JPH11269199A - New protein and dna thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new protein consisting of a protein having a specific amino acid sequence, having lecithin : cholesterol acyltransferase-like activity, and usable as e.g. a preventive/therapeutic agent for diseases such as arteriosclerosis, hyperlipemia and hypercaloric disease. SOLUTION: This new protein is such one as to have lecithin:cholesterol acyltransferase-like activity, such as a protein having the identical or substantially identical amino acid sequence with that shown by formula I, formula II or formula III, or precursor protein thereof, and is useful as a preventive/ therapeutic agent for arteriosclerosis, hyperlipemia, hypercaloric disease, obesity, hypertriglyceride disease, senility, encephalopathy, nephropathy and the like, and for screening compounds promoting or inhibiting lecithin : cholesterol acyltransferase-like activity. The new protein is obtained by the following procedure: a cDNA library or the like derived from e.g. hepatocyte is subjected to PCR using a primer derived from a partial sequence thereof, the resulting gene is integrated into a vector and expressed in host cells.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a novel protein having lecithin: cholesterol acyltransferase-like activity and the like, and its DNA.

[0002]

2. Description of the Related Art Cholesterol is an important lipid that constitutes the cell membrane of animals and regulates its properties, and is an essential substance for life that is indispensable as a precursor of steroid hormones. However, due to changes in eating habits and the environment, adult diseases caused by pathological accumulation of cholesterol such as arteriosclerosis have become a major problem. Is desired. It is thought that high-density lipoprotein (hereinafter sometimes abbreviated as HDL) plays a central role in the export of cellular cholesterol from cells, which is due to the epidemiological inverse correlation between coronary artery disease and plasma HDL, HDL
Causes cholesterol efflux from cultured cells and reduction of intracellular cholesterol (Journal of Lipid Research, 37, 2473)
, 1996). For the reverse transfer of cholesterol from peripheral cells, lecithin: cholesterol acyltransferase (hereinafter sometimes abbreviated as LCAT)
Is greatly involved.

[0003] LCAT transfers the acyl group (fatty acid) at the β-position of lecithin (phosphatidylcholine) to the 3β-OH group of cholesterol, thereby consuming equimolar amounts of lecithin and non-ester cholesterol, and Produces cholesterol esters and lysolecithin (Journal of Lipid Research, 9, 155
P. 1968). LCAT is mostly H in blood.
The cholesterol ester that is present in the DL and shows activity and is produced by the HDL is partially taken up by the liver and metabolized, and part of the cholesterol ester is transferred to the inner core of the HDL particles and is converted into mature HDL having a high cholesterol ester content. Change. HDL
HDL absorbs cholesterol continuously from other cell membranes due to the concentration gradient that results from the consumption of non-ester cholesterol at the same time. Thus, LCAT is HDL
At the same time, the cholesterol is reversely transported to transport cholesterol from peripheral tissues to the liver, and is considered to contribute to the anti-atherosclerotic effect of HDL (Biochim Biofize Acta, 1084, 205, 1991). In the hereditary disease, familial LCAT deficiency, the reverse cholesterol transfer system is deficient, causing specific tissue damage due to cholesterol deposition, corneal opacity, hemolytic anemia associated with abnormal erythrocyte morphology, and proteinuria and kidney due to renal impairment. Failure occurs (Lancet, 388, 778, 1991).
In addition to genetic abnormalities, LCAT activity fluctuates in various conditions involving plasma lipid abnormalities. For example, LCAT activity increases with hypercalorie, obesity, and hypertriglyceridemia (Clinical Science, 38, 593, 197).
0 years), malnutrition, beta-lipoproteinemia, and Tangier's disease. LCAT is a polypeptide composed of 416 amino acid residues synthesized in the liver and exists as a glycoprotein having a molecular weight of 59 to 68 KDa (Journal of Biological Chemistry, 254, 7456, 1979). In the blood,
ApoAI, which is mostly present in HDL and is a major apoprotein of HDL, acts as a cofactor to activate activity and activates LCAT (Feves Letter, 15
Volume, p. 355, 1971). There are various mutations in the LCAT gene, which correspond to various familial LCAT deficiency enzyme deficiencies and changes in clinical picture, and play an important role in plasma lipoprotein metabolism. At present, L
Only one type of CAT has been reported, and the existence of a similar protein having similar activity is not expected. On the other hand, atherosclerosis, thrombus formation, and restenosis after PTCA are closely related to abnormal vascular tonus due to endothelial cell damage, increased inflammatory response, and abnormalities in coagulation and fibrinolytic systems. Vascular remodeling occurs mainly in vascular smooth muscle cell proliferation and transformation. The changes at the molecular level that occur during the process of forming such vascular lesions are being understood at the level of a group of transcription factors that control the expression of individual genes (Kurabayashi et al., New Medicine, 52, 2340, 1997). These specifically expressed genes have a transcription induction system (promoter, enhancer, repressor) that operates only at that time, and the promoter controls the transcription of mRNA that directs protein synthesis. Such promoters are known to be hormone-dependent or growth factor-dependent, and drug screening systems and transgenic animals using these have already been created, and drug screening and analysis of biological functions have been performed. It is used for

[0004]

DISCLOSURE OF THE INVENTION The present invention relates to a novel protein having LCAT activity and the like, a precursor protein thereof, a partial peptide or a salt thereof, a signal peptide, the protein, a precursor protein, a partial protein or a signal peptide. Encoding DNA, recombinant vector, transformant, method for producing the protein, drug containing the protein or DNA, antibody against the protein, screening method / screening kit for compound promoting LCAT activity of the protein ,
Compound obtained by the screening method, drug containing the compound, promoter of novel protein having LCAT activity, etc., screening method / screening kit for compound promoting the promoter activity, compound obtained by the screening method , A medicament containing the compound, and the like.

[0005]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, have found that a human heart,
Successful cloning of cDNAs having novel nucleotide sequences from human kidney and mouse kidney-derived cDNA libraries, respectively, and the proteins encoded thereby are lecithin: cholesterol acyltransferase-like protein (hereinafter referred to as LCAT-like protein) Is). Further, the L
By cloning the genomic DNA of the CAT-like protein and testing the promoter activity,
An AT protein promoter was found. The present inventors have further studied based on these findings, and have completed the present invention.

That is, the present invention relates to (1) SEQ ID NO:
1, a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3, its precursor protein or a salt thereof, (2) SEQ ID NO: 4 to SEQ ID NO: 8. The protein according to item (1), which has the amino acid sequence represented by any one of SEQ ID NOs: 8 and its precursor protein, (3)
Lecithin: the protein or its precursor protein according to item (1) having cholesterol acyltransferase-like activity, (4) a partial peptide of the protein according to item (1) or a salt thereof, (5) SEQ ID NO: 9, sequence A signal peptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 10 or SEQ ID NO: 11, (6) having a base sequence encoding the protein or the precursor protein described in (1). DNA containing DNA, (7) DNA according to (6) having a base sequence represented by any one of SEQ ID NOs: 12 to 19, and (8) DNA according to (5). DNA containing a DNA encoding a signal peptide, (9) a base sequence represented by SEQ ID NO: 20 to SEQ ID NO: 22 DNA of the (8) above, wherein the, (1
0) a recombinant vector containing the DNA according to (6), (11) a transformant transformed with the recombinant vector according to (10), (12) a transformant according to (11). Culturing the transformant to produce and accumulate the protein or precursor protein described in (1), and collecting the collected protein or precursor protein, or a precursor protein or a salt thereof; Manufacturing method,

(13) A medicine containing the protein of (1), its precursor protein or a salt thereof, (14) a medicine containing the DNA of (6), (15) ) The medicament according to item (13) or (14), which is a therapeutic / prophylactic agent for arteriosclerosis, obesity, aging, brain disease, renal disorder or hyperlipidemia, (16) item (1) The protein or its precursor protein according to (4);
Antibody against the partial peptide or a salt thereof according to the above,
(17) The protein according to (1) or the partial peptide according to (4), wherein the protein according to (1), the partial peptide according to (4) or a salt thereof is used. Or a method for screening a compound or a salt thereof which promotes or inhibits lecithin: cholesterol acyltransferase-like activity of a salt thereof, or (18)
The protein according to item (1), the partial peptide according to item (4) or a salt thereof comprising the protein according to item (1), the partial peptide according to item (4) or a salt thereof. A screening kit for a compound or a salt thereof that promotes or inhibits lecithin: cholesterol acyltransferase-like activity, (19) obtained using the screening method according to (17) or the screening kit according to (18), A compound or a salt thereof that promotes or inhibits the lecithin: cholesterol acyltransferase-like activity of the protein described in (1), the partial peptide described in (4) or a salt thereof, and (20) the (17). Obtained by using the screening method of item (1) or the screening kit according to item (18). (21) SEQ ID NO: (1) SEQ ID NO: a protein comprising the protein described in (1), the partial peptide described in (4) or a salt thereof, a compound or a salt thereof which promotes or inhibits lecithin: cholesterol acyltransferase-like activity. : A promoter DNA having the same or substantially the same nucleotide sequence as the nucleotide sequence represented by 38 or a partial DN thereof having promoter activity
A. The DNA according to (21) containing A, and (2)
2) A method for screening a compound or a salt thereof that promotes or inhibits the promoter activity of the DNA described in (21) is provided.

Further, the present invention provides (23) SEQ ID NO:
1, an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3
1, about 50% or more (preferably about 60% or more, more preferably about 70% or more, more preferably about 80% or more, particularly preferably about 90% or more of the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: Above, most preferably about 9
(5% or more) having the homology of (1) or the precursor protein thereof, and (24) the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. A substantially identical amino acid sequence is obtained by deleting one or more (preferably about 1 to 30) amino acids in the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. The amino acid sequence represented by the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 and one or more (preferably, about 1 to 30) amino acids added thereto; 1, amino acids in which one or more (preferably about 1 to 30) amino acids in the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3 have been substituted with other amino acids Paragraph (1), wherein the protein or precursor protein is the amino acid sequence acid sequence, or a combination thereof, (25) SEQ ID NO: first 3-25 amino acid sequence represented by 1,
27th to 36th, 43rd to 66th, 68th to 86th, 92nd to 98th, 107th to 153rd, 15th
5th to 168th, 172nd to 180th, 189th to 2nd
40th, 256-262, 268-275, 277-287, 295-306, 308-332, 336-347 or (and) 351-377 amino acid sequence The peptide according to item (4), which comprises an amino acid sequence having
6) DNA containing a DNA having a base sequence hybridizable under high stringent conditions with a base sequence represented by any one of SEQ ID NOs: 12 to 19, (27) No. (26) (28) a transformant transformed with the recombinant vector according to (27), (29) culturing the transformant according to (28), A protein encoded by the DNA according to item (26), a precursor protein thereof, wherein the protein encoded by the DNA according to item (26) or a precursor protein thereof is produced, accumulated, and collected. (30) A method for producing a body protein or a salt thereof, (30) a protein encoded by the DNA according to (26), which is produced by the method according to (29), or a precursor thereof. Park protein or a salt thereof,

(31) (i) Lecithin and non-ester cholesterol are brought into contact with the protein described in (1), the partial peptide described in (4) or a salt thereof; Lecithin: cholesterol acyltransferase-like activities when lecithin, non-ester type cholesterol and a test compound are brought into contact with the protein described in 1), the partial peptide described in 4) or a salt thereof, are measured and compared. (17) The screening method according to (17),
(32) The protein according to (1), the partial peptide according to (4) or a partial peptide thereof obtained by using the screening method according to (17) or the screening kit according to (18). Lecithin as a salt: a medicament containing a compound that promotes cholesterol acyltransferase-like activity or a salt thereof, (33) an agent for treating or preventing arteriosclerosis, obesity, aging, brain disease, renal disorder or hyperlipidemia (34) the medicament according to (32), which is an agent for treating or preventing hypercalorie, obesity or hypertriglyceridosis; (35) the medicament according to (35),
Lecithin: cholesterol of the protein according to (1), the partial peptide according to (4) or a salt thereof obtained by using the screening method according to (7) or the screening kit according to (18). (36) a medicament comprising a compound that inhibits acyltransferase-like activity or a salt thereof, (36) an agent for treating or preventing malnutrition, β-lipoproteinemia or Tangier's disease,
5) The medicament according to the above,

(37) The antibody described in (16) is competitive with the test solution and the labeled protein described in (1), the partial peptide described in (4) or a salt thereof. And labeled (1) bound to the antibody.
The protein according to item (1), the protein according to item (4), wherein the proportion of the protein according to item (4), the partial peptide according to item (4), or a salt thereof is measured. (38) A method for quantifying a peptide or a salt thereof, (38) reacting a test solution with an antibody described in (16) and a labeled antibody described in (16) insolubilized on a carrier simultaneously or successively Measuring the activity of the labeling agent on the insolubilized carrier after the reaction, wherein the protein according to item (1), the partial peptide according to item (4), or a salt thereof is determined in a test solution. (39) a medicament comprising the antibody according to (16), (40) a medicament according to (39), which is a therapeutic / prophylactic agent for malnutrition, β-lipoproteinemia or Tangier's disease; (41) The DNA complementary to the DNA described in (6) or (26). Is an antisense DNA having a substantially complementary base sequence and capable of suppressing the expression of the DNA, (42) substantially complementary to the DNA according to the item (6) or (26). Base sequence is the DNA
A base sequence having about 70% or more (preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more) homology with the entire base sequence or a partial base sequence of the base sequence complementary to (41) an antisense DNA according to (41), a pharmaceutical comprising the antisense DNA according to (41), and (44) malnutrition;
The medicament according to (43), which is an agent for treating or preventing β-lipoproteinemia or Tangier's disease.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION A protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 of the present invention (hereinafter referred to as the protein of the present invention) ) Is a human or warm-blooded animal (eg, guinea pig, rat, mouse, chicken, rabbit, pig, sheep, cow, monkey, etc.) cells (eg, hepatocytes, spleen cells, nerve cells, glial cells,
Pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts, fiber cells, muscle cells, adipocytes, immune cells (eg, macrophages, T cells, B cells, natural killers) cell,
Mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, osteocytes, osteoblasts, osteoclasts, mammary cells, hepatocytes or stromal cells, Or precursors of these cells, such as stem cells or cancer cells) or any tissue in which those cells are present, such as the brain,
Various parts of the brain (eg, olfactory bulb, amygdala, basal sphere, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla, cerebellum), spinal cord, pituitary, stomach, pancreas, kidney, liver, gonad, thyroid, gall bladder,
Bone marrow, adrenal gland, skin, muscle, lung, digestive tract (eg, large intestine, small intestine), blood vessels, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testicle, ovary, placenta, uterus, bone, joint, skeleton Muscles,
Or cells of blood cell lineage or cultured cells thereof (for example, M
EL, M1, CTLL-2, HT-2, WEHI-3,
HL-60, JOSK-1, K562, ML-1, MO
LT-3, MOLT-4, MOLT-10, CCRF-
CEM, TALL-1, Jurkat, CCRT-HS
B-2, KE-37, SKW-3, HUT-78, HU
T-102, H9, U937, THP-1, HEL, J
K-1, CMK, KO-812, MEG-01, etc.) or a synthetic protein.

The amino acid sequence represented by SEQ ID NO: 2 is located between the 63rd position (Leu) and 64th position (Val) of the amino acid sequence represented by SEQ ID NO: 1.
64th (Glu)-of the amino acid sequence represented by 2
It is the insertion of the 95th (Leu) 32 amino acid residues. The amino acid sequences substantially identical to the amino acid sequences represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 include SEQ ID NO: 1, SEQ ID NO:
2 or the amino acid sequence represented by SEQ ID NO: 3 and about 5
0% or more, preferably about 60% or more, more preferably about 70% or more, more preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more amino acid sequence or the like. Is mentioned. In particular, the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 includes amino acids 3 to 2 of the amino acid sequence represented by SEQ ID NO: 1.
Fifth, 27th to 36th, 43rd to 66th, 68th
86th, 92nd to 98th, 107th to 153rd, 155th to 168th, 172th to 180th, 189th to 240th, 256th to 262nd, 268th
275th, 277th to 287th, 295th to 30th
An amino acid sequence having the sixth, 308th to 332nd, 336th to 347th or (and) 351th to 377th amino acid sequence. Examples of the protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 of the present invention include, for example, the aforementioned SEQ ID NO: 1, SEQ ID NO: 2 or Having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 3,
A protein having substantially the same activity as the protein having the amino acid sequence represented by 1, SEQ ID NO: 2 or SEQ ID NO: 3 is preferred. In the present specification, the lecithin: cholesterol acyltransferase-like activity (may be abbreviated as LCAT-like activity) refers to the aforementioned L
CAT activity (LCAT activity), phospholipid: cholesterol acyltransferase activity, phospholipase activity, lysophosphatidylcholine esterification to phosphatidylcholine, lysophospharipase activity, PAF hydrolysis And transesterification, hydrolysis of fatty acid esters, etc., such as hydrolysis of oxidized phospholipids and esterification of oxidized cholesterol, metabolic regulation activity of high density lipoprotein (HDL) or lipase activity. As substantially the same activity,
For example, LCAT-like activity and the like can be mentioned. Substantially the same quality means that their activity is qualitatively (eg, physiochemically,
Or pharmacologically) homogeneous. Therefore, activities such as LCAT-like activity are equivalent (eg, about 0.1 to
(100-fold, preferably about 0.5- to 10-fold, more preferably 0.5- to 2-fold), but the quantitative factors such as the degree of these activities and the molecular weight of the protein may be different. . The activity such as LCAT-like activity can be measured according to a method known per se.
It can be measured according to the screening method described below.

The proteins of the present invention include, for example, SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
In the amino acid sequence represented by 1 or 2 or more (preferably about 1 to 30, preferably about 1 to 10,
More preferably, the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 in which the number (1 to 5) of amino acids has been deleted is 1 or 2 or more (preferably 1 or more). About 30 pieces, preferably 1 to 1
One or more (preferably) the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 to which about 0, more preferably number (1 to 5) amino acids have been added. Is about 1 to 30, preferably about 1 to 10, and more preferably a number (1 to 5)
Of the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 in which one or more (preferably 1 to
A so-called protein such as a protein containing an amino acid sequence in which about 30, preferably about 1 to 10, and more preferably a number (1 to 5) amino acids have been substituted with another amino acid, or an amino acid sequence obtained by combining them. Mutein is also included. When the amino acid sequence is inserted, deleted or substituted as described above, the position of the insertion, deletion or substitution is not particularly limited, but may be SEQ ID NO: 1,
Positions other than the amino acid sequence common to the amino acid sequences represented by SEQ ID NOs: 2 and 3 are listed. Specific positions of the insertion, deletion or substitution include, for example, the 3rd to 25th amino acids in the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3,
27th to 36th, 43rd to 66th, 68th to 86th, 92nd to 98th, 107th to 153rd, 15th
5th to 168th, 172nd to 180th, 189th to 2nd
40th, 256th to 262nd, 268th to 275th, 277th to 287th, 295th to 306th, 308th to 332nd, 336th to 347th or 3rd
Positions other than the amino acid sequence at positions 51 to 377 and positions other than the amino acid sequence corresponding to the common sequence in the amino acid sequence represented by SEQ ID NO: 2 are exemplified. In addition, as the insertion, deletion or substitution position, in the amino acid sequence represented by SEQ ID NO: 1, positions other than the 163rd Ala to 167th Gly, and in the amino acid sequence represented by SEQ ID NO: 2 195th Ala to 199th position other than Gly, 96th V in the amino acid sequence represented by SEQ ID NO: 2
al-position of Asp at position 127, of the amino acid sequence represented by SEQ ID NO: 3, Ala at position 163
And positions other than the １６167th Gly. In addition, in the amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence from Ala at position 163 to Gly at position 167 is the amino acid sequence of the active center of the protein of the present invention.

Further, specific positions of the insertion, deletion or substitution include, for example, the second, 26th, and 37th amino acids of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3. 42nd, 67th, 87th, 91st, 99th to 100th, 106th,
154th, 169th, 171st, 181st
, 188th, 241st, 245th, 2nd
51st, 255th, 263rd to 264th, 2nd
67th, 276th, 288th to 289th, 2nd
The position of the 94th, 307th, 333rd, 335th, 348th, 350th or 378th amino acids, the position of the amino acid sequence in the amino acid sequence represented by SEQ ID NO: 2, etc. Is mentioned. On the other hand, when the amino acid sequence is added as described above,
Specifically, the amino acid sequence represented by SEQ ID NO: 4 or the amino acid sequence represented by SEQ ID NO: 2 obtained by adding three more amino acids to the N-terminal of the amino acid sequence represented by SEQ ID NO: 1
The amino acid sequence represented by SEQ ID NO: 5 with three more amino acids added to the N-terminal of the amino acid sequence represented by Therefore, the protein of the present invention may be a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 4 or SEQ ID NO: 5. “Substantially the same” has the same meaning as described above. The amino acid sequence represented by SEQ ID NO: 5 is located between the 66th position (Leu) and the 67th position (Val) of the amino acid sequence represented by SEQ ID NO: 4 and the 67th amino acid sequence represented by SEQ ID NO: 5.
It is the one in which the thirty-second (Glu) to 98th (Leu) 32 amino acid residues have been inserted.

The protein in the present specification has an N-terminal (amino terminal) at the left end and a C-terminal (carboxyl terminal) at the right end according to the convention of peptide labeling. The protein of the present invention, including the protein containing the amino acid sequence represented by SEQ ID NO: 1, usually has a carboxyl group (-COOH) or a carboxylate (-CO
O ⁻ ), but the C-terminal may be an amide (—CONH ₂ ) or an ester (—COOR). Here, as R in the ester, for example, methyl, ethyl, n-
C such as propyl, isopropyl or n-butyl
_1-6 alkyl groups, for example, C _3-8 cycloalkyl groups such as cyclopentyl and cyclohexyl, for example, C _6-12 aryl groups such as phenyl and α-naphthyl, for example, phenyl-C _1-2 such as benzyl and phenethyl An alkyl group or α-naphthyl-C such as α-naphthylmethyl
_In addition to C _7-14 aralkyl groups such as a _1-2 alkyl group, a pivaloyloxymethyl group widely used as an oral ester and the like are used. When the protein of the present invention has a carboxyl group (or carboxylate) other than the C-terminus, the protein of the present invention includes a carboxyl group amidated or esterified. As the ester in this case, for example, the above-mentioned C-terminal ester and the like are used. Furthermore, in the protein of the present invention, the amino group of the N-terminal amino acid residue (eg, a methionine residue) has a protecting group (eg, a C _1-6 acyl such as a C _1-6 alkanoyl such as a formyl group and an acetyl group). Group), N-terminal glutamine residue generated by cleavage in vivo, pyroglutamine oxidation, substituent on the side chain of amino acid in the molecule (for example, -OH, -SH,
Amino group, imidazole group, indole group, guanidino group, etc. protected with a suitable protecting group (eg, C _1-6 acyl group such as C _1-6 alkanoyl group such as formyl group, acetyl group) Or a complex protein such as a so-called glycoprotein having a sugar chain bonded thereto. Specific examples of the protein of the present invention include, for example, a protein derived from a human heart having the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 4 [FIG. 1], represented by SEQ ID NO: 2 or SEQ ID NO: 5 Human kidney-derived protein having an amino acid sequence (FIG. 2), SEQ ID NO:
A mouse kidney-derived protein having the amino acid sequence represented by No. 3 is used (FIG. 3).

The precursor protein of the present invention may be, for example, the N-terminal or (and) C
It is a protein having one or more, preferably about 1 to 10, more preferably about 1 to 100, and still more preferably about 1 to 200 amino acids bound to the terminal. Specifically, as the precursor protein of the present invention, a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 is used. The precursor protein of the present invention may be, for example, the above-mentioned human or warm-blooded animal (eg, guinea pig, rat, mouse, chicken, rabbit, pig, sheep, cow, monkey, etc.) cells or any tissue in which those cells are present. Or a synthetic protein. The amino acid sequence substantially identical to SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 is about 50% or more of the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8, The amino acid sequence preferably has about 60% or more, more preferably about 70% or more, more preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more homology. More specifically, a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, and capable of producing the above-described protein of the present invention Any one may be used. Thus, quantitative factors such as the molecular weight of the protein may be different.

The precursor protein of the present invention may be, for example, one or more (preferably 1 to 30) of the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8. Degree, preferably 1-10
About one, more preferably several) of the amino acid sequences represented by SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 in which one or more (preferably 1 to 30) amino acids have been deleted. About one, preferably 1 to 10
1 or more (preferably 1 to 30) to the amino acid sequence represented by the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 to which about (preferably several) amino acids have been added. Degree, preferably 1-10
(Preferably several amino acids), 1 or 2 in the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8.
Or more (preferably about 1 to 30, preferably 1 to
A protein containing an amino acid sequence in which about 10 amino acids (more preferably several amino acids) are substituted with another amino acid, or an amino acid sequence obtained by combining them is also included. When the amino acid sequence is inserted, deleted or substituted as described above, the position of the insertion, deletion or substitution is not particularly limited. For example, in the amino acid sequence represented by SEQ ID NO: 6, Positions other than 196th Ala to 200th position Gly, among amino acid sequences represented by SEQ ID NO: 7, positions 228th position other than Ala to 232th position other than Gly, among amino acid sequences represented by SEQ ID NO: 7 Positions from 129th Val to 160th Asp, SEQ ID NO: 8
In the amino acid sequence represented by
a to 200th positions other than Gly.
Further, the precursor protein of the present invention usually has a carboxyl group (-COOH) or a carboxylate (-C
OO ^-) a but, as in the protein of the present invention described above, C-terminal, it may be an amide (-CONH ₂₎ or ester (-COOR).

Further, the precursor protein of the present invention includes:
Similarly to the above-described protein of the present invention, one in which the amino group of the N-terminal amino acid residue (eg, methionine residue) is protected by a protecting group, and the glutamine residue formed by cleavage of the N-terminal side in vivo is used. Pyroglutamine-oxidized products, those in which the substituent on the side chain of the amino acid in the molecule is protected with an appropriate protecting group, and those complex proteins such as so-called glycoproteins to which sugar chains are bound are also included. Specific examples of the precursor protein of the present invention include, for example, the present invention having the amino acid sequence represented by SEQ ID NO: 1 at the N-terminus of the protein of the present invention having the amino acid sequence represented by SEQ ID NO: 1 (Ie, a protein having the amino acid sequence represented by SEQ ID NO: 6) to which the signal peptide of SEQ ID NO: 6 is bound, SEQ ID NO:
A protein in which the signal peptide of the present invention having the amino acid sequence of SEQ ID NO: 10 is bound to the N-terminal of the protein of the present invention containing the amino acid sequence of SEQ ID NO: 2 (ie, represented by SEQ ID NO: 7) And a protein having an amino acid sequence).
The amino acid sequence represented by SEQ ID NO: 7 is
6, between positions 96 (Leu) and 97 (Val) of the amino acid sequence represented by SEQ ID NO: 97, positions 97 (Glu) to 128 (Lu)
eu) with 32 amino acid residues inserted. Since the precursor protein of the present invention has, for example, a signal peptide described below, the protein of the present invention can be efficiently secreted extracellularly. Further, it is useful as an intermediate for producing the protein of the present invention.
Furthermore, the precursor protein of the present invention can exert the same function as the protein of the present invention, and thus has the same utility as the protein of the present invention.

The partial peptide of the protein of the present invention is a partial peptide of the above-mentioned protein of the present invention, and preferably has the same activity (eg, LCAT-like activity) as the above-mentioned protein of the present invention. Any of them may be used. For example, at least 20 or more, preferably 50 or more, more preferably 70 or more, more preferably 100 or more, most preferably 200 or more amino acid sequences of the constituent amino acid sequences of the protein of the present invention, Peptides having LCAT-like activity are used. Among these peptides, for example, the 3rd to 25th amino acids of the amino acid sequence represented by SEQ ID NO: 1,
27th to 36th, 43rd to 66th, 68th to 86th, 92nd to 98th, 107th to 153rd, 15th
5th to 168th, 172nd to 180th, 189th to 2nd
Amino acid sequence at position 40, 256-262, 268-275, 277-287, 295-306, 308-332, 336-347 or (and) 351-377 For example, a peptide containing an amino acid sequence having the formula: The 16th amino acid sequence represented by SEQ ID NO: 1
An amino acid sequence consisting of the 3rd Ala to the 167th Gly (the 19th amino acid sequence represented by SEQ ID NO: 2);
An amino acid sequence consisting of the 5th Ala to the 199th Gly, the 16th amino acid sequence represented by SEQ ID NO: 3;
Peptides having an amino acid sequence consisting of the third Ala to the 167th Gly) are also suitable. Further, the partial peptide of the present invention has 1 or 2 amino acids in its amino acid sequence.
Or more (preferably about 1 to 10, more preferably number (1 to 5)) amino acids are deleted, or 1 or 2 or more (preferably 1 to 2)
About 0, more preferably about 1 to 10, and still more preferably number (1 to 5) amino acids are added, or
One or more (preferably 1)
About 20 amino acids, more preferably about 1 to 10 amino acids, still more preferably about 1 to 5 amino acids, or 1 or 2 or more amino acids (preferably 1 to 10 amino acids) in the amino acid sequence. (About, more preferably several, more preferably about 1 to 5) amino acids may be substituted with another amino acid.

The partial peptide of the present invention usually has a carboxyl group (-COOH) or a carboxylate (-COO-) at the C-terminus, but has an amide (-CONH ₂ ) Or an ester (—COOR). Furthermore, the partial peptide of the present invention has a structure in which the amino group of the N-terminal amino acid residue (eg, methionine residue) is protected by a protecting group, and Glutamine residues generated by cleavage in the body are pyroglutamine-oxidized, those in which the substituents on the side chains of amino acids in the molecule are protected with appropriate protecting groups, and those in which sugar chains are bonded, such as so-called glycopeptides Complex peptides and the like are also included. Since the partial peptide of the present invention can be used as an antigen for preparing an antibody, it does not necessarily need to have LCAT-like activity.

As the signal peptide of the present invention, for example, a peptide containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11 is used. The signal peptide of the present invention includes, for example, cells of the above-mentioned humans and warm-blooded animals (eg, guinea pigs, rats, mice, chickens, rabbits, pigs, sheep, cows, monkeys, etc.), and any tissues in which those cells are present. Or a synthetic peptide. SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 1
The amino acid sequence substantially identical to the amino acid sequence represented by No. 1 is at least about 70%, preferably at least about 80%, more than the amino acid sequence represented by SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11. Preferably about 90% or more,
Most preferably, it shows an amino acid sequence having about 95% or more homology. More specifically, any peptide having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11 and capable of exhibiting a function as a signal peptide Any one may be used. Thus, quantitative factors such as the molecular weight of the protein may be different.

The signal peptide of the present invention may contain one or more (preferably 1 to 2)
About 10, preferably 1 to 5, more preferably 1
~ 3 amino acids are deleted, or 1 or 2 or more (preferably about 1 to 10)
(Preferably about 1 to 5, more preferably 1 to 3)
Or one or more (preferably about 1 to 10, preferably about 1 to 5, more preferably 1 to 3) amino acids are inserted into the amino acid sequence, or , One or two or more (preferably about 1 to 10, preferably 1
(About 5 to about 5, more preferably 1 to 3) amino acids may be substituted with another amino acid. In the signal peptide of the present invention, the C-terminus usually has a carboxyl group (-C
OOH) or carboxylate (—COO ⁻ ), but the C-terminal may be amide (—CONH ₂ ) or ester (—COOR) as in the protein of the present invention described above. Furthermore, the signal peptide of the present invention has a N-terminal amino acid residue (eg, a methionine residue) in which the amino group is protected by a protecting group, and a N-terminal side, similar to the above-described protein of the present invention. Glutamine residues generated by cleavage in the body are pyroglutamine-oxidized, those in which the substituents on the side chains of amino acids in the molecule are protected with appropriate protecting groups, and those in which sugar chains are bonded, such as so-called glycopeptides Complex peptides and the like are also included. As a specific example of the signal peptide of the present invention, for example, the protein of the present invention having the amino acid sequence represented by SEQ ID NO: 4 was removed from the precursor protein of the present invention having the amino acid sequence represented by SEQ ID NO: 6. A peptide comprising the amino acid sequence represented by SEQ ID NO: 9,
It contains the amino acid sequence represented by SEQ ID NO: 10 by removing the protein of the present invention having the amino acid sequence represented by SEQ ID NO: 1 from the precursor protein having the amino acid sequence represented by SEQ ID NO: 6 From the precursor protein of the present invention having the amino acid sequence represented by the peptide SEQ ID NO: 8, SEQ ID NO: 3
A peptide containing the amino acid sequence represented by SEQ ID NO: 11 from which the protein of the present invention having the amino acid sequence represented by The signal peptide of the present invention includes the protein of the present invention,
Various extracellular secretory proteins can be efficiently extracellularly secreted.

A protein of the present invention, a precursor protein,
As salts of partial peptides or signal peptides, salts with physiologically acceptable acids (eg, inorganic acids, organic acids) and bases (eg, alkali metal salts) are used, and especially physiologically acceptable. Acid addition salts are preferred. Such salts include, for example, inorganic acids (eg, hydrochloric acid, phosphoric acid,
Salts with hydrobromic acid, sulfuric acid, or organic acids (for example,
Examples thereof include salts with acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, and benzenesulfonic acid. The protein of the present invention, its precursor or a salt thereof can be produced from the above-mentioned human or warm-blooded animal cell or tissue by a protein purification method known per se, or contains a DNA encoding the protein described below. Can also be produced by culturing a transformant to be prepared. Further, it can also be produced according to the peptide synthesis method described later. When producing from human or mammalian tissues or cells, after homogenizing human or mammalian tissues or cells, perform extraction with an acid or the like,
The extract can be purified and isolated by combining chromatography such as reverse phase chromatography and ion exchange chromatography.

A protein of the present invention, a precursor protein,
Partial peptide, signal peptide or salt thereof,
Alternatively, a commercially available resin for protein synthesis can be used for the synthesis of these amides. Examples of such a resin include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin,
Methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethylphenylacetamidomethyl resin,
Polyacrylamide resin, 4- (2 ′, 4′-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2 ′,
4'-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin. Using such a resin, an amino acid having an α-amino group and a side chain functional group appropriately protected is condensed on the resin in accordance with the sequence of the target protein according to various known condensation methods. At the end of the reaction, the protein is cleaved from the resin, and at the same time, various protecting groups are removed. Further, an intramolecular disulfide bond formation reaction is carried out in a highly diluted solution to obtain a target protein or an amide thereof. For the condensation of the protected amino acids described above, various activating reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable.
As the carbodiimides, DCC, N, N′-diisopropylcarbodiimide, N-ethyl-N ′-(3-dimethylaminoprolyl) carbodiimide and the like are used. Activation by these requires a racemization inhibitor additive (eg, HOBt, HOOBt)
Together with the protected amino acid directly to the resin, or
The symmetric acid anhydride, HOBt ester or HOOBt ester can be added to the resin after activation of the protected amino acid in advance.

The solvent used for activating the protected amino acid or condensing with the resin can be appropriately selected from solvents known to be usable for the protein condensation reaction. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol,
Sulfoxides such as dimethyl sulfoxide, ethers such as pyridine, dioxane, and tetrahydrofuran; nitriles such as acetonitrile and propionitrile; esters such as methyl acetate and ethyl acetate; and an appropriate mixture thereof are used. The reaction temperature is appropriately selected from a range known to be usable for a protein bond formation reaction, and is usually appropriately selected from a range of about -20 ° C to 50 ° C. The activated amino acid derivative is usually used in a 1.5 to 4-fold excess. As a result of the test using the ninhydrin reaction, when the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. When a sufficient condensation cannot be obtained by repeating the reaction, the unreacted amino acid can be acetylated using acetic anhydride or acetylimidazole so as not to affect the subsequent reaction.

Examples of the protecting group for the amino group of the starting material include Z, Boc, t-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, Trifluoroacetyl, phthaloyl, formyl, 2
-Nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like are used. The carboxyl group is
For example, alkyl esterification (eg, methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl,
Cyclohexyl, cycloheptyl, cyclooctyl, 2
Linear, branched or cyclic alkyl esterification such as adamantyl), aralkyl esterification (eg, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification) , Phenacyl esterification, benzyloxycarbonyl hydrazide, t-butoxycarbonyl hydrazide, trityl hydrazide and the like. The hydroxyl group of serine is
For example, it can be protected by esterification or etherification. Suitable groups for this esterification include, for example, lower (C _1-6 ) alkanoyl groups such as acetyl groups, aroyl groups such as benzoyl groups, and groups derived from carbonic acid such as benzyloxycarbonyl groups and ethoxycarbonyl groups. Used. Examples of a group suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group. Examples of groups for protecting the phenolic hydroxyl group of tyrosine, for example, Bzl, C ^l2 -Bzl, 2-
Nitrobenzyl, Br-Z, t-butyl and the like are used.
As a protecting group for imidazole of histidine, for example,
Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc
Are used.

The activated carboxyl group of the raw material includes, for example, a corresponding acid anhydride, azide, active ester [alcohol (eg, pentachlorophenol, 2,4,5-trichlorophenol, 2,4- Dinitrophenol, cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, and esters with HOBt). As the activated amino group of the raw material, for example, a corresponding phosphoric amide is used. As a method for removing (eliminating) the protecting group, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon,
Also, acid treatment with anhydrous hydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., and reduction with sodium in liquid ammonia Also used. The elimination reaction by the above acid treatment is generally performed at a temperature of about -20 ° C to 40 ° C,
In the acid treatment, for example, anisole, phenol,
It is effective to add a cation scavenger such as thioanisole, metacresol, paracresol, dimethylsulfide, 1,4-butanedithiol, 1,2-ethanedithiol and the like. In addition, the 2,4-dinitrophenyl group used as the imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as the indole protecting group of tryptophan is 1,2-ethanedithiol, 1,4-butane described above. In addition to deprotection by acid treatment in the presence of dithiol or the like, it is also removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia or the like.

The protection of a functional group which should not be involved in the reaction of the raw materials, the protecting group, the elimination of the protective group, the activation of the functional group involved in the reaction, and the like can be appropriately selected from known groups or known means. . As another method for obtaining an amide form of a protein, for example, first, after protecting the α-carboxyl group of the carboxy terminal amino acid by amidation, the peptide (protein) chain is extended to a desired chain length on the amino group side, A protein from which only the protecting group for the N-terminal α-amino group of the peptide chain has been removed and a protein from which only the protecting group for the carboxyl group at the C-terminal has been removed, and these proteins are mixed in a mixed solvent as described above. To condense. Details of the condensation reaction are the same as described above. After purifying the protected protein obtained by the condensation, all the protecting groups are removed by the above-mentioned method, and a desired crude protein can be obtained. The crude protein is purified by various known purification means, and the main fraction is freeze-dried to obtain an amide of the desired protein. In order to obtain an ester of a protein, for example, after condensing the α-carboxyl group of the carboxy terminal amino acid with a desired alcohol to form an amino acid ester, an ester of the desired protein is obtained in the same manner as the amide of the protein be able to.

The partial peptide, signal peptide or salt thereof of the present invention can be produced according to a peptide synthesis method known per se, or by cleaving the protein of the present invention or its precursor protein with an appropriate peptidase. . As a method for synthesizing a peptide, for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, the partial peptide or amino acid that can constitute the partial peptide or signal peptide of the present invention is condensed with the remaining portion, and when the product has a protecting group, the protecting group is eliminated to produce the desired peptide. Can be. Known condensation methods and elimination of protecting groups include:
For example, the following methods can be mentioned. M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publisher
s, New York (1966) Schroeder and Luebke, The Peptid
e), Academic Press, NewYork (1965) Nobuo Izumiya et al., Fundamentals and experiments of peptide synthesis, Maruzen Co., Ltd.
(1975) Haruaki Yajima and Shunpei Sakakibara, Laboratory for Biochemistry 1, Protein Chemistry IV, 205, (1977) Supervised by Haruaki Yajima, Development of Continuing Drugs, Volume 14, Peptide Synthesis, Hirokawa Shoten Thereafter, the partial peptide of the present invention can be purified and isolated by a combination of ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization. When the partial peptide or signal peptide obtained by the above method is in a free form, it can be converted to an appropriate salt by a known method or a method analogous thereto, and conversely, when it is obtained by a salt, a known method Alternatively, it can be converted to a free form or another salt by a method analogous thereto.

The DNA encoding the protein of the present invention may be any DNA containing the above-described nucleotide sequence encoding the protein of the present invention. Genomic DNA, genomic DNA library,
Any of the above-described cell / tissue-derived cDNA, the above-described cell / tissue-derived cDNA library, and synthetic DNA may be used. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. In addition, from the cells and tissues described above,
Reverse Transcriptase Polymerase Chain React directly using prepared total RNA or mRNA fraction
It can also be amplified by ion (hereinafter abbreviated as RT-PCR method). DN encoding the protein of the present invention
A includes, for example, a DNA containing the base sequence represented by SEQ ID NO: 12 or a base sequence that hybridizes under high stringent conditions to the base sequence represented by SEQ ID NO: 12, and DNA encoding a protein having substantially the same activity as the protein (eg, LCAT-like activity), DNA containing the nucleotide sequence represented by SEQ ID NO: 13, or DNA encoding the nucleotide sequence represented by SEQ ID NO: 13 DNA encoding a protein having a nucleotide sequence that hybridizes under stringent conditions and having substantially the same activity as the protein of the present invention, DNA containing the nucleotide sequence represented by SEQ ID NO: 14, or A base sequence that hybridizes with the base sequence represented by No. 14 under high stringent conditions. , DN encoding a protein having a protein substantially the same activity of the present invention
A, a DNA containing the nucleotide sequence represented by SEQ ID NO: 15 or a nucleotide sequence hybridizable under high stringent conditions to the nucleotide sequence represented by SEQ ID NO: 15; DNA encoding a protein having the same activity as described above, DNA containing the nucleotide sequence represented by SEQ ID NO: 16 or a nucleotide sequence hybridizing under high stringent conditions with the nucleotide sequence represented by SEQ ID NO: 16 Any DNA may be used as long as it encodes a protein having the same activity as the protein of the present invention.

DNAs that can hybridize under high stringent conditions to the base sequence represented by any one of SEQ ID NOs: 12 to 16 include:
For example, the nucleotide sequence represented by any one of SEQ ID NOs: 12 to 16 is about 70% or more, preferably about 80% or more, and more preferably about 90% or more.
% Or more, and most preferably about 95% or more. Hybridization is performed by a method known per se or a method analogous thereto, for example, molecular cloning (Molecu
lar Cloning) 2nd (J. Sambrook etal., Cold Spring)
Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. More preferably, it can be performed under high stringent conditions. High stringency conditions include, for example, a sodium concentration of about 19 to
40 mM, preferably about 19-20 mM, at a temperature of about 5
0 to 70 ° C, preferably about 60 to 65 ° C.
Particularly, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C. is most preferable. More specifically, the DNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 1 includes a DNA having the base sequence represented by SEQ ID NO: 12, and the like. As a DNA encoding a protein having the same, a DNA having the base sequence of SEQ ID NO: 13
DNA encoding the protein having the amino acid sequence represented by NA, SEQ ID NO: 3 includes SEQ ID NO: 1
Examples of the DNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 4, such as the DNA having the nucleotide sequence represented by SEQ ID NO: 4, include the DNA having the nucleotide sequence represented by SEQ ID NO: 15, SEQ ID NO: 5 D encoding a protein having the amino acid sequence represented by
As the NA, a DNA having the base sequence represented by SEQ ID NO: 16 or the like is used.

D encoding the precursor protein of the present invention
The NA may be any as long as it contains a base sequence encoding the precursor protein of the present invention described above. In addition, genomic DNA, genomic DNA library, cDNA derived from the aforementioned cells / tissues, cDNA library derived from the aforementioned cells / tissues, synthetic DN
Any of A may be used. As the DNA encoding the precursor protein of the present invention, for example, a DNA having the base sequence represented by SEQ ID NO: 17 or SEQ ID NO: 1
DNA which has a base sequence hybridizable under high stringent conditions with the base sequence represented by No. 7 and encodes a protein capable of producing the above-mentioned precursor protein of the present invention, the base sequence represented by SEQ ID NO: 18 Or a DNA having a base sequence that hybridizes with the base sequence represented by SEQ ID NO: 18 under high stringent conditions, and encoding a protein capable of producing the precursor protein of the present invention described above. DNA having the nucleotide sequence represented by SEQ ID NO: 19, or having the nucleotide sequence hybridizable under high stringent conditions to the nucleotide sequence represented by SEQ ID NO: 19, producing the precursor protein of the present invention described above. DNA encoding the protein to be obtained is used. Examples of the DNA that can hybridize under high stringent conditions to the base sequence represented by any one of SEQ ID NOs: 17 to 19 include, for example, SEQ ID NO:
17 to SEQ ID NO: 19 and about 70% or more, preferably about 80% or more,
More preferably about 90% or more, most preferably about 95%
For example, DNA containing a base sequence having a homology of at least% is used. The same hybridization method and high stringency conditions as described above are used. More specifically, the DNA encoding the precursor protein of the present invention having the amino acid sequence represented by SEQ ID NO: 6 includes a DNA containing the DNA having the base sequence represented by SEQ ID NO: 17, 7
As the DNA encoding the precursor protein of the present invention having the amino acid sequence represented by the following, a DNA containing the DNA having the base sequence represented by SEQ ID NO: 18;
As the DNA encoding the precursor protein of the present invention having the amino acid sequence represented by SEQ ID NO: 8, a DNA containing the DNA having the base sequence represented by SEQ ID NO: 19 or the like is used.

DNA encoding the partial peptide of the present invention
May be any as long as it contains a base sequence encoding the above-described partial peptide of the present invention. In addition, any of genomic DNA, genomic DNA library, cDNA derived from the above-mentioned cells / tissues, cDNA library derived from the aforementioned cells / tissues, and synthetic DNA may be used. DNA encoding the partial peptide of the present invention
For example, a DNA having a partial nucleotide sequence of the DNA having the nucleotide sequence of SEQ ID NO: 12 or a nucleotide sequence that hybridizes with the nucleotide sequence of SEQ ID NO: 12 under high stringent conditions A DNA having a partial nucleotide sequence of a DNA encoding a protein having substantially the same activity as the protein of the present invention, a DNA having a partial nucleotide sequence of a DNA having a nucleotide sequence represented by SEQ ID NO: 13, or A DNA having a base sequence hybridizable with the base sequence represented by No. 13 under high stringent conditions, and having a partial base sequence of DNA encoding a protein having substantially the same activity of the present invention, Number: 1
A DNA having a partial nucleotide sequence of the DNA having the nucleotide sequence represented by SEQ ID NO: 4, or a nucleotide sequence hybridizing under high stringent conditions to the nucleotide sequence represented by SEQ ID NO: 14; DNA having a partial base sequence of DNA encoding a protein having substantially the same activity, and D having a partial base sequence of DNA having a base sequence represented by SEQ ID NO: 15
NA or a partial nucleotide sequence of a DNA having a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence represented by SEQ ID NO: 15 and encoding the protein having substantially the same activity of the present invention. Having DN
A, D having the base sequence represented by SEQ ID NO: 16
DNA having a partial base sequence of NA, or SEQ ID NO:
DN having a nucleotide sequence that hybridizes under high stringent conditions to the nucleotide sequence represented by No. 16 and encoding a protein having substantially the same activity of the present invention.
DNA having the partial base sequence of A is used. DNA that can hybridize with the base sequence represented by any one of SEQ ID NOs: 12 to 16 is as follows:
The meaning is as defined above. The same hybridization method and high stringency conditions as described above are used.

More specifically, in the amino acid sequence represented by SEQ ID NO: 1, the 3rd to 25th positions, the 27th to 36th positions,
43-66, 68-86, 92-98, 107-153, 155-168, 172-180, 189-240, 256
262nd, 268th to 275th, 277th to 28th
7th, 295th to 306th, 308th to 332nd, 336th to 347th or (and) 351st to 351st
As a DNA encoding a peptide containing an amino acid sequence having the 377th amino acid sequence, for example, the 7th to 75th nucleotides of the nucleotide sequence represented by SEQ ID NO: 12,
79th to 108th, 127th to 198th, 202nd
258th, 274th to 294th, 319th to 45th
9th, 463th to 504th, 514th to 540th, 565th to 720th, 766th to 786th, 802th to 825th, 829th to 861st, 883th to 883th
918th, 922th to 996th, 1006th to 10th
For example, DNA having the 41st or (and) the 1051 to 1131st nucleotide sequence is used.

D encoding the signal peptide of the present invention
The NA may be any as long as it contains the above-described nucleotide sequence encoding the signal peptide of the present invention. In addition, genomic DNA, genomic DNA library, cDNA derived from the aforementioned cells / tissues, cDNA library derived from the aforementioned cells / tissues, synthetic DN
Any of A may be used. As the DNA encoding the signal peptide of the present invention, for example, a DNA having the base sequence represented by SEQ ID NO: 20 or SEQ ID NO: 2
D having a nucleotide sequence that hybridizes with the nucleotide sequence represented by 0 under high stringent conditions and encoding a peptide capable of exhibiting a function as a signal peptide
NA, a DNA having the nucleotide sequence represented by SEQ ID NO: 21 or a nucleotide sequence hybridizing with the nucleotide sequence represented by SEQ ID NO: 21 under high stringency conditions, and exhibits a function as a signal peptide DNA encoding the peptide to be obtained, DNA having the base sequence represented by SEQ ID NO: 22, or SEQ ID NO:
DNA encoding a peptide having a base sequence that hybridizes with the base sequence represented by No. 22 under high stringency conditions and capable of exhibiting a function as a signal peptide is used. SEQ ID NO: 20 to SEQ ID NO:
Examples of the DNA capable of hybridizing with the nucleotide sequence represented by any one of SEQ ID NOs: 22 include, for example, SEQ ID NO:
A nucleotide sequence having about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more homology with the nucleotide sequence represented by any one of SEQ ID NOS: 20 to 22. DNA or the like is used. The same hybridization method and high stringency conditions as described above are used. More specifically, the DNA encoding the signal peptide of the present invention having the amino acid sequence represented by SEQ ID NO: 9 includes a DNA containing a DNA having the base sequence represented by SEQ ID NO: 20, Examples of the DNA encoding the signal peptide of the present invention having the amino acid sequence represented include a DNA containing the DNA having the nucleotide sequence represented by SEQ ID NO: 21, and SEQ ID NO: 1
As the DNA encoding the signal peptide of the present invention having the amino acid sequence represented by 1, a DNA containing the DNA having the base sequence represented by SEQ ID NO: 22 or the like is used.

A protein of the present invention, a precursor protein,
As a means for cloning a DNA that completely encodes a partial peptide or a signal peptide (hereinafter, in the description of cloning and expression of DNAs encoding these proteins and the like, these proteins and the like are simply abbreviated as the protein of the present invention), PC using synthetic DNA primer having partial base sequence of protein of the present invention
It can be amplified by the R method, or selected by hybridization with DNA incorporated into an appropriate vector and labeled with a DNA fragment or a synthetic DNA encoding a part or the entire region of the protein of the present invention. . The hybridization method is
For example, molecular cloning (Molecular Clon
ing) 2nd (J. Sambrook et al., Cold Spring Harbor
Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. Conversion of the base sequence of DNA can be performed using a known kit,
For example, using MutantTM-G (Takara Shuzo Co., Ltd.), MutantTM-K (Takara Shuzo Co., Ltd.), etc., the Gupped duplex method and Kunkel
The method can be carried out according to a method known per se such as a method or a method analogous thereto. The DNA encoding the cloned protein can be used as it is depending on the purpose, or may be used after digesting with a restriction enzyme or adding a linker, if desired. The DNA has ATG as a translation initiation codon at its 5 'end and TAA, TGA or TGA as a translation stop codon at its 3' end.
It may have AG. These translation initiation codon and translation termination codon can also be added using a suitable synthetic DNA adapter. The expression vector for the protein of the present invention can be prepared, for example, by (A) cutting out a DNA fragment of interest from DNA encoding the protein of the present invention;
It can be produced by ligating the DNA fragment downstream of a promoter in an appropriate expression vector.

As a vector, a plasmid derived from Escherichia coli (eg, pBR322, pBR325, pUC12,
UC13), a plasmid derived from Bacillus subtilis (eg, pUB11)
0, pTP5, pC194), yeast-derived plasmids (eg, pSH19, pSH15), bacteriophages such as λ phage, animal viruses such as retrovirus, vaccinia virus, baculovirus, etc.
A1-11, pXT1, pRc / CMV, pRc / RS
V, pcDNAI / Neo, etc. are used. The promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression. For example, when animal cells are used as hosts, the SRα promoter, SV40 promoter, L
TR promoter, CMV promoter, HSV-TK
Promoters and the like. Of these, CMV
(Cytomegalovirus) promoter, SRα promoter and the like are preferably used. When the host is a bacterium belonging to the genus Escherichia, a trp promoter, a lac promoter, a recA promoter, a λPL promoter,
When the host is a bacterium of the genus Bacillus, the SPO1 promoter, the S7 promoter,
When the host is yeast, such as a PO2 promoter and a penP promoter, a PHO5 promoter, a PGK promoter, a GAP promoter, an ADH promoter, and the like are preferable. When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable.

[0038] In addition to the above, an expression vector containing an enhancer, a splicing signal, a polyA addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori), and the like, if desired, may be used. Can be used. As a selection marker, for example, dihydrofolate reductase (hereinafter, dhfr)
Gene (sometimes abbreviated as “methotrexate (M
TX) resistance], an ampicillin resistance gene (hereinafter referred to as Amp
^r ), a neomycin resistance gene (hereinafter sometimes abbreviated as Neo ^r , G418 resistance), and the like. In particular, when the dhfr gene is used as a selection marker using dhfr gene-deficient Chinese hamster cells, the target gene can be selected using a thymidine-free medium. If necessary, a signal sequence suitable for the host is added to the N-terminal side of the protein of the present invention. When the host is a genus Escherichia, a PhoA signal sequence, an OmpA signal sequence, etc., and when the host is a Bacillus genus, an α-amylase signal sequence, a subtilisin signal sequence, etc., and the host is yeast. In some cases, the MFα signal sequence,
When the host is an animal cell, such as an SUC2 signal sequence, an insulin signal sequence, an α-interferon signal sequence, an antibody molecule / signal sequence, etc. can be used. A transformant can be produced using the vector containing the DNA encoding the protein of the present invention thus constructed.

As the host, for example, Escherichia, Bacillus, yeast, insect cells, insects, animal cells and the like are used. Specific examples of Escherichia bacteria include
For example, Escherichia coli K1
2. DH1 [Procedures of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 60]
Vol. 160 (1968)], JM103 [Nukuirek
Acids Research, (Nucleic Acids Research), 9
309 (1981)], JA 221 [Journal of Molecule
ar Biology)], 120, 517 (1978)], HB
101 [Journal of Molecular Biology, Vol. 41, 459 (1969)], C600 [Genetics, Vol. 39, 440 (1954)] and the like are used. Examples of Bacillus spp. Include, for example, Bacillus subtilis MI114
[Gene, 24, 255 (1983)], 207-21
[Journal of Biochemistry
Biochemistry), 95, 87 (1984)]. As yeast, for example, Saccharomyces
Saccharomyces cerevisiae AH22, A
^{H22R -, NA87-11A, DKD-5D} , 20B
-12, Schizosacchamyces pombe
romyces pombe) NCYC1913, NCYC203
6. Pichia pastoris KM71
Are used.

As insect cells, for example, the virus is A
In the case of cNPV, a cell line derived from night larvae of moth (Spodop
tera frugiperda cell (Sf cell), Trichoplusia ni
MG1 cells from the midgut, H from the eggs of Trichoplusia ni
igh FiveTM cells, cells from Mamestra brassicae or
Estigmena acrea-derived cells and the like are used. When the virus is BmNPV, a silkworm-derived cell line (Bombyx mor
iN cells; BmN cells) and the like. As the Sf cells, for example, Sf9 cells (ATCC CRL1711), Sf9
21 cells (Vaughn, JL et al., In Vivo
ivo), 13, 213-217, (1977)). As insects, for example, silkworm larvae are used [Maeda et al., Nature, 315, 592 (198).
5)]. As animal cells, for example, monkey cells COS-
7, Vero, Chinese hamster cell CHO (hereinafter abbreviated as CHO cell), dhfr gene-deficient Chinese hamster cell CHO (hereinafter, CHO (dhf)
r ⁻ ) cells), mouse L cells, mouse AtT-2
0, mouse myeloma cells, rat GH3, human FL cells, and the like. In order to transform Escherichia, for example, Processings of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 69
Vol. 2110 (1972) and Gene, Vol. 17, 1
07 (1982).

For transforming Bacillus sp., For example, Molecular & General Genetics, Vol.
111 (1979). To transform yeast, for example, Methods in Enzymolog
y), 194, 182-187 (1991), Processings of the National Academy of Sciences of the USA (Proc. Na)
Acad. Sci. USA), 75, 1929 (197).
8) and the like. In order to transform insect cells or insects, for example, Bio / Technology, 6, 47-55 (1988))
And the like. To transform animal cells, for example, see Cell Engineering Separate Volume 8, New Cell Engineering Experiment Protocol. 263-267 (1995)
(Published by Shujunsha), Virology, Volume 52,
456 (1973). Thus, the DNA encoding the protein
A transformant transformed with an expression vector containing When culturing a transformant whose host is a genus Escherichia or Bacillus, a liquid medium is suitable as a medium used for the cultivation. Nitrogen sources, inorganic substances, etc. are contained. As a carbon source, for example, glucose, dextrin, soluble starch, sucrose, etc.As a nitrogen source, for example, ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal, potato extract and the like Examples of the inorganic or organic substance and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride. In addition, yeast, vitamins, growth promoting factors and the like may be added. The pH of the medium is preferably about 5 to 8.

As a medium for culturing the genus Escherichia, for example, M9 medium containing glucose and casamino acid [Miller, Journal of Experiments in Molecular Genetics (Journa)
l of Experiments in Molecular Genetics), 431-
433, Cold Spring Harbor Laboratory, New York 1
972] is preferred. If necessary, an agent such as 3β-indolylacrylic acid can be added in order to make the promoter work efficiently. When the host is a bacterium belonging to the genus Escherichia, the cultivation is usually performed at about 15 to 43 ° C. for about 3 to 24 hours, and if necessary, aeration and stirring may be added. When the host is a bacterium belonging to the genus Bacillus, the cultivation is usually carried out at about 30 to 40 ° C. for about 6 to 24 hours, and if necessary, aeration and stirring can be added. When culturing a transformant whose host is yeast, for example, a Burkholder minimum medium [Bostian, KL
Processing of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 77, 450
5 (1980)] or an SD medium containing 0.5% casamino acid [Bitter, GA et al., Processings of the.
National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. US
A), 81, 5330 (1984)].
Preferably, the pH of the medium is adjusted to about 5-8. The cultivation is usually performed at about 20 ° C. to 35 ° C. for about 24 to 72 hours, and if necessary, aeration or stirring is added. When culturing a transformant in which the host is an insect cell or an insect, the medium used is Grac.
e's Insect Medium (Grace, TCC, Nature (Natur
e), 195, 788 (1962)) to which an additive such as immobilized 10% bovine serum is appropriately added. Medium pH
Is preferably adjusted to about 6.2 to 6.4. The cultivation is usually performed at about 27 ° C. for about 3 to 5 days, and aeration and / or agitation are added as necessary. When culturing a transformant in which the host is an animal cell, the medium may be, for example, a MEM medium containing about 5 to 20% fetal bovine serum [Seience, 12
2, 501 (1952)], DMEM medium [Virology, 8, 396 (1959)], RPMI
1640 medium [Journal of the American
Medical Association (The Jounal of the Am
erican Medical Association) 199, 519 (19
67)], 199 medium [Proceeding of the Society for the Biological Med.
icine), Vol. 73, 1 (1950)].
Preferably, the pH is between about 6-8. Culture is usually about 30
The reaction is carried out at a temperature of from 40 ° C. to 40 ° C. for about 15 to 60 hours. As described above, the protein of the present invention can be produced on the cell membrane of the transformant.

The protein of the present invention can be separated and purified from the above culture by, for example, the following method. When extracting the protein of the present invention from cultured cells or cells, after culturing, the cells or cells are collected by a known method, suspended in an appropriate buffer, and subjected to sonication, lysozyme and / or freeze-thawing. After the cells or cells are destroyed by the method, a method of obtaining a crude extract of the protein by centrifugation or filtration is used as appropriate. The buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 ^™ . When the protein is secreted into the culture solution, after completion of the culture, the supernatant is separated from the cells or cells by a method known per se, and the supernatant is collected. The protein contained in the culture supernatant or extract obtained in this manner can be purified by appropriately combining known separation and purification methods. These known separation and purification methods include methods using solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SD.
A method using mainly a difference in molecular weight such as S-polyacrylamide gel electrophoresis, a method using a difference in charge such as ion exchange chromatography, a method using specific affinity such as affinity chromatography,
A method using a difference in hydrophobicity such as reversed phase high performance liquid chromatography, a method using a difference in isoelectric point such as isoelectric focusing, and the like are used.

When the protein thus obtained is obtained in a free form, it can be converted to a salt by a method known per se or a method analogous thereto, and conversely, when the protein is obtained in a salt, a method known per se or It can be converted to a free form or another salt by a method analogous thereto. The protein produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by applying an appropriate protein-modifying enzyme before or after purification. As the protein modifying enzyme, for example, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used. The presence or activity of the thus-produced protein of the present invention or a salt thereof can be measured by a binding experiment with a labeled ligand, an enzyme immunoassay using a specific antibody, or the like.

A protein of the present invention, a precursor protein,
The antibody against the partial peptide or a salt thereof may be any of a polyclonal antibody and a monoclonal antibody as long as it can recognize the protein, precursor protein, partial peptide or a salt thereof of the present invention. An antibody against the protein, the precursor protein, the partial peptide or a salt thereof (hereinafter, these proteins and the like are simply abbreviated as the protein of the present invention) using the protein of the present invention as an antigen, It can be produced according to a method for producing an antibody or antiserum known per se. [Preparation of Monoclonal Antibody] (a) Preparation of Monoclonal Antibody-Producing Cell The protein of the present invention is administered to a warm-blooded animal by itself or together with a carrier and a diluent at a site where the antibody can be produced by administration. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. The administration is usually performed once every 2 to 6 weeks, about 2 to 10 times in total. Examples of the warm-blooded animal used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, and chickens, and mice and rats are preferably used. When preparing monoclonal antibody-producing cells, a warm-blooded animal immunized with an antigen, for example, an individual having an antibody titer is selected from a mouse, and the spleen or lymph node is collected 2 to 5 days after the final immunization and contained in the spleen or lymph node. By fusing the antibody-producing cells thus obtained with myeloma cells of the same or different species, a monoclonal antibody-producing hybridoma can be prepared. The antibody titer in the antiserum can be measured, for example, by reacting a labeled protein described below with the antiserum, and then measuring the activity of a labeling agent bound to the antibody. The fusion operation is performed by known methods, for example, the method of Kohler and Milstein [Nature, 256, 495 (1
975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, and PEG is preferably used.

Examples of myeloma cells include NS-1,
Examples include warm-blooded animal myeloma cells such as P3U1, SP2 / 0, and AP-1, but P3U1 is preferably used. The preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1,
PEG (preferably PEG1000-PEG6000)
Is added at a concentration of about 10 to 80%,
Preferably, cell fusion can be carried out efficiently by incubating at 30 to 37 ° C for 1 to 10 minutes. Various methods can be used to screen for monoclonal antibody-producing hybridomas. For example, a hybridoma culture supernatant is added to a solid phase (eg, a microplate) on which a protein antigen is adsorbed directly or together with a carrier, and then radioactive substances or An anti-immunoglobulin antibody labeled with an enzyme or the like (if the cell used for cell fusion is a mouse, an anti-mouse immunoglobulin antibody is used) or protein A
A method for detecting a monoclonal antibody bound to a solid phase, adding a hybridoma culture supernatant to a solid phase to which an anti-immunoglobulin antibody or protein A is adsorbed, adding a protein labeled with a radioactive substance or an enzyme, and A method for detecting a monoclonal antibody bound to a phase is exemplified. The selection of the monoclonal antibody can be performed according to a method known per se or a method analogous thereto. Normal H
It can be performed in a medium for animal cells to which AT (hypoxanthine, aminopterin, thymidine) is added. As a selection and breeding medium, any medium can be used as long as a hybridoma can grow. For example, 1
RPMI 1640 medium containing 10-20%, preferably 10-20% fetal calf serum, GIT medium containing 1-10% fetal calf serum (Wako Pure Chemical Industries, Ltd.) or serum-free medium for hybridoma culture (SFM) -101, Nissui Pharmaceutical Co., Ltd.) can be used. The culturing temperature is usually 20 to 40 ° C, preferably about 37 ° C. The culturing time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks. The culture can be usually performed under 5% carbon dioxide. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.

(B) Purification of Monoclonal Antibody Separation and purification of the monoclonal antibody can be performed by a method known per se, for example, a separation and purification method of immunoglobulin [eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis Method, adsorption and desorption with an ion exchanger (eg, DEAE), ultracentrifugation, gel filtration, antigen-bound solid phase or an active adsorbent such as protein A or protein G to collect only antibodies and dissociate the bond. Specific Purification Method for Obtaining Antibody].

[Preparation of Polyclonal Antibody] The polyclonal antibody of the present invention can be produced according to a method known per se or a method analogous thereto. For example, an immunizing antigen (protein antigen) itself or a complex thereof with a carrier protein is formed, and immunization is performed on a warm-blooded animal in the same manner as in the above-described method for producing a monoclonal antibody. It can be produced by collecting a substance and separating and purifying the antibody. Regarding the complex of an immunizing antigen and a carrier protein used to immunize a warm-blooded animal, the type of the carrier protein and the mixing ratio of the carrier and the hapten are such that the antibody is efficiently cross-linked to the hapten immunized by cross-linking the carrier. If possible, any material may be cross-linked at any ratio.
On the other hand, a method of coupling at a rate of about 0.1 to 20, preferably about 1 to 5 is used. Various coupling agents can be used for coupling the hapten and the carrier. For example, glutaraldehyde, carbodiimide, a maleimide active ester, an active ester reagent containing a thiol group or a dithioviridyl group, or the like is used. The condensation product is administered to a warm-blooded animal itself or together with a carrier or diluent at a site where antibody production is possible. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. Administration is usually once every about 2 to 6 weeks, for a total of about 3 to 1
It is performed about 0 times. The polyclonal antibody can be collected from the blood, ascites, etc., preferably from the blood of a warm-blooded animal immunized by the above method. The measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the measurement of the antibody titer in the antiserum described above. The separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as the above-described separation and purification of the monoclonal antibody.

[0049] DNAs encoding the protein, the precursor protein or the partial peptide of the present invention (hereinafter, these DNAs are abbreviated as the DNA of the present invention in the description of antisense DNA) or are substantially complementary thereto. As an antisense DNA having a complementary base sequence, any antisense DNA having a base sequence complementary or substantially complementary to the DNA of the present invention and having an action capable of suppressing the expression of the DNA can be used. And any antisense DNA. The nucleotide sequence substantially complementary to the DNA of the present invention is, for example, about 70% of the total nucleotide sequence or a partial nucleotide sequence of the nucleotide sequence complementary to the DNA of the present invention (that is, the complementary strand of the DNA of the present invention). % Or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 9% or more.
A base sequence having a homology of 5% or more is exemplified.
In particular, of the entire base sequence of the complementary strand of the DNA of the present invention, the base sequence of the portion encoding the N-terminal portion of the protein of the present invention (for example, a base sequence near the start codon, etc.) is at least about 70% or more of the complementary strand. An antisense DNA having a homology of preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more is suitable. These antisense DNAs can be produced using a known DNA synthesizer or the like.

A protein of the present invention, a precursor protein,
The partial peptide or a salt thereof has an LCAT-like activity, transfers an acyl group (fatty acid) at the β-position of lecithin (phosphatidylcholine) to a 3β-OH group of cholesterol, and equimolar lecithin and non-ester cholesterol. To produce the same moles of cholesterol ester and lysolecithin. In the following, a protein, a precursor protein, a partial peptide or a salt thereof (hereinafter, may be abbreviated as the protein of the present invention) of the present invention, a DNA encoding the protein, the precursor protein or the partial peptide of the present invention ( Hereinafter, the DNA of the present invention
), Antibodies against the protein, the precursor protein, the partial peptide or a salt thereof of the present invention (hereinafter sometimes abbreviated as the antibody of the present invention), and the use of antisense DNA.

(1) Therapeutic / prophylactic agent for various diseases related to the protein of the present invention LCAT contributes to cholesterol metabolism.
When DNA encoding CAT is abnormal or defective or LCAT expression level is decreased, for example, arteriosclerosis, hyperlipidemia, hypercalorie, obesity, aging, brain disease And various diseases such as renal disorder and hypertriglyceridemia. Therefore, the protein and the like of the present invention and the DNA of the present invention include, for example, arteriosclerosis, hyperlipidemia, hypercalorie, obesity, aging, brain disease,
It can be used as a medicament such as an agent for treating or preventing various diseases such as renal disorder and hypertriglyceridemia. For example, because LCAT is reduced or deficient in vivo, cholesterol metabolism in cells is sufficient,
Or, if there is a patient who does not work properly,
By administering the DNA of the present invention to the patient and expressing the protein or the like of the present invention in vivo, (b) inserting the DNA of the present invention into cells and expressing the protein or the like of the present invention, The role of the protein or the like of the present invention in the patient can be sufficiently or normally exerted by transplanting the cells into the patient or (c) administering the protein or the like of the present invention to the patient. . When the DNA of the present invention is used as the above-mentioned therapeutic / prophylactic agent, the DNA may be inserted alone or into a suitable vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, and the like, followed by a conventional method. It can be administered to humans or warm-blooded animals. The DNA of the present invention, as it is,
Alternatively, it can be formulated with a physiologically acceptable carrier such as an auxiliary for promoting ingestion, and administered by a gene gun or a catheter such as a hydrogel catheter. When the protein or the like of the present invention is used as the above therapeutic / prophylactic agent, at least 90%, preferably 95% or more,
It is more preferable to use those that have been purified to 98% or more, more preferably 99% or more.

The proteins and the like of the present invention include, for example, sugar-coated tablets, capsules, elixirs,
It can be used orally as microcapsules or the like, or parenterally in the form of injections such as sterile solutions or suspensions with water or other pharmaceutically acceptable liquids. For example, the protein or the like of the present invention is mixed with physiologically acceptable carriers, flavors, excipients, vehicles, preservatives, stabilizers, binders, and the like in the unit dosage form generally required for the practice of the formulation. Can be manufactured. The amount of the active ingredient in these preparations is such that an appropriate dose in the specified range can be obtained. Examples of additives that can be mixed with tablets, capsules, and the like include binders such as gelatin, corn starch, tragacanth, and acacia, excipients such as crystalline cellulose, corn starch, gelatin, and alginic acid. Useful bulking agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, reddish oil or cherry and the like are used. When the preparation unit form is a capsule, a liquid carrier such as oil and fat can be further contained in the above-mentioned type of material. Sterile compositions for injection can be formulated according to normal pharmaceutical practice such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil, coconut oil and the like. Aqueous liquids for injection include, for example, physiological saline, isotonic solutions containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.), and suitable solubilizing agents. For example, it may be used in combination with an alcohol (eg, ethanol, etc.), a polyalcohol (eg, propylene glycol, polyethylene glycol, etc.), a nonionic surfactant (eg, Polysorbate 80 ™, HCO-50, etc.). Examples of the oily liquid include sesame oil and soybean oil, and may be used in combination with a solubilizing agent such as benzyl benzoate or benzyl alcohol. In addition, buffers (eg, phosphate buffer, sodium acetate buffer, etc.), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.), Preservatives (for example,
Benzyl alcohol, phenol, etc.), antioxidants and the like. The prepared injection is usually filled in an appropriate ampoule. The vector into which the DNA of the present invention has been inserted is also formulated in the same manner as described above, and is usually used parenterally.

The preparations thus obtained are safe and have low toxicity, and are, for example, human or warm-blooded animals (eg, rat, mouse, guinea pig, rabbit, bird, sheep, pig, cow, horse, cat, Dogs, monkeys, chimpanzees, etc.). The dose of the protein or the like of the present invention varies depending on the target disease, the subject of administration, the administration route, and the like. 60 kg), about 0.1 mg to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg of the protein or the like per day.
Administer. In the case of parenteral administration, the single dose of the protein or the like varies depending on the administration subject, the target disease, and the like. (As a body weight of 60 kg), the protein or the like should be administered in an amount of about 0.01 to about
It is convenient to administer about 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg by injecting into the affected area. In the case of other animals, the dose can be administered in terms of 60 kg.

(2) Screening of drug candidate compounds for diseases Since the protein of the present invention has LCAT-like activity,
Compounds or salts thereof that promote the function of the protein or the like (eg, LCAT-like activity) of the present invention include arteriosclerosis, hyperlipidemia, hypercalorie, obesity, aging, brain disease, renal impairment, and high triglyceride. It can be used as a medicament such as an agent for treating or preventing diseases such as diseases (preferably, it can be used as an agent for treating or preventing arteriosclerosis, hyperlipidemia, obesity, aging, cerebral disease, renal disorder, etc.). On the other hand, a compound or a salt thereof that inhibits the function of the protein or the like of the present invention has low nutrition,
It can be used as a medicament such as an agent for treating or preventing β-lipoproteinemia, inflammatory diseases, Tangier's disease and the like. Therefore,
The protein or the like of the present invention is useful as a reagent for screening a compound or a salt thereof that promotes or inhibits the function of the protein or the like of the present invention. That is, the present invention provides (1) a function of the protein of the present invention, its partial peptide or a salt thereof (for example, LCAT-like activity and the like), which comprises using the protein of the present invention, its partial peptide or a salt thereof. Or a salt thereof (hereinafter sometimes abbreviated as an accelerator), or a compound inhibiting the function of the protein, its partial peptide or a salt thereof of the present invention (hereinafter sometimes abbreviated as an inhibitor) And (2) (i) contacting the protein of the present invention, its partial peptide or a salt thereof with lecithin and non-ester cholesterol, and (ii) Lecithin, non-ester cholesterol and a test compound are added to the protein of the present invention, its partial peptide or a salt thereof. Possible to compare the case where is catalyst provides a screening method of promoters or inhibitors, characterized in. Specifically, in the screening method, for example,
It is characterized in that the LCAT-like activities of the protein or the like of the present invention in the cases (i) and (ii) are measured and compared.

As lecithin, for example, commercially available egg white lecithin (Sigma) is used. As the non-ester cholesterol, for example, [ ¹⁴ C] -labeled cholesterol (Amersham) is used. In the screening method of the present invention, it is preferable to use a proteoliposome solution containing these lecithin and non-ester cholesterol. The proteoribosome solution comprises:
It contains apoA-1, [ ¹⁴ C] cholesterol and egg white lecithin in a molar ratio of 0.8: 12.5: 250. Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like. Or a known compound. To carry out the above-mentioned screening method, a sample such as the protein of the present invention is prepared by suspending the protein or the like of the present invention in a buffer suitable for screening. PH buffer
Any buffer that does not inhibit the reaction between the protein or the like of the present invention and lecithin and non-ester cholesterol, such as a phosphate buffer having a pH of about 4 to 10 (preferably, a pH of about 6 to 8) and a Tris-HCl buffer. Good.

The LCAT-like activity of the protein or the like of the present invention can be determined by a method known per se, for example, lipoprotein analysis (Converse, CA and Skinner, RE, eds., 1992).
187 of the year, IRL Press, Oxford)
To 201 (by Gillett, MPT and Owen, JS) or a method analogous thereto. For example, LCA in case (ii) above
T-like activity is about 20% or more compared to the case of the above (i),
Preferably, a test compound which increases the test compound by at least 30% or more, more preferably about 50% or more by LCAT such as the protein of the present invention.
On the other hand, as a compound which promotes the activity similar to the above, the LCAT-like activity in the case of the above (ii) is about 20% or more, preferably 30% or more, more preferably about 50% or more compared with the case of the above (i). A test compound that inhibits can be selected as a compound that inhibits LCAT-like activity such as the protein of the present invention.

The screening kit of the present invention contains a protein, a precursor protein, a partial peptide or a salt thereof of the present invention. Examples of the screening kit of the present invention include the following. [Reagent for screening] Measurement buffer pH 7.4 Tris-HCl buffer, human serum albumin Protein preparation Protein and precursor protein of the present invention. Partial peptide or salt thereof Proteoliposome solution [ ¹⁴ C] cholesterol (10 ⁵ cpm / ml), ApoA-1 / [ ¹⁴ C] cholesterol / egg lecithin (molar ratio 0.8: 12.5: 250) Thin layer chromatography [Measurement method] LCAT-like activity can be measured by using the protein solution of the present invention and a proteoliposome solution. For example, the proteoliposome solution contains apo A-1 and [ ¹⁴ C] cholesterol and egg white lecithin in a molar ratio of 0.1.
It can be obtained by mixing at 8: 12.5: 250 and incubating at 37 ° C. for 30 minutes. After incubating the protein solution of the present invention with the proteoliposome and the test compound for 1 hour at 37 ° C., the formed cholesterol ester was subjected to thin layer chromatography using a developing solution hexane / diethyl ether / acetic acid (83: 16: 1). After isolation, measure. The results can be expressed in nanomoles of free esterified cholesterol per hour.

The compounds or salts thereof obtained by using the screening method or the screening kit of the present invention may be any of the test compounds described above, for example, peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, It is a compound selected from plant extracts, animal tissue extracts, plasma, and the like, and is a compound that promotes or inhibits the function of the protein or the like of the present invention (eg, LCAT-like activity and the like). As the salt of the compound, those similar to the aforementioned salts of the protein of the present invention are used. Compounds that promote the function of the protein or the like (eg, LCAT-like activity) of the present invention include, for example, arteriosclerosis, hyperlipidemia, hypercalorie, obesity, aging, brain disease, renal disorder, triglyceridosis, etc. It can be used as a medicine such as a therapeutic / prophylactic agent for diseases. On the other hand, compounds that inhibit the function of the protein or the like of the present invention are useful as medicaments such as therapeutic or preventive agents for diseases such as malnutrition, β-lipoproteinemia, inflammatory diseases, and Tangier's disease.

When a compound obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned therapeutic / prophylactic agent, it can be carried out according to conventional means. For example, tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions and the like can be prepared in the same manner as in the above-mentioned medicine containing the protein of the present invention. The preparations obtained in this way are safe and low toxic, for example, human or warm-blooded animals (eg, mouse, rat, rabbit, sheep, pig, cow, horse, bird, cat, dog, monkey, chimpanzee, etc.) ) Can be administered. The dose of the compound or a salt thereof depends on its action, target disease, subject to be administered,
Although there is a difference depending on the administration route and the like, for example, when a compound that promotes the function of the protein or the like of the present invention is orally administered for the purpose of treating hyperlipidemia, generally, an adult (body weight 60 kg) is used.
)), The compound is used in an amount of about 0.1 to about 0.1 per day.
Administer 100 mg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg. When administered parenterally, a single dose of the compound may vary depending on the administration subject, target disease, and the like. For example, a compound that promotes the function of the protein or the like of the present invention for the purpose of treating hyperlipidemia may be used. When usually administered to adults (as 60 kg) in the form of injections,
About 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 20 mg of the compound per day.
It is convenient to administer about 1 to 10 mg by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg. On the other hand, when a compound that inhibits the function of the protein or the like of the present invention is orally administered for the purpose of treating β-lipoproteinemia, generally an adult (body weight 60 kg) is used.
)), The compound is used in an amount of about 0.1 to about 0.1 per day.
Administer 100 mg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease and the like, but, for example, inhibits the function of the protein of the present invention for the purpose of treating β-lipoproteinemia. When the compound is usually administered to an adult (as 60 kg) in the form of an injection, about 0.01 to 30 mg of the compound per day is administered.
It is convenient to administer intravenously, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg. In the case of other animals, the dose can be administered in terms of 60 kg.

(3) Quantification of the protein of the present invention, its partial peptide, or a salt thereof An antibody against the protein or the like of the present invention (hereinafter sometimes abbreviated as the antibody of the present invention) is specific for the protein or the like of the present invention. Therefore, it can be used for quantification of the protein of the present invention in a test solution, particularly for quantification by sandwich immunoassay. That is, the present invention provides (i) a method of competitively reacting an antibody of the present invention with a test solution and a labeled protein of the present invention, and the like. A method for quantifying the protein or the like of the present invention in a test solution, which comprises measuring the ratio of the test solution, and (ii) a method for separating the test solution from the antibody of the present invention insolubilized on a carrier and the labeled present invention. A method for quantifying the protein or the like of the present invention in a test solution, comprising reacting the antibody of the present invention with the antibody simultaneously or continuously, and then measuring the activity of the labeling agent on the insolubilized carrier. (Ii) above
In the quantitative method, it is desirable that one antibody is an antibody that recognizes the N-terminal of the protein or the like of the present invention, and the other antibody is an antibody that reacts with the C-terminal of the protein or the like of the present invention.

In addition to the quantification of the protein of the present invention using a monoclonal antibody against the protein of the present invention (hereinafter sometimes referred to as the monoclonal antibody of the present invention), detection by tissue staining or the like is performed. Can also. For these purposes, the antibody molecule itself may be used, or F (ab ') 2, Fab', or Fab fraction of the antibody molecule may be used. The method for quantifying the protein or the like of the present invention using the antibody of the present invention is not particularly limited. Any method can be used as long as it is a method for detecting the amount of the compound by chemical or physical means and calculating it from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephelometry, competition method, immunometric method and sandwich method are preferably used,
In terms of sensitivity and specificity, it is particularly preferable to use a sandwich method described later. As a labeling agent used in a measurement method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like are used. Examples of radioisotopes include [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [
¹⁴ C]. As the above enzyme, a stable enzyme having a large specific activity is preferable. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, a luminol derivative, luciferin, lucigenin and the like are used. Further, a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.

For insolubilization of the antigen or antibody, physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing and immobilizing proteins or enzymes may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose; synthetic resins such as polystyrene, polyacrylamide, and silicon; and glass. In the sandwich method, the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further reacted with another labeled monoclonal antibody of the present invention (secondary reaction). By measuring the activity of the labeling agent, the amount of the protein of the present invention in the test solution can be determined. The primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at staggered times. The labeling agent and the method of insolubilization can be in accordance with those described above. In addition, in the immunoassay by the sandwich method,
The antibody used for the solid phase antibody or the labeling antibody is not necessarily one kind, and a mixture of two or more kinds of antibodies may be used for the purpose of improving measurement sensitivity and the like. In the method for measuring the protein or the like of the present invention by the sandwich method of the present invention, the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction is preferably an antibody having a different binding site to the protein or the like of the present invention. It is. That is, the antibody used in the primary reaction and the secondary reaction are preferably used, for example, when the antibody used in the secondary reaction recognizes the C-terminal of the protein or the like of the present invention. Is an antibody that recognizes other than the C-terminal, for example, the N-terminal.

The monoclonal antibody of the present invention can be used in a measurement system other than the sandwich method, for example, a competition method, an immunometric method, or a nephrometry. In the competition method, an antigen in a test solution and a labeled antigen are allowed to react competitively with an antibody, and then the unreacted labeled antigen is reacted.
(F) and the labeled antigen (B) bound to the antibody are separated (B / F separation), the amount of labeling of either B or F is measured, and the amount of antigen in the test solution is quantified. In this reaction method, a soluble antibody is used as the antibody, B / F separation is performed using polyethylene glycol, a liquid phase method using a second antibody against the antibody, or a solid phase antibody is used as the first antibody, or A solid-phase method using a soluble first antibody and a solid-phased antibody as the second antibody is used. In the immunometric method, an antigen in a test solution and a solid-phased antigen are subjected to a competitive reaction with a fixed amount of a labeled antibody, and then the solid phase and the liquid phase are separated. Is reacted with an excess amount of the labeled antibody, and then the immobilized antigen is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated.
Next, the amount of label in any phase is measured to determine the amount of antigen in the test solution. In nephelometry, the amount of insoluble precipitates generated as a result of an antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test solution is small and only a small amount of sediment is obtained, laser nephrometry utilizing laser scattering is preferably used.

In applying these individual immunoassays to the quantification method of the present invention, no special conditions, operations, and the like need to be set. What is necessary is just to construct the measuring system of the protein etc. of the present invention by adding ordinary technical considerations of those skilled in the art to ordinary conditions and operation methods in each method. For details of these general technical means, reference can be made to reviews, books, and the like. For example, Hiroe Irie, "Radio Immunoassay" (Kodansha, published in 1974), Hiroshi Irie, "Radio Immunoassay" (Kodansha, published in 1979), Eiji Ishikawa et al., "Enzyme Immunoassay" (Medical Shoin, Showa Ed. Ishikawa et al., “Enzyme Immunoassay” (2nd edition) (Issue Shoin, 1982), Eiji Ishikawa et al. “Enzyme Immunoassay” (Third Edition), 3rd Ed. Published in 1962, "Methods in ENZYMOLOGY" Vol. 70 (Immunochem
ical Techniques (Part A)), ibid.Vol. 73 (Immunochem
ical Techniques (Part B)), ibid.Vol. 74 (Immunochem
ical Techniques (Part C)), ibid.Vol. 84 (Immunochem
ical Techniques (Part D: Selected Immunoassays)),
Ibid.Vol. 92 (Immunochemical Techniques (Part E: Mono
clonal Antibodies and General Immunoassay Method
s)), ibid.Vol. 121 (Immunochemical Techniques (Part
I: Hybridoma Technology and Monoclonal Antibodie
s)) (above, published by Academic Press). As described above, the protein and the like of the present invention can be quantified with high sensitivity by using the antibody of the present invention. Furthermore, by quantifying the concentration of the protein of the present invention using the antibody of the present invention,
(1) When a decrease in the concentration of the protein or the like of the present invention is detected, for example, arteriosclerosis, hyperlipidemia, hypercalorie,
Obesity, aging, brain disease, renal disorder, hypertriglyceridemia or other diseases, or the possibility of future disease is high; For example, malnutrition, beta-lipoproteinemia,
It can be diagnosed as a disease such as an inflammatory disease, Tangier's disease, or is likely to be affected in the future. Also,
The antibody of the present invention can be used for detecting the protein or the like of the present invention present in a subject such as a body fluid or a tissue. In addition, preparation of an antibody column used for purifying the protein of the present invention, detection of the protein of the present invention in each fraction at the time of purification, analysis of the behavior of the protein of the present invention in test cells, and the like Can be used for

(4) Gene Diagnostic Agent The DNA of the present invention can be used, for example, as a probe to produce a human or warm-blooded animal (eg, rat, mouse, guinea pig, rabbit, bird, sheep, pig, cow,
DNA or mRNA abnormalities (genetic abnormalities) encoding the protein of the present invention or its partial peptide in horses, cats, dogs, monkeys, chimpanzees, etc.) can be detected.
It is useful as an agent for genetic diagnosis such as damage, mutation or decreased expression of DNA, and increase or excessive expression of the DNA or mRNA. The above-mentioned genetic diagnosis using the DNA of the present invention can be performed, for example, by the known Northern hybridization or PCR-SSCP method (Genomics (Genomics)).
s), Volume 5, pp. 874-879 (1989), Proceedings of the National Academy of Sciences of USA (Proceeding)
s ofthe Natinal Academy of Sciences of the United
States of America), Vol. 86, 2766-2770
(1989)).
For example, when a decrease in expression is detected by Northern hybridization or when DNA is
If a mutation is detected, for example, arteriosclerosis,
It can be diagnosed that there is a high possibility that the disease is hyperlipidemia, hypercalorie, obesity, aging, brain disease, renal disorder, hypertriglyceridemia and the like. On the other hand, when overexpression is detected by Northern hybridization, for example, malnutrition, β-lipoproteinemia, inflammatory disease, Tangie
It can be diagnosed that there is a high possibility of a disease such as r disease.

(5) Pharmaceuticals Containing Antisense DNA The antisense DNA capable of complementarily binding to the DNA of the present invention and suppressing the expression of the DNA is a protein or the like of the present invention in a living body or the present invention. Can be used as a therapeutic / prophylactic agent for, for example, undernutrition, β-lipoproteinemia, inflammatory disease, Tangier's disease and the like. Antisense D above
When NA is used as the above-mentioned therapeutic / prophylactic agent, it can be carried out in the same manner as the aforementioned therapeutic / prophylactic agent for various diseases containing the DNA of the present invention. For example, when the antisense DNA is used, the method can be carried out according to a conventional method after inserting the antisense DNA alone or into an appropriate vector such as a retrovirus vector, an adenovirus vector, and an adenovirus associated virus vector. The antisense DNA can be administered as it is or in the form of a formulation together with a physiologically acceptable carrier such as an adjuvant for promoting uptake, and administered with a gene gun or a catheter such as a hydrogel catheter. Furthermore, the antisense DNA can also be used as a diagnostic oligonucleotide probe for examining the presence or expression of the DNA of the present invention in tissues or cells.

(6) Pharmaceutical Containing Antibody of the Present Invention The antibody of the present invention, which has the activity of neutralizing the activity of the protein or the like of the present invention, includes, for example, malnutrition, β-lipoproteinemia,
It can be used as a medicine such as an agent for treating or preventing various diseases such as Tangier disease. The therapeutic / prophylactic agent for the above-mentioned diseases containing the antibody of the present invention can be used directly as a liquid or as a pharmaceutical composition in an appropriate dosage form as a human or mammal (eg, rat, rabbit, sheep, pig, cow, cat, Dogs, monkeys, etc.) can be administered orally or parenterally. The dose varies depending on the administration subject, target disease, symptoms, administration route, and the like. For example, when used for treatment or prevention of β-lipoproteinemia in adults, the antibody of the present invention is administered once. As an amount, usually 0.01 to
About 20 mg / kg body weight, preferably 0.1 to 10 mg
/ Kg body weight, more preferably 0.1 to 5 mg / k
g about 1 to 5 times a day, preferably 1 to 5 times a day
It is convenient to administer by intravenous injection about three times.
In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If the symptoms are particularly severe, the dose may be increased according to the symptoms. The antibodies of the present invention can be administered as such or as a suitable pharmaceutical composition. The pharmaceutical composition used for the above administration contains the above or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient. Such compositions are provided in dosage forms suitable for oral or parenteral administration. That is,
For example, compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, and capsules (including soft capsules). Syrup, emulsion, suspension and the like. Such a composition is produced by a method known per se and contains a carrier, diluent or excipient commonly used in the field of pharmaceuticals. For example, carriers for tablets, excipients include lactose, starch,
Sucrose, magnesium stearate and the like are used.

Examples of the composition for parenteral administration include injections, suppositories and the like. Injections include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections and the like. In the dosage form. Such injections are prepared according to a method known per se, for example, by dissolving, suspending or emulsifying the antibody or a salt thereof in a sterile aqueous or oily liquid commonly used for injections. As an aqueous liquid for injection, for example, physiological saline, isotonic solution containing glucose and other auxiliary agents and the like are used, and a suitable solubilizing agent,
For example, alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant [eg, polysorbate 80,
HCO-50 (polyoxyethylene (50mol) adduct of
hydrogenated castor oil)].
As the oily liquid, for example, sesame oil, soybean oil, and the like are used, and benzyl benzoate, benzyl alcohol, and the like may be used in combination as a solubilizing agent. The prepared injection is usually filled in an appropriate ampoule. Suppositories to be used for rectal administration are prepared by mixing the above antibody or a salt thereof with a conventional suppository base. The above-mentioned oral or parenteral pharmaceutical compositions are conveniently prepared in dosage unit form so as to be compatible with the dosage of the active ingredient.
Examples of such dosage unit dosage forms include tablets, pills, capsules, injections (ampoules), suppositories and the like, and usually 5 to 500 mg, especially 5 to 100 mg for each dosage unit, 10-2 for other dosage forms
Preferably, it contains 50 mg of the above antibody.
Each of the above-mentioned compositions may contain another active ingredient as long as the composition does not cause an undesirable interaction with the above-mentioned antibody.

(7) DNA-Transferred Animal The present invention relates to a DNA encoding the exogenous protein of the present invention (hereinafter abbreviated as the exogenous DNA of the present invention) or a mutant DNA thereof (the exogenous mutant DNA of the present invention) Which may be abbreviated as such). That is, the present invention provides (1) a non-human mammal having the exogenous DNA of the present invention or a mutant DNA thereof, (2) the animal according to (1), wherein the non-human mammal is a rodent, (3)
(2) The animal according to (2), wherein the rodent is a mouse or a rat; and (4) a recombinant vector containing the exogenous DNA of the present invention or a mutant DNA thereof and capable of being expressed in a mammal. . Exogenous DNA of the present invention
Or a non-human mammal having the mutant DNA thereof (hereinafter, referred to as
The DNA-transferred animal of the present invention) is preferably used for non-fertilized eggs, fertilized eggs, germ cells including spermatozoa and their progenitor cells, etc., preferably at the stage of embryo development in non-human mammal development (more preferably Is the single cell or fertilized egg cell stage and generally before the 8 cell stage), calcium phosphate method, electric pulse method, lipofection method, aggregation method, microinjection method, particle gun method, DE
It can be produced by transferring the target DNA by the AE-dextran method or the like. In addition, the D
By the NA transfer method, the exogenous DNA of the present invention can be transferred to somatic cells, organs of living organisms, tissue cells, and the like, and can be used for cell culture, tissue culture, and the like. The DNA-transferred animal of the present invention can also be produced by fusing cells with a cell fusion method known per se.

Examples of the non-human mammal include, for example, bovine,
Pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, mice, rats and the like are used. Above all, rodents, which are relatively short in ontogeny and biological cycle in terms of preparation of disease animal model systems, and are easy to breed, especially mice (for example, C57 as a pure strain)
As a hybrid line such as BL / 6 line and DBA2 line, B6 line
C3F ₁ system, BDF ₁ system, B6D2F ₁ system, BAL
B / c strain, ICR strain, etc.) or rat (for example,
Wistar, SD, etc.) are preferred. "Mammals" in recombinant vectors that can be expressed in mammals
Examples thereof include humans in addition to the above-mentioned non-human mammals. The exogenous DNA of the present invention refers to the DNA of the present invention once isolated and extracted from a mammal, not the DNA of the present invention originally possessed by a non-human mammal. As the mutant DNA of the present invention, those having a mutation (for example, mutation) in the base sequence of the original DNA of the present invention, specifically, base addition, deletion, substitution with another base, etc. The resulting DNA or the like is used, and also includes abnormal DNA. The abnormal DNA means a DNA that expresses an abnormal protein of the present invention.
NA or the like is used. The exogenous DNA of the present invention may be derived from mammals of the same species or different species from the target animal. In transferring the DNA of the present invention to a target animal, it is generally advantageous to use the DNA as a DNA construct linked downstream of a promoter capable of being expressed in animal cells. For example, when transferring the human DNA of the present invention, various mammals (eg, rabbits,
A DNA construct (eg, vector, etc.) in which the human DNA of the present invention is bound downstream of various promoters capable of expressing DNA derived from dogs, cats, guinea pigs, hamsters, rats, mice, etc .;
For example, a DNA-transferred mammal that highly expresses the DNA of the present invention can be produced by microinjection into a mouse fertilized egg.

Examples of expression vectors for the protein of the present invention include a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis, a plasmid derived from yeast, a bacteriophage such as λ phage, a retrovirus such as Moloney leukemia virus, a vaccinia virus or a baculovirus. Animal viruses and the like are used. Among them, a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis or a plasmid derived from yeast is preferably used. The above DN
Examples of promoters that regulate A expression include, for example,
Promoters of DNAs derived from viruses (eg, Simian virus, cytomegalovirus, Moloney leukemia virus, JC virus, breast cancer virus, poliovirus, etc.), various mammals (human, rabbit, dog, cat,
Guinea pig, hamster, rat, mouse, etc.), for example, albumin, insulin II,
Uroplakin II, elastase, erythropoietin,
Endothelin, muscle creatine kinase, glial fibrillary acidic protein, glutathione S-transferase, platelet-derived growth factor β, keratins K1, K10 and K14, collagen types I and II, cyclic AMP-dependent protein kinase βI subunit, dystrophin, Tartaric acid-resistant alkaline phosphatase,
Atrial natriuretic factor, endothelial receptor tyrosine kinase (generally abbreviated as Tie2), sodium potassium adenosine 3 kinase (Na, K-ATPas)
e), neurofilament light chain, metallothionein I
And IIA, metalloproteinase 1 tissue inhibitor, MHC class I antigen (H-2L), H-ras, renin, dopamine β-hydroxylase, thyroid peroxidase (TPO), polypeptide chain elongation factor 1α (EF-
1α), β actin, α and β myosin heavy chains, myosin light chains 1 and 2, myelin basic protein, thyroglobulin, Thy-1, immunoglobulin, heavy chain variable region (V
NP), serum amyloid P component, myoglobin, troponin C, smooth muscle α-actin, preproenkephalin A, vasopressin and other promoters. Among them, a cytomegalovirus promoter capable of high expression throughout the whole body, a human polypeptide chain elongation factor 1α (EF-1α) promoter, a human and chicken β-actin promoter and the like are preferable.

The above-mentioned vector preferably has a sequence that terminates transcription of a messenger RNA of interest in a DNA-transferred mammal (generally called a terminator). And preferably a Simian virus SV40 terminator or the like. In addition, a splicing signal of each DNA for the purpose of further expressing the desired foreign DNA,
It is also possible to link an enhancer region, a part of an intron of eukaryotic DNA, etc. 5 ′ upstream of the promoter region, between the promoter region and the translation region, or 3 ′ downstream of the translation region. The normal translation region of the protein of the present invention can be derived from liver, kidney, thyroid cells, fibroblast-derived DNA from humans or various mammals (eg, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.) and commercially available DNAs. From the various genomic DNA libraries described above, or as a starting material, a complementary DNA prepared by a known method from RNA derived from liver, kidney, thyroid cells, and fibroblasts. In addition, a translation region obtained by mutating a translation region of a normal protein obtained from the above-described cells or tissues by the point mutagenesis method can be used for the exogenous abnormal DNA. The translation region can be prepared as a DNA construct that can be expressed in a transgenic animal by a conventional DNA engineering technique in which it is ligated downstream of the promoter and, if desired, upstream of the transcription termination site. Transfer of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal. The presence of the exogenous DNA of the present invention in the germinal cells of the produced animal after the DNA transfer means that all the progeny of the produced animal retain the exogenous DNA of the present invention in all of the germ cells and somatic cells. means. The progeny of such animals that have inherited the foreign DNA of the present invention have the foreign DNA of the present invention in all of their germinal and somatic cells.

The non-human mammal to which the exogenous normal DNA of the present invention has been transferred is confirmed to stably maintain the exogenous DNA by mating, and is subcultured as an animal having the DNA in a normal breeding environment. I can do it. Transfer of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in excess in all germ cells and somatic cells of the target mammal. Excessive presence of the exogenous DNA of the present invention in germ cells of a produced animal after DNA transfer means that all offspring of the produced animal have an excessive amount of the exogenous DNA of the present invention in all of their germ cells and somatic cells. Means The offspring of such an animal inheriting the exogenous DNA of the present invention will have the exogenous DNA of the present invention in all of its germ cells and somatic cells.
In excess. By obtaining a homozygous animal having the introduced DNA on both homologous chromosomes and mating the male and female animals, all offspring can be bred to have the DNA in excess. The non-human mammal having the normal DNA of the present invention, in which the normal DNA of the present invention is highly expressed, promotes the function of the endogenous normal DNA, and eventually causes hyperactivity of the protein of the present invention. It may develop and can be used as a disease model animal. For example, using the normal DNA-transferred animal of the present invention, it is possible to elucidate the pathological mechanism of hyperactivity of the protein of the present invention and diseases associated with the protein of the present invention, and to examine a method for treating these diseases. It is possible. In addition, since the mammal into which the exogenous normal DNA of the present invention has been transferred has an increased symptom of the released protein of the present invention, it can be used for screening tests for therapeutic drugs for diseases related to the protein of the present invention. is there.

On the other hand, the non-human mammal having the exogenous abnormal DNA of the present invention should be subcultured in a normal breeding environment as an animal having the DNA after confirming that the exogenous DNA is stably maintained by mating. Can be done. Furthermore, the desired foreign DNA can be incorporated into the above-described plasmid and used as a source material. The DNA construct with the promoter can be prepared by a usual DNA engineering technique. Abnormal D of the present invention at the fertilized egg cell stage
NA transfer is ensured to be present in all germinal and somatic cells of the target mammal. The presence of the abnormal DNA of the present invention in the germinal cells of the produced animal after DNA transfer means that all the offspring of the produced animal have the abnormal DNA of the present invention in all of its germinal and somatic cells. The offspring of such animals that have inherited the exogenous DNA of the present invention have the abnormal DNA of the present invention in all of their germinal and somatic cells. A homozygous animal having the introduced DNA on both homologous chromosomes is obtained, and by crossing these male and female animals, it is possible to breed so that all offspring have the DNA. The non-human mammal having the abnormal DNA of the present invention, in which the abnormal DNA of the present invention is highly expressed, inhibits the function of the endogenous normal DNA, and finally, a functionally inactive form of the protein of the present invention. It may become refractory and can be used as a disease model animal. For example, using the abnormal DNA-transferred animal of the present invention, it is possible to elucidate the pathological mechanism of the function-inactive refractory of the protein of the present invention and to examine a method for treating this disease. In addition, as a specific possibility, an animal in which the abnormal DNA of the present invention is highly expressed can be used to inhibit the function of a normal protein by the abnormal protein of the present invention (dominant negat) in the inactive refractory disease of the protein of the present invention.
ive effect). In addition, since the mammal into which the foreign abnormal DNA of the present invention has been transferred has an increased symptom of the released protein of the present invention, it can be used for a therapeutic drug screening test for inactive refractory disease of the protein of the present invention. It is.

Other possible uses of the above two DNA-transferred animals of the present invention include, for example, use as a cell source for tissue culture, and DNA or R in the tissues of the DNA-transferred animal of the present invention.
Analysis of the relationship with proteins specifically expressed or activated by the protein of the present invention by directly analyzing NA or analyzing protein tissues expressed by DNA; Cultured by standard tissue culture techniques, using these to study the function of cells from tissues that are generally difficult to culture, screening for agents that enhance cell function by using the cells described above, and Isolation and purification of the mutant protein and production of its antibody can be considered. Furthermore, using the DNA-transferred animal of the present invention, it is possible to examine clinical symptoms of a disease related to the protein of the present invention, including a functionally inactive refractory disease of the protein of the present invention, etc. More detailed pathological findings in each organ of a disease model related to the disease can be obtained, which can contribute to the development of a new treatment method, and further to the research and treatment of a secondary disease caused by the disease. In addition, each organ was taken out from the DNA-transferred animal of the present invention, cut into small pieces, and D was released by a protease such as trypsin.
It is possible to obtain NA-transferred cells, culture them, or systematize the cultured cells. Furthermore, it is possible to investigate the specificity of the protein-producing cell of the present invention, its relationship with apoptosis, differentiation or proliferation, or its signal transduction mechanism, and to investigate its abnormalities. It is an effective research material for Furthermore, using the DNA-transferred animal of the present invention, the protein of the present invention includes a functionally inactive refractory disease,
In order to develop therapeutic agents for diseases related to the protein of the present invention, using the above-described test methods and quantitative methods,
It is possible to provide an effective and rapid screening method for the therapeutic agent for the disease. Further, using the transgenic animal of the present invention or the exogenous DNA expression vector of the present invention, a method for treating a DNA associated with the protein of the present invention was investigated.
It is possible to develop.

(8) Knockout Animal The present invention provides a non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated and a non-human mammal deficient in expression of the DNA of the present invention. That is, the present invention provides (1) a non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated,
(2) the embryonic stem cell according to (1), wherein the DNA is inactivated by introducing a reporter gene (eg, a β-galactosidase gene derived from Escherichia coli); (4) The embryonic stem cell according to (1), wherein the non-human mammal is a rodent, (5) the embryonic stem cell according to (4), wherein the rodent is a mouse, 6) a non-human mammal deficient in expression of the DNA in which the DNA of the present invention is inactivated; (7) the DNA is inactivated by introducing a reporter gene (eg, a β-galactosidase gene derived from Escherichia coli); The non-human mammal according to (6), wherein the reporter gene can be expressed under the control of a promoter for the DNA of the present invention,
(8) The non-human mammal according to (6), wherein the non-human mammal is a rodent, (9) the non-human mammal according to (8), wherein the rodent is a mouse, and (10) D) administering the test compound to the animal described in (7) and detecting the expression of the reporter gene;
A method for screening a compound or a salt thereof that promotes or inhibits a promoter activity for NA is provided.

A non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated is defined as a DNA obtained by artificially mutating the DNA of the present invention possessed by the non-human mammal to suppress the expression ability of the DNA. Alternatively, the DNA substantially does not have the ability to express the protein of the present invention by substantially losing the activity of the protein of the present invention encoded by the DNA (hereinafter referred to as the knockout DNA of the present invention). Embryonic stem cells (hereinafter sometimes referred to as ES) of a non-human mammal.
(Abbreviated as cell). As the non-human mammal, the same one as described above is used. As a method for artificially mutating the DNA of the present invention, for example, deletion of a part or all of the DNA sequence by genetic engineering techniques,
By inserting or substituting With these mutations, for example, the knockout DNA of the present invention may be prepared by shifting the codon reading frame or disrupting the function of the promoter or exon. Specific examples of the non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated (hereinafter abbreviated as the DNA-inactivated ES cells of the present invention or the knockout ES cells of the present invention) include, for example, The DNA of the present invention possessed by a non-human mammal is isolated, and its exon portion is a drug resistance gene typified by a neomycin resistance gene, a hygromycin resistance gene, or lacZ (β-galactosidase gene), cat (chloramphenicol acetyl). A DNA sequence (eg, polyA) that disrupts the function of exons by inserting a reporter gene or the like represented by a transferase gene, or terminates transcription of the gene in an intron between exons.
Additional messenger R)
By disabling the synthesis of NA, a DNA strand having a DNA sequence constructed so as to eventually destroy the gene (hereinafter abbreviated as a targeting vector) is introduced into the chromosome of the animal by, for example, homologous recombination. The obtained ES cells were analyzed by Southern hybridization analysis using a DNA sequence on or near the DNA of the present invention as a probe, or D on a targeting vector.
The NA sequence and the DNA sequence of the neighboring region other than the DNA of the present invention used for the preparation of the targeting vector were analyzed by a PCR method using primers, and the knockout E of the present invention was analyzed.
It can be obtained by selecting S cells.

Further, the DN of the present invention can be obtained by the homologous recombination method or the like.
As the ES cells from which A is inactivated, for example, those already established as described above may be used.
It may be newly established according to the method of Evans and Kaufma. For example, in the case of mouse ES cells, currently, 129 line ES cells are generally used. However, since the immunological background is not clear, a pure line instead of this is used as an immunological genetic background. For example, C57BL / 6 mice or C57BL /
Well as the number of oocytes retrieved the lack of 6 DBA / 2 and BDF ₁ mice improved by crossing (C57BL / 6 and F ₁ and DBA / 2) which were established by using the usable good. B
DF ₁ mice are often egg number and egg can be changed to that of a tough, since with C57BL / 6 mice in the background, when the ES cells obtained therefrom, which were generated pathological model mice , C57BL / 6 mice can be advantageously used in that their genetic background can be replaced with C57BL / 6 mice by backcrossing. Also,
When ES cells are established, blastocysts 3.5 days after fertilization are generally used. In addition to this, a large number of initial cells can be efficiently prepared by collecting 8-cell embryos and culturing them up to blastocysts. Embryos can be obtained. Either male or female ES cells may be used, but generally male ES cells are more convenient for producing a germline chimera. It is also desirable to discriminate between males and females as soon as possible in order to reduce cumbersome culture labor. As a method for determining the sex of ES cells, for example, a method of amplifying and detecting a gene in the sex-determining region on the Y chromosome by PCR can be mentioned. By using this method, the number of ES cells of about 1 colony (about 50 cells) is sufficient, whereas the conventional method required about 10 ⁶ cells for karyotype analysis. The primary selection of ES cells in the early stage can be performed by discriminating between male and female, and by enabling selection of male cells at an early stage, labor in the initial stage of culture can be greatly reduced.

The secondary selection can be performed, for example, by confirming the number of chromosomes by the G-banding method. The number of chromosomes in the obtained ES cells is desirably 100% of the normal number. However, when it is difficult due to physical operations or the like at the time of establishment, after knocking out the gene of the ES cells, the cells can be knocked out of normal cells (for example, chromosome in mice). Number is 2n
= Cells). The embryonic stem cell line obtained in this way usually has very good growth properties, but must be carefully subcultured because it tends to lose its ontogenetic ability. For example, on a suitable feeder cell such as STO fibroblast, in a carbon dioxide incubator (preferably 5% carbon dioxide, 95% air or 5%) in the presence of LIF (1-10000 U / ml).
% Oxygen, 5% carbon dioxide gas, 90% air) at about 37 ° C., and at the time of subculture, for example, trypsin / EDTA solution (usually 0.001-0.5% trypsin / 0.5%). (1-5 mM EDTA, preferably about 0.1% trypsin / 1 mM EDTA) treatment to form a single cell;
A method of seeding on newly prepared feeder cells is used. Such passage is usually performed every 1 to 3 days. At this time, it is desirable to observe the cells and, if any morphologically abnormal cells are found, discard the cultured cells. ES cells are differentiated into various types of cells, such as parietal, visceral, and cardiac muscles, by monolayer culture up to high density or suspension culture until cell clumps are formed under appropriate conditions. [MJ
Evans and MH Kaufman, Nature 292
Vol. 154, 1981; GR Martin Proceedings of National Academy of Sciences USA (Proc. Natl. Acad. Sci. USA) No. 7
8, 7634, 1981; TC Doetschman et al., Journal of Embryology and Experimental Morphology, 87, 27, 1985], obtained by differentiating the ES cell of the present invention. The cells deficient in expressing the DNA of the present invention are useful in in vitro cell biology of the protein of the present invention. DNA of the present invention
An expression-defective non-human mammal can be distinguished from a normal animal by measuring the mRNA level of the animal using a known method and indirectly comparing the expression level. As the non-human mammal, those similar to the aforementioned can be used.

The non-human mammal deficient in expression of the DNA of the present invention may be prepared, for example, by introducing the targeting vector prepared as described above into mouse embryonic stem cells or mouse egg cells,
By knocking out the DNA of the present invention, the DNA sequence in which NA has been inactivated is replaced with the DNA of the present invention on the chromosome of mouse embryonic stem cells or mouse egg cells by gene homologous recombination. it can. The cells in which the DNA of the present invention has been knocked out are the DNA sequence of the present invention derived from the mouse used for the targeting vector and the DNA sequence of the target for the Southern hybridization analysis or the DNA sequence on or near the DNA of the present invention. It can be determined by analysis by PCR using a DNA sequence of a nearby region other than DNA as a primer. When a non-human mammalian embryonic stem cell is used, a cell line in which the DNA of the present invention has been inactivated by gene homologous recombination is cloned, and the cell is cultured at an appropriate time, for example, at the 8-cell stage of a non-human mammalian stem cell. The chimeric embryo is injected into an animal embryo or a blastocyst, and the produced chimeric embryo is transplanted into the uterus of the pseudopregnant non-human mammal. The produced animal is a chimeric animal comprising both cells having the normal DNA locus of the present invention and cells having the artificially mutated DNA locus of the present invention. When a part of the germ cells of the chimeric animal has a mutated DNA locus of the present invention, all tissues are artificially mutated from a population obtained by crossing such a chimeric individual with a normal individual. D of the present invention added
It can be obtained by selecting individuals composed of cells having NA loci, for example, by judging coat color or the like. The individual thus obtained is usually an individual heterozygously deficient in expression of the protein of the present invention, mated with an individual deficient in heteroexpression of the protein of the present invention, and homozygously expressing the protein of the present invention from their offspring. Defective individuals can be obtained. When using an egg cell, for example, a transgenic non-human mammal having a targeting vector introduced into a chromosome by injecting a DNA solution into the egg cell nucleus by a microinjection method can be obtained,
Compared to these transgenic non-human mammals,
It can be obtained by selecting a DNA locus of the present invention having a mutation by homologous recombination.

In the individual in which the DNA of the present invention has been knocked out as described above, it is necessary to confirm that the DNA of the animal obtained by the crossing is also knocked out, and to carry out rearing in an ordinary breeding environment. Can be. further,
The germline can be obtained and maintained in accordance with a conventional method. That is, by mating male and female animals having the inactivated DNA, homozygous animals having the inactivated DNA on both homologous chromosomes can be obtained. The obtained homozygous animals were compared to normal animals 1
It can be obtained efficiently by breeding in a state where there are a plurality of homozygotes. By mating male and female heterozygous animals, homozygous and heterozygous animals having the inactivated DNA are bred and subcultured. The non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated are extremely useful for producing a non-human mammal deficient in expressing the DNA of the present invention. In addition, the DN of the present invention
Since non-human mammals deficient in A expression lack various biological activities that can be induced by the protein of the present invention, they can serve as models for diseases caused by inactivation of the biological activity of the protein of the present invention. It is useful for investigating the cause of disease and examining treatment methods.

(8a) Screening Method for Compound Having a Therapeutic / Prophylactic Effect on Diseases Caused by DNA Defects or Damage of the Present Invention The non-human mammal deficient in DNA expression of the present invention is
It can be used to screen for compounds that have a therapeutic or preventive effect on diseases caused by NA deficiency or damage (eg, arteriosclerosis, hyperlipidemia, hypercalorie, obesity, hypertriglyceridemia, etc.). it can. That is, the present invention is characterized by administering a test compound to a non-human mammal deficient in expressing the DNA of the present invention, and observing and measuring changes in the animal. Provided is a method for screening a compound or a salt thereof having a therapeutic / prophylactic effect against a disease. Examples of the non-human mammal deficient in expressing DNA of the present invention used in the screening method include the same ones as described above. Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts,
Examples include plant extracts, animal tissue extracts, and plasma. These compounds may be novel compounds or known compounds. Specifically, a non-human mammal deficient in expression of the DNA of the present invention is treated with a test compound, compared with a non-treated control animal, and tested using changes in each organ, tissue, disease symptoms, etc. of the animal as an index. The therapeutic / preventive effects of the compounds can be tested. As a method of treating a test animal with a test compound, for example, oral administration, intravenous injection and the like are used, and it can be appropriately selected according to the symptoms of the test animal, properties of the test compound, and the like. The dose of the test compound can be appropriately selected according to the administration method, the properties of the test compound, and the like. For example, when screening for a compound having a therapeutic / preventive effect on arteriosclerosis, a glucose tolerance treatment is performed on a non-human mammal deficient in expressing DNA of the present invention, and a test compound is administered before or after glucose tolerance treatment, The blood glucose level and weight change of the animal are measured over time. In the screening method, when a test compound is administered to a test animal, the test animal has a blood glucose level of about 1
When it is reduced by 0% or more, preferably about 30% or more, more preferably about 50% or more, the test compound can be selected as a compound having a therapeutic / preventive effect on arteriosclerosis.

The compound obtained by using the screening method of the present invention is a compound selected from the test compounds described above, and is a disease (eg, arteriosclerosis, etc.) caused by deficiency or damage of the protein, etc. of the present invention. It can be used as a medicament such as a safe and low-toxic therapeutic or prophylactic agent for the disease. Furthermore, compounds derived from the compounds obtained by the above screening can be used in the same manner. The compound obtained by the screening method may form a salt, and the salt of the compound may include a physiologically acceptable acid (eg, an inorganic acid, an organic acid) and a base (eg, an alkali metal). And particularly preferably a physiologically acceptable acid addition salt. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid) Acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like are used. A drug containing the compound or a salt thereof obtained by the screening method can be produced in the same manner as the above-mentioned drug containing the protein of the present invention. The preparations obtained in this way are safe and have low toxicity, for example, humans or mammals (eg, rats, mice, guinea pigs,
Rabbit, sheep, pig, cow, horse, cat, dog, monkey, etc.). The dose of the compound or a salt thereof varies depending on the target disease, the administration subject, the administration route, and the like. For example, when the compound is orally administered for the purpose of treating arteriosclerosis, it is generally adult (body weight 6).
0 kg), from about 0.1 to 100 mg, preferably from about 1.0 to 50 mg of the compound per day,
More preferably, about 1.0 to 20 mg is administered. When administered parenterally, a single dose of the compound should be administered to the subject,
For example, the compound is usually administered in the form of an injection to an adult (60 k
g)), the compound is administered in an amount of about 0.01 to 30 mg per day, preferably about 0.1 to 20 mg per day.
Conveniently, about g, more preferably about 0.1 to 10 mg, will be administered by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg.

(8b) Method for Screening Compound Promoting or Inhibiting the Activity of Promoter for DNA of the Present Invention The present invention relates to a non-human mammal deficient in expressing the DNA of the present invention.
The present invention provides a method for screening a compound or a salt thereof which promotes or inhibits the activity of a promoter for DNA of the present invention, which comprises administering a test compound and detecting the expression of a reporter gene. In the above-mentioned screening method, the non-human mammal deficient in expression of DNA of the present invention may be a non-human mammal deficient in expressing DNA of the present invention in which the DNA of the present invention is inactivated by introducing a reporter gene, A reporter gene that can be expressed under the control of a promoter for the DNA of the present invention is used. Examples of the test compound include the same compounds as described above. As the reporter gene, the same one as described above is used, and a β-galactosidase gene (lacZ), a soluble alkaline phosphatase gene, a luciferase gene and the like are preferable. The DN of the present invention in which the DNA of the present invention is replaced with a reporter gene
In a non-human mammal deficient in A expression, since the reporter gene is under the control of the promoter for the DNA of the present invention, the activity of the promoter can be detected by tracing the expression of a substance encoded by the reporter gene.

For example, when a part of the DNA region encoding the protein of the present invention is replaced with the β-galactosidase gene (lacZ) derived from Escherichia coli, the tissue of the present invention which originally expresses the protein of the present invention is used. Β-galactosidase is expressed instead of protein. Thus, for example, 5-bromo-4-chloro-3-indolyl-β-
By staining with a reagent serving as a substrate for β-galactosidase such as galactopyranoside (X-gal), the expression state of the protein of the present invention in an animal body can be easily observed. More specifically, the protein-deficient mouse of the present invention or a tissue section thereof is fixed with glutaraldehyde or the like, and the buffered phosphate buffered saline (PB
After washing in S), the mixture is reacted with a staining solution containing X-gal at room temperature or about 37 ° C. for about 30 minutes to 1 hour, and then the tissue specimen is washed with a 1 mM EDTA / PBS solution to obtain β-galactosidase. The reaction may be stopped and the color may be observed. Further, mRNA encoding lacZ may be detected according to a conventional method.

The compound or a salt thereof obtained by using the above-mentioned screening method is a compound selected from the above-mentioned test compounds, and is a compound that promotes or inhibits the promoter activity for the DNA of the present invention. The compound obtained by the screening method may form a salt,
Salts of the compound include physiologically acceptable acids (eg,
Salts with inorganic acids) and bases (eg, organic acids),
Particularly preferred are physiologically acceptable acid addition salts. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid) Acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like are used. Since the compound or a salt thereof that promotes the promoter activity for the DNA of the present invention can promote the expression of the protein of the present invention and promote the function of the protein, for example, arteriosclerosis, hyperlipidemia,
It is useful as a drug such as a safe and low-toxic therapeutic or prophylactic agent for diseases such as hypercalorie, obesity, aging, brain disease, renal disorder, and hypertriglyceridemia. On the other hand, compounds or salts thereof that inhibit the promoter activity of the DNA of the present invention can inhibit the expression of the protein of the present invention and inhibit the function of the protein.
It is useful as a medicament such as a safe and low toxic therapeutic / prophylactic agent for diseases such as malnutrition, β-lipoproteinemia, inflammatory diseases, and Tangier's disease. Furthermore, compounds derived from the compounds obtained by the above screening can be used in the same manner.

A drug containing a compound or a salt thereof obtained by the screening method can be produced in the same manner as the above-mentioned drug containing a protein of the present invention or a salt thereof. The preparation obtained in this way is safe and low toxic, and thus can be used, for example, in humans or mammals (eg, rat, mouse, guinea pig, rabbit, sheep, pig, cow, horse, cat, dog, monkey, etc.). Can be administered. The dose of the compound or a salt thereof,
Although there is a difference depending on the target disease, the administration subject, the administration route and the like, for example, when the compound for promoting the promoter activity to DNA of the present invention is orally administered for the purpose of treating arteriosclerosis, generally the adult (with a body weight of 60 kg) In the above, about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to about 100 mg of the compound per day
Administer 20 mg. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease and the like. For example, the DN of the present invention may be used for the treatment of arteriosclerosis.
When a compound that promotes the promoter activity of A is administered to an adult (as 60 kg) in the form of an injection, the compound is administered in an amount of about 0.01 to 30 mg, preferably about 0.1 to 20 mg per day. Preferably about 0.1
It is convenient to administer about 10 to 10 mg by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg. On the other hand, for example, when a compound that inhibits the promoter activity of the DNA of the present invention is orally administered for the purpose of treating β-lipoproteinemia, generally, in an adult (assuming a body weight of 60 kg), the compound is used in an amount of about 0.1 to 100 mg, preferably about 1.0 to
The dose is 50 mg, more preferably about 1.0 to 20 mg. When administered parenterally, a single dose of the compound varies depending on the administration subject, target disease, and the like. For example, it inhibits the promoter activity of the DNA of the present invention for the purpose of treating β-lipoproteinemia. When the compound is administered to an adult (as 60 kg) in the form of an injection, the compound is usually administered in an amount of about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 20 mg per day. 1
It is convenient to administer about 0 mg by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg. As described above, the non-human mammal deficient in expression of the DNA of the present invention is extremely useful for screening for a compound or a salt thereof that promotes or inhibits the activity of the promoter of the DNA of the present invention. It can greatly contribute to the investigation of the cause of various diseases caused by the disease or the development of preventive / therapeutic drugs. In addition, a DNA containing the promoter region of the LCAT-like protein of the present invention (for example, a DNA having the same or substantially the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 38 or a DNA containing a partial DNA thereof) )
Using, a gene encoding various proteins is ligated downstream, and this is injected into the egg cell of an animal to produce a so-called transgenic animal (transgenic animal). It will also be possible to study its effects on living organisms. Furthermore, by linking an appropriate reporter gene to the above promoter portion and establishing a cell line in which this is expressed, a low-molecular-weight compound having the action of specifically promoting or inhibiting the production of LCAT-like protein itself in the body can be obtained. Can be used as a search system. By analyzing the promoter portion, it is also possible to find a new cis element and a transcription factor binding thereto. As a DNA containing a nucleotide sequence substantially identical to the nucleotide sequence represented by SEQ ID NO: 38,
For example, it has a nucleotide sequence that hybridizes under high stringent conditions to the nucleotide sequence represented by SEQ ID NO: 38, and has substantially the same promoter activity as DNA containing the nucleotide sequence represented by SEQ ID NO: 38. Any DNA may be used as long as it has DNA. SEQ ID NO: 38
Examples of a DNA that can hybridize with the base sequence represented by SEQ ID NO: 38 under high stringent conditions include, for example, a base sequence represented by SEQ ID NO: 38 and about 70%
Or more, preferably about 80% or more, more preferably about 9%
DNA containing a base sequence having a homology of 0% or more, most preferably about 95% or more is used. Hybridization can be performed according to the method known per se or a method analogous thereto.

In the present specification and drawings, when bases, amino acids and the like are represented by abbreviations, IUPAC-IUB
Abbreviations based on Commision on Biochemical Nomenclature or common abbreviations in the field,
An example is given below. When an amino acid can have an optical isomer, the L-form is indicated unless otherwise specified. DNA: deoxyribonucleic acid cDNA: complementary deoxyribonucleic acid A: adenine T: thymine G: guanine C: cytosine RNA: ribonucleic acid mRNA: messenger ribonucleic acid dATP: deoxyadenosine triphosphate dTTP: deoxythymidine triphosphate dGTP: deoxyguanosine triphosphate Phosphate dCTP: Deoxycytidine triphosphate ATP: Adenosine triphosphate EDTA: Ethylenediaminetetraacetic acid SDS: Sodium dodecyl sulfate

Gly: glycine Ala: alanine Val: valine Leu: leucine Ile: isoleucine Ser: serine Thr: threonine Cys: cysteine Met: methionine Glu: glutamic acid Asp: aspartic acid Lysine arginine arginine : Tyrosine Trp: Tryptophan Pro: Proline Asn: Asparagine Gln: Glutamine pGlu: Pyroglutamic acid

The substituents, protecting groups and reagents frequently used in the present specification are represented by the following symbols. Me: methyl group Et: ethyl group Bu: butyl group Ph: phenyl group TC: thiazolidine-4 (R) -carboxamide group Tos: p-toluenesulfonyl CHO: formyl Bzl: benzyl ^Cl2 Bzl: 2,6-dichlorobenzyl Bom : Benzyloxymethyl Z: benzyloxycarbonyl Cl-Z: 2-chlorobenzyloxycarbonyl Br-Z: 2-bromobenzyloxycarbonyl Boc: t-butoxycarbonyl DNP: dinitrophenyl Trt: trityl Bum: t-butoxymethyl Fmoc: N-9-fluorenylmethoxycarbonyl HOBt: 1-hydroxybenztriazole HOOBT: 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine HONB: 1-hydroxy-5-nor Bornene-2,3-dicarboximide DCC: N, N'-dicyclohexylcarbodiimide

The sequence numbers in the sequence listing in the present specification indicate the following sequences. [SEQ ID NO: 1] This shows the amino acid sequence of the human heart-derived protein (mature) of the present invention. [SEQ ID NO: 2] This shows the amino acid sequence of the protein (mature) derived from human kidney of the present invention. Between the 63rd position (Leu) and the 64th position (Val) of the amino acid sequence represented by SEQ ID NO: 1, the 64th position (Glu) to the 95th position (Leu) of the amino acid sequence represented by SEQ ID NO: 2 In which 32 amino acid residues have been inserted. [SEQ ID NO: 3] This shows the amino acid sequence of the mouse kidney-derived protein (mature) of the present invention. [SEQ ID NO: 4] This shows the amino acid sequence of the human heart-derived protein (mature) of the present invention. [SEQ ID NO: 5] This shows the amino acid sequence of the human kidney-derived protein (mature) of the present invention. Between the 66th position (Leu) and the 67th position (Val) of the amino acid sequence represented by SEQ ID NO: 4, the 67th position (Glu) to 98th position (Leu) of the amino acid sequence represented by SEQ ID NO: 5 In which 32 amino acid residues have been inserted. [SEQ ID NO: 6] This shows the amino acid sequence of the precursor protein of the human heart-derived protein of the present invention. [SEQ ID NO: 7] This shows the amino acid sequence of the precursor protein of the human kidney-derived protein of the present invention. No. 96 (Leu) and No. 9 of the amino acid sequence represented by SEQ ID NO: 6
Between the 7th position (Val) and the 97th position (Glu) to 128th position (L) of the amino acid sequence represented by SEQ ID NO: 7
eu) with 32 amino acid residues inserted. [SEQ ID NO: 8] This shows the amino acid sequence of the precursor protein of the mouse kidney-derived protein of the present invention. [SEQ ID NO: 9] This shows the amino acid sequence of the signal peptide of the present invention. [SEQ ID NO: 10] This shows the amino acid sequence of the signal peptide of the present invention. [SEQ ID NO: 11] This shows the amino acid sequence of the signal peptide of the present invention.

[SEQ ID NO: 12] This shows the base sequence of DNA encoding the protein (mature) derived from human heart of the present invention having the amino acid sequence represented by SEQ ID NO: 1. [SEQ ID NO: 13] This shows the base sequence of DNA encoding the human kidney-derived protein (mature) of the present invention having the amino acid sequence represented by SEQ ID NO: 2. [SEQ ID NO: 14] Mouse-derived protein of the present invention having the amino acid sequence represented by SEQ ID NO: 3 (mature)
Shows the nucleotide sequence of DNA encoding [SEQ ID NO: 15] This shows the base sequence of DNA encoding the human heart-derived protein (mature) of the present invention having the amino acid sequence represented by SEQ ID NO: 4. [SEQ ID NO: 16] This shows the base sequence of DNA encoding the human kidney-derived protein (mature) of the present invention having the amino acid sequence represented by SEQ ID NO: 5. [SEQ ID NO: 17] This shows the base sequence of DNA encoding the precursor protein of the human heart-derived protein of the present invention having the amino acid sequence represented by SEQ ID NO: 6. [SEQ ID NO: 18] This shows the base sequence of DNA encoding the precursor protein of the human kidney-derived protein of the present invention having the amino acid sequence represented by SEQ ID NO: 7. [SEQ ID NO: 19] This shows the base sequence of DNA encoding the precursor protein of the mouse kidney-derived protein of the present invention having the amino acid sequence represented by SEQ ID NO: 8. [SEQ ID NO: 20] D encoding the signal peptide of the present invention having the amino acid sequence represented by SEQ ID NO: 9
Shows the base sequence of NA. [SEQ ID NO: 21] This shows the base sequence of DNA encoding the signal peptide of the present invention having the amino acid sequence represented by SEQ ID NO: 10. [SEQ ID NO: 22] This shows the base sequence of DNA encoding the signal peptide of the present invention having the amino acid sequence represented by SEQ ID NO: 11.

[SEQ ID NO: 23] This shows the partial base sequence of the DNA encoding the human-derived protein of the present invention used in Example 1 for cloning the full-length DNA encoding the human-derived protein of the present invention. [SEQ ID NO: 24] This shows the base sequence of the synthetic primer used in the cloning of the DNA encoding the human-derived protein of the present invention in Example 1. [SEQ ID NO: 25] This shows the base sequence of the synthetic primer used in Example 1 for cloning DNA encoding the human-derived protein of the present invention. [SEQ ID NO: 26] This shows the base sequence of the synthetic primer used in Example 1 for cloning DNA encoding the human-derived protein of the present invention. [SEQ ID NO: 27] This shows the base sequence of the synthetic primer used in Example 2 for cloning the DNA encoding the mouse-derived protein of the present invention. [SEQ ID NO: 28] This shows the base sequence of the synthetic primer used in Example 2 for cloning DNA encoding the mouse-derived protein of the present invention. [SEQ ID NO: 29] This shows the partial base sequence of the DNA encoding the human-derived protein of the present invention used for cloning of the full-length DNA encoding the mouse-derived protein of the present invention in Example 2. [SEQ ID NO: 30] This shows the base sequence of the synthetic primer used in Example 2 for cloning DNA encoding the mouse-derived protein of the present invention. [SEQ ID NO: 31] This shows the base sequence of the synthetic primer used in Example 2 for cloning DNA encoding the mouse-derived protein of the present invention. [SEQ ID NO: 32] This shows the base sequence of the synthetic primer used in Example 2 for cloning DNA encoding the mouse-derived protein of the present invention. [SEQ ID NO: 33] This shows the base sequence of the synthetic primer used in Example 3 for cloning genomic DNA encoding the human-derived protein of the present invention. [SEQ ID NO: 34] This shows the base sequence of the synthetic primer used in Example 3 for cloning genomic DNA encoding the human-derived protein of the present invention. [SEQ ID NO: 35] This shows the base sequence of the adapter used in Example 3 for cloning genomic DNA encoding the human-derived protein of the present invention. [SEQ ID NO: 36] This shows the base sequence of the synthetic primer used in Example 3 for cloning genomic DNA encoding the human-derived protein of the present invention. [SEQ ID NO: 37] This shows the base sequence of the synthetic primer used in Example 3 for cloning genomic DNA encoding the human-derived protein of the present invention. [SEQ ID NO: 38] This shows the 5 ′ upstream sequence (promoter region) of cDNA encoding the human-derived protein of the present invention. [SEQ ID NO: 39] This shows the amino acid sequence of the partial peptide of the human LCAT-like protein of the present invention chemically synthesized in Example 5. [SEQ ID NO: 40] This shows the amino acid sequence of a partial peptide of the human LCAT-like protein of the present invention chemically synthesized in Example 5. [SEQ ID NO: 41] FLA prepared in Example 7
The base sequence of the synthetic primer used for cloning the DNA encoding the human LCAT-like protein of the present invention fused with the G peptide is shown. [SEQ ID NO: 42] FLA prepared in Example 7
The base sequence of the synthetic primer used for cloning the DNA encoding the human LCAT-like protein of the present invention fused with the G peptide is shown. [SEQ ID NO: 43] This shows the amino acid sequence of the partial peptide of Example 11.

The transformant Escherichia coli DH10B / pT obtained in Example 1 described later.
B1972 was deposited on April 7, 1997 with the Ministry of International Trade and Industry at the National Institute of Advanced Industrial Science and Technology (NIBH).
ERM BP-5900 was deposited with the Fermentation Research Institute (IFO) on April 9, 1997 as deposit number IFO16.
No. 072. In addition, the transformant Escherichia coli (Escherichia coli) obtained in Example 1 described later is used.
coli) DH10B / pTB1973 was purchased on April 7, 1997.
From April 9, 1997, the Fermentation Research Institute (IF) was deposited as the deposit number FERM BP-5901 with the National Institute of Advanced Industrial Science and Technology (NIBH)
O) and deposit number IFO 16073. Further, the transformant Escherichia coli DH10B / pTB obtained in Example 2 described later.
2010 was deposited on July 8, 1997 with the Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology (NIBH) under the deposit number FE.
Deposit No. IFO 16 as RM BP-6011 from the Fermentation Research Institute (IFO) from July 9, 1997.
No. 111 has been deposited. The transformant Escherichiac (Escherichiac) obtained in Example 4 described later is used.
oli) DH5α / pTB2022 was obtained on January 20, 1998.
Deposited from the Ministry of International Trade and Industry at the National Institute of Advanced Industrial Science and Technology (NIBH) under the deposit number FERM BP-6227, and from January 19, 1998 deposited at the Fermentation Research Institute (IFO) under the deposit number IFO 16154. Have been.

[0095]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the present invention is limited thereto. In addition, the genetic manipulation method using Escherichia coli followed the method described in Molecular cloning.

Example 1 Cloning of Gene Encoding Human-Derived LCAT-Like Protein of the Present Invention (1) THP-1 cells were made macrophage-like by two methods and then expressed differently between foamed cells mR
Concentration by subtraction of NA The foaming of THP-1 cells (Dainippon Pharmaceutical Co., Ltd.) was mainly performed by the method of A. Rodriguez et al. (Journal of Lipid Research, 35, 1909, 1994). Followed. Treating THP-1 cells with phorbol myristate acetate (PMA), 400 ng / ml for 3 days,
After making the cells macrophage-like, β VLDL (very low de
nsity lipoprotein) 0.2 mg TC (total cholesterol) / ml was loaded with cholesterol for 1 day.
-1 sample of 400 ng-treated foamed cells derived from cells. Similarly, the cells were treated with PMA, 5 ng / ml for 5 days to form macrophages, and then β VLDL 0.2 mg TC
/ Ml for 1 day to obtain a sample of 5 ng-treated foamed cells derived from THP-1 cells. For both samples, total RNA was extracted using guanidine isothiocyanate (Pharmacia), and further passed through an oligo-dT cellulose column (Pharmacia) to obtain poly (A) ⁺ RN.
A was purified. Each of these poly (A) ⁺ RNA
g as a starting material, a cDNA fragment (a part of cDNA was specifically amplified by PCR) specifically expressed in 5 ng-treated foamed cells by subtraction using a PCR-select cDNA subtraction kit (manufactured by Clontech). Fragments) were collected. The sequence of the adapter for subtraction added to both ends of the obtained PCR fragment is removed by digestion with a restriction enzyme RsaI,
After the blunt-ended DNA fragment was obtained, this fragment was pCR-Sc
It was subcloned into ript (Stratagene).
The DNA base sequence of the subcloned cDNA fragment was decoded, and blasting was performed using the public base database Genemble database based on the determined base sequence.
A homology search by N was performed. As a result, the obtained clone 4S-086 (SEQ ID NO: 23) is a known human LCAT (Biochimica et Biophysica Acta) vol. 910, 142
-148, 1987) and a novel DNA base sequence having about 61% homology.

(2) Isolation of full-length cDNA of 4S-086 cDNA fragment Using the obtained clone 4S-086 as a probe, Northern blot analysis was performed using a commercially available MTN membrane (Clontech). As a result, this gene is
It was presumed to be expressed in skeletal muscle, kidney and testis. Therefore, this full-length cDNA was to be obtained from cDNA libraries derived from human heart and human kidney. Cloning of the cDNA was performed using a Genetrapper positive selection system (Gibco BRL). CDN from human heart and human kidney
Escherichia coli D from the A library (Gibcobee RL)
The H12S strain was isolated from each Terrific Broth (12 g / l bacto-
tryptone (Difco), 24g / l bacto-yeast ext
ract (Difco), 2.3 g / l monopotassium phosphate,
After culturing at 12.5 g / l dipotassium phosphate) at 30 ° C. for 16 hours, a Qiagen plasmid kit (Qiagen)
Was used to purify and extract the plasmid cDNA library. The purified plasmid cDNA library was
NeII and ExoIII (both from Gibco BRL) were digested to prepare a single-stranded cDNA library. On the other hand, as a probe, a synthetic oligonucleotide: 5′-GCTGCTGCCCTACACACTACACAT-3 ′ (SEQ ID NO: 24) was used for screening of a cDNA library. The probe was labeled by biotinylating the 3 ′ end using TdT, biotin-14-dCTP (Gibco BRL). 95 single-stranded cDNA libraries
After treatment at 1 ° C. for 1 minute, the mixture was rapidly cooled in ice, a biotinylated probe was added, and hybridization was carried out at 37 ° C. for 1 hour at room temperature. After hybridization, Genetrapper Positive Selection System / Magnetic Beads (Gibco BRL) was added and left at room temperature for 30 minutes with stirring every 2 minutes. Then, it was placed in a Genetrapper Positive Selection System magnet rack (Gibco BRL) and left for 2 minutes. The supernatant was discarded, and the magnetic beads were washed with Gentra Wrapper positive selection system wash buffer. Washing with the wash buffer was performed three times. Thereafter, the mixture was left in a magnet rack, and the supernatant was discarded. The Genetrapper Positive Selection System / elution buffer was added, and the mixture was left at room temperature for 5 minutes. After placing in a magnet rack for 5 minutes, the DNA of the supernatant was
The solution was collected.

A synthetic oligonucleotide: 5'-GCTGCTGCCCTACAAACTACACAT-3 '(SEQ ID NO: 24) was added as a primer to the obtained DNA solution and treated at 95 ° C. for 1 minute. Gene Trapper Positive Selection System with Repair Enzyme
The mixture was allowed to stand for 2 minutes to synthesize double-stranded DNA. The synthesized double-stranded DNA was introduced into Escherichia coli DH10B using an electroporation apparatus (Bio-Rad). Using the obtained transformant, screening by colony PCR was carried out using two oligonucleotides: 5′-TATCCGGGCCTTTCGTGTCA-3 ′ (SEQ ID NO: 25) and 5′-TCAAAGCCGATGTCTCTGGAAGAACTTGC-3 ′ (SEQ ID NO: 26) as primers. Done. A colony in which an amplified fragment of about 220 bp was formed by PCR was used as a positive clone, and a total of four strains each of two strains were selected from cDNA libraries derived from human heart and human kidney. After culturing each selected E. coli,
Extract DNA and use ABI PRISH Dye Terminator Cycle Seq
uencing Ready Reaction Kit with AmpliTaq DNA polym
The reaction was performed using erase, FS (Perkin Elmer), and the nucleotide sequence of each cDNA fragment was determined using a 377 DNA sequencer (Perkin Elmer). Further, an alignment was performed based on the determined base sequences. As a result, one of the two strains derived from the human heart and one strain derived from the human kidney had 1271 base sequences including the base sequence represented by SEQ ID NO: 17. The remaining one strain derived from human kidney has SEQ ID NO: 18 in which 96 bases have been inserted into the base sequence of the two strains derived from human heart.
And 1335 base sequences including the base sequence represented by The former cDNA fragment encodes 412 amino acids represented by SEQ ID NO: 6, and the latter cDNA fragment encodes 444 amino acids represented by SEQ ID NO: 7, and both encode novel LCAT-like proteins. It had been. Plasmid pT carrying DNA encoding the two novel LCAT-like proteins of the present invention
B1972 and pTB1973 were transformed into Escherichia coli (Escherichi
a.) DH10B to obtain transformants: Escherichia coli DH10B / pTB1972 and DH10B / pTB1973.

Example 2 cD encoding a novel LCAT-like protein derived from mouse
Cloning of NA The partial sequence of a novel LCAT-like protein derived from a mouse was cloned by PCR. Escherichia coli DH12S strain of a mouse kidney-derived cDNA library (Gibco BRL) was added to Terrific B
roth (12g / l bacto-tryptone (Difco), 24g
/ l bacto-yeast extract (Difco), 2.3g / l
The cells were cultured at 30 ° C. for 16 hours with monopotassium phosphate (12.5 g / l dipotassium phosphate), and a plasmid cDNA library was prepared using a Qiagen plasmid kit (Qiagen) and used as a template. New LCA derived from human
From the sequence information of the T-like protein, the following two synthetic primers were prepared and used as primers. 5′-GTGGTGCTGGTCCCCTGGTGATTTG-3 ′ (SEQ ID NO: 27) 5′-GGTGGCCCTGGATGTTTTGTTG-3 ′ (SEQ ID NO: 28) The PCR reaction is a system containing TaKaRa Ex Taq (Takara Shuzo Co., Ltd.) and a thermal cycler (GeneAmp PCR System). 240
0, Perkin Elmer) at 94 ° C for 30 seconds.
The cycle was performed at 55 ° C., 30 seconds, 72 ° C., and 1 minute for 30 cycles at 4 ° C. The obtained amplified fragment was inserted into pT7 blue T-vector (Novagen) using DNA ligation kit version 2 (Takara Shuzo),
It was introduced into E. coli DH5α strain. Plasmid DNA was extracted from the resulting transformant and reacted using Dye Terminator Cycle Sequence FS Ready Reaction Kit (Perkin Elmer), and the nucleotide sequence of the cDNA fragment was determined using a 377 DNA sequencer (Perkin Elmer). did. The obtained clone had 198 base sequences represented by SEQ ID NO: 29.
Therefore, this full-length cDNA was converted to mouse kidney-derived cDN.
A library. Cloning of the cDNA was performed using a Genetrapper positive selection system (Gibco BRL).

The plasmid cDNA library used above was digested with GeneII and ExoIII (both from Gibco BRL) to prepare a single-stranded cDNA library. On the other hand, as a probe, a synthetic oligonucleotide: 5′-GGTTGTACACTACCTTTGCTCCAAG-3 ′ (SEQ ID NO: 30) was used for screening a cDNA library. The probe was labeled by biotinylating the 3 ′ end using TdT, biotin-14-dCTP (Gibco BRL). 95 single-stranded cDNA libraries
After treatment at 1 ° C. for 1 minute, the mixture was rapidly cooled in ice, a biotinylated probe was added, and hybridization was carried out at 37 ° C. for 1 hour at room temperature. After hybridization, Genetrapper Positive Selection System / Magnetic Beads (Gibco BRL) was added and left at room temperature for 30 minutes with stirring every 2 minutes. Then, it was placed in a Genetrapper Positive Selection System magnet rack (Gibco BRL) and left for 2 minutes. The supernatant was discarded, and the magnetic beads were washed with Gentra Wrapper positive selection system wash buffer. Washing with the wash buffer was performed three times. Thereafter, the mixture was left in a magnet rack, and the supernatant was discarded. The Genetrapper Positive Selection System / elution buffer was added, and the mixture was left at room temperature for 5 minutes. After placing in a magnet rack for 5 minutes, the DNA of the supernatant was
The solution was collected.

A synthetic oligonucleotide: 5'-GGTTGTACACTACCTTTGTCCCAAG-3 '(SEQ ID NO: 30) was added as a primer to the obtained DNA solution, and the mixture was treated at 95 ° C for 1 minute. Gene Trapper Positive Selection System with Repair Enzyme
The mixture was allowed to stand for 2 minutes to synthesize double-stranded DNA. The synthesized double-stranded DNA was introduced into Escherichia coli DH10B using an electroporation apparatus (Bio-Rad). Using the obtained transformant, screening by colony PCR was carried out using two oligonucleotides: 5′-GGTAACCAGTTGGAAGCAAAG-3 ′ (SEQ ID NO: 31) and 5′-ATCCAGCAGTCAATGATAACA-3 ′ (SEQ ID NO: 32) as primers. Done. A colony in which an amplified fragment of about 130 bp was formed by PCR was used as a positive clone, and a total of three strains were selected from a mouse kidney-derived cDNA library. After culturing the selected Escherichia coli, DNA is extracted and ABI PRISH
Dye Terminator Cycle Sequencing Ready Reaction Kit
The reaction was carried out using with AmpliTaq DNA polymerase, FS (Perkin Elmer), and the nucleotide sequence of each cDNA fragment was determined using a 377 DNA sequencer (Perkin Elmer). Further, an alignment was performed based on the determined base sequences. As a result, the obtained three clones contained the same DNA fragment, and SEQ ID NO: 19
And 2734 base sequences including the base sequence represented by The cDNA fragment encoded 412 amino acids represented by SEQ ID NO: 8, and encoded a novel mouse-derived LCAT-like protein. This novel mouse-derived LCAT-like protein is different from the novel human-derived LCAT-like protein having the amino acid sequence represented by SEQ ID NO: 1 obtained in Example 1 by 8 bases.
It had a homology of 5.0% and 88.1% at the amino acid level (FIG. 4). The plasmid pTB2010 carrying the DNA encoding the novel LCAT derived from the mouse of the present invention is introduced into Escherichia coli DH10B, and a transformant: Escherichia coli DH10B / p
TB2010 was obtained.

Example 3 Cloning of Genomic DNA of Human-Derived New LCAT-like Protein Sequence analysis near the start codon of DNA encoding the human-derived protein of the present invention was performed using a promoter finder DNA walking kit (Clontech). Was. The used human genomic DNA was previously digested with Ssp I restriction enzymes, and primers were added to the 5 ′ and 3 ′ ends.
AP 1 [5'-GTAATACGACTCACTATAGGGC-3 '(SEQ ID NO:
33) (Clonetech)] and primer AP 2 [5 '
-ACTATAGGGCACGCGTGGT-3 '(SEQ ID NO: 34) (Clontech)]]
TAATACGACTCACTATAGGGCACGCGTGGTCGACGGCCCGGGCTGGT-
3 ′ (SEQ ID NO: 35)] is linked. First, synthetic oligonucleotide GSP 1 [5'-ATCCGGGAGCAGCCCCAC
ACGGTAGG-3 '(SEQ ID NO: 36)] and synthetic oligonucleotide GSP 2 [5'-GGTGTACGACGGTCGCCGCAGGTC-
3 ′ (SEQ ID NO: 37)], a cD encoding a novel LCAT-like protein derived from human
Oligonucleotides from base numbers +45 to +20 and base numbers -1 to -24 of the 5 ′ upstream region sequence of NA (FIG. 5) were synthesized for reference. The first PCR reaction was performed using this human genomic DNA solution, TaKaRa LA PCR kit version 2 (Takara Shuzo Co., Ltd.), AP1, and synthetic oligonucleotide GSP1, and performed a thermal cycler (GeneAmpR PCR S
ystem 2400, PerkinElmer) at 95 ° C for 10 seconds,
The test was performed at 65 ° C. for 30 seconds and 72 ° C. for 5 minutes under the conditions of 30 cycles. Next, this reaction solution was diluted 50-fold with sterile water,
Used for the second PCR reaction. The second PCR reaction was performed using the first PCR reaction solution, TaKaRa LA PCR kit version 2
(Takara Shuzo Co., Ltd.), AP 2, synthetic oligonucleotide GSP
2 using a thermal cycler (GeneAmpR PCR System
2400, Perkin Elmer) at 95 ° C for 10 seconds, 65
The test was performed at 25 ° C. for 30 seconds and 72 ° C. for 5 minutes for 25 cycles. The amplified fragment of about 2.9 kbp obtained from the genomic DNA solution digested with Ssp I was ligated to the pT7 blue T-vector (Novagen) using a DNA ligation kit version 2
(Takara Shuzo Co., Ltd.) and introduced into Escherichia coli DH5α strain to obtain a transformed strain. From the obtained transformant,
The plasmid DNA was extracted and reacted using Dye Terminator Cycle Sequence FS Ready Reaction Kit (Perkin Elmer), and the base sequence of the amplified fragment was determined using a 377 DNA sequencer (Perkin Elmer). This is shown in FIGS. In the obtained clone, a sequence completely matching nucleotide numbers −81 to −25 of the 5 ′ upstream region sequence of the novel human-derived LCAT-like protein cDNA was represented by nucleotide numbers 2788 to 2843 of the nucleotide sequence shown in FIGS. Thus, it was confirmed that this sequence was a sequence of the 5 'upstream region of a novel human-derived LCAT-like protein gene. The analysis of the cis element in this upstream region was examined using GENETYX biodatabase software Ver. 32.0 (software development).

Example 4 Assay for Promoter Activity of Novel LCAT-like Protein Gene Derived from Human In order to confirm that the genomic DNA fragment cloned in Example 3 has promoter activity, alkaline phosphatase, one of the promoter activity test methods, was used. Activity measurements were performed. FIG. 8 shows a method for constructing a plasmid using alkaline phosphatase as a reporter gene. First, using a Mlu I-Hind III fragment, a 2.9 kbp fragment was obtained from a plasmid obtained by cloning about 2.9 kbp from base numbers 1 to 2867 of the base sequence shown in FIGS. 6 and 7 into the pT7 blue T-vector. Release the upstream Ml of the alkaline phosphatase gene of pSEAP-Basic (Clontech)
u Introduced to I-Hind III site. This is E. coli DH 5
The resulting plasmid was introduced into α strain to produce an expression plasmid pTB-2022.
At this time, a 2.9 kbp fragment was isolated using HindIII-EcoRI and introduced into the HindIII-EcoRI site upstream of the alkaline phosphatase gene of pSEAP-Basic, whereby the expression plasmid inserted in the reverse direction was also obtained. It was constructed. This was one of the negative controls. WI38 VA1
Transformation of plasmid into 3 cells can be performed using Trans ITTM-LT1 (Mir
(US company). That is, cells added at a cell concentration of 1 × 10 ⁵ cells / 962 mm ² (6-well plate) were cultured for 24 hours, and then replaced with a serum-free medium, each plasmid 3 μg / well and Trans IT-LTI 10 μl.
/ μg DNA, and introduced by culturing for 4 hours. After the transformation, the cells were cultured in a DMEM medium containing 10% FBS (fetal calf serum), and the cell supernatant was collected 72 hours later.
The alkaline phosphatase activity of the cell supernatant was measured. For measuring alkaline phosphatase activity,
Using Great EscAPeTMSEAP Reporter System 2 (Clontech), an experiment was performed at n = 3 according to this manual. That is, 10 μl of the cell supernatant centrifuged at 12000 × g for 10 minutes was diluted 20-fold with 1 × dilution buffer and treated at 65 ° C. for 30 minutes. 60 μl of the sample was placed in a 96-well microtiter plate, and 60 μl of
ay buffer and 60 μl CSPD Chemiluminescent substra
After adding te and reacting for 15 minutes, alkaline phosphatase activity was measured by LuminoScan / RS (Lab Systems). Next, in order to correct for errors caused by transfection efficiency, pGV-C2 (Nippon Gene) was transfected simultaneously with each expression plasmid, and
After culturing for a period of time, the cell extract was lightly centrifuged and diluted 10-fold. Then, 100 μl of Picagene luminescent substrate (Nippon Gene) was added to 20 μl thereof, and luciferase activity was measured by Luminoscan / RS (Lab Systems). Therefore, the promoter activity was calculated by dividing the value of alkaline phosphatase activity by the value of luciferase activity (Lu
minescence, RLU). FIG. 9 shows the results. As shown in FIG. 9, promoter activity was not observed in the transformant introduced in the reverse direction and in the transformant introduced with pSEAP-Basic, whereas the transformant introduced with pTB-2022 was observed. Showed a significant difference in promoter activity. From these, it was confirmed that a DNA fragment having a functional promoter activity was present in the genomic DNA fragment.

Example 5 Preparation of Rabbit Polyclonal Antibody Partial peptide of human LCAT-like protein [peptide-
I, H-CEDVRGAPYDWRRAPNENGP-
OH (SEQ ID NO: 39)] and a partial peptide [peptide-II, H-PVIGPLKIREQQRSAVSTC
-NH ₂ (SEQ ID NO: 40)] was chemically synthesized, conventional method as K
It bound to LH (Keyhole Limpet Hemocyanin). After mixing Freund's complete adjuvant (FCA) and 500 μg of the above peptide dissolved in physiological saline to form a uniform emulsion, two rabbits each (New Zealand)
White) was injected subcutaneously on the back. Two weeks later, as an additional immunization, Freund's incomplete adjuvant (FIA), the above-mentioned peptide dissolved in physiological saline, and a conjugate of KLH were mixed to form a uniform emulsion, which was then injected subcutaneously into the back of the rabbit. The measurement of the antibody titer was performed as follows.
Four weeks after the final immunization, blood was collected from the vein of the rabbit ear, the blood was kept at 37 ° C. for 30 minutes, left at 4 ° C. for 24 hours, and centrifuged to obtain antiserum. Dilute the antiserum to each concentration,
100 μl of avidin-fixed polystyrene 96-well microplate with biotin-labeled peptides-I and II attached
And incubated at 4 ° C. overnight. After removing the antiserum and washing, an HRP-labeled goat anti-rabbit IgG antibody was added and incubated at 20 ° C. for 1 hour. After the wells were sufficiently washed, a reaction substrate was added and a color reaction was performed. After stopping the reaction by adding 100 μl of an enzyme reaction stop solution, the absorbance at 450 nm was measured using a colorimeter for microplate. The antibody was prepared as follows. Antiserum of anti-peptide-I was converted to peptide-I-Sepharose 6B
The column was further loaded with anti-peptide-II antiserum.
After loading onto an II-Sepharose 6B column and washing with PBS buffer (10 mM, pH 7.2) and physiological saline,
Each was eluted with glycine hydrochloride buffer (100 mM, pH 2.5). The eluate was neutralized by adding a 0.1 M sodium hydroxide solution, and then used as a purified antibody.

Example 6 Localization of LCAT-like Protein in Human Apolipoprotein Fraction by Rabbit Polyclonal Anti-LCAT-like Protein-Peptide Antibody The human apolipoprotein fraction was prepared using two types of IgG antibodies prepared by the method of Example 5. The presence of an LCAT-like protein in it was examined. That is, after adjusting the specific gravity of human plasma with KBr, ultracentrifugation is performed to separate a fraction having a specific gravity of 1.063 g / ml to 1.21 g / ml, and a TBS buffer (10 mM TrisHCl, pH7.
(4, 150 mM NaCl), and then defatted with ethanol / ether. This sample was treated with Apo HDL protein (12.8m
g / ml), and as a result of analysis by Western blotting using two types of IgG antibodies, a product having a molecular weight of about 50,000 which was similarly recognized by each antibody was detected (FIG. 1).
0). Further, in order to examine the presence in other lipoprotein fractions, the specific gravity of human plasma was adjusted with KBr, and then ultracentrifugation was performed to collect a fraction having a specific gravity of 1.21 g / ml or less (lipoprotein fraction). After dialysis, F using Superose6HR (Pharmacia)
Purification was performed by PLC. The elution pattern is shown in FIG. Each of these fractions is TCA (trichloroacetic acid)
After sedimenting and defatting with acetone, the result of analysis by Western blotting using an anti-peptide-II antibody was as shown in FIG.
As shown in the figure, a product with a molecular weight of about 50,000 was detected only in the apoHDL protein fraction, and other apoVLDL proteins and apoLDL
This product was not detected in the protein fraction.

Example 7 Preparation of Recombinant DNA for Expressing Human LCAT-Like Protein / FLAG Fusion Gene in Insect Cells In order to prepare a protein in which the FLAG peptide was fused to the C terminal, FLAG was added to the 5 ′ terminal. (Sequence: DYKDDD
DK) Primer complementary to the upper strand to which the peptide-encoding sequence and the Xho I linker sequence were added [Primer I, 5'-CCGCCTCGAGTCACTTG
TCATC-GTCGGTCGTCCTTGTAGTCG
GGCCCAAGGAGCACACTTTCAG-
3 ′ (SEQ ID NO: 41)] and a primer on the upper strand side upstream of BamHI [Primer II, 5′-GGAG
ACAACC-AACCGGATCCCAGTCATC
GGG-3 ′ (SEQ ID NO: 42)], and the plasmid pTB1973 was used as a template in PC as shown in FIG.
R was performed to obtain a DNA fragment encoding the C-terminal side of the protein, and its nucleotide sequence was confirmed. Eco of pTB1973
A DNA fragment encoding the N-terminal obtained by digestion with RI and BamHI was obtained, and both fragments were pFAST Bac1 (Gibco BRL)
To obtain a target donor plasmid. Example 8 Preparation of Recombinant DNA for Expressing Human LCAT-like Protein Gene in Insect Cells About 1.5 Kb of DNA encoding full-length human LCAT-like protein gene obtained by digesting plasmid pTB1973 with EcoRI and XbaI The fragment was isolated, and this fragment was ligated with pFAST Bac1 digested with the same EcoRI and XbaI to obtain the target donor plasmid.

Example 9 Expression of Human LCAT-Like Protein / FLAG Fusion Gene in Insect Cells Using the donor plasmid described in Example 7, insect cell S
From f9, the transformant virus was transformed into a Bac-To-Bac Baculovirus Expression System (Bac-To-Bac Baculovirus Expression System).
tem; GIBCO BRL). The resulting recombinant virus and insect cell Sf9 moi (number of virus per cell) were infected to adjust to 0.1 and infected.
Cultured for days. When the cell supernatant was collected and subjected to Western blotting, it reacted with both the anti-peptide-II antibody obtained in Example 5 and the anti-FLAG M2 monoclonal antibody (mouse, Cosmo Bio) at a molecular weight of about 47,000. Specific bands were identified. Example 10 Expression of Human LCAT-like Protein Gene in Insect Cells Using the donor plasmid described in Example 8, insect cells S
From f9, the transformant virus was transformed into a Bac-To-Bac Baculovirus Expression System (Bac-To-Bac Baculovirus Expression System).
tem; GIBCO BRL). The recombinant virus thus obtained and insect cell Sf9
The cells were infected with moi (number of viruses per cell) at 0.1 and cultured for 3 days. When the cell supernatant was collected and subjected to Western blotting, a specific band reacting with the anti-peptide-II antibody obtained in Example 5 was confirmed at a molecular weight of about 45,000.

Example 11 Purification of human LCAT-like protein / FLAG fusion protein and determination of N-terminal amino acid sequence Human LCAT-like protein / FLA obtained in Example 10
1 ml of the G fusion protein producing recombinant virus solution was
Insect cells cultured in Excell 400 (JRH)
ive (2.4 × 10 ⁶ cells / ml) for infection. Post-infection cultures were performed at 27 ° C. in spinner flasks for 3 days. The culture supernatant was collected (all the following operations were performed at 4 ° C.), sterilized with a 0.22 μm filter, and dialyzed overnight against TBS buffer (10 mM Tris-HCl, 150 mM NaCl, pH 7.5). , Anti-FLAG M2 affinity column (0.7x10c
m) (Cosmo Bio). After washing with 30 ml of TBS buffer, elution was performed with a FLAG peptide TBS buffer solution (25 to 75 μg / ml) to obtain about 170 μg of the target protein. A part of this solution is subjected to SDS-PAGE and CBB
Staining confirmed that it was a one band at a molecular weight of about 47,000, and that the product was consistent with the band detected by Western blot using an anti-FLAG M2 monoclonal antibody. Also, a part of this solution was SDS-
After PAGE, the amino acid sequence of the product transferred to the IPVH membrane was determined. As a result of analysis by a peptide sequencer (HPG1005A), SEQ ID NO: 43 (AGRHPPVVLV) was obtained. The N-terminus of this protein was the same as the sequence cleaved between positions 33 and 34 of the precursor protein of SEQ ID NO: 6. It turned out to be. Example 12 Crude Purification of Human LCAT-like Protein 1 ml of the human LCAT-like protein-producing recombinant virus solution obtained in Example 10 was added to 50 ml of Excell 400 (JRH).
High Five (2.0 × 10 ⁶ cells / ml) insect cells cultured in a medium
And infected. Post-infection cultures were performed at 27 ° C. in spinner flasks for 3 days. The culture supernatant was collected (all the following operations were performed at 4 ° C.), sterilized with a 0.22 μm filter, and then buffered (4 mM sodium phosphate, 0.5 MN).
aCl, pH 7.4), dialyzed overnight, and equilibrated with the same buffer in a phenyl sepharose HP column (1.5 x 10 cm).
(Manufactured by Pharmacia). After washing with 100 ml of the same buffer, the target protein was eluted while fractionating 2 ml each with about 25 ml of water. FIG. 14 shows the UV absorbance and the elution pattern by Western blot using an anti-peptide-II antibody. It was confirmed that a specific band reacting with the antibody obtained in Example 5 was concentrated around a molecular weight of 45,000.

Example 13 Human LCAT-like protein / FL using fatty acid ester
Determination of esterase activity of AG fusion protein and human LCAT-like protein Bonelli, FS and Jonas, A. [Journal of Biological Chemistry, No. 26]
4, 14723-14728 (1989)]. That is, a reaction buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 0.0
Acetonitrile solution (final concentration: 5 mM to 50 μM) of para-nitrophenyl butyrate (pNPB) was added to 1% EDTA, 1 mM NaN ₃ ) as a substrate, and then human LCAT obtained in Example 9 was added. Protein / FLAG
Add fusion protein (final concentration: 3.4 μg / ml) and add 1 ml total volume
And After incubation at 37 ° C. for 20 minutes, the absorbance at 400 nm was measured every 2 minutes. The result is shown in FIG. Further, the initial velocity was determined from the change rate of the absorbance at each substrate concentration, and Vmax and Km were calculated according to the method of Lineweaver-Burk (FIG. 16). Both the values obtained almost matched the values of LCAT derived from human plasma. In addition, 50 μl of the enzyme consisting of fractions 6 and 7 obtained in Example 12 (FIG. 14) in 4 ml of a crudely purified solution was similarly examined for activity at a substrate concentration of 100 μM, and was confirmed to have esterase activity (FIG. 14). 17). Example 14 Effect of human LCAT-like protein / FLAG fusion protein on esterase activity using DFP or DTNB PNPB Before adding the substrate of Example 13, DFP [Diisopropylfluoro
phosphate] or DTNB [5,5'-Dithiobis- (2-nitrobenzoi
c acid)] and incubated at 25 ° C. for 1 hour to examine whether the activity was inhibited. Substrate concentration is 50
At 0 μM, human LCAT-like protein / FLAG fusion protein had a final concentration of 3.4 μg / ml. As a result, the activity was inhibited by both inhibitors (FIG. 18), suggesting that serine and cysteine are involved in this esterase activity.

[0110]

The protein of the present invention and the DNA encoding the same are useful for treating diseases such as arteriosclerosis, hyperlipidemia, hypercalorie, obesity and hypertriglyceridosis.
It can be used as a prophylactic agent. Further, the protein of the present invention is useful as a reagent for screening a compound or a salt thereof that promotes or inhibits the LCAT-like activity of the protein of the present invention. Furthermore, since an antibody against the protein of the present invention can specifically recognize the protein of the present invention, it can be used for quantification of the protein of the present invention in a test solution.

[0111]

[Sequence list]

[SEQ ID NO: 1] Sequence length: 379 Sequence type: amino acid Topology: linear Sequence type: protein sequence Ala Gly Arg His Pro Pro Val Val Leu Val Pro Gly Asp Leu Gly Asn 1 5 10 15 Gln Leu Glu Ala Lys Leu Asp Lys Pro Thr Val Val His Tyr Leu Cys 20 25 30 Ser Lys Lys Thr Glu Ser Tyr Phe Thr Ile Trp Leu Asn Leu Glu Leu 35 40 45 Leu Leu Pro Val Ile Ile Asp Cys Trp Ile Asp Asn Ile Arg Leu Val 50 55 60 Tyr Asn Lys Thr Ser Arg Ala Thr Gln Phe Pro Asp Gly Val Asp Val 65 70 75 80 Arg Val Pro Gly Phe Gly Lys Thr Phe Ser Leu Glu Plu Leu Asp Pro 85 90 95 Ser Lys Ser Ser Val Gly Ser Tyr Phe His Thr Met Val Glu Ser Leu 100 105 110 Val Gly Trp Gly Tyr Thr Arg Gly Glu Asp Val Arg Gly Ala Pro Tyr 115 120 125 Asp Trp Arg Arg Ala Pro Asn Glu Asn Gly Pro Tyr Phe Leu Ala Leu 130 135 140 Arg Glu Met Ile Glu Glu Met Tyr Gln Leu Tyr Gly Gly Pro Val Val 145 150 155 160 Leu Val Ala His Ser Met Gly Asn Met Tyr Thr Leu Tyr Phe Leu Gln 165 170 175 Arg Gln Pro Gln Ala Trp Lys Asp Lys Tyr Ile Arg Ala Phe Val Ser 180 185 190 Leu Gly Ala Pro Trp Gly Gly Val Ala Lys Thr Leu Arg Val Leu Ala 195 200 205 Ser Gly Asp Asn Asn Arg Ile Pro Val Ile Gly Pro Leu Lys Ile Arg 210 215 220 Glu Gln Gln Arg Ser Ala Val Ser Thr Ser Trp Leu Leu Pro Tyr Asn 225 230 235 240 Tyr Thr Trp Ser Pro Glu Lys Val Phe Val Gln Thr Pro Thr Ile Asn 245 250 255 Tyr Thr Leu Arg Asp Tyr Arg Lys Phe Phe Gln Asp Ile Gly Phe Glu 260 265 270 270 Asp Gly Trp Leu Met Arg Gln Asp Thr Glu Gly Leu Val Glu Ala Thr 275 280 285 Met Pro Pro Gly Val Gln Leu His Cys Leu Tyr Gly Thr Gly Val Pro 290 295 300 Thr Pro Asp Ser Phe Tyr Tyr Glu Ser Phe Pro Asp Arg Asp Pro Lys 305 310 315 320 Ile Cys Phe Gly Asp Gly Asp Gly Thr Val Asn Leu Lys Ser Ala Leu 325 330 335 Gln Cys Gln Ala Trp Gln Ser Arg Gln Glu His Gln Val Leu Leu Gln 340 345 350 Glu Leu Pro Gly Ser Glu His Ile Glu Met Leu Ala Asn Ala Thr Thr 355 360 365 Leu Ala Tyr Leu Lys Arg Val Leu Leu Gly Pro 370 375

[0112]

[SEQ ID NO: 2] Sequence length: 411 Sequence type: amino acid Topology: linear Sequence type: protein sequence Ala Gly Arg His Pro Pro Val Val Leu Val Pro Gly Asp Leu Gly Asn 1 5 10 15 Gln Leu Glu Ala Lys Leu Asp Lys Pro Thr Val Val His Tyr Leu Cys 20 25 30 Ser Lys Lys Thr Glu Ser Tyr Phe Thr Ile Trp Leu Asn Leu Glu Leu 35 40 45 Leu Leu Pro Val Ile Ile Asp Cys Trp Ile Asp Asn Ile Arg Leu Glu 50 55 60 Cys Ser Gly Ala Ile Ser Ala His Tyr Thr Ser Ala Ser Gln Ala Gln 65 70 75 80 Ala Leu Leu Leu Pro Gln Thr Pro Asp Asn Trp Asp Tyr Arg Leu Val 85 90 95 Tyr Asn Lys Thr Ser Arg Ala Thr Gln Phe Pro Asp Gly Val Asp Val 100 105 110 Arg Val Pro Gly Phe Gly Lys Thr Phe Ser Leu Glu Phe Leu Asp Pro 115 120 125 Ser Lys Ser Ser Val Gly Ser Tyr Phe His Thr Met Val Glu Ser Leu 130 135 140 Val Gly Trp Gly Tyr Thr Arg Gly Glu Asp Val Arg Gly Ala Pro Tyr 145 150 155 160 Asp Trp Arg Arg Ala Pro Asn Glu Asn Gly Pro Tyr Phe Leu Ala Leu 165 170 175 Arg Glu Met Ile Glu Glu Met Tyr Gln Leu Tyr Gly Gly Pro Val Val 180 185 190 Leu Val Ala His Ser Met Gly Asn Met Tyr Thr Leu Tyr Phe Leu Gln 195 200 205 Arg Gln Pro Gln Ala Trp Lys Asp Lys Tyr Ile Arg Ala Phe Val Ser 210 215 220 Leu Gly Ala Pro Trp Gly Gly Val Ala Lys Thr Leu Arg Val Leu Ala 225 230 235 240 Ser Gly Asp Asn Asn Arg Ile Pro Val Ile Gly Pro Leu Lys Ile Arg 245 250 255 Glu Gln Gln Arg Ser Ala Val Ser Thr Thr Ser Trp Leu Leu Pro Tyr Asn 260 265 270 Tyr Thr Trp Ser Pro Glu Lys Val Phe Val Gln Thr Pro Thr Ile Asn 275 280 285 Tyr Thr Leu Arg Asp Tyr Arg Lys Phe Phe Gln Asp Ile Gly Phe Glu 290 295 300 Asp Gly Trp Leu Met Arg Gln Asp Thr Glu Gly Leu Val Glu Ala Thr 305 310 315 320 Met Pro Pro Gly Val Gln Leu His Cys Leu Tyr Gly Thr Gly Val Pro 325 330 335 Thr Pro Asp Ser Phe Tyr Tyr Glu Ser Phe Pro Asp Arg Asp Pro Lys 340 345 350 350 Ile Cys Phe Gly Asp Gly Asp Gly Thr Val Asn Leu Lys Ser Ala Leu 355 360 365 Gln Cys Gln Ala Trp Gln Ser Arg Gln Glu His Gln Val Leu Leu Gln 370 375 380 380 Glu Leu Pro Gly Ser Glu HisIle Glu Met Leu Ala Asn Ala Thr Thr 385 390 395 400 Leu Ala Tyr Leu Lys Arg Val Leu Leu Gly Pro 405 410

[0113]

[SEQ ID NO: 3] Sequence length: 379 Sequence type: amino acid Topology: linear Sequence type: protein sequence Ala Gln Arg His Pro Pro Val Val Leu Val Pro Gly Asp Leu Gly Asn 1 5 10 15 Gln Leu Glu Ala Lys Leu Asp Lys Pro Lys Val Val His Tyr Leu Cys 20 25 30 Ser Lys Lys Thr Asp Ser Tyr Phe Thr Leu Trp Leu Asn Leu Glu Leu 35 40 45 Leu Leu Pro Val Ile Ile Asp Cys Trp Ile Asp Asn Ile Arg Leu Val 50 55 60 Tyr Asn Arg Thr Ser Arg Ala Thr Gln Phe Pro Asp Gly Val Asp Val 65 70 75 80 Arg Val Pro Gly Phe Gly Glu Thr Phe Ser Met Glu Phe Leu Asp Pro 85 90 95 Ser Lys Arg Asn Val Gly Ser Tyr Phe Tyr Thr Met Val Glu Ser Leu 100 105 110 Val Gly Trp Gly Tyr Thr Arg Gly Glu Asp Val Arg Gly Ala Pro Tyr 115 120 125 Asp Trp Arg Arg Ala Pro Asn Glu Asn Gly Pro Tyr Phe Leu Ala Leu 130 135 140 Arg Glu Met Ile Glu Glu Met Tyr Gln Met Tyr Gly Gly Pro Val Val 145 150 155 160 Leu Val Ala His Ser Met Gly Asn Val Tyr Met Leu Tyr Phe Leu Gln 165 170 175 Arg Gln Pro Gln Val Trp Lys Asp Lys Tyr Ile His Ala Phe Val Ser 180 185 190 Leu Gly Ala Pro Trp Gly Gly Val Ala Lys Thr Leu Arg Val Leu Ala 195 200 205 Ser Gly Asp Asn Asn Arg Ile Pro Val Ile Gly Pro Leu Lys Ile Arg 210 215 220 Glu Gln Gln Arg Ser Ala Val Ser Thr Ser Trp Leu Leu Pro Tyr Asn 225 230 235 240 His Thr Trp Ser His Glu Lys Val Phe Val Tyr Thr Pro Thr Thr Asn 245 250 255 Tyr Thr Leu Arg Asp Tyr His Arg Phe Phe Arg Asp Ile Gly Phe Glu 260 265 270 Asp Gly Trp Phe Met Arg Gln Asp Thr Glu Gly Leu Val Glu Ala Met 275 280 285 Thr Pro Pro Gly Val Glu Leu His Cys Leu Tyr Gly Thr Gly Val Pro 290 295 300 Thr Pro Asn Ser Phe Tyr Tyr Glu Ser Phe Pro Asp Arg Asp Pro Lys 305 310 315 320 Ile Cys Phe Gly Asp Gly Asp Gly Thr Val Asn Leu Glu Ser Val Leu 325 330 335 Gln Cys Gln Ala Trp Gln Ser Arg Gln Glu His Arg Val Ser Leu Gln 340 345 350 Glu Leu Pro Gly Ser Glu His Ile Glu Met Leu Ala Asn Ala Thr Thr 355 360 365 Leu Ala Tyr Leu Lys Arg Val Leu Leu Glu Pro 370 375

[0114]

[SEQ ID NO: 4] Sequence length: 382 Sequence type: amino acid Topology: linear Sequence type: protein sequence Ala Leu Pro Ala Gly Arg His Pro Pro Val Val Leu Val Pro Gly Asp 1 5 10 15 Leu Gly Asn Gln Leu Glu Ala Lys Leu Asp Lys Pro Thr Val Val His 20 25 30 Tyr Leu Cys Ser Lys Lys Thr Glu Ser Tyr Phe Thr Ile Trp Leu Asn 35 40 45 Leu Glu Leu Leu Leu Pro Val Ile Ile Asp Cys Trp Ile Asp Asn Ile 50 55 60 Arg Leu Val Tyr Asn Lys Thr Ser Arg Ala Thr Gln Phe Pro Asp Gly 65 70 75 80 Val Asp Val Arg Val Pro Gly Phe Gly Lys Thr Phe Ser Leu Glu Phe 85 90 95 Leu Asp Pro Ser Lys Ser Ser Val Gly Ser Tyr Phe His Thr Met Val 100 105 110 Glu Ser Leu Val Gly Trp Gly Tyr Thr Arg Gly Glu Asp Val Arg Gly 115 120 125 Ala Pro Tyr Asp Trp Arg Arg Ala Pro Asn Glu Asn Gly Pro Tyr Phe 130 135 140 Leu Ala Leu Arg Glu Met Ile Glu Glu Met Tyr Gln Leu Tyr Gly Gly 145 150 155 160 Pro Val Val Leu Val Ala His Ser Met Gly Asn Met Tyr Thr Leu Tyr 165 170 175 Phe Leu Gln Arg Gln Pro Gln Ala Trp Lys Asp Lys Tyr Ile Arg Ala 180 185 190 Phe Val Ser Leu Gly Ala Pro Trp Gly Gly Val Ala Lys Thr Leu Arg 195 200 205 Val Leu Ala Ser Gly Asp Asn Asn Arg Ile Pro Val Ile Gly Pro Leu 210 215 220 Lys Ile Arg Glu Gln Gln Arg Ser Ala Val Ser Thr Ser Trp Leu Leu 225 230 235 240 Pro Tyr Asn Tyr Thr Trp Ser Pro Glu Lys Val Phe Val Gln Thr Pro 245 250 255 Thr Ile Asn Tyr Thr Leu Arg Asp Tyr Arg Lys Phe Phe Gln Asp Ile 260 265 270 Gly Phe Glu Asp Gly Trp Leu Met Arg Gln Asp Thr Glu Gly Leu Val 275 280 285 Glu Ala Thr Met Pro Pro Gly Val Gln Leu His Cys Leu Tyr Gly Thr 290 295 300 Gly Val Pro Thr Pro Asp Ser Phe Tyr Tyr Glu Ser Phe Pro Asp Arg 305 310 315 320 Asp Pro Lys Ile Cys Phe Gly Asp Gly Asp Gly Thr Val Asn Leu Lys 325 330 335 Ser Ala Leu Gln Cys Gln Ala Trp Gln Ser Arg Gln Glu His Gln Val 340 345 350 Leu Leu Gln Glu Leu Pro Gly Ser Glu His Ile Glu Met Leu Ala Asn 355 360 365 Ala Thr Thr Leu Ala Tyr Leu Lys Arg Val Leu Leu Gly Pro 370 375 380

[0115]

[SEQ ID NO: 5] Sequence length: 414 Sequence type: amino acid Topology: linear Sequence type: protein sequence Ala Leu Pro Ala Gly Arg His Pro Pro Val Val Leu Val Pro Gly Asp 1 5 10 15 Leu Gly Asn Gln Leu Glu Ala Lys Leu Asp Lys Pro Thr Val Val His 20 25 30 Tyr Leu Cys Ser Lys Lys Thr Glu Ser Tyr Phe Thr Ile Trp Leu Asn 35 40 45 Leu Glu Leu Leu Leu Pro Val Ile Ile Asp Cys Trp Ile Asp Asn Ile 50 55 60 Arg Leu Glu Cys Ser Gly Ala Ile Ser Ala His Tyr Thr Ser Ala Ser 65 70 75 80 Gln Ala Gln Ala Leu Leu Leu Pro Gln Thr Pro Asp Asn Trp Asp Tyr 85 90 95 Arg Leu Val Tyr Asn Lys Thr Ser Arg Ala Thr Gln Phe Pro Asp Gly 100 105 110 Val Asp Val Arg Val Pro Gly Phe Gly Lys Thr Phe Ser Leu Glu Phe 115 120 125 Leu Asp Pro Ser Lys Ser Ser Val Gly Ser Tyr Phe His Thr Met Val 130 135 140 Glu Ser Leu Val Gly Trp Gly Tyr Thr Arg Gly Glu Asp Val Arg Gly 145 150 155 160 Ala Pro Tyr Asp Trp Arg Arg Ala Pro Asn Glu Asn Gly Pro Tyr Phe 165 170 175 Leu Ala Leu Arg Glu Met Ile Glu Glu Met Tyr Gln Leu Tyr Gly Gly 180 185 190 Pro Val Val Leu Val Ala His Ser Met Gly Asn Met Tyr Thr Leu Tyr 195 200 205 Phe Leu Gln Arg Gln Pro Gln Ala Trp Lys Asp Lys Tyr Ile Arg Ala 210 215 220 Phe Val Ser Leu Gly Ala Pro Trp Gly Gly Val Ala Lys Thr Leu Arg 225 230 235 240 Val Leu Ala Ser Gly Asp Asn Asn Arg Ile Pro Val Ile Gly Pro Leu 245 250 255 Lys Ile Arg Glu Gln Gln Arg Ser Ala Val Ser Thr Ser Trp Leu Leu 260 265 270 Pro Tyr Asn Tyr Thr Trp Ser Pro Glu Lys Val Phe Val Gln Thr Pro 275 280 285 Thr Ile Asn Tyr Thr Leu Arg Asp Tyr Arg Lys Phe Phe Gln Asp Ile 290 295 300 Gly Phe Glu Asp Gly Trp Leu Met Arg Gln Asp Thr Glu Gly Leu Val 305 310 315 320 Glu Ala Thr Met Pro Pro Gly Val Gln Leu His Cys Leu Tyr Gly Thr 325 330 335 Gly Val Pro Thr Pro Asp Ser Phe Tyr Tyr Glu Ser Phe Pro Asp Arg 340 345 350 Asp Pro Lys Ile Cys Phe Gly Asp Gly Asp Gly Thr Val Asn Leu Lys 355 360 365 Ser Ala Leu Gln Cys Gln Ala Trp Gln Ser Arg Gln Glu His Gln Val 370 375 380 380 Leu Leu Gln Glu Leu Pro GlySer Glu His Ile Glu Met Leu Ala Asn 385 390 395 400 Ala Thr Thr Leu Ala Tyr Leu Lys Arg Val Leu Leu Gly Pro 405 410

[0116]

[SEQ ID NO: 6] Sequence length: 412 Sequence type: amino acid Topology: linear Sequence type: protein sequence Met Gly Leu His Leu Arg Pro Tyr Arg Val Gly Leu Leu Pro Asp Gly 1 5 10 15 Leu Leu Phe Leu Leu Leu Leu Leu Met Leu Leu Ala Asp Pro Ala Leu 20 25 30 Pro Ala Gly Arg His Pro Pro Val Val Leu Val Pro Gly Asp Leu Gly 35 40 45 Asn Gln Leu Glu Ala Lys Leu Asp Lys Pro Thr Val Val His Tyr Leu 50 55 60 Cys Ser Lys Lys Thr Glu Ser Tyr Phe Thr Ile Trp Leu Asn Leu Glu 65 70 75 80 Leu Leu Leu Pro Val Ile Ile Asp Cys Trp Ile Asp Asn Ile Arg Leu 85 90 95 Val Tyr Asn Lys Thr Ser Arg Ala Thr Gln Phe Pro Asp Gly Val Asp 100 105 110 Val Arg Val Pro Gly Phe Gly Lys Thr Phe Ser Leu Glu Phe Leu Asp 115 120 125 Pro Ser Lys Ser Ser Val Gly Ser Tyr Phe His Thr Met Val Glu Ser 130 135 140 Leu Val Gly Trp Gly Tyr Thr Arg Gly Glu Asp Val Arg Gly Ala Pro 145 150 155 160 Tyr Asp Trp Arg Arg Ala Pro Asn Glu Asn Gly Pro Tyr Phe Leu Ala 165 170 175 Leu Arg Glu Met Ile Glu Glu Met Tyr Gln Leu Tyr Gly Gly Pro Val 180 185 190 Val Leu Val Ala His Ser Met Gly Asn Met Tyr Thr Leu Tyr Phe Leu 195 200 205 Gln Arg Gln Pro Gln Ala Trp Lys Asp Lys Tyr Ile Arg Ala Phe Val 210 215 220 Ser Leu Gly Ala Pro Trp Gly Gly Val Ala Lys Thr Leu Arg Val Leu 225 230 235 240 Ala Ser Gly Asp Asn Asn Arg Ile Pro Val Ile Gly Pro Leu Lys Ile 245 250 255 Arg Glu Gln Gln Arg Ser Ala Val Ser Thr Ser Trp Leu Leu Pro Tyr 260 265 270 Asn Tyr Thr Trp Ser Pro Glu Lys Val Phe Val Gln Thr Pro Thr Ile 275 280 285 Asn Tyr Thr Leu Arg Asp Tyr Arg Lys Phe Phe Gln Asp Ile Gly Phe 290 295 300 Glu Asp Gly Trp Leu Met Arg Gln Asp Thr Glu Gly Leu Val Glu Ala 305 310 315 320 Thr Met Pro Pro Gly Val Gln Leu His Cys Leu Tyr Gly Thr Gly Val 325 330 335 Pro Thr Pro Asp Ser Phe Tyr Tyr Glu Ser Phe Pro Asp Arg Asp Pro 340 345 350 Lys Ile Cys Phe Gly Asp Gly Asp Gly Thr Val Asn Leu Lys Ser Ala 355 360 365 Leu Gln Cys Gln Ala Trp Gln Ser Arg Gln Glu His Gln Val Leu Leu 370 375 380 380 Gln Glu Leu Pro Gly Ser GluHis Ile Glu Met Leu Ala Asn Ala Thr 385 390 395 400 Thr Leu Ala Tyr Leu Lys Arg Val Leu Leu Gly Pro 405 410

[0117]

[SEQ ID NO: 7] Sequence length: 444 Sequence type: amino acid Topology: linear Sequence type: protein sequence Met Gly Leu His Leu Arg Pro Tyr Arg Val Gly Leu Leu Pro Asp Gly 1 5 10 15 Leu Leu Phe Leu Leu Leu Leu Leu Met Leu Leu Ala Asp Pro Ala Leu 20 25 30 Pro Ala Gly Arg His Pro Pro Val Val Leu Val Pro Gly Asp Leu Gly 35 40 45 Asn Gln Leu Glu Ala Lys Leu Asp Lys Pro Thr Val Val His Tyr Leu 50 55 60 Cys Ser Lys Lys Thr Glu Ser Tyr Phe Thr Ile Trp Leu Asn Leu Glu 65 70 75 80 Leu Leu Leu Pro Val Ile Ile Asp Cys Trp Ile Asp Asn Ile Arg Leu 85 90 95 Glu Cys Ser Gly Ala Ile Ser Ala His Tyr Thr Ser Ala Ser Gln Ala 100 105 110 Gln Ala Leu Leu Leu Pro Gln Thr Pro Asp Asn Trp Asp Tyr Arg Leu 115 120 125 Val Tyr Asn Lys Thr Ser Arg Ala Thr Gln Phe Pro Asp Gly Val Asp 130 135 140 Val Arg Val Pro Gly Phe Gly Lys Thr Phe Ser Leu Glu Phe Leu Asp 145 150 155 160 Pro Ser Lys Ser Ser Val Gly Ser Tyr Phe His Thr Met Val Glu Ser 165 170 175 Leu Val Gly Trp Gly Tyr Thr Arg Gly Glu Asp Val Arg Gly Ala Pro 180 185 190 Tyr Asp Trp Arg Arg Ala Pro Asn Glu Asn Gly Pro Tyr Phe Leu Ala 195 200 205 Leu Arg Glu Met Ile Glu Glu Met Tyr Gln Leu Tyr Gly Gly Pro Val 210 215 220 Val Leu Val Ala His Ser Met Gly Asn Met Tyr Thr Leu Tyr Phe Leu 225 230 235 240 Gln Arg Gln Pro Gln Ala Trp Lys Asp Lys Tyr Ile Arg Ala Phe Val 245 250 255 Ser Leu Gly Ala Pro Trp Gly Gly Val Ala Lys Thr Leu Arg Val Leu 260 265 270 Ala Ser Gly Asp Asn Asn Arg Ile Pro Val Ile Gly Pro Leu Lys Ile 275 280 285 Arg Glu Gln Gln Arg Ser Ala Val Ser Thr Ser Trp Leu Leu Pro Tyr 290 295 300 Asn Tyr Thr Trp Ser Pro Glu Lys Val Phe Val Gln Thr Pro Thr Ile 305 310 315 320 Asn Tyr Thr Leu Arg Asp Tyr Arg Lys Phe Phe Gln Asp Ile Gly Phe 325 330 335 Glu Asp Gly Trp Leu Met Arg Gln Asp Thr Glu Gly Leu Val Glu Ala 340 345 350 Thr Met Pro Pro Gly Val Gln Leu His Cys Leu Tyr Gly Thr Gly Val 355 360 365 Pro Thr Pro Asp Ser Phe Tyr Tyr Glu Ser Phe Pro Asp Arg Asp Pro 370 375 380 Lys Ile Cys Phe Gly Asp GlyAsp Gly Thr Val Asn Leu Lys Ser Ala 285 390 395 400 400 Leu Gln Cys Gln Ala Trp Gln Ser Arg Gln Glu His Gln Val Leu Leu 405 410 415 Gln Glu Leu Pro Gly Ser Glu His Ile Glu Met Leu Ala Asn Ala Thr 420 425 430 Thr Leu Ala Tyr Leu Lys Arg Val Leu Leu Gly Pro 435 440

[0118]

[SEQ ID NO: 8] Sequence length: 412 Sequence type: amino acid Topology: linear Sequence type: protein sequence Met Asp Arg His Leu Cys Thr Cys Arg Glu Thr Gln Leu Arg Ser Gly 1 5 10 15 Leu Leu Leu Pro Leu Phe Leu Leu Met Met Leu Ala Asp Leu Thr Leu 20 25 30 Pro Ala Gln Arg His Pro Pro Val Val Leu Val Pro Gly Asp Leu Gly 35 40 45 Asn Gln Leu Glu Ala Lys Leu Asp Lys Pro Lys Val Val His Tyr Leu 50 55 60 Cys Ser Lys Lys Thr Asp Ser Tyr Phe Thr Leu Trp Leu Asn Leu Glu 65 70 75 80 Leu Leu Leu Pro Val Ile Ile Asp Cys Trp Ile Asp Asn Ile Arg Leu 85 90 95 Val Tyr Asn Arg Thr Ser Arg Ala Thr Gln Phe Pro Asp Gly Val Asp 100 105 110 Val Arg Val Pro Gly Phe Gly Glu Thr Phe Ser Met Glu Phe Leu Asp 115 120 125 Pro Ser Lys Arg Asn Val Gly Ser Tyr Phe Tyr Thr Met Val Glu Ser 130 135 140 Leu Val Gly Trp Gly Tyr Thr Arg Gly Glu Asp Val Arg Gly Ala Pro 145 150 155 160 Tyr Asp Trp Arg Arg Ala Pro Asn Glu Asn Gly Pro Tyr Phe Leu Ala 165 170 175 Leu Arg Glu Met Ile Glu Glu Met Tyr Gln Met Tyr Gly Gly Pro Val 180 185 190 Val Leu Val Ala His Ser Met Gly Asn Val Tyr Met Leu Tyr Phe Leu 195 200 205 Gln Arg Gln Pro Gln Val Trp Lys Asp Lys Tyr Ile His Ala Phe Val 210 215 220 Ser Leu Gly Ala Pro Trp Gly Gly Val Ala Lys Thr Leu Arg Val Leu 225 230 235 240 Ala Ser Gly Asp Asn Asn Arg Ile Pro Val Ile Gly Pro Leu Lys Ile 245 250 255 Arg Glu Gln Gln Arg Ser Ala Val Ser Thr Ser Trp Leu Leu Pro Tyr 260 265 270 Asn His Thr Trp Ser His Glu Lys Val Phe Val Tyr Thr Pro Thr Thr 275 280 285 Asn Tyr Thr Leu Arg Asp Tyr His Arg Phe Phe Arg Asp Ile Gly Phe 290 295 300 Glu Asp Gly Trp Phe Met Arg Gln Asp Thr Glu Gly Leu Val Glu Ala 305 310 315 320 Met Thr Pro Pro Gly Val Glu Leu His Cys Leu Tyr Gly Thr Gly Val 325 330 335 Pro Thr Pro Asn Ser Phe Tyr Tyr Glu Ser Phe Pro Asp Arg Asp Pro 340 345 350 350 Lys Ile Cys Phe Gly Asp Gly Asp Gly Thr Val Asn Leu Glu Ser Val 355 360 365 Leu Gln Cys Gln Ala Trp Gln Ser Arg Gln Glu His Arg Val Ser Leu 370 375 380 Gln Glu Leu Pro Gly Ser GluHis Ile Glu Met Leu Ala Asn Ala Thr 385 390 395 400 Thr Leu Ala Tyr Leu Lys Arg Val Leu Leu Glu Pro 405 410

[0119]

[SEQ ID NO: 9] Sequence length: 30 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Met Gly Leu His Leu Arg Pro Tyr Arg
Val Gly Leu Leu Pro Asp Gly 15
10 15 Leu Leu Phe Leu Leu Leu Leu Leu Met
Leu Leu Ala Asp Pro 20 25
30

[0120]

[SEQ ID NO: 10] Sequence length: 33 Sequence type: amino acid Topology: Linear Sequence type: Peptide sequence Met Gly Leu His Leu Arg Pro Tyr Arg
Val Gly Leu Leu Pro Asp Gly 15
10 15 Leu Leu Phe Leu Leu Leu Leu Leu Met
Leu Leu Ala Asp Pro Ala Leu 20 25
30 Pro

[0121]

[SEQ ID NO: 11] Sequence length: 33 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Met Asp Arg His Leu Cys Thr Cys Arg Glu Thr Gln Leu Arg Ser Gly 1 5 10 15 Leu Leu Leu Pro Leu Phe Leu Leu Met Met Leu Ala Asp Leu Thr Leu 20 25 30 Pro

[0122]

[SEQ ID NO: 12] Sequence length: 1137 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA sequence GCCGGACGTC ACCCCCCAGT GGTGCTGGTC CCTGGTGATT TGGGTAACCA ACTGGAAGCC 60 AAGCTGGACA AGCCGACAGT GGTGCACTAC CTCTGCTCAGCAACT TGAACCTGGA ACTGCTGCTG CCTGTCATCA TTGACTGCTG GATTGACAAT 180 ATCAGGCTGG TTTACAACAA AACATCCAGG GCCACCCAGT TTCCTGATGG TGTGGATGTA 240 CGTGTCCCTG GCTTTGGGAA GACCTTCTCA CTGGAGTTCC TGGACCCCAG CAAAAGCAGC 300 GTGGGTTCCT ATTTCCACAC CATGGTGGAG AGCCTTGTGG GCTGGGGCTA CACACGGGGT 360 GAGGATGTCC GAGGGGCTCC CTATGACTGG CGCCGAGCCC CAAATGAAAA CGGGCCCTAC 420 TTCCTGGCCC TCCGCGAGAT GATCGAGGAG ATGTACCAGC TGTATGGGGG CCCCGTGGTG 480 CTGGTTGCCC ACAGTATGGG CAACATGTAC ACGCTCTACT TTCTGCAGCG GCAGCCGCAG 540 GCCTGGAAGG ACAAGTATAT CCGGGCCTTC GTGTCACTGG GTGCGCCCTG GGGGGGCGTG 600 GCCAAGACCC TGCGCGTCCT GGCTTCAGGA GACAACAACC GGATCCCAGT CATCGGGCCC 660 CTGAAGATCC GGGAGCAGCA GCGGTCAGCT GTCTCCACCA GCTGGCTGCT GCCCTAC AAC 720 TACACATGGT CACCTGAGAA GGTGTTCGTG CAGACACCCA CAATCAACTA CACACTGCGG 780 GACTACCGCA AGTTCTTCCA GGACATCGGC TTTGAAGATG GCTGGCTCAT GCGGCAGGAC 840 ACAGAAGGGC TGGTGGAAGC CACGATGCCA CCTGGCGTGC AGCTGCACTG CCTCTATGGC 900 ACTGGCGTCC CCACACCAGA CTCCTTCTAC TATGAGAGCT TCCCTGACCG TGACCCTAAA 960 ATCTGCTTTG GTGACGGCGA TGGTACTGTG AACTTGAAGA GTGCCCTGCA GTGCCAGGCC 1020 TGGCAGAGCC GCCAGGAGCA CCAAGTGTTG CTGCAGGAGC TGCCAGGCAG CGAGCACATC 1080 GAGATGCTGG CCAACGCCAC CACCCTGGCC TATCTGAAAC GTGTGCTCCT TGGGCCC 1137

[0123]

[SEQ ID NO: 13] Sequence length: 1233 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA sequence GCCGGACGTC ACCCCCCAGT GGTGCTGGTC CCTGGTGATT TGGGTAACCA ACTGGAAGCC 60 AAGCTGGACA AGCCGACAGT GGTGCACTCACTCTGCTCAGCTAGACT TGAACCTGGA ACTGCTGCTG CCTGTCATCA TTGACTGCTG GATTGACAAT 180 ATCAGGCTGG AGTGCAGTGG CGCAATCTCG GCTCACTACA CCTCTGCCTC CCAGGCTCAA 240 GCACTTCTCC TGCCTCAGAC TCCGGATAAC TGGGATTACA GGCTGGTTTA CAACAAAACA 300 TCCAGGGCCA CCCAGTTTCC TGATGGTGTG GATGTACGTG TCCCTGGCTT TGGGAAGACC 360 TTCTCACTGG AGTTCCTGGA CCCCAGCAAA AGCAGCGTGG GTTCCTATTT CCACACCATG 420 GTGGAGAGCC TTGTGGGCTG GGGCTACACA CGGGGTGAGG ATGTCCGAGG GGCTCCCTAT 480 GACTGGCGCC GAGCCCCAAA TGAAAACGGG CCCTACTTCC TGGCCCTCCG CGAGATGATC 540 GAGGAGATGT ACCAGCTGTA TGGGGGCCCC GTGGTGCTGG TTGCCCACAG TATGGGCAAC 600 ATGTACACGC TCTACTTTCT GCAGCGGCAG CCGCAGGCCT GGAAGGACAA GTATATCCGG 660 GCCTTCGTGT CACTGGGTGC GCCCTGGGGG GGCGTGGCCA AGACCCTGCG CGTCCTG GCT 720 TCAGGAGACA ACAACCGGAT CCCAGTCATC GGGCCCCTGA AGATCCGGGA GCAGCAGCGG 780 TCAGCTGTCT CCACCAGCTG GCTGCTGCCC TACAACTACA CATGGTCACC TGAGAAGGTG 840 TTCGTGCAGA CACCCACAAT CAACTACACA CTGCGGGACT ACCGCAAGTT CTTCCAGGAC 900 ATCGGCTTTG AAGATGGCTG GCTCATGCGG CAGGACACAG AAGGGCTGGT GGAAGCCACG 960 ATGCCACCTG GCGTGCAGCT GCACTGCCTC TATGGTACTG GCGTCCCCAC ACCAGACTCC 1020 TTCTACTATG AGAGCTTCCC TGACCGTGAC CCTAAAATCT GCTTTGGTGA CGGCGATGGT 1080 ACTGTGAACT TGAAGAGTGC CCTGCAGTGC CAGGCCTGGC AGAGCCGCCA GGAGCACCAA 1140 GTGTTGCTGC AGGAGCTGCC AGGCAGCGAG CACATCGAGA TGCTGGCCAA CGCCACCACC 1200 CTGGCCTATC TGAAACGTGT GCTCCTTGGG CCC 1233

[0124]

[SEQ ID NO: 14] Sequence length: 1137 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA sequence GCCCAACGTC ACCCCCCGGT GGTGCTGGTG CCTGGTGATT TGGGTAACCA GTTGGAAGCA 60 AAGCTGGATA AGCCAAAGGT TGTACACTACCTCGTC 120 TGAATCTGGA ACTGCTTCTG CCTGTTATCA TTGACTGCTG GATTGACAAT 180 ATCAGGCTGG TTTACAACAG AACATCTCGG GCCACCCAGT TTCCCGATGG TGTGGACGTG 240 CGTGTCCCTG GCTTTGGGGA AACATTTTCT ATGGAATTCC TAGACCCCAG CAAGAGGAAT 300 GTGGGTTCCT ATTTCTACAC TATGGTGGAG AGCCTTGTGG GCTGGGGCTA CACACGGGGT 360 GAAGACGTTC GAGGTGCTCC CTATGATTGG CGGCGAGCCC CAAATGAAAA CGGGCCCTAC 420 TTCTTGGCCC TGCGAGAGAT GATCGAGGAG ATGTACCAGA TGTATGGGGG CCCCGTGGTG 480 CTGGTCGCCC ACAGCATGGG CAACGTGTAC ATGCTCTACT TTCTGCAGCG GCAGCCACAA 540 GTCTGGAAGG ACAAATATAT CCATGCCTTC GTCTCACTGG GGGCGCCCTG GGGGGGCGTG 600 GCCAAGACGC TGCGTGTCCT GGCCTCAGGA GACAACAATC GCATTCCCGT CATTGGGCCA 660 CTGAAGATCC GGGAACAGCA GCGATCTGCC GTCTCTACCA GCTGGCTACT GCCATAC AAC 720 CACACTTGGT CACATGAAAA GGTATTTGTA TACACACCCA CGACTAACTA CACGCTCCGG 780 GACTATCACC GGTTCTTCCG GGACATCGGT TTCGAAGATG GCTGGTTCAT GCGGCAGGAC 840 ACAGAAGGGC TGGTTGAAGC CATGACGCCA CCCGGGGTGG AGCTGCACTG CTTGTATGGC 900 ACTGGTGTTC CCACGCCAAA CTCTTTCTAC TACGAGAGCT TTCCTGATCG GGACCCCAAA 960 ATCTGCTTCG GCGATGGTGA CGGCACGGTG AACCTGGAGA GCGTCCTGCA GTGCCAAGCC 1020 TGGCAGAGCC GCCAAGAGCA CAGAGTATCA TTGCAGGAGC TGCCGGGAAG CGAGCACATT 1080 GAGATGCTAG CCAATGCCAC CACCTTGGCT TATCTGAAAC GTGTGCTTCT GGAACCT 1137

[0125]

[SEQ ID NO: 15] Sequence length: 1146 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA sequence GCGCTCCCGG CCGGACGTCA CCCCCCAGTG GTGCTGGTCC CTGGTGATTT GGGTAACCAA 60 CTGGAAGCCA AGCTGGACAA GCCGACAGTG GTGCACTACC TCTGTCA CCAG GAACT CAATCTGGCT GAACCTGGAA CTGCTGCTGC CTGTCATCAT TGACTGCTGG 180 ATTGACAATA TCAGGCTGGT TTACAACAAA ACATCCAGGG CCACCCAGTT TCCTGATGGT 240 GTGGATGTAC GTGTCCCTGG CTTTGGGAAG ACCTTCTCAC TGGAGTTCCT GGACCCCAGC 300 AAAAGCAGCG TGGGTTCCTA TTTCCACACC ATGGTGGAGA GCCTTGTGGG CTGGGGCTAC 360 ACACGGGGTG AGGATGTCCG AGGGGCTCCC TATGACTGGC GCCGAGCCCC AAATGAAAAC 420 GGGCCCTACT TCCTGGCCCT CCGCGAGATG ATCGAGGAGA TGTACCAGCT GTATGGGGGC 480 CCCGTGGTGC TGGTTGCCCA CAGTATGGGC AACATGTACA CGCTCTACTT TCTGCAGCGG 540 CAGCCGCAGG CCTGGAAGGA CAAGTATATC CGGGCCTTCG TGTCACTGGG TGCGCCCTGG 600 GGGGGCGTGG CCAAGACCCT GCGCGTCCTG GCTTCAGGAG ACAACAACCG GATCCCAGTC 660 ATCGGGCCCC TGAAGATCCG GGAGCAGCAG CGGTCAGCTG TCTCCACCAG CTGGCTG CTG 720 CCCTACAACT ACACATGGTC ACCTGAGAAG GTGTTCGTGC AGACACCCAC AATCAACTAC 780 ACACTGCGGG ACTACCGCAA GTTCTTCCAG GACATCGGCT TTGAAGATGG CTGGCTCATG 840 CGGCAGGACA CAGAAGGGCT GGTGGAAGCC ACGATGCCAC CTGGCGTGCA GCTGCACTGC 900 CTCTATGGCA CTGGCGTCCC CACACCAGAC TCCTTCTACT ATGAGAGCTT CCCTGACCGT 960 GACCCTAAAA TCTGCTTTGG TGACGGCGAT GGTACTGTGA ACTTGAAGAG TGCCCTGCAG 1020 TGCCAGGCCT GGCAGAGCCG CCAGGAGCAC CAAGTGTTGC TGCAGGAGCT GCCAGGCAGC 1080 GAGCACATCG AGATGCTGGC CAACGCCACC ACCCTGGCCT ATCTGAAACG TGTGCTCCTT 1140 GGGCCC 1146

[0126]

[SEQ ID NO: 16] Sequence length: 1242 Sequence type: nucleic acid Number of strands: double strand Topology: linear Sequence type: cDNA sequence GCGCTCCCGG CCGGACGTCA CCCCCCAGTG GTGCTGGTCC CTGGTGATTT GGGTAACCAA 60 CTGGAAGCCA AGCTGGACAA GCCGACAGTG GTGCACTACC TCTGCTCCAGCT CAATCTGGCT GAACCTGGAA CTGCTGCTGC CTGTCATCAT TGACTGCTGG 180 ATTGACAATA TCAGGCTGGA GTGCAGTGGC GCAATCTCGG CTCACTACAC CTCTGCCTCC 240 CAGGCTCAAG CACTTCTCCT GCCTCAGACT CCGGATAACT GGGATTACAG GCTGGTTTAC 300 AACAAAACAT CCAGGGCCAC CCAGTTTCCT GATGGTGTGG ATGTACGTGT CCCTGGCTTT 360 GGGAAGACCT TCTCACTGGA GTTCCTGGAC CCCAGCAAAA GCAGCGTGGG TTCCTATTTC 420 CACACCATGG TGGAGAGCCT TGTGGGCTGG GGCTACACAC GGGGTGAGGA TGTCCGAGGG 480 GCTCCCTATG ACTGGCGCCG AGCCCCAAAT GAAAACGGGC CCTACTTCCT GGCCCTCCGC 540 GAGATGATCG AGGAGATGTA CCAGCTGTAT GGGGGCCCCG TGGTGCTGGT TGCCCACAGT 600 ATGGGCAACA TGTACACGCT CTACTTTCTG CAGCGGCAGC CGCAGGCCTG GAAGGACAAG 660 TATATCCGGG CCTTCGTGTC ACTGGGTGCG CCCTGGGGGG GCGTGGCCAA GACCCTG CGC 720 GTCCTGGCTT CAGGAGACAA CAACCGGATC CCAGTCATCG GGCCCCTGAA GATCCGGGAG 780 CAGCAGCGGT CAGCTGTCTC CACCAGCTGG CTGCTGCCCT ACAACTACAC ATGGTCACCT 840 GAGAAGGTGT TCGTGCAGAC ACCCACAATC AACTACACAC TGCGGGACTA CCGCAAGTTC 900 TTCCAGGACA TCGGCTTTGA AGATGGCTGG CTCATGCGGC AGGACACAGA AGGGCTGGTG 960 GAAGCCACGA TGCCACCTGG CGTGCAGCTG CACTGCCTCT ATGGTACTGG CGTCCCCACA 1020 CCAGACTCCT TCTACTATGA GAGCTTCCCT GACCGTGACC CTAAAATCTG CTTTGGTGAC 1080 GGCGATGGTA CTGTGAACTT GAAGAGTGCC CTGCAGTGCC AGGCCTGGCA GAGCCGCCAG 1140 GAGCACCAAG TGTTGCTGCA GGAGCTGCCA GGCAGCGAGC ACATCGAGAT GCTGGCCAAC 1200 GCCACCACCC TGGCCTATCT GAAACGTGTG CTCCTTGGGC CC 1242

[0127]

[SEQ ID NO: 17] Sequence length: 1236 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA sequence ATGGGCCTCC ACCTCCGCCC CTACCGTGTG GGGCTGCTCC CGGATGGCCT CCTGTTCCTC 60 TTGCTGCTGC TAATGCTGCT CGCGGACCCA GCGCTCCCGGCCAG CTGGTGATTT GGGTAACCAA CTGGAAGCCA AGCTGGACAA GCCGACAGTG 180 GTGCACTACC TCTGCTCCAA GAAGACCGAA AGCTACTTCA CAATCTGGCT GAACCTGGAA 240 CTGCTGCTGC CTGTCATCAT TGACTGCTGG ATTGACAATA TCAGGCTGGT TTACAACAAA 300 ACATCCAGGG CCACCCAGTT TCCTGATGGT GTGGATGTAC GTGTCCCTGG CTTTGGGAAG 360 ACCTTCTCAC TGGAGTTCCT GGACCCCAGC AAAAGCAGCG TGGGTTCCTA TTTCCACACC 420 ATGGTGGAGA GCCTTGTGGG CTGGGGCTAC ACACGGGGTG AGGATGTCCG AGGGGCTCCC 480 TATGACTGGC GCCGAGCCCC AAATGAAAAC GGGCCCTACT TCCTGGCCCT CCGCGAGATG 540 ATCGAGGAGA TGTACCAGCT GTATGGGGGC CCCGTGGTGC TGGTTGCCCA CAGTATGGGC 600 AACATGTACA CGCTCTACTT TCTGCAGCGG CAGCCGCAGG CCTGGAAGGA CAAGTATATC 660 CGGGCCTTCG TGTCACTGGG TGCGCCCTGG GGGGGCGTGG CCAAGACCCT GCGCGTC CTG 720 GCTTCAGGAG ACAACAACCG GATCCCAGTC ATCGGGCCCC TGAAGATCCG GGAGCAGCAG 780 CGGTCAGCTG TCTCCACCAG CTGGCTGCTG CCCTACAACT ACACATGGTC ACCTGAGAAG 840 GTGTTCGTGC AGACACCCAC AATCAACTAC ACACTGCGGG ACTACCGCAA GTTCTTCCAG 900 GACATCGGCT TTGAAGATGG CTGGCTCATG CGGCAGGACA CAGAAGGGCT GGTGGAAGCC 960 ACGATGCCAC CTGGCGTGCA GCTGCACTGC CTCTATGGCA CTGGCGTCCC CACACCAGAC 1020 TCCTTCTACT ATGAGAGCTT CCCTGACCGT GACCCTAAAA TCTGCTTTGG TGACGGCGAT 1080 GGTACTGTGA ACTTGAAGAG TGCCCTGCAG TGCCAGGCCT GGCAGAGCCG CCAGGAGCAC 1140 CAAGTGTTGC TGCAGGAGCT GCCAGGCAGC GAGCACATCG AGATGCTGGC CAACGCCACC 1200 ACCCTGGCCT ATCTGAAACG TGTGCTCCTT GGGCCC 1236

[0128]

[SEQ ID NO: 18] Sequence length: 1332 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA sequence ATGGGCCTCC ACCTCCGCCC CTACCGTGTG GGGCTGCTCC CGGATGGCCT CCTGTTCCTC 60 TTGCTGCTGC TAATGCTGCT CGCGGCCCAGCGCCCCCCCCCCCCCCCGG CTGGTGATTT GGGTAACCAA CTGGAAGCCA AGCTGGACAA GCCGACAGTG 180 GTGCACTACC TCTGCTCCAA GAAGACCGAA AGCTACTTCA CAATCTGGCT GAACCTGGAA 240 CTGCTGCTGC CTGTCATCAT TGACTGCTGG ATTGACAATA TCAGGCTGGA GTGCAGTGGC 300 GCAATCTCGG CTCACTACAC CTCTGCCTCC CAGGCTCAAG CACTTCTCCT GCCTCAGACT 360 CCGGATAACT GGGATTACAG GCTGGTTTAC AACAAAACAT CCAGGGCCAC CCAGTTTCCT 420 GATGGTGTGG ATGTACGTGT CCCTGGCTTT GGGAAGACCT TCTCACTGGA GTTCCTGGAC 480 CCCAGCAAAA GCAGCGTGGG TTCCTATTTC CACACCATGG TGGAGAGCCT TGTGGGCTGG 540 GGCTACACAC GGGGTGAGGA TGTCCGAGGG GCTCCCTATG ACTGGCGCCG AGCCCCAAAT 600 GAAAACGGGC CCTACTTCCT GGCCCTCCGC GAGATGATCG AGGAGATGTA CCAGCTGTAT 660 GGGGGCCCCG TGGTGCTGGT TGCCCACAGT ATGGGCAACA TGTACACGCT CTACTTT CTG 720 CAGCGGCAGC CGCAGGCCTG GAAGGACAAG TATATCCGGG CCTTCGTGTC ACTGGGTGCG 780 CCCTGGGGGG GCGTGGCCAA GACCCTGCGC GTCCTGGCTT CAGGAGACAA CAACCGGATC 840 CCAGTCATCG GGCCCCTGAA GATCCGGGAG CAGCAGCGGT CAGCTGTCTC CACCAGCTGG 900 CTGCTGCCCT ACAACTACAC ATGGTCACCT GAGAAGGTGT TCGTGCAGAC ACCCACAATC 960 AACTACACAC TGCGGGACTA CCGCAAGTTC TTCCAGGACA TCGGCTTTGA AGATGGCTGG 1020 CTCATGCGGC AGGACACAGA AGGGCTGGTG GAAGCCACGA TGCCACCTGG CGTGCAGCTG 1080 CACTGCCTCT ATGGTACTGG CGTCCCCACA CCAGACTCCT TCTACTATGA GAGCTTCCCT 1140 GACCGTGACC CTAAAATCTG CTTTGGTGAC GGCGATGGTA CTGTGAACTT GAAGAGTGCC 1200 CTGCAGTGCC AGGCCTGGCA GAGCCGCCAG GAGCACCAAG TGTTGCTGCA GGAGCTGCCA 1260 GGCAGCGAGC ACATCGAGAT GCTGGCGCACAC GCCACCTCC TGGCCTTGCCGACGTCCGCCTG

[0129]

[SEQ ID NO: 19] Sequence length: 1236 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA sequence ATGGATCGCC ATCTCTGCAC CTGTCGCGAG ACCCAGCTCC GGAGTGGCCT CCTGTTACCT 60 CTGTTTCTAC TAATGATGCT GGCAGACCTG ACGCTCCCCCGCGTCAGT CTGGTGATTT GGGTAACCAG TTGGAAGCAA AGCTGGATAA GCCAAAGGTT 180 GTACACTACC TTTGCTCCAA GAAGACGGAC AGCTACTTCA CACTCTGGCT GAATCTGGAA 240 CTGCTTCTGC CTGTTATCAT TGACTGCTGG ATTGACAATA TCAGGCTGGT TTACAACAGA 300 ACATCTCGGG CCACCCAGTT TCCCGATGGT GTGGACGTGC GTGTCCCTGG CTTTGGGGAA 360 ACATTTTCTA TGGAATTCCT AGACCCCAGC AAGAGGAATG TGGGTTCCTA TTTCTACACT 420 ATGGTGGAGA GCCTTGTGGG CTGGGGCTAC ACACGGGGTG AAGACGTTCG AGGTGCTCCC 480 TATGATTGGC GGCGAGCCCC AAATGAAAAC GGGCCCTACT TCTTGGCCCT GCGAGAGATG 540 ATCGAGGAGA TGTACCAGAT GTATGGGGGC CCCGTGGTGC TGGTCGCCCA CAGCATGGGC 600 AACGTGTACA TGCTCTACTT TCTGCAGCGG CAGCCACAAG TCTGGAAGGA CAAATATATC 660 CATGCCTTCG TCTCACTGGG GGCGCCCTGG GGGGGCGTGG CCAAGACGCT GCGTGTC CTG 720 GCCTCAGGAG ACAACAATCG CATTCCCGTC ATTGGGCCAC TGAAGATCCG GGAACAGCAG 780 CGATCTGCCG TCTCTACCAG CTGGCTACTG CCATACAACC ACACTTGGTC ACATGAAAAG 840 GTATTTGTAT ACACACCCAC GACTAACTAC ACGCTCCGGG ACTATCACCG GTTCTTCCGG 900 GACATCGGTT TCGAAGATGG CTGGTTCATG CGGCAGGACA CAGAAGGGCT GGTTGAAGCC 960 ATGACGCCAC CCGGGGTGGA GCTGCACTGC TTGTATGGCA CTGGTGTTCC CACGCCAAAC 1020 TCTTTCTACT ACGAGAGCTT TCCTGATCGG GACCCCAAAA TCTGCTTCGG CGATGGTGAC 1080 GGCACGGTGA ACCTGGAGAG CGTCCTGCAG TGCCAAGCCT GGCAGAGCCG CCAAGAGCAC 1140 AGAGTATCAT TGCAGGAGCT GCCGGGAAGC GAGCACATTG AGATGCTAGC CAATGCCACC 1200 ACCTTGGCTT ATCTGAAACG TGTGCTTCTG GAACCT 1236

[0130]

[SEQ ID NO: 20] Sequence length: 90 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA sequence ATGGGCCTCC ACCTCCGCCCCC CTACCGTGTG GGGCTGCTCC CGGATGGCCT CCTGTTCCTC 60 TTGCTGCTGC TAATGCTGCT CGCGGACCCA 90

[0131]

[SEQ ID NO: 21] Sequence length: 99 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA sequence ATGGGCCTCC ACCTCCGCCC CTACCGTGTG GGGCTGCTCC CGGATGGCCT CCTGTTCCTC 60 TTGCTGCTGC TAATGCTGCT CGCGGACCCA GCGCTCCCG 99

[0132]

[SEQ ID NO: 22] Sequence length: 99 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA sequence ATGGATCGCC ATCTCTGCAC CTGTCGCGAG ACCCAGCTCC GGAGTGGCCT CCTGTTACCT 60 CTGTTTCTAC TAATGATGCT GGCAGACCTG ACGCTCCCG

[0133]

[SEQ ID NO: 23] Sequence length: 392 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA sequence ACACGCTCTA CTTTCTGCAG CGGCAGCCGC AGCCTGGTAA GGACAAGTAT ATCCGGGCCT 60 TCGTGTCACT GGGTGCGC TGGGCGCACGCCTGCGGCACCT CCGGATCCCA GTCATCGGGC CCCTGAAGAT CCGGGAGCAG CAGCGGTCAG 180 CTGTCTCCAC CAGCTGGCTG CTGCCCTACA ACTACACATG GTCACCTGAG AAGGTGTTCG 240 TGCAGACACC CACAATCAAC TACACACTGC GGGACTACCG CAAGTTCT GCCAGCAG GCGA TCGATGCAG GCGA TCGATGCAG GG

[0134]

[SEQ ID NO: 24] Sequence length: 23 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA sequence GCTGCTGCCC TACAACTACA CAT 23

[0135]

[SEQ ID NO: 25] Sequence length: 19 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA sequence TATCCGGGCC TTCGTGTCA 19

[0136]

[SEQ ID NO: 26] Sequence length: 28 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA sequence TCAAAGCCGA TGTCCTGGAA GAACTTGC 28

[0137]

[SEQ ID NO: 27] Sequence length: 24 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA sequence GTGGTGCTGG TCCCTGGTGA TTTG 24

[0138]

[SEQ ID NO: 28] Sequence length: 22 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: synthetic DNA sequence GGTGGCCCTG GATGTTTTGT TG 22

[0139]

[SEQ ID NO: 29] Sequence length: 198 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: cDNA sequence GTGGTGCTGG TCCCTGGTGA TTTGGGTAAC CAGTTGGAAG CAAAGCTGGA TAAGCCAAAG 60 GTTGTACACT ACCTTTGCTC CAAGAAGACG GACAGCTACT TCACACTTCTG GCACTACTG TGCCTGTTAT CATTGACTGC TGGATTGACA ATATCAGGCT GGTTTACAAC 180 AAAACATCCA GGGCCACC 198

[0140]

[SEQ ID NO: 30] Sequence length: 25 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA sequence GGTTGTACAC TACCTTTGCT CCAAG 25

[0141]

[SEQ ID NO: 31] Sequence length: 21 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA sequence GGTAACCAGT TGGAAGCAAA G 21

[0142]

[SEQ ID NO: 32] Sequence length: 21 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA sequence ATCCAGCAGT CAATGATAAC A 21

[0143]

[SEQ ID NO: 33] Sequence length: 22 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA sequence GTAATACGAC TCACTATAGG GC 22

[0144]

[SEQ ID NO: 34] Sequence length: 19 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA sequence ACTATAGGGC ACGCGTGGT 19

[0145]

[SEQ ID NO: 35] Sequence length: 48 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: synthetic DNA sequence GTAATACGAC TCACTATAGG GCACGCGTGG TCGACGGCCC GGGCTGGT 48

[0146]

[SEQ ID NO: 36] Sequence length: 26 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA sequence ATCCGGGAGC AGCCCCACAC GGTAGG 26

[0147]

[SEQ ID NO: 37] Sequence length: 24 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: synthetic DNA sequence GGTGTACGAC GGTCGCCGCA GGTC 24

[0148]

[SEQ ID NO: 38] Sequence length: 2867 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA sequence ACTATAGGGC ACGCGTGGTC GACGGCCCGG GCTGGTATTA AAAAAAAAAA TCAGGGCCGG 60 GTGTGATGGC TCATACCTGT AATCCCAGCA CTTTGGGAGATCTCGAGG GTTCAAGACC AGCCTGGCCA ACATGGCGAA ATCCCGTCTC TACTAAAAAA 180 TACAAAAATC AGCTGGGCGT GGTGGCGGGT GCCTGTAATC CCAGCTATTC AGGAGGCTGA 240 GGCAGGAGAA TCGCTTGAAC CCAGGAGGCA GAGGTTGCAG TGAGCCGGGA TCACGCCACT 300 GCTCTCCAGC CTGGGTGACA GAGCAAAACT CTGTCTCAAA AAAAAAAAAA AGGTGTCAGC 360 CTGGCATGTG GAGAACGACC CACAGGAACG AGGGCGTGCA TTGGGACATC AGTGACGAGG 420 CTGTTGTGGG AATAGGGTAG TGTGGTTTGG GGAGTGTAGA GCTGGCAAGC CCTTATGACC 480 ACCTGAGTTG TGGTTCTGAG AAGCATGGAA GCATCCAGAG CTCAGGATGA TGCCAAGTCT 540 GCAGCCTGGG GGATCAGGTG GATGGCAGAG TCATTGTGAA AAGGGAGGAC CCTCACTTTC 600 TGACCCTTCT CCACAGTGCC AGCATGGGTC ATTGCTGACC AGGCCTTGCC ATCCTGCCCC 660 TAATGGCTGT GGTTCCTAAC ACATGCAGGG CCTGTGGGGT TGAAGCACCA AGGAACC CCT 720 CTTGAGGACA GGGCTACCCT TCCAGGGGCC CATGGTCACC GGATGCTGCT GGGCCGGCAA 780 GACATTTAGA CTGTGGCCAG AGTCCAAGGT GGCCCAGCAC CTCTTGATCC TTCTCTTCCT 840 CCACATAACC TTTGACTGGA CTTCTGCCCG TCCCTAGGCC TGCAGAAGAG TCTCTGGTCT 900 CCCACGCTGG GTTTTCACCA GATGGGTCTT CACTGATCTT CTGTTGGGTC ACGGGTGAAG 960 GTGGGGGAGG CAGGGGCTTT GGGAGTGGGA GTTCTGAGCC AGGGCCTTAG CGGGAGATGG 1020 CTGGACCTTA AGAGAGGGTG GGGCTGGTCA CAGTGGCACA CATCTGTAAT CTCAGCACTT 1080 TGGGAGGCTG AGGCAGGTGG ATCACTTAAG GCCAGGAGTT CAAGACTGGC CTGGGCAACA 1140 TAGTGAGACC CCAACTCTAC AAAAAAAAAA ACTAGCTGAG CTTGGAGGTG TACACTTGTA 1200 GTCCCAGCTA CTCAGGAGGC TGAGGTGGGA AGGATTGCTT GAGCCTGAGA GGTCAAGGAT 1260 GCAGTGAGCC GTGATTGCGC TACTGCACTT GGGTGACAGA GAGACCCTTT CTCAAAAAAA 1320 AAAAAAAAAA AAAGGAAAGA AAAGAAAAAG GGGCCGGGCA CAGTAGCTCA CGCCTGTAAT 1380 CCCAGCACTT TGGGAGGCCG AGGCAGGTGG ATCACCTGAG GTTGCGAGTT CAAGACCAGC 1440 CTGACTAACA TGGAGAAACC CCGTCTCTAC TAAAAATACA AAATTAGCCA GGGTGGTGGC 1500 GCATGCCTGC AATCCCAGCT ACTCGGGAGG CTGAGGCAGG AGAATTGCTT GAATCCAGGA 1560 GA CGGAGGTT ACGGTGAGCC AAGATTGCAC CATTGCACTC CAGCCTGGGC AACAAGAGCG 1620 AAACTCTGTC TCAAAAAAAA AAAAGAAAAA GAAAAAGAAA AAAGAAAGAG GGAGGGTGGT 1680 GGTAGCCCAG TCACCAACAT GTTTCACTAT AAGAACTCGA GAAGGGCAGG GCAAGATAGT 1740 GGCTTCATAG CCAGGTCAGC TGCTTACCAA GAAGAAGGAA GGAAGGGGCA GGACAAATTT 1800 CTTGGGACCA GGTGGGATGA CCAGGGTGCA GCTGCCCCTT CGAAGGGGTG GGTGTGTGGA 1860 GGATCAAGAC CTCTATTTCC CAAATACTCT CGTCCCTCTA TCCCACAGTG ACCTATGGTG 1920 CTGGCATATA ACCAGCTGTC AGGTCTTTGC CCACTCTGTT CGCCCCTGCT TCCTGGCGCA 1980 GGGAGTCCAT GTCCTCTCTG GTTCCCCAGG TTTGCGAGAG TGGAGGGGGA CCACGAGCTC 2040 CCGATGCCTC TCCTGCTCTG CAGGGGAACT TGCAGATGGC CCATGGCGCA GGGTCGAGAC 2100 TCAAGCCCAC TCCCAACCCC GCGCCCGAAC TGCCCGGACT GGCGGGGTGA CGCTGCACTC 2160 TGCGCCCCTA AAACGAACAG ATTAACCCCT CTCCTGGGAA CTGAACATGC TGACCTGGCC 2220 TCTCCCGGTT CCCCCCGCAT CTGTAACCCC GGGGCAGAGT TACAGGGGCT GACTGGCCGC 2280 ACCCAGGTGC CCTCGGGGCA GGGTGTGCTA AGAATTGGTG TGGGGGCTGC ACAAAGGTCC 2340 TGGTCAGCTC CTGGTCACCT GAGGCCCAAG AACTGTCCGG GACTCACTTC CTCTCTTCTT 2400 GCTTTAAC CG GGGTCGCTCA GCAGCGGCCA GCGCTGCACC CCTTATCCTC TCCCGGTCTT 2460 GTCCGTTCCA GATCCTCCAG GTCAGGGGGT CGCCAAGTGA GAGCTGCGCA GCGTGGATTT 2520 CGGGTACCCA GGGCTGGGCG GGGTACAGCA GCGGCGAGCT GGGTTCCCGG GTGGGCGACT 2580 GACAGCCCGG AGCCAGGCGA TACCTCGATC CATCGATGCG CTCGGCGCTC AGCGTGGTCC 2640 AGGAAGCAGG GGGTTGGGCA AGGGCGGGGC GGCGACCTCC GACTGGGAGG GGCGTATATG 2700 GCGGCGAGTC CCTATTGGCC AGCCATTTGC GGGAGGCGGG CCCTGATTGG CCGGGGGGAT 2760 GCGGGGGATG CGGGCGGCGG GGTTAAGCGC GTCGCCACCG CCCCCGCCTA GGCGAGAGCC 2820 CAGAGAGCTG AACCTGCATC CCGGACCTGC GGCGACCGTC GTACACC 2867

[0149]

[SEQ ID NO: 39] Sequence length: 20 Sequence type: amino acid Topology: linear Sequence type: protein sequence Cys Glu Asp Val Arg Gly Ala Pro Tyr Asp Trp Arg Arg Ala Pro Asn 1 5 10 15 Glu Asn Gly Pro 20

[SEQ ID NO: 40] Sequence length: 19 Sequence type: amino acid Topology: linear Sequence type: protein sequence Pro Val Ile Gly Pro Leu Lys Ile Arg Glu Gln Gln Arg Ser Ala Val 1 5 10 15 Ser Thr Cys

[SEQ ID NO: 41] Sequence length: 63 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: cDNA sequence CCGCTCGAGT CACTTGTCAT CGTCGTCGTC CTTGTAGTCG GGCCCAAGGA GCACACGTTT 60 CAG 63

[SEQ ID NO: 42] Sequence length: 31 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: cDNA sequence GGAGACAACC AACCGGATCC CAGTCATCGG G 31

[SEQ ID NO: 43] Sequence length: 10 Sequence type: amino acid Topology: linear Sequence type: protein sequence Ala Gly Arg His Pro Pro Val Val Leu Val 1 5 10

[Brief description of the drawings]

FIG. 1 shows the nucleotide sequence of the DNA encoding the human heart-derived protein of the present invention and the amino acid sequence encoded thereby.

FIG. 2 shows the nucleotide sequence of the DNA encoding the human kidney-derived protein of the present invention and the amino acid sequence encoded thereby.

FIG. 3 shows the nucleotide sequence of the DNA encoding the mouse kidney-derived protein of the present invention and the amino acid sequence encoded thereby.

FIG. 4 shows a comparison of the amino acid sequence of the human heart-derived protein and the mouse kidney-derived protein of the present invention. hCLP is a protein derived from human heart, m
CLP indicates a mouse kidney-derived protein. The blacked-out portion indicates a portion where amino acid residues are different in both amino acid sequences.

FIG. 5 shows the sequence of the 5 ′ upstream region of cDNA encoding the human-derived LCAT-like protein of the present invention.

FIG. 6 shows the 1st to 1500th sequence of the 5 ′ upstream region sequence (promoter region) of cDNA encoding the human-derived LCAT-like protein of the present invention (FIG. 7)
followed by).

FIG. 7 shows a sequence at positions 1501 to 2867 of the 5 ′ upstream region sequence (promoter region) of cDNA encoding the human-derived LCAT-like protein of the present invention (starting codon ATG is defined as a sequence at positions 2868 to 2870) (Continued from FIG. 6).

FIG. 8 shows a construction diagram of pTB 2022 and antisense pTB 2022 obtained in Example 4.

FIG. 9 shows the results of measuring the promoter activity of a transformant into which pTB 2022 has been introduced.

FIG. 10 shows the results of Western blotting performed in Example 6 (electropherogram). In the figure, lane 1 shows human HDL protein (12.75 μg), lane 2 shows human HDL protein (25.5 μg), and lane 3 shows a recombinant human LCAT-like protein derived from insect cells.

FIG. 11 shows an elution pattern of purification by FPLC performed in Example 6.

FIG. 12 shows the results of Western blotting performed in Example 6 (electropherogram). In the figure, lanes 4 and 6
Is the apo VLDL fraction, lanes 8, 10, 12, and 14 are
apo LDL fraction, lanes 16, 18, 20, 22
2 shows the HDL fraction.

FIG. 13 shows a method for constructing a donor plasmid obtained in Example 7.

FIG. 14 shows the results of UV absorbance and Western blotting in protein elution performed in Example 12 (electropherogram).

FIG. 15 shows the results of a PNPB assay performed in Example 13. In the figure, the substrate concentration-◆-is 5.0E-03.
M,-■-is 2.0E-03M,-△-is 1.0E-04
M and + indicate 5.0E-05M.

FIG. 16 shows human LCAT-like protein and human LC calculated based on the Lineweaver-Burk method described in Example 13.
The Vmax (nmol / min) value and the Km (M) value of the AT protein are shown.

FIG. 17 shows the results of a PNPB assay performed in Example 13. In the figure,-◆-indicates 1.0E-04.

FIG. 18: PNPB of human LCAT-like protein after chemical modification of serine and cysteine residues performed in Example 14.
The following shows a comparison. In the figure,-■-is DFP, -x- is DTN.
B is shown.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI A61K 48/00 ABX C07K 19/00 AGZ C12N 1/19 C07K 16/40 1/21 19/00 9/10 C12N 1/19 9 / 99 1/21 C12P 21/08 5/10 C12Q 1/48 9/10 G01N 33/566 9/99 A61K 37/52 AAM 15/09 ZNA ACV C12P 21/08 ADN C12Q 1/48 C12N 5/00 B G01N 33/566 15/00 ZNAA // (C12N 1/19 C12R 1: 865) (C12N 1/21 C12R 1:19) (C12N 1/21 C12R 1: 125) (C12N 5/10 C12R 1:91) (C12N 9/10 C12R 1:19)

Claims (22)

[Claims] 1. A protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, its precursor protein or a salt thereof. 2. The protein according to claim 1, which has an amino acid sequence represented by any one of SEQ ID NOs: 4 to 8, or a precursor protein thereof. 3. The protein according to claim 1, which has lecithin: cholesterol acyltransferase-like activity, or a precursor protein thereof. 4. A partial peptide of the protein according to claim 1, or a salt thereof. 5. A signal peptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11. 6. A DNA containing a DNA having a base sequence encoding the protein or the precursor protein according to claim 1. 7. The DNA according to claim 6, which has a base sequence represented by any one of SEQ ID NOs: 12 to 19. 8. A DNA containing the DNA encoding the signal peptide according to claim 5. 9. The DNA according to claim 8, which has a base sequence represented by SEQ ID NO: 20 to SEQ ID NO: 22. 10. A recombinant vector containing the DNA according to claim 6. [11] A transformant transformed with the recombinant vector according to [10]. (12) culturing the transformant according to (11),
A method for producing the protein, the precursor protein or a salt thereof according to claim 1, wherein the protein or the precursor protein according to claim 1 is produced, accumulated, and collected. [13] A pharmaceutical comprising the protein according to [1], its precursor protein or a salt thereof. [14] A pharmaceutical comprising the DNA according to [6]. 15. The medicament according to claim 13 or 14, which is a therapeutic / prophylactic agent for arteriosclerosis, obesity, aging, brain disease, kidney disease or hyperlipidemia. 16. An antibody against the protein according to claim 1 or a precursor protein thereof, the partial peptide according to claim 4, or a salt thereof. 17. A protein according to claim 1, a partial peptide according to claim 4, or a salt thereof, wherein the protein according to claim 1, a partial peptide according to claim 4, or a salt thereof is lecithin. : A method for screening a compound or a salt thereof that promotes or inhibits cholesterol acyltransferase-like activity. 18. A protein according to claim 1, comprising the partial peptide according to claim 4 or a salt thereof, lecithin: cholesterol of the protein according to claim 1, a partial peptide according to claim 4 or a salt thereof. A screening kit for a compound or a salt thereof that promotes or inhibits an acyltransferase-like activity. (19) a protein according to (1), a partial peptide according to (4), or a lecithin: cholesterol acyl salt thereof obtained by using the screening method according to (17) or the screening kit according to (18); A compound or a salt thereof that promotes or inhibits transferase-like activity. 20. The protein according to claim 1, obtained by using the screening method according to claim 17 or the screening kit according to claim 18, lecithin: cholesterol acyltransferase of the partial peptide according to claim 4 or a salt thereof. Comprising a compound or a salt thereof that promotes or inhibits the like activity. 21. A promoter DNA having the same or substantially the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 38 or a partial DN thereof having promoter activity
DNA containing A. 22. A method for screening a compound or a salt thereof which promotes or inhibits the promoter activity of DNA according to claim 21.