JP3418416B2 - Nucleic acid diagnostics - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel method for testing cancer, and more specifically, a method for testing cancer characterized by performing Northern blotting using MK gene as a probe. The present invention relates to a method for examining cancer, which comprises amplifying DNA to be measured by PCR using a fragment oligonucleotide of the MK gene as a primer, and detecting the presence or absence of the MK gene in the measurement target.

[0002]

BACKGROUND ART Various types of cancer-related antigens such as antigens, enzymes, or oncogene product proteins, which are specifically expressed in cancer cells, have been discovered so far, and each gene encoding these many antigens is used to Histological diagnosis of cancer has been made and is being attempted. An example of an oncogene is the ras gene, m
A typical example is the yc gene. Genes encoding cancer-related antigens that appear or exist in cancer cells and leukemia cells of various organs in the body, as well as cancer cells in various growth cycles and processes, will continue to be discovered through research efforts, and these new tumors will be found. Using multiple genetic markers in combination,
The diagnostic results of cancer are expected to improve.

The present inventors have previously found that mouse teratocarcino
Finding new growth differentiation factors derived from
Quality, Midkine, and Midkine (hereinafter abbreviated as MK)
The MK gene encoding
(Tomomura, M. et al, J. Biol Chem.,26
Five, 10765-10770, 1990). This Midkine has 118 amino acid residues
Subsequent research shows that MK
Induces neurite outgrowth, survival maintenance, and maturation of seed neurons
Has been shown to have an action (Muramatsu, H.
and Muramatsu, T., Biochem. Biophys. Res. Commun.,
 177, 652-658, 1991).

The present inventors then cloned the human MK gene using the mouse MK gene (Tsutsui, J., Bioch.
em. Biophys. Res. Commun., 176 , 792-797, 1991),
Furthermore, these genes were successfully expressed in animal cells or Escherichia coli to successfully prepare MK, and a patent application was filed (Japanese Patent Application No. 3-195397). Furthermore, the present inventors immunized mice and rabbits with this MK to produce antibodies, and
A pure antibody was prepared by affinity purification of the ude antibody using the above-mentioned MK in which a large amount of protein was expressed.This anti-MK antibody was used to detect some cancer cells in addition to nerve cells. Histochemical staining was tried, and anti-MK antibody was added to the cancer cell culture system. Surprisingly, most of the cancer cells were stained with the anti-MK antibody, and Wi
In the culture system of cancer cells such as lms' tumor cells, it was found that the growth of cancer cells was significantly inhibited, and it was revealed that MK is a cancer-related antigen. Filed another patent application.

[0005]

DISCLOSURE OF THE INVENTION In addition to the above findings, the present inventors further prepared an MK gene encoding MK as a probe by using a chemical synthesizer or the like or by preparing by a PCR method or the like, and using this, Northern blotting was performed on cells and the various Wilms' tumor cells and other cancer cells, and most of the cancer cells other than nerve cells strongly expressed the MK gene, and further biopsy tissue cells of cancer lesions of cancer patients were detected. Many are MK
Although the gene was strongly expressed, it was surprisingly revealed that normal cells did not express the MK gene at all or hardly expressed. The test method of the present invention comprises Northern blotting using the MK gene encoding midkine (Midkine) as a probe for liver cancer, renal cancer, gastric cancer and lung cancer.
It is an inspection method. Another test method of the present invention detects the presence or absence of the MK gene in the measurement target by amplifying the measurement target DNA by PCR using a fragment from the MK gene encoding midkine (Midkine) as a primer. A method for examining liver cancer, renal cancer, gastric cancer, and lung cancer characterized by the following.

[0006] The present invention has been achieved through intensive research based on such remarkable findings, and the object of the present invention is to perform cancer blotting on tissue cells of a living body by Northern blotting using the MK gene as a probe. Whether or not
It is to provide a method of testing . Another object of the present invention is to PCR the DNA to be measured using the MK gene as a primer.
It is intended to provide a test method for testing for the presence or absence of the MK gene in a measurement target after amplification by.

MK gene probe and PC used in the present invention
The R primer can be prepared by a conventional method.
For example, it is manufactured using Applied Biosystems Inc. or the like.

[0008]

The present invention will be described in more detail with reference to Reference Examples, Examples and Experimental Examples, but the present invention is not limited to these. Reference example Preparation of MK gene probe Mauze MK cDNA (Tomomura, M. et al, J. Biol. Chem.,
265 , 10765-10770, 1990) and the Msp1 fragment (nucleotide numbers -38 to 444) was cut out and used. Human MK cDNA (Ts
utsui, J. et al, Biochem. Biophys. Res. Commun., 1
76 , 792-797, 1991) was used.

Example 1 RNA Separation from Cells and Northern Blotting Total cellular RNA of organs, tissues and cells of patients, healthy people and experimental animals was analyzed by the guanidinium isothiocyanate-cesium chloride method (Chirgwin, JM et al., Biochemistry). , 18 , 5
294-5299, 1979). 10μ for each RNA
Each g was placed on a 1% glyoxal agarose gel and electrophoresed at 1.5 volts / cm for 16 hours (Maniatis, T. et.
al, Molecular Cloning; Cold Spring Harbor Laborat
ory, 1982). After transfer to a nitrocellulose filter, MK-specific RNA was detected with a [ ³² P] -labeled MK probe (random oligonucleotide label). Northern hybridization conditions are 42 ℃, 50mM Tris HCl
Buffer, pH 8.0 (1M NaCl, 10mM EDTA, 0.1% SDS, 0.2%
Polyvinylpyrrolidone, 0.2% Ficoll, 0.2% bovine serum albumin included) for 15 hours.

Example 2 PCR Mouse MK cDNA (Tomomura, M., J. Biol. Chem., 265, 1
0765-10770, 1990) 12-31 bases and antisense strand 464-4
89 was synthesized and used as a primer, and PCR was performed using cDNA from mRNA of various mouse cancer tissues / cells as a template.
Denaturation 93 ° C 1 min, annealing 50 ° C 2 min, extension 72 ° C 3
In 25 minutes, 25 cycles were performed. After the PCR product is subjected to 1% agarose gel electrophoresis, Southern hybridization (condition: according to Northern hybridization) is performed with an MK-specific probe to detect the presence of MK cDNA, that is, the presence of MkmRNA.
Identified. Human MKcDN was added to human cancer tissues and cells by the same method.
The presence of A, that is, the presence of MK mRNA was detected and identified.

Experimental Example The results shown in Table 1 were obtained by carrying out Example 1. MK gene is well expressed in liver cancer, colorectal cancer, gastric cancer, lung cancer, renal cancer, Wilms' tumor, etc., while expression in normal tissues is almost absent or weak, which makes it useful as a cancer-specific marker. There is.

[0012]

[Table 1]

[0013]

EFFECT OF THE INVENTION Using the MK gene fragment as a primer, the DNA to be measured can be amplified by PCR, and the cancer tissue-derived DNA present in the test sample can be detected and identified.

─────────────────────────────────────────────────── ─── Continued Front Page (56) References European Patent Application Publication 476233 (EP, A 1) J. Cell. Biochem. (1992), Suppl. 0,16, Part F 1992, p. 80 (58) Fields surveyed (Int.Cl. ⁷ , DB name) BIOSIS (DIALOG) WPI (DIALOG)

Claims (2)

(57) [Claims] 1. A method for examining liver cancer, kidney cancer, gastric cancer and lung cancer, which comprises performing Northern blotting using a MK gene encoding midkine (Midkine) as a probe. 2. The presence or absence of the MK gene in the measurement target is detected by amplifying the measurement target DNA by PCR using a fragment from the MK gene encoding midkine (Midkine) as a primer. A method for examining liver cancer, kidney cancer, gastric cancer and lung cancer.