JPS60100597A - Protein - Google Patents

Abstract

PURPOSE:A protein existing in the brain of mouse, having specific molecular weight and isoelectric point measured by a two-dimensional electrophoresis, useful as an essential factor for the growth of the tissue cell of the cerebral nervous system and as a marker for tumor, and appearing and dissapearing at a specific growth stage of mouse. CONSTITUTION:The homogenate of the brain embryo of a mouse from the 11- days old embryo to 12 weeks after birth, is subjected to a high-speed centrigugal separation. The obtained supernatant liquid is added with an alcohol, and the precipitated fraction is treated by gel-filtration and ion exchange chromatography. Each protein component is separated by two-dimensional electrophoresis to obtain the objective protein SN-a-SN-g having a molecular weight of 47,000-68,000 and an isoelectric point of 5.0-5.7.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a protein that exists in the mouse brain and changes in a period-specific manner during the growth process of the mouse.

Cancer-related antigens such as α-fetoprotein and carcinoembryonic antigen (CEA) have been used as tumor markers in the diagnosis and diagnosis of cancer3, and with the development and progression of various cancers,
Substances that are different from those used in the past are ebbing and flowing in living bodies, and attempts are being made to establish new cancer diagnostic methods using these as markers.

The present inventor is conducting research on substances that appear only in the early stages of embryonic development in animals, and as a result of intensive research on proteins that appear and disappear over time during the development and differentiation of the mouse brain nervous system. found that certain proteins appear in a time-specific manner and are absent in normal adult animals. We have completed the present invention based on the belief that these proteins are specific to proliferating cells and can serve as markers for excessive proliferation.

The protein of the present invention is present in the mouse brain and has a molecular weight of 47. OOO or 68.00
It is a protein characterized by having an isoelectric point of 0 and an isoelectric point of 5.0 to 5.7, which waxes and wanes in a period-specific manner during the growth process of mice.

The present inventor has developed an I.I.
As a result of analyzing the brain homosyne-1 components of CR mice by two-dimensional electrophoresis, there are 20 major proteins in the brain.
It was confirmed that the proteins of the present invention were classified into species, and 7 of them are proteins of the present invention that change in a period-specific manner during the mouse growth process, while the other 13 types do not change over time and exist constantly. did. The properties of the protein of the present invention will be described below.

Molecule Jn: G 8. Appears in embryo on day +1111. OO
Protein with O1 isoelectric point of 5.7 (hereinafter abbreviated as 5N-a)
exists, and this substance disappears in the 15th day embryo.

(Proteins that appear in the embryo on day 2112 and have a molecule JjI of 68.000 and an isoelectric point of 5.6 and 5.55 (hereinafter 5, respectively)
N-b, abbreviated as 5N-c) is IJIE, and 5N-b is 1
5N-c in day 5 embryos disappears in day 17 embryos.

Appears on day +3+13 embryo and has a molecular weight of 1i 8. OOO1
A protein with an isoelectric point of 5.45 (hereinafter abbreviated as 5N-d) exists and disappears in the 18th day embryo.

Appears on day +4112 embryo, molecule IIi; 52.00
0.

Isoelectric point 5.1 and molecular weight 50. OOU, isoelectric point 5.0
5N-(!, SN (abbreviated)) exist, and 5N-e gradually increases until day 15 of the embryo and remains unchanged thereafter. 8N-r increases until day 16 of the embryo. shows a decreasing trend from the 17th day onward until the 1FJth day after birth.After that, it gradually increases until the 7th day after birth, the increase stops, and thereafter there is no change.

(5) A protein that is not observed at all during embryonic development, appears at 12 weeks of age, and has a molecular weight of 47,000 and an isoelectric point of 5.25 (hereinafter abbreviated as SN-g).

Among these proteins, 5N-b, 5N-C.

When a brain homogenate extract of 8N-d is analyzed by two-dimensional electrophoresis after treatment with neuraminidase, the spot of 8N-d disappears and the spot of SN-a increases. This thing 5N-b, 5N-c15N-d
It has been found that each protein is a sialoglycoprotein, and SNa is a prototype thereof. 5N-
ILSSN-e.

5N-f and SN'-g are not changed by neuraminidase treatment.

In addition to mouse brain embryos, we followed the differentiation process of six embryos, liver embryos, kidney liver embryos, and intestinal embryos, and analyzed their homogenate extracts by two-dimensional electrophoresis. Proteins 5N-a, SN-, and g are not present in the intestinal embryo. Each protein was detected in 6 embryos, liver embryos, and kidney liver, although in very small amounts. However, these almost completely disappeared before birth. Furthermore, 8N-g was slightly present in the heart at 12 weeks of age. This suggests that each of the proteins psN-a to SN-g exists in particularly large quantities only in the brain.

The protein of the present invention can be separated and purified using known techniques. For example, each protein can be separated and purified by centrifuging a mouse brain embryo homogenate at high speed, adding alcohol to the resulting supernatant, and treating the precipitated fraction with gel filtration and ion exchange chromatography.

The protein of the present invention, which is specifically present in large amounts in the brain and undergoes phase-specific decline during the process of development and differentiation, acts as an essential factor or promoter of tissue cell growth in the cranial nervous system, and can be delivered to living organisms. Yes, it's effective. IH again! They are useful as tumor markers, and it is expected that antibodies against them will be used in the diagnosis and treatment of cancer.

This will be explained in more detail below with reference to Examples.

Example 1 Mice were washed with 10mM Tris-HCl buffer containing 2% Triton
The mixture was homogenized at pH 7.6) and extracted at 4°C for 30 minutes. Then high-speed centrifugation (40,000 rpm)
was carried out for 60 minutes, 5 times the amount of ethanol was added to the obtained supernatant, centrifuged for 5 minutes (3,000 rpm), and the supernatant was removed. The resulting precipitate was treated with 9.5 M urea, 2% NP-
40 (manufactured by Hiraisha), 2-thian holine (PH3,5~
10) (manufactured by Pharmacia), 5% mercaptoethanol, and 0.25t I6 sodium dodecyl sulfate (abbreviated as Hayashita SDS) to prepare a solution.

Next, each protein was separated from this solution by two-dimensional electrophoresis, and its molecular weight and isoelectric point were measured. Bovine serum albumin (67.000 in total) as a control protein;
Egg white aldimine (43,000) and aldolase (
3.1.0011) was used.

The following formulation was used for first-dimensional electrophoresis. [
Urea 2.76y, 30% acrylamide 067-110
俤NJ)-40'l shoulder t1 amhorine (P I-13
, 5-10) 0.25 ml, 10 μt of 10% ammonia persulfate, sti'IsuMEDo, 14i.

water 0.91aiJ) (for 6 bottles). Samples were loaded onto this one-dimensional gel with 10 nLM Hs PO as the anolyte.
4. Using 20 m M Na OIf as the catholyte, electrophoresis was performed at 400 V for 13 hours, then at 10 V for 1 hour.Then, the gel after electrophoresis was
S sample buffer (xol glycerin, 5-mercagtoethanol, 23% SDS.

Equilibrate with 62.5mM) Lis-HCl buffer (PH6,8) and run a 7.5m acrylamide slab gel (25mM).
M lis, 192++tM glycine, ol, %
SDS) and second-dimensional electrophoresis was performed at 120 V for 4 hours. Each protein separated by J is 0.0
After coloring the teeth for 1 hour with 5% Kumasi Blue, 10% methyl alcohol, and 10% acetic acid, 10 yen of methyl alcohol, 10
After decolorizing with thiacetic acid, each spot was measured.

Two to four mice each were used: 111 days after fertilization to 188 days after fertilization, 1 day after birth, 7 days after birth, and 122 days after birth.

There are seven types of proteins that change over time (from 5N-a to SN-g), and their characteristics are the same as described in the text.

There are 13 other proteins that give major spots, but all of these proteins do not change over time and are constantly present throughout the entire period from the beginning of development.

Its characteristics are described for reference.

Number Molecular weight Isoelectric point 5N-167,0005,7 8N-267,0005,45 SN-353,0005,75 SN-453,0005,65 SN-552,0005,3 SN-650,0005,2 SN-752,0004,9 SN-851,0006,05 SN 9 51,000 5.95 SN-1047,0006,0 8N-1147,0005,9 8N 12 46.000 5.65 8N 13 45,000 5.45 Example 2゜ A mouse brain extract was obtained in the same manner as in Example 1.

0.1 unit of neuraminidase (manufactured by Senkin Soy Sauce Co., Ltd.) was added to the extract at 100°C, and the mixture was kept at 37°C for 60 minutes. Next, two-dimensional electrophoresis was performed in the same manner as in Example 1 to separate the extracted proteins.

As a result, SN-a or SN as shown in the text
-g properties were confirmed.

Patent applicant: Mitsui Pharmaceutical Industries, Ltd.

Claims (1)

[Claims] It exists in the mouse brain and has a molecular weight of 4 according to two-dimensional electrophoresis.
A protein characterized by having an isoelectric point of 7,000 to 68,000 and an isoelectric point of 50 to 57, which changes in a period-specific manner during the growth process of mice.