: 1 may consist of an amino acid sequence having 95, 96%, 97%, 98%, 99% or more sequence homology.

The present inventors used a composition comprising an antibody that specifically binds to the IPNH (Inosineuridine preferring nucleoside hydrolase) protein and confirmed the effectiveness for differentiation and diagnosis of keratitis patients.

In one embodiment of the invention, Acanthamoeba IPNH (Inosineuridine preferring nucleoside hydrolase) using the antibody human corneal cells (Human corneal epithelial cell, HCE), non-pathogenic Acanthamoeba (Acanthamoeba castellanii # 30011-nonpathogenic) and pathogenic Acanthamoeba (Acanthamoeba castellanii #30868-pathogenic), as a result of performing western blotting, it was confirmed that IPNH was specifically detected only in Acanthamoeba (see Example 3), and also in human corneal epithelial cells (HCE). , Acanthamoeba castellanii #30011-nonpathogenic, Acanthamoeba castellanii #30868-pathogenic, Clinical isolate ( Acanthamoebas spp .), F.solani, P.aeruginosa and S.aureus As a result of performing western blotting, it was confirmed that the IPNH antibody was specifically detected only in the type of Acanthamoeba (see Example 4).

From the above results, it was confirmed that when a composition containing an inosineuridine preferring nucleoside hydrolase (IPNH) antibody of the present invention is used, it can be usefully used for the differential diagnosis of keratitis in patients with aciamoebic keratitis.

As another aspect of the present invention, the present invention provides a kit for differential diagnosis of keratitis comprising the composition.

In one embodiment of the present invention, the kit for differential diagnosis of aciamoebic keratitis may be composed of one or more other component compositions, solutions, or devices suitable for the analysis method.

In another aspect of the present invention, the present invention provides an antigen-antibody reaction of an antibody specific to an Inosineuridine preferring nucleoside hydrolase (IPNH) protein with respect to a biological sample derived from a subject, comprising measuring the antibody response of a patient Provides an information provision method for differential diagnosis of keratitis.

As used in the present invention, the term "information providing method for differential diagnosis of keratitis in keratitis patients" is a diagnosis or differential step to provide objective information necessary for diagnosing keratitis disease, and the clinical judgment or opinion of a doctor is excluded. The biological sample derived from the subject is not limited thereto, but may be, for example, a tissue or a cell, preferably a tissue or cell derived from a keratitis patient.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.

[Example]

Example 1. Securing IPNH (Inosineuridine preferring nucleoside hydrolase) in Acanthamoeba secreted protein

1-1.Inosineuridine preferring nucleoside hydrolase (IPNH) ORF

Inosineuridine preferring nucleoside hydrolase (IPNH) ORF among the secreted proteins of pathogenic Acanthomoebae (ATCC#30868) was secured and 1,107bp nucleotides of IPNH (Inosineuridine preferring nucleoside hydrolase) as shown in FIG. 369 amino acids of preferring nucleoside hydrolase) were identified.

1-2. Expression confirmation and purification of IPNH (Inosineuridine preferring nucleoside hydrolase)

After cloning IPNH (Inosineuridine preferring nucleoside hydrolase) obtained in Example 1-1 into pGEX 4T-3, 1 mM IPNH (Inosineuridine preferring nucleoside hydrolase) was added and incubated at 25° C. for 4 hours.

As a result, as shown in FIG. 2a, IPNH (Inosineuridine preferring nucleoside hydrolase) protein was not expressed in the control group, but IPNH (Inosineuridine preferring nucleoside hydrolase) protein was expressed in Total, soluble and insoluble. Similarly, the eluted protein corresponding to line 3 was separated and purified to obtain a large amount.

In addition, as shown in FIG. 3, 50 μg of the isolated and purified IPNH (Inosineuridine preferring nucleoside hydrolase) protein was mixed with 50 μl of adjuvant, and inoculated 3 times with 2 mice at 3-week intervals to generate an antibody.

Example 2. Confirmation of similarity of IPNH (Inosineuridine preferring nucleoside hydrolase) protein in keratitis causative bacteria

Information on the Inosineuridine preferring nucleoside hydrolase (IPNH) protein of Fusarium solani, Staphylococcus aureus, Pseudomonas aeruginosa and Acanthamoeba castellanii , which can cause keratitis, was obtained from GenBank, and the similarity of the proteins was compared, and the differences in protein as shown in FIG. 4 were obtained. A result indicating that was derived.

Example 3. Confirmation of IPNH antibody detection in cell culture medium

Example Human corneal cells (Human corneal epithelial cell, HCE) using the protein antibodies of the first Acanthamoeba IPNH (Inosineuridine preferring nucleoside hydrolase) generated by the, non-pathogenic Acanthamoeba (Acanthamoeba castellanii # 30011-nonpathogenic) and pathogenic Acanthamoeba (Acanthamoeba As a result of performing western blotting in the culture medium of castellanii #30868-pathogenic), as shown in FIG. 5 , the antigen of IPNH was not detected in human corneal cells (line 1), and Acanthamoeba (line 2) and Acanthamoeba culture medium ( It was confirmed that it was specifically detected only in line 3).

Example 4. Confirmation of IPNH antibody detection of Acanthamoeba in the keratitis causative strain

Western blot was performed on P. aeruginosa, S. aureus, F. solani and A. castellanii, which are the causes of keratitis, using the protein antibody of Inosineuridine preferring nucleoside hydrolase (IPNH) produced in Example 1. As shown in 6, it was confirmed that the IPNH antibody was specifically detected only in Acanthamoeba (lines 2, 3 and 4).

From the above results, it was confirmed that the antibody of the present invention specifically binds only to Acanthamoeba.

The description of the present invention stated above is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. There will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

<110> University-Industry Cooperation Group of Kyung Hee University <120> Composition comprising antibody of IPNH protein for diagnosing Acanthamoeba keratitis and diagnosis kit of Acanthamoeba keratitis using the same <130> PD19-305 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 368 <212> PRT <213> IPNH (Inosineuridine preferring nucleoside hydrolase) protein <400> 1 Met Thr Ala Ser Lys Ser Phe Ile Val Val Val Ala Met Leu Cys Leu 1 5 10 15 Met Ala Thr Val Ala Leu Ala His Arg Pro Ser Pro Ser Pro Ser Pro 20 25 30 Ser Pro Lys Pro Thr Val Phe Val Phe Thr Asp Val Asn Thr Asp Asp 35 40 45 Phe Met Ala Leu Lys Leu Ile Leu Lys Tyr Ser Asn Val Lys Val Gly 50 55 60 Gly Phe Val Ala Gly Cys Ala Gly Phe Cys Asn Met Gly Pro Gly Ile 65 70 75 80 Gln Asn Leu Phe Gly Leu Leu Lys Phe Met Asp Arg Asp Asp Val Pro 85 90 95 Val Trp Ala Gly Glu Ala Tyr Ala Ser Thr Glu Ile Asp Ser Gly Asn 100 105 110 Tyr Ser Cys Thr Phe Gln Lys Thr Val Pro Leu Phe Pro Lys Gly Lys 115 120 125 Thr Trp Ala Asp Thr Ile Leu Gly Leu Asn Gln Arg Tyr Pro Arg Leu 130 135 140 Thr Asp Pro Gln Arg Asn Tyr Tyr Pro Gly Phe Pro Gln Val Tyr Glu 145 150 155 160 Pro Leu Arg Ala Ala Ile Ala Ala Leu Asp Gly Pro Val Glu Phe Leu 165 170 175 Ser Leu Gly Thr Leu Thr Glu Ile Asp Tyr Leu Phe Arg Arg Tyr Pro 180 185 190 Asp Leu Lys Gln Arg Val Arg Arg Ile Thr Ile Met Gly Gly Ala Val 195 200 205 His Val Pro Gly Asn Leu Phe Phe Pro Arg Gly Ser Ala Pro Asn Thr 210 215 220 Val Ala Glu Cys Asn Ile Tyr Leu Asp Pro His Ser Ala Arg Asn Val 225 230 235 240 Phe Val Ser Gly Val Pro Ile Arg Leu Val Pro Leu Asp Ala Thr Asn 245 250 255 Ala Phe Gln Leu Ser Trp Glu Phe Leu Asn Glu Phe Asp Asp Val Ala 260 265 270 Arg Thr Lys Glu Ala Lys Phe Val Lys Asp Leu Leu Ile Leu Ile Lys 275 280 285 Asn Asn Ser Ala Ala Thr Ser Met Tyr Ser Leu Trp Asp Pro Leu Thr 290 295 300 Ala Ala Ile Leu Ala Asp His Ser Ile Ile Phe Glu Glu Glu Thr Val 305 310 315 320 Asn Met Thr Val Val Thr Ser Gly Ser Gln Ala Gly Arg Thr Val Ile 325 330 335 Asp Asn Val Glu Gly Lys Pro Val Gln Val Val Leu Thr Ala Asp Pro 340 345 350 Asp Phe Tyr Asp Ile Phe Ile Asp Lys Leu Asn Arg Arg Arg Arg Trp 355 360 365

Claims (5)

A composition for differential diagnosis of aciamoebic keratitis, comprising an antibody that specifically binds to an inosineuridine preferring nucleoside hydrolase (IPNH) protein, wherein the inosineuridine preferring nucleoside hydrolase (IPNH) protein comprises the amino acid sequence of SEQ ID NO: 1 , A composition for differential diagnosis of aciamoebic keratitis. delete According to claim 1,
The IPNH (Inosineuridine preferring nucleoside hydrolase) protein is characterized in that the secretion from the pathogenic Acanthomoebae, the composition. A kit for differential diagnosis of Acanthamoebaic keratitis, comprising the composition of claim 1 or 3. An information providing method for differential diagnosis of keratitis in patients with Acanthomoebic keratitis, comprising measuring an antigen-antibody reaction of an antibody specific for Inosineuridine preferring nucleoside hydrolase (IPNH) protein with respect to a biological sample derived from a subject, the method comprising: IPNH (Inosineuridine preferring nucleoside hydrolase) protein is characterized in that consisting of the amino acid sequence of SEQ ID NO: 1, Information providing method for differential diagnosis of keratitis in patients with Acanthomoebic keratitis.

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