JPH0746983A - Production of human tissue plasminogen activating factor derived from human normal cell - Google Patents

Abstract

PURPOSE:To provide a method for producing human tissue plasminogen activating factor derived from human normal cells. CONSTITUTION:A DNA sequence coding mouse metallothionein promoter or human metallothionein promoter and the subject activating factor composed of the 530 amino acids beginning from glycine at the N-terminal includes a DNA sequence given in sequence No:1 of the sequence table, and produces the objective activating factor using microbial or animal cells transformed by a recombinant DNA vector functionally linked to the above-mentioned promoter and having self-replicating ability in animal cells.

Description

Detailed Description of the Invention

The present invention provides a new recombinant vector transformed with a new expression vector containing a new DNA sequence encoding a tissue plasminogen activator (hereinafter abbreviated as t-PA) produced by normal human cells. The present invention relates to a method for producing a tissue plasminogen activator produced by normal human cells using host cells.

Although the present invention is specifically set forth as follows, the background of the invention and related important references are cited in the text by reference and at the end by reference.

A) Tissue plasminogen activator produced by normal human cells. T-PA according to the present invention is produced and secreted by vascular endothelial cells and various tissue cells and dissolves fibrin, which is the main body of thrombus. When it does, it plays the leading role.
Currently, urokinase and streptokinase are used for the treatment of thrombosis for convenience. However, they have bleeding and antigenicity problems and also have a weak affinity for fibrin. Moreover, since urokinase is purified from human urine, which is difficult to obtain, the process is unsanitary, complicated, and expensive, and leaves many problems. Therefore, the development of new medicines is desired. Recently, human cancer cells, Bowes melanoma
It has been reported that a relatively large amount of t-PA is secreted and produced by the (melanoma) strain (Reference 1). However, since its origin is malignant melanoma cells, there are various problems in developing t-PA derived therefrom as a medicine. For example, in order to obtain t-PA by the cell culture method of the Bowes melanoma strain which is a malignant tumor cell, the safety of the process itself must be carefully examined. In addition, recently, a method for incorporating t-PA from this Bowes melanoma strain into an expression vector to transform a host cell and producing t-PA using this recombinant host cell has been announced (Reference 2). Here, t-PA derived from the Bowes melanoma strain, which is a cancer cell, is similar to t-PA isolated from normal human tissue in terms of immunological and amino acid composition.
It is stated that there is no distinction. However, the carcinogenic mechanism has not yet been completely scientifically elucidated. Under such circumstances, there is no answer as to whether or not there is any risk in producing t-PA using a gene isolated from cancer cells, let alone applying it as a medicine. For this reason, the present inventor has no concern about the danger as described above.
As a result of searching human normal cells producing PA, cells having relatively high activity were found. Furthermore, a new DN encoding t-PA from this normal human cell using recombinant DNA technology
The present invention was achieved by isolating the A sequence, incorporating this DNA sequence into a replicable vector capable of expressing t-PA, and transforming a host cell to obtain a recombinant host cell. By using this recombinant host cell, a process for mass-producing t-PA derived from normal human cells without risk of danger was established.

B) Gene manipulation technology B-1) Screening of human tissue plasminogen activator-producing cells Many cancer cells are screened and t-PA-secreting cells exist because of the ease of the culture method. It was already known to do (Reference 3), but its essence remains largely unknown. The present inventor has cultured various human normal cells, preferably human fetal epidermal cells, fetal lung tissue cells, fetal kidney cells, etc.
As described in the method of ranell-Piperno, Reich (Reference 4), they were screened for their ability to produce plasminogen activator using the activity of dissolving fibrin on a fibrin plate as an index. As a result, a strain that secreted and produced t-PA in a relatively large amount could be selected and used as a starting material. All of these strains are human normal diploid cells having a chromosome number of 2n = 46 and a finite number of passages.

B-2) Purification and size fractionation of mRNA One of normal cells derived from human fetal epithelium that produce a high amount of t-PA screened by the present invention (MTC017).
Can be cultured in a large amount to extract mRNA from the cultured cells. Specifically, from the cell, first of all RN
Extract A. Next, poly (A) plus mRNA is separated and collected from total RNA using oligo (dT) cellulose. The resulting mRNA is electrophoresed on an agarose gel and fractionated according to its size. Each fraction was microinjected into Xenopus Iaevis oocytes.
and culture in the medium of Barth (Reference 5). Thereafter, this medium is placed on a fibrin plate prepared according to the method of Granerie Perno and Reich (Reference 4), and the dissolution zone thereof is measured, whereby the fraction having t-PA activity can be selected. The fact that this fraction contains mRNA encoding t-PA can be confirmed by the fact that anti-t-PA serum inhibits the reaction on the fibrin plate.

B-3) Preparation of colony library containing t-PA gene Fractions containing t-PA-encoding mRNA were subjected to cDNA according to the method of Gubler and Gubler and Hoffman (Reference 6). And plasmid pU
Insert into C13. Then E. coli HB101 (A
TCC33694) is transformed to obtain ampicillin resistant colonies. Whatman (Wh
atman) 541 filter, and then the DNA of each colony is fixed on the filter by the method of Gergen et al. (Reference 7).

B-4) Preparation of DNA probe As described in JP-A-59-80613, the present inventor screened by a combination of ion exchange chromatography, affinity chromatography, gel filtration and the like. t-PA is purified from normal human cells producing t-PA. To prepare a probe, the partial amino acid sequence of this t-PA is determined as follows. Degradation products obtained by partially hydrolyzing t-PA protein with trypsin are separated and purified, and the amino acid sequence of each is determined from the amino terminus. As a result, the optimum region for probe preparation was set to Tyr-Cys-Trp-C.
It was set to ys-Asn. D encoding this amino acid sequence
Eight types of 14 nucleotides (14m) complementary to the NA sequence
er) 5'-dTTnCACCAnCAnTA (n is A
Alternatively, G) is synthesized by the solid phase phosphotriester method (Reference 8).

B-5) Separation and Identification of Bacterial Clone Containing t-PA cDNA Sequence Eight 14-nucleotide bodies (14) synthesized in section B-4)
The mixture mer) by T4- poly quinuclidine retinyl de kinase to label the 5 'end with ³² p. Using this as a probe, hybridization is carried out with a filter on which the cDNA library prepared in section B-3) is fixed. From the colonies that showed a positive hybridization reaction, the burn boy and the dolly (Birn bomb, Do
ly) method (Reference 9) to isolate plasmid DNA,
The nucleotide sequence of the cDNA insert is determined by the method of Maxam and Gilbert (Reference 10).

B-6) Expression of t-PA gene in animal cells as a host DNA encoding t-PA isolated from normal human cells
The sequence had 9 differences from the DNA sequence encoding t-PA isolated from the melanoma cell line, Bowes melanoma strain. In addition, when the entire amino acid sequence of normal cell-derived t-PA determined from the sequence was examined, t-PA
It was presumed that the PA molecule had four sites to which sugar chains could be added. The t-PA producing strain MTC01 was prepared by adding tunicamycin, which is an antibiotic that actually inhibits glycosylation.
When 7 is cultivated, the molecular weight of t-PA is reduced to about 50,000, suggesting that about 30% of the molecular weight is occupied by sugar. It is considered that the addition of sugar chains is involved in the stable function of the enzyme in vivo, and the absence of sugar chains may show antigenicity in vivo. Therefore, by expressing the cloned t-PA gene in animal cells, a natural type of t with a sugar chain added
-We thought it was a good idea to produce PA. A desirable expression vector for expressing the t-PA gene in a host animal cell includes a promoter region, a ribosome binding site, a transcription termination sequence, a polyadenylation site, an origin of replication, and a host cell transfected with the vector. It sometimes contains genes that serve as selectable markers. As for the promoter region and ribosome binding site, generally, those derived from animal cell viruses can be used. For example, the promoter region and ribosome binding site possessed by papovavirus, adenovirus, retrovirus, human cytomegalovirus, etc. can be used. Further, a promoter region and a ribosome binding site located in front of an animal cell-derived gene can also be used. In that case, the promoter of a gene that is strongly expressed is generally considered to be a strong promoter region for other genes as well, so it is advantageous to use it. For example, it is considered to be advantageous to utilize a gene for mouse, human or other animal metallothionein, immunoglobulin, insulin, albumin, etc. in the promoter region and ribosome binding site. Regarding the transcription termination sequence and polyadenylation site, those derived from the same virus or various genes can be used. For the origin of replication, the viruses mentioned above, in particular the bovine papilloma virus (Bovine Pa
The vector may be constructed so as to contain an exogenous origin that can be derived from P. piloma virus (hereinafter abbreviated as BPV) or Simian virus 40 (hereinafter abbreviated as SV 40), or the expression vector of interest is fragmented into the host cell chromosome. May be used to utilize the chromosome replication mechanism. A gene serving as a selectable marker when a vector is transfected into a host cell is not always necessary, but it is advantageous to have a gene for rapid and accurate selection when selecting a transformed cell. For example, isolated herpesvirus (HS
V) has a thymidine kinase gene (TK) and a mouse dihydrofolate reductase gene (DHFR). In these cases, the host cell is a thymidine kinase deficient strain (tK).
^-) or dihydrofolate reductase-deficient strain (dhfr ^-) to use. Further, a xanthine guanine phosphoribosyl transferase gene (Eco-gpt) of Escherichia coli which shows resistance to mycophenolic acid in animal cells and a neomycin resistance gene (neo) derived from transposon 5 which shows resistance to the antibiotic G418 in animal cells can also be used. t-P
Depending on the vector of the present invention containing the DNA sequence encoding A, the host cell to be transfected can be selected from a large number of cells currently established. Among them, preferable examples include mouse L cells (Reference 11), thymidine kinase deficient L cells (Reference 12), dihydrofolate reductase deficient Chinese hamster ovary (CHO) cells (Reference 13), mouse C127I cells (ATCC CRL1616). ), Mouse NIH3T3 cells (ATCC CRL1658), mouse FM3A cells (Reference 14), human Hela cells (AT
CC CCL2), African green monkey COS1 cells (ATCC CCL1 650), various mouse and human myeloma cells, such as mouse P.
3 / NS1 / 1-Ag4-1 (NS-1) cells (ATC
CTIB18), mouse P3X63Ag8 cells (AT
CC TIB 9), mouse J558 (ATCC TI
B 6) and the like are used, but this is an example, and the use of other animal cells is also possible. For transfecting host cells with the vector of the present invention containing a DNA sequence encoding t-PA, generally the calcium phosphate method (Reference 15) may be used. In addition, methods such as a nuclear injection method (Reference 16), a protoplast fusion method (Reference 17), an electric shock introduction method (Reference 18), and a DEAE-dextran method (Reference 19) can also be used. After transfection by the above method, the resulting transformed cells are separately cultured, and the t-PA activity of the culture supernatant is measured, whereby it can be confirmed that human normal cell-derived t-PA is produced. .

[0010]

【Example】

1) Screening of normal human cells that produce t-PA Various normal human cells shown in Table 1 (Table 1) were used as screening targets. The treated cells are confluent (c
The cells were cultured until they became onfluent. T-PA activity was quantified using 15 μl of the culture supernatant. The fibrin plate prepared by the method of Granerie Piperno and Reich (Reference 4) was allowed to stand at 37 ° C. for 16 hours,
The dissolution zone was measured. At this time, a sample was prepared by mixing each culture supernatant with anti-urokinase serum or anti-t-PA serum, and the dissolution zone thereof was also measured at the same time for comparison. For the activity, the purified urokinase was used as a standard, and the dissolution zone of the diluted solution was simultaneously measured to prepare a standard curve, and the activity was quantified in terms of urokinase international units. A part of the screened cells and their t-PA activity and urokinase activity were as shown in Table 1 (Table 1). As a result, since the MTC017 strain secretes and produces a relatively large amount of t-PA, this cell was used as a material for t-PA gene isolation. This cell is a normal diploid cell derived from human fetal epithelium having a chromosome number of 2n = 46 and having a passage number of about 80 generations.

[0011]

[Table 1]

2) Purification of mRNA and size fractionation The MTC017 strain was cultured in a large amount and 5 × 10 ⁸ cells were collected. The collected cells were washed with 20 ml of phosphate buffered saline (PBS) and then 20 ml of 10 mM Tris-
HCl (pH 8.6), 10 mM KCl, 1.5 mM
Resuspended in MgCl ² , 3 mM dithiothreitol solution. Surfactant Nonidet P-40 (final concentration 0.5%) was added to lyse the cells, and after centrifugation, total RN
The supernatant containing A was further purified by phenol / chloroform extraction. Make the aqueous layer 0.3 M sodium acetate solution,
Next, 2 volumes of ethanol was added to precipitate the total RNA. Using oligo (dT) cellulose, m from total RNA
RNA was purified (Reference 20). The yield is 5 × 1
0 ⁸ total RNA and 100 from cultured cells of 4~5mg of
150 μg of poly (A) plus mRNA was obtained. Poly (A) plus mRNA using low melting point agarose gel
Was fractionated (Reference 21). 1.5% low melting point agarose, 50 mM boric acid, 5 mM sodium borate, 1
A slab gel containing 0 mM sodium sulfate was used. Poly (A) plus mRNA treated with methylmercuric hydroxide was electrophoresed on this gel. After the electrophoresis, the gel was immersed in a 0.1 M dithiothreitol solution for 30 minutes and then divided according to the size of the molecular weight. 4M capacity of 0.5M for each slice
After adding ammonium acetate, the mixture was heated to 65 ° C. to dissolve the gel. After that, phenol extraction and chloroform extraction were repeated to remove the agarose component, and then each mRNA fraction was collected by ethanol precipitation. Next 500 ng
Each of the mRNA fractions was isolated by microinjection into Xenopus oocytes (Reference 5). The oocytes injected with mRNA are statically cultured at 20 ° C. for 24 hours in a medium of Barth. Next, a part of this medium was placed on a fibrin plate prepared according to the method of Granerie Piperno and Reich (Reference 4), and the medium was placed at 37 ° C. for 16 hours.
After incubation for a period of time, the lysis zone was measured. At this time, a sample in which anti-t-PA serum was mixed with medium was also incubated. As a result, an mRNA fraction was selected which had the largest lysis zone and whose lysis was suppressed when the anti-t-PA serum was mixed.

3) Preparation of colony library containing t-PA gene mRNA fraction 5 having t-PA synthesis ability selected in 2)
Double-stranded cDNA was prepared by the method of Gubler and Hoffman (Reference 6) using μg. A plasmid pUC13 1 in which a deoxy (C) residue was ligated to this cDNA and a deoxy (G) residue was similarly ligated to the pstI site at the end
Annealed with μg. With the annealed mixture E. col
i HB101 and (ATCC 33694) was transformed to obtain colonies of 5000 ampicillin-resistant.
After attaching this colony to Whatman 541 filter by the method of Jergen et al. (Reference 7), alkali treatment,
Neutralize, wash, and dry the DNA of each colony
Was fixed to the filter.

4) Preparation of DNA probe As described in JP-A-59-80613, a method combining ion exchange chromatography, affinity chromatography, gel filtration method and the like can be used.
T-PA is purified from human normal cell line MTC017.
The partial amino acid sequence of this t-PA was determined as follows to prepare a probe. Purified t-PA1m
g to 3 ml of 0.1 M ammonium bicarbonate (pH 7.
5), trypsin (TPCK, L-1-tosylamide-2-phenylethylchloromethylketone treated trypsin) was added at a molar ratio of 100.000: 1, and partially hydrolyzed at room temperature for 2 hours. Was done. Next, the reaction was stopped by adding DFP (diisopropyl fluorophosphate) in an amount of 10.000 times that of the added trypsin. The reaction product was dialyzed against 5 mM formic acid and then lyophilized. The protein was then loaded with 0.56 M Tris-HCl (pH 8.
6), dissolved in 5 ml of a solution containing 8 M urea and 5 mM EDTA. The disulfide bond was reduced by adding 0.1 ml of β-mercaptoethanol. Reaction is under nitrogen 45
It was carried out at ℃ for 2 hours. Then 1.4M iodoacetic acid and 1N
The reducing disulfide was carboxymethylated by adding 1.0 ml of a solution containing NaOH. After leaving it at room temperature for 20 minutes, dialyzing it against 0.01% Tween 80 at room temperature,
Lyophilized. The resulting lyophilized carboxymethylated protein was dissolved in 5 ml of a 0.1 M ammonium bicarbonate (pH 7.2) buffer containing 0.1 M ammonium thiocyanate, and then Red-Sepha equilibrated with the same buffer.
After passing through the rose column and washing the column with the same buffer,
The concentration of ammonium thiocyanate was linearly increased to 1.0 M to elute the adsorbed protein. as a result,
Protein peaks were obtained in the non-adsorbed fraction and the adsorbed fraction. SD
As a result of analysis by S-acrylamide gel electrophoresis, each showed a single protein band and both had a molecular weight of about 35.00.
It was 0. Collect each protein peak and
After dialysis against M ammonium bicarbonate (pH 7.5), each was freeze-dried. The purified t-PA and the protein obtained by the above method were each subjected to amino acid sequence determination using a gas phase protein sequencer (manufactured by Applied Biosystems). For t-PAI, G from the N-terminal
The amino acid sequence of 38 residues was determined in the order of ly-Ala-Arg-Ser-Tyr-Gln. The amino acid sequence of the non-adsorbed fraction protein is Ile-Lys-Gly-Gly.
-Leu-, and for adsorbed fraction proteins, Ser-Asn-Arg-Val-Glu-T
yr-Cys-Trp-Cys-Asn-Ser-Gl
The amino acid sequence of y-Arg- was determined. It was confirmed that the N-terminal Ser of this adsorbed protein was the 31st position from the N-terminal of t-PA. As a result, the optimum region for probe production was set to Tyr-Cys-Trp-Cys-Asn.
And Eight kinds of 14 nucleotides (14mer) 5'- complementary to the DNA sequence encoding this amino acid sequence
dTTnCACCAnCAnTA (n represents A or G) was chemically synthesized by the solid phase phosphotriester method (Reference 8).

5) Bacteria containing the t-PA cDNA sequence
Isolation and identification of clones 8 types of 14 nucleotides (14 me) synthesized in section 4)
r) mixture by T4-polynucleotide kinase
The 5'end³²Labeled with P. 6 x SSC (1 x SS
C; 0.15M NaCl, 0.015M sodium succinate
Lithium pH 7.0), 10 × Denhardt's solution
(1 x Denhardt's solution; 0.02% bovine serum al
Bumin (fraction V), 0.02% polyvinylpyrrolidone
(3.6 x 10 ³~ 4 x 10^FourDalton), 0.02%
Ficoll (4 x 10^FiveDalton)), 0.1% SD
S, containing 100 μg / ml sonicated salmon sperm DNA
In solution, use the colony library prepared in section C-3)
Prehybridize the containing filter for 2 hours at room temperature
It was Then 6x SSC, 10x Denhardt, and 5
× 10 ⁶Contains labeled probe at a concentration of count / min ml
Hybridized overnight in solution at room temperature. Then fill
At room temperature in 6 × SSC and 0.1% SDS solution.
Wash 3 times for 5 minutes, then in the same solution at 30 ° C
Washing was repeated 3 times for 5 minutes. The washed filter
After air-drying, DuPont Cronex Lightnin
G-Plus (Dupont Cronex Light
Ning Plus) intensifying screen
The film was exposed for 16 hours to Kodak XR-5.
14 showed a positive hybridization reaction
Burnboim and Dolly's method from individual colonies (Reference)
Plasmid DNA was isolated by a modified method of 9). The plush
The nucleotide sequence of the cDNA insert with Maxum
It was determined by the Gilbert method (Reference 10). As a result, black
Amino acid sequence determined from the DNA base sequence of T-1.
Column and the amino acid sequence of purified t-PA confirmed in section 4).
Clone encodes t-PA by comparison with the column
It was found to contain cDNA. This c is shown in FIG.
The nucleotide sequence of the DNA insert is shown. This insert
It has a length of 2170bP and an ohmic length of 1686bP.
Includes Punreading Frame, 76bP
5'side untranslated region and 408bP 3'side untranslated region
Contains. In addition, this DNA sequence is
DNA sequence encoding t-PA isolated from S. marcescens strain
Compared with (Reference 2), 3 places in the 5'-side untranslated region,
One region in the coding region of t-PA protein, 3'untranslated
There were 5 differences in the area.

6) Expression of t-PA gene using animal cells as host 6-1) Expression of t-PA using mouse metallothionein (MMT) promoter 6-1-1) Construction of plasmid pMT-1 Fig. 1 ( A plasmid to be introduced into animal cells was prepared by the procedure shown in Fig. 1). First, from clone T-1, plasmid p
T-1 was isolated. For the purpose of removing the double-stranded GC base pair produced during cDNA synthesis, 25 μg of pT
-1 was cleaved with Hind III, followed by 0.4 U Bal3
The reaction was performed at 37 ° C. for 2 minutes. Next, the HindIII linker was ligated with T4 DNA ligase, and then cut with HindIII and BamHI, and the DNA fragment containing the t-PA gene was separated and collected by polyacrylamide gel electrophoresis.
On the other hand, pUC13 was cleaved with HindIII and BamHI, and then a DNA fragment of about 2700 bp was separated and collected by low melting point agarose gel electrophoresis. This DNA fragment and the previously prepared DNA fragment containing the t-PA gene were ligated to each other with T4 DNA ligase, and then E. coli HB101
Was transformed. A plasmid was prepared from 10 of the obtained ampicillin-resistant colonies, and the DNA base sequence of the 5'untranslated region of the t-PA gene was determined. Plasmid pT406 prepared from clone T-406 contains 3
It contained a 9 bp 5'untranslated region. 20 μg pT
406 was cleaved with HindIII and BamHI, and a DNA fragment containing a gene of about 2200 bP was separated and collected by polyacrylamide gel electrophoresis. On the other hand, 10 μg of plasmid pSV2-dhfr (ATCC 37146) was added to H
A DNA fragment of about 4200 bP, which had been cleaved with indIII and BamHI and had the dhfr gene removed, was separated and collected by low melting point agarose gel electrophoresis. 0.2 μg of this DNA fragment
And 0.1 μg of the approximately 2200 bP fragment containing the t-PA gene isolated above were ligated to each other with T4 DNA ligase, and then E. E. coli HB101 was transformed to obtain ampicillin resistant colonies. A small amount of a plasmid was isolated from these colonies, and a clone having the desired plasmid pMT-1 was identified based on the restriction enzyme cleavage pattern.

6-1-2) Preparation of plasmid pMT-2 After a large amount of plasmid was isolated from the clone having plasmid pMT-1, 10 μg of pMT-1 was added to HindII.
It was cleaved with I and BamHI, and a DNA fragment of about 3 KbP containing the t-PA gene and the poly (A) signal of SV40 was separated and collected by low melting point agarose gel electrophoresis. on the other hand,
Plasmid pdBPV-MMTneo (342-12)
From (Reference 22), a DNA fragment containing PML (Reference 23) and MMT promoter was prepared. 10 μg pdBPV
After cutting MMMneo (342-12) with BglII, 5 units of DNA polymerase I Klenov (K
lenpw) fragment, as well as 0.1 mM dATP each,
Add dCTP, dGTP and TTP, and add 10 at 37 ° C.
After incubation for a minute, the sticky ends were made blunt ends. After purification by phenol extraction, chloroform treatment and ethanol precipitation, 0.05 μg of Hind III Lincoln was added and ligated with T4 DNA ligase. Next Bam
Cleaved with HI and partially cleaved with HindIII pML
The fragment containing the and MMT promoter was separated and collected by low melting point agarose gel electrophoresis. This DNA fragment 0.05μ
g, a 3 Kbp DNA fragment containing the previously prepared t-PA gene and SV40 poly (A) signal, 0.04 μ
After ligation to each other with gOT4 DNA ligase, E. c
oli HB101 was transformed to obtain ampicillin-resistant colonies. A small amount of a plasmid was isolated from these colonies, and a clone having the desired plasmid pMT-2 was identified based on the restriction enzyme digestion pattern of the plasmid.

6-1-3) Construction of plasmid pMT-3 After a large amount of plasmid was isolated from a clone having plasmid pMT-2, it was cleaved with BamHI and subsequently calf intestine-derived alkaline phosphatase (hereinafter abbreviated as CIP). )
And a 5'-terminal dephosphorylation reaction was performed. on the other hand,
pdBPV-1 (142-6) (ATCC 3713)
After cutting 4) with BamHI, about 8 Kbp of BPV
_{The 100T} DNA fragment was separated and collected by low melting point agarose gel electrophoresis. Next, 0.05 μg of BPV _100T DNA fragment and pMT digested with BamHI and treated with CIP
-20.02 μg were ligated together with T4dna ligase and then E. E. coli HB101 was transformed to obtain ampicillin resistant colonies. A small amount of plasminogen was isolated from these colonies, and the target plasmid pMT was determined from the restriction enzyme digestion pattern of the plasmid.
A clone with -3 was identified.

6-1-4) Construction of plasmid pMT-4 The plasmid pMT-2 was partially cleaved with SalI and then CIP-treated, and the cohesive end was made blunt with a Klenov fragment of DNA polymerase. This DNA fragment was used as a vector. On the other hand, PdBPV-MMTneo (34
2-12) was cut with EcoRI and BamHI. Next, after changing the sticky end to a blunt end with Klenov fragment of DNA polymerase I, a DNA fragment containing the nor gene of about 4 Kbp was separated and collected by low melting point agarose gel electrophoresis. After 0.05 μg of this dna fragment and 0.05 μg of the previously prepared vector were ligated to each other with T4 DNA ligase, E. E. coli HB101 was transformed. Thereafter, a clone having the desired plasmid pMT-4 was identified in the same manner as in the previous section.

6-1-5) Construction of plasmid pMT-5 The plasmid pMT-4 was cleaved with BamHI and then CIP.
Processed. 0.05 μg of this DNA fragment and 6-1-3)
0.05 μg of BPV _100T DNA fragment prepared in the above section was added to T4.
After ligation to each other with DNA ligase, E. coli
HB101 was transformed. Thereafter, a clone having the desired plasmid pMT-5 was identified in the same manner as in the previous section.

6-1-6) Introduction of plasmid having MMT promoter into animal cells and expression of t-PA t having the MMT promoter prepared in 6-1-2) to 6-1-5) above -PA expression plasmid was introduced into animal cells. The introduction method was a modification of the calcium phosphate precipitation method, which is the method of Graham and Van der Eb (Reference 16). That is, 2 × HBS (50 mM Hepes
(PH 7.1), 280 mM NaCl), 1.4 mM
While sucking sterile air into the solution containing NaH ₂ PO ₄ ,
Add an equal volume of a DNA solution containing 50 mM CaCl ₂
The NA-CaPO ₄ precipitates was prepared. After the dropping, the mixture was allowed to stand for 20 minutes, then the coprecipitate was well suspended with a pipette and added dropwise to the host cells cultured in the flask. CO _{2 at} 37 ° C for several hours
After culturing in an incubator, the medium was removed and 15% Gl was added.
Add a solution containing ycerol and 2xHBS, 37 ℃
And allowed to stand for 3 minutes. After removing this solution, a new medium was added and the medium was kept for 2 days in a CO ₂ incubator. The medium was replaced with a new medium and the medium was used for 1 week. In this case, since the neo resistance gene was used as a selectable marker for the transformant, a medium containing the antibiotic G418 was used. Next, after removing the medium, according to the method of Jones et al. (Reference 24), a casein agar layer was used in place of fibrin, and this was overlaid on the cells and cultured overnight. A t-PA producing strain showing a Tomei lysis zone in the casein agar layer was selected, each clone was cultured, and the t-PA activity of the culture supernatant was measured to obtain a t-PA high producing strain. A part of the result is shown in Table 2 (Table 2). In addition, the t-PA activity of the medium supernatant obtained by culturing this high-producing strain in a medium containing 1 μM Cd ²⁺ or 50 μM Zn ²⁺ is also shown in the same table. As a result, clone MT-325 exhibited the highest t-PA activity, 110 units / ml / day without adding Cd ²⁺ or Zn ^2+, with the addition of Cd ²⁺ or Zn ²⁺ 305 or 3
10 units / ml / day of t-PA was produced.

[0022]

[Table 2] 6-2) Expression of t-PA using human metallothionein (hMT) promoter 6-2-1) Preparation of plasmid phT-1 As shown in FIG. 3 (FIG. 3), a plasmid using the hMT promoter was prepared. . First, the plasmid pMTS
VPA (University of South America
rnCalifomia, School of Med
icine's M. Obtained from Dr. Karin) XbaI
After cutting with, the cohesive ends were made blunt with the Klenov fragment of DNA polymerase I. H to this DNA fragment
The ind III linker was ligated with T4 DNA ligase and then cut with Hind III. Approximately 840 bpD of hMT promoter part by low melting point agarose gel electrophoresis
The NA fragment was separated and collected. On the other hand, the plasmid pMT-2
Was cleaved with Hind III, treated with CIP, and then treated with low melting point agarose gel electrophoresis from plasmid pTM-2 to MM.
A DNA fragment of about 5.5 bp excluding the T promoter was separated and collected. This DNA fragment and the previously prepared DNA fragment having the hMT promoter were ligated to each other with T4 DNA ligase, and E. E. coli HB101 was transformed.
A small amount of a plasmid was isolated from the obtained ampicillin-resistant colonies, and a clone having the desired plasmid phT-1 was identified from the digestion pattern of the plasmid with a restriction enzyme.

6-2-2) Preparation of plasmid phT-2 This was prepared in the same manner as the plasmid phT-1 except that pMT-4 was used instead of the plasmid pMT-2. 6-2-3) Preparation of plasmid phT-3 The plasmid phT-1 was cleaved with BamHI and treated with CIP. This DNA fragment and the approximately 8 kbp BPV _100T DNA fragment prepared when the plasmid pMT-3 was prepared were subjected to T4D.
Binding to each other with NA ligase, E. coli HB10
1 was transformed. Hereinafter, a clone having phT-3 was identified by the same method as in 6-2-1).

6-2-4) Preparation of plasmid phT-4 A plasmid phT-3 was prepared in the same manner as the plasmid phT-3 except that phT-2 was used in place of the plasmid phT-1.

[0025]

[Table 3]

6-2-5) A model having an nMT promoter
Introduction of rasmid into animal cells and expression of t-PA Prepared in 6-2-1) to 6-2-4) above
Plasmid for expressing t-PA having a hMT promoter
Was introduced into animal cells. Introduction method and t-PA high-producing strain
The screening method is performed in the same manner as in Section 6-1-6).
It was A part of the result is shown in Table 3 (Table 3). Also,
This high-producing strain has 1 μM Cd²⁺Or 50 μM Zn²⁺or
Is 1 μM dexamethasone
The same is true for the t-PA activity of the culture supernatant cultured in the medium containing e).
As shown. As a result, clone hT-382 is the best
shows t-PA activity, Cd²⁺, Zn ²⁺Or dexamethasone
105 units / ml day, Cd²⁺, Z
n²⁺, Or 330-340 when dexamethasone is added
Unit / ml / day of t-PA was produced.

References 1. D. C. Rijken and D.M. Colle
n, J. BiolChem. , 256 p. 7035
(1981) 2. JP-A-59-42321. E. L. Wilson et al, Cancer
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513 (1979) 10. A. M. Maxam and W.D. Gilber
t Proc. Natl. Acad. Sci. U. S.
A 74 p. 560 (1977) 11. W. R. Earle, J.M. Natl. Cancel
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1 (1984) 19. P. Sheldrick et al, Pro
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Cold Spring Harbor Laborat
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[0028]

[Sequence list]

SEQ ID NO: 1 Sequence length: 1590 Sequence type: Nucleic acid Number of strands: Double-strand Topology: Linear Sequence type: cDNA to genomic DNA Origin organism name: Human (homo sapiens) Tissue type : features 1-1590 E mat peptide sequence of fetal epithelial sequence GGAGCCAGAT CTTACCAAGT GATCTGCAGA GATGAAAAAA CGCAGATGAT ATACCAGCAA 60 CATCAGTCAT GGCTGCGCCC TGTGCTCAGA AGCAACCGGG TGGAATATTG CTGGTGCAAC 120 AGTGGCAGGG CACAGTGCCA CTCAGTGCCT GTCAAAAGTT GCAGCGAGCC AAGGTGTTTC 180 AACGGGGGCA CCTGCCAGCA GGCCCTGTAC TTCTCAGATT TCGTGTGCCA GTGCCCCGAA 240 GGATTTGCTG GGAAGTGCTG TGAAATAGAT ACCAGGGCCA CGTGCTACGA GGACCAGGGC 300 ATCAGCTACA GGGGCACGTG GAGCACAGCG GAGAGTGGCG CCGAGTGCAC CAACTGGAAC 360 AGCAGCGCGT TGGCCCAGAA GCCCTACAGC GGGCGGAGGC CAGACGCCAT CAGGCTGGGC 420 CTGGGGAACC ACAACTACTG CAGAAACCCA GATCGAGACT CAAAGCCCTG GTGCTACGTC 480 TTTAAGGCGG GGAAGTACAG CTCAGAGTTC TGCAGCACCC CTGCCTGCTC TGAGGGAAAC 540 AGTGACTGCT ACTTTGGGAA TGGGTCAGCC TACCGTGGCA CGCACAGCCT CACCGAGTCG 600 GGTGCCTCCT GCCTC CCGTG GAATTCCATG ATCCTGATAG GCAAGGTTTA CACAGCACAG 660 AACCCCAGTG CCCAGGCACT GGGCCTGGGC AAACATAATT ACTGCCGGAA TCCTGATGGG 720 GATGCCAAGC CCTGGTGCCA CGTGCTGAAG AACCGCAGGC TGACGTGGGA GTACTGTGAT 780 GTGCCCTCCT GCTCCACCTG CGGCCTGAGA CAGTACAGCC AGCCTCAGTT TCGCATCAAA 840 GGAGGGCTCT TCGCCGACAT CGCCTCCCAC CCCTGGCAGG CTGCCATCTT TGCCAAGCAC 900 AGGAGGTCGC CCGGAGAGCG GTTCCTGTGC GGGGGCATAC TCATCAGCTC CTGCTGGATT 960 CTCTCTGCCG CCCACTGCTT CCAGGAGAGG TTTCCGCCCC ACCACCTGAC GGTGATCTTG 1020 GGCAGAACAT ACCGGGTGGT CCCTGGCGAG GAGGAGCAGA AATTTGAAGT CGAAAAATAC 1080 ATTGTCCATA AGGAATTCGA TGATGACACT TACGACAATG ACATTGCGCT GCTGCAGCTG 1140 AAATCGGATT CGTCCCGCTG TGCCCAGGAG AGCAGCGTGG TCCGCACTGT GTGCCTTCCC 1200 CCGGCGGACC TGCAGCTGCC GGACTGGACG GAGTGTGAGC TCTCCGGCTA CGGCAAGCAT 1260 GAGGCCTTGT CTCCTTTCTA TTCGGAGCGG CTGAAGGAGG CTCATGTCAG ACTGTACCCA 1320 TCCAGCCGCT GCACATCACA ACATTTACTT AACAGAACAG TCACCGACAA CATGCTGTGT 1380 GCTGGAGACA CTCGGAGCGG CGGGCCCCAG GCAAACTTGC ACGACGCCTG CCAGGGCGAT 1440 TCGGGAGGCC CCCTGGTGTG TCTGAA CGAT GGCCGCATGA CTTTGGTGGG CATCATCAGC 1500 TGGGGCCTGG GCTGTGGACA GAAGGATGTC CCGGGTGTGT ACACCAAGGT TACCAACTAC 1560 CTAGACTGGA TTCGTGACAA CATGCGACCG 1590

SEQ ID NO: 2 Sequence length: 2170 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to genomic DNA Origin organism name: Human (homo sapiens) Tissue type : Fetal epithelial sequence characteristics 1-76 E 5'UTR 77-1762 E CDS 77-172 E sig peptide 173-1762 E mat peptide 1763-1762 E 3'UTR sequence CTAGGGCTGG AGAGAAAACC TCTGCGAGGA AAGGGAAGGA GCAAGCCGTG AATTTAAGGG 60 ACGCTGTGAA GCAATCATGG ATGCAATGAAGAGAGT TGCTGCTGTG 120 TGGAGCAGTC TTCGTTTCGC CCAGCCAGGA AATCCATGCC CGATTCAGAA GAGGAGCCAG 180 ATCTTACCAA GTGATCTGCA GAGATGAAAA AACGCAGATG ATATACCAGC AACATCAGTC 240 ATGGCTGCGC CCTGTGCTCA GAAGCAACCG GGTGGAATAT TGCTGGTGCA ACAGTGGCAG 300 GGCACAGTGC CACTCAGTGC CTGTCAAAAG TTGCAGCGAG CCAAGGTGTT TCAACGGGGG 360 CACCTGCCAG CAGGCCCTGT ACTTCTCAGA TTTCGTGTGC CAGTGCCCCG AAGGATTTGC 420 TGGGAAGTGC TGTGAAATAG ATACCAGGGC CACGTGCTAC GAGGACCAGG GCATCAGCTA 480 CAGGGGCACG TGGAGCACAG CGGAGAGTGG CGCCGAGTGC ACCAACTGGA ACAGCAGCGC 540 GTTGGCCCAG AAGCCCTACA GCGGGCGGAG GCCAGACGCC ATCAGGCTGG GCCTGGGGAA 600 CCACAACTAC TGCAGAAACC CAGATCGAGA CTCAAAGCCC TGGTGCTACG TCTTTAAGGC 660 GGGGAAGTAC AGCTCAGAGT TCTGCAGCAC CCCTGCCTGC TCTGAGGGAA ACAGTGACTG 720 CTACTTTGGG AATGGGTCAG CCTACCGTGG CACGCACAGC CTCACCGAGT CGGGTGCCTC 780 CTGCCTCCCG TGGAATTCCA TGATCCTGAT AGGCAAGGTT TACACAGCAC AGAACCCCAG 840 TGCCCAGGCA CTGGGCCTGG GCAAACATAA TTACTGCCGG AATCCTGATG GGGATGCCAA 900 GCCCTGGTGC CACGTGCTGA AGAACCGCAG GCTGACGTGG GAGTACTGTG ATGTGCCCTC 960 CTGCTCCACC TGCGGCCTGA GACAGTACAG CCAGCCTCAG TTTCGCATCA AAGGAGGGCT 1020 CTTCGCCGAC ATCGCCTCCC ACCCCTGGCA GGCTGCCATC TTTGCCAAGC ACAGGAGGTC 1080 GCCCGGAGAG CGGTTCCTGT GCGGGGGCAT ACTCATCAGC TCCTGCTGGA TTCTCTCTGC 1140 CGCCCACTGC TTCCAGGAGA GGTTTCCGCC CCACCACCTG ACGGTGATCT TGGGCAGAAC 1200 ATACCGGGTG GTCCCTGGCG AGGAGGAGCA GAAATTTGAA GTCGAAAAAT ACATTGTCCA 1260 TAAGGAATTC GATGATGACA CTTACGACAA TGACATTGCG CTGCTGCAGC TGAAATCGGA 1320 TTCGTCCCGC TGTGCCCAGG AGAGCAGCGT GGTCCGCACT GTGTGCCTTC CCCCGGCGGA 1380 CCTGCAGCTG CCGGACTGGA C GGAGTGTGA GCTCTCCGGC TACGGCAAGC ATGAGGCCTT 1440 GTCTCCTTTC TATTCGGAGC GGCTGAAGGA GGCTCATGTC AGACTGTACC CATCCAGCCG 1500 CTGCACATCA CAACATTTAC TTAACAGAAC AGTCACCGAC AACATGCTGT GTGCTGGAGA 1560 CACTCGGAGC GGCGGGCCCC AGGCAAACTT GCACGACGCC TGCCAGGGCG ATTCGGGAGG 1620 CCCCCTGGTG TGTCTGAACG ATGGCCGCAT GACTTTGGTG GGCATCATCA GCTGGGGCCT 1680 GGGCTGTGGA CAGAAGGATG TCCCGGGTGT GTACACCAAG GTTACCAACT ACCTAGACTG 1740 GATTCGTGAC AACATGCGAC CGTGACCAGG AACACCCGAC TCCTCAAAAG CAAATGAGAT 1800 CCCGCCTCTT CTTCTTCAGA AGACACTGCA AAGGCGCAGT GCTTCTCTAC AGACTTCTCC 1860 AGACCCACCA CACCGCAGAA GCGGGACGAG ACCCTACAGG AGAGGGAAGA GTGCATTTTC 1920 CCAGATACTT CCCATTTTGG AAGTTTTCAG GACTTTGTCT GATTTCAGGA TACTCTGTCA 1980 GATGGGAAGA CATGAATGCA CACTAGCCTC TCCAGGAATG CCTCCTCCCT GGGCAGAAAT 2040 GGCCATGCCA CCCTGTTTTC GCTAAAGCCC AACCTCCTGA CCTGTCACCG TGAGCAGCTT 2100 TGGAAACAGG ACCACAAAAA TGAAAGCATG TCTCAATAGT AAAAGATAAC AAGATCTTTC 2160 AGGAAAGACG 2170

[Brief description of drawings]

FIG. 1 is a diagram showing a procedure for preparing a plasmid pMT-1 shown in Example 6-1-1).

FIG. 2 is a diagram showing a procedure for preparing the plasmid phT-1 shown in Example 6-2-1).

Front page continuation (72) Inventor Fumio Ozen 1 90 Machiho, Mobara-shi, Chiba

Claims (2)

[Claims] 1. A DNA sequence encoding a human tissue plasminogen activator derived from human normal cells, which comprises a mouse metallothionein promoter or a human metallothionein promoter and 530 amino acids starting from glycine at the N-terminus, which is a DNA sequence. In an animal cell, the sequence of which comprises a DNA sequence as set forth in SEQ ID NO: 1 of the Sequence Listing, the DNA sequence encoding the tissue plasminogen activator being operably linked to the above promoter. A method for producing a human tissue plasminogen activator derived from a normal human cell using a microorganism or an animal cell transformed with a recombinant DNA vector capable of self-replication. 2. A DNA sequence encoding a human tissue plasminogen activator derived from a human normal cell, which comprises a mouse metallothionein promoter or a human metallothionein promoter and 530 amino acids starting from glycine at the N-terminal, which is a DNA sequence. In an animal cell, the sequence of which comprises a DNA sequence as set forth in SEQ ID NO: 1 of the Sequence Listing, the DNA sequence encoding the tissue plasminogen activator being operably linked to the above promoter. Human derived from normal human cells produced by a method for producing human tissue plasminogen activator derived from normal human cells using a microorganism or animal cell transformed with a recombinant DNA vector having the ability of self-replication Tissue plasminogen activator.