JPWO2006134960A1 - Screening method for anti-inflammatory agents - Google Patents

Abstract

A simple screening method for obtaining an anti-inflammatory drug is provided. The polypeptide used in the screening method of the present invention is involved in the expression of cell adhesion molecules and plays an important role in the adhesion of vascular endothelial cells to leukocytes. By evaluating, a vascular endothelial cell adhesion inhibitor can be screened.

Description

  The present invention relates to a screening method for a vascular endothelial cell adhesion inhibitor and an anti-inflammatory agent.

  Various inflammatory diseases are caused by accumulation and infiltration of leukocytes in the inflamed lesion. In order for leukocytes to accumulate and infiltrate into lymph nodes or inflamed foci, it must first adhere to vascular endothelial cells. This adhesion process includes the following three stages: 1) rolling of leukocytes on vascular endothelial cells, 2) activation of leukocytes, and 3) strong adhesion of leukocytes to vascular endothelial cells. The first rolling (stage 1)) involves E-selectin, one of the cell adhesion molecules induced and expressed on vascular endothelial cells by inflammatory cytokines, and sialyl Lewis X (sLeX) expressed on leukocytes. It has been clarified that it is caused by binding to a sugar chain antigen. Subsequent activation (stage 2)) and strong adhesion (stage 3)) are cell adhesion molecules VCAM- induced and expressed on vascular endothelial cells by integrins expressed on leukocytes and inflammatory cytokines. 1 (Vascular Cell Adhesion Molecule-1) or cell adhesion molecule ICAM-1 (Intercellular Adhesion Molecule-1) has been revealed to be caused by a cell adhesion molecule belonging to the immunoglobulin superfamily (Non-patent Document 1) ).

  It has also been reported that the expression of various cell adhesion molecules is increased in various inflammatory lesions, and that they are involved in the pathogenesis of chronic inflammation. Yes.

  These phenomena occur in inflammatory diseases with leukocyte infiltration at the lesion site, such as rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, Hashimoto's thyroiditis, nodular periarteritis, ulcerative colitis, etc. It is thought that. In addition, these phenomena are known to cause allergic diseases accompanied by leukocyte infiltration.

Based on the above, suppressing the expression of various cell adhesion molecules induced by inflammatory cytokines at the inflammatory site can prevent the accumulation and infiltration of leukocytes into the inflammatory lesion, thereby enabling treatment of inflammatory diseases. It is considered to be.
Molecular Medicine, 1995, vol. 32, p. 90

  The object of the present invention is to provide various inflammatory diseases that have been difficult to treat with conventional anti-inflammatory agents, which have a novel action mechanism of suppressing the expression of cell adhesion molecules and suppressing adhesion of vascular endothelial cells to leukocytes. The object is to provide a simple screening system for obtaining useful substances having high therapeutic effects on autoimmune diseases.

As a result of diligent research, the present inventors have found that among 6 genes found as genes whose expression was induced in human umbilical vein endothelial cells (HUVEC) by addition of TNF-α, which is a kind of inflammatory cytokine. The present invention was completed by finding that the gene is involved in the expression of cell adhesion molecules such as ICAM-1 and VCAM-1, or that vascular endothelial cells are involved in adhesion to white blood cells. Substances that decrease the expression level of polypeptides encoded by these genes or inhibit the functions of the polypeptides are substances that suppress adhesion between vascular endothelial cells and leukocytes, and these substances are novel It is thought to be an anti-inflammatory drug based on the mechanism.
That is, according to the present invention,
[1]
(1) A method for screening a vascular endothelial cell adhesion inhibitor comprising a step of bringing a vascular endothelial cell into contact with a test substance, and (2) a step of measuring the expression level of the gene described in any of the following:
1) DNA consisting of a base sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 or 23, or 2) SEQ ID NO: 1, 3, 5, 7, (A) a cell adhesion molecule that hybridizes with a DNA comprising the nucleotide sequence represented by 9, 11, 13, 15, 17, 19, 21, or 23 under stringent conditions and in the presence of an inflammatory cytokine. And / or (b) a polypeptide encoding a polypeptide having a function of inducing adhesion between vascular endothelial cells and white blood cells,
[2]
(1) A method for screening a vascular endothelial cell adhesion inhibitor comprising a step of bringing a vascular endothelial cell into contact with a test substance, and (2) a step of measuring the expression level of the polypeptide described in any of the following:
1) Polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24, or 2) SEQ ID NO: 2, 4, 6, 8 In the amino acid sequence represented by 10, 12, 14, 16, 18, 20, 22, or 24, having an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, and / or added; and A polypeptide having a function of inducing expression of a cell adhesion molecule in the presence of an inflammatory cytokine, and / or (b) a function of inducing adhesion between a vascular endothelial cell and a white blood cell,
[3]
The method for screening a vascular endothelial cell adhesion inhibitor according to the above [1] or [2], further comprising a step of selecting a test substance that reduces the expression level of the gene or polypeptide compared to the absence of the test substance,
[4]
(1) A step of contacting a test substance with a cell transformed with an expression vector containing the gene according to [1] above and expressing a polypeptide encoded by the gene,
(2) including the step of contacting white blood cells with the cells, and (3) measuring the amount of adhesion between the white blood cells and the cells.
Screening method for vascular endothelial cell adhesion inhibitor,
[5]
A screening method for a vascular endothelial cell adhesion-suppressing substance according to the above [4], further comprising a step of selecting a test substance that reduces the amount of adhesion between leukocytes and the cells as compared with the absence of the test substance
[6]
(1) a step of bringing a test substance into contact with a cell transformed with an expression vector containing the gene according to [1] and expressing a polypeptide encoded by the gene; and (2) cell adhesion Including the step of measuring the expression level of the molecule,
Screening method for vascular endothelial cell adhesion inhibitor,
[7]
The method for screening a vascular endothelial cell adhesion-suppressing substance according to the above [6], further comprising a step of selecting a test substance that reduces the expression level of the cell adhesion molecule compared to the absence of the test substance;
[8]
(1) contacting the polypeptide according to [2] above with a test substance in the presence of a labeled specific conjugate of the polypeptide, and (2) the polypeptide and the specific conjugate. Including the step of measuring the amount of binding,
Screening method for vascular endothelial cell adhesion inhibitor,
[9]
A screening method for a vascular endothelial cell adhesion inhibitor according to the above [8], further comprising a step of selecting a test substance that reduces the binding amount of the specific conjugate as compared with the absence of the test substance;
[10]
The screening method according to any one of [1] to [9] above, wherein the cell adhesion molecule is any one selected from the group consisting of ICAM-1, VCAM-1, and E-selectin,
[11]
The screening method according to any one of [1] to [10] above, further comprising a step of contacting an inflammatory cytokine;
[12]
The screening method according to any one of the above [1] to [11], which selects a vascular endothelial cell adhesion inhibitor as an anti-inflammatory drug.

  By using the screening method of the present invention, it becomes possible to search for an unprecedented anti-inflammatory agent exhibiting an anti-inflammatory action by suppressing adhesion between vascular endothelial cells and white blood cells.

In the primary screening (Cell-ELISA) of Example 2d), siRNA that showed an expression-inhibitory effect of any one of ICAM-1, VCAM-1, and E-selectin was divided into four series of experiments using Cell-ELISA. The effect of suppressing the expression of ICAM-1 was reevaluated (Example 3a): secondary screening). This graph shows 9 siRNAs (hatched or gray columns) targeting 6 genes determined to be inflammatory cytokine-inducing polynucleotides from the experimental results of Example 3a) -c), negative control siRNA (white column) ) And secondary screening results of positive control siRNA (black column). Each column shows the average value of absorbance at 490 nm obtained by four series analysis, and the error bar shows the standard deviation. The shaded columns indicate siRNAs that showed a positive reaction in suppressing the expression of ICAM-1 in the primary screening, and the gray columns indicate siRNAs that showed a positive result with other adhesion molecules. In the primary screening (Cell-ELISA) of Example 2d), siRNA that showed an expression-inhibitory effect of any one of ICAM-1, VCAM-1, and E-selectin was divided into four series of experiments using Cell-ELISA. The effect of suppressing the expression of VCAM-1 was reevaluated (Example 3a): secondary screening). This graph shows 9 siRNAs (hatched or gray columns) targeting 6 genes determined to be inflammatory cytokine-inducing polynucleotides from the experimental results of Example 3a) -c), negative control siRNA (white column) ) And secondary screening results of positive control siRNA (black column). Each column shows the average value of absorbance at 490 nm obtained by four series analysis, and the error bar shows the standard deviation. The shaded columns indicate siRNAs that showed a positive reaction in suppressing the expression of VCAM-1 in the primary screening, and the gray columns indicate siRNAs that showed positive results for other adhesion molecules. In the primary screening (Cell-ELISA) of Example 2d), siRNA that showed an expression-inhibitory effect of any one of ICAM-1, VCAM-1, and E-selectin was divided into four series of experiments using Cell-ELISA. The effect of suppressing the expression of E-selectin was reevaluated (Example 3a): secondary screening). This graph shows 9 siRNAs (hatched or gray columns) targeting 6 genes determined to be inflammatory cytokine-inducing polynucleotides from the experimental results of Example 3a) -c), negative control siRNA (white column) ) And secondary screening results of positive control siRNA (black column). Each column shows the average value of absorbance at 490 nm obtained by four series analysis, and the error bar shows the standard deviation. The shaded columns indicate siRNAs that showed a positive reaction by suppressing the expression of E-selectin in the primary screening, and the gray columns indicate siRNAs that showed a positive result with other adhesion molecules. In the primary screening (Cell-ELISA) of Example 2d), the suppressive effect of siRNA that showed the suppressive effect of ICAM-1 expression was evaluated at the mRNA expression level by quantitative PCR. This graph shows seven siRNAs (hatched columns) that showed suppression of ICAM-1 expression among siRNAs of 6 genes judged to be inflammatory cytokine-inducing polynucleotides from the experimental results of Examples 3a) to c). The results for negative control siRNA (white column) and positive control siRNA (black column) are shown. In addition, an asterisk (*) represents that 1 or more reduction (50% or more expression reduction) was shown by the difference of (DELTA) Ct compared with negative control siRNA. a. ICAM-1 mRNA expression level (ΔCt value) was evaluated. b. Whether each siRNA of a knocked down its target gene was evaluated by mRNA expression level (ΔCt value). In the primary screening (Cell-ELISA) of Example 2d), the inhibitory effect of siRNA showing an inhibitory effect on VCAM-1 expression was evaluated at the mRNA expression level by quantitative PCR. This graph shows five siRNAs (hatched columns) that showed suppression of VCAM-1 expression among siRNAs of 6 genes determined to be inflammatory cytokine-inducing polynucleotides from the experimental results of Examples 3a) to c). The results for negative control siRNA (white column) and positive control siRNA (black column) are shown. In addition, an asterisk (*) represents that 1 or more reduction (50% or more expression reduction) was shown by the difference of (DELTA) Ct compared with negative control siRNA. a. ICAM-1 mRNA expression level (ΔCt value) was evaluated. b. Whether each siRNA of a knocked down its target gene was evaluated by mRNA expression level (ΔCt value). In the primary screening (Cell-ELISA) of Example 2d), the inhibitory effect of siRNA that showed an E-selectin expression inhibitory effect was evaluated at the mRNA expression level by quantitative PCR. This graph shows 8 siRNAs (hatched columns) that showed suppression of E-selectin expression among 6 siRNAs determined to be inflammatory cytokine-inducing polynucleotides from the experimental results of Example 3a) -c). The results for negative control siRNA (white column) and positive control siRNA (black column) are shown. In addition, an asterisk (*) represents that 1 or more reduction (50% or more expression reduction) was shown by the difference of (DELTA) Ct compared with negative control siRNA. a. ICAM-1 mRNA expression level (ΔCt value) was evaluated. Since the analysis was divided into two plates due to the number of samples, the data in each plate was shown separately. b. Whether each siRNA of a knocked down its target gene was evaluated by mRNA expression level (ΔCt value). In the primary screening (Cell-ELISA) of Example 2d), siRNA showing an expression suppression effect of any of ICAM-1, VCAM-1, and E-selectin was transfected into HUVEC and induced by TNF-α. Whether adhesion with THP-1 cells can be suppressed was examined (4 series of experiments). This graph shows nine siRNAs (shaded columns) targeting six genes determined to be inflammatory cytokine-inducing polynucleotides from the experimental results of Examples 3a) to c), negative control siRNA (white column), And the analysis result of positive control siRNA (black column) is shown. Each column shows the average value of the fluorescence intensity obtained by the four series analysis, and the error bar shows the standard deviation. Moreover, it represents that many THP-1 cells have adhered to HUVEC, so that fluorescence intensity is high. In addition, an asterisk (*) represents that 50% or more of adhesion suppression was shown compared with negative control siRNA. None of the siRNAs examined this time showed a significant THP-1 cell adhesion inhibitory effect. In the primary screening (Cell-ELISA) of Example 2d), siRNA that showed an expression inhibitory effect of either ICAM-1, VCAM-1, or E-selectin was transfected into HUVEC and induced by TNF-α. Whether adhesion with U937 cells can be suppressed was examined (four series for each experiment). This graph shows nine siRNAs (shaded columns) targeting six genes determined to be inflammatory cytokine-inducing polynucleotides from the experimental results of Examples 3a) to c), negative control siRNA (white column), And the analysis result of positive control siRNA (black column) is shown. Each column shows the average value of the fluorescence intensity obtained by the four series analysis, and the error bar shows the standard deviation. Moreover, it shows that many U937 cells have adhere | attached on HUVEC, so that fluorescence intensity is high. In addition, an asterisk (*) represents that 50% or more of adhesion suppression was shown compared with negative control siRNA. In the primary screening (Cell-ELISA) of Example 2d), siRNA showing an expression suppression effect of any of ICAM-1, VCAM-1, and E-selectin was transfected into HUVEC and induced by TNF-α. Whether adhesion with MOLT-4 cells can be suppressed was examined (4 series for each experiment). This graph shows nine siRNAs (shaded columns) targeting six genes determined to be inflammatory cytokine-inducing polynucleotides from the experimental results of Examples 3a) to c), negative control siRNA (white column), And the analysis result of positive control siRNA (black column) is shown. Each column shows the average value of the fluorescence intensity obtained by the four series analysis, and the error bar shows the standard deviation. Moreover, it represents that many MOLT-4 cells have adhere | attached on HUVEC, so that fluorescence intensity is high. In addition, an asterisk (*) represents that 50% or more of adhesion suppression was shown compared with negative control siRNA. In the primary screening (Cell-ELISA) of Example 2d), siRNA showing an expression suppression effect of any of ICAM-1, VCAM-1, and E-selectin was transfected into HUVEC and induced by TNF-α. Whether adhesion with Jurkat cells can be suppressed was examined (4 series of experiments). This graph shows nine siRNAs (shaded columns) targeting six genes determined to be inflammatory cytokine-inducing polynucleotides from the experimental results of Examples 3a) to c), negative control siRNA (white column), And the analysis result of positive control siRNA (black column) is shown. Each column shows the average value of the fluorescence intensity obtained by the four series analysis, and the error bar shows the standard deviation. Moreover, it shows that many Jurkat cells have adhered to HUVEC, so that fluorescence intensity is high. In addition, an asterisk (*) represents that 50% or more of adhesion suppression was shown compared with negative control siRNA.

1. Polynucleotides whose expression is increased by inflammatory cytokines “Polynucleotides whose expression is increased by inflammatory cytokines” that can be used in the screening method of the present invention refers to inflammatory cytokines of human and / or non-human mammals. Means a polynucleotide whose expression is increased by the addition of (inflammatory cytokine treatment).

  Inflammatory cytokines refer to cytokines that are deeply involved in inflammatory reactions associated with bacterial and viral infections, tumors, tissue damage, and the like. For example, interleukin 1 (IL-1), interleukin 6 (IL-6), interleukin 8 (IL-8), TNF-α (tumor necrosis factor), etc. are known, but preferably TNF-α. It is.

  Specifically, the polynucleotide whose expression is increased by treatment with inflammatory cytokine is specifically the base represented by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 or 23 in the sequence listing A polynucleotide comprising the sequence is included.

  In addition, if the person has ordinary knowledge in the technical field to which the present invention belongs, a part of the sequence of the above-mentioned natural-type polynucleotide may be substituted with another nucleotide or deleted by a genetic engineering technique. Polynucleotides modified by addition or the like can be prepared, and it can be confirmed whether or not they have the same function as a natural polypeptide by an assay method described later. A polynucleotide having a nucleotide sequence in which some nucleotides are substituted, deleted, or added to such a natural nucleotide sequence, and exhibiting an activity equivalent to that of the natural polynucleotide is also a polynucleotide of the present invention. include. Examples of nucleotide sequence modification include deletion introduction by restriction enzymes or DNA exonuclease, mutation introduction by site-directed mutagenesis, modification of nucleotide sequence by PCR using mutation primers, direct introduction of synthetic mutant DNA, etc. It can be done by a method. Specific examples of the polynucleotide whose expression level varies depending on the treatment with inflammatory cytokine are represented by SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 or 23 in the sequence listing. Examples thereof include, but are not limited to, polynucleotides comprising any of the base sequences. The term “polynucleotide” in the present invention includes not only DNA but also mRNA and cDNA. It also includes a full-length gene and an EST (expression sequence tag).

  Unless otherwise specified, genetic engineering techniques may be performed according to known methods (for example, Maniatis, T. et al., “Molecular Cloning-A Laboratory Manual”, Cold Spring Harbor Laboratory, NY, 1982). Is possible.

1.1 Method for Identifying Polynucleotides whose Expression is Increased by Inflammatory Cytokine Treatment The polynucleotide of the present invention whose expression is increased by inflammatory cytokine treatment is a polynucleotide whose expression level is changed by inflammatory cytokine treatment, An increase in expression can be confirmed by treating inflammatory cytokines on endothelial cells. Specifically, for example, after preparing total RNA from vascular endothelial cells by the method shown in Example 1 described later, it can be identified by performing DNA microarray analysis.

As a result of the analysis, it is possible to identify a polynucleotide that is significantly different in the expression level between human umbilical vein endothelial cells treated with inflammatory cytokines and human umbilical vein endothelial cells not treated with inflammatory cytokines as inflammatory cytokine-derived polynucleotides. . The identified polynucleotide is hereinafter referred to as “inflammatory cytokine-inducing polynucleotide”.
Here, “significantly different” means, for example, that the amount of mRNA of a gene when treated with inflammatory cytokines is compared with that when inflammatory cytokines are not treated by analysis using a DNA microarray commercially available from Affymetrix, Agilent, etc. If the amount of mRNA of the gene is treated with inflammatory cytokines in a real-time PCR system (quantitative PCR system) commercially available from Applied Systems, etc. The case where the difference in ΔCT value is 1 or more compared to the above case, that is, the case where the amount of mRNA is increased by 2 times or more, etc.

  Specifically, the inflammatory cytokine-inducing polynucleotide thus identified is a nucleotide sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 or 23 in the sequence listing. The polynucleotide which consists of can be mentioned.

1.2 Inflammatory cytokine-derived polynucleotide In addition to the polynucleotide specified in 1.1 above, polynucleotides that hybridize with each polynucleotide under stringent conditions and whose expression is increased by inflammatory cytokines are also present. It can be included in the polynucleotide of the invention. The polynucleotide thus identified is also referred to as “inflammatory cytokine-inducing polynucleotide”. That is, the inflammatory cytokine-inducing polynucleotide of the present invention is the polynucleotide according to any one of 1) or 2) below.
1) DNA comprising a base sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or 23,
2) It hybridizes under stringent conditions with DNA consisting of the base sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or 23, and inflammation A polypeptide encoding a polypeptide having (a) a function of inducing expression of a cell adhesion molecule and / or (b) a function of inducing adhesion between a vascular endothelial cell and a white blood cell in the presence of a sex cytokine.

  In the present invention, “hybridizes under stringent conditions” means, for example, hybridization at 68 ° C. in a commercially available hybridization solution Express Hybridization Solution (manufactured by Clontech), or DNA After hybridization at 68 ° C. in the presence of 0.7-1.0 M NaCl using a fixed filter, a 0.1-2 fold concentration SSC solution (1 fold concentration SSC is 150 mM NaCl, 15 mM citric acid). And hybridizing under conditions that can be identified by washing at 68 ° C. or equivalent conditions.

1.3 Assay for Inflammatory Cytokine-Induced Polynucleotides Inflammatory cytokine-induced polynucleotides are significantly increased in human umbilical vein endothelial cells by the treatment with inflammatory cytokines compared to the case without treatment. A polynucleotide that affects the expression of cell adhesion molecules and / or changes in adhesion between vascular endothelial cells and leukocytes, and is a substance that affects the expression of cell adhesion molecules and adhesion between vascular endothelial cells and leukocytes. It can be used for screening and the like.

  As an example of a specific assay method, first, a full-length cDNA is obtained from a human cDNA library or the like according to a known method such as a colony hybridization method using the identified inflammatory cytokine-derived polynucleotide fragment as a probe. This full-length cDNA can be assayed by introducing it into an appropriate cell and highly expressing it and examining whether or not the adhesion between vascular endothelial cells and white blood cells is affected.

  In addition, an antisense nucleic acid against the total RNA of the gene to be tested can be introduced into a cell to examine how it affects the function of each target cell. Another method for suppressing gene expression is to use a double-stranded single-stranded RNA (siRNA) (“Genes and Developments”), 2001. January 15, Volume 15, Issue 2, p. 188-200). For example, siRNA can be introduced according to a method described in the literature, and the expression level of a gene targeted for siRNA and the expression level of a cell adhesion molecule can be examined. On the other hand, a transformed cell is produced according to “5. Method for producing transformed cell” described later, and what kind of influence is exerted on the function of the cell, specifically, the expression level of cell adhesion molecules and adhesion to leukocytes. You can consider whether it appears.

1.4 Function for Inducing Expression of Cell Adhesion Molecule of Inflammatory Cytokine-Inducing Polypeptide A decrease in the expression level of cell adhesion molecule is observed in vascular endothelial cells in which expression of inflammatory cytokine-inducing polynucleotide is suppressed. For example, DNA consisting of a base sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 or 23 in the sequence listing, or SEQ ID NO: 1, 3, 5, (A) a cell that hybridizes with a DNA comprising the nucleotide sequence represented by 7, 9, 11, 13, 15, 17, 19, 21, or 23 under stringent conditions and in the presence of an inflammatory cytokine When siRNA against DNA encoding a polypeptide having the function of inducing adhesion molecule expression and / or (b) the function of inducing adhesion between vascular endothelial cells and white blood cells is prepared and transfected into human umbilical vein vascular endothelial cells Compared with the negative control, the expression level of one or more cell adhesion molecules such as ICAM-1, VCAM-1, and E-selectin is significantly reduced. That. That is, an inflammatory cytokine-inducing polypeptide, which is an expression product of an inflammatory cytokine-inducing polynucleotide, plays an important role in inducing cell adhesion molecules (cell adhesion molecule expression inducing function).

1.5 Function of inducing adhesion of vascular endothelial cells to leukocytes by inflammatory cytokine-inducing polypeptide Decrease in the amount of adhesion to leukocytes is observed in vascular endothelial cells in which expression of inflammatory cytokine-inducing polynucleotide is suppressed

  For example, a human umbilical vein vascular endothelial cell, DNA consisting of a base sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 or 23 of the sequence listing, or Hybridizes under stringent conditions with a DNA comprising the nucleotide sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or 23, and is an inflammatory cytokine In the presence of (a) a cell adhesion molecule expression-inducing function and / or (b) a siRNA against a DNA encoding a polypeptide having an adhesion-inducing function between vascular endothelial cells and leukocytes, The amount of adhesion with white blood cells is significantly reduced compared to the negative control. In other words, inflammatory cytokine-inducing polypeptide, an expression product of inflammatory cytokine-inducing polynucleotide, plays an important role in inducing adhesion of vascular endothelial cells to leukocytes (function of inducing adhesion of vascular endothelial cells to leukocytes). .

2. Inflammatory cytokine-inducing polypeptide The “inflammatory cytokine-inducing polypeptide” of the present invention can be used in the screening of the present invention that affects the induction of expression of cell adhesion molecules and adhesion between vascular endothelial cells and leukocytes in vascular endothelial cells. A polypeptide that can be produced. The expression level of the inflammatory cytokine-inducing polypeptide in the present invention is significantly increased in human umbilical vein vascular endothelial cells when treated with inflammatory cytokines compared to the case where the inflammatory cytokine is not treated. The polypeptide encoded by the inflammatory cytokine-inducing polynucleotide of the present invention can be used for screening for detecting a test substance that affects adhesion between vascular endothelial cells and white blood cells.

The inflammatory cytokine-inducing polypeptide of the present invention plays an important role in the function of inducing the expression of cell adhesion molecules and / or the induction of adhesion of vascular endothelial cells to leukocytes as described in 1.4 and 1.5 above. Plays. Specifically, the inflammatory cytokine-inducing polypeptide that can be used in the screening of the present invention is the polypeptide described in either 1) or 2) below.
1) Polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24, or 2) SEQ ID NO: 2, 4, 6, 8 In the amino acid sequence represented by 10, 12, 14, 16, 18, 20, 22, or 24, having an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, and / or added; and A polypeptide having (a) a function of inducing cell adhesion molecule expression and / or (b) a function of inducing adhesion between vascular endothelial cells and white blood cells in the presence of inflammatory cytokines.

  The polypeptide of 2) includes the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24, and (a) a cell adhesion molecule And / or (b) a polypeptide having a function of inducing adhesion between vascular endothelial cells and white blood cells, for example, SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, A polypeptide in which an appropriate marker sequence or the like is added to the N-terminal and / or C-terminal of a polypeptide consisting of the amino acid sequence represented by 18, 20, 22 or 24 (ie, a fusion polypeptide) is also (a) a cell. It is included as long as it has a function of inducing adhesion molecule expression and / or (b) a function of inducing adhesion between vascular endothelial cells and white blood cells. As the marker sequence, a sequence for easily confirming the expression of a polypeptide, confirming subcellular localization, or purification can be used. For example, a FLAG epitope, a hexa-histidine tag, a hemagglutinin A tag, a myc epitope, etc. can be mentioned.

  In the present specification, the term “polypeptide” includes a protein, a protein having a sugar chain added thereto, and a salt.

3. Method for Producing Inflammatory Cytokine-Inducing Polypeptide Inflammatory cytokine-inducing polypeptide can be synthesized in vitro or can be prepared by a known genetic engineering technique using a polynucleotide encoding this polypeptide.

3.1 In Vitro Synthesis Method More specifically, the polynucleotide is synthesized in a solution containing an enzyme, a substrate and an energy substance necessary for transcription and translation after the polynucleotide encoding the target protein is incorporated into an expressible vector. It is. Examples include, but are not limited to, a rapid translation system (RTS) manufactured by Roche diagnostics. Taking the case of using RTS as an example, when a gene of interest is cloned into an expression vector controlled by a T7 promoter and added to an in vitro reaction system, mRNA is first transcribed from the template DNA by T7 RNA polymerase. Thereafter, translation is performed by a ribosome or the like in an E. coli solution, and the target polypeptide is synthesized in the reaction solution (Biochemica, 1, 20-23 (2001), Biochemica, 2, 28-29 (2001)).

3.2 Methods Prepared by Known Genetic Engineering Techniques By transforming transformed cells (ie, other prokaryotic or eukaryotic host cells with an expression vector containing an inflammatory cytokine-inducing polynucleotide, The transformed cells expressing the polypeptide are cultured under conditions that allow the polypeptide to be expressed, and the target protein is separated from the culture by a method generally used for protein separation and purification. And can be prepared by purification. Specifically, it will be described in detail in the section “5. Method for producing transformed cell”.

4). Antibody to Inflammatory Cytokine-Inducing Polypeptide An antibody to inflammatory cytokine-inducing polypeptide specifically recognizes an inflammatory cytokine-inducing polypeptide or a part thereof. Such antibodies can include, for example, antibodies that bind to any of the inflammatory cytokine-inducing polypeptides but do not bind to any other protein. Such an antibody can be used in a screening method for measuring the expression of a polypeptide described in the section “6. Screening method” described later, and can also be used as a pharmaceutical composition containing an antibody.

  The antibody may be prepared by using a conventional method (for example, Shinsei Kagaku Kenkyusho 1, Protein 1, p. 389-397, 1992) to an animal with an antigenic protein or any polypeptide selected from its amino acid sequence. It can be obtained by immunizing and collecting and purifying antibodies produced in vivo. Further, according to a known method (for example, Kohler and Milstein, Nature 256, 495-497, 1975, Kennet, R. ed., Monoclonal Antibody p. 365-367, 1980, Prenum Press, NY) A hybridoma can also be established by fusing an antibody-producing cell that produces an antibody against the polypeptide and a myeloma cell to obtain a monoclonal antibody.

  Examples of the antigen for producing an antibody include a target polypeptide or a polypeptide comprising at least 6 consecutive partial amino acid sequences thereof, or a derivative obtained by adding any amino acid sequence or carrier thereto.

  The antigen polypeptide can be obtained by synthesizing a target polypeptide in vitro or by producing it in a host cell by genetic manipulation. Specifically, after incorporating a polynucleotide encoding the polypeptide of interest into an expressible vector, it is synthesized in a solution containing enzymes, substrates and energy substances necessary for transcription and translation, or other prokaryote, Alternatively, the polynucleotide can be obtained by expressing the polynucleotide by transforming a eukaryotic host cell.

  The polypeptide to be an antigen can be produced by the method described in the section “3. Production of inflammatory cytokine-inducing polypeptide” above.

  The antibody obtained as described above can be used for various immunological measurement methods such as RIA method, ELISA method, fluorescent antibody method, passive hemagglutination reaction method, and immunohistological staining.

  As an example of an antibody that specifically binds to the target polypeptide, a monoclonal antibody that specifically binds to the target polypeptide can be mentioned. The method for obtaining the antibody is as described below.

In producing a monoclonal antibody, the following work steps are generally required. That is,
(A) purification of a biopolymer used as an antigen;
(B) a step of preparing antibody-producing cells after immunization by injecting an antigen into an animal, collecting blood, determining its antibody titer to determine the timing of splenectomy,
(C) Preparation of myeloma cells (hereinafter referred to as “myeloma”),
(D) cell fusion between antibody-producing cells and myeloma,
(E) selection of a hybridoma group producing the target antibody;
(F) Division into single cell clones (cloning),
(G) In some cases, culturing hybridomas for producing a large amount of monoclonal antibodies, or raising animals transplanted with hybridomas,
(H) Examination of the physiological activity of the monoclonal antibody thus produced and its recognition specificity, or the examination of the properties as a labeling reagent.

  Hereinafter, although the production method of a monoclonal antibody is explained in full detail along the said process, the production method of this antibody is not restricted to this, For example, antibody producing cells other than a spleen cell and myeloma can also be used.

(A) Purification of antigen As the antigen, the protein of the present invention prepared by the method as described above or a part thereof can be used. Furthermore, since the primary structure of the protein of the present invention has been clarified by the present invention, a partial peptide of the protein of the present invention can be chemically synthesized using a method well known to those skilled in the art, and this can be used as an antigen. .

(B) Preparation of antibody-producing cells The antigen obtained in step (a) is mixed with Freund's complete or incomplete adjuvant or an auxiliary agent such as potassium alum, and an experimental animal is immunized as an immunogen. As an experimental animal, a mouse is most preferably used, but is not limited thereto.

  The immunogen administration method for mouse immunization may be subcutaneous injection, intraperitoneal injection, intravenous injection, intradermal injection, or intramuscular injection, but subcutaneous injection or intraperitoneal injection is preferred.

  Immunization can be repeated once or multiple times at appropriate intervals (preferably at intervals of 1 to 5 weeks). Thereafter, the antibody titer against the antigen in the serum of the immunized animal is measured, and if the animal having a sufficiently high antibody titer is used as a source of antibody-producing cells, the effect of subsequent operations can be enhanced. In general, it is preferable to use animal-derived antibody-producing cells 3 to 5 days after the final immunization for subsequent cell fusion.

  The antibody titer used here includes radioisotope immunoassay (hereinafter referred to as “RIA method”), solid-phase enzyme immunoassay (hereinafter referred to as “ELISA method”), fluorescent antibody method, passive hemagglutination reaction. Various known techniques such as the method can be mentioned, but the RIA method or the ELISA method is more preferable from the viewpoint of detection sensitivity, rapidity, accuracy, possibility of automation of operation, and the like.

  The antibody titer in the present invention can be measured according to the procedure described below, for example, according to the ELISA method. First, the purified or partially purified antigen is adsorbed on a solid phase surface such as a 96-well plate for ELISA, and the solid phase surface to which no antigen is adsorbed is a protein unrelated to the antigen, such as bovine serum albumin (hereinafter referred to as “BSA”). After the surface is washed, it is contacted with a serially diluted sample (for example, mouse serum) as the first antibody, and the monoclonal antibody in the sample is bound to the antigen. Further, an antibody against a mouse antibody labeled with an enzyme as a second antibody is added and bound to the mouse antibody. After washing, the substrate of the enzyme is added, and the change in absorbance due to color development based on the decomposition of the substrate is measured. calculate.

(C) Preparation process of myeloma As a myeloma, generally, a cell line obtained from a mouse, for example, 8-azaguanine resistant mouse (BALB / c origin) myeloma strain P3X63Ag8U. 1 (P3-U1) [Yelton, D.C. E. et al. Current Topics in Microbiology and Immunology, 81, 1-7 (1978)], P3 / NSI / 1-Ag4-1 (NS-1) [Kohler, G. et al. et al. European J.M. Immunology, 6, 511-519 (1976)], Sp2 / O-Ag14 (SP-2) [Shulman, M .; et al. Nature, 276, 269-270 (1978)] or the like is preferably used. These cell lines contain 8-azaguanine in a suitable medium such as 8-azaguanine medium [RPMI-1640 medium supplemented with glutamine, 2-mercaptoethanol, gentamicin, and fetal calf serum (hereinafter referred to as “FCS”). Added medium], Iskov's Modified Dulbecco's Medium (Iscove's Modified Dulbecco's Medium; hereinafter referred to as "IMDM"), or Dulbecco's Modified Eagle Medium (Dulbecco's Modified Eagle Medium; hereinafter referred to as "DMEM"). However, 3 to 4 days before cell fusion, the cells were subcultured in a normal medium [for example, ASF104 medium (manufactured by Ajinomoto Co., Inc.) containing 10% FCS] to secure a cell number of 2 × 10 7 or more on the day of fusion. Keep it.

(D) Cell fusion Antibody-producing cells are plasma cells and their precursor cells, lymphocytes, which may be obtained from any part of an individual, and generally include spleen, lymph nodes, peripheral blood, or these Spleen cells are most commonly used, although they can be obtained from appropriate combinations.

  After the final immunization, a site where antibody-producing cells are present, such as the spleen, is removed from a mouse having a predetermined antibody titer, and spleen cells that are antibody-producing cells are prepared. The most commonly used method for fusing these spleen cells with the myeloma obtained in step (c) is a method using polyethylene glycol, which has a relatively low cytotoxicity and a simple fusion operation. This method includes, for example, the following procedure.

  Spleen cells and myeloma are thoroughly washed with a serum-free medium (for example, RPMI 1640) or phosphate buffered saline (hereinafter referred to as “PBS”), and the ratio of the number of spleen cells to myeloma is 5: 1 to 10: 1. Mix to a suitable degree and centrifuge. After removing the supernatant and loosening the precipitated cells, a serum-free medium containing 1 ml of 50% (w / v) polyethylene glycol (molecular weight 1000 to 4000) is added dropwise with stirring. Thereafter, 10 ml of serum-free medium is slowly added and then centrifuged. The supernatant was discarded again, and the precipitated cells were recovered from a normal medium (hereinafter referred to as “HAT”) containing an appropriate amount of hypoxanthine / aminopterin / thymidine (hereinafter referred to as “HAT”) and mouse interleukin-2 (hereinafter referred to as “IL-2”). The medium is suspended in a medium and dispensed to each well of a culture plate (hereinafter referred to as “plate”) and cultured at 37 ° C. for about 2 weeks in the presence of 5% carbon dioxide gas. HAT medium is supplemented as needed.

(E) Selection of hybridoma group When the myeloma cell is an 8-azaguanine resistant strain, that is, a hypoxanthine / guanine / phosphoribosyltransferase (HGPRT) deficient strain, the myeloma cell that has not fused, and the myeloma Fusion cells between cells cannot survive in a HAT-containing medium. On the other hand, fused cells between antibody-producing cells or hybridomas between antibody-producing cells and myeloma cells can survive, but fused cells between antibody-producing cells have a lifetime. Therefore, by continuing the culture in the HAT-containing medium, only the hybridoma of the antibody producing cell and the myeloma cell survive, and as a result, the hybridoma can be selected.

  About the hybridoma which grew in colony form, culture medium exchange to the culture medium (henceforth "HT culture medium") remove | excluding aminopterin from HAT culture medium is performed. Thereafter, a part of the culture supernatant is collected, and the antibody titer is measured, for example, by ELISA.

  As mentioned above, although the method using the 8-azaguanine resistant cell line was illustrated, other cell lines can also be used according to the selection method of a hybridoma, and the culture medium composition to be used also changes in that case.

(E) Cloning Hybridomas that have been found to produce specific antibodies by measuring antibody titers by the same method as described in step (b) are transferred to another plate for cloning. This cloning method includes a limiting dilution method in which one well of a plate is diluted and cultured so that one hybridoma is contained, a soft agar method in which colonies are collected by culturing in a soft agar medium, and one by one using a micromanipulator. And the like, and a “sorter clone” in which one cell is separated by a cell sorter. However, the limiting dilution method is simple and often used.

  For wells in which antibody titers are recognized, for example, cloning by limiting dilution is repeated 2 to 4 times, and those in which antibody titers are stably recognized are selected as the monoclonal antibody-producing hybridoma strains of the present invention.

(F) Preparation of monoclonal antibody by hybridoma culture The hybridoma that has been cloned is cultured by changing the medium from the HT medium to the normal medium. Mass culture is performed by rotary culture using a large culture bottle or spinner culture. By purifying the supernatant in this mass culture using a method well known to those skilled in the art, such as gel filtration, a monoclonal antibody that specifically binds to the protein of the present invention can be obtained. In addition, ascites containing a large amount of the monoclonal antibody of the present invention can be obtained by growing the hybridoma within the abdominal cavity of the same strain of mice (for example, the above-mentioned BALB / c) or Nu / Nu mice. . As a simple purification method, a commercially available monoclonal antibody purification kit (for example, MAbTrap GII kit; manufactured by Pharmacia) or the like can also be used. The monoclonal antibody thus obtained has a high antigen specificity for the polypeptide of interest.

(G) Assay for monoclonal antibody The isotype and subclass of the monoclonal antibody thus obtained can be determined as follows. First, examples of the identification method include an ochterlony method, an ELISA method, and an RIA method. The octerulony method is simple, but concentration is necessary when the concentration of the monoclonal antibody is low. On the other hand, when the ELISA method or the RIA method is used, the culture supernatant is reacted with the antigen-adsorbing solid phase as it is, and further, antibodies corresponding to various immunoglobulin isotypes and subclasses are used as secondary antibodies. Isotypes and subclasses can be identified. Further, as a simpler method, a commercially available kit for identification (for example, mouse typer kit; manufactured by Bio-Rad) or the like can be used.

  Furthermore, the protein can be quantified by the foreign lowry method and the method of calculating from the absorbance at 280 nm [1.4 (OD280) = immunoglobulin 1 mg / ml].

  The monoclonal antibody of the present invention thus obtained can be used for detection and separation / purification of the target polypeptide utilizing its specificity.

5. Method for producing transformed cell A transformed cell useful for use in the screening of the present invention or for producing a polypeptide of interest is prepared by incorporating an inflammatory cytokine-inducing polynucleotide into an appropriate vector DNA, It can be produced by transforming host cells (prokaryotic cells, eukaryotic cells).

  The method for producing the polynucleotide encoding the target protein is not particularly limited. For example, (I) a method using PCR, (II) a conventional genetic engineering method (that is, a cDNA library) Examples include a method using a method for selecting a transformant containing a desired cDNA from transformed transformants), or (III) a chemical synthesis method, and a method using PCR is preferred. Hereinafter, each manufacturing method will be sequentially described.

(I) Method Using PCR In a method using PCR, for example, a polynucleotide encoding a target polypeptide can be produced by the following procedure. That is, mRNA is extracted from human cells or tissues that have the ability to produce the desired polypeptide. Next, based on the base sequence of the polynucleotide encoding the target polypeptide, a pair of primer sets that can sandwich the entire length of mRNA corresponding to the target polypeptide, or a part of the mRNA region thereof. Create a set of two primer sets that can be sandwiched. By performing reverse transcriptase-polymerase chain reaction (RT-PCR), the full-length cDNA of the target polypeptide or a part thereof can be obtained.

  More specifically, first, total RNA containing mRNA encoding the polypeptide of interest is extracted from the cells or tissues (for example, pancreas) having the ability to produce the polypeptide of interest by a known method. Examples of the extraction method include a guanidine / thiocyanate / hot / phenol method, a guanidine / thiocyanate / guanidine / hydrochloric acid method, or a guanidine / thiocyanate / cesium chloride method, and the guanidine / thiocyanate / cesium chloride method may be used. preferable. The cell or tissue having the ability to produce the target polypeptide is, for example, Northern blotting using a polynucleotide encoding the target polypeptide or a part thereof, or an antibody specific for the target polypeptide. It can be specified by Western blotting or the like.

  Subsequently, the extracted mRNA is purified. The mRNA may be purified by a conventional method. For example, the mRNA can be purified by adsorbing it to an oligo (dT) cellulose column and then eluting it. If desired, mRNA can be further fractionated by sucrose density gradient centrifugation or the like. In addition, commercially available mRNA that has been extracted and purified can be used without extracting mRNA. Next, a reverse transcriptase reaction is performed on the purified mRNA in the presence of, for example, a random primer, an oligo dT primer, and / or a custom-synthesized primer to synthesize a first strand cDNA. This synthesis can be performed by a conventional method. Using the obtained first strand cDNA, PCR can be carried out using two primers sandwiching the full length or a partial region of the target polynucleotide to amplify the target cDNA. The obtained DNA is fractionated by agarose gel electrophoresis or the like. If desired, the desired DNA fragment can be obtained by cleaving the DNA with a restriction enzyme or the like and connecting it. Moreover, the target DNA fragment can also be obtained from genomic DNA.

(II) Method Using Conventional Genetic Engineering Technique In a method using a conventional genetic engineering technique, for example, a polynucleotide encoding a target polypeptide can be produced by the following procedure. First, single-stranded cDNA is synthesized using reverse transcriptase using mRNA prepared by the above-described method using PCR as a template, and then double-stranded cDNA is synthesized from this single-stranded cDNA. Examples of the method include the S1 nuclease method (Efstratidis, A. et al., Cell, 7, 279-288, 1976), the Land method (Land, H. et al., Nucleic Acids Res. 9, 2251-2266, 1981), O. The Joon Yoo method (Yoo, OJ et al., Proc. Natl. Acad. Sci. USA, 79, 1049-1053, 1983), or the Okayama-Berg method (Okayama, H. and Berg, P. Mol. Cell. Biol. 2, 161-170, 1982).

Next, after preparing a recombinant plasmid containing the double-stranded cDNA, it is introduced into E. coli (eg, DH5α strain) and transformed, and, for example, a recombinant is selected using drug resistance to tetracycline or ampicillin as an index. . Transformation of the host cell can be accomplished by, for example, the method of Hanahan (Hanahan, DJ Mol. Biol. 166, 557-580, 1983), ie, CaCl ₂ , MgCl ₂ , or This can be performed by adding the recombinant DNA body to competent cells prepared in the presence of RbCl. In addition to the plasmid, a lambda-type phage vector can also be used as the vector.

  As a method for selecting a transformant having the target cDNA from the transformant thus obtained, for example, the following (A) screening method using a synthetic oligonucleotide probe, (B) prepared by PCR (C) a screening method using a probe, (C) a method of producing and screening a target polypeptide in other animal cells, (D) a method of selecting using an antibody against the target polypeptide, or (E) selective hybridization. A method using a translation system can be employed.

(A) In the screening method using a synthetic oligonucleotide probe, for example, a transformant having the target cDNA can be selected by the following procedure. That is, an oligonucleotide corresponding to all or part of the target polypeptide is synthesized, and this is probed (labeled with ³² P or ³³ P) and hybridized with a nitrocellulose filter on which the DNA of the transformant is denatured and immobilized. The obtained positive strain is searched and selected. When synthesizing an oligonucleotide for a probe, it can be a nucleotide sequence derived using codon usage, or a plurality of nucleotide sequences combining possible nucleotide sequences. . In the latter case, the type can be reduced by including inosine.

(B) In a screening method using a probe prepared by PCR, for example, a transformant having the target cDNA can be selected by the following procedure. That is, the oligonucleotides of the sense primer and the antisense primer corresponding to a part of the target polypeptide are synthesized, and they are combined and PCR is performed to amplify a DNA fragment encoding all or part of the target polypeptide. As the template DNA used here, cDNA synthesized by reverse transcription reaction from mRNA of cells producing the target polypeptide or genomic DNA can be used. The thus prepared DNA fragment is labeled with, for example, 32P or 33P, and this is used as a probe to perform colony hybridization or plaque hybridization, thereby selecting a transformant having the target cDNA.

(C) In the method of producing and screening a target polypeptide in other animal cells, for example, a transformant having the target cDNA can be selected by the following procedure. That is, the transformed strain is cultured, the polynucleotide is amplified, the animal cell is transfected with the polynucleotide, and the polypeptide encoded by the polynucleotide is produced on the cell surface. In this case, either a plasmid capable of self-replication and containing a transcription promoter region, or a plasmid that can be integrated into the chromosome of an animal cell can be used. By detecting the target polypeptide using an antibody against the target polypeptide, a transformant having the target cDNA is selected from the original transformants.

(D) In the method of selecting using an antibody against the target polypeptide, for example, a transformant having the target cDNA can be selected by the following procedure. That is, cDNA is previously incorporated into an expression vector, a polypeptide is produced on the cell surface of the transformed strain, and a desired polypeptide-producing strain is detected using an antibody against the target polypeptide and a secondary antibody against the antibody. Then, a transformant having the target cDNA is selected.

(E) In a method using a selective hybridization translation system, for example, a transformant having a target cDNA can be selected by the following procedure. That is, the cDNA obtained from the transformant is blotted onto a nitrocellulose filter or the like, and after separately hybridizing mRNA prepared from cells having the ability to produce the desired polypeptide, the mRNA bound to the cDNA is dissociated, to recover. The recovered mRNA is injected into a suitable polypeptide translation system, for example, Xenopus oocytes, or translated into a polypeptide using a cell-free system such as rabbit reticulocyte lysate or wheat germ. A transformant having the target cDNA is selected by detection using an antibody against the target polypeptide.

  A method for collecting a polynucleotide encoding a target polypeptide from the obtained transformant of interest is a known method (for example, Maniatis, T. et al., “Molecular Cloning-A Laboratory Manual”, Cold Spring Harbor Laboratory, NY, 1982). For example, it can be performed by separating a fraction corresponding to plasmid DNA from cells and cutting out the cDNA region from the obtained plasmid DNA.

(III) A method using a chemical synthesis method In a method using a chemical synthesis method, for example, a polynucleotide encoding a target polypeptide can be produced by binding a DNA fragment produced by the chemical synthesis method. . Each DNA can be synthesized using a DNA synthesizer [for example, Oligo 1000M DNA Synthesizer (manufactured by Beckman) or 394 DNA / RNA Synthesizer (manufactured by Applied Biosystems)]. In addition, the polynucleotide encoding the polypeptide of interest is based on information on the polypeptide of interest, such as the phosphite triester method (Hunkapiller, M. et al., Nature, 10, 105-111, 1984), etc. It can also be produced by chemical synthesis of nucleic acids according to conventional methods. The codons for the desired amino acid are known per se and may be selected arbitrarily, and can be determined according to a conventional method in consideration of, for example, the codon usage of the host to be used (Crantham, R. et al., Nucleic Acids Res. 9, 43-74, 1981). Furthermore, partial modification of the codons of these base sequences is performed by site-specific mutagenesis (Mark specific DF) using primers comprising synthetic oligonucleotides encoding the desired modification according to a conventional method (Mark, DF). Et al., Proc. Natl. Acad. Sci. USA, 81, 5562-5666, 1984) and the like.

  Sequencing of the DNA thus obtained can be performed, for example, by the chemical modification method of Maxam-Gilbert (Maxam, AM and Gilbert, W. “Methods in Enzymology”, 65, 499-559, 1980) or dideoxynucleotide. The chain termination method (Messing, J. and Vieira, J. Gene, 19, 269-276, 1982) can be used.

  Examples of the prokaryotic host include Escherichia coli and Bacillus subtilis. To transform a gene of interest into these host cells, the host cell is transformed with a plasmid vector containing a replicon or origin of replication from a species compatible with the host and regulatory sequences. The vector preferably has a sequence capable of imparting phenotypic (phenotypic) selectivity to transformed cells.

  For example, K12 strain and the like are often used as E. coli, and pBR322 and pUC-type plasmids are generally used as vectors. However, the present invention is not limited thereto, and any known various strains and vectors can be used.

  Examples of promoters in E. coli include tryptophan (trp) promoter, lactose (lac) promoter, tryptophan lactose (tac) promoter, lipoprotein (lpp) promoter, polypeptide chain elongation factor Tu (tufB) promoter, Any promoter can be used to produce the polypeptide of interest.

  As Bacillus subtilis, for example, the strain 207-25 is preferable, and pTUB228 (Ohmura, K. et al. (1984) J. Biochem. 95, 87-93) and the like are used as a vector. is not. By ligating a DNA sequence encoding the signal peptide sequence of Bacillus subtilis α-amylase, secretion expression outside the cell can also be achieved.

  Examples of eukaryotic host cells include vertebrates, insects, and yeast cells. Examples of vertebrate cells include COS cells (Gluzman, Y. (1981) Cell 23, 175-) that are monkey cells. 182, ATCC: CRL-1650) and Chinese hamster ovary cells (CHO cells, ATCC: CCL-61) dihydrofolate reductase deficient strain (Urlauub, G. and Chasin, LA (1980) Proc. Natl. Acad. Sci. USA 77, 4126-4220) is often used, but is not limited thereto.

  As an expression promoter for vertebrate cells, a promoter that is usually located upstream of the gene to be expressed, an RNA splice site, a polyadenylation site, a transcription termination sequence, and the like can be used. You may have. Examples of the expression vector include pCR3.1 having a cytomegalovirus early promoter (manufactured by Invitrogen), pSV2dhfr having a SV40 early promoter (Subramani, S. et al. (1981) Mol. Cell. Biol. 1, 854-864) and the like, but is not limited thereto.

  For example, when a COS cell is used as a host cell, the expression vector has an SV40 origin of replication, and can self-propagate in the COS cell. Furthermore, a transcription promoter, a transcription termination signal, and an RNA splice site The thing provided with can be used. The expression vectors include diethylaminoethyl (DEAE) -dextran method (Luthman, H. and Magnusson, G. (1983) Nucleic Acids Res. 11, 1295-1308), calcium phosphate-DNA coprecipitation method (Graham, FL. and van der Eb, AJ (1973) Virology 52, 456-457), and electropulse perforation (Neumann, E. et al. (1982) EMBO J. 1, 841-845). Thus, the desired transformed cells can be obtained. When a CHO cell is used as a host cell, an expression vector and a vector capable of expressing a neo gene that functions as an antibiotic G418 resistance marker, such as pRSVneo (Sambrook, J. et al. (1989): “Molecular Cloning”. A Laboratory Manual "Cold Spring Harbor Laboratory, NY) and pSV2neo (Southern, PJ and Berg, P. (1982) J. Mol. Appl. Genet. 1, 327-341), etc. By selecting G418-resistant colonies, transformed cells that stably produce the target polypeptide can be obtained.

  When insect cells are used as host cells, cell lines derived from Spodoptera frugiperda ovarian cells (Sf-9 or Sf-21) or Trichoplusia ni egg cells High Five cells (Wickham, T. J. et al.). al, (1992) Biotechnol.Prog.I: 391-396) and the like, and pVL1392 using a polyhedrin protein promoter of autographer nuclear polyhedrosis virus (AcNPV) as a baculovirus transfer vector. / 1393 is often used (Kidd, IM and VC Emery (1993) The use of baculoviruses as expression vectors. pplied Biochemistry and Biotechnology 42,137-159). In addition, vectors using baculovirus P10 and promoters of the same basic protein can also be used. Furthermore, it is also possible to express a recombinant protein as a secreted protein by linking the secretory signal sequence of the envelope surface protein GP67 of AcNPV to the N-terminal side of the target protein (Zhe-mei Wang, et al. (1998). Biol.Chem.379, 167-174).

  As an expression system using a eukaryotic microorganism as a host cell, yeast is generally well known, and among them, Saccharomyces yeasts such as baker's yeast Saccharomyces cerevisiae and petroleum yeast Pichia pastoris are preferable. Examples of expression vectors for eukaryotic microorganisms such as yeast include promoters of alcohol dehydrogenase genes (Bennetzen, JL and Hall, Bd (1982) J. Biol. Chem. 257, 3018-3025. ) And acid phosphatase gene promoter (Miyanohara, A. et al. (1983) Proc. Natl. Acad. Sci. USA 80, 1-5) and the like can be preferably used. When expressed as a secretory protein, it can also be expressed as a recombinant having a secretory signal sequence and an endogenous protease possessed by the host cell or a known protease cleavage site on the N-terminal side. For example, in a system in which human mast cell tryptase of a trypsin type serine protease is expressed in petroleum yeast, it is activated by connecting the yeast α-factor secretion signal sequence to the N-terminal side of the yeast yeast factor KEX2 protease cleavage site. Type tryptase is known to be secreted into the medium (Andrew, L. Niles, et al. (1998) Biotechnol. Appl. Biochem. 28, 125-131).

  The transformant obtained as described above can be cultured according to a conventional method, and the desired polypeptide is produced intracellularly or extracellularly by the culture. As a medium that can be used for the culture, various media that are conventionally used can be appropriately selected depending on the employed host cells. For example, in the case of COS cells, for example, a medium in which a serum component such as fetal bovine serum (FBS) is added to a medium such as RPMI-1640 medium or Dulbecco's modified Eagle minimum essential medium (DMEM), if necessary, is used. can do. In the case of 293-EBNA cells, a medium obtained by adding G418 to a medium such as Dulbecco's modified Eagle's minimum essential medium (DMEM) to which a serum component such as fetal bovine serum (FBS) is added can be used.

  Recombinants produced inside or outside of the transformant by the above culture are separated and purified by various known separation procedures utilizing the physical and chemical properties of the target polypeptide. can do. Specific examples of the method include treatment with an ordinary protein precipitant, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography ( Examples thereof include various liquid chromatography such as HPLC), dialysis methods, and combinations thereof. Further, by connecting histidine consisting of 6 residues to the recombinant protein to be expressed, it can be efficiently purified with a nickel affinity column. By combining the above methods, the target polypeptide can be easily produced in large quantities with high yield and high purity. Further, the molecular weight of the purified polypeptide can be determined by a usual method such as mass spectrometry or SDS-PAGE. Although splicing variants may exist, these polypeptides are also included in the target polypeptide as long as they have the same function (biological activity) as the target polypeptide.

  By culturing the transformed cell, the target polypeptide produced inside or outside the cell is separated by various known separation methods utilizing the physical properties and biochemical properties of the polypeptide. Can be purified. Specifically, for example, by culturing cells expressing the target polypeptide, suspending them in a buffer, homogenizing, and centrifuging, a fraction containing the target polypeptide can be obtained. . After solubilizing the obtained fraction, treatment with a normal protein precipitating agent, ultrafiltration, various liquid chromatography [eg, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchanger chromatography, affinity The target polypeptide can be purified by chromatography, high performance liquid chromatography (HPLC), etc.], dialysis, or a combination thereof. In addition, when solubilizing the cell membrane fraction, the characteristics of the receptor should be maintained after solubilization by using a solubilizing agent that is as gentle as possible (for example, CHAPS, Triton X-100, or dichitonin). Can do.

  The target polypeptide is fused with the marker sequence in frame and expressed, thereby facilitating confirmation of polypeptide expression, confirmation of intracellular localization, purification, and the like. Examples of the marker sequence include a FLAG epitope, a hexa-histidine tag, a hemagglutinin tag, or a myc epitope. Further, by inserting a specific amino acid sequence recognized by a protease (for example, enterokinase, factor Xa, thrombin, etc.) between the marker sequence and the polypeptide of interest, the marker sequence portion is bound by these proteases. It is possible to cut and remove. For example, there is a report in which a muscarinic acetylcholine receptor and a hexahistidine tag are linked with a thrombin recognition sequence (Hayashi, MK and Haga, TJ Biochem. 120, 1232-1238, 1996).

6). Screening method of the present invention The polypeptide used in the screening method of the present invention was found as a polypeptide encoded by a gene whose expression level was increased by adding TNF-α, which is a kind of inflammatory cytokine, to vascular endothelial cells. Is. As shown in the Examples, it can be seen that it is involved in the induction of expression of cell adhesion molecules such as ICAM-1, VCAM-1, E-selectin, and adhesion of vascular endothelial cells to leukocytes.

  Therefore, a substance that suppresses the function of the polypeptide suppresses the expression of cell adhesion molecules, and consequently inhibits adhesion of vascular endothelial cells to white blood cells (hereinafter referred to as “vascular endothelial cell adhesion inhibitor”). It becomes. Such substances inhibit adhesion between vascular endothelial cells and white blood cells, and can be applied to inflammatory diseases, autoimmune diseases, asthma, psoriasis, rejection in transplants, rheumatoid arthritis, etc. Indications for inflammatory diseases are most preferred.

  In addition, an anti-inflammatory drug can be screened by measuring the expression level of at least one of the polypeptide or a polynucleotide encoding the polypeptide.

  The “test substance” used in this screening method refers to a substance to be examined for the effect of promoting or suppressing the function of the polypeptide of the present invention by the screening method of the present invention. Examples of the test substance include compounds, microbial metabolites, plant and animal tissue extracts, derivatives thereof, and mixtures thereof. In addition, nucleic acids or derivatives thereof (including antisense oligonucleotides, ribozymes, siRNA, etc.) designed to increase the expression level of the polynucleotide of the present invention can be used as test substances. The dose and concentration of the test substance can be set as appropriate, or multiple types of doses can be set, for example, by creating a dilution series, and can be administered in an appropriate state such as an individual or liquid. May be dissolved in an appropriate buffer, or a stabilizer may be added. In the case of a screening method using cultured cells, it can be added to a medium and cultured. When added to the medium, it may be added from the beginning of the culture or may be added during the culture, and the number of additions is not limited to one. Although the period of culturing in the presence of the test substance may be set as appropriate, it is preferably 30 minutes to 48 hours. When administering a test substance to a mammal individual, the administration form such as oral administration, intravenous injection, intraperitoneal injection, transdermal administration, and subcutaneous injection is properly used depending on the physical properties of the test substance. Moreover, the time from administration to obtaining a sample can be appropriately selected.

  Cells used in this screening method express inflammatory cytokine-inducing polypeptides, and are similar to those produced in vascular endothelial cells by stimulation with vascular endothelial cells, cells derived from vascular endothelial cells, or inflammatory cytokines such as TNF-α. Any cell that exhibits an inflammatory reaction can be used without particular limitation. Moreover, if it shows the same reaction as a human, it can be used without being restricted to the animal species of a cell. Specifically, a human vascular endothelial cell line that stably expresses the inflammatory cytokine-inducing polypeptide of the present invention, or human umbilical vein intravascular cells (HUVEC) in which the inflammatory cytokine-inducing polypeptide is forcibly expressed in a retrovirus vector or the like. ) And human microvascular endothelial cells (HMEC-1) or the like cultured in vascular endothelial cell medium (manufactured by Cell Applications) or MCDB131 medium (manufactured by Invitrogen) containing 10% fetal bovine serum (FBS) it can.

  When vascular endothelial cells are used in carrying out this screening method, it is desirable to stimulate the cultured cells in order to reproduce the inflammatory state in the living body. Specific examples include a method of adding inflammatory cytokines, LPS, pertussis toxin, and the like to the medium. Inflammatory cytokines refer to cytokines that are deeply involved in inflammatory reactions associated with bacterial and viral infections, tumors, tissue damage, and the like. Examples include interleukin 1 (IL-1), interleukin 6 (IL-6), tumor necrosis factor (TNF-α), and the like, and tumor necrosis factor (TNF-α) is preferable.

  As a material for preparing a sample for this embodiment, a whole cell extract or a nuclear extract fraction of cultured cells cultured in the presence or absence of a test substance can be used. Is preferred. The whole cell extract is subjected to a step of preparing an ELISA / RIA sample or a Western blot sample after removing insoluble substances by high-speed centrifugation as necessary.

6.1 Screening Method Utilizing Expression Level Measurement Inflammatory cytokine-inducing polypeptides induce cell adhesion with white blood cells in vascular endothelial cells by inducing the expression of vascular adhesion molecules. Therefore, a substance that decreases the expression level of the polypeptide is a substance having an ability to suppress vascular endothelial cell adhesion. Specifically, these substances can be obtained by a screening method including the following steps.

A-1. When measuring gene expression level (1) Inhibiting vascular endothelial cell adhesion, including the step of contacting vascular endothelial cells with a test substance, and (2) the step of measuring the level of gene expression described in any of the following Substance screening method:
1) DNA consisting of a base sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or 23, or 2) SEQ ID NO: 1, 3, 5, 7, (A) a cell adhesion molecule that hybridizes with a DNA comprising the base sequence represented by 9, 11, 13, 15, 17, 19, 21, or 23 under stringent conditions and in the presence of an inflammatory cytokine. And / or (b) a polypeptide encoding a polypeptide having a function of inducing adhesion between vascular endothelial cells and white blood cells.
A-2. When measuring the expression level of a polypeptide (1) A step of contacting a vascular endothelial cell with a test substance, and (2) a step of measuring the expression level of the polypeptide according to any of the following: Screening method for adhesion inhibitors:
1) Polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24, or 2) SEQ ID NO: 2, 4, 6, 8 In the amino acid sequence represented by 10, 12, 14, 16, 18, 20, 22, or 24, having an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, and / or added; and In the presence of inflammatory cytokines, (a) a function of inducing cell adhesion molecule expression and / or (b) a function of inducing adhesion between vascular endothelial cells and white blood cells.
B. When using transformed cells (1) 1) DNA comprising the nucleotide sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 or 23, or 2) sequence It hybridizes under stringent conditions with DNA comprising the nucleotide sequence represented by the numbers 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 or 23, and In the presence, transformed with an expression vector comprising (a) a cell adhesion molecule expression-inducing function, and / or (b) a polypeptide encoding a function-inducing function between vascular endothelial cells and white blood cells, Contacting a cell expressing a polypeptide encoded by the gene with a test substance, and (2) measuring the expression level of the gene.
A screening method for a vascular endothelial cell adhesion inhibitor.

6.1.1 Method for Measuring Polynucleotide As an embodiment of the screening method of the present invention, there is a method for detection using an inflammatory cytokine-inducing polynucleotide as an index. As such a polynucleotide, a polynucleotide having the sequence of all or a part of the sequences shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23 of the sequence listing Is mentioned. The polynucleotide can be measured according to the following method.

  The cultured cells used in the method of the present invention may be cultured under any conditions as long as no test substance is added as long as any one of the target polynucleotides can be expressed. For example, culture conditions established for the cultured cells are known, and when the cells express the target polynucleotide under the conditions, the cells may be cultured under the conditions.

  In measuring a polynucleotide, it is first necessary to extract total RNA. RNA extraction methods include guanidine thiocyanate / cesium chloride ultracentrifugation, guanidine thiocyanate / hot phenol method, guanidine hydrochloride method, acidic guanidine thiocyanate / phenol / chloroform method (Chomczynski, P. and Sacchi, N., ( 1987) Anal.Biochem., 162, 156-159) can be employed, but the acidic guanidine thiocyanate / phenol / chloroform method is preferred. Moreover, commercially available reagents for RNA extraction (for example, ISOGEN (manufactured by Nippon Gene), TRIZOL reagent (manufactured by Invitrogen)) and the like can also be used according to the protocol attached to the reagent.

  It is preferable that the obtained total RNA is further purified to mRNA alone if necessary. Although the purification method is not particularly limited, it is known that most mRNAs present in the cytoplasm of eukaryotic cells have a poly (A) sequence at the 3 ′ end. By adsorbing mRNA to the oligo (dT) probe, capturing the mRNA on the paramagnetic particles immobilized with streptavidin using the binding between biotin / streptavidin, washing and then eluting the mRNA. MRNA can be purified. Alternatively, a method may be employed in which mRNA is adsorbed on an oligo (dT) cellulose column and then purified by elution. Furthermore, mRNA can be further fractionated by sucrose density gradient centrifugation or the like. However, for the method of the present invention, these mRNA purification steps are not essential, and as long as the expression of the polynucleotide encoding the inflammatory cytokine-inducing polypeptide can be detected, the total RNA is used in the subsequent steps. It can also be used.

  In the steps included in the screening method, the expression level of the inflammatory cytokine-derived polynucleotide can be measured by nucleic acid hybridization using a solid phase sample such as RT-PCR, ribonuclease protection assay, run-on assay, gene chip, etc. However, it is particularly preferable to use RT-PCR.

  First, a reverse transcriptase reaction using mRNA of the inflammatory cytokine-inducing polypeptide of the present invention as a template is performed, and then PCR is performed to specifically amplify a DNA fragment. In this method, in order to specifically amplify the nucleotide sequence of interest, an antisense primer complementary to a specific partial sequence of the mRNA of interest, and the sequence of cDNA generated from the antisense primer by reverse transcriptase A sense primer complementary to a specific partial sequence is used. RT-PCR can be performed using a commercially available kit (for example, RNA PCR kit AMV ver2.1: manufactured by Takara Bio Inc.) according to the manual attached to the kit, but can also be performed by the following method.

  The antisense primer used for both reverse transcriptase reaction and PCR is a contiguous 15 to 40 nucleotides in the antisense sequence of the polynucleotide sequence encoding the inflammatory cytokine-inducing polypeptide of the invention. , Preferably consisting of a nucleotide sequence of at least 18 to 30 nucleotides, more preferably 23 to 30 nucleotides.

  On the other hand, the sequence of the sense primer used in PCR is the nucleotide sequence of the polynucleotide encoding the inflammatory cytokine-inducing polypeptide of the present invention, from the position at the most 5 ′ end in the sequence corresponding to the complementary strand of the antisense primer. Furthermore, it consists of an arbitrary partial sequence of 15 to 40 nucleotides, preferably 18 to 35 nucleotides, more preferably 21 to 30 nucleotides in the sequence existing in the 5 ′ terminal region. However, if there are sequences complementary to each other in the sense primer and the antisense primer, annealing of the primers will amplify a non-specific sequence, which may hinder specific polynucleotide detection. It is preferable to design primers that avoid such combinations.

  These antisense primer and sense primer are both linked to the 5 ′ end of the nucleotide sequence defined above by a nucleotide sequence unrelated to the nucleotide sequence of the polynucleotide encoding the inflammatory cytokine-inducing polypeptide of the present invention. It may be added as. However, it is preferable that the linker does not cause non-specific annealing with the nucleic acid in the reaction solution during the reaction so as not to hinder specific polynucleotide detection. The RT-PCR reaction can be performed by a conventional method. In addition, the sample for detection by RT-PCR does not need to be normally refined to poly (A) + RNA.

  After completion of PCR, the reaction solution is electrophoresed to detect whether a band of a desired size is amplified. In order to perform quantitative detection, PCR is performed under the same conditions using a serially diluted cDNA clone as a standard template DNA, and the number of temperature cycles capable of quantitative detection is determined, or for example, every 5 cycles. A part of the reaction solution is sampled and subjected to electrophoresis. Further, for example, by using radiolabeled dCTP at the time of PCR reaction, the amount of radioactivity incorporated into the band can be quantified as an index. As a method for improving the reliability of polynucleotide quantification, a competitive RT-PCR method (Souaze et al. (1996) BioTechniques 21, 280-285) improved from the RT-PCR method described above, and a TaqMan PCR method (Heid et al. 1996) Genom.Res.6, 986-994) can also be used.

  Comparison of detection results between a cell-derived sample cultured in the presence of the test substance and a cell-derived sample cultured in the absence of the test substance, and as a result, the test in which the expression level of the target polynucleotide was reduced The substance is a substance that suppresses the function of the polypeptide of the present invention, and can suppress the adhesion of vascular endothelial cells due to the expression of the polypeptide, and thus can be a therapeutic or preventive agent for an anti-inflammatory agent.

6.1.2 Method for Measuring Polypeptide As another embodiment of the screening method of the present invention, there is a method for detecting an inflammatory cytokine-inducing polypeptide (protein). Specifically, the expression level of the polypeptide can be measured by the following embodiment.

  As a preferred embodiment of the method for measuring the polypeptide expression level, a method using an antibody will be described. The antibody used at this time can be prepared according to the description in “4. Antibody to inflammatory cytokine-inducing polypeptide” above. First, an antibody that specifically recognizes an inflammatory cytokine-inducing polypeptide is immobilized after the polypeptide in the sample is solid-phased on the inner bottom surface of a multi-well plate such as a 96-well plate or a 384-well plate or a membrane. The detection used is performed. Of these, the use of multiwell plates such as 96-well plates and 384-well plates is a method generally called solid-phase enzyme immunoassay (ELISA method) or radioisotope immunoassay (RIA method). On the other hand, as a method of immobilizing the membrane, a method of transferring the polypeptide to the membrane through polyacrylamide electrophoresis of the sample (Western blotting) or a so-called dot in which the sample or its diluted solution is directly impregnated into the membrane. Examples include blotting and slot blotting.

(1) Sample Preparation As a material for preparing a sample for this embodiment, a whole cell extract or a nuclear extract fraction of cultured cells cultured in the presence or absence of a test substance can be used. A whole cell extract is preferred. The whole cell extract is subjected to a step of preparing an ELISA / RIA sample or a Western blot sample after removing insoluble substances by high-speed centrifugation as necessary.

  As a sample for ELISA / RIA, for example, a whole cell extract of cultured cells cultured in the presence or absence of a test substance is used as it is or a sample diluted appropriately with a buffer is used. For the sample for Western blotting (for electrophoresis), for example, the whole cell extract of cultured cells cultured in the presence or absence of a test substance is used as it is, or appropriately diluted with a buffer, and subjected to SDS-polyacrylamide electrophoresis. Mix with sample buffer solution (manufactured by Sigma, etc.) containing 2-mercatol ethanol. In the case of dot / slot blotting, for example, the whole cell extract of cultured cells cultured in the presence or absence of the test substance, or an appropriate dilution with a buffer solution can be used directly by using a blotting device. Adsorb to.

(2) Immobilization of the sample In order to specifically detect the polypeptide in the sample obtained as described above, the sample is precipitated by immunoprecipitation, a method using ligand binding, etc., Detection can be performed without immobilization, or the sample to be detected as it is can be immobilized. In the case of immobilizing a polypeptide, as a membrane used for Western blotting, dot blotting or slot blotting, nitrocellulose membrane (for example, manufactured by Bio-Rad), nylon membrane (for example, High Bond-ECL (Amersham) (Manufactured by Pharmacia)) or polyvinylidene difluoride (PVDF) membrane (for example, manufactured by Bio-Rad).

  As a so-called blotting method for transferring a polypeptide from a gel after electrophoresis to a membrane, a wet blotting method (CURRENT PROTOCOLS IN IMMUNOLOGY volume 2 ed by J. E. Coligan, A. M. Kruisbek, D. H. Marg. EM Shevach, W. Strober), semi-dry blotting method (see CURRENT PROTOCOLS IN IMMUNOLOGY volume 2), and the like. Equipment for dot blotting and slot blotting is also commercially available (eg, Biodot).

  On the other hand, in order to perform detection and quantification by the ELISA method / RIA method, a sample or a diluted solution thereof (for example, 0.05% azide) is placed on a dedicated 96-well plate (for example, immunoplate maxi soap (manufactured by Nunk)). The inner bottom surface of the well by placing phosphate buffered saline (so-called “PBS”) containing sodium chloride and allowing to stand at 4 ° C. to room temperature overnight or at 37 ° C. for 1 to 3 hours. The polypeptide is adsorbed on to immobilize.

(3) Detection The antibody is directly labeled, or the antibody is used as a primary antibody in cooperation with a labeled secondary antibody that specifically recognizes the antibody (recognizes an antibody derived from the animal that produced the antibody). Used for detection.

  Preferred examples of the type of label include an enzyme (alkaline phosphatase or horseradish peroxidase) or biotin (however, an operation for binding an enzyme-labeled streptavidin to the biotin of the secondary antibody is added), but is not limited thereto. Various pre-labeled antibodies (or streptavidin) are commercially available for methods using labeled secondary antibodies (or labeled streptavidin). In the case of RIA, an antibody labeled with a radioisotope such as I125 is used, and the measurement is performed using a liquid scintillation counter or the like.

  By detecting the activity of these labeled enzymes, the amount of polypeptide as an antigen is measured. In the case of alkaline phosphatase or horseradish peroxidase, a substrate that develops color or a substrate that emits light by the catalyst of the enzyme is commercially available.

  When a substrate that develops color is used, it can be detected visually in Western blotting or dot / slot blotting. In the ELISA method, quantification is preferably performed by measuring the absorbance of each well (measurement wavelength varies depending on the substrate) using a commercially available microplate reader. In addition, preferably, a dilution series of the antigen used for antibody production in (3) above is prepared, and this is used as a standard antigen sample, and the detection operation is performed simultaneously with other samples, and the standard antigen concentration and the measured value are plotted. By creating a curve, it is possible to quantify the antigen concentration in other samples.

  On the other hand, if a substrate that emits light is used, it can be detected by autoradiography using an X-ray film or imaging plate in Western blotting or dot / slot blotting, or by photography using an instant camera. Quantification using densitometry, a molecular imager Fx system (manufactured by Bio-Rad) or the like is also possible. When a luminescent substrate is used in the ELISA method, the enzyme activity is measured using a luminescent microplate reader (for example, manufactured by Bio-Rad).

(4) Measurement procedure i) In the case of Western blot, dot blot or slot blot First, in order to prevent non-specific adsorption of antibodies, the membrane is previously blocked with substances that inhibit such non-specific adsorption (skimmed milk, casein, bovine An operation (blocking) of immersing in a buffer solution containing serum albumin, gelatin, polyvinylpyrrolidone, etc.) for a certain period of time is performed. As the composition of the blocking solution, for example, phosphate buffered saline (PBS) or Tris buffered saline (TBS) containing 5% skim milk, 0.05 to 0.1% Tween 20 is used. Instead of skim milk, Block Ace (Dainippon Pharmaceutical Co., Ltd.), 1 to 10% bovine serum albumin, 0.5 to 3% gelatin or 1% polyvinylpyrrolidone may be used. The blocking time is 16 to 24 hours at 4 ° C. or 1 to 3 hours at room temperature.

  Next, the membrane was washed with PBS or TBS containing 0.05 to 0.1% Tween 20 (hereinafter referred to as “washing solution”) to remove excess blocking solution, and then prepared by the method described in (3) above. The antibody is immersed in a solution appropriately diluted with a blocking solution for a certain period of time to allow the antibody to bind to the antigen on the membrane. The dilution ratio of the antibody at this time can be determined, for example, by conducting a preliminary Western blotting experiment using a serially diluted recombinant antigen described in 3) above as a sample. This antibody reaction operation is preferably performed at room temperature for 2 hours. After completion of the antibody reaction operation, the membrane is washed with a washing solution. Here, when the antibody used is labeled, the detection operation can be performed immediately. When an unlabeled antibody is used, a secondary antibody reaction is subsequently performed. For example, when using a commercially available product, the labeled secondary antibody is diluted 2000 to 20000 times with a blocking solution (if a suitable dilution factor is described in the attached instructions, follow that description). The membrane after the primary antibody is washed away is immersed in the secondary antibody solution at room temperature for 45 minutes to 1 hour, washed with a washing solution, and then a detection operation according to the labeling method is performed. The washing operation is performed, for example, by first shaking the membrane in the washing solution for 15 minutes, then exchanging the washing solution with a new one and shaking for 5 minutes, and then changing the washing solution again and shaking for 5 minutes. You may wash | clean by replacing | exchanging washing | cleaning liquid further as needed.

ii) ELISA / RIA
First, in order to prevent non-specific adsorption of the antibody to the inner bottom surface of the well on which the sample is solid-phased by the method (2), blocking is performed in advance as in the case of Western blot. The blocking conditions are as described in the Western blot section.

  Next, after the wells were washed with PBS or TBS (hereinafter referred to as “washing solution”) containing 0.05 to 0.1% Tween 20 to remove excess blocking solution, the well was prepared by the method described in (3) above. A solution obtained by appropriately diluting the prepared antibody with a washing solution is dispensed and incubated for a predetermined time to allow the antibody to bind to the antigen. The dilution ratio of the antibody at this time can be determined by conducting a preliminary ELISA experiment using, for example, a serial dilution of the recombinant antigen described in (3) above as a sample. This antibody reaction operation is preferably performed at room temperature for about 1 hour. After the antibody reaction operation is completed, the inside of the well is washed with a washing solution. Here, when the antibody used is labeled, the detection operation can be performed immediately. When an unlabeled antibody is used, a secondary antibody reaction is subsequently performed. For example, when using a commercially available product, the labeled secondary antibody is diluted 2000 to 20000 times with a washing solution (when a suitable dilution factor is described in the attached instructions, follow the description). The secondary antibody solution is dispensed into the well after the primary antibody has been removed by washing, incubated at room temperature for 1 to 3 hours, washed with a washing solution, and then subjected to a detection operation according to the labeling method. The washing operation is performed, for example, by first dispensing the washing solution into the well and shaking for 5 minutes, then replacing the washing solution with a new one and shaking for 5 minutes, and then changing the washing solution again and shaking for 5 minutes. You may wash | clean by replacing | exchanging washing | cleaning liquid further as needed.

  In the present invention, the so-called sandwich ELISA can be carried out, for example, by the method described below. First, in any one of the amino acid sequences of the inflammatory cytokine-derived polypeptide of the present invention, two regions that are rich in hydrophilicity are selected, and a partial peptide consisting of 6 or more amino acids in each region is synthesized, Two types of antibodies using the partial peptide as an antigen are obtained. One of these antibodies is labeled as described in (4) above. The unlabeled antibody is immobilized on the inner bottom surface of a 96-well ELISA plate according to the method described in (2) above. After blocking, the sample solution is placed in a well and incubated at room temperature for 1 hour. After washing the well, the labeled antibody dilution is dispensed into each well and incubated. After washing the well again, a detection operation is performed according to the labeling method.

(5) Determination The detection result is compared between a sample derived from a cell cultured in the presence of the test substance and a sample derived from a cell cultured in the absence of the test substance, and as a result, the production amount of the polypeptide is reduced. The test substance is a substance that suppresses the function of the target polypeptide, and can suppress the adhesion of vascular endothelial cells due to the expression of the polypeptide, and thus can be a therapeutic or preventive agent for an anti-inflammatory agent.

6.2 Screening by measurement of function The inflammatory cytokine-inducing polypeptide has the activity of inducing the expression of cell adhesion molecules by stimulating inflammatory cytokines and / or the activity of inducing adhesion between vascular endothelial cells and leukocytes. Therefore, the substance that suppresses the function of the polypeptide is a substance having an ability to suppress adhesion of vascular endothelial cells. Specifically, these substances can be obtained by a screening method including the following steps.

A. Screening method using adhesion to leukocytes (1) 1) DNA comprising a nucleotide sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 or 23, or 2) It hybridizes under stringent conditions with DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or 23, and inflammation In the presence of sex cytokines (a) encoding a polypeptide having a function of inducing the expression of cell adhesion molecules, and / or (b) a function of inducing adhesion between vascular endothelial cells and white blood cells,
A step of contacting a test substance with a cell that is transformed with an expression vector containing the polypeptide and expressing the polypeptide,
(2) A step of bringing leukocytes into contact with the cells, and (3) a step of measuring an adhesion amount between leukocytes and the cells.

B. Screening method using expression induction activity of cell adhesion molecule (1) 1) Consists of a base sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 or 23 DNA or 2) hybridizes under stringent conditions with DNA consisting of the base sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 or 23, And an expression vector comprising (a) a cell adhesion molecule expression inducing function in the presence of TNF-α and / or (b) a polypeptide encoding a polypeptide having a function of inducing adhesion between vascular endothelial cells and leukocytes. The step of contacting the test substance with a cell that is transformed with and expressing the polypeptide, and (2) measuring the expression level of the cell adhesion molecule,
A screening method for a vascular endothelial cell adhesion inhibitor.

  Specifically, a test substance is added to the culture supernatant and cultured for an appropriate period of time. Intercellular adhesion molecule (ICAM) -1, vascular cell adhesion molecule (VCAM) -1, and E-selectin are induced in vascular endothelial cells during inflammation. The expression of adhesion molecules is evaluated as an index. The expression of each adhesion molecule on the cell surface is detected by a technique using a flow cytometer after separating the endothelial cells from the incubator, staining with a fluorescent labeled monoclonal antibody (manufactured by BD Bioscience) against each adhesion molecule. be able to. Alternatively, it can be directly quantified on a microplate well by a method simulating enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody that recognizes each adhesion molecule. Specifically, the culture supernatant of endothelial cells cultured for an appropriate period of time with a test substance added on a 96-well microplate is removed, and the endothelial cells are fixed with a fixing solution of methanol: ethanol (3: 1 mixing ratio). After blocking with PBS containing 1% bovine serum albumin (BSA: Sigma), the endothelial cells were treated with anti-human ICAM-1, anti-VCAM-1, anti-E-selectin mouse IgG1 monoclonal antibody (manufactured by BD Bioscience). After reacting with a diluted solution containing PBS containing 0.1% BSA, washing with PBS containing 0.05% Tween-20 and blocking again with PBS containing 1% bovine serum albumin (BSA: Sigma), 0. Horse radish peroxidase (HRP) -labeled anti-mouse IgG antibody (Kirke) diluted in PBS containing 1% BSA aard & Perry Laboratories) and then washed with PBS containing 0.05% Tween-20, and each microplate using HRP was developed using peroxidase coloring kit O (Sumitomo Bakelite) according to the protocol of the instruction manual. By measuring the absorbance of the wells at 490 nm, the expression of each adhesion molecule on the endothelial cell surface can be quantified. Moreover, the free adhesion molecule secreted into the culture supernatant can also be measured with a Quantikine ELISA kit (manufactured by R & D Systems) according to the attached instruction manual. In the above method, it is determined that the test substance in which the expression of any adhesion molecule is reduced is a substance that suppresses the function of the inflammatory cytokine-inducing polypeptide of the present invention as compared to control (no test substance added) cells. be able to.

  On the other hand, the method for measuring the adhesion between white blood cells and vascular endothelial cells is performed by fluorescent labeling using 96 Vybrant Cell Adhesion Assay Kit on cells cultured for a suitable time with a test substance added on a 96-well microplate. T lymphocyte cultured cells (MOLT-4, Jurkat, etc.), monocyte cultured cells (THP-1, U937, etc.) or leukocytes isolated from human peripheral venous blood were added and adhered to endothelial cells after washing The fluorescence of white blood cells can be quantified by measuring with a fluorescent microplate reader. In the above method, the test substance with reduced adhesion of any white blood cell compared to control (no test substance added) cells is a substance that suppresses the function of the inflammatory cytokine-inducing polypeptide of the present invention. Can be determined.

6.3 Screening method using specific conjugate Here, specific conjugate includes proteins (including antibodies), aptamers, low-molecular compounds, etc. that can specifically bind to inflammatory cytokine-inducing polypeptides. . Inflammatory cytokine-inducing polypeptides are thought to regulate their activity by binding directly or indirectly to other proteins inside or outside the cell. Therefore, a substance that inhibits the binding between the polypeptide and the protein is highly likely to be a substance that regulates vascular endothelial cell adhesion. Specifically, these substances can be obtained by a screening method including the following steps.
(1) 1) Polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24, or 2) SEQ ID NO: 2, 4, In the amino acid sequence represented by 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24, it has an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, and / or added. And in the presence of an inflammatory cytokine, (a) a cell adhesion molecule expression-inducing function, and / or (b) a polypeptide showing an adhesion-inducing function between vascular endothelial cells and white blood cells, and a test substance, In the presence of a labeled specific conjugate of the polypeptide, and (2) measuring the amount of binding between the polypeptide and the specific conjugate.
A screening method for a vascular endothelial cell adhesion inhibitor.

  Examples of a method for obtaining a protein that can specifically bind include a method of performing affinity purification of a protein that binds to a purified inflammatory cytokine-inducing polypeptide. As an example of a specific method, an inflammatory cytokine-inducing polypeptide fused with a sequence consisting of 6 histidines as an affinity tag is prepared, and this is extracted with a cell extract (previously charged on a nickel-agarose column). The column is passed through the column) and incubated at 4 ° C. for 12 hours, and then nickel-agarose carrier is separately added to the mixture and incubated at 4 ° C. for 1 hour. After sufficiently washing the nickel-agarose carrier with a washing buffer, 100 mM imidazole is added to elute and purify the protein in the cell extract that specifically binds to the inflammatory cytokine-inducing polypeptide, and determine this structure. . Thus, it forms a complex with a protein that directly binds to an inflammatory cytokine-inducing polypeptide and a protein that does not have a binding activity to an inflammatory cytokine-inducing polypeptide but directly binds to an inflammatory cytokine-inducing polypeptide. Thus, a protein that indirectly binds to an inflammatory cytokine-inducing polypeptide can be purified [Experimental Medicine separate volume, Biomanual Series 5 “Transcription Factor Research Method”, pp215-219 (published by Yodosha)].

  Other methods include Far Western blotting [Experimental medicine separate volume, “New Genetic Engineering Handbook” p76-81 (published by Yodosha)] and two-hybrid system method using yeast and mammalian cells [Experimental Medicine separate volume, Cloning by “New Genetic Engineering Handbook” p66-75 (published by Yodosha) and “Checkmate Manmarian Two-Hybrid System” (manufactured by Promega) is also possible, but it is not limited to these methods.

  If a cDNA of a protein that interacts directly or indirectly with the inflammatory cytokine-inducing polypeptide of the present invention can be obtained by these methods, screening for a substance that inhibits the interaction between the inflammatory cytokine-inducing polypeptide and the protein is performed. Can be used. Specifically, first, a lysate (preferably a purified preparation of an inflammatory cytokine-inducing polypeptide) prepared by disrupting cells such as transformed cells in which the inflammatory cytokine-inducing polypeptide is expressed is prepared. Screening conditions such as buffer, ion, and / or pH are optimized, and the lysate and labeled protein that interacts with the inflammatory cytokine-inducing polypeptide in the optimized buffer together with the test compound for a period of time. Incubate. After the reaction, the solution is filtered with a nitrocellulose filter or the like, washed with an appropriate amount of buffer, and then the radioactivity remaining on the filter is measured with a liquid scintillation counter or the like. A compound that inhibits the binding of an inflammatory cytokine-inducing polypeptide and a protein that interacts with the polypeptide can be selected using the binding inhibition of the obtained label as an index. Whether this compound is an agonist or an antagonist can be confirmed by other screening methods described in this section.

  In addition, when the protein that can specifically bind is an antibody, it can be obtained according to the description in “4. Antibody to inflammatory cytokine-inducing polypeptide”.

  When the inflammatory cytokine-inducing polypeptide is indirectly bound to the protein and is mediated via some other factor, the above screening is performed in the same manner in the presence of a cell extract containing the factor, for example. I do. In this case, a substance that acts on the factor may be selected.

The specific binder can be labeled with various substances, usually in order to make its behavior detectable. The protein can be labeled by using a conventional method described in, for example, “Molecular Cell Biology Basic Experimental Method” (Takeichi Horie et al., 1994). Examples of various substances include chemiluminescent substances, enzymes, fluorescent substances, colored beads, radioisotopes, elements, metals, and biotin. Specific examples are shown below, but are not limited thereto. The chemiluminescent substance refers to, for example, luminol and acridinium ester. Examples of enzymes include β-galactosidase, alkaline phosphatase and peroxidase. Examples of the fluorescent substance include europium cryptate, FITC (fluorescein isothiocyanate) and RITC (tetramethylrhodamine isothiocynate). Examples of the colored beads include protein A beads, wheat germ agglutinin (WGA) beads, and streptavidin beads. Radioisotope refers to, for example, ¹⁴ C, ¹²⁵ I, ³ H, and the like. The element refers to a lanthanide element such as europium. Examples of metals include ferritin and gold colloid.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited at all by these.

Search for candidate inflammatory cytokine-inducing polynucleotides by DNA microarray analysis Vascular endothelial cells have important functions for inducing inflammatory reactions such as leukocyte infiltration and cytokine secretion. Therefore, in order to find a gene that is expressed in vascular endothelial cells and plays an important role in the inflammatory reaction, firstly, a gene whose expression is increased in vascular endothelial cells by stimulation of a typical inflammatory cytokine, tumor necrosis factor (TNF) -α. The search was performed by DNA microarray analysis according to the following procedure.

a) Preparation of Total RNA of TNF-α Stimulated Vascular Endothelial Cells Purified cryopreserved human umbilical vein vascular endothelial cells (HUVEC: manufactured by Cambrex BioScience Walkersville) according to the attached instruction manual (EGM-2) : Cambrex BioScience Walkersville Inc.) with EGM-2 additive factor set (Cambrex BioScience Walkersville Inc.) added in 15 ml culture solution (hereinafter EGM-2) in a 75 cm ² culture flask at 37 ° C. and CO ₂ concentration of 5% To 30% HEPES buffer (hereinafter referred to as HEPES solution: manufactured by Cambrex BioScience Walkersville), 0.025% trypsin / 0.01% E TA solution (hereinafter trypsin solution: Cambrex BioScienceWalkersville Co.), the cells were harvested using trypsin neutralizing solution (Cambrex BioScienceWalkersville Co.). This was centrifuged at 230 xg for 5 minutes at room temperature, suspended in a cell banker (manufactured by Toji Field Co., Ltd.) so that the cell concentration was 4.5 x 10 ⁵ cells / ml, and 1 ml each was dispensed into a serum tube. After being poured and stored frozen at −80 ° C., it was used as a stock in the following experiments. Four of the stock cells were resuspended in 15 ml of EGM-2, seeded in a 75 cm ² bottom area culture flask, and the culture supernatant was replaced with fresh 15 ml of EGM-2 every day for confluence. The culture was continued until Next, the cells are detached with HEPES solution, trypsin solution, and trypsin neutralization solution, and the cells are centrifuged at 230 × g at room temperature for 5 minutes, 10 ml of EGM-2 is added, and 2.5 ml each of 17.5 ml of EGM- dispensed into 15cm petri dish 4 Like containing the 2 min, the cells 37 ° C. while exchanging the culture supernatant with fresh EGM-2 of 20ml each day until confluence, CO ₂ concentration of 5% for The culture was continued under. The culture supernatant of 3 petri dishes with confluent cells contains recombinant human TNF-α (GT) 0.1% bovine serum albumin (BSA: Sigma Aldrich) to a concentration of 20 μg / ml 10 μl of a solution dissolved in phosphate buffer (PBS) was replaced with 20 ml of EGM-2 (TNF-α final concentration 10 ng / ml) and changed to 37 ° C. and CO ₂ concentration of 5% for 1 hour. After culturing for 4 hours and 24 hours, the culture supernatant is removed, washed with 10 ml of PBS, 12 ml of TRIzol reagent (Invitrogen) is added, cells are immediately lysed using a cell scraper, and 15 ml of the cell lysate is added. It moved to the centrifuge tube and cryopreserved at -80 degreeC. In addition, as for the cells for the remaining one dish, 20 ml of fresh EGM-2 was added instead of EGM-2 containing TNF-α as TNF-α-untreated cells for control (0-hour treatment). A cell lysate was prepared by the above method and stored at −80 ° C. Thereafter, the cell lysate was thawed at room temperature, and then total RNA was purified according to the instruction manual attached to the TRIzol reagent. RNA was dissolved in 500 μl of diethylpyrocarbonate (DEPC) -treated water.

b) DNA microarray analysis The following DNA microarray analysis is carried out by using an expressed sequence tag (prepared from Microarray Bar Corded Slide Type 7 Star (hereinafter referred to as Type 7 Star: manufactured by Amersham Bioscience)) using 12 human organ-derived cDNAs as templates. ) Including about 5000 sequence PCR fragments (about 500 base pairs in length) spotted using Array Spotter Generation III (Molecular Dynamics) and Kazusa human cDNA nylon microarray (hereinafter referred to as KDR array: Kaken) This was carried out using Genex). In addition, 2 spots of each PCR fragment were mounted on Type 7 Star.
Analysis in Type 7 Star was performed according to the following procedure. MRNA was amplified (aRNA-ized) using Message Amp aRNA Kit (Ambion) using 2.5 μg of the total RNA of a) as a template according to the attached instruction manual. Next, 1 μg of each aRNA at 0, 1, 4 and 24 hours after TNF-α treatment for 1 microarray was used for each microarray, and cDNA was prepared by Atlas Glass Fluorescent Labeling (Clontech) according to the attached instruction and Cy3 labeling or Cy5 labeling After that, a combination of Cy3 labeled cDNA treated with TNF-α treated for 0 hours (control) and Cy5 labeled cDNA treated with TNF-α treated with 1, 4 and 24 hours, and conversely, Cy5 labeled cDNA treated with TNF-α treated for 0 hours (control). Were mixed with a combination of Cy3 labeled cDNA (total 6 types of combinations) for 1, 4 and 24 hours after treatment with TNF-α and purified with MinElute PCR Purification Kit (Qiagen). Then, 50 μl of each mixture of purified labeled cDNA, 50 μl of Hybridization Buffer Version 2 (Amersham Biosciences), and 100 μl of formamide were mixed, and an automatic microarray hybridization processor (Automatic Slide Processor (ASP) (Amersham Biosciences) ) Was used to hybridize and wash one microarray (total 6) for each type of mixed solution according to the instruction manual. These microarrays were scanned with Array Scanner Generation III (manufactured by Molecular Dynamics), and fluorescence signals of Cy3 and Cy5 of the microarray were respectively acquired as image data. Further, with respect to the acquired image data, each spot was gridded using image analysis software ArrayVison (manufactured by Imaging Research), the intensity of both the fluorescence signals of Cy3 and Cy5 was digitized, and the digitized data was stored in a text file. .

Moreover, the analysis by a KDR array was analyzed in the following procedures. 17 μg of total RNA of 0), 1, 4 and 24 hours treated with a) of TNF-α in the above and 8 μg of dT25 oligomer were mixed and diluted with DEPC-treated distilled water to a total volume of 18.8 μl, and 70 ° C. for 10 minutes. After heating, it was cooled rapidly on ice. Superscript First-Strand Synthesis System for RT-PCR (manufactured by Invitrogen) attached to this is 5 × 1st Strand Buffer 10 μl, 0.1M dithothiitol (DTT) 5 μl, TP ) 4 μl each containing 10 mM, 0.1 mM dCTP (prepared by diluting 10 mM manufactured by Invitrogen 100-fold) 0.2 μl, 111 TBq / mmol, 370 Mbq / ml [α- ³³ P] dCTP (manufactured by Amersham Biosciences) ) Add 10 μl, incubate at 42 ° C. for 2 minutes, add 2 μl of SuperScript II reverse transcriptase and incubate at 42 ° C. for 1 hour It was. Next, it was purified by MinElute PCR Purification Kit (manufactured by Qiagen) according to the attached instruction manual, and 10 mg / ml Cot-1 DNA (manufactured by Invitrogen) 2.5 μl, 1 μg / μl polyadenylic acid (manufactured by Sigma Aldrich) 1 μl was added, and the mixture was added to 420 μl of Perfect Hyb Hybridization Solution (manufactured by Toyobo Co., Ltd.) that had been heated to 68 ° C. after heat denaturation at 100 ° C. for 5 minutes, and incubated at 68 ° C. for 30 minutes. At the same time, eight KDR arrays are rolled into a glass vial (Mighty Vial: manufactured by Tech Jam) with a capacity of 5 ml, a barrel diameter of 16.5 mmφ, and a total height of 45 mm. Place a glass rod, add 200 μl of PerfectHybridization Solution to each vial, place the vials in series in a hybridization bottle (Robbins) and rotate at 68 ° C. at 68 ° C. while rotating with a Micro Hybridization Incubator (Robbins). Prehybridization was performed for minutes. Thereafter, the Perfect Hyb Hybridization Solution in the vial was discarded, and 200 μl of Perfect Hyb Hybridization Solution mixed with ³³ P-labeled cDNA mixed with this was added to each KDR array, and rotated at 68 ° C. in the same manner as in prehybridization. Incubate overnight and hybridize. In addition, hybridization was performed on two KDR arrays for each sample of 0, 1, 4, and 24 hours treated with TNF-α. Then, remove the KDR array from the vial and perform 2 × SSC, 1% SDS, 68 ° C., 15 minutes of washing twice, 0.1 × SSC, 1% SDS, 68 ° C., 15 minutes of washing twice. The KDR array was exposed to an imaging plate (IP: manufactured by Fuji Film) for 1, 4 or 7 days, and the radioactivity of the KDR array transferred to the IP was measured with a fluoro image analyzer FLA3000G (manufactured by Fuji Film). Acquired as. Then, using the image data, each spot was gridded by image analysis software ArrayGauge (manufactured by Fuji Film Co., Ltd.), and the radioactivity was digitized and stored in a text file.
Next, numerical data of each spot obtained by the analysis of both microarrays was integrated using GeneSpring (manufactured by Agilent) as microarray data analysis software, and TNF-α1, 4, 24 hours for each spot. Calculate the signal ratio obtained by interrupting the signal intensity of treatment with the signal intensity of TNF-α0 time treatment (TNF-α untreated), that is, how many times the expression changes after 1, 4 and 24 hours of treatment due to TNF-α treatment, Data of 3 spots or more out of a total of 4 spots in which data of fluorescent spots (Cy3, Cy5) are replaced by 2 spots on the microarray in the Type 7 Star, or in the case of Kazusa array, two arrays analyzed in parallel In either point after 1, 4 or 24 hours Gunaru intensity ratio were extracted spot expressing increased 2 or more i.e. more than double. Furthermore, as a result of analyzing the PCR fragment sequence of the extracted spot as a query, BioSCOUT (manufactured by Lion) and European Bioinstitutes Institute (EBI) and Ensembl, which is a public database of Sanger Institute, resulted in a 2-fold increase in expression. Spots determined to be present were finally aggregated into a total of 142 genes, excluding duplicates. Then, information such as full-length mRNA sequences was obtained from the database, and IDs were attached to these in the form of “TNFUP_serial number”. Of these 142 genes, the gene encoding the inflammatory cytokine-inducing polypeptide targeted by the present invention due to the discovery of a novel function in the analysis of Examples 2 and 3 described later is TNFUP_0013 (sequence No. 1), TNFUP — 0064 (SEQ ID NO: 3), TNFUP — 0072 (SEQ ID NO: 5), TNFUP — 0113 (SEQ ID NO: 7), TNFUP — 0114 (SEQ ID NO: 9), and TNFUP — 0142 (SEQ ID NO: 11). At this time, two for TNFUP_0072 (TNFUP_0072; SEQ ID NO: 5, TNFUP_0072B; SEQ ID NO: 13), and six for TNFUP_0142 (TNFUP_0142; SEQ ID NO: 11, TNFUP_0142B; SEQ ID NO: 15, TNFUP_0142C; SEQ ID NO: 17, TNFUP_014; There was a splicing variant of SEQ ID NO: 19, TNFUP — 0142E; SEQ ID NO: 21, TNFUP — 0142F; SEQ ID NO: 23). And as a result of database search, the gene names of these 6 genes are on the Entrez Gene of National Center for Biotechnology Information (NCBI), which is a public database. -Like (IFI44L), TNFUP_0072 is TNFAIP3 interacting protein 1 (TNIP1), TNFUP_0113 is TBC1 domain family, member 4 (TBC1D4), TNFUP_0114 is NCSTN, NCSTN TNFUP — 0142 was SON DNA binding protein (SON).

Functional screening of inflammatory cytokine-derived polypeptides using RNA interference (RNAi) (primary screening)
By knocking down each gene with a small interfering RNA (siRNA), it is determined whether 142 up-regulated genes extracted from the DNA microarray analysis of Example 1 have an important function in inflammation in endothelial cells. The following procedure was used to verify whether the induction of adhesion molecule expression that occurs in HUVEC upon stimulation can be suppressed. That is, when the expression of an adhesion molecule is suppressed by siRNA, the gene targeted by the siRNA becomes an inflammatory cytokine-inducing polynucleotide candidate.

a) Preparation of siRNA For functional screening of inflammatory cytokine-inducing polypeptides using RNAi, in vitro transcription synthesis of siRNA was performed using CUGA7 in vitro siRNA Synthesis Kit (Nippon Gene). In addition, among the siRNAs synthesized this time, a prominent adhesion molecule expression inhibitory effect was confirmed in Example 2d) described later, and an inflammatory cytokine-inducing polypeptide 6 that was confirmed to have a novel function by analysis of Example 3 described later. Table 1 shows examples of siRNAs for genes, target sequences of negative control siRNAs and positive control siRNAs, synthesis oligo DNA sequences, and synthesized siRNA sequences. Specific siRNA synthesis / preparation was performed as follows. For the up-regulated gene (142 gene) found in Example 1b), an open reading frame (ORF) was extracted from the full-length mRNA sequence, and if there was a splicing variant, only the base sequence of the common portion was extracted. . Next, the siRNA sense strand 3 ′ overhang 2 base portion and the siRNA double strand portion (double strand region in the table) 19 base portion extracted from the base sequence extracted as shown in the examples of Table 1 and Table 2 below, and A 23-base region (Target Sequence: target sequence in the table) consisting of 2 base portions of the 3 ′ overhang portion of the siRNA antisense strand was selected for each gene, and the target sequence was determined according to the CUGA7 in vitro siRNA Synthesis Kit instruction manual. Sense strand template oligo DNA and antisense strand template oligo DNA, for example, in Table 1 in which a complementary sequence (5'-CTATAGTGAGTCGTATTA-3 ') of the promoter sequence of T7 RNA polymerase is added to 3' Designed by chemical synthesis (manufactured by Sigma-Genosys). SEQ ID NO: 25-30 is TNFUP_0013, SEQ ID NO: 31-32 is TNFUP_0064, SEQ ID NO: 33-34 is TNFUP_0072, SEQ ID NO: 35-36 is TNFUP_0113, SEQ ID NO: 37-38 is TNFUP_0114, and SEQ ID NO: 39-42 is TNFUP_0142. It is the base sequence of oligo DNA for siRNA synthesis. SEQ ID NOs: 45 and 46 are intercellular adhesion molecule (ICAM) -1, SEQ ID NOs: 47 and 48 are vascular cell adhesion molecule (VCAM) -1, SEQ ID NOs: 49 and 50 are E-selectin, and SEQ ID NOs: 51 and 52 are tumors. Oligo DNA for synthesizing positive control siRNA against necrosis factor receptor 1 (TNFR1) (previously confirmed that the siRNA of the product has a high knockdown effect) and SEQ ID NOs: 43 and 44 hit any gene in the Blast search against GenBank This is a base sequence of an oligo DNA for negative control siRNA having a 19-base sequence designed in the target region in the target region. Some modifications were made to the instructions using the CUGA7 in vitro siRNA Synthesis Kit kit using as a template all the oligo DNAs designed for the 142 gene and control gene detected in Example 1 described above. The method was used for in vitro transcription synthesis and purification of siRNA. The modification is to suppress the interferon response induced in cells transfected with siRNA transcribed in vitro by removing the phosphate group at the 5 ′ end. Kim et al. (Nature Biotechnology, 2004, 22, p321-325). This is an improved version of the method reported by. Specifically, after carrying out in vitro transcription reaction and nuclease reaction in half of the standard protocol of the kit, 7 μl of StrataClean Resin (manufactured by Stratagene) was added to the 50 μl reaction solution and stirred for 1 minute with a vortex mixer. The mixture was allowed to stand at room temperature, centrifuged at 12000 rpm for 1 minute at room temperature, and the supernatant was purified with MicroSpin G-25 (Amersham Bioscience), and then 20 units (2 μl) of calf intestinal, alkaline phosphate (CIP: New England Biolabs). ), 6 μl of 10 × reaction buffer (attached to CIP) and 2 μl of RNase Free distilled water (total amount 60 μl), stirred by tapping and stirred at 37 ° C. for 1 hour. The mixture was incubated, 40 μl of RNase Free distilled water was added, and finally siRNA was purified by phenol / chloroform according to the protocol of CUGA7 in vitro siRNA Synthesis Kit. Then, the concentration of siRNA was measured using a spectrophotometer and adjusted to 100 ng / μl with RNA Free distilled water. When siRNAs are synthesized by the above method using oligo DNAs having the sequences of SEQ ID NOs: 25 to 52, those having the sequences shown in siRNA Sequences in Tables 1 and 2 are synthesized.

b) Culture and preparation of HUVEC for functional screening In the culture flask (Sumitomo Bakelite Co., Ltd.) having a bottom area of 75 cm ² coated with bovine collagen type I according to the instruction manual using the purchased frozen storage HUVEC (manufactured by Cell Applications) In an endothelial cell growth medium (manufactured by Cell Applications), the cells were cultured under conditions of 5% CO ₂ and 37 ° C., and the confluent cells were subcultured using subculture set A (manufactured by Cell Applications). Peel and start subculturing, and if the cells from one culture flask are subcultured into two, the first subculture cell is confluent after 7 to 8 passages Is removed using subculture set A, centrifuged at 200 xg for 5 minutes, and then 2 Suspended in l cell bunker (Ju慈 Field Inc.), which was used in the following experiments the cells were cryopreserved two serum tubes each 1ml as stock. Cells from one frozen stock are added to a bovine collagen type I-coated bottom area 75 cm ² culture flask containing 15 ml of medium, and after 6 to 18 hours, replaced with 25 ml of fresh medium and cultured for 3 days. Cells were collected using subculture set A, centrifuged at 200 × g for 5 minutes, and a suspension was prepared in endothelial cell growth medium to a cell concentration of 1 × 10 ⁵ cells / ml. 100 μl (1 × 10 ⁴ cells) was dispensed into each well of a 96-well microplate (Sumitomo Bakelite Co., Ltd.) coated with bovine collagen type I. After culturing this for 18 to 24 hours, the cells in each well reached 95-100% confluence. A total of three 96-well microplates with analysis for primary screening were prepared for ICAM-1, VCAM-1, and E-selectin analysis.

c) siRNA transfection The siRNA synthesized in the above a) was transfected into the cells in each well of the above b) by using the Targetfect-siRNA transfection kit (manufactured by Targeting Systems) by the following operation. In addition, for each analysis of screening, 92 siRNAs, 3 positive control siRNAs (for ICAM-1, VCAM-1, and E-selectin) and 3 negative siRNAs for 96 siRNAs (for ICAM-1 measurement, Transfection was performed on a 96-well plate for VCAM-1 measurement and E-Selectin measurement.
Each 100 ng / μl of siRNA was dispensed into a well of a 96-well PCR plate in a volume of 3.1 μl (0.31 μg), and 120 μl of SolnA, 75 μl of SolnB, and Dulbecco 'containing 4500 mg / l glucose contained in the kit. 15.5 ml of a mixed solution of s modified Eagle's medium (DMEM: Sigma-Aldrich) was added to each well of the PCR plate containing siRNA, and the PCR plate was sealed with ABsolute QPCR Seal (ABgene). Thereafter, the mixture was mixed by inversion, and the mixture was spun down with a microplate centrifuge and incubated at 37 ° C. for 25 minutes. Then, the culture supernatant of the 96-well microplate in which the cells of the above b) are cultured is extracted with an 8-series type aspirator, and immediately, 51 μl of siRNA mixed solution in each well of the 96-well PCR plate is used with an 8-series micropipette. (Corresponding to 0.1 μg siRNA per well) was added and incubated for 30 minutes at 37 ° C. Immediately thereafter, 100 μl of fresh endothelial cell growth medium was added by an 8-series micropipette and incubated for 16 hours under conditions of 5% CO ₂ concentration and 37 ° C. Thereafter, 126 μl of the supernatant in each well was removed, 75 μl of fresh endothelial cell growth medium was added to make the total amount of the supernatant 100 μl, and the ICAM-1 measurement plate and VCAM-1 measurement plate were used for E-selectin measurement for 8 hours. The plate was incubated for 16 hours under the same conditions. Thereafter, 50 μl (final concentration: 0.25 ng / ml) of human TNF-α (manufactured by GT) diluted with endothelial cell growth medium to a concentration of 0.75 ng / ml was added to each well of the microplate for ICAM-1. 18.5 hours, each well of the VCAM-1 measurement plate was added with 50 μl (final concentration 10 ng / ml) of 30 ng / μl of TNF-α dilution, 18.5 hours, and each well of the E-selectin measurement plate was added. Added 30 μg / μl of TNF-α dilution (final concentration 10 ng / ml) and incubated for 2.5 hours under conditions of 5% CO ₂ and 37 ° C.

d) Cell-enzyme-linked immunosorbent assay (Cell-ELISA)
Next, each adhesion molecule expressed on the HUVEC surface was detected on a microplate by a method simulating enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody that recognizes the adhesion molecule. Specifically, remove the supernatant of the 96-well microplate that was incubated after siRNA transfection in c) above, immediately add 100 μl of methanol: ethanol (3: 1 mixture ratio) fixative, and let stand at room temperature for 1 hour. After washing with PBS containing 0.05% Tween-20 three times using a plate washer, 100 μl of PBS containing 1% BSA was added, blocking was performed at room temperature for 30 minutes, the supernatant was removed, and anti-human ICAM-1 was removed. , Anti-VCAM-1, anti-E-selectin mouse IgG1 monoclonal antibody (manufactured by BD Bioscience) diluted with PBS containing 0.1% BSA 500 times (final concentration 1 μg / ml) in each well of a corresponding microplate Add 100 μl each and allow the antibody to react for 30 minutes at room temperature. Washer washed 4 times with 0.05% Tween-20 containing PBS using. Then, after blocking again with 100 μl of PBS containing 1% BSA at room temperature for 30 minutes and removing the supernatant, the horse radish peroxidase (HRP) -labeled anti-mouse diluted 1000 times with PBS containing 0.1% BSA (final concentration 1 μg / ml) 100 μl of IgG antibody (manufactured by Kirkegaard & Perry Laboratories) was added and reacted, then washed 5 times with PBS containing 0.05% Tween-20 using a microplate washer, the supernatant was completely removed, and HRP was removed. The peroxidase color development kit O (manufactured by Sumitomo Bakelite Co., Ltd.) was used for color development reaction according to the protocol in the instruction manual, and the absorbance at 490 nm of each microplate well was measured to evaluate the expression of each adhesion molecule on the endothelial cell surface. That is, when the absorbance is low, the expression of the adhesion molecule is more strongly suppressed. Furthermore, the mean value (mean) and standard deviation (SD) of absorbance at 490 nm for 92 siRNAs were calculated for each of the ICAM-1, VCAM-1, and E-selectin analysis plates, and the absorbance below “mean-2 × SD” was calculated. The indicated siRNA was determined to be positive. As a result, 21 siRNAs corresponding to 18 genes showed an expression suppression effect of any one of ICAM-1, VCAM-1, and E-selectin, and were determined to be positive. Selected as a nucleotide candidate.

Verification of Inflammatory Cytokine-Induced Polynucleotide Candidates The effect of 21 siRNAs determined to be positive in Example 2d) was re-verified by the following procedure. As a result of this revalidation, TNFUP — 0013 (SEQ ID NO: 1), TNFUP — 0064 (SEQ ID NO: 3), TNFUP — 0072 (SEQ ID NO: 5), TNFUP — 0113 (SEQ ID NO: 7), TNFUP — 0114 (SEQ ID NO: 9), TNFUP — 0142 ( Six genes of SEQ ID NO: 11) were narrowed down.

a) Reproducibility of adhesion molecule expression suppression by Cell-ELISA (secondary screening)
In order to confirm the reproducibility of the results of the primary screening described in Example 2d), the number of analyzes for 21 siRNAs that showed a positive reaction was increased in 4 series (4 wells), and the same method as in Example 2 was repeated. Analysis was performed. As a result, most of the siRNA adhesion inhibitory effect detected by the primary screening was reproduced.
FIGS. 1 to 3 show a total of 9 genes among 21 siRNAs for which 6 secondary genes were confirmed to have an inhibitory effect on the expression of adhesion molecules in this secondary screening, and were judged to be highly related to inflammation in b) and c) described later. Results are shown for one siRNA and negative and positive control siRNA. In addition, the vertical axis | shaft of a figure shows the light absorbency of 490 nm at the time of transfecting each siRNA, and a shaded column shows that it is siRNA which the expression suppression effect of the applicable adhesion molecule was positive in the primary screening of d). . The gray column indicates that the corresponding adhesion molecule is not positive but is positive for other adhesion molecules, and the white and black columns indicate data in siRNAs of the negative control and positive control, respectively. Each data shows the average value of the data of 4 wells, and the error bar shows the standard deviation of the data of 4 wells.
As for the expression of ICAM-1 (FIG. 1), it is considered that ICAM-1 was detected higher than usual in the assay of this batch when estimated from the data of the positive control siRNA (ICAM1-si). Although it is difficult to understand, three siRNAs against TNFUP_0013 (TNFUP_0013-1, 3, 4) and siRNA against TNFUP_0064 (TNFUP_0064-4) showed an expression suppression effect of about 40% reduction at 490 nm absorbance than the negative control siRNA. Moreover, about 30% of expression suppression was recognized by siRNA (TNFUP_0114-3) with respect to TNFUP_0114. Furthermore, for TNFUP — 0142, only one of the two siRNAs (TNFUP — 0142-3) of which the inhibitory effect was detected in the primary screening showed an inhibitory effect of about 30%, but one siRNA (TNFUP — 0142-2) showed about 40%. An inhibitory effect was observed.
Regarding the expression of VCAM-1 (FIG. 2), siRNA against TNFUP_0064 (TNFUP — 0064-4), siRNA against TNFUP — 0114 (TNFUP — 0114-3), and two siRNAs against TNFUP — 0142 (TNFUP — 0142-2, 3) are more 490 nm absorbance than negative control siRNA. The effect of suppressing the expression was reduced by 50% or more. The siRNA against TNFUP_0072 (TNFUP_0072-2) was found to have an inhibitory effect of about 50%, although the effect was slightly weaker than these.
Furthermore, regarding the expression of E-selectin (FIG. 3), about 50% decrease in absorbance at 490 nm in two siRNAs against TNFUP_0013 (TNFUP_0013-3, 4) in which the suppressive effect was detected in the primary screening, compared to the negative control siRNA. The expression suppression effect was shown. In addition, siRNA against TNFUP_0072 (TNFUP — 0072-2), siRNA against TNFUP — 0113 (TNFUP — 0113-4), and siRNA against TNFUP — 0114 (TNFUP — 0114-3) were found to have an inhibitory effect of 50% or more. And about TNFUP_0142, the suppression effect of 50% or more was recognized by one siRNA (TNFUP_0142-2) among two siRNA which detected the suppression effect by the primary screening.
From these results, TNFUP_0013, which corresponds to the siRNA shown in FIGS. 1 to 3, is ICAM-1 and E-selectin, TNFUP_0064 is ICAM-1 and VCAM-1, TNFUP_0072 is VCAM-1 and E-selectin, and TNFUP_0113 is ENF. -Selectin, TNFUP_0114 is considered to be highly likely to be involved in the expression induction of ICAM-1, VCAM-1 and E-selectin, and TNFUP_0142 is ICAM-1, VCAM-1 and E-selectin, so-called inflammatory cytokine-derived polynucleotide It became clear that.

b) Evaluation of gene variation by quantitative PCR Whether or not the expression of each adhesion molecule varies at the mRNA level due to 21 siRNAs positive in Example 2d), and the gene targeted by the siRNA is actually Whether it was knocked down was evaluated by quantitative PCR according to the following procedure. One HUVEC stock cell cryopreserved after 8 passages in a) above is added to a 75 cm ² bottom area culture flask coated with bovine collagen type I containing 15 ml of endothelial cell growth medium. After culturing for 6 to 18 hours under conditions of CO ₂ concentration 5% and 37 ° C., the medium was replaced with 25 ml of fresh medium, and confluent cells were collected using subculture set A after 3 days of culture. After centrifuging at 200 × g for 5 minutes, a suspension was prepared in an endothelial cell growth medium to a cell concentration of 1 × 10 ⁵ cells / ml. 2 ml (2 × 10 ⁵ cells) was dispensed into each well of a 6-well culture plate coated with bovine collagen type I (manufactured by Sumitomo Bakelite), and overnight under conditions of 5% CO ₂ and 37 ° C. Incubated and cultured to 95-100% confluent. Next, 21 siRNA or negative control or positive control siRNA that was positive in Example 2d) was obtained by mixing 8 μl of SolnA, 5 μl of SolnB and 987 μl of DMEM containing 4500 mg / l glucose included in the Targetfect-siRNA transcription kit kit. (100 ng / μl each), added to 20 μl, incubated for 25 minutes at 37 ° C., replaced with 2 ml of HUVEC culture supernatant, immediately incubated for 30 minutes at 37 ° C. with a CO ₂ concentration of 5%, fresh 2 ml of an endothelial cell growth medium was added, and the mixture was incubated for 14-15 hours under the conditions of 5% CO ₂ concentration and 37 ° C. Then, the culture supernatant was replaced with 1.9 ml of fresh endothelial cell growth medium, and for measurement of ICAM-1, 10 ng after replacement of the medium, 100 μl of 5 ng / ml TNF-α (final concentration: 0.25 ng / ml), VCAM-1 For measurement, 100 μl of 200 ng / ml TNF-α (final concentration 10 ng / ml) 10 hours after the medium replacement, and for E-selectin 100 μl of 200 ng / ml TNF-α 15.5 hours after the medium replacement (final concentration 10 ng) / Ml) was added. Then, after incubation for 14 hours in the case of ICAM-1 and VCAM-1 measurement, and 2.5 hours in the case of E-selectin measurement under the conditions of 5% CO ₂ concentration and 37 ° C., RNeasy Micro Kit (manufactured by Qiagen) ) Was used to extract total RNA according to the attached instruction manual. Next, 0.5 μg of total RNA was used for each sample, and cDNA was synthesized using an oligo dT primer with a superscript first strand system (manufactured by Invitrogen) according to the instructions for use, and this was synthesized using QIAquick PCR Purification Kit (Qiagen). (CDNA is eluted with 30 μl elution buffer), and 1 μl of cDNA per reaction is used as a template, and PCR primer is used at a concentration of 0.2 μM with SYBR Green PCR Master Mix (manufactured by Applied Biosystems). In accordance with the above, a reaction solution (each reaction is in two series) is prepared, heated at 95 ° C. for 10 minutes with GeneAmp 5700 (Applied Biosystems), and then at 95 ° C. for 15 seconds. The amplification curve was prepared by repeating the reaction at 60 ° C. for 1 minute 40 times. Of the 18 gene PCR primers corresponding to the 21 siRNAs used this time, the PCR primer for 6 genes encoding the inflammatory cytokine-inducing polypeptide found by the analysis of Example 3a) -c) is TNFUP_0013: sequence. No. 53-54, TNFUP_0064: SEQ ID NO: 55-56, TNFUP_0072: SEQ ID NO: 57-58, TNFUP — 0113: SEQ ID NO: 59-60, TNFUP — 0114: SEQ ID NO: 61-62, TNFUP — 0142: SEQ ID NO: 63-64. PCR primers for adhesion molecules are ICAM-1: SEQ ID NO: 65-66, VCAM-1: SEQ ID NO: 67-68, E-selectin: SEQ ID NO: 69-70, and PCR primer for endogenous control gene is β-actin: sequence No. 71-72, glyeraldehydre-3-phosphate dehydrogenase (GAPDH): SEQ ID NOs: 73-74 (see Table 3). The Ct value of each well was obtained from the obtained amplification curve using GeneAmp 5700 SDS software (Applied Biosystems), and each gene was calculated from the average value of Ct of GAPDH and β-actin for each siRNA transfected sample. The ΔCt value obtained by subtracting the Ct value in γ was determined, and this was compared with the ΔCt value when the negative control siRNA was transfected to evaluate the variation in expression of each gene. Among the 21 siRNA data that were positive in Example 2d), 6 genes (genes encoding inflammatory cytokine-inducing polypeptides) judged to be highly relevant to inflammation from the results of Examples 3a) to c) The variation in the expression of mRNA of each adhesion molecule and the target gene of each siRNA when 9 siRNAs are transfected with (a) is shown in FIG. 4 ab, FIG. 5 ab, and FIG. 6 ab. Although some of the siRNAs shown in FIGS. 4a, 5a, and 6a have a small range of fluctuation in the expression of adhesion molecule mRNA, the difference between the negative control and the ΔCt value in most of the siRNAs shown in FIGS. An expression decrease of 1 or more, ie 50%, was observed, and the results of Example 2d) and Example 3a) were reproduced at the mRNA level.
Specifically, for mRNA expression of ICAM-1 (FIG. 4a), 2 out of 3 siRNAs against TNFUP_0013 (TNFUP_0013-1, 3), siRNA against TNFUP_0064 (TNFUP_0064-4), siRNA against TNFUP_0114 (TNFUP — 0114-3) In one of the two siRNAs against TNFUP — 0142 (TNFUP — 0142-2), an expression decrease of 50% or more was observed.
As for mRNA expression of VCAM-1 (FIG. 5a), siRNA for TNFUP_0064 (TNFUP_0064-4), siRNA for TNFUP_0072 (TNFUP_0072-2), siRNA for TNFUP_0114 (TNFUP_0114-3), and two siRNAs for TNFUP_0142 (TNFUP01-01) 3), a decrease in expression of 50% or more was observed.
Furthermore, regarding mRNA expression of E-selectin (FIG. 6a), 50% or more in three siRNAs against TNFUP_0013 (TNFUP_0013-1, 3, 4), siRNA against TNFUP_0113 (TNFUP_0113-4), and siRNA against TNFUP_0114 (TNFUP_0114-3) Decreased expression. In addition, an expression decrease close to 50% was observed in siRNA against TNFUP_0072 (TNFUP_0072-2) and one of the two siRNAs against TNFUP_0142 (TNFUP_0142-2).
As shown in FIGS. 4b, 5b, and 6b, siRNAs against these 6 genes knock down the mRNA expression of the target gene by 1 or more, that is, 50% or more, in the difference between the negative control and the ΔCt value. It was also confirmed.
Thus, knocking down these 6 genes with their siRNA reveals that TNF-α-induced adhesion molecule expression induction is also suppressed by mRNA, and these 6 genes are related to the inflammatory response of vascular endothelial cells. It was reconfirmed that it is a highly gene (inflammatory cytokine-inducing polynucleotide).

c) Leukocyte cell-HUVEC adhesion analysis It was verified whether or not the adhesion between leukocyte cells and HUVEC induced by TNF-α was actually suppressed by 21 siRNAs that were positive in the primary screening of Example 2d). Specifically, according to the procedure of Example 2a), the cells were cultured and prepared on four black clear bottom 96-well microplates (manufactured by BD Bioscience) coated with bovine collagen type I. Next, 16.4 μl (1.64 μg) of 21 siRNAs (100 ng / μl) dispensed into 96-well format sterilized microtubes, SolnA 164 μl and SolnB 102.5 μl contained in the Targetfect-siRNA transfection kit. And 2500 ml of Dulbecco's modified Eagle's medium (DMEM: Sigma-Aldrich) containing 4500 mg / l glucose were added and mixed by inversion, followed by spin-down and incubation at 37 ° C. for 25 minutes. Then, the culture supernatant of the 96-well microplate in which HUVEC was cultured was extracted with an 8-spin type aspirator, and immediately using an 8-strip micropipette, 51 μl each of the above siRNA mixture was equivalent to 1 μg siRNA per well. (Added to a total of 16 wells of 4 wells × 4 plates per siRNA) and incubated at 37 ° C. for 30 minutes. Immediately thereafter, 100 μl of fresh endothelial cell growth medium was added by 8 micropipettes and incubated for 15 hours at a CO ₂ concentration of 5% at 37 ° C. Thereafter, 126 μl of the supernatant in each well was removed, 75 μl of fresh endothelial cell growth medium was added to make the total amount of the supernatant 100 μl, and incubation was continued under the same conditions for 9 hours. Thereafter, 50 μl (final concentration 10 ng / ml) of 30 ng / μl of TNF-α dilution was added and incubated for 18 hours under the conditions of 5% CO ₂ concentration and 37 ° C. On the other hand, T lymphocyte cultured cells (MOLT-4, Jurkat), monocyte cultured cells (THP-1, U937) subcultured in RPMI 1640 medium (Sigma Aldrich) containing 10% fetal bovine serum (FBS) Each of 7 × 10 ⁷ cells was fluorescently labeled using a Vybrant Cell Adhesion Assay Kit according to the instructions for use, and a suspension at a concentration of 5 × 10 ⁶ cells / ml was prepared using an FBS-free RPMI1640 medium. Then, the supernatant of each well of the above-mentioned microplate after the TNF-α treatment was extracted with an 8-type aspirator, and immediately 100 μl (5 × 10 ⁵ cells) of each fluorescently labeled white blood cell was added to each microplate. Was added using an 8 micropipette and incubated at 37 ° C. for 1 hour. Next, 100 μl of FBS-free RPMI1640 medium is added to each well, and immediately washed twice with 200 μl of FBS-free RPMI1640 medium using an 8-strip aspirator and 8-strip multi-micropipette, and finally 200 μl of PBS is added. In addition, the fluorescence was measured with a fluorescence microplate reader (excitation / fluorescence: 485 nm / 535 nm), and the average value of 4 series (4 wells) data for each siRNA was determined. This fluorescence intensity is proportional to the number of white blood cells remaining on the microplate (adhered to HUVEC). And the case where the fluorescence intensity was 50% or less (adhesion inhibition of 50% or less) of the fluorescence intensity in the negative control siRNA was determined as positive. FIGS. 7 to 10 show data on 9 siRNAs corresponding to 6 genes determined as inflammatory cytokine-derived polynucleotides from the results of Examples 3a) to c) among 21 siRNAs analyzed, and negative and positive control siRNAs. Show. As shown in FIGS. 7 to 10, among the siRNAs analyzed this time, TNFUP — 0064-4 is MOLT-4 and Jurkat, TNFUP — 0114-3 is U937, MOLT-4 and Jurkat, TNFUP — 0142-2 is MOLT-4, Jurkat, and TNFUP — 0142− 3 was shown to inhibit the adhesion of MOLT-4 by 50% or more, and it became clear that 3 genes of TNFUP_0064, TNFUP_0114, and TNFUP_0142 are involved in adhesion to at least the white blood cells examined this time. These results suggest that endothelial cells play an important role in the induction of leukocyte adhesion and infiltration. In addition, in the cells examined this time, TNFUP_0013, TNFUP_0072, and TNFUP_0113 did not show an adhesion-suppressing effect with the siRNA, but a) and b) proved to suppress the expression of adhesion molecules. Because the mode of adhesion suppression was different depending on the combination of siRNA and leukocyte cell type in this analysis, there is a possibility that it is related to the adhesion of leukocytes of other leukocytes not used this time or peripheral blood. I think enough.

d) Summary of Verification of Inflammatory Cytokine-Induced Polynucleotide Candidates To summarize this verification experiment, at least in HUVEC, TNFUP_0013 is a gene related to the expression of ICAM-1 and E-selectin, and TNFUP_0064 is an ICAM-1 and VCAM-1 gene. TNFUP_0072 is a gene related to the expression of the T lymphocyte lineage cell (MOLT-4, Jurkat), TCAMUP_0072 is related to the expression of VCAM-1 and E-selectin, and TNFUP — 0113 is related to the expression of E-selectin TNFUP — 0114 is a gene associated with the expression of ICAM-1, VCAM-1 and E-selectin and associated with the adhesion of monocyte cells (U937) and T lymphocyte cells (MOLT-4, Jurkat) , TNFUP — 0142 was found to be a gene associated with the expression of ICAM-1, VCAM-1 and E-selectin and associated with adhesion of T lymphocyte cells (MOLT-4, Jurkat).

SEQ ID NO: 1 is the gene sequence of TNFUP_0013.
SEQ ID NO: 2 is the amino acid sequence encoded by the gene sequence of TNFUP_0013.
SEQ ID NO: 3 is the gene sequence of TNFUP_0064.
SEQ ID NO: 4 is the amino acid sequence encoded by the gene sequence of TNFUP_0064.
SEQ ID NO: 5 is the gene sequence of TNFUP_0072.
SEQ ID NO: 6 is the amino acid sequence encoded by the gene sequence of TNFUP_0072.
SEQ ID NO: 7 is the gene sequence of TNFUP — 0113.
SEQ ID NO: 8 is the amino acid sequence encoded by the gene sequence of TNFUP — 0113.
SEQ ID NO: 9 is the gene sequence of TNFUP — 0114.
SEQ ID NO: 10 is the amino acid sequence encoded by the gene sequence of TNFUP — 0114.
SEQ ID NO: 11 is the gene sequence of TNFUP — 0142.
SEQ ID NO: 12 is the amino acid sequence encoded by the gene sequence of TNFUP — 0142.
SEQ ID NO: 13 is the gene sequence of TNFUP_0072B, which is a splicing variant of TNFUP_0072.
SEQ ID NO: 14 is the amino acid sequence encoded by the gene sequence of TNFUP_0072B, which is a splicing variant of TNFUP_0072.
SEQ ID NO: 15 is the gene sequence of TNFUP — 0142B, which is a splicing variant of TNFUP — 0142.
SEQ ID NO: 16 is the amino acid sequence encoded by the gene sequence of TNFUP — 0142B, which is a splicing variant of TNFUP — 0142.
SEQ ID NO: 17 is the gene sequence of TNFUP — 0142C, which is a splicing variant of TNFUP — 0142.
SEQ ID NO: 18 is the amino acid sequence encoded by the gene sequence of TNFUP — 0142C, which is a splicing variant of TNFUP — 0142C.
SEQ ID NO: 19 is the gene sequence of TNFUP — 0142D, which is a splicing variant of TNFUP — 0142.
SEQ ID NO: 20 is the amino acid sequence encoded by the gene sequence of TNFUP — 0142D, which is a splicing variant of TNFUP — 0142.
SEQ ID NO: 21 is the gene sequence of TNFUP — 0142E, which is a splicing variant of TNFUP — 0142E.
SEQ ID NO: 22 is the amino acid sequence encoded by the gene sequence of TNFUP — 0142E, which is a splicing variant of TNFUP — 0142E.
SEQ ID NO: 23 is the gene sequence of TNFUP — 0142F, which is a splicing variant of TNFUP — 0142.
SEQ ID NO: 24 is the amino acid sequence encoded by the gene sequence of TNFUP — 0142F, which is a splicing variant of TNFUP — 0142.
SEQ ID NO: 25 is the base sequence of an oligo DNA for sense strand synthesis of siRNA (TNFUP_0013-1) for TNFUP_0013.
SEQ ID NO: 26 is the base sequence of oligo DNA for antisense strand synthesis of siRNA (TNFUP_0013-1) for TNFUP_0013.
SEQ ID NO: 27 is the base sequence of an oligo DNA for sense strand synthesis of siRNA (TNFUP_0013-3) for TNFUP_0013.
SEQ ID NO: 28 is the base sequence of oligo DNA for antisense strand synthesis of siRNA against TNFUP_0013 (TNFUP_0013-3).
SEQ ID NO: 29 is the base sequence of the sense strand synthesis oligo DNA for siRNA (TNFUP — 0013-4) for TNFUP — 0013.
SEQ ID NO: 30 is the base sequence of oligo DNA for antisense strand synthesis of siRNA against TNFUP_0013 (TNFUP_0013-4).
SEQ ID NO: 31 is the base sequence of the sense strand synthesis oligo DNA for siRNA against TNFUP_0064 (TNFUP_0064-4).
SEQ ID NO: 32 is the base sequence of an oligo DNA for antisense strand synthesis of siRNA (TNFUP — 0064-4) for TNFUP — 0064.
SEQ ID NO: 33 is the base sequence of an oligo DNA for sense strand synthesis of siRNA (TNFUP_0072-2) for TNFUP_0072.
SEQ ID NO: 34 is the base sequence of an antisense strand oligo DNA for siRNA against TNFUP_0072 (TNFUP_0072-2).
SEQ ID NO: 35 is the base sequence of an oligo DNA for sense strand synthesis of siRNA (TNFUP — 0113-4) for TNFUP — 0113.
SEQ ID NO: 36 is the base sequence of the oligo-DNA for antisense strand synthesis of siRNA against TNFUP — 0113 (TNFUP — 0113-4).
SEQ ID NO: 37 is the base sequence of oligo DNA for sense strand synthesis of siRNA (TNFUP — 0114-3) for TNFUP — 0114.
SEQ ID NO: 38 is the base sequence of oligo DNA for antisense strand synthesis of siRNA (TNFUP — 0114-3) for TNFUP — 0114.
SEQ ID NO: 39 is the base sequence of an oligo DNA for sense strand synthesis of siRNA (TNFUP — 0142-2) for TNFUP — 0142.
SEQ ID NO: 40 is the base sequence of oligo DNA for antisense strand synthesis of siRNA (TNFUP — 0142-2) for TNFUP — 0142.
SEQ ID NO: 41 is the base sequence of oligo DNA for sense strand synthesis of siRNA (TNFUP — 0142-3) for TNFUP — 0142.
SEQ ID NO: 42 is the base sequence of an oligo DNA for antisense strand synthesis of siRNA (TNFUP — 0142-3) for TNFUP — 0042.
SEQ ID NO: 43 is the base sequence of the sense strand oligo DNA of negative control siRNA (N. Ctrl-si).
SEQ ID NO: 44 is the base sequence of the oligo DNA for antisense strand of negative control siRNA (N. Ctrl-si).
SEQ ID NO: 45 is the base sequence of the sense strand oligo DNA of positive control siRNA (ICAM1-si) for ICAM-1.
SEQ ID NO: 46 is the base sequence of oligo DNA for antisense strand of positive control siRNA (ICAM1-si) against ICAM-1.
SEQ ID NO: 47 is the base sequence of the sense strand oligo DNA of the positive control siRNA against VCAM-1 (VCAM1-si).
SEQ ID NO: 48 is the base sequence of oligo DNA for antisense strand of positive control siRNA against VCAM-1 (VCAM1-si).
SEQ ID NO: 49 is the base sequence of the sense strand oligo DNA of positive control siRNA against E-selectin (E-selectin-si).
SEQ ID NO: 50 is the base sequence of the antisense strand oligo DNA of positive control siRNA against E-selectin (E-selectin-si).
SEQ ID NO: 51 is the base sequence of the sense strand oligo DNA of the positive control siRNA (TNFR1-si) for TNFR1.
SEQ ID NO: 52 is the base sequence of the antisense strand oligo DNA of the positive control siRNA (TNFR1-si) for TNFR1.
SEQ ID NO: 53 is the base sequence of the PCR primer for quantification of TNFUP_0013.
SEQ ID NO: 54 is the base sequence of PCR primer for TNFUP_0013 quantification.
SEQ ID NO: 55 is the base sequence of PCR primer for quantification of TNFUP_0064.
SEQ ID NO: 56 is the base sequence of PCR primer for quantification of TNFUP — 0064.
SEQ ID NO: 57 is the base sequence of PCR primer for quantification of TNFUP_0072.
SEQ ID NO: 58 is the base sequence of PCR primer for TNFUP_0072 quantification.
SEQ ID NO: 59 is the base sequence of the PCR primer for quantification of TNFUP — 0113.
SEQ ID NO: 60 is the base sequence of the PCR primer for quantification of TNFUP — 0113.
SEQ ID NO: 61 is the base sequence of PCR primer for quantification of TNFUP — 0114.
SEQ ID NO: 62 is the base sequence of PCR primer for quantification of TNFUP — 0114.
SEQ ID NO: 63 is the base sequence of the PCR primer for quantifying TNFUP — 0142.
SEQ ID NO: 64 is the base sequence of PCR primer for quantification of TNFUP — 0142.
SEQ ID NO: 65 is the base sequence of the PCR primer for ICAM-1 quantification.
SEQ ID NO: 66 is the base sequence of the PCR primer for quantifying ICAM-1.
SEQ ID NO: 67 is the base sequence of PCR primer for quantification of VCAM-1.
SEQ ID NO: 68 is the base sequence of the PCR primer for quantification of VCAM-1.
SEQ ID NO: 69 is the base sequence of the PCR primer for quantifying E-selectin.
SEQ ID NO: 70 is the base sequence of the PCR primer for E-selectin quantification.
SEQ ID NO: 71 is the base sequence of PCR primer for β-actin quantification.
SEQ ID NO: 72 is the base sequence of the PCR primer for β-actin quantification.
SEQ ID NO: 73 is the base sequence of PCR primer for quantification of GAPDH.
SEQ ID NO: 74 is the base sequence of the PCR primer for GAPDH quantification.

Claims (12)

(1) A method for screening a vascular endothelial cell adhesion inhibitor comprising a step of bringing a vascular endothelial cell into contact with a test substance, and (2) a step of measuring the expression level of the gene described in any of the following:
1) DNA consisting of a base sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or 23, or 2) SEQ ID NO: 1, 3, 5, 7, (A) a cell adhesion molecule that hybridizes with a DNA comprising the base sequence represented by 9, 11, 13, 15, 17, 19, 21, or 23 under stringent conditions and in the presence of an inflammatory cytokine. And / or (b) a polypeptide encoding a polypeptide having a function of inducing adhesion between vascular endothelial cells and white blood cells. (1) A method for screening a vascular endothelial cell adhesion inhibitor comprising a step of bringing a vascular endothelial cell into contact with a test substance, and (2) a step of measuring the expression level of the polypeptide described in any of the following:
1) Polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24, or 2) SEQ ID NO: 2, 4, 6, 8 In the amino acid sequence represented by 10, 12, 14, 16, 18, 20, 22, or 24, having an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, and / or added; and A polypeptide having (a) a function of inducing expression of cell adhesion molecules and / or (b) a function of inducing adhesion between vascular endothelial cells and white blood cells in the presence of inflammatory cytokines.   The method for screening a vascular endothelial cell adhesion inhibitor according to claim 1 or 2, further comprising a step of selecting a test substance that reduces the expression level of the gene or polypeptide as compared to the absence of the test substance. (1) contacting a test substance with a cell transformed with an expression vector containing the gene of claim 1 and expressing a polypeptide encoded by the gene;
(2) including the step of contacting white blood cells with the cells, and (3) measuring the amount of adhesion between the white blood cells and the cells.
A screening method for a vascular endothelial cell adhesion inhibitor. The screening method for a vascular endothelial cell adhesion inhibitor according to claim 4, further comprising a step of selecting a test substance that reduces the amount of adhesion between leukocytes and the cells compared to the absence of the test substance. (1) a step of bringing a test substance into contact with a cell transformed with an expression vector containing the gene of claim 1 and expressing a polypeptide encoded by the gene; and (2) a cell adhesion molecule. Including the step of measuring the expression level of
A screening method for a vascular endothelial cell adhesion inhibitor.   The screening method for a vascular endothelial cell adhesion inhibitor according to claim 6, further comprising a step of selecting a test substance that reduces the expression level of the cell adhesion molecule as compared with the absence of the test substance. (1) contacting the polypeptide of claim 2 with a test substance in the presence of a labeled specific conjugate of the polypeptide; and (2) binding of the polypeptide and the specific conjugate. Including the step of measuring the quantity,
A screening method for a vascular endothelial cell adhesion inhibitor.   9. The screening method for a vascular endothelial cell adhesion inhibitor according to claim 8, further comprising a step of selecting a test substance that reduces the binding amount of the specific binder as compared to the absence of the test substance.   The screening method according to any one of claims 1 to 9, further comprising a step of contacting an inflammatory cytokine.   The screening method according to any one of claims 1 to 10, wherein the cell adhesion molecule is any one selected from the group consisting of ICAM-1, VCAM-1, and E-selectin. The screening method according to any one of claims 1 to 11, wherein a vascular endothelial cell adhesion inhibitor is selected as an anti-inflammatory drug.

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