JPS6188879A - Plasmid vector, its preparation and use - Google Patents

Abstract

PURPOSE:To provide a plasmid vector having a specific promoter dimer, and capable of expressing the inserted DNA in a culture animal cell irrespective of the insertion direction of the exogeneous DNA. CONSTITUTION:A plasmid containing SV40 early promoter and replication initiation point is digested with a restriction enzyme to obtain a DNA fragment having blunt end at the downstream end of the promoter. The fragment is introduced into Escherichia coli to obtain a transformed strain containing said DNA fragment. pKCR(Hpa) is separated from the E.coli, and a promoter DNA fragment is separated therefrom. Two promoter DNA fragments are bonded with an adaptor DNA. The plasmid pML-RIIG is modified to produce a vector for the insertion of the promoter dimer, and the early promoter is introduced into the vector to obtain a plasmid pDE-1.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a novel plasmid vector using recombinant DNA techniques, and its production and use.

Conventional technology, such as lymphinization, is used to analyze all messenger RNA (mR
mRNA Δ encoding a specific phospho-7okine in NA)
If the content is extremely low, use the known pheasant formation translation assay method (hereinafter referred to as HT assay method) [e.g.
ord et al., Nucleic Ac1d
SRes, L 2039-2053 (1978)
;Taniguchi at Ja,.

Proc, Jpn, ^cad,, MM3-, 464
-469 (1979): Nagata et Dan...
Nature, +1316-320 (1980)]
It is extremely difficult to accurately clone cDNA. In addition, a method using a DNA blow j synthesized based on the primary structure of a protein [e.g.
an I upper n, Proc, Natl, 8c
ad, Sci,, W, 5036-5040 (+
979); allace e-engineering 3J,,
Nucleic 8cidsRes,, L 8
79-894 (1981)] cannot be applied to substances whose primary protein structure is unknown. As a way to overcome these difficulties, cDNA can be linked to specific promoter DNA.
It has been considered to directly measure the expression of the cDNA by cloning using a vector containing the fragment, thereby searching and identifying the clone of interest. Various vectors for this purpose have already been proposed. [Example, T
aniguchietn, , Nature, , 12
．． 305-310 (1983): Okayamat
t, al,, Mat, Ce11. [l1ol
,, 2. 280-289 (1983)].

In this case, the vector used is mainly derived from the S40 virus, but unless the cDNA is inserted downstream of the promoter in the correct direction and the cDNA contains the entire coding sequence, it is difficult to express the cDNA. It is.

Problems to be Solved by the Invention The purpose of the present invention is to
The object of the present invention is to provide a plasmid vector capable of efficiently expressing cDNA in mammalian cells and microbial cells, regardless of the direction of insertion of NA, and a method for producing and using the same.

Means for Solving the Problem In order to achieve this object, the present invention is characterized in that bromo has promoter-dimers which are linked to face each other via mammalian-derived adapter DNA. provides a plasmid vector for

The plasmid vector according to the invention is facilitated by the method of bromo-mediated ligation to form a promoter dimer, which is then inserted into the plasmid.

Next, the present invention provides a method for producing a recombinant plasmid, which comprises a step of removing the adapter DNA of the plasmid vector according to the present invention using a restriction enzyme and inserting a gene involved in the biosynthesis of a desired physiologically active substance instead. be done.

Furthermore, the present invention provides a gene cloning method comprising the steps of introducing the above-mentioned recombinant plasmid into cells derived from a microorganism or mammal and culturing the cells in a medium.

Furthermore, a desired physiologically active substance can be efficiently produced by culturing the above microorganism or mammalian-derived cells in a medium and collecting the physiologically active substance produced and accumulated in the culture.

Plasmid vector according to the present invention (hereinafter referred to as plasmid p
DE-1) and the recombinant plasmid (hereinafter referred to as plasmid pDE-2) have the following characteristics.

(1,) Both plasmids are pML CLu5k
y & []otchan.

Nature 293.79-81 (1981)] as a backbone, there are no sequences that would interfere with replication in cultured animals, making it an efficient DNA
and early promoter
A pair of DNA fragments containing the adapter DNA face each other with both ends of the adapter DNA in between. This adapter DNA is conjugated with, for example, Hpa I (for pDE-1) or EcoRI.
(in case of pDE-2) and replaced with cD
If you insert NA, you can change the direction of the cDNA (orientat
ion) is expected to be expressed regardless of the Also, cDN
Vectors in which A was not inserted are self-ligated (Self
-ligation) for example 5V40DN
A sequence has a direct palindromic structure (palindromic 5e
This has the advantage that it cannot be expanded with E. coli and the background can be kept low.

(3) Both plasmids have a pair of DNA fragments with a replication origin, so CO8 cells [Gluzman.

In T antigen-positive cells such as Ce1l 23.175-182 (+981)], the gene ffi (gene dosa
ge) and enhanced gene expression are expected.

(4) As shown in Figure 5, the plasmid pDE-2 is made by preliminarily preparing the full-length cDNA by sucrose density gradient centrifugation, agarose electrophoresis, etc.
-fan); Lh) and clone it. Furthermore, by connecting CcoRI linkers to both ends of the cDNA and inserting it into a vector, the cloned cDNA can be directly inserted into the vector. In summary, the method of the present invention for cutting out the vector ~ consists of the following steps.

(11) Preparation of plasmid pKCR (Hpa) Digest the plasmid containing the SV40 early bromocouple and replication origin with an appropriate restriction enzyme to create a DNA fragment with a blunt end downstream of the promoter. to create a transformed strain containing the above DNA fragment.

(2) Collect pKCR (Hpa) from E. coli and isolate a promoter DNA fragment from it.

(3) Two promoter DNA fragments and adapter DNA
Combine with .

(4) Modify plasmid pML-RIIG to create a vector for inserting 21 promoters.

(5) Introduce the early promoter into the vector to obtain plasmid pDE-1.

(6) E c o Rl81Ui of plasmid vector
Digestion of li-1 DNA with restriction enzymes is usually 0.1~
100μg of DNA into 2-22-2O0 preferably 10
~40mM) of Tris(hydroquinemethyl)aminometh-7-HCfl (hereinafter referred to as Tris-HCj!) (
pH 6.0-9.5, preferably pH 7.0-8.0
), 0.1 to 300 units of restriction enzyme (preferably 1 to 3 units of enzyme S per 1 μg of DNA) in M g CR2 of 1-150 mM NaC1,2-22-2O, preferably 5-10 mM). ) at 18 to 42°C (preferably 32 to 38°C) for 15 minutes to 30 hours. The reaction is usually stopped by applying VV at 55-75°C (preferably 63-70°C) for 5-30 minutes, but the restriction enzyme can be inactivated with a reagent such as phenol or soethylbirocarbonate. A method of serving can also be used.

Synthetic oligonucleotides were prepared using the diethyl phosphate method (H,G,
Khorana et Ll, :J, Mat.
&iol, , fl, 209 (1972)), triethyl phosphate method [R, Crea et al.
: Proc, Natl, 8cad,
Sci, Il, S, A, Q.

5765 (+978>) or phosphite method [-+, D! "Jatteucci Dan" top: J,
Am, Chem, Soc, ill, 3185
(+981) ].

To phosphorylate the synthesized oligonucleotide, 2-
22-2O0 preferably 10-10-7O Tri
5-HCR (pH 6,0-9,5, preferably pH',
, o-8,0), 3-20mM (preferably 4-14
-1O Vj g Cj! 2. Carry out the reaction using 0.1-100 units of T4 polynucleotide kinase in 1-10 mM dithiothreitol at 20-40°C (preferably 35-38°C) for 5 minutes to 2 hours. .

When binding DNA fragments, 2 to 200mM (preferably 10.-70mM) of Tr+5-HCf (pH 6,
0-9.5, preferably pH 7.0-8.0), 2-
22-2O (preferably 5-10mM) MgCl2.0
．． 1-10mM (preferably 0.5-2mM) AT
P, 15 at 1-37°C (preferably 3-20°C) using 0,1-10 units of T4 DNA ligase in 1-50mM (preferably 5-15mM) dithiothreitol.
The binding reaction is carried out for minutes to 72 hours (preferably 2 to 20 hours).

Fill-in reaction of DNA ends is performed from 1 to
50mM Tris-acetic acid (pH 6,0-9,5), 1
0-100mM potassium acetate, 1-10mM dithiothreitol, 1-20mM magnesium acetate and 0.1-10mM dATP each.

dCTPSdGTPOyoU d'FT P ChuteT
It is carried out using 0.1 to 20 units of ID NA Polymer 7 at 1 to 40°C for 10 to 60 minutes.

DNAI! Purification of fr fragments, recombinant plasmids, etc. is performed using known agarose gel electrophoresis methods such as CL and W+esla.
nder:Analytical Biochemistry
try 98.305 (1979) by E.

Transformation can be carried out, for example, as in the Examples below (N.

Cohen et al.'s method CProc, Natl, Ac
ad, Sci,, L! S^69, 2110 (197
2)]. The transformed strain can be used as an ampicillin-resistant strain.

As a host microorganism, Escherichia coli (for example, Escherichia coli
A derivative of strain HBIOI or C3R603) is used.

As host animal cells, cell lines of clear-breasted animals such as mice, rats, guinea pigs, rabbits, monkeys, sheep, goats, horses, and cows are used. As a specific preferred example, monkey CO8 cells are used.

Isolation of plasmids from microorganisms can be carried out, for example, in 14. as in the following example. C., Birnboim et al.: Nuc.
leicAcids Research 7.1513
(+979).

Actual bh example 1 (1) Production of plasmid pKCR(llpa) (
1st [21j plasmid pK CRC Flare,
K, eL aj.

Proc, Na1l, Acad, S
ci,, 78. 1527-1531 (198
] 10 μg l OmM Tr + 5-1-
ICI! (1)II7.5), 100mM Na(1
! , 5mM V+gCI2 and lOf'-position Ha
The mixture was reacted in 50 μβ of a solution containing m I-II at 37° C. for 2 hours. The reaction solution was extracted with phenol and chloroform, and then precipitated with ethanol. Precipitate 33
mM Tris, -acetic acid (pH 7,9), 66mM potassium chlorate, 5mM nothiothreitol, 10mM magunium acetate, 0.5mM each dATP, dCT
In a reaction 1100 μβ consisting of P, dGTP and dTTP, and T4 DNA polymerase at position 12.5 MA,
Incubate at 37°C for 20 minutes. The reaction solution was extracted with phenol and chloroform, and then precipitated with ethanol. Thus, 03 μg of plasmid with the BamHI cut end turned into a blunt end.
passed away. This was mixed with 66mM Trys-HCj!
(pH 7,6), 10mM dithiothreitol, 1mM
ATP, 7mM Mg (L12.3, 5 x I
O-'0.0. Incubate overnight at 5° C. in 30 μβ of a reaction solution consisting of a phosphorylated Hpa 1 linker and a 12-unit DNA ligase.

Using this reaction mixture, E. coli HBIOI was purified by conventional CP.
roc, Natl, Acad, Sc3.69.2
110 (1972)] according to the method described in
12 transformed strains (Ambi/Lin resistant, Δm pG)
It's tan. This transformed strain was transformed using a conventional method (MolecularC).
loning-A Laboratory Manua
A plasmid pKCR (Hpa) having l-Cold Sprang<Hpa1 site was obtained.

(2) Isolation of SV40 early promoter fragment from pKCR(Hpa): pKCR(Hpa)lIg was dissolved in 10mM Trys.
-H(1 (pH 7,5), 10mM NaCR16m
M MgCj! 2 and 500 units each of EcoRI
The mixture was incubated at 37°C for 2 hours in a reaction solution containing (manufactured by BRL) and lpaT (manufactured by Takara Shuzo). The reaction solution was extracted with phenol and chloroform, and then precipitated with ethanol.

Precipitate with 5-25% sugar, 50mM Tris-H
Using a solution containing CR (pH 7.5) and 1mM EDTΔ, incubate at 26 Kr with an Inkman 5W280-tar.
P, m, sucrose density gradient centrifugation at 4°C for 12 hours yielded approximately 50 promoter DNA fragments of approximately 400 bp.
μg were isolated from the vector. To further purify this DNA, 1% agarose gel electrophoresis was performed to obtain approximately 2
0 μg of EcoR1-Hpal DNA fragment was recovered.

(3) Preparation of 21 promoters 1 μg of EcoRI-Hpal DNAΔ fragment was added to 66 mM
Tri 5-HC1! (pH 7,6), 10mM
Dithiothreitol, 1mM Δ′'P17mVI
40μ of reaction solution containing WIgCRxb and 12 units of T4 DNA ligase! The cells were incubated overnight at 4°C.

After extraction with phenol and chloroform, DNA was recovered by ethanol precipitation. This DNA is E
This is DNA in which coRI-Hpal fragments are linked together.

This DNAjμg was transferred to lomM Tris-HC1!
(pH 7,5), 100mM Na (1!

6 m M M g CR2 and EcoRl 5
The mixture was incubated at 37° C. for 1 hour in 30 μβ of a reaction solution containing the unit. After phenol and chloroform extraction, DNA was recovered by ethanol precipitation. This DNA is E
It had a c o R1 truncated end.

This DNA was mixed with 33mM Tris-acetic acid (pH 7,
9), 66mM potassium acetate, 10mM magnesium acetate, 6mM dithiothreitol, each 0.5mM d
ATP, dCTP, dGTP and dTTP, and T
30μβ reaction solution containing 2.5 units of 4DNAΔ polymerase
The cells were incubated for 20 minutes at 37°C. After extraction with phenol and chloroform, DNA was recovered by ethanol precipitation. The DNA obtained here is
There are two types having the structures shown in FIG.

(4) Preparation of vector for promoter-dimer insertion Two plasmids pML-RIfG CLu5ky
& [1otchan.

Nature 293.79-81 (1981)) was modified to create a vector for promoter-dimer insertion.

In plasmid pML-RIIG, 5VII
Since a DNA fragment containing the origin of replication (ori) of the ODNΔ genome was present, the following reaction was performed to remove the fragment.

2μg pML-RITG, 10mM Trys, -
HCβ (pH 7,5), 6mM Mg ('1..10
40 μm of reaction solution containing 0 mM NaCR and 2 units of EcoRi was incubated at 37° C. for 2 hours.

From this reaction solution, 1% γ-garose gel electrophoresis was performed.
The EcoRIf fragment containing ori was removed, and DNA corresponding to the punk bone of the plasmid was recovered. This DNA 0
2 μg was added to 66 m MTri 5-HCA (pH
7,6), lomM dithiodithiostol, 1mM
DNA self-ligation (self
gat+on) Saota. Using the reaction solution, E. coli HBIO
I was transformed according to a conventional method. Transformed strain (8mp
') was cultured using a conventional method, and the plasmid was recovered.

One of the plasmids containing a single EcoR1 site and DNA encoding 8mpm from pML-RnG with the 5V40 DNA fragment removed was named pMLO-1.

(5) SV40 early promoter-2 derivatives on plasmid p
Preparation of plasmid pDE-1 introduced into Y/ILO-1 10 μg of plasmid pMLO-1 was added to 10 mM T
ris-HCA (pH 7,5), 6mM MgCj! 2,
100mM NaCf1 and 10? n position EcoR1
100μj of reaction solution containing! After incubating at 37° C. for 2 hours, the plasmid pMLO-] was cut with EcoRI. 5. After extracting the eluate with phenope chloroform, ethanol precipitation was performed to recover DNA.

1.5 μg of this DNA was added to 33 mM Trys-acetic acid (pH 7,9), 66 mM potassium acetate, 10 mM magnesium acetate, 6 mM dithiothreitol, 0.5 μg each.
m M dATP, dCTP, aGTP and dTT
The EcoRI end of the DNA was made into a plant end by incubation at 37° C. for 20 minutes in a reaction solution of 40 μβ containing P and 25 units of T 4 DNA polymerase. After the reaction solution was extracted with phenol and chloroform, ethanol precipitation was performed to recover DNA.

Approximately 1.5 μg of this DNA was added to 10mM Tris-
HCj! (pH 7,5), 7mM MgCA2.5
65°C in a 40μρ reaction solution containing 0mM NaCl, 6mM mercaptoethanol, and 0.5 units of alkaline phosphatase (manufactured by Boehringer Mannheim).
After incubation for 1 hour, the plasmid DNA was subjected to a dephosphorylation reaction.

This dephosphorylation reaction prevents ligation between the two ends of the plasmid. The reaction solution was extracted with phenol and chloroform, and precipitated with ethanol to obtain DNA.

0.22 μg of this DNA and 0.22 μg of the promoter dimer (3) were mixed with 66 mM Tris-H (
1 (pH 7,6), 10mM dithiothreitol, 1m
Both DNAs were incubated overnight at 4° C. in a 30 μβ reaction solution containing MATP, 7 mM MgCL, and 1.2 units of DNA IJ gauze to bind both DNAs.

The reaction mixture was used to transform Escherichia coli HBIOI by the method of (:ohen et al.).
Using Hpa I promoter DNA fragment as a probe,
in 5 itu hybridization/Yon method CGruns
tein & llogness, Proc,
Natl 8cadSci,, 70.233
0-2334 (1975)), promoter-2
We searched for plasmid clones in which the mer was inserted. As a result, plasmid pDE-1 as shown in FIG. 3 was obtained.

Plasmid pDE-1 is obtained by inserting an NA fragment into pMLO-1 as shown in FIG. 2B in (3). Although we screened several hundred colonies, (
As shown in Figure 2 in 3), no plasmid into which the NA fragment had been inserted was detected.

Escherichia coli containing plasmid pDE-1 was obtained from Escheri
chia coli εDE-1 (FERM BP-
623) and was deposited with the Institute of Microbial Technology (Feikokuken) of the Agency of Industrial Science and Technology on January 1, 1981.

Example 2 Preparation of plasmid pDE-2 by introducing EcoRI intervention site into plasmid pDE-1 A new E
The following reaction was performed to introduce a coRI site.

Plasmid pDE-1(7)I CJugI (1mM
Tri 5-HCl (pH'A5), 100mN
a Plasmid pDE-1 was cleaved with Hpa 1 by reacting at 37° C. for 1 hour in 50 μp of a reaction solution containing CR15m M M gCea and 10 units of Hpa I. A plasmid DNA fragment from which the adapter DNA had been removed was recovered from the reaction solution by 1% agarose gel electrophoresis. This plasmid DNA fragment was subjected to a dephosphorylation reaction as follows. That is, 3 μg of DNA is
DmM Tris-HCj! (pl(7,5), 7m
The cells were incubated at 65° C. for 1 hour in 40 μp of anti-bif&ml; containing M MgCL, 50 mM NaCl, 6 mM mercaptoethanol, and 1 volume of alkaline 7-osphatase. After extracting the reaction solution with alcohol and chloroform, ethanol precipitation was performed to recover the DNA.

This DNA was ligated with an EC0RI linker, human inter 7, and Ron-β gene DNA fragment.

1-Nawaji, 0.21 μg of plasmid DNA was added to 0.2 μg of plasmid DNA.
031μg EcoRl linker (manufactured by Takaraj Nishizo),
02 μg of human interferon-β gene DNA fragment (], 55 Kb JP-A-57-776511>, 66
mM Tri, 5-)ICJ' (pH 7,6), 10
mM dithiothreitol, 1mM ATP, 7mM
MgCj'2 and 1.2 units (7) T4DNAI
The mixture was incubated overnight at 4° C. in 30 μβ of a reaction solution containing J gauze. Using the reaction mixture, large lI! Bacteria HB101
was transformed according to a conventional method. The obtained transformed strain (8m
The plasmid pw) was isolated and analyzed, and the plasmid pDE-2 shown in FIG. 4 was obtained. Also, plasmid pD
Plasmid pDE-2i, which is a plasmid similar to E-2, but with the interferon gene inserted in the opposite direction, was also used in the same way (see above).The human interferon-β gene inserted into these plasmids is
plays the role of

Escherichia coli containing plasmid pDE-2 was obtained from Escheri
chla coli EDE-2 (FERl,! BP
-624) and was deposited with Koukoken on Z 1, 1988.

Example 3 Expression of interferon-β gene by plasmid pDE-2: Plasmids pDE-2 and pDE-2i obtained by the method of Example 2 were expressed in a conventional manner [Taniguchi at, L.
L.

Nature, 302.305-310 (1983)
) was introduced into monkey COS cultured cells and examined for interferon-βa transgenic expression. In both cases, interferon-β of 5,000 to 10,000 units/ml was introduced into CO8 cultured Kamiura cells. Secreted.

Effects of the Invention The plasmid of the present invention is very advantageous as an expression vector because the inserted DNA can be expressed in cultured animal cells regardless of the direction in which the foreign DNA is inserted.

[Brief explanation of the drawing]

FIG. 1 shows the construction process of plasmid pKcR (Hpa). FIG. 2 shows the structure of 21 SV40 promoters. A is adapter DNA when there is no adapter DNA, B is adapter DNA
This is the case when there is A. In the figure, the horizontal arrow +1 indicates the SV/In promoter, and the 3rd U21 is the plasmid pDE-
The structure of 1 is shown. Figure 4 shows the structure of plasmid pDE-2. FIG. 5 shows the engineering for cDNA glueing and expression using plasmid pDE-2. Patent applicant: Tsuyoshi Taniguchi Toshiyuki Shohamaoka Figure 2 A B Figure 3 pML bassophone No. 5

Claims (9)

[Claims] (1) A promoter dimer is formed by linking a pair of virus- or mammal-derived DNA fragments having a promoter and a DNA replication origin so that they face each other via microorganism- or mammal-derived adapter DNA. A plasmid vector comprising: (2) The plasmid vector according to claim 1, wherein the virus is SV40 or a retrovirus. (3) The plasmid vector according to claim 1, wherein the microorganism is Escherichia coli. (4) The perasmid vector according to claim 1, wherein the mammal is a mouse, rat, guinea pig, rabbit, goat, sheep, horse, cow, or monkey. (5) A pair of virus- or mammal-derived DNA fragments having a promoter and a DNA replication origin are linked to face each other via a microorganism- or mammal-derived adapter DNA.
A method for producing plasmid vectors, which consists of the steps of forming a vector and inserting it into a plasmid. (6) A recombinant plasmid comprising the step of removing the adapter DNA in the plasmid vector described in claim 1 using a restriction enzyme and incorporating a gene involved in the biosynthesis of a desired physiologically active substance instead. manufacturing method. (7) The method according to claim 6, wherein the physiologically active substance is human interferon. (8) A gene cloning method comprising the step of introducing the recombinant plasmid obtained by the method described in claim 6 into cells of microorganism or mammalian origin and culturing the cells to express the gene. . (9) The recombinant plasmid obtained by the method described in claim 6 is introduced into cells derived from microorganisms or mammals, and the cells are cultured in a medium to remove physiologically active substances accumulated in the culture. A method for producing physiologically active substances that consists of a collection process.