JP2008206483A - Diagnostic markers for early diagnosis of invasive periodontitis and therapeutic genes for invasive periodontitis - Google Patents

Abstract

An effective diagnostic marker enabling early diagnosis, an early diagnostic method using the diagnostic marker, and treating invasive periodontitis for invasive periodontitis for which there is no effective early diagnosis method Nucleic acids, antibodies or compositions for doing so are provided.
A gene marker encoding caldesmon, lactoferrin, heat shock 70 KDa protein, which is highly expressed in neutrophils of patients with invasive periodontitis associated with reduced neutrophil function, and a diagnostic method using the same Diagnostic composition. Furthermore, a method for treating the disease and a composition therefor by regulating the expression level of the gene.
[Selection figure] None

Description

  The present invention relates to a diagnostic marker for early diagnosis of invasive periodontitis and a therapeutic gene for invasive periodontitis. The present invention also relates to diagnostic markers for neutrophil hypofunction and therapeutic genes for neutrophil hypofunction.

  In all diseases, the onset is thought to involve genetic predisposition, environmental factors, or both. For example, genetic predisposition occupies all the causes of the onset of one-factor hereditary diseases such as hemophilia. On the other hand, environmental factors such as external force are mostly responsible for trauma. Both genetic predisposition and environmental factors are involved in the development of lifestyle-related diseases such as diabetes or arteriosclerosis. Infectious diseases are the result of interactions between host defenses, which are hereditary predispositions, and bacteria, which are environmental factors. Periodontitis is an infection that develops due to bacterial infection. Periodontitis is roughly classified into chronic periodontitis and invasive periodontitis (Non-patent Document 1). The cause of the onset of chronic periodontitis is thought to be mainly caused by bacteria, which are environmental factors. On the other hand, genetic predisposition is considered to be deeply related to the onset of invasive periodontitis compared to chronic periodontitis.

  Invasive periodontitis occurs in young people in their teens or twenties, and it is a periodontitis that rapidly causes significant periodontal tissue destruction, despite the relatively mild presence of plaque and gingival inflammation. is there. Invasive periodontitis is classified into a localized type in which periodontal tissue destruction is limited to the front teeth or the first molar part, and a wide range of periodontal tissue destruction to the entire jaw. Periodontitis previously classified as juvenile periodontitis or rapidly progressive periodontitis is now included in invasive periodontitis. Although there are many reports on invasive periodontitis, the cause of the onset at the molecular or genetic level has not been fully elucidated. Among patients with invasive periodontitis, it is known that there are patients with neutrophil dysfunction, and that there is a familial onset group. It is used as one.

  Until now, for neutrophil dysfunction in patients with invasive periodontitis, decreased chemotaxis, increased superoxide production, decreased phagocytosis and bactericidal activity against Actinobacillus actinomycetemcomitans, decreased expression of receptors for chemotactic factors, A decrease in the synthesis of leukotriene B4 or abnormalities in the intracellular signal transduction system have been reported. In particular, with regard to abnormalities in the neutrophil signaling system, many studies have been conducted on inositol triphosphate metabolic pathways or signaling pathways involving calcium ions, including reduction of DAG kinase, reduction of calcium influx factors, Abnormal intracellular calcium ion concentration or decrease in protein kinase C has been reported. However, since the causative factors of invasive periodontitis are considered to be diverse, these reports are considered to be only a part of the causative factors of invasive periodontitis.

  As described above, invasive periodontitis is periodontitis that develops early and involves rapid periodontal tissue destruction. In invasive periodontitis, alveolar bone resorption occurs in the early days without inflammatory symptoms and little clinical evidence. In accordance with this, inflammation develops and progresses rapidly, resulting in rapid tissue destruction, forming deep periodontal pockets with subbony pockets. Although the onset and progression of invasive periodontitis are thought to involve abnormal host defense functions, there are variations in the causes of each patient. There is a family accumulation, and a genetic predisposition may be greatly involved. Because of its rapid progression, it can even become a complete denture before adulthood. In addition, it is known that invasive periodontitis is difficult to treat in general treatment of chronic periodontitis. Therefore, it is necessary to diagnose invasive periodontitis early and to predict and prevent periodontitis.

  Invasive periodontitis is a disease that develops early and rapidly destroys periodontal tissue. When a patient visits a dental clinic when he / she notices tooth movement or gingival swelling, if the degree of periodontal tissue destruction is large, extraction may occur. There are many cases in which gingiva is lowered by periodontal treatment for preserving teeth and causes aesthetic disorders even when the extraction is not performed. It is difficult to say that there is a satisfactory dental treatment for invasive periodontitis patients in their teens to 30s. Therefore, before invasive periodontitis develops, it is the best treatment for the patient to predict its onset and prevent its onset.

  Among patients with invasive periodontitis, it is known that there are patients who recognize a decrease in neutrophil function, and this is used as one of the indicators of diagnosis of invasive periodontitis. Neutrophils are granulocytes that play a central role in host defense against pathogens (bacteria, fungi, protozoa, etc.) that enter the body. Neutrophils have various functions such as chemotaxis, phagocytosis, adhesion, active oxygen production, and degranulation. A method of diagnosing neutrophil function at an early stage is an indicator of not only invasive periodontitis but also various diseases (for example, congenital granulocytopenia and leukocyte adhesion disorder (LAD)). Is needed.

  Despite the need for an effective method that enables early diagnosis of invasive periodontitis and neutrophil function decline, it has not yet been realized. .

1999 International Works for a Classification of Periodic Diseases and Conditions. Papers. Oak Brook, Illinois, October 30-1999, January 2nd. Ann Periodontol 1999; 4: 1-6

  In view of the above problems, the present invention provides an effective diagnostic marker that enables early diagnosis of invasive periodontitis for which no effective early diagnosis method currently exists, and early diagnosis using the diagnostic marker The challenge is to provide a law. Another object of the present invention is to provide a nucleic acid, an antibody or a composition for treating invasive periodontitis.

  It is another object of the present invention to provide a diagnostic marker that enables early diagnosis of neutrophil functional decline and an early diagnostic method using the diagnostic marker. Another object of the present invention is to provide a nucleic acid, antibody or composition for inhibiting and / or suppressing the decrease in neutrophil function.

  The present inventors have completed the present invention by newly identifying a gene that is highly expressed in neutrophils of patients with invasive periodontitis accompanied by a decrease in neutrophil function. These genes are markers that enable early diagnosis of invasive periodontitis and reduced neutrophil function. Therefore, the present invention has a remarkable effect of solving the problem of providing an early diagnosis of invasive periodontitis and neutrophil function decline that did not exist conventionally.

Accordingly, the present invention provides the following.
(Item 1)
A diagnostic marker for assessing the presence and / or likelihood of invasive periodontitis, comprising:
A nucleic acid that hybridizes under stringent conditions with a complementary strand of a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 1, and that encodes a physiologically active polypeptide;
A nucleic acid comprising a sequence at least 80% homologous to the nucleic acid sequence set forth in SEQ ID NO: 1 and encoding a polypeptide having physiological activity;
A nucleic acid comprising a fragment of the nucleic acid sequence set forth in SEQ ID NO: 1;
A pair of nucleic acids comprising a fragment of the nucleic acid sequence set forth in SEQ ID NO: 1 and having a length of at least 10 bases and capable of amplifying at least a part of the nucleic acid sequence set forth in SEQ ID NO: 1 as a PCR primer ,
An antibody that reacts with a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2,
A nucleic acid that hybridizes under stringent conditions with a complementary strand of a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 3, and that encodes a physiologically active polypeptide;
A nucleic acid comprising a sequence that is at least 80% homologous to the nucleic acid sequence set forth in SEQ ID NO: 3 and that encodes a physiologically active polypeptide;
A nucleic acid comprising a fragment of the nucleic acid sequence set forth in SEQ ID NO: 3,
A pair of nucleic acids comprising a fragment of the nucleic acid sequence set forth in SEQ ID NO: 3 and having a length of at least 10 bases and capable of amplifying at least a part of the nucleic acid sequence set forth in SEQ ID NO: 1 as a PCR primer ,
An antibody that reacts with a polypeptide having the amino acid sequence of SEQ ID NO: 4,
A nucleic acid that hybridizes under stringent conditions with a complementary strand of a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 5, and that encodes a physiologically active polypeptide;
A nucleic acid comprising a sequence at least 80% homologous to the nucleic acid sequence set forth in SEQ ID NO: 5 and encoding a polypeptide having physiological activity;
A nucleic acid comprising a fragment of the nucleic acid sequence set forth in SEQ ID NO: 5,
A pair of nucleic acids comprising a fragment of the nucleic acid sequence set forth in SEQ ID NO: 5 and having a length of at least 10 bases and capable of amplifying at least a part of the nucleic acid sequence set forth in SEQ ID NO: 1 as a PCR primer ,
An antibody that reacts with a polypeptide having the amino acid sequence of SEQ ID NO: 6,
A nucleic acid that hybridizes under stringent conditions with a complementary strand of a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 7, and that encodes a physiologically active polypeptide;
A nucleic acid comprising a sequence at least 80% homologous to the nucleic acid sequence set forth in SEQ ID NO: 7, which encodes a polypeptide having physiological activity;
A nucleic acid comprising a fragment of the nucleic acid sequence set forth in SEQ ID NO: 7,
A pair of nucleic acids comprising a fragment of the nucleic acid sequence set forth in SEQ ID NO: 7 and having a length of at least 10 bases and capable of amplifying at least a part of the nucleic acid sequence set forth in SEQ ID NO: 1 as a PCR primer ,
An antibody that reacts with a polypeptide having the amino acid sequence of SEQ ID NO: 8,
An invasive periodontitis diagnostic marker comprising a nucleic acid or an antibody selected from the group consisting of:
(Item 2)
The diagnostic marker according to item 1, comprising:
A nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 1;
A nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 3,
A nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 5, and a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 7,
A diagnostic marker comprising a nucleic acid selected from the group consisting of:
(Item 3)
A diagnostic marker according to item 1, comprising a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 1.
(Item 4)
The diagnostic marker according to item 1, comprising:
An antibody that reacts with a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2,
An antibody that reacts with a polypeptide having the amino acid sequence of SEQ ID NO: 4,
An antibody that reacts with the polypeptide having the amino acid sequence set forth in SEQ ID NO: 6, and an antibody that reacts with the polypeptide having the amino acid sequence set forth in SEQ ID NO: 8,
A diagnostic marker comprising an antibody selected from the group consisting of:
(Item 5)
A diagnostic marker according to item 1, comprising an antibody that reacts with a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2.
(Item 6)
A method for assessing the presence and / or likelihood of invasive periodontitis in a subject comprising:
(A) (i) a nucleic acid comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7, or (ii) a stringent and complementary strand of the nucleic acid of (i) A nucleic acid that hybridizes under conditions, the nucleic acid encoding a polypeptide having physiological activity,
Measuring the expression level of in a subject-derived biological sample, and (b) evaluating the prognosis and / or malignancy in the subject using the expression level as an index,
Including the method.
(Item 7)
A method for assessing the presence and / or likelihood of invasive periodontitis in a subject comprising:
(A) (i) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8, or (ii) SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 And a polypeptide having an amino acid sequence containing one or several mutations, substitutions, insertions or deletions in an amino acid sequence selected from the group consisting of SEQ ID NO: 8, and having physiological activity,
Measuring the expression level of in a subject-derived biological sample, and (b) evaluating the prognosis and / or malignancy in the subject using the expression level as an index,
Including the method.
(Item 8)
A diagnostic marker for assessing neutrophil functional decline, the following:
A nucleic acid that hybridizes under stringent conditions with a complementary strand of a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 1, and that encodes a physiologically active polypeptide;
A nucleic acid comprising a sequence at least 80% homologous to the nucleic acid sequence set forth in SEQ ID NO: 1 and encoding a polypeptide having physiological activity;
A nucleic acid comprising a fragment of the nucleic acid sequence set forth in SEQ ID NO: 1;
A pair of nucleic acids comprising a fragment of the nucleic acid sequence set forth in SEQ ID NO: 1 and having a length of at least 10 bases and capable of amplifying at least a part of the nucleic acid sequence set forth in SEQ ID NO: 1 as a PCR primer ,
An antibody that reacts with a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2,
A nucleic acid that hybridizes under stringent conditions with a complementary strand of a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 3, and that encodes a physiologically active polypeptide;
A nucleic acid comprising a sequence that is at least 80% homologous to the nucleic acid sequence set forth in SEQ ID NO: 3 and that encodes a physiologically active polypeptide;
A nucleic acid comprising a fragment of the nucleic acid sequence set forth in SEQ ID NO: 3,
A pair of nucleic acids comprising a fragment of the nucleic acid sequence set forth in SEQ ID NO: 3 and having a length of at least 10 bases and capable of amplifying at least a part of the nucleic acid sequence set forth in SEQ ID NO: 1 as a PCR primer ,
An antibody that reacts with a polypeptide having the amino acid sequence of SEQ ID NO: 4,
A nucleic acid that hybridizes under stringent conditions with a complementary strand of a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 5, and that encodes a physiologically active polypeptide;
A nucleic acid comprising a sequence at least 80% homologous to the nucleic acid sequence set forth in SEQ ID NO: 5 and encoding a polypeptide having physiological activity;
A nucleic acid comprising a fragment of the nucleic acid sequence set forth in SEQ ID NO: 5,
A pair of nucleic acids comprising a fragment of the nucleic acid sequence set forth in SEQ ID NO: 5 and having a length of at least 10 bases and capable of amplifying at least a part of the nucleic acid sequence set forth in SEQ ID NO: 1 as a PCR primer ,
An antibody that reacts with a polypeptide having the amino acid sequence of SEQ ID NO: 6,
A nucleic acid that hybridizes under stringent conditions with a complementary strand of a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 7, and that encodes a physiologically active polypeptide;
A nucleic acid comprising a sequence at least 80% homologous to the nucleic acid sequence set forth in SEQ ID NO: 7, which encodes a polypeptide having physiological activity;
A nucleic acid comprising a fragment of the nucleic acid sequence set forth in SEQ ID NO: 7,
A pair of nucleic acids comprising a fragment of the nucleic acid sequence set forth in SEQ ID NO: 7 and having a length of at least 10 bases and capable of amplifying at least a part of the nucleic acid sequence set forth in SEQ ID NO: 1 as a PCR primer ,
An antibody that reacts with a polypeptide having the amino acid sequence of SEQ ID NO: 8,
A diagnostic marker for decreased neutrophil function, comprising a nucleic acid or antibody selected from the group consisting of:
(Item 9)
The diagnostic marker according to item 8, comprising:
A nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 1;
A nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 3,
A nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 5, and a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 7,
A diagnostic marker comprising a nucleic acid selected from the group consisting of:
(Item 10)
9. The diagnostic marker according to item 8, comprising a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 1.
(Item 11)
The diagnostic marker according to item 8, comprising:
An antibody that reacts with a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2,
An antibody that reacts with a polypeptide having the amino acid sequence of SEQ ID NO: 4,
An antibody that reacts with the polypeptide having the amino acid sequence set forth in SEQ ID NO: 6, and an antibody that reacts with the polypeptide having the amino acid sequence set forth in SEQ ID NO: 8,
A diagnostic marker comprising an antibody selected from the group consisting of:
(Item 12)
9. The diagnostic marker according to item 8, comprising an antibody that reacts with a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2.
(Item 13)
A method for assessing neutrophil functional decline in a subject comprising:
(A) (i) a nucleic acid comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7, or (ii) a stringent and complementary strand of the nucleic acid of (i) A nucleic acid that hybridizes under conditions, the nucleic acid encoding a polypeptide having physiological activity,
A step of measuring the expression level of the neutrophil in the subject using the expression level as an index, and a step of measuring the expression level of the neutrophil in the subject,
Including the method.
(Item 14)
A method for assessing neutrophil functional decline in a subject comprising:
(A) (i) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8, or (ii) SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 And a polypeptide having an amino acid sequence containing one or several mutations, substitutions, insertions or deletions in an amino acid sequence selected from the group consisting of SEQ ID NO: 8, and having physiological activity,
A step of measuring the expression level of the neutrophil in the subject using the expression level as an index, and a step of measuring the expression level of the neutrophil in the subject,
Including the method.
(Item 15)
A composition for treating invasive periodontitis, comprising:
An antibody that reacts with a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2,
An antibody that reacts with a polypeptide having the amino acid sequence of SEQ ID NO: 4,
An antibody that reacts with the polypeptide having the amino acid sequence set forth in SEQ ID NO: 6, and an antibody that reacts with the polypeptide having the amino acid sequence set forth in SEQ ID NO: 8,
A composition comprising an antibody selected from the group consisting of:
(Item 16)
A composition for treating invasive periodontitis, comprising:
An antibody that reacts with a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2,
A composition comprising:
(Item 17)
The composition according to item 15 or 16, further comprising an anti-inflammatory agent.
(Item 18)
Item 18. The composition according to Item 17, wherein the anti-inflammatory agent is minocycline (Periocrine (registered trademark)).
(Item 19)
A composition that inhibits and / or suppresses neutrophil functional decline,
An antibody that reacts with a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2,
An antibody that reacts with a polypeptide having the amino acid sequence of SEQ ID NO: 4,
An antibody that reacts with the polypeptide having the amino acid sequence set forth in SEQ ID NO: 6, and an antibody that reacts with the polypeptide having the amino acid sequence set forth in SEQ ID NO: 8,
A composition comprising an antibody selected from the group consisting of:
(Item 20)
A composition that inhibits and / or suppresses neutrophil functional decline,
An antibody that reacts with a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2,
A composition comprising:
(Item 21)
21. The composition according to item 19 or 20, further comprising a neutrophil function-reducing inhibitor.
(Item 22)
A composition for treating invasive periodontitis, the composition comprising:
The nucleic acid sequence set forth in SEQ ID NO: 1,
The nucleic acid sequence set forth in SEQ ID NO: 3,
The nucleic acid sequence set forth in SEQ ID NO: 5, and the nucleic acid sequence set forth in SEQ ID NO: 7,
A nucleic acid that reduces the expression level of a gene comprising a nucleic acid sequence selected from the group consisting of:
Composition.
(Item 23)
23. The composition according to item 22,
Comprising a nucleic acid that reduces the expression level of a gene comprising the nucleic acid sequence of SEQ ID NO: 1.
Composition.
(Item 24)
The nucleic acid is
A nucleic acid that hybridizes under stringent conditions with a complementary strand of a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 1, and is a nucleic acid fragment that encodes a physiologically active polypeptide. A nucleic acid comprising a sequence that is at least 80% homologous to the nucleic acid sequence and is a fragment of a nucleic acid that encodes a physiologically active polypeptide, and a stringent condition with the complementary strand of the nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 3 A nucleic acid that hybridizes underneath and is a fragment of a nucleic acid that encodes a polypeptide having physiological activity,
A nucleic acid comprising a nucleic acid sequence which is at least 80% homologous to the nucleic acid sequence described in SEQ ID NO: 3 and is a fragment of a nucleic acid encoding a polypeptide having physiological activity. Complementation of a nucleic acid comprising the nucleic acid sequence described in SEQ ID NO: 5 A nucleic acid that hybridizes with a strand under stringent conditions, and that is a fragment of a nucleic acid that encodes a physiologically active polypeptide, a nucleic acid comprising a sequence that is at least 80% homologous to the nucleic acid sequence set forth in SEQ ID NO: 5 A nucleic acid that is a fragment of a nucleic acid encoding a polypeptide having biological activity,
A nucleic acid that hybridizes under stringent conditions with a complementary strand of a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 7, a nucleic acid fragment that encodes a polypeptide having physiological activity, and SEQ ID NO: 7 23. The composition of item 22, wherein the composition is selected from the group consisting of nucleic acids that are nucleic acids comprising a sequence that is at least 80% homologous to the nucleic acid sequence described and that is a fragment of a nucleic acid that encodes a biologically active polypeptide.
(Item 25)
The nucleic acid is
A nucleic acid that hybridizes under stringent conditions with a complementary strand of a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 1, and a nucleic acid fragment that encodes a physiologically active polypeptide;
25. The composition according to item 24, wherein
(Item 26)
The composition according to items 22 to 25, further comprising an anti-inflammatory agent.
(Item 27)
27. The composition according to item 26, wherein the anti-inflammatory agent is minocycline (Periocrine (registered trademark)).
(Item 28)
A composition that inhibits and / or suppresses neutrophil functional decline, the composition comprising:
The nucleic acid sequence set forth in SEQ ID NO: 1,
The nucleic acid sequence set forth in SEQ ID NO: 3,
The nucleic acid sequence set forth in SEQ ID NO: 5, and the nucleic acid sequence set forth in SEQ ID NO: 7,
A nucleic acid that reduces the expression level of a gene comprising a nucleic acid sequence selected from the group consisting of:
Composition.
(Item 29)
29. A composition according to item 28,
Comprising a nucleic acid that reduces the expression level of a gene comprising the nucleic acid sequence of SEQ ID NO: 1.
Composition.
(Item 30)
The nucleic acid is
A nucleic acid that hybridizes under stringent conditions with a complementary strand of a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 1, and is a nucleic acid fragment that encodes a physiologically active polypeptide. A nucleic acid comprising a sequence that is at least 80% homologous to the nucleic acid sequence and is a fragment of a nucleic acid that encodes a physiologically active polypeptide, and a stringent condition with the complementary strand of the nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 3 A nucleic acid that hybridizes underneath and is a fragment of a nucleic acid that encodes a polypeptide having physiological activity,
A nucleic acid comprising a nucleic acid sequence which is at least 80% homologous to the nucleic acid sequence described in SEQ ID NO: 3 and is a fragment of a nucleic acid encoding a polypeptide having physiological activity. Complementation of a nucleic acid comprising the nucleic acid sequence described in SEQ ID NO: 5 A nucleic acid that hybridizes with a strand under stringent conditions, and that is a fragment of a nucleic acid that encodes a physiologically active polypeptide, a nucleic acid comprising a sequence that is at least 80% homologous to the nucleic acid sequence set forth in SEQ ID NO: 5 A nucleic acid that is a fragment of a nucleic acid encoding a polypeptide having biological activity,
A nucleic acid that hybridizes under stringent conditions with a complementary strand of a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 7, a nucleic acid fragment that encodes a polypeptide having physiological activity, and SEQ ID NO: 7 29. The composition according to item 28, wherein the composition is selected from the group consisting of nucleic acids that are nucleic acids comprising a sequence that is at least 80% homologous to the described nucleic acid sequence and that encodes a physiologically active polypeptide.
(Item 31)
The nucleic acid is
30. The composition according to item 29, which is a nucleic acid that hybridizes under stringent conditions with a complementary strand of a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 1, and is a fragment of a nucleic acid that encodes a physiologically active polypeptide. .
(Item 32)
32. The composition according to items 28 to 31, further comprising a neutrophil function-lowering inhibitor.
(Item 33)
A method for screening an anti-inflammatory agent, the method comprising:
(A) (i) a nucleic acid comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7, or (ii) a stringent and complementary strand of the nucleic acid of (i) A nucleic acid that hybridizes under conditions, the nucleic acid encoding a polypeptide having physiological activity,
Contacting a cell expressing a candidate compound with a candidate compound;
(B) measuring the expression level of the nucleic acid in the cell; and (c) selecting a compound having anti-inflammatory activity from the candidate compounds using the measured expression level as an index;
Including the method.
(Item 34)
A method for screening an anti-inflammatory agent, the method comprising:
(A) (i) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8, or (ii) SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 And a polypeptide having an amino acid sequence containing one or several mutations, substitutions, insertions or deletions in an amino acid sequence selected from the group consisting of SEQ ID NO: 8, and having physiological activity,
Contacting a cell expressing a candidate compound with a candidate compound;
(B) measuring the expression level of the polypeptide in the cell; and (c) selecting a compound having anti-inflammatory activity from the candidate compounds using the measured expression level as an index;
Including the method.
(Item 35)
A method for screening an anti-inflammatory agent, the method comprising:
(A) (i) a nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 1, or (ii) a nucleic acid that hybridizes with a complementary strand of the nucleic acid of (i) under stringent conditions, and having a physiologically active polypeptide Nucleic acid encoding,
Contacting a cell expressing a candidate compound with a candidate compound;
(B) measuring the expression level of the nucleic acid in the cell; and (c) selecting a compound having anti-inflammatory activity from the candidate compounds using the measured expression level as an index;
Including the method.
(Item 36)
A method for screening an anti-inflammatory agent, the method comprising:
(A) (i) a polypeptide having the amino acid sequence of SEQ ID NO: 2, or (ii) an amino acid sequence comprising one or several mutations, substitutions, insertions or deletions in the amino acid sequence of SEQ ID NO: 2, and A polypeptide having physiological activity,
Contacting a cell expressing a candidate compound with a candidate compound;
(B) measuring the expression level of the polypeptide in the cell; and (c) selecting a compound having anti-inflammatory activity from the candidate compounds using the measured expression level as an index;
Including the method.
(Item 37)
A method for screening a neutrophil function-reducing inhibitor, the method comprising:
(A) (i) a nucleic acid comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7, or (ii) a stringent and complementary strand of the nucleic acid of (i) A nucleic acid that hybridizes under conditions, the nucleic acid encoding a polypeptide having physiological activity,
Contacting a cell expressing a candidate compound with a candidate compound;
(B) measuring the expression level of the nucleic acid in the cell; and (c) selecting a compound having anti-inflammatory activity from the candidate compounds using the measured expression level as an index;
Including the method.
(Item 38)
A method for screening a neutrophil function-reducing inhibitor, the method comprising:
(A) (i) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8, or (ii) SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 And a polypeptide having an amino acid sequence containing one or several mutations, substitutions, insertions or deletions in an amino acid sequence selected from the group consisting of SEQ ID NO: 8, and having physiological activity,
Contacting a cell expressing a candidate compound with a candidate compound;
(B) measuring the expression level of the polypeptide in the cell; and (c) selecting a compound having anti-inflammatory activity from the candidate compounds using the measured expression level as an index;
Including the method.
(Item 39)
A method for screening a neutrophil function-reducing inhibitor, the method comprising:
(A) (i) a nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 1, or (ii) a nucleic acid that hybridizes with a complementary strand of the nucleic acid of (i) under stringent conditions, and having a physiologically active polypeptide Nucleic acid encoding,
Contacting a cell expressing a candidate compound with a candidate compound;
(B) measuring the expression level of the nucleic acid in the cell; and (c) selecting a compound having anti-inflammatory activity from the candidate compounds using the measured expression level as an index;
Including the method.
(Item 40)
A method for screening a neutrophil function-reducing inhibitor, the method comprising:
(A) (i) a polypeptide having the amino acid sequence of SEQ ID NO: 2, or (ii) an amino acid sequence comprising one or several mutations, substitutions, insertions or deletions in the amino acid sequence of SEQ ID NO: 2, and A polypeptide having physiological activity,
Contacting a cell expressing a candidate compound with a candidate compound;
(B) measuring the expression level of the polypeptide in the cell; and (c) selecting a compound having anti-inflammatory activity from the candidate compounds using the measured expression level as an index;
Including the method.
(Item 41)
A kit for assessing the possibility of invasive periodontitis,
The composition according to any one of items 1 to 5, and instructions for evaluating the possibility of periodontitis,
A kit comprising:
(Item 42)
A kit for evaluating the possibility of neutrophil function decline, the kit comprising:
13. The composition according to any one of items 8 to 12, and instructions for measuring the possibility of neutrophil function decline,
A kit comprising:

  These and other advantages of the present invention will be apparent to those of ordinary skill in the art upon reading and understanding the following detailed description with reference to the accompanying drawings.

  The present invention provides an effective diagnostic marker that enables early diagnosis of invasive periodontitis and an early diagnosis method using the diagnostic marker. Nucleic acids, antibodies or compositions for treating invasive periodontitis are also provided by the present invention.

  Furthermore, the present invention provides a diagnostic marker that enables early diagnosis of neutrophil functional decline and an early diagnosis method using the diagnostic marker. Nucleic acids, antibodies or compositions for inhibiting and / or suppressing neutrophil functional decline are also provided by the present invention.

  Therefore, the present invention has a remarkable effect of solving the problem of providing an early diagnosis of invasive periodontitis and neutrophil function decline that did not exist conventionally.

  The invention will now be described by way of example, with reference to the accompanying drawings, where appropriate. The present invention will be described below. Throughout this specification, it should be understood that the singular forms also include the plural concept unless specifically stated otherwise. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

  The embodiments provided below are provided for a better understanding of the present invention, and the scope of the present invention should not be limited to the following description. It will be apparent to those skilled in the art that modifications can be made as appropriate within the scope of the present invention in consideration of the description in the present specification.

  The present inventors have completed the present invention by newly identifying a gene that is highly expressed in neutrophils of patients with invasive periodontitis accompanied by a decrease in neutrophil function. These genes are markers that enable early diagnosis of invasive periodontitis and reduced neutrophil function. Therefore, the present invention has a remarkable effect of solving the problem of providing an early diagnosis of invasive periodontitis and neutrophil function decline that did not exist conventionally.

  Specifically, the present inventors isolated neutrophils from patients with invasive periodontitis associated with reduced neutrophil function, and identified genes that showed high expression at the protein level by proteomic analysis. The present invention has been completed. The genes identified as markers in the present invention are Caldesmon (SEQ ID NO: 1 shows the nucleic acid sequence, SEQ ID NO: 2 shows the amino acid sequence), Lactoferrin (SEQ ID NO: 3 shows the nucleic acid sequence, SEQ ID NO: 4 shows the amino acid sequence, respectively) 4) of Heat shock 70 kDa protein (SEQ ID NO: 5 shows the nucleic acid sequence, SEQ ID NO: 6 shows the amino acid sequence), and Stac (SEQ ID NO: 7 shows the nucleic acid sequence, and SEQ ID NO: 8 shows the amino acid sequence). It was one.

  Caldesmon is a calmodulin-binding protein and is known to play an important role in secretion or organelle movement while being involved in intracellular signal transduction.

  Lactoferrin is a protein that exhibits antibacterial, antifungal, virovirus, anticancer, anti-inflammatory, immunomodulatory activity and the like. About Lactoferrin, the example which shows a different genotype between a healthy subject and a invasive periodontitis patient is reported. In addition, the amount of lactoferrin in the saliva of patients with localized invasive periodontitis (anterior teeth and molars (back teeth) (especially periodontitis in which symptoms appear only in the front teeth and first molars)) is higher than that in healthy subjects. It has been reported that Lactoferrin has a higher iron-binding ability than normal subjects in patients with localized invasive periodontitis.

  The heat shock 70 kDa protein is a protein that is required for the maintenance of a living body and is produced as a result of a biological defense reaction. Heat shock 60 kDa and 65 kDa proteins have been reported to be involved in the periodontal disease process.

  Although the function of Stac is hardly known at present, it is suggested that it may be involved in intracellular signal transmission in nerve cells.

(Definition of terms)
Listed below are definitions of terms particularly used in the present specification.

  “Periodontitis” refers to an inflammatory disease in which lesions spread to periodontal tissues or alveolar bone among lesions appearing in periodontal tissues. Periodontitis is generally roughly classified into two types: chronic periodontitis and invasive periodontitis. The “periodontitis” in the present invention particularly refers to invasive periodontitis, but is not limited thereto, and the present invention can also be applied to chronic periodontitis.

  “Presence of periodontitis” refers to the patient or subject suffering from periodontitis. If "periodontitis is present" in a patient or subject, the patient or subject is suffering from periodontitis; conversely, if "periodontitis is absent" in the patient or subject, the patient or subject The subject is not suffering from periodontitis. In the present specification, “existence of periodontitis” refers to periodontal tissue examination, radiographic examination, serum antibody titer examination, periodontal pathogenic bacteria examination using saliva and gingival crevicular fluid, neutrophil Determined by various tests of functional tests.

  “Possibility of periodontitis” refers to the likelihood, risk, or likelihood of onset of periodontitis in a patient or subject.

  As used herein, “assessing the presence and / or likelihood of periodontitis” means whether or not the patient or subject suffers from periodontitis, and the patient or subject develops periodontitis It means to evaluate whether or not there is a high possibility. In the present invention, the expression level of the marker gene is generally higher in the biological sample derived from the subject than in the control (for example, 20 times, 15 times, 10 times, 9 times, 8 times the expression level of the control). , 7 times, 6 times, 5 times, 4 times, 3 times, 2 times, 1.9 times, 1.8 times, 1.7 times, 1.6 times, 1.5 times) The body is evaluated as “existing or likely to develop periodontitis” and comparable to controls (eg, 1 and 0.9 times the onset of controls) 0.8 times, 0.7 times, 0.6 or 0.5 times to 2 times, 1.9 times, 1.8 times, 1.7 times, 1.6 times, 1.5 times, 1. 4 times, 1.3 times, 1.2 times, or 1.1 times), the subject "does not have periodontitis or is not likely to develop periodontitis" And comment And low compared to the control (eg, less than 1.5 times, less than 1.4 times, less than 1.3 times, less than 1.2 times, less than 1.1 times, or 1 time the control expression level) The subject is assessed as “there is less or less likely to develop periodontitis”. Examples of the control used in the present invention include, but are not limited to. The control is, for example, the expression level of the marker gene after normalization by the expression level of the housekeeping gene in a biological sample derived from a healthy person.

  “Marker” refers to a macromolecular, microscopic, or molecular feature of a biological agent that unambiguously identifies the biological agent. In the present invention, “markers” are the Caldesmon gene, the Lactoferrin gene, the Heat shock 70 kDa protein gene, and the Stac gene.

  The “expression amount” of a marker gene refers to the amount of nucleic acid or polypeptide expressed from the marker gene. Methods for measuring the amount of nucleic acid or polypeptide are well known in the art.

  Treating periodontitis means treating periodontitis, reducing the severity of periodontitis, preventing the onset of periodontitis, or preventing recurrence of periodontitis Say things.

  In this specification, the definition of “neutrophil” is generally the same as the definition in the art. Neutrophils have a variety of powerful functions such as chemotaxis, phagocytosis, adhesion, active oxygen production, and degranulation, and are the most important granulocytes in host defense against pathogens that enter the body. is there. In invasive periodontitis, this pathogen is in particular Actinobacillus actinomycetemcomitans. This A. Actinomycetemcomitans is said to produce a leukocyte toxin called leukotoxin and reduce the function of neutrophils or other immune cells.

  “Degraded neutrophil function” means that neutrophil functions (eg, chemotaxis, phagocytosis, adhesion, active oxygen production, degranulation, etc.) are compared to healthy neutrophils. It means to decrease. “Evaluating the functional decline of neutrophils” means determining whether or not there is a decline in the function of the target neutrophils compared to healthy neutrophils. In one aspect of the present invention, the decrease in neutrophil function refers to a decrease in chemotaxis ability. Neutrophil chemotaxis is assayed, for example, by the methods shown in the general procedure of the examples.

  As used herein, “assessing neutrophil hypofunction” refers to determining whether neutrophil hypofunction has occurred in a patient or subject. In one aspect of the present invention, this neutrophil function is chemotaxis. In the present invention, generally, the expression level of the marker gene is higher in the biological sample derived from the subject than in the control (for example, 20 times, 15 times, 10 times, 9 times, 8 times the expression level of the control). 7 times, 6 times, 5 times, 4 times, 3 times, 2 times, 1.9 times, 1.8 times, 1.7 times, 1.6 times, 1.5 times) Is evaluated as “the function of neutrophils is reduced” and comparable to that of the control (for example, 1 times, 0.9 times, 0.8 times, 0.7 times the expression level of the control, 0.6 or 0.5 times to 2 times, 1.9 times, 1.8 times, 1.7 times, 1.6 times, 1.5 times, 1.4 times, 1.3 times, 1.2 Times or 1.1 times), the subject is assessed as “no neutrophil function is not reduced”. The control is, for example, the expression level of the marker gene after normalization by the expression level of the housekeeping gene in a biological sample derived from a healthy person.

  “Inhibiting and / or suppressing neutrophil functional decline” means preventing neutrophil functional decline, preventing neutrophil functional decline, or restoring degraded neutrophil function It means to make it. In the present invention, the function of the reduced function of neutrophils is up to 50%, up to 60%, up to 70%, up to 80%, up to 85%, up to 90%, up to 95% compared to healthy neutrophils. Up to, and preferably up to 100%, and even more preferably up to 100% or more, it is said to inhibit and / or suppress a decrease in neutrophil function.

  As used herein, “physiological activity” refers to an activity that causes any physiological or pharmacological action. In one embodiment of the present invention, the physiological activity is neutrophil function-reducing activity. In one embodiment of the present invention, the physiological activity is neutrophil chemotaxis reducing activity.

  A “polynucleotide”, “nucleic acid” or “nucleic acid molecule” is a ribonucleotide (adenosine, guanosine, uridine, or cytidine in the form of a phosphate ester polymer that is in single-stranded, double-stranded, or other form; “RNA molecule”) or deoxyribonucleotides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine (DNA molecule)), or any phosphoester analog thereof (eg, phosphorothioate and thioester).

  A “polynucleotide sequence”, “nucleic acid sequence” or “nucleotide sequence” is a series of nucleotide bases (also referred to as “nucleosides”) in a nucleic acid (eg, DNA or RNA) and any chain of two or more nucleotides. Or its complementary strand. Preferred nucleic acids of the present invention include the nucleic acids set forth in any of SEQ ID NOs: 1, 3, 5 or 7 and their complementary strands, variants and fragments.

  “Complementary strand” means a strand of nucleotides capable of forming base pairs with a nucleic acid sequence. For example, each strand of double-stranded DNA has a complementary base sequence, and the other strand is a complementary strand when viewed from one strand.

  A “coding sequence” or a sequence that “encodes” an expression product (eg, RNA, polypeptide, protein, or enzyme) is a nucleotide sequence that, when expressed, results in the production of that product.

  A “protein”, “peptide” or “polypeptide” includes a contiguous sequence of two or more amino acids. Preferred peptides of the present invention include the peptide shown in any of SEQ ID NOs: 2, 4, 6 or 8, and variants and fragments thereof.

  A “protein sequence”, “peptide sequence”, or “polypeptide sequence” or “amino acid sequence” refers to a series of two or more amino acids in a protein, peptide, or polypeptide.

  As used herein, “homology” of genes (eg, nucleic acid sequences, amino acid sequences, etc.) refers to the degree of identity of two or more gene sequences with each other. In this specification, the identity of sequences (nucleic acid sequences, amino acid sequences, etc.) refers to the degree of two or more comparable sequences that are identical to each other (individual nucleic acids, amino acids, etc.). Therefore, the higher the homology between two genes, the higher the sequence identity or similarity. Whether two genes have homology can be examined by direct sequence comparison or, in the case of nucleic acids, hybridization methods under stringent conditions. When directly comparing two gene sequences, the DNA sequence between the gene sequences is typically at least 50% identical, preferably at least 70% identical, more preferably at least 80%, 90% , 95%, 96%, 97%, 98% or 99% are identical, the genes are homologous. In the present specification, “similarity” of genes (for example, nucleic acid sequences, amino acid sequences, etc.) refers to two or more gene sequences when the conservative substitution is regarded as positive (identical) in the above homology. The degree of identity with each other. Thus, when there is a conservative substitution, homology and similarity differ depending on the presence of the conservative substitution. When there is no conservative substitution, homology and similarity indicate the same numerical value.

  In this specification, the comparison of similarity, identity and homology between amino acid sequences and base sequences is calculated using FASTA, which is a sequence analysis tool, and using default parameters.

  As used herein, “fragment” refers to a polypeptide or polynucleotide having a sequence length of 1 to n−1 with respect to a full-length polypeptide or polynucleotide (length is n). The length of the fragment can be appropriately changed according to the purpose. For example, the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10, Examples include 15, 20, 25, 30, 40, 50 and more amino acids, and lengths expressed in integers not specifically listed here (eg, 11 etc.) are also suitable as lower limits. obtain. In the case of polynucleotides, examples include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides. Non-integer lengths (eg, 11 etc.) may also be appropriate as a lower limit. In the present specification, the lengths of polypeptides and polynucleotides can be represented by the number of amino acids or nucleic acids, respectively, as described above, but the above numbers are not absolute, and the upper limit is not limited as long as they have the same function. Alternatively, the above-mentioned number as the lower limit is intended to include the upper and lower numbers (or, for example, up and down 10%) of the number. In order to express such intention, in this specification, “about” may be added before the number. However, it should be understood herein that the presence or absence of “about” does not affect the interpretation of the value. The length of a fragment useful herein can be determined by whether or not at least one of the functions of a full-length protein that serves as a reference for the fragment is retained.

  As used herein, an “isolated” biological factor (eg, a nucleic acid or protein, etc.) refers to other biological factors within the cells of the organism in which it is naturally occurring (eg, In the case of a nucleic acid, it is substantially separated from a nucleic acid containing a non-nucleic acid factor and a nucleic acid sequence other than the target nucleic acid; in the case of a protein, a protein containing a non-protein factor and an amino acid sequence other than the target protein). Or it is purified. “Isolated” nucleic acids and proteins include nucleic acids and proteins purified by standard purification methods. Thus, isolated nucleic acids and proteins include chemically synthesized nucleic acids and proteins.

  As used herein, a “purified” biological agent (such as a nucleic acid or protein) refers to a product from which at least part of the factors naturally associated with the biological agent has been removed. Thus, typically the purity of the biological agent in a purified biological agent is higher (ie, enriched) than the state in which the biological agent is normally present.

  The terms “purified” and “isolated” as used herein are preferably at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and most preferably Means that at least 98% by weight of the same type of biological agent is present.

  In the present specification, “polynucleotide hybridizing under stringent conditions” refers to well-known conditions commonly used in the art. Such a polynucleotide can be obtained by using colony hybridization method, plaque hybridization method, Southern blot hybridization method or the like using a polynucleotide selected from among the polynucleotides of the present invention as a probe. Specifically, hybridization was performed at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which DNA derived from colonies or plaques was immobilized, and then 0.1 to 2 times the concentration. Means a polynucleotide that can be identified by washing the filter under conditions of 65 ° C. using a SSC (saline-sodium citrate) solution (composition of 1-fold concentration of SSC solution is 150 mM sodium chloride, 15 mM sodium citrate). To do. Hybridization was performed in Molecular Cloning 2nd ed. , Current Protocols in Molecular Biology, Supplements 1-38, DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford Universe. Here, the sequence containing only the A sequence or only the T sequence is preferably excluded from the sequences that hybridize under stringent conditions. The “hybridizable polynucleotide” refers to a polynucleotide that can hybridize to another polynucleotide under the above hybridization conditions. Specifically, the hybridizable polynucleotide is a polynucleotide having at least 60% homology with the base sequence of DNA encoding a polypeptide having the amino acid sequence specifically shown in the present invention, preferably 80% The polynucleotide which has the above homology, More preferably, the polynucleotide which has 95% or more of homology can be mentioned.

As used herein, “highly stringent conditions” are designed to allow hybridization of DNA strands having a high degree of complementarity in nucleic acid sequences and to exclude hybridization of DNA having significant mismatches. Say conditions. Hybridization stringency is primarily determined by temperature, ionic strength, and the conditions of denaturing agents such as formamide. Examples of “highly stringent conditions” for such hybridization and washing are 0.0015 M sodium chloride, 0.0015 M sodium citrate, 65-68 ° C., or 0.015 M sodium chloride, 0.0015 M citric acid. Sodium, and 50% formamide, 42 ° C. For such highly stringent conditions, see Sambrook et al. , Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory (Cold Spring Harbor, N, Y. 1989); and Anderson et al. Nucleic Acid Hybridization: a Practical Approach, IV, IRL Press Limited (Oxford, England). See Limited, Oxford, England. If necessary, more stringent conditions (eg, higher temperature, lower ionic strength, higher formamide, or other denaturing agents) may be used. Other agents can be included in the hybridization and wash buffers for the purpose of reducing non-specific hybridization and / or background hybridization. Examples of such other agents include 0.1% bovine serum albumin, 0.1% polyvinyl pyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecyl sulfate (NaDodSO ₄ or SDS), Ficoll, Denhardt's solution, sonicated salmon sperm DNA (or another non-complementary DNA) and dextran sulfate, but other suitable agents can also be used. The concentration and type of these additives can be varied without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are usually performed at pH 6.8-7.4; however, at typical ionic strength conditions, the rate of hybridization is almost pH independent. Anderson et al. , Nucleic Acid Hybridization: a Practical Approach, Chapter 4, IRL Press Limited (Oxford, England).

Factors that affect the stability of the DNA duplex include base composition, length, and degree of base pair mismatch. Hybridization conditions can be adjusted by one of ordinary skill in the art to apply these variables and to allow different sequence related DNAs to form hybrids. The melting temperature of a perfectly matched DNA duplex can be estimated by the following equation:
T _m (° C.) = 81.5 + 16.6 (log [Na ⁺ ]) + 0.41 (% G + C) −600 / N−0.72 (% formamide)
Where N is the length of the duplex formed, [Na ⁺ ] is the molar concentration of sodium ions in the hybridization or wash solution, and% G + C is the (guanine + in the hybrid Cytosine) base percentage. For imperfectly matched hybrids, the melting temperature decreases by about 1 ° C. for each 1% mismatch.

As used herein, “moderately stringent conditions” refers to conditions under which a DNA duplex having a higher degree of base pair mismatch than can be generated under “highly stringent conditions”. . Examples of representative “moderately stringent conditions” are 0.015 M sodium chloride, 0.0015 M sodium citrate, 50-65 ° C., or 0.015
M sodium chloride, 0.0015 M sodium citrate, and 20% formamide, 37-50 ° C. By way of example, “moderately stringent” conditions at 50 ° C. in 0.015 M sodium ion tolerate a mismatch of about 21%.

  It will be appreciated by those skilled in the art that there may not be a complete distinction between “highly” stringent conditions and “moderate” stringent conditions herein. For example, at 0.015M sodium ion (no formamide), the melting temperature of a perfectly matched long DNA is about 71 ° C. In washing at 65 ° C. (same ionic strength), this allows about 6% mismatch. In order to capture more distant related sequences, one of ordinary skill in the art can simply decrease the temperature or increase the ionic strength.

  For oligonucleotide probes up to about 20 nucleotides, a reasonable estimate of the melting temperature in 1M NaCl is provided by Tm = (2 ° C. for 1 AT base) + (4 ° C. for 1 GC base pair) . The sodium ion concentration in 6 × sodium citrate (SSC) is 1 M (see Suggs et al., Developmental Biology Using Purified Genes, page 683, Brown and Fox (ed.) (1981)).

A natural nucleic acid encoding a protein such as a polypeptide having the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8 or a variant or fragment thereof is, for example, one of the nucleic acid sequences of SEQ ID NO: 1, 3, 5 or 7, respectively. It is easily separated from a cDNA library having PCR primers and hybridization probes containing portions or variants thereof. A nucleic acid encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8 or a variant or fragment thereof is essentially 1% bovine serum albumin (BSA); 500 mM sodium phosphate (NaPO ₄ ); 1 mM EDTA; defined by hybridization buffer containing 7% SDS at a temperature of 42 ° C., and essentially 2 × SSC (600 mM NaCl; 60 mM sodium citrate); wash buffer containing 0.1% SDS at 50 ° C. 1% bovine serum albumin (BSA), preferably at essentially a temperature of 50 ° C .; 500 mM sodium phosphate (NaPO ₄ ); 15% formamide; 1 mM EDTA; 7% SDS Hybridization buffer, and essentially 50 ° C 1 × SSC (300 mM NaCl; 30 mM sodium citrate); 1% bovine serum albumin (most preferably essentially at a temperature of 50 ° C.) under low stringent conditions defined by a wash buffer containing 1% SDS. BSA); 200 mM sodium phosphate (NaPO ₄ ); 15% formamide; 1 mM EDTA; hybridization buffer containing 7% SDS, and essentially 0.5 × SSC at 65 ° C. (150 mM NaCl; 15 mM sodium citrate) Capable of hybridizing with one or a portion of the sequence shown in SEQ ID NO: 1 under low stringency conditions defined by a wash buffer containing 0.1% SDS.

  As used herein, the percentage of “identity”, “homology” and “similarity” of sequences (such as amino acids or nucleic acids) is determined by comparing two sequences that are optimally aligned in a comparison window. . Here, the reference sequence for optimal alignment of the two sequences (a gap may occur if the other sequences contain additions, in the portion of the polynucleotide or polypeptide sequence within the comparison window, Reference sequences herein shall include no additions or deletions (ie, gaps) as compared to the reference). Find the number of match positions by determining the number of positions where the same nucleobase or amino acid residue is found in both sequences, and divide the number of match positions by the total number of positions in the comparison window. Is multiplied by 100 to calculate the percent identity. When used in a search, homology is assessed using an appropriate one from a variety of sequence comparison algorithms and programs well known in the art. Such algorithms and programs include TBLASTN, BLASTP, FASTA, TFASTA and CLUSTALW (Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85 (8): 2444-2448, Altschul et al., 1990 ,. J. Mol.Biol.215 (3): 403-410, Thompson et al., 1994, Nucleic Acids Res.22 (2): 4673-4680, Higgins et al., 1996, Methods Enzymol.266: 383-402. Altschul et al., 1990, J. Mol.Biol.215 (3): 403-410, Altschul et al. 1993, Nature Genetics 3: 266-272), but is not limited thereto. In a particularly preferred embodiment, the Basic Local Alignment Tool (BLAST) known in the prior art (eg, Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2267-2268, Altschul et al., 1990). J. Mol. Biol. 215: 403-410, Altschul et al., 1993, Nature Genetics 3: 266-272, Altschul et al., 1997, Nuc. Acids Res.25: 3389-3402). Used to assess protein and nucleic acid sequence homology. In particular, a comparison or search can be achieved by performing the following operations using five dedicated BLAST programs.

(1) Compare amino acid query sequence with protein sequence database in BLASTP and BLAST3;
(2) Compare nucleotide query sequence with nucleotide sequence database at BLASTN;
(3) Comparison of a conceptual translation product obtained by converting a nucleotide query sequence (both strands) with BLASTX in six reading frames with a protein sequence database;
(4) Compare protein query sequence with TBLASTN to nucleotide sequence database converted in all six reading frames (both strands);
(5) Comparison of nucleotide query sequence converted by TBLASTX in 6 reading frames with nucleotide sequence database converted in 6 reading frames.

  The BLAST program identifies similar segments called “high-score segment pairs” between an amino acid query sequence or nucleic acid query sequence, and preferably a test sequence obtained from a protein sequence database or nucleic acid sequence database. Thus, a homologous sequence is identified. High score segment pairs are preferably identified (ie, aligned) by a scoring matrix well known in the art. Preferably, a BLOSUM62 matrix (Gonnet et al., 1992, Science 256: 1443-1445, Henikoff and Henikoff, 1993, Proteins 17: 49-61) is used as the scoring matrix. Although not as preferred as this matrix, a PAM or PAM250 matrix can also be used (see, for example, Schwartz and Dayhoff, eds., 1978, Matrixes for Detection Distance Relationships: Atlas of Protein Sequence and Stance and Stench. thing). The BLAST program evaluates the statistical significance of all identified high score segment pairs and preferably selects segments that meet a user-defined threshold level of significance, such as a user-specific homology rate. It is preferred to evaluate the statistical significance of high score segment pairs using Karlin's formula for statistical significance (see Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2267-2268). thing).

  In the present specification, the “primer” refers to a substance necessary for initiation of a reaction of a polymer compound to be synthesized in a polymer synthase reaction. In the synthesis reaction of a nucleic acid molecule, a nucleic acid molecule (for example, DNA or RNA) complementary to a partial sequence of a polymer compound to be synthesized can be used.

  Examples of nucleic acid molecules that are usually used as primers include those having a nucleic acid sequence of at least 8 consecutive nucleotides that is complementary to the nucleic acid sequence of the target gene. Such a nucleic acid sequence is preferably at least 9 contiguous nucleotides long, more preferably 10 contiguous nucleotides long, even more preferably 11 contiguous nucleotides long, 12 contiguous nucleotides long, 13 19 contiguous nucleotide lengths, 14 contiguous nucleotide lengths, 15 contiguous nucleotide lengths, 16 contiguous nucleotide lengths, 17 contiguous nucleotide lengths, 18 contiguous nucleotide lengths, 19 contiguous lengths It can be a nucleic acid sequence of nucleotide length, 20 contiguous nucleotide lengths, 25 contiguous nucleotide lengths, 30 contiguous nucleotide lengths, 40 contiguous nucleotide lengths, 50 contiguous nucleotide lengths. The nucleic acid sequence used as a primer is a nucleic acid that is at least 70% homologous, more preferably at least 80% homologous, more preferably 90% homologous, most preferably 95% homologous to the sequence described above. Contains an array. A sequence suitable as a primer may vary depending on the nature of the sequence intended for synthesis (amplification), but those skilled in the art can appropriately design a primer according to the intended sequence. Such primer design is well known in the art, and may be performed manually or using a computer program (eg, LASERGENE, PrimerSelect, DNAStar).

  As used herein, “substitution, addition or deletion” of a polypeptide or polynucleotide is a substitution of an amino acid or a substitute thereof, or a nucleotide or a substitute thereof, respectively, with respect to the original polypeptide or polynucleotide. , Adding or removing. Such substitution, addition, or deletion techniques are well known in the art, and examples of such techniques include site-directed mutagenesis techniques. The number of substitutions, additions or deletions may be any number as long as it is one or more, and such number is a function (for example, information on hormones, cytokines) in a variant having the substitution, addition or deletion. As long as the transmission function is maintained, the number can be increased. For example, such a number can be one or several, and preferably can be within 20%, within 10%, or less than 100, less than 50, less than 25, etc. of the total length.

  Molecular biological techniques, biochemical techniques, and microbiological techniques used in this specification are well known and commonly used in the art, and are described in, for example, Sambrook J. et al. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, F .; M.M. (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, F .; M.M. (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associate ES and Wiley-Interscience; A. (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, F .; M.M. (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, F .; M.M. (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, M .; A. et al. (1995). PCR Strategies, Academic Press; Ausubel, F .; M.M. (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; J. et al. et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press, “Experimental Methods for Gene Transfer & Expression Analysis”, Yodosha, 1997, etc., which are related in this specification (may be all) Is incorporated by reference.

  For DNA synthesis technology and nucleic acid chemistry for producing artificially synthesized genes, see, for example, Gait, M. et al. J. et al. (1985). Oligonucleotide Synthesis: A Practical Approach, IRLPpress; Gait, M .; J. et al. (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F .; (1991). Oligonucleotides and Analogues: A Practical Approac, IRL Press; Adams, R .; L. et al. (1992). The Biochemistry of the Nucleic Acids, Chapman &Hall; Shabarova, Z. et al. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, G .; M.M. et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, G .; T.A. (I996). Bioconjugate Technologies, Academic Press, etc., which are incorporated herein by reference in the relevant part.

  In order to confirm the presence of the nucleic acid in the present specification, it can be evaluated by any appropriate method including a molecular biological measurement method such as radioactivity method, fluorescence method, Northern blot method, dot blot method, PCR method and the like.

  As used herein, “biological sample” refers to, but is not limited to, for example, blood, serum, urine, and / or biopsy tissue. A preferred biological sample in the present invention is blood (particularly peripheral blood), but is not limited thereto. Another preferred biological sample in the present invention is neutrophils.

“Antibody” includes, but is not limited to, antibodies and fragments thereof (preferably antigen-binding fragments). The terms include monoclonal antibodies, polyclonal antibodies, bispecific antibodies, Fab antibody fragments, F (ab) ₂ antibody fragments, Fv antibody fragments (eg, V _H or V _L ), single chain Fv antibody fragments, and dsFv antibodies. Includes fragments. Furthermore, the antibody molecules of the invention can be fully human antibodies, mouse antibodies, rat antibodies, rabbit antibodies, goat antibodies, chicken antibodies, humanized antibodies, or chimeric antibodies. The antibody of the present invention specifically reacts with a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8.

  “Neutralization” refers to a phenomenon in which a substance having biological activity loses its activity by binding to an antibody against it. In the present context, this biological activity is in particular a decrease in neutrophil function. In one aspect of the invention, this function of neutrophils is chemotaxis.

  “Neutralizing antibody” refers to an antibody that neutralizes biological activity. The “neutralizing antibody” in the present specification particularly refers to an antibody that neutralizes a decrease in neutrophil function. In one aspect of the present invention, this neutrophil function is chemotaxis.

  The nucleic acids or antibodies of the invention can be used in combination with anti-inflammatory agents. This anti-inflammatory agent includes, but is not limited to, any anti-inflammatory agent known in the art. A particularly useful anti-inflammatory agent used in combination with the composition of the present invention is minocycline (Periocrine®).

  In the present specification, the “neutrophil function-lowering inhibitor” refers to any factor that can inhibit and / or suppress the function decrease of neutrophils.

  The gene of the present invention can be knocked down (suppressed) using siRNA. A method for preparing siRNA from a predetermined gene is well known, and for example, it can be prepared by a method as described in Examples. Furthermore, (1) G or C is not continuously present 4 or more, (2) A or T is not continuously 4 or more, (3) G or C is not present 9 or more bases, etc. May be added. The siRNA of the present invention has 19 base length, 20 base length, 21 base length, 22 base length, 23 base length, 24 base length, 25 base length, 26 base length, 27 base length, 28 base length, 29 base length, Or it is 30 base length. The siRNA of the present invention is preferably 19 bases long. The siRNA of the present invention is also preferably 20 bases long. The siRNA of the present invention is also preferably 21 bases in length. The siRNA of the present invention is also preferably 22 bases in length. The siRNA of the present invention is also preferably 23 bases in length. The siRNA of the present invention is also preferably 24 bases in length. For the preparation method of siRNA, see the general procedure section of the Examples.

  The terms “express” and “expression” allow or cause information in a gene, RNA sequence or DNA sequence to become apparent (eg, cells involved in transcription and translation of the corresponding gene) To produce a protein by activating the function). A DNA sequence is expressed in or by a cell to form an “expression product” (eg, RNA (eg, mRNA) or protein). The expression product itself may also be said to be “expressed” by the cell.

  The term “transformation” means the introduction of a nucleic acid into a cell. The introduced gene or sequence may be referred to as a “clone”. A host cell that receives the introduced DNA or RNA has been “transformed” and is a “transformant” or a “clone”. The DNA or RNA introduced into the host cell can be from any source, and can be from a cell of the same genus or species as the host cell or from a cell of a different genus or species.

  The term “vector” refers to a medium (eg, a plasmid) into which a DNA or RNA sequence can be introduced into a host cell so as to transform the host and facilitate expression and / or replication of the introduced sequence as needed. ).

  Vectors that can be used in the present invention include plasmids, viruses, bacteriophages, integratable DNA fragments, and other media that can facilitate the introduction of nucleic acids into the host genome. A plasmid is the most commonly used form of vector, although all other forms of vectors that provide similar functions and are known or known in the art are wild-type herein. Suitable for use. See, for example, Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985 and Supplements, Elsevier, N .; Y. And Rodriguez et al., (Eds.), Vectors: A Survey of Molecular Cloning Vectors and Their Uses 1988, Buttersworth, Boston, MA.

  The term “expression system” means a host cell and compatible vector that, under appropriate conditions, can express a protein or nucleic acid carried by the vector and introduced into the host cell. Common expression systems include E. coli. E. coli host cells and plasmid vectors, insect host cells and baculovirus vectors, and mammalian host cells and vectors.

  Expression of the nucleic acid encoding the polypeptide set forth in SEQ ID NO: 2, 4, 6 or 8 of the present invention can be performed by conventional methods in either prokaryotic or eukaryotic cells. E. Although E. coli host cells are most frequently used in prokaryotic systems, many other bacteria (eg, various Pseudomonas and Bacillus strains) are known in the art, and these are used as well. obtain. Suitable host cells for expressing the nucleic acid encoding the polypeptide set forth in SEQ ID NO: 2, 4, 6 or 8 include prokaryotes and higher eukaryotes. Prokaryotes include both gram negative and gram positive organisms (eg, E. coli and B. subtilis). Higher eukaryotes are established tissue culture cells derived from animal cells (animal cells of both non-mammalian origin (eg, insect cells) and mammalian origin (eg, human, primate, and rodent)). Includes strains.

  Prokaryotic host-vector systems encompass a wide variety of vectors for many different species. Typical vectors for amplifying DNA are pBR322 or many of its derivatives (eg, pUC18 or pUC19). Vectors that can be used to express the polypeptide of SEQ ID NO: 2, 4, 6 or 8 include vectors containing the lac promoter (pUC series); vectors containing the trp promoter (pBR322-trp); Ipp promoter Vectors including (pIN series); vectors including λ-pP promoter or λ-pR promoter (pOTS); or vectors including hybrid promoter (eg, ptac) (pDR540), but are not limited thereto. Brousius et al., “Expression Vectors Employing Lambda-, trp-, lac-, and Ipp-derived Promoters”, Rodriguez and Denhardt (Ed.) Vectors: A Survey. See 205-236. Many polypeptides are described in US Pat. Nos. 4,952,496, 5,693,489, and 5,869,320, and Davanloo, P. et al. Et al. (1984) Proc. Natl. Acad. Sci. USA 81: 2035-2039; W. Et al. (1986) J. MoI. Mol. Biol. 189: 113-130; Rosenberg, A .; H. (1987) Gene 56: 125-135; and Dunn, J. et al. J. et al. (1988) Gene 68: 259, as disclosed in E. It can be expressed at high levels in the E. coli / T7 expression system.

  Higher eukaryotic tissue culture cells can also be used for recombinant production of the polypeptides set forth in SEQ ID NOs: 2, 4, 6 or 8 of the present invention. Although any higher eukaryotic tissue culture cell line (including insect baculovirus expression systems) can be used, mammalian cells are preferred. Such transformation, transfection and propagation of cells has become a routine procedure. Examples of useful cell lines include HeLa cells, Chinese hamster ovary (CHO) cell lines, J774 cells, Caco2 cells, rat infant kidney (BRK) cell lines, insect cell lines, avian cell lines, and monkey (COS) cells. Strains. Expression vectors for such cell lines usually include an origin of replication, a promoter, a translation initiation site, an RNA splice site (if genomic DNA is used), a polyadenylation site, and a transcription termination site. These vectors also usually contain a selection gene or amplification gene. Suitable expression vectors can be plasmids, viruses, or retroviruses that carry a promoter derived from a source such as, for example, adenovirus, SV40, parvovirus, vaccinia virus, or cytomegalovirus. Examples of expression vectors include pCR® 3.1, pCDNA1, pCD (Okayama et al. (1985) Mol. Cell Biol. 5: 1136), pMC1neo Poly-A (Thomas et al., (1987) Cell 51: 503), pREP8, pSVSPORT and derivatives thereof, and baculovirus vectors (eg, pAC373 or pAC610).

The present invention also provides the polypeptide of SEQ ID NO: 2, 4, 6 or 8 of the present invention and the polynucleotide of SEQ ID NO: 1, 3, 5 or 7, and the second polypeptide portion or the second polynucleotide portion. (Which may be referred to as “tags”). The fusion polypeptide of the present invention can be easily constructed, for example, by inserting the polynucleotide of the present invention or a fragment thereof into an expression vector. The fusions of the invention can include a tag that facilitates purification or detection. Such tags include glutathione-S-transferase (GST), hexahistidine (His6) tag, maltose binding protein (MBP) tag, hemagglutinin (HA) tag, cellulose binding protein (CBP) tag, and myc tag. Is mentioned. Detectable tags (eg, ³² P, ³⁵ S, ³ H, ^99m Tc, ¹²³ I, ¹¹¹ In, ⁶⁸ Ga, ¹⁸ F, ¹²⁵ I, ¹³¹ I, ^{113 m} In, ⁷⁶ Br, ⁶⁷ Ga, ^99m Tc, ¹²³ I, ¹¹¹ In and ⁶⁸ Ga) can also be used to label the polypeptides and polynucleotides of the invention. Methods for constructing and using such fusions are well known in the art.

  As used herein, “operably linked” refers to a transcriptional translational regulatory sequence (eg, promoter, enhancer, etc.) or a translational regulatory sequence that has the expression (operation) of a desired sequence. That means. In order for a promoter to be operably linked to a gene, the promoter is usually placed immediately upstream of the gene, but need not necessarily be adjacent.

  In this specification, any technique may be used for introducing a nucleic acid molecule into a cell, and examples thereof include transformation, transduction, and transfection. Such a technique for introducing a nucleic acid molecule is well known in the art and commonly used. For example, Ausubel F. et al. A. (1988), Current Protocols in Molecular Biology, Wiley, New York, NY; Sambrook J et al. (1987) Molecular Cloning: A Laboratory Manual, 2nd Ed. And its third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, a separate volume of experimental medicine “Gene Transfer & Expression Analysis Experimental Method” Yodosha, 1997, and the like. Gene transfer can be confirmed using the methods described herein, such as Northern blot, Western blot analysis, or other well-known conventional techniques.

  Moreover, as a method for introducing a vector, any of the above-described methods for introducing DNA into cells can be used. For example, transfection, transduction, transformation, etc. (for example, calcium phosphate method, liposome method, DEAE dextran method, electroporation method, method using particle gun (gene gun), etc.).

  As used herein, “pharmaceutically acceptable carrier” refers to a substance that is used when producing agricultural chemicals such as pharmaceuticals or animal drugs, and does not adversely affect active ingredients. Such pharmaceutically acceptable carriers include, for example, but are not limited to: antioxidants, preservatives, colorants, flavors, and diluents, emulsifiers, suspending agents, solvents , Fillers, bulking agents, buffering agents, delivery vehicles, excipients and / or pharmaceutical adjuvants.

  The type and amount of the drug used in the treatment method of the present invention is based on the information obtained by the method of the present invention (for example, information on the disease), the purpose of use, the target disease (type, severity, etc.), A person skilled in the art can easily determine the patient's age, weight, sex, medical history, the form or type of the site of the subject to be administered, and the like. The frequency with which the monitoring method of the present invention is applied to a subject (or patient) also depends on the purpose of use, target disease (type, severity, etc.), subject's age, weight, gender, medical history, treatment course, etc. Can be easily determined by those skilled in the art. The frequency of monitoring the disease state includes, for example, daily-once every several months (for example, once a week-once a month). It is preferable to perform monitoring once a week to once a month while observing the progress.

  If desired, more than one drug may be used in the treatment of the present invention. When two or more drugs are used, substances with similar properties or origins may be used, or drugs with different properties or origins may be used. Information regarding disease levels for methods of administering two or more such drugs can also be obtained by the methods of the present invention.

  A “subject” or “patient” is a human or dog, cat, marmoset, horse, cow, pig, sheep, goat, elephant, giraffe, chicken, lion, monkey, owl, rat, squirrel, hostolith, mouse, etc. Means vertebrates including As used herein, a “subject” or “patient” is preferably a mammal, more preferably a human.

(Pharmaceutical composition)
The polynucleotide of the present invention, an expression vector containing the polynucleotide, can be used as a component of a pharmaceutical composition for the treatment of invasive periodontitis and the inhibition of neutrophil function decline.

  In the present specification, the “effective amount” of a drug refers to an amount capable of exerting the intended drug effect of the drug. In the present specification, among such effective amounts, the minimum concentration may be referred to as the minimum effective amount. Such minimum effective amounts are well known in the art, and usually the minimum effective amount of a drug is determined by those skilled in the art or can be determined as appropriate by those skilled in the art. In order to determine such an effective amount, an animal model or the like can be used in addition to actual administration. The present invention is also useful in determining such effective amounts.

  As used herein, “pharmaceutically acceptable carrier” refers to a substance that is used when producing agricultural chemicals such as pharmaceuticals or animal drugs, and does not adversely affect active ingredients. Such pharmaceutically acceptable carriers include, for example, but are not limited to: antioxidants, preservatives, colorants, flavors, and diluents, emulsifiers, suspending agents, solvents , Fillers, bulking agents, buffering agents, delivery vehicles, excipients and / or agricultural or pharmaceutical adjuvants.

  The type and amount of the drug used in the treatment method of the present invention is based on the information obtained by the method of the present invention (for example, information on the disease), the purpose of use, the target disease (type, severity, etc.), A person skilled in the art can easily determine the patient's age, weight, sex, medical history, the form or type of the site of the subject to be administered, and the like. The frequency with which the monitoring method of the present invention is applied to a subject (or patient) also depends on the purpose of use, target disease (type, severity, etc.), patient age, weight, gender, medical history, treatment course, etc. In view of this, it can be easily determined by those skilled in the art. The frequency of monitoring the disease state includes, for example, daily-once every several months (for example, once a week-once a month). It is preferable to perform monitoring once a week to once a month while observing the progress.

  If desired, more than one drug may be used in the treatment of the present invention. When two or more drugs are used, substances with similar properties or origins may be used, or drugs with different properties or origins may be used. Information regarding disease levels for methods of administering two or more such drugs can also be obtained by the methods of the present invention.

  In the present invention, the expression level of the gene of the present invention, the presence and / or possibility of invasive periodontitis, and the like, with respect to organisms, cultured cells, tissues, etc. of similar types (for example, mice against humans) once. When neutrophil function decline is correlated, the analysis of the corresponding gene expression level can be correlated with the presence and / or likelihood of invasive periodontitis, and neutrophil decline. Those skilled in the art will readily understand.

  As used herein, a “kit” includes a plurality of containers and manufacturer's instructions, and each container includes a pharmaceutical composition of the present invention, other agents, and a carrier. A product.

  Hereinafter, the present invention will be described in detail with reference to examples and the like, but the present invention is not limited thereto.

(General method)
(Measurement of neutrophil chemotaxis)
6 ml of peripheral blood was collected from the midline brachial vein of a subject using a heparinized vacuum blood collection tube, and then 120 μl was dropped on a cover glass and incubated at 37 ° C. under 5% CO ₂ gas phase for 45 minutes. After incubation, neutrophils adhered on a cover glass were used after washing three times with phosphate buffered saline (PBS, pH 7.5 Nissui Pharmaceutical, Tokyo).

Chemotaxis was measured by the Zigmond chamber method. The Zigmond chamber method is a method in which a Zigmond chamber is used, a chemotactic factor and a culture solution are placed in each chamber, and neutrophils that migrate toward the chemotactic factor side are counted and measured. 10-8M N-formyl-methyl-phenylalanine (fMLP, SIGMA, MO. USA) as chemotactic factor, Hank's balanced salt solution (HBSS, pH 7.5 Nissui Pharmaceutical, Tokyo) and 10% as control A culture solution in which the geratin solution was mixed and adjusted to pH 7.2 with 1 M NaHCO ₃ was used. fMLP stimulation was performed at 37 ° C. under 5% CO ₂ gas phase for 20 minutes. Thereafter, the morphology of neutrophils stimulated with fMLP was observed using a phase-contrast microscope, and neutrophils that migrated neutrophils that changed their shape into spindles, neutrophils that did not migrate neutrophils that did not change their shape Sphere. Chemotaxis is shown as 100 percent of the number of neutrophils changed to spindle type relative to the total number of neutrophils observed.

(Separation of neutrophils)
Eighty milliliters of peripheral blood was collected from the midline brachial vein into a heparinized vacuum blood collection tube. A 15 ml disposable centrifuge tube (CORNING, USA) was overlaid with 3.5 ml of Hystapaque 1119 (SIGMA) and 1.5 ml of Hystapaque 1077 (SIGMA) on top of it. On top of that, 4-6 ml of blood was overlaid and centrifuged at 450 × g for 20 minutes. The neutrophil layer was collected and PBS was added to 50 ml. Furthermore, it was washed by centrifugation at 90 × g for 10 minutes, and neutrophils were collected as a precipitate. 10 ml of lysing buffer (NH ₂ Cl, KHCO ₃ , Na ₂ EDTA, pH 7.4, 4 ° C.) was added and suspended, and then allowed to stand for 6-10 minutes. After standing, PBS was added to a final volume of 50 ml and centrifuged at 90 × g for 10 minutes. After removing the supernatant, the hypotonic solution treatment was performed again in the same manner to remove mixed red blood cells. 50 ml of PBS was added to the obtained precipitate, and the number of cells was counted with a hemocytometer.

(Proteome analysis)
Neutrophils were suspended in 480 μl of RPMI1640-HEPES (SIGMA, USA) in 5 × 10 ⁶ separated neutrophils, and preincubated for 5 minutes at 37 ° C. in 5% CO ₂ gas phase. Next, 4 × 10 ⁻⁸ M fMLP was added and further incubated at 37 ° C., 5% CO ₂ gas phase conditions for 20 minutes.

  The fMLP-stimulated neutrophils were suspended in a cell wash buffer (10 mM TrisCl, 5 mM Magnesium acetate) and centrifuged at 4 ° C. and 3,000 × g for 4 minutes. After washing the cells three times by centrifugation, the precipitate was suspended in cell lysis buffer (30 mM Tris Cl, 2M Thiourea, 7M Urea, 4% CHAPS). The suspension was sonicated on ice and then centrifuged at 12,000 × g for 10 minutes at 4 ° C. to remove insoluble fractions. The protein prepared using pH INDICATOR PAPER pH 4.5-10 (Whatman, Germany) was confirmed to be pH 8.0-9.0.

  Immobiline Drystrip (pH 3-10 NL, 24 cm, Amersham Bioscience) was swollen with (8M Urea, 4% CHAPS, 1% Pharmalytes 3-10 for IEF, 13 mM Dithiothreitol, BroC5) The mixture was swelled with a solution (450 μl) at 20 ° C. for 12 hours. After swelling the strips, isoelectric focusing was performed at 61 kV-h. At the same time, as a gel for protein excision, Immobiline Drystrip was swollen with 450 μl of a mixed solution of a swelling solution and an unlabeled 500 μg protein sample, followed by isoelectric focusing. Note that IPGphor (Amersham Bioscience) was used for swelling and isoelectric focusing.

  Next, equilibration was performed in order to convert the Immobiline Drystrip after isoelectric focusing to the SDS buffer system. Strips gel was immersed in an equilibration solution (50 mM Tris Cl, 6M Urea, 30% Glycerol, 2% SDS, Bromophenol Blue) and 0.5% Dithiothreol for 10 minutes for equilibration. Further, the strip gel was immersed for 10 minutes in a solution obtained by adding 4.5% Iodoacetamide to the equilibration solution. After equilibration, the gel surface was lightly passed through SDS running buffer (25 mM Tris, 192 mM Glycine, 0.2% SDS) for the purpose of smoothing the gel surface.

  Equilibrated strip gels for analysis and cutting were placed on a 25.5 × 20.5 cm, 12.5% acrylamide gel, and conditions of 71 V, 168 mA, 12 W, 30 minutes, 239 V, 399 mA, 95 W, 5 hours. SDS-PAGE was performed. In order to fix the gel to the glass plate, the acrylamide gel for protein excision was subjected to Bind-Silane treatment on one side of the glass plate before gel preparation. SDS-PAGE was performed using Ettan Daltsix (Amersham Bioscience). All operations in this section were performed under light shielding.

  The protein excision gel is stained with Deep Purple Total Protein Stain (Amersham Bioscience), the gel is scanned with Typhoon 9400 (Amersham Bioscience), and the automated image analysis software DeCycler (Ascham) Data detection, digitization, spot matching, and protein expression analysis were performed.

(Identification of protein by MALDI-TOF-MS)
Mass spectrometry was performed with a matrix-assisted laser desorption ionization (MALDI-TOF-MS) method using biflex IV (BRUKER DALTONICS, USA). A mixed solution of a sample solution and a matrix (α-cyano-4-hydroxycinnamic acid) is dropped on a target plate (MTP 384 massive target gold plated T (BRUKER DALTONICS, USA)) and dried by natural drying. A mixed crystal was formed. The target plate was attached to the ion source, and the spectrum was detected by applying laser light under vacuum. In addition, Peptide calibration standard (BRUKER DALTONICS) was used as a standard. A database search was performed using Mascot Search.

(Purification of total RNA)
Total RNA was extracted from the neutrophils stimulated with fMLP by the acidphenol method using ISOGEN (Nippon Gene, Tokyo) and purified.

(Real-time PCR)
Based on 1.0 μg of total RNA, 1st strand cDNA was synthesized. The reverse transcription reaction was performed using Transscriptor First Strand cDNA Synthesis Kit (Roche, Germany). Using 1.0 μg of the obtained cDNA, monitoring was performed in real time using ABI PRISM 7700 system (Applied Biosystems), and quantitative analysis of mRNA expression was performed.

(Preparation of siRNA)
An annealed double-stranded synthetic siRNA is obtained from a siRNA provider known in the art (for example, Nippon Easy Tea, Toyama, Japan). The synthetic siRNA is dissolved in an RNAse-free solution, adjusted to a final concentration of 20 μM, and then introduced into cells.

(Example 1 Identification of Caldesmon gene, Lactoferrin gene, Heat shock 70 kDa protein gene, and Stac gene as marker genes)
With the log value (Average Ratio) 1.5 of the ratio between the average value of the protein spot expression in the invasive periodontitis patient group and the average value of the healthy person as the boundary, the expression level is changed between the invasive periodontitis patient and the healthy person. Different proteins were identified.

  As a result, caldesmon (P = 0.26, Average.Ratio = 1.99), lactoferrin (P = 0.033, Average.Ratio = 1.92), heat shock 70 kDa protein (P = 0.04, Average. Ratio = 1.50) and stac (P = 0.17, Average.Ratio = 1.50) are compared to healthy neutrophils, showing an invasive periodontal period that shows reduced neutrophil chemotaxis It was identified as a protein that was significantly expressed in neutrophils of patients with inflammation.

(Example 2) Expression analysis of marker gene at gene level
Of the four marker genes identified in Example 1, focusing on Caldesmon, Caldesmon expression kinetics in neutrophils was analyzed at the mRNA level. Patients with invasive periodontitis patients with reduced neutrophil chemotaxis, invasive periodontitis with chronic neutrophil chemotaxis, chronic periodontitis patients, and healthy subjects It was.

The neutrophils derived from these four groups of subjects were subjected to quantitative analysis of mRNA expression by real-time PCR using the following primers and probes.
Caldesmon forward primer;
GGTGAATGCCCCAGAACAGGTGT (SEQ ID NO: 9)
Caldesmon reverse primer;
GCGTTTTTGGCGTCTTTCC (SEQ ID NO: 10)
Taqman probe;
AGGCCAAGACAACCACCACAACAACT (SEQ ID NO: 11).

  The results are shown in FIG. As is clear from FIG. 1, the invasive periodontitis patient group showing a decrease in neutrophil chemotaxis ability is an invasive periodontitis patient group and a chronic periodontitis patient group showing no decrease in neutrophil chemotaxis ability. Compared with the healthy group, Caldesmon mRNA expression was clearly higher.

(Example 3 Promotion of reduced neutrophil chemotaxis by overexpression of Caldesmon)
Only the Caldesmon expression plasmid and the vector are introduced into neutrophils derived from healthy subjects, and the neutrophil chemotaxis is evaluated by the method described above. Chemotaxis is shown as 100 percent of the number of neutrophils changed to spindle type relative to the total number of neutrophils observed. In Caldesmon overexpressing neutrophils, chemotaxis ability is significantly reduced compared to neutrophils into which only the vector has been introduced. This result reveals that Caldesmon induces a decrease in neutrophil chemotaxis and that Caldesmon is a marker of neutrophil functional decline and invasive periodontitis.

(Example 4 Knockdown of Caldesmon gene by siRNA)
Caldesmon siRNA prepared as described above is introduced into neutrophils derived from patients with invasive periodontitis that show reduced neutrophil chemotaxis, and the chemotaxis is observed. In neutrophils knocked down by Caldesmon, a significant suppression and / or inhibition of a decrease in chemotaxis is observed compared to neutrophils into which siRNA has not been introduced. This result reveals that Caldesmon may be a target for the treatment of invasive periodontitis and neutrophil hypofunction.

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, this invention should not be limited and limited to this embodiment. It is understood that the scope of the present invention should be construed only by the claims. It is understood that those skilled in the art can implement an equivalent range based on the description of the present invention and the common general technical knowledge from the description of specific preferred embodiments of the present invention.

FIG. 1 shows an invasive periodontitis patient group showing a decrease in neutrophil chemotaxis ability, an invasive periodontitis patient group showing no reduction in neutrophil chemotaxis ability, a chronic periodontitis patient group, and a healthy person group Shows a comparison of Caldesmon mRNA expression levels of neutrophils. Bars show mean values + SD.

SEQ ID NO: 1 = Caldesmon nucleic acid sequence SEQ ID NO: 2 = Caldesmon amino acid sequence SEQ ID NO: 3 = Lactoferrin nucleic acid sequence SEQ ID NO: 4 = Lactoferrin amino acid sequence SEQ ID NO: 5 = Heat shock 70 kDa protein nucleic acid SEQ ID NO: 6 = Heat shock 70 kDa protein amino acid sequence SEQ ID NO: 7 = Stac nucleic acid sequence SEQ ID NO: 8 = Stac amino acid sequence SEQ ID NO: 9 = real-time PCR forward primer for Caldesmon SEQ ID NO: 10 = real-time PCR reverse primer for Caldesmon SEQ ID NO: 11 = Taqman probe

Claims (42)

A diagnostic marker for assessing the presence and / or likelihood of invasive periodontitis, comprising:
A nucleic acid that hybridizes under stringent conditions with a complementary strand of a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 1, and that encodes a physiologically active polypeptide;
A nucleic acid comprising a sequence at least 80% homologous to the nucleic acid sequence set forth in SEQ ID NO: 1 and encoding a polypeptide having physiological activity;
A nucleic acid comprising a fragment of the nucleic acid sequence set forth in SEQ ID NO: 1;
A pair of nucleic acids comprising a fragment of the nucleic acid sequence set forth in SEQ ID NO: 1 and having a length of at least 10 bases and capable of amplifying at least a part of the nucleic acid sequence set forth in SEQ ID NO: 1 as a PCR primer ,
An antibody that reacts with a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2,
A nucleic acid that hybridizes under stringent conditions with a complementary strand of a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 3, and that encodes a physiologically active polypeptide;
A nucleic acid comprising a sequence that is at least 80% homologous to the nucleic acid sequence set forth in SEQ ID NO: 3 and that encodes a physiologically active polypeptide;
A nucleic acid comprising a fragment of the nucleic acid sequence set forth in SEQ ID NO: 3,
A pair of nucleic acids comprising a fragment of the nucleic acid sequence set forth in SEQ ID NO: 3 and having a length of at least 10 bases and capable of amplifying at least a part of the nucleic acid sequence set forth in SEQ ID NO: 1 as a PCR primer ,
An antibody that reacts with a polypeptide having the amino acid sequence of SEQ ID NO: 4,
A nucleic acid that hybridizes under stringent conditions with a complementary strand of a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 5, and that encodes a physiologically active polypeptide;
A nucleic acid comprising a sequence at least 80% homologous to the nucleic acid sequence set forth in SEQ ID NO: 5 and encoding a polypeptide having physiological activity;
A nucleic acid comprising a fragment of the nucleic acid sequence set forth in SEQ ID NO: 5,
A pair of nucleic acids comprising a fragment of the nucleic acid sequence set forth in SEQ ID NO: 5 and having a length of at least 10 bases and capable of amplifying at least a part of the nucleic acid sequence set forth in SEQ ID NO: 1 as a PCR primer ,
An antibody that reacts with a polypeptide having the amino acid sequence of SEQ ID NO: 6,
A nucleic acid that hybridizes under stringent conditions with a complementary strand of a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 7, and that encodes a physiologically active polypeptide;
A nucleic acid comprising a sequence at least 80% homologous to the nucleic acid sequence set forth in SEQ ID NO: 7, which encodes a polypeptide having physiological activity;
A nucleic acid comprising a fragment of the nucleic acid sequence set forth in SEQ ID NO: 7,
A pair of nucleic acids comprising a fragment of the nucleic acid sequence set forth in SEQ ID NO: 7 and having a length of at least 10 bases and capable of amplifying at least a part of the nucleic acid sequence set forth in SEQ ID NO: 1 as a PCR primer ,
An antibody that reacts with a polypeptide having the amino acid sequence of SEQ ID NO: 8,
An invasive periodontitis diagnostic marker comprising a nucleic acid or an antibody selected from the group consisting of: The diagnostic marker according to claim 1, wherein:
A nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 1;
A nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 3,
A nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 5, and a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 7,
A diagnostic marker comprising a nucleic acid selected from the group consisting of: A diagnostic marker according to claim 1, comprising a nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 1. The diagnostic marker according to claim 1, wherein:
An antibody that reacts with a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2,
An antibody that reacts with a polypeptide having the amino acid sequence of SEQ ID NO: 4,
An antibody that reacts with the polypeptide having the amino acid sequence set forth in SEQ ID NO: 6, and an antibody that reacts with the polypeptide having the amino acid sequence set forth in SEQ ID NO: 8,
A diagnostic marker comprising an antibody selected from the group consisting of: The diagnostic marker according to claim 1, comprising an antibody that reacts with a polypeptide having the amino acid sequence of SEQ ID NO: 2. A method for assessing the presence and / or likelihood of invasive periodontitis in a subject comprising:
(A) (i) a nucleic acid comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7, or (ii) a stringent and complementary strand of the nucleic acid of (i) A nucleic acid that hybridizes under conditions, the nucleic acid encoding a polypeptide having physiological activity,
Measuring the expression level of a biological sample derived from the subject, and (b) evaluating the prognosis and / or malignancy in the subject using the expression level as an index,
Including the method. A method for assessing the presence and / or likelihood of invasive periodontitis in a subject comprising:
(A) (i) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8, or (ii) SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 And a polypeptide having an amino acid sequence containing one or several mutations, substitutions, insertions or deletions in an amino acid sequence selected from the group consisting of SEQ ID NO: 8, and having physiological activity,
Measuring the expression level of a biological sample derived from the subject, and (b) evaluating the prognosis and / or malignancy in the subject using the expression level as an index,
Including the method. A diagnostic marker for assessing neutrophil functional decline, the following:
A nucleic acid that hybridizes under stringent conditions with a complementary strand of a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 1, and that encodes a physiologically active polypeptide;
A nucleic acid comprising a sequence at least 80% homologous to the nucleic acid sequence set forth in SEQ ID NO: 1 and encoding a polypeptide having physiological activity;
A nucleic acid comprising a fragment of the nucleic acid sequence set forth in SEQ ID NO: 1;
A pair of nucleic acids comprising a fragment of the nucleic acid sequence set forth in SEQ ID NO: 1 and having a length of at least 10 bases and capable of amplifying at least a part of the nucleic acid sequence set forth in SEQ ID NO: 1 as a PCR primer ,
An antibody that reacts with a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2,
A nucleic acid that hybridizes under stringent conditions with a complementary strand of a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 3, and that encodes a physiologically active polypeptide;
A nucleic acid comprising a sequence that is at least 80% homologous to the nucleic acid sequence set forth in SEQ ID NO: 3 and that encodes a physiologically active polypeptide;
A nucleic acid comprising a fragment of the nucleic acid sequence set forth in SEQ ID NO: 3,
A pair of nucleic acids comprising a fragment of the nucleic acid sequence set forth in SEQ ID NO: 3 and having a length of at least 10 bases and capable of amplifying at least a part of the nucleic acid sequence set forth in SEQ ID NO: 1 as a PCR primer ,
An antibody that reacts with a polypeptide having the amino acid sequence of SEQ ID NO: 4,
A nucleic acid that hybridizes under stringent conditions with a complementary strand of a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 5, and that encodes a physiologically active polypeptide;
A nucleic acid comprising a sequence at least 80% homologous to the nucleic acid sequence set forth in SEQ ID NO: 5 and encoding a polypeptide having physiological activity;
A nucleic acid comprising a fragment of the nucleic acid sequence set forth in SEQ ID NO: 5,
A pair of nucleic acids comprising a fragment of the nucleic acid sequence set forth in SEQ ID NO: 5 and having a length of at least 10 bases and capable of amplifying at least a part of the nucleic acid sequence set forth in SEQ ID NO: 1 as a PCR primer ,
An antibody that reacts with a polypeptide having the amino acid sequence of SEQ ID NO: 6,
A nucleic acid that hybridizes under stringent conditions with a complementary strand of a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 7, and that encodes a physiologically active polypeptide;
A nucleic acid comprising a sequence at least 80% homologous to the nucleic acid sequence set forth in SEQ ID NO: 7, which encodes a polypeptide having physiological activity;
A nucleic acid comprising a fragment of the nucleic acid sequence set forth in SEQ ID NO: 7,
A pair of nucleic acids comprising a fragment of the nucleic acid sequence set forth in SEQ ID NO: 7 and having a length of at least 10 bases and capable of amplifying at least a part of the nucleic acid sequence set forth in SEQ ID NO: 1 as a PCR primer ,
An antibody that reacts with a polypeptide having the amino acid sequence of SEQ ID NO: 8,
A diagnostic marker for decreased neutrophil function, comprising a nucleic acid or antibody selected from the group consisting of: The diagnostic marker according to claim 8, wherein:
A nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 1;
A nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 3,
A nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 5, and a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 7,
A diagnostic marker comprising a nucleic acid selected from the group consisting of: A diagnostic marker according to claim 8, comprising a nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 1. The diagnostic marker according to claim 8, wherein:
An antibody that reacts with a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2,
An antibody that reacts with a polypeptide having the amino acid sequence of SEQ ID NO: 4,
An antibody that reacts with the polypeptide having the amino acid sequence set forth in SEQ ID NO: 6, and an antibody that reacts with the polypeptide having the amino acid sequence set forth in SEQ ID NO: 8,
A diagnostic marker comprising an antibody selected from the group consisting of: The diagnostic marker according to claim 8, comprising an antibody that reacts with a polypeptide having the amino acid sequence of SEQ ID NO: 2. A method for assessing neutrophil functional decline in a subject comprising:
(A) (i) a nucleic acid comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7, or (ii) a stringent and complementary strand of the nucleic acid of (i) A nucleic acid that hybridizes under conditions, the nucleic acid encoding a polypeptide having physiological activity,
Measuring the expression level of a biological sample derived from the subject, and (b) evaluating a decrease in the function of the neutrophil in the subject using the expression level as an index.
Including the method. A method for assessing neutrophil functional decline in a subject comprising:
(A) (i) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8, or (ii) SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 And a polypeptide having an amino acid sequence containing one or several mutations, substitutions, insertions or deletions in an amino acid sequence selected from the group consisting of SEQ ID NO: 8, and having physiological activity,
Measuring the expression level of a biological sample derived from the subject, and (b) evaluating a decrease in the function of the neutrophil in the subject using the expression level as an index.
Including the method. A composition for treating invasive periodontitis, comprising:
An antibody that reacts with a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2,
An antibody that reacts with a polypeptide having the amino acid sequence of SEQ ID NO: 4,
An antibody that reacts with the polypeptide having the amino acid sequence set forth in SEQ ID NO: 6, and an antibody that reacts with the polypeptide having the amino acid sequence set forth in SEQ ID NO: 8,
A composition comprising an antibody selected from the group consisting of: A composition for treating invasive periodontitis, comprising:
An antibody that reacts with a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2,
A composition comprising: The composition according to claim 15 or 16, further comprising an anti-inflammatory agent. The composition according to claim 17, wherein the anti-inflammatory agent is minocycline (Periocrine®). A composition that inhibits and / or suppresses neutrophil functional decline,
An antibody that reacts with a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2,
An antibody that reacts with a polypeptide having the amino acid sequence of SEQ ID NO: 4,
An antibody that reacts with the polypeptide having the amino acid sequence set forth in SEQ ID NO: 6, and an antibody that reacts with the polypeptide having the amino acid sequence set forth in SEQ ID NO: 8,
A composition comprising an antibody selected from the group consisting of: A composition that inhibits and / or suppresses neutrophil functional decline,
An antibody that reacts with a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2,
A composition comprising: 21. The composition according to claim 19 or 20, further comprising a neutrophil function-reducing inhibitor. A composition for treating invasive periodontitis, the composition comprising:
The nucleic acid sequence set forth in SEQ ID NO: 1,
The nucleic acid sequence set forth in SEQ ID NO: 3,
The nucleic acid sequence set forth in SEQ ID NO: 5, and the nucleic acid sequence set forth in SEQ ID NO: 7,
A nucleic acid that reduces the expression level of a gene comprising a nucleic acid sequence selected from the group consisting of:
Composition. 23. The composition of claim 22, comprising
Comprising a nucleic acid that reduces the expression level of a gene comprising the nucleic acid sequence of SEQ ID NO: 1.
Composition. The nucleic acid is
A nucleic acid that hybridizes under stringent conditions with a complementary strand of a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 1, and is a nucleic acid fragment that encodes a physiologically active polypeptide. A nucleic acid comprising a sequence that is at least 80% homologous to the nucleic acid sequence and is a fragment of a nucleic acid that encodes a physiologically active polypeptide, and a stringent condition with the complementary strand of the nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 3 A nucleic acid that hybridizes underneath and is a fragment of a nucleic acid that encodes a polypeptide having physiological activity,
A nucleic acid comprising a nucleic acid sequence which is at least 80% homologous to the nucleic acid sequence described in SEQ ID NO: 3 and which is a fragment of a nucleic acid encoding a polypeptide having physiological activity. Complementation of a nucleic acid comprising the nucleic acid sequence described in SEQ ID NO: 5 A nucleic acid that hybridizes with a strand under stringent conditions, and that is a fragment of a nucleic acid that encodes a physiologically active polypeptide, a nucleic acid comprising a sequence that is at least 80% homologous to the nucleic acid sequence set forth in SEQ ID NO: 5 A nucleic acid that is a fragment of a nucleic acid encoding a polypeptide having biological activity,
A nucleic acid that hybridizes under stringent conditions with a complementary strand of a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 7, a nucleic acid fragment that encodes a polypeptide having physiological activity, and SEQ ID NO: 7 23. The composition of claim 22, wherein the composition is a nucleic acid comprising a sequence that is at least 80% homologous to the described nucleic acid sequence and is a fragment of a nucleic acid that encodes a biologically active polypeptide. The nucleic acid is
A nucleic acid that hybridizes under stringent conditions with a complementary strand of a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 1, and a nucleic acid fragment that encodes a physiologically active polypeptide;
25. The composition of claim 24, wherein 26. The composition of claims 22-25, further comprising an anti-inflammatory agent. 27. The composition of claim 26, wherein the anti-inflammatory agent is minocycline (Periocrine <(R)>). A composition that inhibits and / or suppresses neutrophil functional decline, the composition comprising:
The nucleic acid sequence set forth in SEQ ID NO: 1,
The nucleic acid sequence set forth in SEQ ID NO: 3,
The nucleic acid sequence set forth in SEQ ID NO: 5, and the nucleic acid sequence set forth in SEQ ID NO: 7,
A nucleic acid that reduces the expression level of a gene comprising a nucleic acid sequence selected from the group consisting of:
Composition. 30. The composition of claim 28, wherein
Comprising a nucleic acid that reduces the expression level of a gene comprising the nucleic acid sequence of SEQ ID NO: 1.
Composition. The nucleic acid is
A nucleic acid that hybridizes under stringent conditions with a complementary strand of a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 1, and is a nucleic acid fragment that encodes a physiologically active polypeptide. A nucleic acid comprising a sequence that is at least 80% homologous to the nucleic acid sequence and is a fragment of a nucleic acid that encodes a physiologically active polypeptide, and a stringent condition with the complementary strand of the nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 3 A nucleic acid that hybridizes underneath and is a fragment of a nucleic acid that encodes a polypeptide having physiological activity,
A nucleic acid comprising a nucleic acid sequence which is at least 80% homologous to the nucleic acid sequence described in SEQ ID NO: 3 and which is a fragment of a nucleic acid encoding a polypeptide having physiological activity. Complementation of a nucleic acid comprising the nucleic acid sequence described in SEQ ID NO: 5 A nucleic acid that hybridizes with a strand under stringent conditions, and that is a fragment of a nucleic acid that encodes a physiologically active polypeptide, a nucleic acid comprising a sequence that is at least 80% homologous to the nucleic acid sequence set forth in SEQ ID NO: 5 A nucleic acid that is a fragment of a nucleic acid encoding a polypeptide having biological activity,
A nucleic acid that hybridizes under stringent conditions with a complementary strand of a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 7, a nucleic acid fragment that encodes a polypeptide having physiological activity, and SEQ ID NO: 7 29. The composition of claim 28, wherein the composition is selected from the group consisting of nucleic acids that are nucleic acids comprising a sequence that is at least 80% homologous to the nucleic acid sequence described and is a fragment of a nucleic acid that encodes a biologically active polypeptide. The nucleic acid is
30. The composition of claim 29, which is a nucleic acid that hybridizes under stringent conditions with a complementary strand of a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 1 and that encodes a physiologically active polypeptide. object. The composition according to any one of claims 28 to 31, further comprising a neutrophil function-reducing inhibitor. A screening method for an anti-inflammatory agent, the method comprising:
(A) (i) a nucleic acid comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7, or (ii) a stringent and complementary strand of the nucleic acid of (i) A nucleic acid that hybridizes under conditions, the nucleic acid encoding a polypeptide having physiological activity,
Contacting a cell expressing a candidate compound with a candidate compound;
(B) measuring the expression level of the nucleic acid in the cell; and (c) selecting a compound having anti-inflammatory activity from the candidate compounds using the measured expression level as an index;
Including the method. A screening method for an anti-inflammatory agent, the method comprising:
(A) (i) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8, or (ii) SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 And a polypeptide having an amino acid sequence containing one or several mutations, substitutions, insertions or deletions in an amino acid sequence selected from the group consisting of SEQ ID NO: 8, and having physiological activity,
Contacting a cell expressing a candidate compound with a candidate compound;
(B) measuring the expression level of the polypeptide in the cell; and (c) selecting a compound having anti-inflammatory activity from the candidate compounds using the measured expression level as an index;
Including the method. A screening method for an anti-inflammatory agent, the method comprising:
(A) (i) a nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 1, or (ii) a nucleic acid that hybridizes with a complementary strand of the nucleic acid of (i) under stringent conditions, and having a physiologically active polypeptide Nucleic acid encoding,
Contacting a cell expressing a candidate compound with a candidate compound;
(B) measuring the expression level of the nucleic acid in the cell; and (c) selecting a compound having anti-inflammatory activity from the candidate compounds using the measured expression level as an index;
Including the method. A screening method for an anti-inflammatory agent, the method comprising:
(A) (i) a polypeptide having the amino acid sequence of SEQ ID NO: 2, or (ii) an amino acid sequence comprising one or several mutations, substitutions, insertions or deletions in the amino acid sequence of SEQ ID NO: 2, and A polypeptide having physiological activity,
Contacting a cell expressing a candidate compound with a candidate compound;
(B) measuring the expression level of the polypeptide in the cell; and (c) selecting a compound having anti-inflammatory activity from the candidate compounds using the measured expression level as an index;
Including the method. A method for screening a neutrophil function-reducing inhibitor, the method comprising:
(A) (i) a nucleic acid comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7, or (ii) a stringent and complementary strand of the nucleic acid of (i) A nucleic acid that hybridizes under conditions, the nucleic acid encoding a polypeptide having physiological activity,
Contacting a cell expressing a candidate compound with a candidate compound;
(B) measuring the expression level of the nucleic acid in the cell; and (c) selecting a compound having anti-inflammatory activity from the candidate compounds using the measured expression level as an index;
Including the method. A method for screening a neutrophil function-reducing inhibitor, the method comprising:
(A) (i) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8, or (ii) SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 And a polypeptide having an amino acid sequence containing one or several mutations, substitutions, insertions or deletions in an amino acid sequence selected from the group consisting of SEQ ID NO: 8, and having physiological activity,
Contacting a cell expressing a candidate compound with a candidate compound;
(B) measuring the expression level of the polypeptide in the cell; and (c) selecting a compound having anti-inflammatory activity from the candidate compounds using the measured expression level as an index;
Including the method. A method for screening a neutrophil function-reducing inhibitor, the method comprising:
(A) (i) a nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 1, or (ii) a nucleic acid that hybridizes with a complementary strand of the nucleic acid of (i) under stringent conditions, and having a physiologically active polypeptide Nucleic acid encoding,
Contacting a cell expressing a candidate compound with a candidate compound;
(B) measuring the expression level of the nucleic acid in the cell; and (c) selecting a compound having anti-inflammatory activity from the candidate compounds using the measured expression level as an index;
Including the method. A method for screening a neutrophil function-reducing inhibitor, the method comprising:
(A) (i) a polypeptide having the amino acid sequence of SEQ ID NO: 2, or (ii) an amino acid sequence comprising one or several mutations, substitutions, insertions or deletions in the amino acid sequence of SEQ ID NO: 2, and A polypeptide having physiological activity,
Contacting a cell expressing a candidate compound with a candidate compound;
(B) measuring the expression level of the polypeptide in the cell; and (c) selecting a compound having anti-inflammatory activity from the candidate compounds using the measured expression level as an index;
Including the method. A kit for assessing the possibility of invasive periodontitis, the kit comprising:
A composition according to any one of claims 1 to 5, and instructions for evaluating the possibility of periodontitis,
A kit comprising: A kit for evaluating the possibility of neutrophil function decline, the kit comprising:
A composition according to any one of claims 8 to 12, and instructions for measuring the possibility of functional decline of the neutrophils,
A kit comprising: