JP3700791B2 - Fas antigen fusion protein, DNA encoding the protein, vector containing the DNA, host cell transformed with the vector, therapeutic agent containing the vector as an active ingredient, and screening method using the host cell - Google Patents

Description

[0001]
[Industrial application fields]
The present invention includes (i) a fusion protein in which an amino sequence containing a ligand binding region of a nuclear receptor is bound to a C terminal of an amino acid sequence containing a transmembrane region and a functional expression region of Fas antigen, (ii) the fusion DNA encoding a protein, (iii) an expression vector containing the DNA, (iv) a transformant transformed with the expression vector, (v) treatment of cancer or autoimmune disease containing the expression vector as an active ingredient And (vi) a method for detecting a ligand of a nuclear receptor using the transformant.
[0002]
BACKGROUND OF THE INVENTION
Recently, receptors for humoral factors such as cytokines and hormones having important physiological activities are being elucidated by genetic manipulation. For example, receptors for fat-soluble hormones collectively called intracellular receptors. Analysis of the amino acid sequences of various nuclear receptors revealed that they are ligand-dependent transcription factors having a common basic structure and form one gene family. That is, a steroid receptor such as a glucocorticoid receptor, a brodiesterone receptor, a mineralcorticoid receptor, an androgenic receptor, an estrogen receptor, a retinoid receptor such as a retinoid X receptor, a retinoic acid receptor, and vitamin D_ThreeA family consisting of receptors and thyroid hormone receptors. For these receptors, it is known that functional regions such as a ligand binding site and a site recognizing a target DNA sequence are clearly separated [see Science, 240, 889 (1988). However, there are also receptors whose ligands are still unknown.
[0003]
In general, the functional expression of a nuclear receptor is controlled by a ligand binding region. For example, in a steroid receptor in the absence of a ligand (steroid), the ligand binding region forms an inactive state in a complex with a protein called HSP90, but when steroid binds to the ligand binding region, The complex dissociates, and the functional expression region (DNA recognition site) of the receptor binds to the target DNA to regulate the expression of the target gene.
[0004]
On the other hand, although the mechanism of function expression in the retinoid receptor, vitamin D3 receptor and thyroid hormone receptor has not been sufficiently elucidated, it is considered that at least complex formation with HSP90 does not occur. This is a very different point from steroid receptors. Other retinoid receptors, vitamin D_ThreeReceptors and thyroid hormone receptors have several properties that are different from steroid receptors (for example, the A / B region is shorter than steroid receptors, and the overall molecular weight is therefore reduced, etc.) Therefore, it has been proposed to position these receptors as subfamilies within a large family.
[0005]
On the other hand, Fas antigen is a membrane protein clearly shown to be involved in programmed cell death (apoptosis). Yonehara et al. Produced monoclonal antibodies against human cell surface antigens and obtained anti-Fas antibodies showing lethal activity against various human cells [J. Exp. Med.,169, 1747 (1989)]. The cDNA of the cell surface molecule recognized by the anti-Fas antibody was isolated and the structure of the human Fas antigen was determined [Cell,66, 233 (1991)]. The Fas antigen consists of 335 amino acids, and the 16 amino acids on the N-terminal side are presumed to be signal peptides. A transmembrane region consisting of 17 hydrophobic amino acids is present at the center of the molecule, and 157 amino acids on the N-terminal side are considered to be extracellular regions and 145 amino acids on the C-terminal side are considered to be cytoplasmic regions.
[0006]
Since the structure of Fas antigen is similar to the receptor for cancer necrosis factor (TNF), it is presumed that apoptosis by Fas antibody occurs by a mechanism similar to the action of TNF. The functional region of the Fas antigen has also been gradually clarified, and it is known that the region essential for apoptosis signal transduction (functional expression region) is the amino acid sequence portion from the 175th to the 304th [J. Biol. Chem.,268, 10932 (1993)]. Furthermore, the amino acid sequence of mouse Fas antigen has also been clarified [J. Immunology,148, 1274 (1992)], and has a total of 49.3% homology to the human Fas antigen. The functional expression region is considered to be the amino acid sequence from the 166th to the 291st corresponding to the region of the human Fas antigen.
[0007]
[Prior art]
The receptor has a ligand binding region and a signal transduction region. When a ligand binds to the ligand binding region, the three-dimensional structure of the signal transmission region changes, and the signal is transmitted to another protein or DNA.
Recently, research on signal transduction in a fusion protein in which a ligand binding region of a receptor and a signal transduction region of a heterologous protein are combined has been actively conducted.
For example, a cell transformed with a gene encoding a fusion protein of a ligand (ie, estrogen) binding region of an estrogen receptor, which is one of steroid receptors, and a signal transduction region of human cancer gene c-Myc, Diene stimulation revealed that the cells became cancerous and proliferated [Nature,340,
66 (1989)].
[0008]
Similar experiments have been performed for the ligand-binding regions of the receptors for glucocorticoid, mineralcorticoid, and estrogen and E1A (adenovirus), c-Fos, v-Myb, C / EBP, v-Rel, GATA-1, 3, GAL4-VP16, Rev (HIV), c-Ab1, in the fusion protein with the functional expression region of each protein [Cell,54, 1073 (1988), Proc. Natl. Acad. Sci. USA,88, 5114 (1991), EMBO J.,Ten, 3713 (1991), Science,251, 288 (1991), EMBO J.,11, 4641 (1992), Genes Dev. (In press), Proc. Natl. Acad. Sci. USA,90, 1657 (1993), ibid.,87, 7787 (1990), EMBO J.,12, 2809 (1993)], it has been confirmed that signals are transmitted by binding of ligands corresponding to nuclear receptors.
[0009]
OBJECT OF THE INVENTION
As a result of repeated studies for the purpose of applying the above-described fusion protein to cancer therapy, the present inventors have decided to use Fas antigen as a signal transduction protein. In the experiment, first, the possibility was examined using mouse Fas antigen.
The functional expression region of the mouse Fas antigen is considered to be the amino acid sequence portion from the 166th to 291st positions. However, even when this portion was bound to ligand binding regions of various hormone receptors as ligand binding proteins, the Fas antigen signal was not transmitted (see Example 4). Therefore, we succeeded in transmitting a signal for the first time by fusing the 136th to 305th amino acid sequence including not only the function expression region but also the transmembrane region with the ligand binding region of the hormone receptor. Completed the invention.
[0010]
Various signal transduction proteins used in the prior art are all those that act as transcription factors except that c-Ab1 acts as a kinase, and control the expression of the function of the target protein. In this respect, since the Fas antigen used in the present invention is a cell membrane receptor, it is considered that signal transmission is not performed unless it is bound to the membrane. This was completely unexpected from the prior art. Until now, no fusion protein using Fas antigen as a signal transduction protein has been known. Therefore, the fusion protein of the present invention and the DNA encoding the protein are considered to be a novel invention. In the present invention, it was also confirmed for the first time that signal transduction can be performed even if a subfamily receptor other than a steroid receptor is used as a nuclear receptor used as a ligand binding region.
[0011]
Whether or not this subfamily receptor is signaled in a fusion protein that binds the ligand binding region of the subfamily receptor, considering the difference in structure and signal transduction mechanism from the steroid receptor. It was unknown. The present invention proves that not only steroid receptors but also any nuclear receptors can be used as a ligand-binding region for signal transduction, which is significant.
Furthermore, the present inventors have found that screening for ligand agonist / antagonist can be performed by using cells transformed with a plasmid containing DNA encoding the fusion protein.
[0012]
DISCLOSURE OF THE INVENTION
The present invention
(i) a fusion protein in which an amino acid sequence containing a ligand binding region of a nuclear receptor is bound to the C-terminus of an amino acid sequence containing a transmembrane region and a functional expression region of Fas antigen,
(ii) DNA encoding the fusion protein,
(iii) a replication or expression vector containing the DNA,
(iv) a host cell transformed with the replication or expression vector,
(v) a cancer or autoimmune disease therapeutic agent containing the expression vector as an active ingredient, or
(vi) a method for screening an agonist or antagonist for a nuclear receptor, characterized by using the host cell
About.
[0013]
As the Fas antigen and the nuclear receptor constituting the fusion protein of the present invention, those derived from mammals such as those derived from humans, monkeys, dogs, cats, mice, rats, and guinea pigs are used. The Fas antigen and the nuclear receptor are preferably the same species as each other, but different species may be used. In the present invention, it has been confirmed that signal transmission can be performed even if different species are used. For example, combination of human Fas antigen and human nuclear receptor, combination of mouse Fas antigen and human nuclear receptor, combination of human Fas antigen and rat nuclear receptor, mouse Fas antigen and rat nuclear receptor A combination of a nuclear receptor, a human Fas antigen and a mouse nuclear receptor, or a mouse Fas antigen and a mouse nuclear receptor is preferably used.
[0014]
As the amino acid sequence containing the transmembrane region and functional expression region of Fas antigen used in the present invention and the amino acid sequence containing the ligand binding region of the nuclear receptor, only the portion corresponding to each region may be used. In addition, flanking regions (regions not involved in functional regions, several tens of amino acid sequences, preferably 60 or less) are included at both ends (N-terminal and / or C-terminal) of the portion corresponding to each region. You may use the thing. Furthermore, if desired, an amino acid sequence including a signal peptide region may be bound to the N-terminus of the transmembrane region of the Fas antigen. The amino acid sequence corresponding to each region includes not only the natural amino acid sequence, but also amino acid deletions, substitutions, additions and additions to such an extent that the apoptotic function of the original Fas antigen or the ligand binding function of the nuclear receptor is not impaired. Inserted ones can be used.
[0015]
Among the Fas antigens used in the present invention, the amino acid sequence including the transmembrane region and functional expression region of human Fas antigen is the 319th to 319th Val, and the mouse Fas antigen has a 305th to 305th from the 136th Ser. Sequences up to the second Leu are preferably used. As the amino acid sequence including the signal peptide region, the -16th to 23rd sequences are preferably used for human Fas antigen, and the -21st to 14th sequences are used for mouse Fas antigen.
[0016]
Examples of nuclear receptors used in the present invention include glucocorticoid receptors, brodiesterone receptors, mineralocorticoid receptors, androgenene receptors, estrogen receptors, steroid receptors such as retinoid X receptors, and retinoic acid receptors. And the like, retinoid receptors, vitamin D3 receptors, or thyroid hormone receptors. Preferably, human or rat estrogen receptor, human or mouse retinoid X receptor α, retinoid X receptor β or retinoid X receptor γ or human or mouse retinoic acid receptor α, retinoic acid receptor β or Retinoic acid receptor γ is used.
[0017]
The ligand binding region of each receptor is already known [Science,240, 889 (1988). ] For example, human glucocorticoid receptor from 528 to 777, human brodiesterone receptor from 680 to 934, human mineralocorticoid receptor from 734 to 984, human androgen receptor From 676 to 919, from 311 to 551 for human estrogen receptor, from 307 to 557 for rat estrogen receptor, from 225 to 462 for human retinoid X receptor α, human retinoid X receptor β from 297 to 526, mouse retinoid X receptor α from 230 to 467, mouse retinoid X receptor β from 171 to 410, mouse retinoid X receptor γ from 229th Up to 463rd, 198 to 462 for human retinoic acid receptor α, 191 to 448 for human retinoic acid receptor β, 200 to 454 for human retinoic acid receptor γ, and 198 for mouse retinoic acid receptor α. No. 462 to No. 462, mouse retinoic acid receptor β from 190 to 448, mouse retinoic acid receptor γ from 200 to 458, human vitamin D_ThreeIt is the amino acid from the 192nd to the 427th amino acid in the receptor, from the 183rd to the 410th amino acid in the human thyroid hormone receptor α, and from the 232nd to the 456th amino acid in the human thyroid hormone receptor β.
[0018]
Preferably, human estrogen receptor 311 to 551, rat estrogen receptor 307 to 557, human retinoid X receptor α 225 to 462, human retinoid X receptor β From 297 to 526, mouse retinoid X receptor α from 230 to 467, mouse retinoid X receptor β from 171 to 410, mouse retinoid X receptor γ from 229 to 463, From 198th to 462th of human retinoic acid receptor α, from 191st to 448th of human retinoic acid receptor β, from 200th to 454th of human retinoic acid receptor γ, from 198 of mouse retinoic acid receptor α. No. 190 to No. 462 of mouse retinoic acid receptor β From to 448 th, sequence from 200 th mouse retinoic acid receptor γ to 458 th is used. More preferably, human estrogen receptor 281 to 595, rat estrogen receptor 286 to 600, human retinoic acid receptor α 176 to 462, or mouse retinoic acid receptor The sequence from α 177 to 458 is used.
[0019]
Among the fusion proteins of the present invention, preferred are:
(i) a fusion protein in which the amino acid sequence from 311 to 551 of the human estrodiene receptor is linked to the C-terminus of the amino acid sequence from 145 to 319 of human Fas antigen;
(ii) a fusion protein in which the amino acid sequence from the 307th to 557th of the rat estrogen receptor is bound to the C-terminal of the amino acid sequence from the 145th to 319th of the human Fas antigen;
(iii) a fusion protein in which the amino acid sequence of human retinoid X receptor α is bound to the amino acid sequence of positions 225 to 462 to the C-terminal of the amino acid sequence of positions 145 to 319 of human Fas antigen;
(iv) a fusion protein in which the amino acid sequence from 297th to 526th of human retinoid X receptor β is linked to the C-terminal of the amino acid sequence from 145th to 319th of human Fas antigen;
(v) a fusion protein in which the amino acid sequence from the 230th to 467th positions of the mouse retinoid X receptor α is bound to the C-terminal of the amino acid sequence from the 145th to 319th positions of the human Fas antigen;
[0020]
(vi) a fusion protein in which the amino acid sequence from 171st to 410th of mouse retinoid X receptor β is bound to the C-terminal of the amino acid sequence from 145th to 319th of human Fas antigen;
(vii) a fusion protein in which the amino acid sequence from 229th to 463rd of mouse retinoid X receptor γ is bound to the C-terminal of the amino acid sequence from 145th to 319th of human Fas antigen;
(viii) a fusion protein in which the amino acid sequence from 198th to 462th of human retinoic acid receptor α is bound to the C-terminal of the amino acid sequence from 145th to 319th of human Fas antigen;
(ix) a fusion protein in which the amino acid sequence from 191st to 448th of human retinoic acid receptor β is bound to the C-terminal of the amino acid sequence from 145th to 319th of human Fas antigen;
(x) a fusion protein in which the amino acid sequence from the 200th to the 454th positions of the human retinoic acid receptor γ is bound to the C-terminal of the amino acid sequence from the 145th to 319th positions of the human Fas antigen;
[0021]
(xi) a fusion protein in which the amino acid sequence from the 198th to the 462nd of the mouse retinoic acid receptor α is bound to the C-terminal of the amino acid sequence from the 145th to 319th of the human Fas antigen;
(xii) a fusion protein in which the amino acid sequence from 190th to 448th of mouse retinoic acid receptor β is bound to the C-terminal of the amino acid sequence from 145th to 319th of human Fas antigen, or
(xiii) A fusion protein in which the amino acid sequence from the 200th to the 458th positions of the mouse retinoic acid receptor γ is bound to the C-terminal of the amino acid sequence from the 145th to 319th positions of the human Fas antigen.
[0022]
Moreover, as another preferable thing,
(i) a fusion protein in which the amino acid sequence from 311 to 551 of the human estrodiene receptor is bound to the C-terminal of the amino acid sequence from 136 to 305 of mouse Fas antigen;
(ii) a fusion protein in which the amino acid sequence from the 307th to 557th of the rat estrogen receptor is bound to the C-terminal of the amino acid sequence from the 136th to 305th of the mouse Fas antigen;
(iii) a fusion protein in which the amino acid sequence from the 225th to 462th positions of the human retinoid X receptor α is bound to the C-terminal of the amino acid sequence from the 136th to 305th positions of the mouse Fas antigen;
(iv) a fusion protein in which the amino acid sequence from the 297th to 526th positions of the human retinoid X receptor β is bound to the C-terminal of the amino acid sequence from the 136th to 305th positions of the mouse Fas antigen;
[0023]
(v) a fusion protein in which the amino acid sequence from mouse positions 230 to 467 of mouse retinoid X receptor α is bound to the C-terminal of the amino acid sequence from positions 136 to 305 of mouse Fas antigen;
(vi) a fusion protein in which the amino acid sequence from the 171st to 410th positions of the mouse retinoid X receptor β is bound to the C-terminal of the amino acid sequence from the 136th to 305th positions of the mouse Fas antigen;
(vii) a fusion protein in which the amino acid sequence from the 229th to 463rd positions of the mouse retinoid X receptor γ is bound to the C-terminal of the 136th to 305th amino acid sequences of the mouse Fas antigen;
(viii) a fusion protein in which the amino acid sequence from the 198th to the 462nd amino acid sequence of the human retinoic acid receptor α is bound to the C-terminal of the amino acid sequence from the 136th to the 305th amino acid sequence of the mouse Fas antigen;
(ix) a fusion protein in which the amino acid sequence from 191st to 448th of human retinoic acid receptor β is bound to the C-terminal of the amino acid sequence from 136th to 305th of mouse Fas antigen;
[0024]
(x) a fusion protein in which the amino acid sequence from the 200th position to the 454th position of the human retinoic acid receptor γ is bound to the C-terminal of the amino acid sequence from the 136th position to the 305th position of the mouse Fas antigen;
(xi) a fusion protein in which the amino acid sequence from mouse positions 198 to 462 of mouse retinoic acid receptor α is linked to the C-terminal of the amino acid sequence from position 136 to position 305 of mouse Fas antigen;
(xii) a fusion protein in which the amino acid sequence from mouse positions 190 to 448 of mouse retinoic acid receptor β is bound to the C-terminal of the amino acid sequence from mouse positions 136 to 305 of mouse Fas antigen, or
(xiii) A fusion protein in which the amino acid sequence from the 200th position to the 458th position of the mouse retinoic acid receptor γ is bound to the C-terminal of the amino acid sequence from the 136th position to the 305th position of the mouse Fas antigen.
[0025]
Among the fusion proteins of the present invention, more preferred are:
(i) a fusion protein in which the amino acid sequence of 281 to 595 of the human estrodiene receptor is linked to the C terminus of the amino acid sequence of 145 to 319 of human Fas antigen;
(ii) a fusion protein in which the amino acid sequence from the 286th to the 600th amino acid sequence of the rat estrogen receptor is bound to the C-terminal of the amino acid sequence from the 145th to 319th amino acid of the human Fas antigen;
(iii) a fusion protein in which the amino acid sequence from 176th to 462th of human retinoic acid receptor α is bound to the C-terminal of the amino acid sequence from 145th to 319th of human Fas antigen;
(iv) a fusion protein in which the amino acid sequence from 177th to 458th of mouse retinoic acid receptor α is bound to the C-terminal of the amino acid sequence from 145th to 319th of human Fas antigen;
[0026]
(v) a fusion protein in which the amino acid sequence of 281 to 595 of the human estrogen receptor is bound to the C-terminal of the amino acid sequence of 136 to 305 of mouse Fas antigen;
(vi) a fusion protein in which the amino acid sequence from the 286th to the 600th amino acid sequence of the rat estrogen receptor is bound to the C-terminal of the amino acid sequence from the 136th to the 305th amino acid of the mouse Fas antigen;
(vii) a fusion protein in which the amino acid sequence from the 176th to 462th positions of the human retinoic acid receptor α is bound to the C-terminal of the amino acid sequence from the 136th to 305th positions of the mouse Fas antigen;
(viii) A fusion protein in which the amino acid sequence from the 177th to the 458th of the mouse retinoic acid receptor α is bound to the C-terminal of the amino acid sequence from the 136th to the 305th of the mouse Fas antigen.
[0027]
The present invention also includes DNA encoding the fusion protein of the present invention.
As is well known, 1 to 6 types of codons encoding one amino acid (for example, one type of methionine (Met) and six types of leucine (Leu)) are known. Therefore, the base sequence of DNA can be changed without changing the amino acid sequence.
[0028]
The present invention includes all base sequence groups encoding the fusion protein of the present invention. The productivity of the fusion protein may be improved by changing the base sequence.
The DNA encoding the fusion protein of the present invention can be prepared according to the following method.
That is,
(i) Amplifying a DNA encoding an amino acid sequence including a necessary region out of the amino acid sequences of Fas antigen and nuclear receptor by a polymerase chain reaction (PCR) method,
(ii) It is carried out by incorporating a PCR product obtained from the Fas antigen into an appropriate vector, and subsequently incorporating a PCR product obtained from the intracellular receptor.
[0029]
In more detail,
Step (i) is a step of amplifying the components necessary for fusion by the PCR method. In the case of Fas antigen, a base sequence encoding an amino acid sequence including a transmembrane region and a functional expression region is chemically synthesized and used as a primer. In the case of an intracellular receptor, a base sequence encoding an amino acid sequence including a ligand binding region is chemically synthesized and used as a primer.
A specific restriction enzyme site is provided at the 5 'end of the obtained primer. As a template for the PCR method, mRNA isolated and purified from cells or cell lines of corresponding mammals such as humans and mice can be used. The PCR method is performed by a known method. Recently, since a PCR automation apparatus is also widespread, this apparatus is preferably used.
[0030]
Step (ii) is a step of fusing both parts in a vector. As a plasmid vector used in this step, there are many known plasmid vectors that function in E. coli (for example, pBR322) and those that function in Bacillus subtilis (for example, pUB110). .
It can also be directly incorporated into an expression vector. For example, in the case of expressing in E. coli, the PCR product of Fas antigen is connected downstream of an appropriate promoter (eg, trp promoter, lac promoter, λPL promoter, T7 promoter, etc.), and then received downstream in the cell. The PCR product of the body is connected and inserted into a vector that functions in E. coli (for example, pUC18, pUC19, etc.) to create an expression vector. For expression in mammalian cells, an appropriate promoter (eg, SV40 promoter, LTR promoter, metallothionein) in an appropriate vector (eg, retrovirus vector, papilloma virus vector, vaccinia virus vector, SV40 vector, etc.). An expression vector is prepared by inserting the PCR product of the Fas antigen and then the PCR product of the intracellular receptor downstream of the promoter or the like. Preferably, pCMX (Cell,66663 (1991). ) Is used.
[0031]
When expressed in mammalian cells, a PCR product having a base sequence encoding the signal peptide region of Fas antigen can be inserted immediately downstream of the promoter, if desired.
The present invention includes a replication or expression vector comprising the DNA of the present invention. Examples of the vector include a plasmid, virus, or phage vector comprising an ori region and, if necessary, a promoter for expression of the DNA, a promoter control factor, and the like. The vector may contain one or more selective marker genes (eg, neomycin resistance gene, blasticidin S resistance gene).
[0032]
Examples of the expression system (host-vector system) for producing the fusion protein of the present invention include bacterial, yeast, insect cell and mammalian cell expression systems.
For example, when expressing in E. coli, E. coli transformed with an expression vector that functions in E. coli (for example, E. coli DH1, E. coli JM109, E. coli HB101 strain, etc.) is cultured in an appropriate medium. Thus, the desired polypeptide can be obtained from the cells. In addition, if a bacterial signal peptide (for example, a pelB signal peptide) is used, the target fusion protein can be secreted into the periplasm.
[0033]
In addition, when expressed in mammalian cells, suitable mammalian cells (for example, monkey COS-7 cells, Chinese hamster CHO cells, mouse L cells, mouse fibroblasts) using the above-described expression vector that functions in mammalian cells. Cells, human cancer cells, etc.) are transformed, and the transformant is cultured in an appropriate medium to produce the target fusion protein. The polypeptide obtained as described above can be isolated and purified by a general biochemical method.
The present invention includes host cells transformed with a replication or expression vector comprising the DNA of the present invention.
[0034]
【The invention's effect】
The fusion protein of the present invention thus obtained can be used as a therapeutic agent for cancer or autoimmune diseases. That is, an expression vector containing DNA encoding the fusion protein of the present invention is locally administered to a cancer lesion or autoimmune disease lesion by a targeting method (for example, encapsulated in liposomes). The vector enters the cancer cell at the lesion site, enters the gene, and continuously expresses the fusion protein. Thereafter, a ligand corresponding to a nuclear receptor constituting a part of the fusion protein of the present invention (for example, 9-cis-retinoic acid or retinoic acid receptor when a retinoid X receptor is used as the receptor) Vitamin A) is administered if A ligand binds to the fusion protein of the present invention produced in cancer cells, and an apoptosis signal is transmitted. Thus, cancer cells die.
[0035]
Furthermore, the fusion protein of the present invention can be widely used as a screening method for agonists or antagonists for intracellular nuclear receptors. That is, a specimen is added to cells that always express the fusion protein of the present invention. If the cell dies, the specimen indicates that it has agonist activity, and if the cell survives in the presence of the agonist, the specimen is found to have antagonist activity.
The screening method of the present invention not only can process a large amount of samples in a short time, but also exhibits an effect (result) as cell death. It can be said that it is a law.
[0036]
【Example】
Hereinafter, the present invention will be described in more detail and more specifically by way of examples, but of course the present invention is not limited thereto.
[0037]
Example 1 Preparation of PCR products
FIG. 1 shows a conceptual diagram of a PCR primer preparation method. In the figure, region A shows the signal peptide region of mouse Fas antigen, region B shows the extracellular region of mouse Fas antigen, region C shows the transmembrane region of mouse Fas antigen, and region D shows the cytoplasm of mouse Fas antigen. Region E represents the ligand binding region of human retinoic acid receptor α, and region F represents the ligand binding region of rat estrogen receptor.
[0038]
(1) Preparation of PCR product of mouse Fas antigen
A report by Fukunaga et al. (J. Immunology,1481274 (1992)), the following primers were prepared according to the structure of the mouse Fas antigen.
F1: Amino acid sequence from the 131st position to the 143rd position: a primer that corresponds to the structure of Pro Cys Thr Ala Thr Ser Asn Thr Asn Cys Arg Lys Gln and in which restriction enzyme NcoI and KpnI sites are introduced into a part of 5 ′ SEQ ID NO: 1),
[0039]
5'-CACCATGGGTACCAGCAATACAAACTGCAGGAAAC-3 '
Was synthesized.
F2: Amino acid sequence from position 209 to position 221: primer corresponding to the structure of Glu Asp Met Thr Ile Gln Glu Ala Lys Lys Phe Ala Arg with restriction enzyme NcoI and KpnI sites introduced into a part of 5 ′ side ( SEQ ID NO: 2),
[0040]
5'-GAAGCCATGGGTACCCAGGAAGCTAAAAAATTTGCTCGA-3 '
Was synthesized.
F3: Amino acid sequence from 298th to 306th (C-terminal): Asn Glu Asn Glu Gly Gln Cys Leu Glu and corresponding to a part of 3 'untranslated region, and further BamHI site in part of 3'- side Introduced 3′-primer (SEQ ID NO: 3),
[0041]
5'-TCAGGATCCAGACATTGTCCTTCATTTTCATT-3 '
Was synthesized.
S1: A part of the 5 'untranslated region of Fas antigen cDNA, including amino acid sequence from -21st to -14th: Corresponds to the structure of Met Leu Trp Ile Trp Ala Val Leu 5'-side restriction enzyme HindIII and A primer (SEQ ID NO: 4) introduced with a NotI site;
[0042]
5'-CTGAAGCTTCGCGGCCGCACCATGCTGTGGATCTGGGCTGTCCTG-3 '
Was synthesized.
S2: part of cDNA of Fas antigen, amino acid sequence from 5th to 14th: 3′-primer corresponding to Ser Ile Ser Glu Ser Leu Lys Leu Arg Arg and having a HindIII site introduced in part of 3 ′ side (SEQ ID NO: 5),
[0043]
5'-CCTCCTAAGCTTTAAACTCTCGGAGATGCT-3 '
Was synthesized.
Mouse T cell line, RLM-11 (Cell,68, 1109 (1992)) as a template, using F1 and F3 PCR products as primers, and using a thermal sequencer (model TSR-300, manufactured by Iwaki Glass Co., Ltd.) at 95 ° C. As a result of RT-PCR under conditions of 180 seconds × 1 → (95 ° C. for 60 seconds, 55 ° C. for 60 seconds, 72 ° C. for 60 seconds) × 15 → 72 ° C. for 180 seconds, a fragment of about 520 bp (Referred to as Fas-TM). Next, PCR was performed under the same conditions as described above using the same mRNA as above as a template and PCR products of F2 and F3 as primers. As a result, a fragment of about 280 bp (hereinafter referred to as Fas-ΔTM) I wrote.) Furthermore, as a result of PCR under the same conditions as described above using the same mRNA as the template and the PCR products of S1 and S2 as primers, a fragment of about 120 bp (hereinafter referred to as Fas-sig) is obtained. Obtained.
[0044]
(2) Preparation of PCR product of human retinoic acid α receptor
Giguere et al. (Nature,330, 624 (1987)), the following primers were prepared from the structure of the receptor for human retinoic acid α.
R1: Amino acid sequence from 173rd to 182nd: Primer corresponding to the structure of Glu Cys Ser Glu Ser Tyr Thr Leu Thr Pro, and a restriction enzyme Bgl II site introduced into a part of 5 ′ side (SEQ ID NO: 6) ,
[0045]
5'-GGCAGATCTCTCTGAGAGCTACACGCTGACGCCG-3 '
Was synthesized.
R2: Amino acid sequence from the 456th position to the 462nd position (C terminus): Ser Pro Ala Thr His Ser Pro and corresponding to a part of the 3 ′ untranslated region 3 ′ with a BamHI site introduced into a part of the 3 ′ side A primer (SEQ ID NO: 7),
[0046]
5'-CGTGGATCCTCACGGGGAGTGGGTGGCCGGGCT-3 '
Was synthesized.
Example 1- (1) Using a thermal sequencer (model TSR-300) using mRNA obtained from HL-60 of human prebone myeloid leukemia cells as a template and using R1 and R2 as primers. As a result of RT-PCR under the same conditions as above, a fragment of about 870 bp (hereinafter referred to as RARα) was obtained.
[0047]
(3) Preparation of rat estrogen receptor PCR product
Koike et al. (Nucleic Acid Research,15, 2499 (1987)), the following primers were prepared from the structure of the rat estrogen receptor.
R3: Amino acid sequence from the 282nd to the 292nd: Primer corresponding to the structure of Arg Asn Glu Met Gly Thr Ser Gly Asp Met Arg, in which a restriction enzyme BamHI site was introduced in part of the 5 ′ side (SEQ ID NO: 8) ,
[0048]
5'-CGAAAGGATCCGGGCACTTCAGGAGACATGAGA-3 '
Was synthesized. R4: amino acid sequence from the 598th to the 600th (C-terminal): Asn Thr Ile and 3 ′ primer corresponding to a part of the 3 ′ untranslated region and a BamHI site introduced into a part on the 3 ′ side (SEQ ID NO: 9),
[0049]
5'-TGGGGTACCTGGGAGTTCTCAGATGGTGTT-3 '
Was synthesized.
PUCER6 (Granted by Professor Masami Muramatsu, Saitama Medical University, Nucleic Acid Research,15, 2499 (described in 1987) using the mRNA obtained as a template, R3 and R4 as primers, and using a thermal sequencer (model TSR-300), the same conditions as in Example 1- (1) As a result of PCR, a fragment of about 970 bp (hereinafter referred to as ER) was obtained.
[0050]
Example 2 Construction of cDNA encoding fusion protein
An expression vector pCMXEPI containing a part of the structure of influenza hemagglutinin (Epi) was prepared as a sequence for detecting the fusion protein. A construction diagram of cDNA encoding the fusion protein is shown in FIG. 2 (in the figure, * indicates (NheI) when the DNA of the fusion sequence 5 is prepared).
First, pCMX vector (Cell,66, 663 (1991)) with HindIII-KpnI and a synthetic nucleotide (SEQ ID NO: 10) having the following sequence:
[0051]
5'-AGCTTGGCGCCACCATGGTGTACCCCTACGACGTGCCCGACTATGCCAGCCTGGGTAC-3 '
(PCMXEPI), the vector is opened with KpnI-BamHI, and a PCR product (Fas-TM) containing the transmembrane region and functional expression region of Fas antigen and a PCR product (Fas-Δ) containing only the functional expression region TM) (both prepared in Example 1 (1)) each KpnI-BamHI fragment was incorporated. As a result, a sequence in which Epi was directly fused with the transmembrane region and functional expression region of Fas antigen or the functional expression region was obtained. The vector was then opened with BamHI to incorporate the Bgl II-BamHI fragment of the ligand binding region of the retinoic acid receptor (RARα, prepared in Example 1 (2)). Epi-Fas-RARα fusion sequence was obtained from the plasmid integrated in the required direction (hereinafter Epi-Fas-TM-RARα was fused sequence 1, and Epi-Fas-ΔTM-RARα was fused sequence 2). ). On the other hand, a Fas antigen signal sequence (Fas-sig) having the same end was introduced into the Hind III site in the upstream region of each of the fusion sequences 1 and 2. The directions were confirmed, and fusion sequences 3 and 4 having a Fas antigen signal sequence corresponding to fusion sequences 1 and 2, respectively, were obtained.
Similarly, a fusion sequence of Epi-Fas-TM-ER was obtained (hereinafter referred to as fusion sequence 5).
[0052]
Example 3 Construction of a fusion protein expression system
The concept of the fusion protein expression system is shown in FIG. 3 (in the figure, * indicates (NheI) when producing DNA of the fusion sequence 5).
Vector pSV2bsr (Exp. Cell. Res., Reported by Izumi et al.,197, 229, (1991)), a blasticidin S resistant portion (EcoRI-PvuII fragment) was excised. Expression vector pEFBOS (Nuc. Acid Res.,18, 5322 (1990)), only the one located on the 3 ′ side of the EF-1α promoter is deleted, and the above-mentioned fragment (the blasticidin S resistant portion) is blunt-ended at the remaining site. It was introduced after becoming. Fusion sequences 1 and 2 were cut with HindIII-BamHI, fusion sequences 3 and 4 with NotI-BamHI, and further fusion sequence 5 with HindIII-NheI and smoothed. The BstXI site in the multi-cloning site of the vector pEFBOSbsr was blunted and the above fusion sequence was introduced.
[0053]
The obtained expression vector was linearized with EcoRI and introduced into mouse fibroblasts, L929, human cancer cells, and HeLa by the usual calcium phosphate method. Exp. Cell. Res.,197, 229, (1991) (plastisidine S selection) were used to select cells that always express the fusion protein.
[0054]
Example 4 Cell killing assay with vitamin A
Cells constantly expressing each fusion protein, eg L929, 2.5 × 10^FourThe cells were suspended in DMEM (Dulbecco's modified Eagle's medium) containing 10% FCS (fetal calf serum) at a ratio of 1/100 μl and distributed to a 96-well microtiter plate. The cells were cultured in 5% carbon dioxide gas at 37 ° C. for 24 hours. A sample with a concentration twice the final concentration of vitamin A was prepared in the same culture and 100 μl was added. After further incubation for 15 hours and discarding the supernatant, the living cells were stained with crystal violet (0.75% crystal violet in 50% ethanol, 0.25% NaCl, 1.75% formaldehyde, room temperature, 20 minutes) (Cell ,66, 233 (1991)) and Itoh et al. After washing twice with PBS (phosphate buffer saline), the absorbance at 540 nm was directly measured with a micro ELISA reader.
[0055]
FIG. 4 shows changes with time in the number of viable cells during vitamin A treatment. FIG. 5 shows changes in vitamin A concentration (in the figure, black circles are cells into which the fusion protein expression system has been introduced, and white circles are cells into which the fusion protein expression system has not been introduced). These results were obtained from experiments using cells into which the fusion sequence 1 was introduced. Cell death induced by 1 μM vitamin A is more than 90% complete after 8 hours. In addition, the action of vitamin A in the assay after 15 hours was concentration-dependent, and the 50% action concentration (IC50) was about 0.1 μM.
[0056]
In the cells into which the fusion sequence 2 or 4 was introduced, the above “induction of ligand-specific cell death” phenomenon was not observed at all. The result of the fusion sequence 3 to which the Fas antigen signal sequence (Sig) was added is the same as the result of the fusion sequence 1 described above, and the effects of both can be distinguished in the concentration dependency of vitamin A and the change in the reaction over time. It was not.
[0057]
The above results are different from previously reported fusion proteins using transcription factors, etc. In the case of Fas antigen, signal transduction is not performed even if only the functional expression region is fused, and the transmembrane region is essential. Is shown. Moreover, it is shown that a signal is also transmitted by a fusion protein bound with a ligand binding region of a subfamily of intracellular receptors. Furthermore, it was confirmed that a signal is transmitted even if the Fas antigen and the nuclear receptor are from different animals, and that apoptosis occurs in the cells of the different animals.
[0058]
Example 5 Cell killing assay with estrogen
In the same manner as in Example 4, cells constantly expressing each fusion protein were cultured, and after adding estrogen, the cells were cultured and stained, and the absorbance was measured.
FIG. 6 shows changes in estrogen concentration (in the figure, black squares are cells into which the fusion protein expression system has been introduced, and white squares are cells into which the fusion protein expression system has not been introduced). The effect of estrogen in the assay after 15 hours was concentration dependent and its 50% working concentration (IC50) was about 0.1 nM. This result indicates that the apoptotic function of Fas antigen is also expressed in a fusion protein bound with a ligand binding region of a steroid receptor.
[0059]
[Sequence Listing]
SEQ ID NO: 1
Sequence length: 35
Sequence type: Nucleic acid
Number of chains: 1 strand
Topology: Linear
Array
CACCATGGGT ACCAGCAATA CAAACTGCAG GAAAC
[0060]
SEQ ID NO: 2
Sequence length: 39
Sequence type: Nucleic acid
Number of chains: 1 strand
Topology: Linear
Array
GAAGCCATGG GTACCCAGGA AGCTAAAAAA TTTGCTCGA
[0061]
SEQ ID NO: 3
Sequence length: 32
Sequence type: Nucleic acid
Number of chains: 1 strand
Topology: Linear
Array
TCAGGATCCA GACATTGTCC TTCATTTTCA TT
[0062]
SEQ ID NO: 4
Sequence length: 45
Sequence type: Nucleic acid
Number of chains: 1 strand
Topology: Linear
Array
CTGAAGCTTC GCGGCCGCAC CATGCTGTGG ATCTGGGCTG TCCTG
[0063]
SEQ ID NO: 5
Sequence length: 30
Sequence type: Nucleic acid
Number of chains: 1 strand
Topology: Linear
Array
CCTCCTAAGC TTTAAACTCT CGGAGATGCT
[0064]
SEQ ID NO: 6
Sequence length: 34
Sequence type: Nucleic acid
Number of chains: 1 strand
Topology: Linear
Array
GGCAGATCTC TCTGAGAGCT ACACGCTGAC GCCG
[0065]
SEQ ID NO: 7
Sequence length: 33
Sequence type: Nucleic acid
Number of chains: 1 strand
Topology: Linear
Array
CGTGGATCCT CACGGGGAGT GGGTGGCCGG GCT
[0066]
SEQ ID NO: 8
Sequence length: 33
Sequence type: Nucleic acid
Number of chains: 1 strand
Topology: Linear
Array
CGAAAGGATC CGGGCACTTC AGGAGACATG AGA
[0067]
SEQ ID NO: 9
Sequence length: 30
Sequence type: Nucleic acid
Number of chains: 1 strand
Topology: Linear
Array
TGGGGTACCT GGGAGTTCTC AGATGGTGTT
[0068]
SEQ ID NO: 10
Sequence length: 58
Sequence type: Nucleic acid
Number of chains: 1 strand
Topology: Linear
Array
AGCTTGGCGC CACCATGGTG TACCCCTACG ACGTGCCCGA CTATGCCAGC CTGGGTAC
[Brief description of the drawings]
FIG. 1 is a conceptual diagram of a PCR primer preparation method.
FIG. 2 is a construction diagram of cDNA encoding a fusion protein.
FIG. 3 is a conceptual diagram of a fusion protein expression system.
FIG. 4 is a graph showing the relationship between the administration time and the number of living cells in an apoptosis assay using vitamin A.
FIG. 5 is a graph showing the relationship between the concentration of vitamin A and the number of living cells in an apoptosis assay using vitamin A.
FIG. 6 is a graph showing the relationship between the concentration of estrogen and the number of living cells in an apoptosis assay using estrogen.

Claims (22)

  At the C-terminus of the amino acid sequence of the mouse Fas antigen from the 136th to the 305th amino acid sequence or the amino acid sequence of the human Fas antigen from the 145th to 319th amino acid sequence (but not including the extracellular region of the Fas antigen), A fusion protein in which an amino acid sequence containing a ligand binding region of a nuclear receptor is bound.   At the N-terminal of the amino acid sequence from the 136th to the 305th amino acid sequence of the mouse Fas antigen or the amino acid sequence from the 145th to 319th amino acid sequence of the human Fas antigen (excluding the extracellular region of the Fas antigen), The fusion protein according to claim 1, wherein the amino acid sequence from -21st to 14th of mouse Fas antigen or the amino acid sequence from -16th to 23rd of human Fas antigen is bound.   The fusion protein according to claim 1 or 2, wherein the nuclear receptor is a steroid receptor.   The fusion protein according to claim 3, wherein the nuclear receptor is an estrogen receptor.   5. The amino acid sequence containing the ligand binding region of the nuclear receptor is the amino acid sequence from 311 to 551 of the human estrogen receptor, or the amino acid sequence from 307 to 557 of the rat estrogen receptor. The fusion protein described.   The fusion protein according to claim 1 or 2, wherein the nuclear receptor is a retinoid receptor.   The fusion protein according to claim 6, wherein the nuclear receptor is a retinoid X receptor.   The amino acid sequence including the ligand binding region of the nuclear receptor is the amino acid sequence from the 225th to the 462th position of the human retinoid X receptor α, the amino acid sequence from the 297th to the 526th position of the human retinoid X receptor β, the mouse retinoid The amino acid sequence from the 230th to the 467th position of the X receptor α, the amino acid sequence from the 171st to the 410th position of the mouse retinoid X receptor β, or the amino acid sequence from the 229th to the 463rd position of the mouse retinoid X receptor γ The fusion protein according to claim 7.   The fusion protein according to claim 6, wherein the nuclear receptor is a retinoic acid receptor.   The amino acid sequence including the ligand binding region of the nuclear receptor is the amino acid sequence from the 198th to the 462nd human retinoic acid receptor α, the amino acid sequence from the 191st to 448th human retinoic acid receptor β, human retinoin 200 to 454 amino acid sequence of acid receptor γ, 198 to 462 amino acid sequence of mouse retinoic acid receptor α, 190 to 448 amino acid sequence of mouse retinoic acid receptor β, or The fusion protein according to claim 9, which has an amino acid sequence from the 200th position to the 458th position of mouse retinoic acid receptor γ. Fusion protein according to claim 1 or 2 wherein the cell nuclear receptor is a vitamin D ₃ receptor.   The fusion protein according to claim 1 or 2, wherein the nuclear receptor is a thyroid hormone receptor.   From the C-terminal of the amino acid sequence 136 to 305 of the mouse Fas antigen, or the C-terminal of the amino acid sequence 145 to 319 of the human Fas antigen, the 281st to 595th of the human estrogen receptor The fusion protein according to claim 1, wherein the amino acid sequence or the amino acid sequence from the 286th to the 600th amino acid sequence of the rat estrogen receptor is bound.   From the 176th to 462th positions of the human retinoic acid receptor α at the C-terminal of the amino acid sequence from the 136th to 305th positions of the mouse Fas antigen or the C-terminal of the amino acid sequence from the 145th to 319th positions of the human Fas antigen The fusion protein according to claim 1, wherein the amino acid sequence of 177 or 458 of mouse retinoic acid receptor α is bound.   A DNA encoding the fusion protein according to claim 1.   A DNA encoding the fusion protein according to any one of claims 1 to 12.   A DNA encoding the fusion protein according to claim 13 or 14.   A replication or expression vector comprising the DNA of claim 15.   A host cell transformed with a replication or expression vector according to claim 18.   A therapeutic agent for cancer or autoimmune disease comprising the expression vector according to claim 18 as an active ingredient.   A method for screening an agonist or antagonist for a nuclear receptor, comprising using the host cell according to claim 19.   A method for screening an agonist or antagonist for an estrogen receptor, a retinoid X receptor or a retinoic acid receptor, wherein the host cell according to claim 19 is mouse fibroblast L929 or human cancer cell HeLa.

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