JP2003310257A - Dna micro array of chlamydia pneumoniae, method for detecting whether infection with chlamydia pneumoniae occur or not, and method for screening medicine - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To prepare a DNA micro array method of Chlamydia pneumoniae and to detect whether or not a cell is infected with Chlamydia pneumoniae from an expression file of a Chlamydia pneumoniae gene by using the DNA micro array method and to apply the DNA micro method to a method for screening medicines. <P>SOLUTION: About 1,000 kinds of gene DNAs of Chlamydia pneumoniae analyzed by a genome project are amplified and arranged on a glass plate to prepare a DNA micro array device. Thereby, expression variation analysis of the Chlamydia pneumoniae gene is made possible. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD OF THE INVENTION Chlamydia pneumoniae
pneumoniae DNA microarray and Chlamydia pneumoniae DNA microarray, and a method for examining the presence or absence of Chlamydia pneumoniae infection from a gene expression profile, and a method for screening a drug useful as a therapeutic agent for a disease caused by Chlamydia pneumoniae.

[0002]

Chlamydia pneumoniae is well known as a causative bacterium of respiratory infections such as pneumonia and bronchitis. Recently, analysis of antibodies and genes shows that it is associated with various diseases such as ischemic heart disease and arteriosclerosis, and autoimmune diseases such as asthma and multiple sclerosis with chronic infection. It has become clear. Thereby, a diagnostic method using an antigen of Chlamydia pneumoniae, an anti-Chlamydia pneumoniae antibody, and a diagnostic method using the presence or absence of Chlamydia pneumoniae-derived DNA as an index have been reported (JP 2001-28985 A).
7).

Although a diagnostic method using an antibody or antigen of Chlamydia pneumoniae has an advantage as a simple test method, it has a problem in terms of detection sensitivity and reliability of results. For example, in the case of Chlamydia, it is roughly divided into Chlamydia pneumoniae and Chlamydia trachomatis.
), And Chlamydia psittaci), and are known to have common antigenicity. Therefore, careful attention should be paid to the presence or absence of cross-reactivity. Needless to say, the gene level test is more advantageous in terms of detection sensitivity and reliability.

Chlamydia pneumoniae, unlike common bacteria, cannot grow in in vitro cell-free systems. It is an obligate cytoparasitic bacterium that can grow only in living host cells. Therefore, the development of gene manipulation systems has been delayed. Currently, due to the progress of genome analysis, in CWL029 strain in the US and J138 strain in Japan,
The entire genome sequence of Chlamydia pneumoniae has been reported (Shir
ai M. et al., NucleicAcids Res., 2000; 28: 2311-4
). In addition, DNA microarray (DNA chip)
Has attracted attention as a method capable of simultaneously detecting the expression of a large number of genes.

[0005]

Problems to be Solved by the Invention DN of Chlamydia pneumoniae
It is a subject to prepare an A microarray to grasp the expression profile of the Chlamydia pneumoniae gene and to apply it to a method for examining the presence or absence of Chlamydia pneumoniae infection and a drug screening method.

[0006]

[Means for Solving the Problems] A DNA microarray is a device in which DNA is arranged at high density on a glass substrate, and the gene expression profile of an organism can be grasped in detail by hybridization of this array glass and cDNA. You can The present invention amplifies about 1,000 types of Chlamydia pneumoniae gene DNAs analyzed by the Genome Project and arranges them on a glass plate to form D
An NA microarray device was produced to enable analysis of chlamydia pneumoniae gene expression variation.

Chlamydia bacteria are known to be pathogenic bacteria with extremely high morbidity, and show various symptoms depending on the species. Chlamydia pneumoniae, which was designated as a new species in 1989,
Not only respiratory infections, but also arteriosclerosis, heart disease, arthritis,
It is also suspected of being associated with systemic diseases such as sarcoidosis. Although the whole genome analysis was completed in 1999, and various genetic studies are progressing by this, much is unknown at present regarding the elucidation of functions such as pathogenic factors and parasitic mechanism to host cells.

Chlamydia pneumoniae is capable of growing only in living host cells (an obligatory intracellular lifec
is an obligate cell parasitic bacterium. Despite its clinical importance, the unclear biological and biochemical pleiotropic aspects of Chlamydia pneumoniae are due to an obligatory intracellular life cycle. The present inventors have revealed the entire genomic sequence of Chlamydia pneumoniae J138 strain (Shirai M. et al., Nu.
cleic Acids Res., 2000; 28: 2311-4). Chlamydia pneumoniae gene DNA analyzed by this genome project
About 1,000 kinds were amplified by PCR. In addition, Chlamydia pneumoniae P that has this gene arrayed on a glass plate
The NA microarray was developed for the first time in the world. Using this Chlamydia pneumoniae DNA microarray technology, almost all ORFs (open re
ading frame) was analyzed.

By using the Chlamydia pneumoniae DNA microarray device according to the present invention, it is possible to identify the expression profiles of almost all genes of Chlamydia pneumoniae, and thus it is possible to identify genes essential for the infection process and new genes relating to transcriptional regulation. It became possible to elucidate a series of signal transduction pathways and elucidate the parasitic mechanism. Moreover, since the efficacy and mechanism of action of therapeutic agents can be grasped using this device, it is considered to play a major role in the development of new therapeutic and diagnostic methods for chlamydia infection. The present invention is a new device that forms the basis of gene therapy technology.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described based on examples with reference to the drawings, but the present invention is not limited to these examples.

[0011]

EXAMPLE A DNA microarray glass having almost all the genes of Chlamydia pneumoniae J138 strain was developed according to the production procedure outlined in FIG. First,
DNA by PCR using the plasmid vector used for Chlamydia pneumoniae whole genome sequence as a template
It was made. The amplified DNA was spotted on a slide glass coated with poly-L-lysine,
After cross-linking with UV irradiation of J, containing succinic anhydride 1
It was immersed in a -methyl-2-pyrrolidinone solution, sodium borate was added, and blocking was performed at room temperature for 30 minutes. Finally, after washing with ultrapure water and boiling ultrapure water, a drying step was performed to prepare a Chlamydia pneumoniae DNA microarray.

The Chlamydia DNA microarray produced in this example covers almost all of all ORFs (1070) on the genome. The total number of spots was about 2,300, and 888 kinds of DNA fragments were spotted in an overlapping manner. From the clones sequenced at the time of genome sequencing, 762 appropriate clones corresponding to each ORF or operon were selected. For 121 ORFs, individual clones were created and spotted (Table 1). The human gene GAPDH (glyceraldehyde-3-pho), which is the host, is used.
sphate dehydrogenase) and β-actin (β-actin) as positive controls, and Pseudomonas putida mt-
The gene for adenylate kinase derived from 2 is spotted as a negative control.

[0013]

[Table 1]

Total RNA was extracted from cells in the early stage of Chlamydia pneumoniae infection and cells in the late stage of Chlamydia pneumoniae infection, and using this as a template, Cy-3 or Cy-5.
Each cDNA labeled with was prepared. Synthesized Cy
-3 labeled cDNA and Cy-5 labeled cDNA were mixed,
Hybridized to Chlamydia pneumoniae DNA microarray. After hybridization, washing and drying
The fluorescence intensity of the A microarray was read by a scanner (Fig. 2). Normalization of fluorescence intensity was performed with GAPDH and β-actin which are host genes. An example of the result of fluorescence detection by scanning and the result of gene expression analysis by computer is shown in FIG. Similarly, a human DNA microarray was also used to test the expression of human genes under infection conditions.

When fluorescence-labeled cDNA prepared from uninfected cells was used to hybridize with a Chlamydia pneumoniae DNA microarray, no fluorescent signal spot was detected on this DNA microarray. This indicates that the DNA microarray of the present invention has a low background and high specificity.

In order to understand the change in gene expression profile due to Chlamydia pneumoniae infection, comparison was made 24 hours after the infection with cells in the early stage of infection and 48 hours after the infection with cells in the late stage of infection. CDNA prepared from HEp-2 cells 24 and 48 hours after infection was analyzed by Chlamydia pneumoniae DNA microarray.
There were 194 genes out of a total of 1070 ORFs whose signal was more than doubled after 48 hours compared to after 4 hours. At each time, the number of ORFs contained in the spots higher than the value obtained by adding the standard deviation to the average signal of the negative control spots was only one at 24 hours (the ORF of the 692nd, MOMP, MOMP).
Code), there were 312 in 48 hours. Table 2 shows
The 312 genes were classified by function, and the number of ORFs on the genome contained in each category, the number of genes whose expression was detected by the microarray this time, and the genomic ORF
The ratio (%) of the detected number to the number is shown. In addition, some of the interesting genes in the detected ORF
Listed in Table 3.

[0017]

[Table 2]

[0018]

[Table 3]

At 48 hours post infection, the presence of the fluorescent signal indicated that 194 ORFs were present on the DNA microarray. These OR
In F, ADP / ATP translocase, OmpA, OmpB, IncB, I
ncC, Pmp, serine / threoninekinase, SET domain prote
The genes for in and many other housekeeping proteins were included.

From the above results, the DNA microarray of Chlamydia pneumoniae according to the present invention was obtained from Chlamydia pneumoniae J1.
It was found that almost all ORFs of 38 strains were covered and the detection sensitivity was sufficiently high. This system detected transcripts for 194 genes, including many genes of interest and housekeeping genes. It also showed high specificity for the Chlamydia gene. Therefore, it was shown that the Chlamydia pneumoniae DNA microarray is useful for analyzing the gene expression of Chlamydia pneumoniae, and is useful for elucidating the growth cycle of Chlamydia pneumoniae and the anti-Chlamydia pneumoniae effect of various drugs.

[0021]

By using the Chlamydia pneumoniae DNA microarray according to the present invention, the gene expression profile of almost all Chlamydia pneumoniae can be grasped. Further, it can be applied to a method for examining the presence or absence of Chlamydia pneumoniae infection from a gene expression profile and a method for screening a drug useful as a therapeutic drug for a disease caused by Chlamydia pneumoniae.

[Brief description of drawings]

FIG. 1 is a diagram showing a procedure for producing a Chlamydia pneumoniae DNA microarray.

FIG. 2 is a diagram showing an example of use of a Chlamydia pneumoniae DNA microarray.

FIG. 3 is a diagram showing an example of results of gene expression analysis by scanning and computer for fluorescence detection.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 33/50 G01N 33/53 M 33/53 33/569 F 33/569 37/00 102 37/00 102 C12N 15/00 F (72) Inventor Hidehiro Fuji, 1-1-1 Minamikogushi, Ube City, Yamaguchi Prefecture Yamaguchi University School of Medicine (72) Manabu Sugii 1-1-1 Minamikogushi, Ube City, Yamaguchi Prefecture Yamaguchi University School of Medicine F Term (reference) 2G045 AA25 AA28 AA40 CB01 CB21 DA12 DA13 DA14 DA77 FB02 FB07 FB12 FB16 GC15 4B024 AA19 CA01 CA09 HA14 HA19 4B029 AA07 FA12 4B063 QA13 QA18 QA19 QQ08 QQ42 QR32 QR25 QR62 QR77 QR77

Claims (10)

[Claims] 1. A DNA microarray of Chlamydia pneumoniae. 2. A DNA microarray of Chlamydia pneumoniae J138 strain. 3. The DNA microarray according to claim 1, which covers almost all of the Chlamydia pneumoniae gene. 4. The DN according to any one of claims 1 to 3.
A method for examining the presence or absence of Chlamydia pneumoniae infection from the expression profile of the Chlamydia pneumoniae gene using A microarray. 5. The examination method according to claim 4, wherein the presence or absence of Chlamydia pneumoniae infection is examined by targeting Chlamydia pneumoniae-infected cells isolated from a living tissue. 6. The DN according to any one of claims 1 to 3.
A method for screening a drug useful as a therapeutic agent for a disease caused by Chlamydia pneumoniae, using A microarray. 7. The screening method according to claim 6, wherein cells infected with Chlamydia pneumoniae are used. 8. The screening method according to claim 6, wherein a cell line infected with Chlamydia pneumoniae is used. 9. The screening method according to claim 6, wherein the cell lines infected with Chlamydia pneumoniae are used to compare the expression profiles of Chlamydia pneumoniae genes at 24 hours and 48 hours after infection. 10. The screening method according to claim 6, wherein Chlamydia pneumoniae-infected cells isolated from living tissue are used.