JPH03290184A - Cell of scavenger receptor-producing animal - Google Patents

Abstract

NEW MATERIAL:Animal cell able to manifesting I-type or II-type human scavenger receptor transformed by a vector containing gene coding I-type or II-type human scavenger receptor under the control of promoter capable of functioning in animal cell. USE:Used for a remedy of arterial sclerosis. PREPARATION:For instance, mRNA is collected from macrophage-like cell divided by cultivation of human leukemia cell-based THP-1 in the presence of phorbol 12-myristate 13-acetate(PMA) and cDNA library is produced from the mRNA by a normal method, then a strain having gene coding I-type or II-type human scavenger receptor is selected by using a probe, thus resultant DNA is collected and connected to a downstream of a promoter to form a recombinant plasmid, then the plasmid is inserted into chinese hamster ovary(CHO), etc., to afford the aimed transformed animal cell.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention provides an animal cell capable of expressing human scavenger receptor type I or type II, and a method for producing human scavenger receptor type I or type II using the cell. Regarding.

(1) (2) [Conventional technology] Arteriosclerosis occurs when macrophages that have taken in modified forms of low-density lipoprotein (LDL), which is a complex of cholesterol and lipoproteins, turn into foam cells and are deposited under the cells lining blood vessels. It is thought to be caused by the accumulation of Scavenger receptors are proteins that exist in the cell membrane of macrophages and function when binding to denatured LDL and taking it into cells. Furthermore, scavenger receptors are involved in the removal of various denatured substances, foreign substances such as viruses, and physiologically active substances such as endotoxins in vivo. Therefore, elucidating the structure and mechanism of action of scavenger receptors is important for elucidating the onset mechanism of arteriosclerosis and developing diagnostic, preventive and therapeutic methods, as well as reagents and drugs for these. Conceivable. It is also important for elucidating the functions of macrophages and the reticuloendothelial system.

T. Kodama et al. (Natare, 343:5
32-535.1990; and Nature, 343
:570-572. (1990) cloned the bovine scavenger receptor gene, deduced the amino acid sequence of the receptor from its base sequence, and further deduced its structure. According to this, there are two types of bovine scavenger receptors: type (1) and type (2), type (2) has a structure in which the C-terminus of type I is shortened, and both exist as trimers.

The human mononuclear leukemia cell line THP-1 was treated with 200 nM phorbol 12-myristate 13-acetate (PMA).
It is known that Po1y (A)” RNA extracted from macrophage-like cells cultured and differentiated in the presence of 100% hybridizes with a bovine scavenger receptor cDNA fragment (T., Koclama et al., Natu
re, 343:531-535.1990>. However, the cloning of the human scavenger receptor gene and the expression of the human scavenger receptor by the cloned gene have not been published.

[Problem to be solved by the invention]

Therefore, the present invention aims to provide an animal cell capable of expressing a human scavenger receptor and a method (3) for producing a human scavenger receptor using the cell.

[Means to solve the problem]

The above problem was achieved by obtaining a gene encoding a human scavenger receptor from cells expressing the receptor, creating a vector by ligating this under the control of a promoter that can function in animal cells, and using this vector to transform animal cells. This is achieved by transforming cells that can stably express large amounts of human scavenger receptors.

[Specific explanation]

It is known that macrophage-like cells are obtained by culturing the human leukemia cell line THP-1 in the presence of PMA, and that mRNA extracted from these cells hybridizes with a bovine scavenger receptor cDNA fragment. Therefore, mRNA is extracted from the macrophage-like cells obtained in this way, and then c
It is possible to create a DNA library (4). The cDNA library can be constructed using any known method for constructing a cDNA library from mRNA, such as the Okayama & Berg method, G.
This can be performed by the Ubler & Loffman method or the like. The cDNA library prepared in this way can be prepared using, for example, any fragment of the bovine scavenger receptor cDNA that has already been cloned, or a synthetic oligonucleotide designed based on the predicted amino acid sequence of the bovine scavenger receptor, as a probe. can be used for screening according to conventional methods. Alternatively, using such probes or using cDNA Air fragments of human scavenger receptor cloned as described above, genomes can be extracted from macrophage-like cells differentiated from cells that produce human scavenger receptors, such as THP-1 cells. DNA can also be obtained.

Next, the human scavenger receptor gene cloned in this way is linked downstream of a promoter that can function in animal cells to create a transformation vector for inserting the gene (5) (6) into animal cells. Create. As a vector for this purpose, any vector that can function in animal cells can be used, such as a cytomegalovirus promoter, a dihydrofluorate reductase promoter, an MMLV promoter, a mecrothionein promoter, etc. can. It is desirable that the transformation vector further contains a selection marker gene for selecting cells transformed by the vector, and examples of such selection marker genes include a neomycin resistance gene, HGPRT gene, DHPR gene, etc. etc. can be used. The vector desirably also contains an origin of replication for replication in animal cells, an E. coli origin of replication for genetic manipulation in E. coli, and an E. coli selection marker gene.

An example of a starting vector for producing such a transformation vector is R,/CMV (obtained from Introgen). This vector contains the cytomegalovirus promoter, multiple cloning site, and CH
5 containing in this order polyA, M13 origin of replication, SV40 origin of replication, Ne, 1-ficin resistance gene, polyASptlc origin of replication, ampicillin resistance gene.
, a 4Kbp shuttle vector. Therefore, by inserting the human scavenger receptor gene into the multiple cloning site of this starting vector, it is possible to place this gene under the control of cytomegalovirus, and to transform the human scavenger receptor gene into a transformation vector made from this starting vector. Transformed animal cells can be selected by culturing in a neomycin-containing medium.

A transformation plasmid (recombinant plasmid) obtained by inserting the human scavenger receptor gene into the starting vector can be transformed into E. coli, for example JM103, and then selected with ampicillin and further amplified. Next, it is purified from E. coli by a conventional method, for example, by CsCj2 ultracentrifugation.

In the present invention, any animal cell line can be used, such as Chinese hamster overly (CHD).
cell'! ? '6S cells, HEPG 2 cells, culture edge (7
) Fibroblasts, cultured vascular endothelial cells, cultured bone marrow cells, etc. can be used.

Transformation of animal cells with transformation vectors can be performed according to conventional methods. That is, for example, F-12 containing 7% fetal bovine serum (FBS) is used to culture animal cells.
Notrient Mixture medium (Ham F-
12:GIBCO) etc. Transformation can be carried out according to any conventional method, such as cationic liposome-mediated transformation (Proc, Natl, Ac
ad, Sci.

USA, 84 Near 413-74'17. 198
7), calcium phosphate method, electropora
tion method, retrovirus vector method, etc. Cationic liposome-mediated transformation was performed using the synthetic cationic lipid N-(1-(2,3-dioleyloxy)
This is carried out by mixing liposomes containing [propyl]-N,N,N-)limethylammonium chloride (DOTMA) and transformation vector DNA to form a complex, and adding this complex to animal cells. This transformation can be carried out, for example, using Opti-MEMI Reduced S
erumMedium (Opti-MEM; GI
BCO) medium.

(8) Cells that have actually been transformed are selected by screening animal cells that have undergone transformation treatment. This selection method depends on the type of selection gene contained in the transformation vector. For example, if the transformation vector contains a neomycin resistance gene, neomycin-resistant colonies can be selected by transforming the cell colonies in a neomycin-containing medium.

Next, cells that express a large amount of human scavenger receptor are selected from the transformed animal cell population. Scavenger macrophages take up denatured (eg, oxidized) low-density riboproteins, but also acetylated low-density lipoproteins (AcLDL), an experimental model of denatured forms. Therefore, by incorporating labeled AcLDL into transformed animal cells and selecting cells that carry many labels, cells that express a large amount of scavenger receptors can be selected. In this case, any label can be used, for example 1°1'-dioctadecyl-3,3,3',3'-te(9) (10) tramethyl-indocarbocyanine perchloride (D
AcLDL (D i I-
AcLDL), cells incorporating Dir-AcLDL can be directly observed using a fluorescence microscope. Alternatively, compactin (40%), mevalonic acid (150-200%) and acetyl LDL
(1 to 10 μg/d) in a selective medium containing scavenger receptors such that only cells containing scavenger receptors survive.

Human scavenger receptor cDNA can be isolated from the expression cells thus obtained as follows. That is, to isolate scavenger receptor cDNA from cells expressing human scavenger receptor, nuclear DNA is prepared from cells expressing human scavenger receptor by a conventional method, and v! By combining appropriate oligonucleotide primers (about 20 bases) from the base sequence shown in 1iRIjM2fIe,
polymerase chain reaction (P
CR) to target human scavenger receptor I
Type or ■ type cDNA can be amplified and isolated. In other words, the expressing cells type ■ or type ■ are φ100 m
The cells were cultured for 3 days in a cell culture of 100 ml, and the cells were collected by trypsin treatment (~7 IX'lO cells). Cells were washed with PBS (p
0 containing 50 pg/d proteinase K.
5% SO3, 10mM EDTA, 15mM NaCL
Suspended in 10mM Tris buffer and incubated at 65°C.
After incubation for an hour, nuclear DNA is prepared by phenol extraction, chloroform extraction, and ethanol precipitation.

l) GATAAAATC to 5'-GAGA8GT as a PCR primer for Nukavenger receptor cDNA.
AGTG -3'2) 5' -ACAATATGT
GTGG 8TTGGAG-3 and 3) 5
' -GTAATTGGAGTAAGAAGAGG-3
', and for type ■, a combination of primers 1) and 2),
For type (2), the desired cDNA fragment can be amplified using a combination of primers 1) and 3). PCR is 100
1 μM of each primer 1 pg of nuclear DNA in the reaction system of p.
, Taq polymerase 2.5 units,
30 cycles of 95℃ 1 minute, 54℃ 1 minute, 72℃ 3 minutes
Repeated times.

The amplified DNA fragments were separated by 0.7% agarose gel electrophoresis and recovered from the gel by a conventional method11). The size of the obtained DNA is 1.8 kilobases for the ■ type and 1.3 kilobases for the ■ type.

〔Effect of the invention〕

The scavenger receptor-expressing cells of the present invention contain modified LDL that is taken into cells from this pathway regarding the scavenger receptor pathway involved in the development of arteriosclerosis.
It can be used to analyze the specificity of (!J Gantt). It is also useful as a model for analyzing the enclocytosis of substances into cells via receptors. Furthermore, the cells of the present invention can be used to screen therapeutic agents for arteriosclerosis, such as LDL degeneration inhibitors, acyl Co-A cholesterol acyltransferase (ACAT) activity inhibitors, and the like during the development process. It can also be used to produce human scavenger receptor proteins with sugar chains. It can also be used as an experimental system for the treatment of foreign substances or denatured substances via scavenger receptors, or as an experimental system for infection with hepatitis B virus (12), which causes infection with denatured albumin.

Example 1. Cloning of human scavenger receptor cDNA The human mononuclear leukemia cell line THP-1 was cultured in RPM11640 medium containing 10% fetal bovine serum. THP
The cells were cultured for 4 hours in the presence of 200 nM phorbol 12-myritete 13-acetate (PMA) to differentiate into macrophage-like cells.

From this cell, Chomczynski (Anal,
Poly(A)'' mRNA was isolated by guanidine thiocyanate/phenol/chloroform extraction according to the method of Biochem, 162:156, 1987). and Hoffm
Double-stranded cDNA was prepared according to the method of An (Gene, 25:263.1783). This cDNA is
coRI adapter and in a 5-20% potassium acetate gradient.
50.n Instruments). ooorp
Size fractionation was performed by centrifugation for 3 hours at m.

In this way, a cDNA fraction with a molecular weight (13) (14) of approximately 800 base pairs or more (according to agarose gel electrophoresis) was obtained, which was ligated to a BcoRI-digested λZAP n (obtained from Stratagene) vector and packaged. and XL-I Bl
ue cells (manual from Stratagene) were infected. λ2AP n containing cDNA is amplified and cDNA
I got the A library.

Plasmid pBSR7 (T, Kodama et al., Natur
e, 343:531-535. 354 bp of Xba corresponding to the collagen-like domain from (1990)
The l-3phl fragment was obtained and radioactively labeled with 32p to prepare a hybridization probe.

5 x 105 plaques of the human scavenger receptor cDNA library were screened with the DNA probe and two positive clones were obtained. This is called typee and type (■). The cDNA base sequences of these positive clones were determined by Sanger's guideeoxy method. All of these contain one long open reading frame, each showing homology with the nucleotide sequence of the bovine scavenger receptor cDNA, and
The base sequences of the type and the type ■ differed on the 3'-end side. These base sequences are shown in Figure 1 (■ type) and Figure 2 (■ type). When the cDNA library of 6 XIP plaques was further screened using the cDNA portions of these clones as probes, 14 clones of type ■ and three clones of type ■ were obtained. Representative clones of type ■ and type ■ were isolated to phSRI and phSRI[
It was bound and used for subsequent experiments.

'1 example 2. Preparation of human scavenger receptor producing cells The plasmid clones phSRI and phSRII obtained as described above were treated as follows. These plasmids were digested with restriction enzymes between ndllI and Xba
The entire region from the translation start codon ATG to the translation stop codon TAA (■ type: 451 amino acids; ■ type: 35
A DNA fragment containing 8 amino acids (equivalent to 8 amino acids) was obtained.

On the other hand, an approximately 5.4 Kbp animal cell expression vector Re/CMV (Invitrogen (
15) was digested with old ndI and XbaI to obtain a linearized vector. By ligating the DNA fragment to this linearized vector, the former nd m (895) and Xba
Human scavenger cDNA between I(986) and
Recombinant plasmids pXhSRI and pXhSR■ into which the fragments were inserted were obtained by ampicillin resistance selection. This recombinant was transfected into Escherichia coli JM103 and amplified, and then purified by CsCII ultracentrifugation.

These recombinants were used to transform Chinese hamster Oliver (C)10-Kl) cells. That is, 5 x 105 CHO-Kl cells/60 mm diameter plate were placed in F-12 containing 7% fetal bovine serum (FBS).
Notient Mixture medium (Ham F-1
2HGIBCD) in an atmosphere of 5% CO2 at 37°C for 3 days to reach 80% confluence. The cultured cells were treated with Opti-MεMIReduced Serum.
Medium (Opti-M [iM; GIBCO
Washed three times with The recombinant DNA in 5 and the lipofectin reagent (BRL) in 50d were added to the cells.
Add Opti-MEM medium 3- containing
02, (16) Cultured at 37°C for 19 hours. Next, Ham F-12 medium 3 containing 15% FBS was added and further cultured overnight to perform transfection. Cultures were harvested from dates by treatment with trypsin-EDTA solution.

Since the vector Rc/CMV used to create the recombinant contains a neomycin resistance gene, transformed cells can be selected by selecting neomycin resistant bacteria. The above cells I x 106/Φ6
Ham F- containing 0mm dates and 7% FBS
After culturing in 12 medium for 6 hours, G413 (Geneti
cin; manufactured by GIBCO) to a final concentration of 470. w/-
Neomycin-resistant cell colonies were selected by culturing in 5% CD□ at 37°C for 12 days. During this culture, the medium was replaced every 4 days. ■
Forty-eight colonies each of type and type ■ were selected and cultured in a 24-well plate.

In order to select cells that express a high amount of human scavenger receptor from these transformed cell lines, (17) (18) 1.1'-dioctadecyl-3,3,3',3'tetramethyl-indocarbo Cyanine Velcrole) (D
acetylated low-density lipoprotein (O
An uptake test of AcLDL into cells was conducted using il-AcLDL). That is, the cells cultured as described above were collected by trypsin treatment, and approximately 700 cells/Φ7 m Teflon-coated slide (manufactured by Bokusui Brown) from each clone were placed in Ham F-12 medium containing 400 g of neomycin. After culturing overnight in
LDL protein/ml was added and further cultured overnight. Wash the cells with phosphate buffer and add D to the cells.
Ir uptake was observed using a fluorescence microscope, and the cell line with the strongest fluorescence intensity was selected. As a result, human scavenger receptor expressing cells 11SRl-28 (type I expression strain) and 11SRII-6CII type expression strain were obtained.

These animal cells) ISRl-28 and H3RI[-6
2837 (FORM BP-)
2 Nairiki and i Koken Joyori'lz8m (FBRM 8P-
28111)) was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology.

Example 4. Purification of scavenger receptor protein from animal cells CH○ cells expressing scavenger receptor were incubated with 5% F.
C3, after mass culturing in Ham F12 medium, collected with a rubber policeman and treated with 20mM Tris (HC, 12pH).
8), 1mMcacL, 150mM NaCj2.1f
homogenized in 1M PMSF solution (solution A),
The precipitated membrane material is collected by ultracentrifugation at 100.000 g for 1 hour and solubilized in solution A containing 1% Tritonx 100 for 30 minutes.

This 7 r 1 ton
a et al. PNAS 85°9238-9242. 1988
) apply L I M NaCI! , 20
mMTris-)ICl3pt18.1mM CaC
j! Recover in a solution containing 2.1% TrltOn X100. Furthermore, an immunoaffinity immunization method using antibodies against human scavenger receptors (T. Kodama et al., PNAS, 85.9238-9242゜1988)
This allows the human scavenger receptor to be detected in pure quantities.

(19) (20) Cells expressing human scavenger receptors were homophased or cell membrane proteins obtained in Example 4 were immobilized (T, Koda
denatured LDL (acetyl L) labeled with 125 or fluorescence (e.g. Dil).
Addition of DL or oxidized LDL) shows some binding to the plate. By adding various specimens (e.g. serum, culture supernatant, etc.) and observing the inhibition of the binding of labeled denatured LDL, it is possible to quantify the amount of scavenger receptor-binding substances in the blood. Become.

[Brief explanation of the drawing]

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GTGVSAEIMAMKEEQV 02 34 CAT TTG GAA CAG GM ATA AA
A GGA GM GTG AAA GTA CTGH
LEQEIKGEVKVL 41 47 AAT AACATCACT AAT GAT CTC
AGA CTG AAA GAT TGG GAANN
Engineering TNDLRLKDWEEIO 60 CAT TCT CAG ACCTTG AGA M
T ATCACT TTA ATT CAA GGTH
8QTLRN Engineering TLIQG19 73 CCT CCT GGA CCCCCG GGT GA
A AAA GGA GAT CGA GGT CCC
PPGPPGEKGDRGP 58 86 ACT GGA GAA AGT GGT CC
A CGA GGA TTT CCA GGT C
CA ATATGESGPRGFPGP E97 99 GGT CCT CCG GGT CTT AA
A GGT GAT CGG GGA GCA
ATT GGCGPPGL'KGDRGA Engineering
G36 12 TTT CCT GGA AGT CGA GGA
C'I'CCCA GGA TAT GCCGGA
AGGFPGSRGLPGYAGR 75 25 CCA GGA AAT TCT GGA CCA
AAA GGCCAG AAA GGG GAA AA
GPGNSGPKGQKGEK 014 38 GGG AGT GGA MCACA TTA AGA
CCA GTA CAA CTCACT GATGS
GNTLRPVQLTD 053 57 CAT ATT AGG GCA GGG CCCTC
T TM GATCAGGTGG GTTGGGCG
G097

Claims (1)

[Scope of Claims] 1. Human scavenger receptor type I or type II transformed with a vector comprising a gene encoding human scavenger receptor type I or type II under the control of a promoter capable of functioning in animal cells. Animal cells capable of expressing type II. 2. The animal cell according to claim 1, wherein the gene encoding human scavenger receptor type I or type II has the base sequence of the coding region in the base sequence shown in FIG. 1 or FIG. 3. The animal cell according to claim 1, wherein the promoter is a cytomegalovirus promoter. 4. The animal cell according to claim 1, wherein the animal cell is a Chinese hamster overly cell. 5. The first enzyme located downstream of the cytomegalovirus promoter
cD containing the coding region in the base sequence shown in the figure or Figure 2
The animal cell according to claim 1, which is a Chinese hamster overly cell transformed with a vector into which an NA fragment has been inserted. 6. A method for producing a human scavenger receptor, which comprises culturing the cell according to any one of claims 1 to 5. 7. A method for detecting denatured lipoproteins or denatured substances in blood using the cell according to any one of claims 1 to 5. 8. Gene encoding human scavenger receptor type II. 9. The gene according to claim 8, which has the nucleotide sequence of the coding region shown in FIG.