JP2007085783A - IMMUNOLOGICAL DETECTION REAGENT OF KAWASAKI DISEASE BY USING PEPTIDE OF PART FROM 186-TH AMINO RESIDE, CALCULATED FROM FIRST AMINO ACID BASE OF nSm-hPAF, THE TOTAL LENGTH OF rSm-hPAF OR N-TERMINAL OF rSm-hPAF TO 205-TH AMINO RESIDUE AS ANTIGEN - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an immunological detection reagent influential in the diagnosing of Kawasaki disease, during a period that is difficult to diagnose as Kawasaki disease in the initial stage of the development of disease. <P>SOLUTION: The immunological detection reagent of the Kawasaki disease, using peptide of 186-205 as residues counted from the total length of nSm-hPAF, the total length of the Sm-hPAF (rSm-hPAF) or the N-terminal of recomination Sm-hPAF as the antigen, is provided. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an immunological detection method that is effective in diagnosing Kawasaki disease. More specifically, the present invention relates to the entire length of nSm-hPAF, rSm-hPAF, or the 205th amino acid residue from the 186th amino acid residue counted from the N-terminal (first amino acid base) of rSm-hPAF. The present invention relates to an immunological detection reagent for Kawasaki disease using a peptide up to the base as an antigen, and an immunological detection method for Kawasaki disease using the same.

  Kawasaki disease, also called "acute fever mucocutaneous fistula syndrome", rapidly fever, rash throughout the body, red eyes, red tongue, swollen cervical lymph glands, swollen limbs, In the recovery period, it presents a disease image such as skin peeling from the fingertip. Conventionally, Kawasaki disease has been diagnosed by clinical findings, and it has not always been easy to distinguish it from other febrile and rash diseases at the initial stage of the onset.

    In view of the above-mentioned conventional problems, the present invention aims to provide an effective serological diagnostic method for diagnosing Kawasaki disease at a time when diagnosis as Kawasaki disease at the early stage of disease onset is difficult. And an immunological detection reagent for Kawasaki disease using an antigen-antibody reaction and an immunological detection method for Kawasaki disease using the antigen-antibody reaction.

    The present invention provides an immunological detection reagent for Kawasaki disease using the full length of nSm-hPAF as an antigen.

    The present invention further provides an immunological detection reagent for Kawasaki disease using the full length of recombinant Sm-hPAF (rSm-hPAF) as an antigen.

    The present invention further provides an immunological detection reagent for Kawasaki disease using a peptide of 186 to 205 residues counted from the N-terminus of recombinant Sm-hPAF as an antigen.

      The present invention provides an immunological detection method for Kawasaki disease by measuring the amount of antibody in plasma or serum collected from a subject to be diagnosed using the full length of nSm-hPAF as an antigen. Is.

    The present invention further provides an immunological detection method for Kawasaki disease by measuring the amount of antibody in plasma or serum collected from a subject to be diagnosed using the full length of recombinant Sm-hPAF as an antigen. It is to provide.

    The present invention further uses the peptide of 186 to 205 residues counted from the N-terminus of recombinant Sm-hPAF as an antigen to determine the amount of antibody in blood plasma or serum collected from a subject to be diagnosed. By measuring, an immunological detection method of Kawasaki disease is provided.

    Furthermore, the present invention provides a peptide consisting of 186 to 205 residues counted from the N-terminus of recombinant Sm-hPAF.

      According to the present invention, a peptide of 186 to 205 residues counted from the full length of nSm-hPAF, the full length of rSm-hPAF, or the N-terminus of recombinant Sm-hPAF is detected against humans suspected of having Kawasaki disease. It is possible to obtain useful information in diagnosing whether or not you are suffering from Kawasaki disease by measuring the amount of antibody in plasma or serum collected from the subject. Together with diagnostic findings from other clinical findings, it becomes possible to more accurately diagnose whether or not the subject has Kawasaki disease.

  The above-mentioned immunological detection reagent for Kawasaki disease is dissolved in an appropriate buffer solution, and the obtained solution is put into a test tube or the like, and the buffer solution is washed and removed to place the immunological detection reagent on the wall of the test tube or the like. Whether the patient has Kawasaki disease by attaching blood plasma or serum from the subject to the test tube or the like and measuring the amount of antibody in the plasma or serum using a conventionally known ELISA method It helps to diagnose whether or not.

    The total length of nSm-hPAF used in the present invention, the total length of rSm-hPAF, and the 205th amino acid residue from the 186th amino acid residue counted from the N-terminal group (first) amino acid base of rSm-hPAF The peptide up to the group is produced by the following method. Although the full length of nSm-hPAF and the full length of rSm-hPAF itself have already been reported, the association with Kawasaki disease was first revealed by the present invention, and the N-terminal group of Sm-hPAF (first It was first identified and identified by the present invention that the peptide from the 186th amino acid residue to the 205th amino acid residue counting from the amino acid base) can be an effective antigen in the diagnosis of Kawasaki disease. It is a thing.

(1) Total length of nSm-hPAF Human platelets (floating in plasma) are contained in the culture supernatant of Streptococcus mitis (abbreviated as S.mitis) Nm-65 obtained from the oral cavity of children with Kawasaki disease. (S. mitis-derived human platelet aggregation factor, native Sm-hPAF, nSm-hPAF) was found and reported. (See Non-Patent Document 1).
Ohkuni, H. et. Al., “Biologically active extracellular products of oral viridans streptococci and the aetiology of Kawasaki disease”, J. Med. Microbiol, Vol. 39, pp352-362, 1993

NSm-hPAF was purified from the metabolite of S. mitis and its properties were examined. The amino acid sequence of 15 residues from the N-terminus of nSm-hPAF was _H Asp-Glu-Gln-Gly-Asn-Arg-Pro -Val-Glu-Thr-Glu-Asn-Ile-Ala-Arg- was determined (Non-patent Document 2).
Ohkuni H. et.al., “Purification and partial characterization of a novel human platelet aggregation factor in extracellular products of Streptococcus mitis, strain Nm-65”, FEMS Immunology and Medical Mircobiology 17 (1997) 121-129

The procedure for determining the entire base sequence of Sm-hPAF is as follows.
(1) Determination of the base sequence of the N-terminal amino acid of nSm-hPAF
Mixed oligonucleotide-primed PCR method (MOP PCR) based on the N-terminal amino acid sequence of nSm-hPAF (above) obtained by purification from the culture supernatant of S. mitis ) And the PCR product was designated as sm-65. This product was subjected to cloning and sequence (gene sequence) analysis using E. coli, JM109, and the nucleotide sequence of the N-terminal amino acid was determined.

(2) Full length of recombinant Sm-hPAF (rSm-hPAF) Next, PCR cassette (a method of amplifying other peripheral regions based on the known nucleotide sequence) was used to primer full length Sm-hPAF gene with BamHI and HindIII sites. As amplified. The amplified gene (sm-hpaf) was inserted into a portion cleaved at the BamHI site and HindIII site of the pQE-9 vector, ligated with the E. coli gene, and a recombinant was prepared using E. coli and JM109. The E. coli was cultured, and recombinant Sm-hPAF (rSm-hPAF) was purified from the cells. The above method is shown in FIG. (Refer nonpatent literature 3).
Hideaki Nagamune et al., “S.mitis human platelet coagulation factor derived from Kawasaki disease”, 77th Annual Meeting of the Bacteriological Society of Japan, 272 pages, April 2004

    The entire base sequence of this rSm-hPAF was determined. The entire base sequence of rSm-hPAF is shown in FIG. A complete sequence of the rSm-hPAF gene was identified, including 252 bases upstream and 98 bases downstream of the open reading frame (ORF) of sm-hpaf. The nucleotide sequence of ORF was able to encode a protein consisting of 665 amino acid residues including its signal sequence. All or 633 amino acid residues were encoded as wild type protein and did not contain cysteine.

    Substitution of the complete sequence of the rSm-hPAF gene with the amino acid sequence and homology search revealed that it was extremely homologous with some of the known hemolytic toxins such as S. intermedius and S. pneumonia. (See FIGS. 3a to 3c, FIGS. 4 and 5). FIG. 5 is a diagram comparing the lengths of amino acid sequences, showing that Sm-hPAF has a longer amino acid sequence than other hemolysates, in other words, has a large molecular weight, and is homologous in Domain1-4. Yes. The domain 0-4 homologous part and non-homogeneous part can be seen from the comparison of amino acid sequences in FIGS. The part of domain 0 in the first half of the base sequence is a unique sequence of rSm-hPAF and is a sequence not found in other hemolysates. On the other hand, the amino acid sequence of rSm-hPAF written in parallel with other hemolysates is a relatively homologous part. The total amino acid sequence based on this base sequence is shown in the top row of rSm-hPAF in FIGS. 3a to 3c.

(3) Peptides from 186 to 205 residues (TQVGDRTAPVVDQTSALKD) counted from the N-terminus (see FIG. 3a)
Secondary amino acid and tertiary structure were predicted from the primary structure of rSm-hPAF amino acid, and various amino acid residue polypeptides were synthesized. In order to establish an effective serological diagnostic method for diagnosing Kawasaki disease, 186-205 amino acid residues from the N-terminus according to the present invention were selected from these polypeptides. The 186-205 amino acid residues (TQVGDRTAPVVDQTSALKD) from the N-terminal are considered to be exposed on the molecular surface on the three-dimensional structure of the protein.

    Antibodies in various diseases including Kawasaki disease and healthy subjects' serum or plasma were measured by ELISA using nSm-hPAF, rSm-hPAF and peptides of 186 to 205 residues counted from the N-terminus as antigens. The specific experimental method is as follows.

First, 0.1 μg (microgram) of the antigen rSm-hPAF or 0.05 μg of the synthetic peptide was placed in a test tube (well of a microplate) and adsorbed on the test tube. Next, 100 μl (0.1 ml) of human serum or plasma diluted 1: 100 or 1: 800 was added to the test tube on which the antigen was adsorbed, and allowed to react at 37 ° C. for a certain period of time. After reaction, add anti-human IgG antibody or anti-human IgM antibody made in mouse, react for a certain time at 37 ° C, add anti-mouse enzyme-labeled anti-IgG antibody or anti-mouse enzyme-labeled IgM antibody made in sheep, Further, the substrate was added to develop color, and the color intensity at a wavelength (OD) of 405 nm was measured with a colorimeter to obtain the antibody value.

FIG. 6 shows the experimental results using rSm-hPAF as the antigen, and FIG. 7 shows the results using the synthetic peptide as the antigen. The antibodies (IgG class) in the acute phase of Kawasaki disease against rSm-hPAF and synthetic peptides showed significantly higher values than those of other febrile diseases and healthy subjects as control groups (FIGS. 6 and 7). . Although not shown, the results were the same when nSm-hPAF was used. Antigenic determinants (epitope, sites where antibodies react directly) in rSm-hPAF antigen are probably present in synthetic peptides. However, it is possible that another antigenic determinant is present in rSm-hPAF.

Absorption experiments with other hemolysates were performed by adding 20 μl of ILY, Ply, SLO and other hemolysates (50 μg / ml) to serum or plasma (220 μl) diluted 1: 100, followed by 30 minutes at 37 ° C., 4 ° C. And overnight incubation followed by the ELISA described above. RSm-hPAF was also used as a control. As a result, as shown in FIG. 10, it was absorbed by rSm-hPAF, but not absorbed by other hemolysates, and it was shown that the antibody value did not decrease.

  For these antigens, there was no significant difference in IgM antibodies (FIGS. 8 and 9). In general, IgM antibody rises in the early stage of infection and then declines, and IgG class antibody rises before and after the IgM antibody declines. The reason why the IgM class antibody was low and not significantly different from the control group in this data is not necessarily clear, but the infection with this bacterium (S. mitis) was established shortly before the onset of Kawasaki disease. If so, the IgM antibody may have increased. However, since IgM antibodies are low, it may be based on measuring the amount of antibodies, perhaps at a time when the infection occurred earlier and the IgG antibodies were elevated instead. Although it is not speculative, the time when IgG antibody was elevated may coincide with the clinical onset of Kawasaki disease. In addition, IgM antibodies may have a low affinity for the antigen Sm-hPAF, as measured by ELISA.

    These antibodies showed a decrease in absorption experiments in S. intermedius-derived Intermedilysin (ILY), S. pneumoniar-derived Pneumolysis (PLY), and S. pyogenes-derived Streptolysin-O (SLO), which have homology to rSm-hPAF. (FIG. 10), it seems to be a highly specific reaction.

    Kawasaki disease is diagnosed from clinical findings, and it is not always easy to distinguish it from other febrile and rash diseases at the beginning of the onset. The measurement method using this antigen can be a powerful serological diagnostic method for suspecting or diagnosing Kawasaki disease in the early stage of disease onset.

A method for constructing an expression vector for recombinant Sm-hPAF is shown. The nucleotide sequence of rSm-hPAF gene is shown. The amino acid sequence of rSm-hPAF is compared with the amino acid sequences of known hemolysin toxins such as S. intermedius and S. pneumonia. The amino acid sequence of rSm-hPAF is compared with the amino acid sequences of known hemolysin toxins such as S. intermedius and S. pneumonia. The percentage of homology between the amino acid sequence of rSm-hPAF and known hemolytic toxins such as S. intermedius and S. pneumonia is shown. It is the figure which compared the length of the amino acid sequence between the amino acid sequence of rSm-hPAF and known hemolysates produced, such as S. intermedius and S. pneumonia. The experimental result using rSm-hPAF as an antigen is shown. The experimental result which used the synthetic peptide as an antigen is shown. The experimental result with respect to IgM antibody about rSm-hPAF antigen is shown. The experimental result with respect to the IgM antibody about a synthetic peptide antigen is shown. The result of the absorption experiment of IgG class antibody in rSm-hPAF, S.intermedius origin Intermedilysin (ILY), S. pneumoniar origin Pneumolysis (PLY), S.pyogenes origin Streptolysin-O (SLO) is shown.

Claims (7)

  An immunological detection reagent for Kawasaki disease using the full length of nSm-hPAF as an antigen.   An immunological detection reagent for Kawasaki disease using the full length of recombinant Sm-hPAF as an antigen.   An immunological detection reagent for Kawasaki disease using a peptide of 186 to 205 residues counted from the N-terminus of Sm-hPAF as an antigen.   An immunological detection method for Kawasaki disease by measuring the amount of antibody in blood plasma or serum collected from a subject to be diagnosed using the full length of nSm-hPAF as an antigen.   An immunological detection method for Kawasaki disease by measuring the amount of antibody in plasma or serum collected from a subject to be diagnosed using the full length of recombinant Sm-hPAF as an antigen.   By measuring the amount of antibody in blood plasma or serum collected from a subject to be diagnosed using a peptide of 186 to 205 residues counted from the N-terminus of recombinant Sm-hPAF as an antigen, Immunological detection method for Kawasaki disease.   A peptide consisting of 186 to 205 residues counted from the N-terminus of recombinant Sm-hPAF.

Images