JP2837348B2 - Method for producing human tissue plasminogen activator derived from human normal cells - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [0001] The present invention relates to a tissue plasmid produced by normal human cells.
Sminogen activator (hereinafter abbreviated as t-PA)
With a new expression vector containing the new DNA sequence
Normal human cells using new transformed host cells
For the production of tissue plasminogen activator produced by yeast
Related. The present invention has been described in detail as described below.
The background of the present invention and relevant important literature
Quotes the numbers in the text and cites the references at the end. A) Tissue plasmid produced by normal human cells
Nogen activator The t-PA according to the present invention is used for vascular endothelial cells and various tissues.
It is produced and secreted from cells and is the main body of thrombus.
It plays a leading role in dissolving Brin.
For the sake of convenience, urokinase is currently used for the treatment of thrombosis.
Streptokinase has been used. But these are
It includes bleeding and antigenic problems,
The affinity to do is weak. And urokinase is complicated to obtain
Process is unsanitary, complicated and high
It is valuable and leaves many problems. This
Therefore, development of a new medicine is desired. Recently
Bowes melanoma (Bowes melanoma)
melanoma) strain secretes relatively large amounts of t-PA
It is reported to be produced (Reference 1). But that
Originate from malignant melanoma cells
There are various problems in developing t-PA as a drug.
Contains. For example, Bowes Mela, a malignant tumor cell
To obtain t-PA by the cell culture method of the Norma strain,
The safety of the process itself must be carefully considered
No. Recently, t-PA was obtained from this Bowes melanoma strain.
The host cell is transformed by integrating the expression vector,
A method for producing t-PA using a transformed host cell has been disclosed.
(Reference 2). Here, Bow cells, a cancer cell
T-PA from the ranoma strain was isolated from normal human tissue.
For the obtained t-PA and immunological and amino acid composition,
It is stated that there is no distinction. However, carcinogenesis mechanism
Are still completely scientifically elucidated
Not necessarily. Under such circumstances, cancer cells
T-PA using the gene isolated from
And the dangers of applying it as a medicine
There is no answer as to whether there is. For these reasons
The present inventor has proposed that t-
As a result of searching for normal human cells that produce PA,
Found cells with high potential. In addition, the human normal cells
New DN encoding t-PA using recombinant DNA technology
A sequence can be isolated and replicable to further express t-PA
This DNA sequence is integrated into a simple vector to transform host cells.
The transformation resulted in a recombinant host cell, leading to the present invention. This recombination
Use of host cells reduces the risk of normal human cells
A process for mass-producing t-PA with no concerns about its properties has been established.
Was. [0004] B) Gene manipulation technology B-1) Human tissue plasminogen activator-producing cell
Screening Because of the ease of culture, many cancer cells
And that cells that secrete t-PA are present.
(3), but its essence remains largely unknown.
Up to. The present inventors have proposed various human normal cells, preferably
Indicates human fetal epidermal cells, fetal lung tissue cells, fetal kidney cells
Cells and the like are cultured, and granelli piperno and Reich (G
(Ranell-Piperno, Reich) Method
As described in (Reference 4), fibrin play
Using the activity to dissolve fibrin on
Is screened by plasminogen activator production performance
I did it. As a result, secretory production of t-PA is relatively large.
Was selected and used as starting material. these
All have 2n = 46 chromosome numbers and finite passage numbers
Human normal diploid cells. B-2) Purification and size fractionation of mRNA Humans Highly Producing t-PA Screened by the Present Invention
One of the normal cells derived from fetal epithelium (MTC017)
Cultivates large quantities and extracts mRNA from the cultured cells.
Can do it. Specifically, first, all RNs are
Extract A. Then use oligo (dT) cellulose
To collect and recover poly (A) plus mRNA from total RNA
I do. The obtained mRNA is subjected to electrophoresis on agarose gel.
Apply and fractionate by size. Africa for each fraction
Clawed frog (Xenopus IaevisOocytes
Microinjection
in the medium of Barth
Culture (Reference 5). Then this medium is granelli
-A file prepared according to the method of Perno and Reich (Reference 4).
Place on a burin plate and measure its dissolution zone.
It is possible to select a fraction having t-PA activity by
come. This fraction contains mRNA encoding t-PA.
The inclusion is that the anti-t-PA serum is
Can be confirmed by inhibiting the reaction. [0006] B-3) Colony containing the t-PA gene
Making Evry The fraction containing the mRNA encoding t-PA was
Raler and Hoffman (Gubler and Hoffman)
According to the method (Reference 6), cDNA is
Insert into C13. nextE. FIG. coli  HB101 (A
TCC33694) to transform ampicillin-resistant
Obtain colonies. A portion of each colony is
atman) After being deposited on a 541 filter,
By the method of Gergen et al.
Immobilize the knee DNA on the filter. B-4) Preparation of DNA probe Ion exchange as described in JP-A-59-80613
Exchange chromatography, affinity chromatography
The combination of gel filtration, gel filtration, etc.
The inventors screened the human positive producing t-PA.
The t-PA is purified from the normal cells. To make a probe
Next, the partial amino acid sequence of this t-PA is
To decide. Partial hydrolysis of t-PA protein with trypsin
Separation and purification of the decomposed products obtained by
The amino acid sequence is determined from the amino terminal. As a result, professional
Tyr-Cys-Trp-C
ys-Asn. D encoding this amino acid sequence
Eight types of 14 nucleotides (14 m
er) 5'-dTTnCACCAnCAnTA (n is A
Or represents G) by the solid-phase phosphotriester method
Synthesize (Reference 8). B-5) A cell containing the t-PA cDNA sequence
Isolation and identification of bacterial clones Eight types of 14 nucleotides (14) synthesized in section B-4)
mer) is reacted with T4-polynucleotide kinase.
The 5 'end³²Label with p. Use this as a probe
The cDNA library prepared in section B-3) was immobilized.
Hybridization with the filter is performed. Positive
Colonies showing positive hybridization reactions
From Birnboim and Dory
ly) (Reference 9).
Maxam and Gilbert base sequences for cDNA inserts
(Maxam, Gilbert) (Reference 10)
decide. B-6) t-PA residue using animal cells as a host
Gene expression DNA encoding t-PA isolated from normal human cells
The sequence was derived from a melanosarcoma cell line, the Bowes melanoma line.
A DNA sequence encoding the isolated t-PA and 9
There was a difference. In addition, normal cells determined from the sequence
When the entire amino acid sequence of the next t-PA was examined,
PA molecule has four sites to which sugar chains can be added
It was estimated. An antibiotic that actually inhibits glycosylation
Tunicamycin was added to produce t-PA producing strain MTC01.
7 reduces the molecular weight of t-PA to about 50,000
Therefore, about 30% of the molecular weight is determined by sugar
I show you. In the living body of the enzyme, the addition of sugar chains
Is considered to be involved in the stable function of
Lack of sugar chain may show antigenicity in vivo
is there. Therefore, the cloned t-PA gene can be transferred to animal cells.
Natural type t with sugar chains added by
-Thought that producing PA was a good method. Movement to be a host
Desirable expression for expressing the t-PA gene in target cells
The current vector is the promoter region, ribosome junction
Position, transcription termination sequence, polyadenylation site, replication origin
Transfection of the vector and host cells.
Contains a gene that becomes a selectable marker when Step
For the motor region and ribosome binding site,
And those derived from animal cells. example
For example, papovavirus, adenovirus, retrovirus
Or the promotion of human cytomegalovirus
Region and a ribosome binding site are available. Again
Region located in front of the gene derived from the target cell
Regions and ribosome binding sites can also be used. That
In general, the promoter of a gene that is strongly expressed
Is a strong promoter region for other genes
It is advantageous to use it. An example
For example, metallothionees from mice, humans or other animals
, Immunoglobulin, insulin, albumin, etc.
Uses promoter region and ribosome binding site for gene
It is considered advantageous to do so. Transcription termination sequence, polyade
Similar virus or various genes
You can use the next one. For the replication origin, see above
Viruses, especially bovine papilloma virus (Bovine Pa)
pilloma Virus: hereinafter abbreviated as BPV)
Is Simian Virus 40 (hereinafter abbreviated as SV 40)
The exogenous origin that can be derived from
May be constructed, or the desired
By incorporating the expression vector as a fragment,
You may make use of canism. Host cells
Is a selectable marker when transfecting
Is not always necessary, but having this
Quick and accurate selection when selecting transformed cells
This is advantageous. For example, isolated herpes virus (HS)
V) Thymidine kinase gene (TK)
There is a drofolate reductase gene (DHFR)
In this case, the host cell is a thymidine kinase-deficient strain (tK
^-) Or dihydrofolate reductase deficient strain (dhfr^-)use
To use. In addition, large cells showing mycophenolic acid resistance in animal cells
Enterobacterial xanthine guanine phosphoribosyl trans
Antibiotics in ferrase gene (Eco-gpt) and animal cells
Neoma derived from transposon 5 showing resistance to substance G418
Isin resistance gene (neo) and the like can also be used. t-P
A vector of the present invention comprising a DNA sequence encoding A
Depending on the host cell to be transfected,
To select from a large number of cells currently established
I can do it. A preferred example is a mouse.
L cells (Reference 11), thymidine kinase deficient L cells
(Reference 12), dihydrofolate reductase deficient chiney
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), mouse and human
Seed myeloma cells, such as mouse P
3 / NS1 / 1-Ag4-1 (NS-1) cells (ATC
C TIB18), mouse P3X63Ag8 cells (AT
CC TIB 9), mouse J558 (ATCC TI
B6) etc. are used, but this is an example and
The use of external animal cells is also possible. Code t-PA
A vector of the invention containing the DNA sequence
For transfection of
The sium method (Reference 15) may be used. Other nuclear injection methods
(Reference 16), protoplast fusion method (Reference 17),
Introduction by Qi shock (Reference 18), DEAE-Deki
A method such as the Strand method (Reference 19) can also be used. the above
Transformation obtained after transfection by the method
After the cultured cells are separated and cultured, the t-PA activity of the culture supernatant is determined.
Is measured to produce human normal cell-derived t-PA.
You can see that they are growing. [0010] 【Example】 1) Screening of normal human cells producing t-PA
The Various human normal cells shown in Table 1 (Table 1) were screened.
Was targeted for Cells treated are confluent (c
The cells were cultured until they were in the onfluent state. That culture
T-PA activity was quantified using 15 μl of the culture supernatant. The
Using the method of Llanelli Piperno and Reich (Reference 4)
The resulting fibrin plate was allowed to stand at 37 ° C. for 16 hours,
The dissolution zone was measured. At this time, add anti-urokina to each culture supernatant.
Preparation of samples mixed with serum or anti-t-PA serum
The dissolution zone was also measured and compared at the same time. In addition, live
The purified urokinase is used as a standard to dissolve the diluted solution.
The solution zone was measured at the same time to create a standard curve,
The activity was quantified in terms of international units. Screeninin
Part of the cell and its t-PA activity and urokiner
The activity was as shown in Table 1 (Table 1). The result
As a result, the MTC017 strain secreted and produced a relatively large amount of t-PA.
Therefore, these cells were used as a material for isolating the t-PA gene.
Was. This cell has 2n = 46 chromosomes and is about 80 generations
Normal diploid cells derived from human fetal epithelium
You. [0011] [Table 1]2) Purification and size fractionation of mRNA The MTC017 strain was cultured in large quantities, and 5 × 10⁸Collection of cells
I did. Collect cells in 20 ml phosphate buffered saline
After washing with (PBS), 20 ml of 10 mM Tris-
HCl (pH 8.6), 10 mM KCl, 1.5 mM
  MgCl^TwoResuspended in 3 mM dithiothreitol solution
did. Surfactant Nonidet P-40 (final concentration
0.5%) to lyse the cells.
The supernatant containing A is updated by phenol / chloroform extraction.
Was purified. The aqueous layer is brought to a 0.3 M sodium acetate solution,
Next, 2 volumes of ethanol were added to precipitate total RNA.
Was. Using oligo (dT) cellulose, m
RNA was purified (Reference 20). The yield was 5 × 1
0⁸4-5 mg of total RNA and 100-
150 μg of poly (A) plus mRNA was obtained. Low
Poly (A) plus mRNA using melting point agarose gel
(Reference 21). 1.5% low melting point agar
Loin, 50 mM boric acid, 5 mM sodium borate, 1
A slab gel containing 0 mM sodium sulfate was used. Hydroxylation
Poly (A) plus mRNA treated with methylmercury
The gel was electrophoresed. After electrophoresis, the gel was washed with 0.1 M dithios
After immersion in Reitol solution for 30 minutes, the molecular weight
Divided according to. 0.5X of 4 times capacity for each slice
After adding ammonium acetate, heat to 65 ° C to dissolve the gel.
Let it go. Then phenol extraction and chloroform extraction
After repeated removal of agarose components, ethanol precipitation
Each mRNA fraction was collected by precipitation. Then 500ng
Of each mRNA fraction from Xenopus laevis oocytes
The microinjection was performed (Reference 5). Note mRNA
Entered oocytes in Barth medium at 24:00
Incubate at 20 ° C for a while. Then part of this medium
According to the method of Granery-Piperno and Reich (Reference 4).
And place on a prepared fibrin plate at 37 ° C for 16 hours.
Incubate for hours and measure the lysis zone. At this time
Simultaneous sample mixed with anti-t-PA serum in medium
Incubated. As a result, the melting zone is the largest
In addition, when anti-t-PA serum is mixed,
The mRNA fraction to be suppressed was selected. 3) A colony line containing the t-PA gene
Making a library MRNA fraction 5 having t-PA combination performance selected in 2)
μg and the method of Gabler and Hoffman (Reference 6)
Double stranded cDNA was prepared. Deoxygenated cDNA
(C) Connect the residues and similarly add deoxy to the pstI site.
(G) Plasmid pUC131 with residues attached to the ends
Annealed with μg. With the annealed mixtureE. FIG. col
i  Transform HB101 (ATCC 33694)
As a result, 5000 ampicillin-resistant colonies were obtained.
This colony was washed by the method of Jergen et al.
Alkaline treatment after attaching to Toman 541 filter,
Perform neutralization, washing, and drying operations to obtain DNA for each colony.
Was fixed to the filter. 4) Preparation of DNA probe As described in JP-A-59-80613, ion
Exchange chromatography, affinity chromatography
Fee, by a method that combines gel filtration, etc.
T-PA is purified from human normal cell line MTC017.
To prepare a probe, the partial amino acid sequence of this t-PA
The columns were determined as follows. Purified t-PA1m
g of 3 ml of 0.1 M ammonium bicarbonate (pH 7.
5) and dissolved in trypsin (TPCK, L-1-tosyl).
Treated with amido-2-phenylethyl chloromethyl ketone
Trypsin) in a molar ratio of 100.000: 1
And partially hydrolyzed at room temperature for 2 hours. next
DFP (diethylene glycol) 10.000 times the amount of trypsin added.
(Sopropyl fluorophosphate) to stop the reaction
did. The reaction was dialyzed against 5 mM formic acid and lyophilized. Tan
Park was then 0.56 M Tris-HCl (pH 8.
6) In 5 ml of a solution containing 8 M urea and 5 mM EDTA
Dissolved. Add 0.1 ml β-mercaptoethanol
To reduce the disulfide bond. The reaction is carried out under nitrogen 45
C. for 2 hours. Next, 1.4M iodoacetic acid and 1N
  1.0 ml of a solution containing NaOH was added to
Fido was carboxymethylated. Leave at room temperature for 20 minutes
Then, it is dialyzed against 0.01% Tween 80 at room temperature,
Lyophilized. The obtained freeze-dried carboxymethylated compound
Add 5 ml of 0.1 M ammonium thiocyanate
Containing 0.1 M ammonium bicarbonate (pH 7.2) buffer
Red-Sepha equilibrated with the same buffer
After passing through a rose column and washing the column with the same buffer,
The concentration of ammonium thiocyanate was increased linearly to 1.0M.
, And the adsorbed protein was eluted. 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,
It shows one protein band, and both have a molecular weight of about 35.00.
It was 0. Collect each protein peak and add 0.1
After dialysis against M ammonium bicarbonate (pH 7.5),
Each was lyophilized. Purified t-PA and obtained by the above method
Each of the proteins obtained is a gas phase protein sequencer.
-(Applied Biosystems)
Sequencing was performed. For t-PAI, G
ly-Ala-Arg-Ser-Tyr-Gln
The amino acid sequence of 38 residues was determined. Non-adsorbed fraction tongue
The amino acid sequence of Park is Ile-Lys-Gly-Gly.
-Leu-
For Ser-Asn-Arg-Val-Glu-T
yr-Cys-Trp-Cys-Asn-Ser-Gl
The amino acid sequence of y-Arg- was determined. This adsorption tank
The N-terminal Ser of the park is the 31st from the N-terminal of t-PA.
Was confirmed. As a result, the probe
The optimal region of Tyr-Cys-Trp-Cys-Asn
And Matches the DNA sequence encoding this amino acid sequence
Complementary eight 14-mer 5'-
dTTnCACCAnCAnTA (n is A or G
Is chemically synthesized by the solid-phase phosphotriester method.
(Reference 8). 5) Bacteria containing t-PA cDNA sequence
Isolation and identification of clones Eight kinds of 14 nucleotides (14me) synthesized in section 4)
r) by T4-polynucleotide kinase
The 5 'end³²Labeled with P. 6 x SSC (1 x SS
C: 0.15M NaCl, 0.015M Nato succinate
Lium pH 7.0), 10 × Denhardt solution
(1 × Denhardt solution; 0.02% bovine serum al
Bumin (fraction V), 0.02% polyvinylpyrrolidone
(3.6 × 10 ^Three~ 4 × 10^FourDalton), 0.02%
Ficoll (4 × 10^FiveDalton)), 0.1% SD
S, containing 100 μg / ml sonicated salmon sperm DNA
In the solution, use the colony library prepared in C-3).
Prehybridize the containing filter for 2 hours at room temperature
Was. Then 6 × SSC, 10 × Denhardt, and 5 ×
× 10 ⁶Contains labeled probe at a concentration of count / min ml
Hybridized in solution overnight at room temperature. Then fill
At room temperature in 6 × SSC and 0.1% SDS solution
Wash three times for 5 minutes, then in the same solution at 30 ° C
Washing was repeated three times for 5 minutes. Clean the filter
After air drying, Dupont Kronex Lightnin
G plus (Dupont Cronex Light)
Ning Plus) using an intensifying screen and X-ray
The film was exposed to a film (Kodak XR-5) for 16 hours.
14 that showed a positive hybridization reaction
Barnboim and Dory's method from individual colonies (literature
Plasmid DNA was isolated by the modification of 9). The Plasmi
The base sequence of the cDNA insert using Maxam
It was determined by the Gilbert method (Reference 10). As a result,
Amino acid sequence determined from the DNA base sequence of
Column and amino acid sequence of purified t-PA identified in section -4)
By comparison with the column, the clone encodes t-PA
It was found to contain cDNA. This c is shown in FIG.
1 shows the nucleotide sequence of a DNA insert. This insert is
It has a length of 2170 bP and an
Includes pun reading frame, 76bP
5 'untranslated region and 3' untranslated region of 408bP
Contains. In addition, this DNA sequence was
DNA sequence encoding t-PA isolated from a strain
Compared to (Reference 2), 3 places in the 5 'untranslated region
One place in the region encoding t-PA protein, 3 'untranslated
There were 5 differences in the area. 6) t-PA inheritance using animal cells as a host
Expression of offspring 6-1) Mouse metallothionein (MMT) promoter
Expression of t-PA using 6-1-1) Preparation of plasmid pMT-1 In addition to the procedure shown in FIG. 1 (FIG. 1),
A mid was made. First, plasmid p was cloned from clone T-1.
T-1 was isolated. GC salt produced during cDNA synthesis
25 μg pT for the purpose of removing the base pair duplex
-1 was cut with Hind III, and then 0.4 U of Bal3
The reaction was performed at 37 ° C. for 2 minutes. Next, Hind III linker
Was ligated with T4 DNA ligase, followed by Hind III
Cleavage with BamHI, DNA fragment containing t-PA gene
The pieces were separated and recovered by polyacrylamide gel electrophoresis.
On the other hand, pUC13 was cut with Hind III and BamHI.
After that, about 2700 bp by low melting point agarose gel electrophoresis
The DNA fragment was separated and collected. Prepare this DNA fragment first
The DNA fragment containing the t-PA gene was ligated with T4 DNA
After binding with each otherE. FIG. coli  HB101
Was transformed. The resulting ampicillin-resistant colony
Plasmids were prepared from 10 of the
The DNA base sequence of the 5 'untranslated region was determined. Claw
Plasmid pT406 prepared from T-406
It contained a 9 bp 5 'untranslated region. 20 μg pT
406 was cut with Hind III and BamHI,
Lyramide gel electrophoresis contains about 2200 bP gene.
DNA fragments were separated and recovered. On the other hand, 10μg plus
Mid pSV2-dhfr (ATCC 37146) was converted to H
Cleavage with indIII and BamHI to remove the dhfr gene
The approximately 4200 bp DNA fragment was converted to a low melting point agarose gel.
And collected by electrophoresis. 0.2 μg of this DNA fragment
And about 2200 bP containing the t-PA gene isolated earlier
0.1 μg of fragments were ligated to each other with T4 DNA ligase.
After lettingE. FIG. coli  HB101 was transformed and
Picillin resistant colonies were obtained. From these colonies
A small amount of plasmid is isolated and its restriction enzyme
Clone containing the desired plasmid pMT-1
Identified. 6-1-2) Preparation of plasmid pMT-2 Plasmids were cloned from clones having plasmid pMT-1.
After large isolation, 10 μg of pMT-1 was replaced with HindII.
I and BamHI cut, low melting point agarose gel electrophoresis
Poly (A) signal of t-PA gene and SV40
And a DNA fragment of about 3 KbP containing the DNA was separated and recovered. on the other hand,
Plasmid pdBPV-MMTneo (342-12)
(Reference 22) to PML (Reference 23) and MMT Promo
A DNA fragment containing the protein was prepared. 10 μg of pdBPV
-MMTneo (342-12) was cut with BglII
Then, 5 units of Klenov DNA polymerase I (K
lenpw) fragments, as well as 0.1 mM dATP each;
Add dCTP, dGTP and TTP and add 10
Incubate for 1 minute to make the sticky ends blunt. H
Enol extraction, chloroform treatment, ethanol precipitation
After purification, 0.05 μg of Hind III Lincoln was added.
Was added and ligated with T4 DNA ligase. Next, Bam
PML cut with HI and partially cut with HindIII
And a fragment containing the MMT promoter and low melting point agarose
And collected by electrophoresis. This DNA fragment 0.05μ
g and the previously prepared t-PA gene and SV40 poly
(A) 0.04 μm of a 3 Kbp DNA fragment containing a signal
After binding to each other with g0T4 DNA ligase,E. FIG. c
oli  HB101 was transformed and ampicillin-resistant
Colonies were obtained. Plasmids are reduced from these colonies.
Amount of the plasmid, and
Clone containing the desired plasmid pMT-2.
Specified. 6-1-3) Preparation of plasmid pMT-3 Plasmids were cloned from clones containing plasmid pMT-2.
After isolation in large quantities, cut with BamHI followed by calf intestine
Derived alkaline phosphatase (hereinafter abbreviated as CIP)
To perform a dephosphorylation reaction at the 5 'end. on the other hand,
pdBPV-1 (142-6) (ATCC 3713
4) After cutting with BamHI, about 8 Kbp of BPV
_100TSeparation of DNA fragments by low melting point agarose gel electrophoresis
Collected. Next, 0.05 μg of BPV_100TDNA fragment
And pMT with BamHI digestion and CIP treatment
20.02 μg ligated together with T4dna ligase
After lettingE. FIG. coli  HB101 was transformed and
A colony resistant to ampicillin was obtained. These colonies
A small amount of plasminogen and isolate the plasmid.
The target plasmid pMT is obtained from the restriction enzyme cleavage pattern.
A clone with -3 was identified. 6-1-4) Preparation of plasmid pMT-4 Plasmid pMT-2 was partially cut with SalI.
Then, CIP treatment is performed, and Klenov DNA polymerase
The cohesive ends of the fragments were made blunt. This DNA fragment is
And On the other hand, PdBPV-MMTneo (34
2-12) was digested with EcoRI and BamHI. next
Blunt cohesive ends with Klenov fragment of DNA polymerase I
After changing to the end, low melting point agarose gel electrophoresis
Separation and recovery of DNA fragment containing 4Kbp nor gene
did. 0.05 μg of this dna fragment and the vector prepared above
Ligated to each other with T4 DNA ligase.
After lettingE. FIG. coli  HB101 was transformed. Less than
Carry the desired plasmid pMT-4 in the same manner as in the previous section.
Clones were identified. 6-1-5) Preparation of plasmid pMT-5 After cutting plasmid pMT-4 with BamHI, CIP
Processed. 0.05 μg of this DNA fragment and 6-1-3)
BPV prepared in section_100T0.05 μg of the DNA fragment was added to T4
After binding to each other with DNA ligase,E. FIG. coli
HB101 was transformed. The purpose is the same as in the previous section
A clone having the plasmid pMT-5 was identified. 6-1-6) Project having an MMT promoter
Introduction of Rasmid into Animal Cells and Expression of t-PA Prepared from 6-1-2) to 6-1-5)
For expression of t-PA having isolated MMT promoter
Was introduced into animal cells. Graham and fans
Graham, Van der Eb
Method (Reference 16) is a modification of the calcium phosphate precipitation method.
went. That is, 2 × HBS (50 mM Hepes
(PH 7.1), 280 mM NaCl), 1.4 mM
NaH_TwoPO_FourWhile inhaling sterile air into the solution containing
50 mM CaCl_TwoOf DNA solution containing
NA-CaPO_FourA precipitate was prepared. 20 minutes after dripping
After this, suspend the coprecipitate well with a pipette.
It was dropped on the cultured host cells. 37 ° C CO for several hours_Two
After culturing in an incubator, the medium was removed and 15% Gl
ycerol and a solution containing 2 × HBS were added, and the mixture was added at 37 ° C.
For 3 minutes. After removing this solution, add fresh medium
And CO for 2 days_TwoThe culture was performed in an incubator. Change
The medium was replaced with a fresh medium for 1 week. in this case
Uses the neo resistance gene as a selectable marker for transformed strains.
Therefore, a medium containing the antibiotic G418 was used. Next
After removing the land, the method of Jones et al.
According to dedication 24), the casein agar layer should be used instead of fibrin.
This was used to overlay the cells and cultured overnight. Casei
T-PA producing strain showing Tomei's melting zone
After culturing each clone individually,
The t-PA activity of Qing was measured to obtain a t-PA high producing strain. So
A part of the results is shown in Table 2 (Table 2). Also this high school student
The production strain was 1 μM Cd²⁺Or 50 μM Zn²⁺Medium containing
Table 1 also shows the t-PA activity of the culture supernatant cultured in the above. This
As a result, clone MT-325 exhibited the highest t-PA activity.
Shows, Cd²⁺Or Zn²⁺110 units without adding
/ Ml / day, Cd²⁺Or Zn²⁺305 or 3
10 units / ml / day of t-PA were produced. [0022] [Table 2]6-2) Human metallothionein (hMT) promoter
Expression of t-PA using 6-2-1) Preparation of plasmid phT-1 As shown in FIG. 3 (FIG. 3), the hMT promoter was used.
The used plasmid was prepared. First, the plasmid pMTS
VPA (University of South America)
rnCaliformia, School of Med
icine M. Obtained from Dr. Karin) XbaI
After cleavage with DNA, the Klenov fragment of DNA polymerase I is
Used to make the sticky ends blunt. This DNA fragment contains H
Ind III linker ligated with T4 DNA ligase
After that, it was cut with Hind III. Low melting point agarose sedge
Approximately 840 bp of hMT promoter
The NA fragment was separated and recovered. On the other hand, plasmid pMT-2
Was cut with Hind III and subjected to CIP treatment.
MM from plasmid pTM-2 by GALOS gel electrophoresis
A DNA fragment of about 5.5 bp excluding the T promoter was separated.
Separated and collected. This DNA fragment and the previously prepared hMT pro
DNA fragments with motors are separated by T4 DNA ligase
Combined withE. FIG. coli  HB101 was transformed.
Plasmids were isolated from the resulting ampicillin-resistant colonies.
Isolate a small amount and pattern the plasmid with restriction enzymes.
Clone containing the desired plasmid phT-1
Identified. 6-2-2) Preparation of plasmid phT-2 After using pMT-4 instead of plasmid pMT-2
Other than the above, the same method as that for preparing the plasmid phT-1 was used.
Prepared. 6-2-3) Preparation of plasmid phT-3 Plasmid phT-1 was cut with BamHI and treated with CIP.
I understood. When preparing this DNA fragment and plasmid pMT-3
About 8 kbp BPV prepared in_100TT4D DNA fragment
Bind each other with NA ligase,E. FIG. coli  HB10
1 was transformed. Hereinafter, the same method as in section 6-2-1)
Identified a clone having phT-3. 6-2-4) Preparation of plasmid phT-4 After using phT-2 instead of plasmid phT-1
Other than the above, the same method as that for preparing the plasmid phT-3
Prepared. [0025] [Table 3] 6-2-5) Projection having nMT promoter
Introduction of Rasmid into Animal Cells and Expression of t-PA Prepared in the above 6-2-1) to 6-2-4)
For expression of t-PA having hMT promoter
Was introduced into animal cells. Introduction method and t-PA high producing strain
Was performed in the same manner as in the paragraph 6-1-6).
Was. Part of the results are shown in Table 3 (Table 3). Also,
This high-producing strain was converted to 1 μM Cd²⁺Or 50 μM Zn²⁺or
Is 1 μM dexamethasone
e) The t-PA activity of the culture supernatant cultured in the medium containing
As shown. As a result, clone hT-382 was the best
shows t-PA activity and Cd²⁺, Zn ²⁺Or dexamethasazo
105 units / ml day without adding Cd²⁺, Z
n²⁺Or dexamethasone when added 330-340
Unit / ml / day of t-PA was produced. References 1. D. C. Rijken and D.S. Colle
n, J. et al. BiolChem. ,256  p. 7035
(1981) 2. JP-A-59-42321 3. E. FIG. L. Wilsonet al, Cancer
Res. ,40p. 933 (1980) 4. A. Granelli-Piperno and
E. FIG. Reich, J .; Exp. Med. ,148  p.
223 (1978) 5. H. Teraoka, M .; Tamaki, K .; Ta
Naka, protein, nucleic acid, enzyme26  p. 602 (1
981) 6. U. Gubler and B.S. J. Hoffma
n, Gene25p. 263 (1983) 7. J. P. Gergen et al Nuclei
c Acids Res. ,7  p. 2115 (197
9) 8. Crea et al, Nucleic Acid
s Res. ,8p. 2331 (1980) 9. H. C. Birnboim and J.M. Dol
y, Nucleic Acids Res. ,7  p. 1
513 (1979) 10. A. M. Maxam and W.S. Gilber
t Proc. Natl. Acad. Sci. U. S.
A74  p. 560 (1977) 11. W. R. Earle, J. et al. Natl. Cance
r Inst.4p. 165 (1943) 12. S. Kit et al, Exp. Cell. R
es. ,31  p. 297 (1963) 13. G. FIG. Urlaub, L .; A. Chasin Pr
oc. Natl. Acad. Sci. U. S. A. ,7
7  p. 4216 (1980) 14． N. Nakano Tohoku, J .; Exp.
Med.88p. 69 (1966) 15. F. L. Graham and A. J Van
  der Eb, Virology52  p. 546
(1978) 16. A. Graessman et al, Meth
ods in Enzymology65  p. 816
(1980) Academic Press, New
  York 17． W. Schaffner Proc. Natl.
Acad Sci. USA,77  p. 2163 (1
980) 18. H. Potter et al Proc. Na
tl. Acad. Sci. USA,81  p. 716
1 (1984) 19. P. Sheldrick et al, Pro
c. Natl. Acad. Sci. USA,70
  p. 3621 (1973) 20. T. Maniatis et al, Mole
cultural Cloning, pp. 197-198
Cold SpringHarbor Laborat
ory, New York (1982) 21. Same as pp. 204-205 22. M. Law et al, Molecular
and CelluarBiology,3  p. 21
10 (1983) 23. M. Lusky and M.S. Botchan,
  Nature293p. 79 (1981) 24. P. Jones et al, Cell,5
  p. 323 (1975) [0028] [Sequence list] SEQ ID NO: 1 Sequence length: 1590 Sequence type: nucleic acid Number of chains: 2 chains Topology: linear Sequence type: cDNA to genomic DNA origin Organism name: human (homo sapiens) Tissue type: fetal epithelium Array features 1-1590 E mat peptide Array 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 GCCTCCCGTG 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 TCTGAACGAT 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 chains: 2 chains Topology: linear Sequence type: cDNA to genomic DNA origin Organism name: human (homo sapiens) Tissue type: fetal epithelium Array features 1-76 E 5'UTR 77-1762 E CDS 77-172 E sig peptide 173-1762 E mat peptide 1763-1762 E 3'UTR Array CTAGGGCTGG AGAGAAAACC TCTGCGAGGA AAGGGAAGGA GCAAGCCGTG AATTTAAGGG 60 ACGCTGTGAA GCAATCATGG ATGCAATGAA GAGAGGGCTC TGCTGTGTGC 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 CGGAGTGTGA 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 THE DRAWINGS FIG. 1 is a diagram showing the procedure for preparing plasmid pMT-1 shown in Example 6-1-1). FIG. 2 is a diagram showing a procedure for preparing a plasmid phT-1 shown in Example 6-2-1).

Continuation of the front page Examiner Midori Tominaga (56) References JP-A-59-42321 (JP, A) Proc. Natl. Acad. Sc i. USA, 81 (17) (1984), p. 5355-5359 FEBS LETTERS, 189 (1) (1985), p. 145-149 Proc. Natl. Acad. Sc i. USA, 82 (13) (1985), pp. 4326-4330 Mol. Cell. Biol. , 5 (6) (1985) p. 139-1357 GENE (AMST), 31 (1-3) (1984), p. 147-154 (58) Fields investigated (Int. Cl. ⁶ , DB name) C12N 15/00-15/90 C12N 9/64 BIOSIS (DIALOG) EPAT (QUESTEL) WPI (DIALOG)

Claims (1)

(57) [Claims] D which encodes a mouse metallothionein promoter or a human metallothionein promoter, and a human tissue plasminogen activator derived from human normal cells consisting of 530 amino acids whose N-terminus starts with glycine
An NA sequence whose DNA sequence corresponds to SEQ ID NO: 1 in the sequence listing
A DNA sequence encoding the tissue plasminogen activator, wherein the DNA sequence is operably linked to the promoter and is capable of autonomously replicating in animal cells. A method for producing human tissue plasminogen activator derived from normal human cells using mouse C127I cells transformed with a vector. 2. D which encodes a mouse metallothionein promoter or a human metallothionein promoter, and a human tissue plasminogen activator derived from human normal cells consisting of 530 amino acids whose N-terminus starts with glycine
An NA sequence whose DNA sequence corresponds to SEQ ID NO: 1 in the sequence listing
A DNA sequence encoding the tissue plasminogen activator, wherein the DNA sequence is operably linked to the promoter and is capable of autonomously replicating in animal cells. A human normal cell-derived human tissue plasminogen activator produced by a method for producing a human normal cell-derived human tissue plasminogen activator using a mouse C127I cell transformed with a vector.