JPH0578397A - Thrombolytic protein - Google Patents

Abstract

PURPOSE:To provide a new thrombolytic protein having high thrombus specificity and exhibiting an anti-coagulative action. CONSTITUTION:The thrombolytic protein of this invention is a hybridized product produced by combining a tissue plasminogen-activator or its modified product with a part or all of the epithelial cell-multiplying factor-like domain of throbomodulin directly or through a linker, and is produced by a genetic recombination method.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a tissue plasminogen activator (hereinafter, t-PA) or its modified form and an epidermal growth factor (hereinafter, EGF) -like domain of thrombomodulin (hereinafter, TM). The present invention relates to a thrombolytic protein formed by binding a part or all of it. For more details,
The present invention relates to a thrombolytic protein that exerts the anticoagulant action of TM and also has the action of t-PA having high thrombosis specificity via TM. Further, the present invention relates to a new thrombolytic protein which is clinically less likely to cause reocclusion and has an effect of reducing side effects.

[0002]

2. Description of the Related Art t-PA is converted into plasmin which is an active enzyme by hydrolyzing plasminogen which is an inactive enzyme precursor existing in plasma. Plasmin decomposes fibrin clots (thrombus) in blood vessels caused by various causes (see Document 1 below)). 1) European Patent Application Publication No. 0041766 A2

[0003] t-PA has a higher affinity for fibrin than other thrombolytic agents, and has attracted attention as a substance that specifically dissolves fibrin and eventually thrombus. However,
When used in clinical practice, it was revealed that recanalization of a blocked coronary artery requires a much higher dose (100 to 150 mg per patient) than expected (Reference 2 below). Therefore, it has been pointed out that the effect of t-PA on thrombus specificity is diminished, resulting in a generalized bleeding tendency. Therefore, a modified t-PA or a hybrid with another protein has been prepared for the purpose of imparting higher thrombus specificity (Reference 3 below).
~ 5)). 2) The TII MI Study Group, N. Ingle. J. Med. 320 618-
627 (1989) (The TIMI study group, NE)
ngl. J. Med. 320 618-627 (198)
9)) 3) European Patent Application Publication No. 292326 4) JP-A-1-85078 5) European Patent Application Publication No. 350692

Another problem with t-PA is reocclusion. If severe stenosis remains even after successful recanalization with thrombolytic therapy, acute reocclusion often occurs (Reference 6 below). Particularly, in the case of t-PA, according to the report of Gold et al., The reocclusion rate was as high as 45% in the acute phase (Reference 7 below), and in other reports, it was reported that the reocclusion rate was 18 to 30%. (Reference 8 below),
9)). 6) Di. Gee. Harrison Etoal. Circulation 69 991 (1984) (DG Harrison et al., Circ).
repetition 69 991 (1984)) 7) H. Kei. Gold Etoal. , Circulation 73 347 (1986) (HK Gold et al., Circulat.
Ion 73 347 (1986)) 8) J. H. Chesebro et al. Circulation 76142 (1987) (JH Chesebro et al., Circ.
ulation 76 142 (1987)) 9) Kizen Shinichi Angiology 29 497 (1989)

On the other hand, TM is a glycoprotein existing on vascular endothelial cells and binds thrombin generated in blood with a very high affinity to form a complex. When TM binds to thrombin, it suppresses the blood coagulation action (activation of fibrin formation catalyst, blood coagulation factor V, and platelet activation) of thrombin. Furthermore, this complex has a rate of conversion of protein C, a vitamin K-dependent protein, into anticoagulant enzyme (activated protein C) of at least 1000.
Doubled. That is, TM has an action of converting thrombin from blood coagulation enzyme to anticoagulation enzyme.

The TM was originally a C.I. T. It was identified by Esmon et al. (Reference 10 below). TM has been isolated and purified from rabbit lung (Reference 11 below), bovine lung (Reference 12 below), and human placenta (Reference 13 below). Human TM cDNA has been reported, and the chemical structure diagram of TM has been provided (reference document 14), 189th.
Page 3). The TM molecule consists of five domains, a hydrophobic region from the amino terminus, a cysteine-rich region, and a potential O-
It is composed of a sequence-rich region with a glycosylation site, a hydrophobic transmembrane region, and a cytosolic tail.

Of these, the region rich in cysteine is EG
It has been shown to have 6 repeating structures similar to the precursor of F (hereinafter referred to as E1, E2, E3, E4, E5, and E6 from the amino terminal side), and is called an EGF-like domain. There is. As an amino acid sequence corresponding to E1 to E6 of natural human TM, for example, the first amino acid sequence of Reference 14) is described.
Natural TM, as disclosed in Fig. 5 on page 894
Amino acid sequence corresponding to positions 227 to 462 (E1
= 227-262, E2 = 263-304, E3 =
305th-344th, E4 = 345-386th, E5 = 3
87-421 position, E6 = 422-462 position). Regarding the functional site of TM, it has been reported that E2-E6 of the EGF-like domains have the ability to bind to thrombin and significantly increase protein C activation (References 15 and 16 below), In addition, in an experiment using recombinant TM derived from human, it has been reported that E4 to E6 (hereinafter, E456) have a function of binding to thrombin and significantly increasing protein C activation (see Reference 1 below).
7), 18)). Studies have also been conducted to develop human-derived recombinant TM containing E456 as a therapeutic agent for DIC (disseminated intravascular coagulation).

10) C. tea. Esmon Etoaru. , Proc. National. Acad. Rhinoceros. USA. 78 2249-2252 (1981) (CT Esmon et al., Proc. Na.
tl. Acad. Sci. U. S. A. 78 2249
-2252 (1981)) 11) N. Elle. Esmon Etoaru. , J. Biol. Chemistry 261 8 59-864
(1982) (NL Esmon et al., J. Biol.
Chemistry 261 8 59-864 (1
982)) 12) H. Vi. Yakubosky Etoaru. , J. Biol. Chemistry 261 3876-3
882 (1986) (HV Jakubowski et al., J. Am.
Biol. Chemistry 261.3876-3
882 (1986)) 13) H. H. Salem Etoal. , J.
Biol. Chemistry 259 12246-1225
1 (1984) (H. H. Salem et al., J. Biol.
Chemistry 259 12246-12251
(1984)) 14) Kei. Suzuki Etoaru. , Embo. J. 6
1891-1897 (1987) (K. Suzuki et al., EMBO J. 6).
1891-1897 (1987)) 15) C.I. tea. Esmon Etoaru. , J.
Biol. Chemistry 6 3352-3356 (1
989) (CT Esmon et al., J. Biol.
Chemistry6 3352-3356 (198
9)) 16) Japanese Patent Application Laid-Open No. 2-19399 17) M. Zushietaru. , J. Biol. Chemistry 26410351-10353 (198
9) M.M. Zushi et al. , J. Biol. Che
history 264 10351-10353 (1
989)) 18) Japanese Patent Application Laid-Open No. 2-255699

[0009]

As described above, the development of a novel thrombolytic protein having a high thrombus specificity and exhibiting an anticoagulant effect is desired in the art, but it is still satisfactory. It is the actual situation that is not obtained. Therefore, an object of the present invention is to approach thrombin that is locally present in a thrombus through the high affinity of TM for thrombin,
Therefore, the thrombolytic action of t-PA is exerted (thrombus-specific lysis), and thrombin-catalyzed blood coagulation reactions such as fibrin formation, blood coagulation factor V activation and platelet activation are blocked, Furthermore, the complex of TM and thrombin is to provide a thrombolytic protein that increases the activation of protein C and promotes an anticoagulant reaction. Furthermore, in the treatment of thrombosis, side effects due to thrombotic-specific lytic action, that is, systemic bleeding is reduced, thrombin activity is blocked after thrombolysis, and reocclusion due to exertion of anticoagulant action associated with increased protein C activation is caused. An object is to provide a therapeutic agent for thrombosis that prevents it.

[0010]

The inventors of the present invention have made extensive studies to solve the above problems, and as a result, have completed the present invention. That is, the gist of the present invention is (1) a thrombolytic protein obtained by binding t-PA or a variant thereof with a part or all of the EGF-like domain of TM, (2) the thrombus according to (1) above A DNA containing a nucleotide sequence encoding a lytic protein, (3) a recombinant expression vector capable of expressing the DNA according to (2) above in a transformed prokaryotic or eukaryotic cell, (4)
A prokaryotic or eukaryotic cell transformed with the recombinant expression vector according to (3) above, and (5) a thrombolytic protein according to (1) above as an active ingredient. The present invention relates to a therapeutic agent for thrombosis.

In the present invention, the source of t-PA is not particularly limited as long as it is of human origin, and examples thereof include Bose melanoma, uterus, human T cell-derived established cells, human fetal lung cells and the like. May be derived from. In the present invention, the modified t-PA means a product which can be produced from natural t-PA by a protein engineering method and has a property of converting plasminogen into plasmin. For example, among five domain structures of t-PA, a mutant (K2A) consisting of a kringle 2 (K2) and an active domain (A), or a kringle 1 (K1), a kringle 2 (K2) and an active domain (A). And a deletion type mutant such as a mutant (K1K2A).

In the present invention, a part of the EGF-like domain of TM is not particularly limited to a gene source as long as TM is of human origin, and examples thereof include placental syncytiotrophoblast cells and human fetal lung cells. , The lumen of lymphatic vessels, and the like. EGF present in such TM
-Like domains (E1-E6), such as E456 and E4
Those including 56 are listed. The entire EGF-like domain refers to E1 to E6.

The thrombolytic protein of the present invention is t-PA.
Or a thrombolytic protein obtained by binding a variant thereof with a part or all of the EGF-like domain of TM, and specifically, an amino acid sequence corresponding to E456 of TM is an amino acid sequence corresponding to K2A, Or TM E
An example is one in which the amino acid sequence corresponding to 456 binds to the amino acid sequence corresponding to K1K2A to form a hybrid. As a mode of binding, both may be bound directly or may be bound via a linker having one or more amino acids. The linker is not particularly limited as long as it does not affect the action of the thrombolytic protein of the present invention, but a peptide consisting of 1 to 20 amino acids is usually used, for example, one consisting of 3 or 5 glycines. It is illustrated.

The amino acid sequence corresponding to K2A of t-PA as used in the present invention is, for example, 176 of natural t-PA.
An amino acid sequence containing an amino acid sequence corresponding to positions 527 to 527 can be mentioned. Furthermore, it may consist of an amino acid sequence obtained by mutating a part of the amino acid sequence without changing the main activity of K2A of t-PA by natural mutation or artificial mutation. In the present invention,
Examples of the amino acid sequence corresponding to K1K2A of t-PA include an amino acid sequence containing an amino acid sequence corresponding to positions 87 to 527 of natural t-PA. Furthermore, t-P may be caused by natural mutation or artificial mutation.
It may consist of an amino acid sequence obtained by mutating a part of the amino acid sequence without changing the main activity of A's K1K2A.

The amino acid sequence corresponding to E456 of TM in the present invention includes, for example, an amino acid sequence (SEQ ID NO: 1) containing an amino acid sequence corresponding to positions 345 to 462 of natural TM. Furthermore, it may consist of an amino acid sequence obtained by mutating a part of the amino acid sequence without changing the main activity of TM E456 by natural mutation or artificial mutation.

[Chemical 1]

The thrombolytic protein of the present invention can be prepared, for example, by E45 of TM between the signal sequence of the cDNA region encoding the deletion mutant of t-PA and the following sequence.
It can be carried out by inserting a gene, cDNA, or synthetic oligonucleotide which encodes the amino acid sequence of 6 or sequences similar thereto.
The gene may be obtained by amplification by the PCR method (described later). The deletion mutant of t-PA encodes t-PA.
It can be prepared by replacing a part of the DNA region with a synthetic oligonucleotide having a nucleotide sequence encoding a deletion mutant or a DNA fragment obtained by an in vitro chain extension reaction.

Furthermore, the preparation of the thrombolytic protein in which the TM and t-PA sequences are linked via a linker is as follows:
It can be carried out by inserting a synthetic oligonucleotide between the sequences of the gene encoding TM and the gene encoding t-PA. The cDNAs encoding these hybrids are used for expression and production of the final target substance of the present invention, such as amplification in a host by ligation with an appropriate plasmid or expression vector. Further, as the host, both prokaryotic organism cells such as Escherichia coli and animal cells, and eukaryotic organism cells such as yeast can be used. The cDNA encoding t-PA necessary for producing the thrombolytic protein of the present invention is shown in the following reference 19).
It can be easily obtained by using the method described in (1) to (30).

19) European Patent Application Publication No. 0093
619 A1 20) Penica Etoal. , Nature 301 214
-221 (1983) (Pennica et a)
l. , Nature 301 214-221 (198).
4)) 21) International publication No. 8703906 22) European Patent Application Publication No. 233013 23) JP 61-233630 A 24) JP 62-48378 25) JP 62-130690 26) JP 62-192323 27) JP 62-269688 Publication 28) JP 62-272976 A 29) JP 63-133988 B 30) JP 63-230083 A

As one of the techniques necessary for preparing the cDNA encoding the thrombolytic protein of the present invention, site-directed mutagenesis (Site-) by Zoller and Smith, Kunkel, or the like. directed mutagen
sis) (see References 31 and 32 below)). Thereby, for example, c of the t-PA deletion mutant
Any restriction enzyme cleavage site can be introduced at any position of DNA. That is, for example, a cDNA encoding the amino acid sequence of a t-PA deletion mutant is incorporated into an M13-based phage vector, and the resulting double-stranded M13 DNA is transformed into a single-stranded form from a culture solution of Escherichia coli. M13 DNA may be prepared, and a synthetic oligonucleotide for mutagenesis having an arbitrary restriction enzyme cleavage site may be used as a primer for annealing and then a complementary strand synthesis reaction may be performed to induce mutation. Such site-directed mutagenesis (Site-directed mutagen)
For example, Mutan-G or Mutan-K of Takara Shuzo Co., Ltd. may be used as the sis system.

31) M. J. Zola Etoal. , Methods in Enzymology 100 4
68 (1983) (M. J. Zoller et al., Method).
s in Enzymology 100 468 (1
983)) 32) Tea. A. Kunkel Etoal. , Methods in Enzymology 154 367 (198
7) (TA. Kunkel et al., Method
s in Enzymology 154 367
(1987))

As a method of gene amplification, polymerase
The chain reaction (PCR) method is mentioned (Reference 33 below). That is, by using the gene or cDNA as a template and two kinds of synthetic oligonucleotides as primers and PCR using Taq polymerase, DNA is prepared.
The gene may be amplified in vitro using an amplification device. 33) H. A. Erlich Etoal. , PC R Technology (1990) (HA Erlich et al., PCR Te)
chnology (1990))

The synthesis of the oligonucleotide by the phosphoamidite method may be carried out using a model 381A DNA synthesizer manufactured by Applied Biosystems Co., Ltd. according to the principle shown in the following document 34). Further, the purification may be carried out using the OPC cartridge for purifying oligonucleotides manufactured by the same company. 34) S. L. View cage Etoal. , Tetrahedron Letters-22 (20) 1859 (19
81) (SL Beaucage et al., Tetr.
ahedron Letters-22 (20) 1
859 (1981))

In order to confirm the desired mutation or the inserted DNA sequence, the DNA base sequence may be determined according to the dideoxy method shown in the following document 35). 35) F. Sanger Etoal. , Prosci. Nashor. Acad. Sai. USA 74 546 3
(1977) (F. Sanger et al., Proc. Nat.
l. Acad. Sci. USA 74 5463 (1
977))

To prepare a deletion mutant of t-PA, t
In order to insert a TM-derived amino acid sequence into the deletion mutant of -PA, or to insert a peptide sequence as a linker between the amino acid sequences of TM and t-PA,
Genes encoding these amino acid sequences, cDNA,
Alternatively, using synthetic oligonucleotides, genes such as cleavage and deletion of DNA by restriction enzymes as described in the examples, separation, recovery, or ligation of DNA fragments produced thereby by agarose or polyacrylamide gel electrophoresis. The operation may be performed, but most of these individual techniques are known, and reference is mainly made to the following document 36). 36) T. Maniatis et al., Molecular Cloning, Laboratory Manual, Cold Pre-Harbor Laboratory (1982) (T. Maniatis et al., Molcu.
lar Cloning, A Laboratory
Manual, Cold Spring Harbor
Laboratory (1982))

From the double-stranded M13DNA containing the cDNA region having the insertion sequence obtained by the above operation to cDNA
It is also possible to cut out the A portion and ligate it with a plasmid or an expression vector according to the above-mentioned document 36). To transform an appropriate host with the expression plasmid thus obtained, the electric pulse method (Reference 37 below) may be used. 37) Shinichiro Takayama, Cell Engineering 6 771 (1987)

For example, by an electric pulse method using an expression plasmid pCDM8-301TMIII containing the cDNA of the thrombolytic protein of the present invention, in which the E456 region of TM is inserted between the signal sequence of the t-PA deletion mutant and K1. COS-1 cells may be transformed. The culture supernatant of the transformed cells contains thrombolytic protein in an amount that can be used as it is for various activity measurements by appropriate dilution.

Various vector DNAs (or plasmid DNAs) used in each process for production and E. coli strains and animal cell strains as their hosts are widely available and easily available unless otherwise specified. For example, vector DNA and E. coli strains are easily available from Takara Shuzo Co., Ltd. or Toyobo Co., Ltd., and animal cell lines are easily available from Dainippon Pharmaceutical Co., Ltd. The expression vector CDM8 (ori ⁻ ) presented herein is a shuttle vector that can be used in prokaryotic and eukaryotic host cells. A host prokaryotic organism suitable for use of this vector has a marker gene containing an amber mutation capable of being reverted by a suppressor (supF) in host cells such as Escherichia coli MC1061 / p3 strain (Reference 38 below). Microbial strains are effective. 38) Bee. Seed, Nature 329 840-8
42 (1987) (B. Seed, Nature 329 840-84).
2 (1987)) and useful as eukaryotic host cells are COS-1, COS-7, COPS, WOP,
And animal cell lines such as Chinese hamster and ovary (CHO) cell lines.

The thrombolytic protein of the present invention thus obtained can be extremely easily obtained as a preparation by dispensing a required amount in a container by a conventional method and freeze-drying. This lyophilizate is dissolved in a pharmaceutically acceptable carrier substance, for example saline, and administered by intravenous or intraarterial or intracardiac injection. The administration method may be continuous intravenous infusion, or a part of the planned dose may be administered first and then the rest.

[0029]

EXAMPLES Examples will be shown below as examples of the present invention, but the present invention is not limited thereto. 1. Cloning of E456 gene Human chromosomal DNA was prepared from human fetal lung-derived WI-38 cells according to the method described in the above reference 36). T
The nucleotide sequence of the M gene is described in the following reference 39), and by comparison with the nucleotide sequence of cDNA complementary to TM mRNA (described in the above reference 14)), the TM gene consists of one exon Is not included. Then, based on the restriction enzyme map described in the following Reference 39) and FIG. 1, the region containing the gene sequence encoding E456 of TM was amplified by PCR reaction. This amplified gene has the same base sequence as the cDNA complementary to the mRNA for the region. 39) Tea. Shiraitoaru. , J. Biochem. 103 281-285 (1988) (T. Shirai et al., J. Bioche.
m. 103 281-285 (1988))

Specifically, first, two kinds of primers P-
Pst1 (SEQ ID NO: 2) and P-Pst2 (SEQ ID NO: 3) were synthesized. Using these two kinds of primers and the human chromosomal DNA obtained by the above method, a PCR reaction was performed,
A gene having PstI sites at both ends was amplified.

[Chemical 2]

[Chemical 3]

The conditions of the PCR reaction were human chromosome DN prepared from human fetal lung-derived WI-38 cells by the above method.
0.1 μg of A, primers P-Pst1 and P-Ps
t2 is 50 pmoles each, dNTP is 600 μM, reaction buffer [KCl 50 mM, Tris.HCl (pH
8.5) 10 mM, MgCl ₂ 2.5 mM, gelatin 0.2 mg / ml], Taq polymerase 2 units
(Manufactured by Takara Shuzo Co., Ltd.) was mixed so as to have a volume of 100 μl, and the reaction was performed at 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 5 minutes.
Cycle, followed by 94 ℃ for 1 minute, 55 ℃ for 1 minute, 72 ℃
One cycle of 10 minutes reaction was performed. Amplified gene is P
Digested with stI, the resulting fragment was named fragment PP and was isolated from polyacrylamide gel by standard techniques. This fragment was ligated with a commercially available plasmid pUC118 (Takara Shuzo Co., Ltd.) digested with PstI and dephosphorylated at the 5'end, and Escherichia coli JM109 strain (Takara Shuzo Co., Ltd.) was transformed with this to transform the plasmid. pUC118-E456 was obtained. FIG. 2 shows a schematic assembly diagram of the plasmid pUC118-E456.

2. Amplification of E456 Gene The gene encoding E456 was amplified by PCR reaction. In order to form a cassette of this amplified gene, a primer P- containing a restriction enzyme recognition sequence inside the sequence
SpeI (SEQ ID NO: 4), P-XbaI (SEQ ID NO: 5)
And P-ET14I (SEQ ID NO: 6) were synthesized.

[Chemical 4]

[Chemical 5]

[Chemical 6]

In the PCR reaction, the plasmid pUC11 was used as a template.
8-E456 was used, and P-SpeI and P were used as primers.
-XbaI and a combination of P-SpeI and P-ET14I were used. The conditions of the PCR reaction were 10 ng of the plasmid pUC118-E456 and 5 primers each.
0 pmoles, dNTPs 200 μM, reaction buffer [KCl 50 mM, Tris · HCl (pH 8.5) 1
0 mM, MgCl ₂ 2.5 mM, gelatin 0.2 mg /
ml], Taq polymerase 2 units (Takara Shuzo Co., Ltd.) were mixed so as to be 100 μl, and 94 ° C.
Min, 55 ° C. 1 min, 72 ° C. 3 min, 29 cycles, followed by 94 ° C. 1 min, 55 ° C., 1 min, 72 ° C. 5 min, 1 cycle. As shown in FIG. 3, the amplified genes were SpeI and XbaI, and SpeI and E, respectively.
digested with coT14I and named these fragments Fragment SX and Fragment SE, respectively,
Isolated from polyacrylamide gel by standard techniques.

3. Construction of Plasmid pCDM8-101 Encoding K2A Mutant In order to facilitate understanding of the contents described below, conceptual diagrams of this construction method are shown in FIGS. 4 to 6. First, two kinds of oligonucleotides D5 (SEQ ID NO: 7) and D2 (SEQ ID NO: 8) were synthesized.

[Chemical 7]

[Chemical 8]

Of these, D5 should be a sequence in which the signal peptide of t-PA and the sequence encoding K2 are linked together, and the 5'ends of both synthetic oligonucleotides D5 and D2 are the restriction enzymes BglII and EcoRI, respectively.
It is designed to be the recognition site of. The two types of oligonucleotides were mixed in equimolar amounts and annealed, and then DNA polymerase I (Klenowfra) was added.
(gment) to extend the complementary strand to form a double strand. This 2
The stranded DNA was digested with EcoRI and BglII and the 98 bp fragment D25 was isolated from a polyacrylamide gel. On the other hand, the commercially available vector pUC19
Vector pUC19H in which the HindIII recognition site in the multicloning site of Escherichia coli was replaced with the BglII recognition site
^- to prepare a B ^+. This vector pUC19H ^- B ⁺ was digested with EcoRI and BglII, the 5'end was dephosphorylated, and the previously prepared fragment D25 was ligated to transform E. coli HB101 strain (Takara Shuzo Co., Ltd.). , Plasmid pUCD25 was obtained (FIG. 4). The insertion sequence region of this plasmid was confirmed by the nucleotide sequence determination method using the dideoxy method.

The plasmid pTZB-000 containing t-PA cDNA was obtained by the method described in Reference 40) below. This plasmid pTZB-000 was digested with EcoRI and BglII, and the plasmid pUCD
25 was digested with EcoRI, BglII and ScaI, and further digested with NarI, and Escherichia coli JM109 strain was transformed with this to obtain plasmid pTZB-101ΔEE. 40) Japanese Patent Application No. 2-268816 The plasmid pTZB-000 was digested with EcoRI and 4
The 72 bp fragment EE was isolated from a polyacrylamide gel. This fragment and plasmid pTZ
B-101ΔEE was digested with EcoRI and ligated with the one obtained by dephosphorylating the 5'end.
The M109 strain was transformed with the plasmid pTZB-10.
1 was obtained (FIG. 5).

This plasmid pTZB-101 was Bam
After digestion with HI, an approximately 1650 bp fragment 101 was isolated from a polyacrylamide gel, and this fragment 101 and the plasmid pCDM8 (Bgl) were digested with BglII and ligated with 5'-terminal dephosphorylated. The MC1061 / p3 strain was transformed to obtain the plasmid pCDM8-101 (FIG. 6). This plasmid pCDM8-101 contains the signal peptide of t-PA and the genes encoding the K2 and A domains. The joint between the signal peptide and the gene encoding the K2 domain and the sequence before and after it are shown in SEQ ID NO: 9.

[Chemical 9]

4. Construction of Plasmid pCDM8-301 Encoding K1K2A Mutant A conceptual diagram of this construction method is shown in FIGS. 7 to 10. First, two oligonucleotides D3 (SEQ ID NO: 10) and D4
(SEQ ID NO: 11) was synthesized.

[Chemical 10]

[Chemical 11]

Of these, D3 should be a sequence in which the signal peptide of t-PA and the sequence encoding K1 are connected, and the 5'ends of both synthetic oligonucleotides D3 and D4 are the restriction enzymes BglII and ApaLI, respectively.
It is designed to be the recognition site of. The two types of oligonucleotides were mixed in equimolar amounts and annealed, and then DNA polymerase I (Klenowfra) was added.
(gment) to extend the complementary strand to form a double strand. This 2
The stranded DNA was digested with BglII and ApaLI and the 89 bp fragment D34 was isolated from a polyacrylamide gel. Also, refer to the plasmid pUC-000 containing the t-PA cDNA (see Reference 41 below).
Was digested with SacI and EcoRI to isolate a fragment ScE of approximately 800 bp from a polyacrylamide gel, which was used to transform plasmid pUC19 with SacI and EcRI.
The product digested with oRI was ligated, and E. coli JM1 was ligated.
The strain 09 was transformed to obtain the plasmid pUC19SE (FIG. 7). 41) Japanese Patent Application No. 2-87005

This plasmid pUC19SE was cloned into ApaL
After digestion with I and EcoRI, the 276 bp fragment ALE was isolated from a polyacrylamide gel. Separately, the plasmid pUC19SE was cloned into BglII and E.
digested with coRI and dephosphorylated the 5'end,
Fragment D34 and Fragment A prepared previously
The LEs were ligated by the three parties, and E. coli JM109
The strain was transformed to obtain the plasmid pUC-301ΔEE (Fig. 8). The insertion sequence region of this plasmid was confirmed by the nucleotide sequence determination method using the dideoxy method.

This plasmid pUC-301ΔEE was added to B
After digestion with glII and EcoRI, the 365 bp fragment BgE was isolated from a polyacrylamide gel.
The above-mentioned plasmid pTZB-000 was digested with BglII and EcoRI, the 5'end was dephosphorylated, and the fragment BgE was ligated, and Escherichia coli JM109 strain was transformed with this plasmid pTZB-.
A 301ΔEE was obtained (FIG. 9).

The plasmid pTZB-301ΔEE was Ec
Digested with oRI and dephosphorylated at the 5'end and the fragment EE described in the previous section were ligated, and E. coli JM109 strain was transformed with this plasmid PT.
ZB-301 was obtained. This plasmid pTZB-301
Was digested with BamHI and a fragment 301 of about 1840 bp was isolated from a polyacrylamide gel. This fragment 301 and plasmid pCDM8 (Bgl) were
After digestion with glII, the 5'-terminal dephosphorylated product was ligated, and E. coli MC1061 / p3 strain was transformed with this to obtain plasmid pCDM8-301 (Fig. 1).
0). This plasmid pCDM8-301 contains the signal peptide of t-PA and the genes encoding the K1, K2 and A domains. Signal peptide and K1
The joint between the genes encoding the domain and the sequences before and after it are shown in SEQ ID NO: 12.

[Chemical formula 12]

5. Construction of plasmid pCDM8-101TM encoding E456-K2A The conceptual diagram of this construction method is shown in FIGS. 11 to 13. First, "CCAAGG" which is the EcoT14I recognition sequence existing between the signal sequence of the plasmid pTZB-101 and the base sequence encoding the K2 sequence was replaced with "CCTAGG". For that, the technique of site-directed mutagenesis was used. pTZB-101 was digested with BamHI and SmaI and the approximately 1.2 Kb fragment BSm was isolated from a polyacrylamide gel. This and M13tv18 digested with BamHI and SmaI were ligated to transform E. coli JM109 strain.
v18BSm was obtained (FIG. 11).

The single-stranded form M13tv18BSm was obtained as a template for site-directed mutagenesis according to the method described in the above-mentioned reference 41). M13tv18BSm (ET) was annealed with the synthesized oligonucleotide M-ET (SEQ ID NO: 13) by using this single-stranded form M13tv18BSm as a template, and Mutan described in the aforementioned References 31) and 39).
-G system (Takara Shuzo Co., Ltd.) was used.
The confirmation of single base substitution was performed by the method of base sequence determination using the dideoxy method. M13mp18BSm (ET) B
About 1.2 K obtained by digestion with amHI and SmaI
The fragment BSm (ET) of b was isolated from polyacrylamide gel by standard techniques. This fragment BSm (ET) and the plasmid pUC118 were added to BamHI.
And digested with SmaI were ligated, and Escherichia coli JM109 strain was transformed with this, and plasmid p
UCBSmET was obtained (Figure 12).

[Chemical 13]

This plasmid pUCBSmET was transformed into Eco
After digestion with T14I, dephosphorylated 5'end,
Ligate the above-mentioned fragment SE to obtain E. coli J
The M109 strain was transformed with the plasmid pUCBSmT.
M was obtained. This plasmid pUCBSmTM was added to BglII.
After digestion with SmaI and SmaI, a fragment BgSm of about 1.4 Kb was obtained from polyacrylamide gel. This and the above-mentioned plasmid pCDM8-101 were digested with BglII and SmaI and ligated with a dephosphorylated 5'end, and Escherichia coli MC1061 / p3 strain was transformed with this to obtain plasmid pCDM8-101TM. (FIG. 13). This plasmid pCDM8-101TM
Is a signal peptide of t-PA from the amino terminal side, T
It contains genes encoding the E4, E5, E6 domains of M and the K2 and A domains of t-PA. The gene encoding the E4, E5, and E6 domains of TM and the sequences before and after it are shown in SEQ ID NO: 14.

[Chemical 14]

[Chemical 15]

6. Construction of plasmid pCDM8-301TMIII encoding E456-K1K2A The conceptual diagram of this construction method is shown in FIGS. 14 to 17. Site-specific using the Mutan-K system (Takara Shuzo Co., Ltd.) described in the above references 32) and 41) for inserting an XbaI site between the signal sequence of the plasmid pTZB-301 and the nucleotide sequence encoding the K1 sequence. Mutagenesis was performed. First, single-stranded pTZB-301 (dU) containing deoxyuridine in the sequence was obtained. The synthesized oligonucleotide M-Xba (SEQ ID NO: 15) was annealed using this single-chain form pTZB-301 (dU) containing deoxyuridine as a template, and pTZB-301X was obtained using the Mutan-K system described above. It was

[Chemical 16] After digesting this with XbaI, the 5'end was dephosphorylated, this was ligated with the above-mentioned fragment SX, and Escherichia coli JM109 strain was transformed to obtain pTZB-301TM (FIG. 14).

After digesting pTZB-301TM with BamHI, a fragment of about 2.1 Kb was obtained from polyacrylamide gel using standard techniques, and this fragment and pCDM8 (Bgl) were digested with BglII to dephosphorize the 5'end. Ligate the oxidised one and use this to transform E. coli MC
The 1061 / p3 strain was transformed into pCDM8-301.
TM was obtained (Fig. 15).

The base sequence of this plasmid pCDM8-301TM was confirmed by digestion with a restriction enzyme or base sequence determination using the dideoxy method.
A single base deletion was recognized at the II recognition site. So pCD
A region containing a single base deletion site of M8-301TM (Hin
The region from dIII to NheI) was replaced with the region from HindIII to NheI inside the region encoding HindIII to E456 in the multiple cloning site of the plasmid pUCBSmTM described in the preceding paragraph to restore the deleted base. That is, the plasmid pUCBSmTM was added to Hi.
After digestion with ndIII and NheI, a fragment HN of about 0.4 Kb was isolated from a polyacrylamide gel.
This fragment HN and the plasmid pCDM8-30
1TM was digested with HindIII and NheI and dephosphorylated at the 5'end to ligate E. coli MC
The 1061 / p3 strain was transformed with the plasmid pCDM.
8-301TMII was obtained (Fig. 16).

Further, the region whose nucleotide sequence has not been confirmed is the plasmid pTZB-0 described in the above-mentioned document 40).
It was replaced with the equivalent area of 00. Plasmid pCDM8-
After digesting 301TMII with HindIII and NarI,
A fragment HNa of about 0.7 Kb was isolated from a polyacrylamide gel. In addition, the plasmid pTZB-00
0 was digested with NarI and BamHI to isolate a fragment NaB of about 1.7 Kb from a polyacrylamide gel. Both fragments and plasmid pCDM8
5'after digestion of (Bgl) with HindIII and BglII
Ligate the dephosphorylated end with the three parties,
The E. coli MC1061 / p3 strain was transformed with this to obtain the plasmid pCDM8-301TMIII (FIG. 1).
7). This plasmid pCDM8-301TMIII is t
-Containing the signal peptide of PA, the E4, E5, E6 domains of TM and the K1, K2 and A domains of t-PA. TM E4, E5, E6
The gene encoding the domain and the sequences before and after it are shown in SEQ ID NO: 16.

[Chemical 17]

[Chemical 18]

[Chemical 19]

7. Plasmids pCDM8-101TM3G, -101TM5G, containing glycine linker-
Construction of 301TM3G, -301TM5G Plasmids pCDM8-101TM3G and pCDM in which a gene (linker) encoding a sequence containing three consecutive or five glycines was inserted between the genes encoding E456 and K2A in pCDM8-101TM.
8-101TM5G was constructed. Also, pCDM8-3
In 01TMIII, plasmids pCDM8-301TM3G and pCDM8-3 in which a gene (linker) encoding a sequence containing 3 or 5 consecutive glycines was inserted between the genes encoding E456 and K1K2A.
01TM5G was constructed. First, four kinds of oligonucleotides having a glycine linker sequence necessary for this construction were synthesized (3A: SEQ ID NO: 17, 3B: SEQ ID NO: 18, 5).
A: SEQ ID NO: 19, 5A: SEQ ID NO: 20).

[Chemical 20]

[Chemical 21]

[Chemical formula 22]

[Chemical formula 23]

The 5'ends of these four kinds of oligonucleotides were phosphorylated and annealed with a combination of 3A and 3B, 5A and 5B to obtain double-stranded linkers 3AB and 5AB. Plasmid pCDM8-101TM3
A conceptual diagram of the construction of G or pCDM8-101TM5G is shown in FIG. The above described plasmid pUC
BSmTM was digested with EcoT14I and dephosphorylated at the 5'end and ligated with linker 3AB or 5AB, which was used to transform Escherichia coli HB101 strain, and plasmid pUC having one linker each.
101TM3G or pUC101TM5G was obtained.
The nucleotide sequences of the regions containing the linker of these plasmids were confirmed by the dideoxy method. Both plasmids are Bgl
II and SmaI, respectively, and the above-mentioned plasmid pCDM8-101 were digested with BglII and SmaI.
Ligated with those digested by
The C1061 / p3 strain was transformed into pCDM8-10.
1TM3G or pCDM8-101TM5G was obtained. This plasmid pCDM8-101TM3G or pCDM8-101TM5G contains t-PA signal peptide, TM E4, E5, E6 domains, glycine linker and genes encoding t-PA K2 and A domains. The gene encoding the glycine linker and the sequences before and after it are pCDM8-101TM.
3G is shown in SEQ ID NO: 21, and is pCDM8-10.
1TM5G is shown in SEQ ID NO: 22.

[Chemical formula 24]

[Chemical 25]

A conceptual diagram of the construction of the plasmid pCDM8-301TM3G or pCDM8-301TM5G is shown in FIGS. First, the plasmid p
A plasmid pUC19AfBg in which the HincII recognition site of UC19 was converted into a BglII recognition site was prepared, digested with SacI and BglII, and the above-described plasmid pCDM8-301TMIII was digested with SacI and BglII.
After digestion with amHI and ligation, B
It was digested with amHI and Escherichia coli HB101 strain was transformed with it to prepare plasmid pUCAfBg-BSc (FIG. 19).

The plasmid pUCAfBg-BSc was added to Xb
Ligation of the above-mentioned linker 3AB or 5AB with a 5'end dephosphorylated by digestion with aI, E. coli HB101 strain was transformed with this, and plasmid pUCAfBg-BSc3 containing one of each linker was ligated.
G or pUCAfBg-BSc5G was obtained. The nucleotide sequences of the regions containing the linker of these plasmids were confirmed by the dideoxy method. The two plasmids were digested with BglII and NarI, respectively, and the above-mentioned plasmid pCDM8-301 was digested with BglII and NarI and dephosphorylated at the 5'end, and ligated,
E. coli MC1061 / p3 strain was transformed with these,
pCDM8-301TM3G or pCDM8-30
1TM5G was obtained (FIG. 20).

This plasmid pCDM8-301TM3
G or pCDM8-301TM5G contains a gene encoding the signal peptide of t-PA, E4, E5, E6 domains of TM, a glycine linker and K1, K2 and A domains of t-PA. The gene encoding the glycine linker and the sequences before and after it are pCDM
8-301TM3G is shown in SEQ ID NO: 23, p
SEQ ID NO: 24 is shown for CDM8-301TM5G.

[Chemical formula 26]

[Chemical 27]

8. COS of thrombolytic proteins of the invention
-1 Expression and secretion in cells pCDM8-101,-
101TM, -101TM3G, -101TM5G,-
301, -301TMIII, -301TM3G, -30
COS- by the above-mentioned electric pulse method using 1TM5G
One cell was transformed. 24 in DMEM medium containing 10% fetal bovine serum and 2.5 μg / ml aprotinin
After culture for a period of time, the medium was serum-free and aprotinin-free DM
The medium was replaced with an EM medium, and the culture was continued for 96 or 120 hours. The culture solution was centrifuged and the supernatant was recovered. 90% or more of the t-PA contained in the culture supernatant thus obtained was present in the form of a single chain. 1/9 volume of DMEM medium containing 1% bovine plasma albumin (BSA) was added to the collected supernatant, and the mixture was frozen and stored. This cryopreserved supernatant was used for the subsequent activity measurement.

9. Quantification of protein amount The amount of each protein in each of the above culture supernatants was measured by enzyme immunoassay (E
The amount of protein in each supernatant was confirmed by measuring by LISA (manufactured by Biopool). Representative examples of measurement results Table 1
Shown in.

10. Assay (Assay) Method The plasminogen activating activity of the thrombolytic protein of the present invention was measured by the method described below. Synthetic chromogenic substrate S-
Plasmin-specific synthetic tripeptide chromogen substrate S-2251 (HD-Val-), which is specific for plasmin, based on the indirect chromogenic assay using 2251 (the method shown in the following Reference 42). Leu-L
ys-pNA · 2HCl · H a ₂ O) was quantitatively measured by using. 42) J. H. Verheigen et al. , Thrombo. Hemost. 48 (3) 266-26
9 (1982) (JH Verheijen et al., Thr.
omb. Haemost. 48 (3) 266-269
(1982)) The measurement using S-2251 was carried out by adding 20 μl of the diluted sample to a reaction mixture [plasminogen 125 nM, S-225.
1 350 μM, fibrin fragment 6 mg / ml
(0.10% (v / v) Tween 80 and 5.0 m
0.15 mM Tris pH 7.8 solution containing g / ml gelatin), and plasmin (2.69 CU / ml)] 20
The mixture was mixed with 0 μl, incubated at 37 ° C., the absorbance at 405 nm was measured with time using a plate reader (manufactured by Molecular Device), and data processing was performed using the analysis software of the company, Softmax. From this result and the measurement result of the above-mentioned protein amount, the activity (specific activity) per constant protein amount was determined. Table 1 shows a typical example.

[Table 1]

[0058]

The thrombolytic protein of the present invention is t-P
A or a variant thereof, which is a hybrid in which part or all of the EGF-like domain of TM is bound,
It has the property of exerting the anticoagulant action of M and simultaneously having the action of t-PA having high thrombus specificity via TM. Therefore, the therapeutic agent for thrombosis containing the thrombolytic protein of the present invention as an active ingredient is expected to have the effects of hardly causing reocclusion in clinical practice and reducing side effects.

[Brief description of drawings]

FIG. 1 Chromosomal DNA of human TM gene and its surroundings
The restriction enzyme map of is shown. The black box is the human TM
Indicates a gene.

FIG. 2 shows a conceptual diagram of construction of plasmid pUC118-E456.

FIG. 3 shows a conceptual diagram of the construction of fragment SX and fragment SE in which a gene portion encoding E456 is formed into a cassette.

FIG. 4. Plasmid pCDM encoding the K2A mutant.
A part of the construction conceptual diagram of 8-101 is shown.

FIG. 5: Plasmid pCDM encoding K2A mutant
A part of the construction conceptual diagram of 8-101 is shown.

FIG. 6: Plasmid pCDM encoding K2A mutant
A part of the construction conceptual diagram of 8-101 is shown.

FIG. 7: Plasmid pC encoding K1K2A mutant
A part of the construction conceptual diagram of DM8-301 is shown.

FIG. 8: Plasmid pC encoding the K1K2A mutant
A part of the construction conceptual diagram of DM8-301 is shown.

FIG. 9: Plasmid pC encoding the K1K2A mutant
A part of the construction conceptual diagram of DM8-301 is shown.

FIG. 10: Plasmid p encoding the K1K2A mutant
A part of the construction conceptual diagram of CDM8-301 is shown.

FIG. 11: Plasmid p encoding E456-K2A
A part of the construction conceptual diagram of CDM8-101TM is shown.

FIG. 12: Plasmid p encoding E456-K2A
A part of the construction conceptual diagram of CDM8-101TM is shown.

FIG. 13: Plasmid p encoding E456-K2A
A part of the construction conceptual diagram of CDM8-101TM is shown.

FIG. 14 shows a part of a conceptual diagram for constructing a plasmid pCDM8-301TMIII encoding E456-K1K2A.

FIG. 15 shows a part of a conceptual diagram of construction of a plasmid pCDM8-301TMIII encoding E456-K1K2A.

FIG. 16 shows a part of a conceptual diagram for constructing a plasmid pCDM8-301TMIII encoding E456-K1K2A.

FIG. 17 shows a part of a conceptual diagram for constructing a plasmid pCDM8-301TMIII encoding E456-K1K2A.

FIG. 18: Plasmid pC containing a glycine linker
DM8-101TM3G, pCDM8-101TM5G
The construction conceptual diagram of is shown.

FIG. 19: Plasmid pC containing a glycine linker
DM8-301TM3G, pCDM8-301TM5G
A part of the construction conceptual diagram of is shown.

FIG. 20: Plasmid pC containing a glycine linker
DM8-301TM3G, pCDM8-301TM5G
A part of the construction conceptual diagram of is shown.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location C12N 1/19 9050-4B 1/21 7236-4B 5/10 9/64 Z 7823-4B 15 / 58 15/62 15/70 15/81 15/85 // C12P 21/02 J 8214-4B (C12N 1/21 C12R 1:19) (C12P 21/02 C12R 1:19) (72) Inventor Hideo Aki Sumitomo Pharmaceutical Co., Ltd. 3-1-198 Kasugade, Konohana-ku, Osaka

Claims (8)

[Claims] 1. A thrombolytic protein comprising a tissue plasminogen activator or a variant thereof bound to a part or all of the epidermal growth factor-like domain of thrombomodulin. 2. A tissue plasminogen activator or a variant thereof is bound to a part or all of the epidermal growth factor-like domain of thrombomodulin via a linker having one or more amino acids. The thrombolytic protein according to claim 1, wherein 3. The variant according to claim 1 or 2, wherein the tissue plasminogen activator has a kringle 2 and an active domain mutant (K2A), or a kringle 1, a kringle 2 and an active domain mutant. The thrombolytic protein according to claim 1 or 2, which is (K1K2A). 4. The thrombolytic protein according to claim 1, 2 or 3, wherein a part of the epidermal growth factor-like domain according to claim 1 or 2 contains E456. 5. A DN containing a nucleotide sequence encoding the thrombolytic protein according to claim 1, 2, 3 or 4.
A. 6. A recombinant expression vector capable of expressing the DNA of claim 5 in a transformed prokaryotic cell or eukaryotic cell. 7. A prokaryotic or eukaryotic cell transformed with the recombinant expression vector according to claim 6. 8. A therapeutic agent for thrombosis, which comprises the thrombolytic protein according to claim 1, 2, 3 or 4 as an active ingredient.