JPH0898690A - Production of gene bank using new cosmid vector - Google Patents

Abstract

PURPOSE: To provide a method for simply and efficiently obtaining a gene bank of extraneous DNA using a cosmid vector prepared by using a gene manipulation technique. CONSTITUTION: Various extraneous DNA fragments having 35-45Kbp lengths are inserted between a light arm and a left arm each having at least 3 cos of lambda phage or lambdoid phase having a same direction obtained by cleaving with two kinds of restriction enzymes and each having at least one or two cos, and the each fragment is changed into transduced phage particles by in vitro packaging and cloned into Escherichia in which lambda phase is lysogenized, and then induced, and the recombinant DNA is changed into transduced phage particles by vivo packaging to produce a gene bank of the extraneous DNA.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to gene manipulation technology,
More specifically, it relates to a method for producing a gene bank using a novel cosmid vector prepared by using a gene manipulation technique.

[0002]

2. Description of the Related Art The gene size of mammals including human is often over 20 kilobase pairs (hereinafter, kilobase pairs are abbreviated as Kbp). Since the size of the DNA that can be cloned into the widely used lambda vector at present is 20 Kbp, it is not possible to clone a gene larger than this in a functional form.

Lambda Fuerge cohesive en
a vector having d site (hereinafter abbreviated as cos),
That is, a cosmid vector can be used to clone a DNA having a size of 35 to 45 Kbp. Of the various vectors, the cosmid vector can clone the longest DNA, but it is difficult to prepare a long genomic DNA, the background of only the vector without insert is high, and the efficiency is not good. As a result, the use of cosmid vectors has not progressed. However, [Ishiyu-Hello It's and Burke (Is
h-Horowicz & Burke)] has succeeded in lowering the background value of only the vector and increasing the cloning efficiency by devising a vector having directionality by using phosphatase. Hollow It's and JF Burke:
Nucleic Acids Research (D.Ish-Ho
rowicz & J.M. F. Burke, Nuclei
c Acids Res. ), 9 , 2889 (198)
1)].

The method for producing a gene bank of genomic DNA by the Ishi-Horowitz & Burke method is as follows (for example, pJB- which is one of the cosmid vectors widely used at present). 8 as an example). pJB-
8 has a size of 5.44 Kbp and has a restriction enzyme BamHI,
It is a cosmid vector having one lambda cos cos which is cleaved at each of HindIII and Sa1I.

(1) pJB-8 is cleaved with HindIII to give a linear DNA, and then a HindIII fragment is inactivated by phosphatase treatment. Then, by cutting with BamHI, two DNAs were obtained.
A fragment was obtained, containing a large DNA fragment containing cos (5.4
Kbp) is isolated by agarose gel electrophoresis. The obtained cos-containing DNA fragment (5.4 Kbp)
However, it corresponds to the left arm of Lambda Fuaj.

(2) pJB-8 is cut with SalI to give a linear DNA, and then treated with phosphatase to inactivate the cut piece of SalI. Then, by cutting with BamHI, two DNAs were obtained.
A fragment was obtained, containing a small DNA fragment containing cos (2.3
4 Kbp) is isolated by agarose gel electrophoresis. The obtained DNA fragment containing cos (2.34 Kb
p) corresponds to the right arm of Lambda Fuge.

(3) For example, human genomic DNA is partially cleaved with a restriction enzyme Sau3A or MboI,
A DNA fragment with a size of 35 to 45 Kbp is fractionated. The DNA fragment having a size of 35 to 45 Kbp was inserted between the left arm and the right arm separately prepared in (1) and (2) with a ligation enzyme (T4-DNA ligase) to give a recombinant DN.
Prepare A. (4) A gene bank of human genomic DNA can be produced by converting the recombinant DNA into transduced phage particles by in vitro packaging and then infecting E. coli.

[0008]

The above is the description of Ishi-Horowicz & Burke.
The method of producing a gene bank of genomic DNA by the Burke method has the following four problems. First, p
JB-8 has only one cos, and the left arm and the right arm must be prepared separately. Second, DNA fragments containing cos must be separated by agarose gel electrophoresis for each of the left and right arms. Third, pJB-
8 has a left arm length of 5.4 Kbp and a right arm length of 2.34.
Since it is Kbp, and the lengths of the left arm and the right arm are different, 35
The probability of ligation to ~ 45 Kbp genomic DNA is also different,
The mixed ratio of left and right arms must be considered.
Fourthly, in the gene bank obtained by this production method, the recombinant DNA is replicated as a plasmid in Escherichia coli and separated and purified as a circular plasmid DNA (ccc-DNA) covalently closed from Escherichia coli. However, the recombinant DNA is used as a transduction phage particle to form a genomic DNA.
It is not possible to manufacture the gene bank of.

[0009]

In order to solve these problems, the present inventors have at least three lambda or lamdoid charge cos having the same direction, and have two types of restrictions. Use of this cosmid vector to prepare a new cosmid vector characterized in that the right arm and the left arm having at least one or two cos and having almost the same length can be equimolar and obtained by cutting with an enzyme. As a result, the method for producing a foreign DNA gene bank was completed.

The cosmid vector used in the present invention is
At least 3 c's with the same orientation in the same molecule
os is present and can be obtained by cutting with two kinds of restriction enzymes. Since the right arm and the left arm having at least one or two cos and having almost the same length can be equimolar, the right arm and the left arm are separated. It does not need to be prepared in advance and need not be separated by agarose gel electrophoresis. Furthermore, since the lengths of the right arm and the left arm are almost equal, the genome DN of 35 to 45 Kbp is
It may be considered that the degree of ligation to A by T4-DNA ligase is almost equal, and an equimolar mixture of the right arm and the left arm formed by cutting with two kinds of restriction enzymes is directly used for 35 to 45
It can be expected that half of the recombinant DNA can be transformed into transduction pharage particles when ligated to the Kbp genomic DNA. As described above, when the cosmid vector of the present invention is used, a DNA fragment of 35 to 45 Kbp can be obtained as a genomic DNA.
The gene bank A can be manufactured more easily and efficiently.

Further, the vector of the present invention comprises at least 3 coss having the same orientation in the same molecule.
Therefore, when the recombinant DNA is transformed into transduction pharage particles by in vitro packaging and infected with E. coli, the recombinant DNA contains two cos in E. coli.
Replicate as a plasmid DNA containing At this stage, it is a gene bank of foreign DNA as plasmid DNA, but when Escherichia coli in which lambda phage is lysogenized is used as a host, it is heated or irradiated with ultraviolet rays to induce it. The lysogenized lambda phage enters the lysis cycle, the lambda phage grows in E. coli, and finally the E. coli lyses and the lambda phage particles come out. At this time, the recombinant DNA, which is present in E. coli as a plasmid DNA having two cos, is also incorporated into the head portion of the lambda phage due to having two cos, so that in v
It is converted to transduced phage particles by iv packaging. In this way, a gene bank of foreign DNA can be produced as transduction phage particles.

In gene transfer into cultured mammalian cells, the method using chromosomes is (1) the efficiency and frequency of gene transfer are always 10 to 100 times higher than those using plasmid DNA or genomic DNA. , (2) It is excellent in that many stable transformant clones are obtained [Rewis, Doubly H, Sriniva Samp Earl: Stoke N and Simon Bitsch, El: Somatocel Geneetics (Lew)
is, W. H. Srinivasan, P .; R. , S
tokoe, N .; , & Siminovitch,
L ,: Somat, cell Genet. , 6 , 33
3, (1980)].

Perhaps this is related to the fact that DNA has a chromatin structure in which chromosomes are densely packed with chromatin proteins and is protected from the attack of deoxyribonuclease (DNase). In addition, the dense chromatin structure may facilitate the uptake into cells. However, DNA cannot be recombined with a chromosome as it is, and it is not easy to prepare a large amount of chromosomes necessary for experiments from cells. Now, paying attention to the structure of lambda phage particles, the lambda DNA is closely packed by the coat protein, and the situation is very similar to that of the chromosomal DNA encapsulated in the chromatin protein.

Further, using a lambda phage vector,
A method for preparing a gene bank as a lambda phage particle by DNA recombination has already been established. Ishiura et al.
Lambda vector Char incorporating the V-1 TK gene
Using the recombinant of on4A (lambda sphere particles),
When the transfer of HSV-1TK gene into Ltk ⁻ cells was examined, (1) the transfer efficiency was 10 ⁻⁵ , and naked D
NA is an order of magnitude higher than that of intact NA, but is one order of magnitude lower than that of chromosomal use. (2) Transformed cell colonies are larger than those obtained with DNA, and most of the clones are non-selective media. However, when it was kept stable, the expected result was obtained [Ishiura Em et al .: Morekiular Cellular Biology (Ishiura, M., et a.
l. , Mol, Cell. Biol. ), 2 , 607
(1982)].

However, the essential problem with the gene bank prepared with the lambda vector is that the maximum size of DNA that can be cloned into the lambda vector is 20 Kbp. That is, the gene cannot be cloned.
Therefore, the cosmid vector having at least 3 cos of the present invention is excellent in that it can clone a foreign DNA of 35 to 45 Kbp, and can produce a gene bank as a transduction phage particle, and thus it is great for gene transfer into cells. It seems to exert power.

[0016] which is one of the novel cosmid vector of the present invention, showing the ^{pTcosAp r / ori / cm r /} neo r in FIG. ^{pTcosAp r / ori / cm r /} neo
^r is cos3 having the same direction derived from pRH (-44) or pLH (+129) which is a cosmid vector.
Individual and [Miwa and Matsubara, Jean, Miwa &
Matsubara, Gene, 20 , 267 (198).
2)], a neomycin resistance gene derived from Tn903 (n
eo ^r) [Oka, Sugisaki and high waves, journal of Morekiyura Biology (Oka, S
ugisaki & Takanami, J. et al. Mol.
Biol. , 147 , 217 (1981)], pNT-.
Chloramphenicol resistance gene (cm ^r ) derived from 31
[Tomizawa and Ito Proceeding of National Academic Accidents of Sciences USA [Tomizawa & Itoh, Proc.
Natl. Acad. Sci. U. S. A. ), 78 ,
6096 (1981)], the ampicillin resistance gene from pBR-327 (Ap ^r) and origin of replication (ori)
[Soveron, Cavalrubias & Bo
Liver, Gene), 9 , 287 (1980)]. The size is 4.9 Kbp and the restriction enzyme Cl
It is cut at one place each by aI and BamHI.
The method for producing a gene bank of genomic DNA (foreign DNA) using this vector is as follows (FIG. 2).

(1) pDcosAp^r/ Ori / cm^r
/ Neo^rA linear D by cutting with ClaI
NA and then ClaI by phosphatase treatment
Inactivate the cut pieces of. Then cut with BamHI
Therefore, at least one cos
Prepares a right arm and a left arm with two equal lengths
It (2) Foreign DNA such as Sau3A or MboI
Digestion with limited digestion and digestion of DNA with a size of 35-45 Kbp
Prepare pieces. (3) 35-45 Kbp genomic DNA
The fragment prepared in (1) was used for T4-DN between the left and right arms.
Recombinant DNA is prepared by insertion with A ligase.
(4) Recombinant DNA in vitro packaging
Change into transduction phasic particles and melt the lambda pharge.
Infect the E. coli that has been prototyping. (5) This transduction
Body DNA is Ap derived from vector^r, Cm^rAnd ori
As it contains ampicillin or chloramphenicol
Recombination with two cos by selecting in
Select only Escherichia coli that has body DNA as a plasmid
You can make use of it. (6) Next, transformant
(E. coli) I said that it would be heated or irradiated with ultraviolet rays.
Lysis of lambda futage that had been lysogenized by inducing eel
By shifting to the cycle
Recombinant recombinant DNA by in vivo packaging
It can be transformed into transduced phage particles. This transduction
-One cos and Ap in the particle ^r, Ori, outside
Recombinant DNA consisting of native DNA is encapsulated. This
Gene van of genomic DNA (foreign DNA)
Can be produced as a transduction fare particle
It

Further, the present inventors have having a selection marker for mammalian cells, cosmid vectors for the purpose of gene transfer into mammalian cells, pTcosAp ^r /
^{ori / TK / cm r / neo} r ( Figure 3) was created.
^{pTcosAp r / ori / TK / cm} r / neo
^r is three cos having the same directionality derived from pRH (−44) or pLH (+129), neo ^r derived from Tn903, cm ^r derived from pNT-31, and pBR-327.
Derived from Ap ^r and ori and TK gene derived from pTK-4 [Ishiura Em et al., Molecular Cellular Biology, Ishiura, M. et al. , Et al. , Mo
l. Cell. Biol. , 2 , 607 (1982)]
Consists of.

This cosmid vector has the above-mentioned pTc.
Like the ^{osAp r / ori / cm r /} neo r, it can be produced of genomic DNA gene bank. That is, there are 3 cos with the same direction, and ClaI and Bam
By cutting with HI, the right arm and the left arm each having one or two cos can be equimolar, and BamHI
Recombinant DNA can be prepared by inserting genomic DNA having a size of 35 to 45 Kbp into the site. Then, this recombinant DNA can be converted into transduced phage particles by in vitro packaging and transferred to Escherichia coli (lambda lysogen is lysogenized). Alternatively, it can be obtained by selecting with chloramphenicol.

Next, a recombinant DNA having two coss was induced by inducing a trasformant (Escherichia coli).
Gene banks can be produced by conversion into transduced phagy particles by vivo packaging. Furthermore, since this transduction phage particle has a TK gene, it can be used as a marker for thymidine kinase (TK) activity when the gene is transferred into cultured mammalian cells so that the recombinant DNA derived from the vector can be obtained. There is an advantage that transfer determination can be performed at the level of cultured cells.

[0021] The present invention relates to a method for producing a gene bank using such a novel cosmid vector. Examples of cos used in the present invention include those derived from lambda or lambda. In particular,
The cos derived from lambda fragrance is mentioned as a preferable thing. Further, in order to produce a gene bank as a transduction phage particle with the cosmid vector of the present invention, it is sufficient if it exists in three cos vectors having the same orientation in the same molecule, but three or more cos. May be included in the vector.

The vector of the present invention has a right arm and a left arm each having at least one cos or two by cutting with two kinds of restriction enzymes. As a combination of these two kinds of restriction enzymes, Is PvuII and Bam
HI, PvuII and Bg1II, ClaI and BamHI, C
laI and BglII can be mentioned. Of course, other combinations can be considered, and the combination is not limited to these. In order to determine that the vector-derived DNA fragment has been inserted into the vector of the present invention, selection by drug resistance is generally performed. Therefore, DNA expressing drug resistance in the vector of the present invention
It is preferred that the fragment is integrated.

Examples of drug resistance include usual antibiotic resistance such as ampicillin resistance, tetracycline resistance, neomycin (kanamycin) resistance, or chloramphenicol resistance. pTcosAp ^r / ori / c
m ^r / neo ^r and, pTcosAp ^r / ori / TK
/ Cm ^r / ne o ^r has an ampicillin resistance gene, a chloramphenicol resistance gene and a neomycin resistance gene. Genomic DNA using the vector of the present invention
When a gene bank of (foreign DNA) is produced and transferred to a cultured cell of a mammal (gene bank), a specific physiological function is expressed in order to determine that a vector-derived DNA fragment has been inserted. It is preferred that the DNA fragment is integrated in the vector of the present invention.

Genes expressing a specific physiological function include aminoglycoside 3'-phosphotransferase (neo) gene [Oka, Sugisaki and Takanami, Jianal Morekiurer Biology (Ok
a, Sugisaki & Takanami, J .; M
ol. Biol. ), 147 , 217 (1981)],
Thymidine kinase (TK) gene [Wiggler, M. et al., Cell (Wigler, M. et al., Cel
l), 14 , 725 (1978), adenine phosphoribosyl transferase (APRT) gene [Wiggler, M et al., Proceedings of National
Academia of Sciences USA (W
igler, M .; , Et al. , Pro. Natl.
Acad. Sci. U. S. A. ), 76 , 1373
(1979)], hypoxanthine guanine phosphoribosyl trantransferase (HGPRT) gene [Graph, L.H.
al. , Somat. Cell. Genet. ),
5 , 1031 (1979)], xanthine guanine phosphoribosyl transferase (XGPRT or gpt).
Gene [Berg, P., et al., Molecular Cellular Biology (Berg, P., et al., Mol.
Cell. Biol. ), 1 , 449 (1981)] and the like. The novel cosmid vector pTc of the present invention
^{osAp r / ori / TK / cm} r / neo r has the TK gene.

Furthermore, in the vector of the present invention, Col
It is preferable to have a gene fraction having a replication function derived from EI. Examples of the foreign DNA used in the present invention include DNA derived from microorganisms such as bacteria, filamentous fungi, yeasts, higher animals and plants, various types of phages, and the like. In addition, the restriction enzyme (DNA
In order to join a DNA cohesive end generated by cleavage with an enzyme having the function of selectively cleaving a specific nucleotide sequence site of a fragment) or a restriction enzyme and another DNA fragment cohesive end having a complementary relationship therewith The enzyme to be used (ligase), the enzyme that makes DNA terminal ends that are not complementary to each other to make sticky ends complementary to each other, or the ligase that connects them is already well known, and many enzymes are known. It is commercially available and the methods using them are widely disclosed [Molecuyler Cloning Cold Spring Harbor Laboratory, Maniathes, Tay, etc. (Molecu
lar Cloning, Cold Spring H
arbor Laboratory, Maniati
S.T. , Et al. ), (1982)], they may be used.

Genomic DNA using the vector of the present invention
By creating a gene bank of (foreign DNA), it is possible to efficiently find a specific gene that expresses a useful substance. This particular gene is a bacterium,
There are DNAs derived from microorganisms such as filamentous fungi and yeasts, higher animals and plants, and various types of phages. Specific useful substances include, for example, various hormones, enzymes, and synthetic enzymes such as antibiotics and amino acids. .

Hereinafter, the present invention will be described in more detail with reference to examples. Example ^{1 pTcosAp r / ori / cm r} / neo
Preparation and preparation of ^r (FIGS. 4, 5 and 6) (a) Preparation and preparation of pHScos (1) Preparation 10 μg of pRH (−44), which is a cosmid vector,
Reaction mixture 100 μl [150 mM NaCl, 6 mM Tr
is-HCl (pH 7.9), 6 mM MgCl ₂ , 10
0 μg / ml BSA], 69 units of restriction enzyme Ba
It was completely digested by reacting with mHI (New England Biolabs) for 2 hours. After the reaction,
Extract with an equal volume of water-saturated phenol, concentrate the aqueous layer with secondary butanol, add 0.1 volumes of 3M sodium acetate and 2 volumes of ethanol to obtain DNA as a precipitate, and wash with 95% ethanol. After that, it was dried with a water-jet pump, and the precipitate was washed with 10 mM Tris-HCl (pH
8.0), an aqueous solution of 0.1 mM EDTA (hereinafter,
It was dissolved in 7 μl (abbreviated as 10 t / 0.1E) (˜1 μg / μl).

Next, 1 μl (to 1 μg) of this BamHI-digested DNA solution was added to 25 μl of the reaction mixture [50 mMT.
ris-HCl (pH 7.2), 10 mM MgSO ₄ ,
0.1 mM DTT, 50 μg / ml BSA, 80 μMd
ATP, 80μMdCTP, 80μMdGTP, 80μ
MdTTP] with 2 units of Klenow enzyme (Bo
Ehringer Mannheim) at 30 ° C. for 30 minutes, and a double-stranded chain D having complementary strands to each other.
The NA fragment (cohesive end) was changed to a double-stranded DNA fragment (blunt end) having both ends aligned. After completion of the reaction, the reaction solution was heat-treated at 65 ° C. for 10 minutes to inactivate the Klenow enzyme.

Next, 10 μl of this DNA solution (0.4 μl
g) and SalI linker [d (pG-G-T-C-G
-A-C-C)] 2 μg and 20 μl of the reaction mixture [50
mMTris-HCl (pH 7.4) 10 mM MgCl
₂ , 10 mM DTT, 1 mM spermidine, 1 mMAT
P, 100 μg / ml BSA] 6 units T4-D
Ligation was performed by reacting with NA ligase at 22 ° C. for 6 hours. Then, phenol extraction was performed to obtain DNA as an ethanol precipitate.

This DNA precipitate was mixed with 600 μl of the reaction mixture.
[150 mM NaCl, 6 mM Tris-HCl (pH
7.9), 6 mM MgCl ₂ , 6 mM DTT, 100 μ
g / ml BSA] 1000 units of restriction enzyme Sa1
I (Takara) was allowed to react at 37 ° C. for 4 hours for digestion and then applied to 0.8% preparative agarose gel for 1
Electrophoresis was carried out at 0 mA for 12 hours. The decomposed DNA fragment becomes visible by ethidium bromide staining and long-wavelength ultraviolet illumination. After cutting out a gel containing a DNA fragment (3.89 Kbp) consisting of cos, ori, and Ap,
5 times the volume of extract [0.5M NH ₄ OAc, 1 mM ED
In the presence of TA (pH 7.5): water-saturated phenol = 1: 1], the gel was ground to elute the DNA.

The eluate was concentrated with secondary butanol, and 0.1 times volume of 3M sodium acetate and 2 times volume of ethanol were added to obtain a DNA precipitate. This D
NA precipitation was reacted with 20 μl of reaction mixture [50 mM Tris-HC
1 (pH 7.4), 10 mM MgCl ₂ , 10 mM DT
T, 1 mM spermidine, 1 mM ATP, 100 μg /
mlBSA], and reacted with 6 units of T4-DNA ligase at 12 ° C. for 17.5 hours to prepare cos and ori,
The DNA fragment consisting of Ap ^r (3.89 Kbp) was cyclized. Then, phenol extraction was performed to obtain DNA as an ethanol precipitate. The precipitate was extracted with 0.1 M Tris-HCl.
It was dissolved in 20 μl of (pH 7.5) solution.

(2) Transformation 10 μl of this DNA solution was added to E. coli HB101 comp.
100 μl of ent cell (E. coli HB101 in logarithmic growth phase at 0-4 ° C. in 0.1 M CaCl ₂ solution
After treatment for 1 minute, 0.1MC of 1/10 volume of the original culture solution
resuspended in NaCl _2/14% glycerol solution, -70
Mix well) and store at 0 ° C for 10 minutes, then 37
After incubating at 0 ° C. for 5 minutes to incorporate DNA into E. coli, L-broth (1% Bactotrypton was added thereto).
e, 0.5% yeast extract, 0.5% NaCl, 0.1%
(Glucose) (2 ml) was added, and the mixture was shake-cultured at 37 ° C. for 1 hour. The cell suspension was spread on an L-plate (a plate containing 1% agar added to L-prose) containing 20 μg / ml of ampicillin, and cultured at 37 ° C. for 24 to 48 hours to obtain ampicillin-resistant clones. It was

3) Small amount culture and small amount DNA preparation Ap ^r clone was cloned in 2 ml of L-broth (Ap20).
The cells were allowed to grow by shaking overnight at 37 ° C. (containing μg / ml). 1.5 ml of the culture solution was placed in an Eppendorf tube, and the cells were collected by a desk centrifuge, and the cells were extracted with an extract [8% sucrose, 0.5% Triton X-100, 5
0 mM EDTA (pH 8.0), 10 mM Tris-H
Cl (pH 8.0)] 0.35 ml. Next, 25 μl of lysozyme solution [10 mg / ml, 10 m
MTris-HCl (pH 8.0)] was added and mixed, and then the tube was placed in boiling water for 40 seconds. Then, immediately put the tube in a centrifuge (21,000 rp at 4 ° C).
m, 15 minutes) to obtain a supernatant liquid, which was RNase
Solution A (20mg / ml, 10t / 0.1E) 0.5μ
l was added and the mixture was kept warm at 37 ° C. for 1 hour to decompose RNA. Then, after deproteinization by phenol extraction, DNA was obtained as an ethanol precipitate, and the precipitate was mixed with 10 t / 0.1E20 μl.
To obtain a plasmid DNA solution.

(4) Characterization of plasmid DNA Then, 1 μl or 2 μl of this plasmid DNA solution was added to the restriction enzymes HindIII, SalI, PvuII and PstI.
After digestion with, etc., 0.4-1.5% agarose gel electrophoresis [40 mM Tris, 20 mM sodium acetate, 2 mM EDTA (pH 8.15); 10 mA, 12
~ 24 hours]. The decomposed DNA fragment can be detected by ethidium bromide staining and long-wavelength ultraviolet irradiation.
In this way, by constructing a restriction map of plasmid DNA, the plasmid D possessed by each clone can be obtained.
The NA was characterized. And Bam of pRH (-44)
DNA fragment between the HI site and the SalI site (0.3
1 Kbp) was deleted, and Escherichia coli harboring a plasmid: pHScos (FIG. 4) in which a SalI linker was inserted at that position was obtained.

(5) Large-scale culture and preparation of plasmid DNA This E. coli was cultured in TYG-P medium (1% Bactortry).
ptone, 0.1% yeast extract, 0.2% glucose, 0.1% NH ₄ Cl, 0.3% NaCl, 0.01
% Na ₂ SO ₄ , 0.08% MgCl ₂ .6H ₂ O,
_{1.52% Na 2 HPO 4 · 12H} 2 O, 0.3% KH
₂ PO ₄ ) in 1 l (containing 20 μg / ml of Ap), 37
After culturing with shaking at 0 ° C., when the absorbance at 600 nm (A ₆₀₀ ) reached 1.0, 100 ml of chloramphenicol was added.
The mixture was added to give a concentration of μg / ml, and the mixture was further cultured by shaking at 37 ° C. for 15 hours. After culturing, the cells were collected by centrifugation (6,000 rpm, 10 minutes),
Next, the cells were treated with 25% sucrose and 50 mM Tris-HCl.
It was resuspended in 18 ml of isotonic buffer consisting of (pH 8.0). To this, a lysozyme solution [5 mg / ml, 0.2
3.6 ml of 5M Tris-HCl (pH 8.0)] was added and gently mixed, and the mixture was kept at 0 ° C. for 5 minutes.
Add 7.2 ml of 25 MEDTA (pH 8.0) solution,
Mix gently and place at 0 ° C. for 5 minutes.

Then, 2.7 ml of 10% SDS solution and 5
Add 8.1 ml of MNaCl solution and mix gently,
After overnight at 0 ° C, centrifuge (20,000 rp
m, 45 minutes) over a crude lysate (cleared l)
Ysate) -39 ml was obtained. RN is added to this crude lysate
aseA solution (20mg / ml, 10t / 0.1E) 2
After adding 0 μl and incubating at 37 ° C. for 1 hour to decompose RNA, deproteinization was carried out by phenol extraction, and then Sepharose 2B was subjected to gel filtration. Plasmid DN
DNA was obtained as an ethanol precipitate from a void claxion containing A, and then the DNA was cesium chloride-ethidium bromide solution [1M Tris-HCl (pH 8.0) 0.3.
75 ml, 0.25 MEDTA (pH 8.0) 0.30
ml, H ₂ O 6.45 ml, CsCl 17.5 g, Et
The solution was subjected to equilibrium density gradient centrifugation (60,000 rpm, 12 hours) with Br solution (4 mg / ml, 0.375 ml).

Then, the covalently closed circular plasmid DNA (ccc-DNA) becomes a band-like band under the fluorescent band of the linear plasmid DNA and the chromosome in the centrifuge tube under long-wavelength ultraviolet irradiation. I can see it. The ccc-DNA band was fractionated, extracted with isopropanol saturated with a CsCl aqueous solution to remove ethidium bromide, and then dialyzed against 10 t / 0.1E1 l. Then, ccc-DNA was obtained as an ethanol precipitate from the dialysate, and the precipitate was dissolved in 10 t / 0.1E2 ml to prepare a pHScos plasmid DNA solution.

(B) Preparation and Preparation of pHBcos 10 μg of pHScos was added to 400 μl of the reaction mixture to prepare 185
It was digested by reacting with SalI of unit at 37 ° C. for 2 hours. After deproteinization by phenol extraction, DNA was obtained as an ethanol precipitate, and the precipitate was 10t / 0.1E7 μl.
(˜1 μg / μl). Next, this SalI
1 μl (~ 1 μg) of DNA solution digested with
5 μl with 2 units of Klenow enzyme and 3 at 22 ° C
After reacting for 0 minutes, cohesive end is blue
changed to nt end. Then, the reaction solution is heated at 65 ° C. for 10 minutes.
The Klenow enzyme was inactivated by heat treatment for minutes.

10 μl (0.4 μg) of this DNA solution
And a BamHI linker [d (pC-G-G-A-T-
C-C-G)] 2 μg were ligated by reacting with 20 μl of the reaction mixture with 6 units of T4-DNA ligase at 22 ° C. for 6 hours. Then, 10 μl of this reaction solution is added to the reaction mixture 5
0 unit of 15 unit T4-DNA ligase and 12 ℃
And reacted for 17.5 hours for ring closure. Then, phenol extraction was performed to obtain DNA as an ethanol precipitate. The precipitate was dissolved in 0.1 M Tris-HCl (pH 7.5) solution 20
It was dissolved in μl.

Next, E. coli (HB101) was transformed with 10 μl of this DNA solution, and then A
A p ^r clone was obtained. Then, the characteristics of the plasmid DNA possessed by these clones were investigated, and Sa of pHScos was examined.
A plasmid having a BamHI linker inserted at the 11 site (in this case, SalI sites are reconstructed on both sides of the BamHI site): Escherichia coli having pHBcos (FIG. 4) was obtained. After this E. coli was grown in 1 liter of TYG-P medium, ccc-DNA was separated and purified from the cells and p.
HBcos plasmid DNA was prepared.

(C) Preparation and preparation of pCHcos 10 μg of cosmid vector pLH (+129) was mixed with 100 μl of reaction mixture [50 mM NaCl, 100 mMT
ris-HCl (pH 7.5), 5 mM MgCl ₂ , 1
00 μg / ml BSA], 69 units of restriction enzyme E
It was digested by reacting with coRI (Takara) at 37 ° C for 2 hours. After deproteinization with phenol extraction, D
NA was obtained as an ethanol precipitate, which was precipitated 10t / 0.1E
It was dissolved in 7 μl (˜1 μg / ml). Next, Eco
1 μl (~ 1 μg) of the DNA solution digested with RI was added to 2 units of Klenow enzyme and 22 μl in a reaction mixture of 25 μl.
After reacting at 0 ° C. for 30 minutes, the cohesive end was changed to the blunt end. Then, the reaction solution is heated to 65 ° C.
The Klenow enzyme was inactivated by heat treatment for 10 minutes.
Next, 10 μl (0.4 μg) of this DNA solution and Cl
aI linker [d (pC-A-T-C-G-A-T-
G)] 2 μg was ligated with 20 μl of the reaction mixture by reacting with 6 units of T4-DNA ligase at 22 ° C. for 6 hours.

Then, 10 μl of this reaction solution was mixed with 50 μl of the reaction mixture and 15 units of T4-DNA ligase and 12
The ring was closed by reacting at 17.5 hours for 1 hour. Then, phenol extraction was performed to obtain DNA as ethanol precipitate,
The precipitate was dissolved in 0.1 M Tris-HCl (pH 7.5) solution 2
It was dissolved in 0 μl. Next, after transforming E. coli (HB101) with 10 μl of this DNA solution, an Ap ^r clone was obtained. Then, the characteristics of the plasmid DNA possessed by these clones were examined, and pLH (+1
29) A plasmid having a ClaI linker inserted at the EcoRI site (in this case, E on both sides of the ClaI site).
The coRI site is reconstructed. ): E. coli with pCHcos (FIG. 4) was obtained. This Escherichia coli was grown in 1 liter of TYG-P medium, and ccc-DNA was separated and purified from the cells to prepare pCHcos plasmid DNA.

(D) pCH / HBcos (pDcos)
Preparation and Preparation of pCHcos 20 μg, Reaction Mixture 200 μl [60 m
MNaCl, 7 mM Tris-HCl (pH 7.4),
7 mM MgCl ₂ , 6 mM DTT, 100 μg / ml B
SA)], a 114 unit restriction enzyme HindIII
(New England Biolabs) and 192 unit PvuII (Takara) and 37
The mixture was reacted at ℃ for 2 hours and digested, then subjected to 0.8% preparative agarose gel electrophoresis to obtain cos and Ap ^r , o.
A DNA fragment containing ri (2.5 Kbp) was separated and purified to obtain DNA as an ethanol precipitate, and the precipitate was collected at 10 t /
It was dissolved in 0.1E8.5 µl (~ 1 µg / µl).

On the other hand, 20 μg of pHBcos was added to the reaction mixture 2
At 00 μl, 114 units of HindIII and 192
After digestion with unit PvuII at 37 ° C. for 2 hours, it was subjected to 0.8% preparative agarose gel electrophoresis to separate and purify the cos-containing DNA fragment (1.6 Kbp) to obtain the DNA as an ethanol precipitate. , Precipitation 10
It was dissolved in t / 0.1E 6.0 μl (˜1 μg / μl). And pCHcos cos and Ap ^r , ori
DNA solution of HindIII / PvuII fragment containing 1 μl
Hind containing cos of pHBcos (up to 1 μg)
1 μl (˜1 μg) of the DNA solution of III / PvuII fragment was ligated by reacting 20 μl of the reaction mixture with 6 units of T4-DNA ligase at 12 ° C. for 17.5 hours. Then, phenol extraction was performed to obtain DNA as an ethanol precipitate, which was precipitated with 0.1 M Tris-HCl (pH 7.
5) Dissolved in 20 μl of solution.

Next, after transforming E. coli (HB101) with 10 μl of this DNA solution, Ap
^{An r} clone was obtained. Next, the characteristics of the plasmid DNA possessed by these clones were examined, and the two cos and Ap
A plasmid having ^r , ori: pDcos (FIG. 4) was obtained. This E. coli was mixed with 1 liter of TYG-P medium.
After proliferating in the medium, ccc-DNA was separated and purified from the cells to prepare pDcos plasmid DNA.

(E) Preparation and preparation of pHC-79 / cm ^r A cosmid vector pHC-79 [Horn & Collins, Gene.
Gene), 11 , 291 (1980)] 10 μl,
Reaction mixture 100 μl [50 mM NaCl, 6 mM Tri
s-HCl (pH 7.9), 6 mM MgCl ₂ , 100
μg / ml BSA], 15 units of restriction enzyme Cla
I (manufactured by Boehringer Mannheim) was reacted at 37 ° C. for 2 hours for digestion. After deproteinization by phenol extraction, DNA was obtained as ethanol precipitate,
The precipitate was dissolved in 10 t / 0.1E7 μl (˜1 μg / ml).

On the other hand, 10 μg of pNT-31 was mixed with 200 μl of a reaction mixture [100 mM NaCl, 10 mM Tris-
HCl (pH 8.4), 6 mM MgCl ₂ , 6 mM DT
T, 100 μg / ml BSA], 40 units of restriction enzyme TaqI (New England Biolab).
(manufactured by s company) at 65 ° C. for 2 hours for digestion,
Cm for 0.8% preparative agarose gel electrophoresis
A DNA fragment containing ^r (0.79 Kbp) was separated and purified to obtain DNA as an ethanol precipitate, and the precipitate was collected at 10 t /
This was dissolved in 0.1E 3.0 μl (-1 μg / μl).
Then, Ta containing 1 μl of DNA solution (˜1 μg) obtained by digesting pHC-79 with ClaI and cm ^{r of} pNT-31.
1 μl (∼1 μg) of the q-I fragment DNA solution was added to 6 units of T4-DNA ligase in 20 μl of the reaction mixture.
The reaction was carried out at 2 ° C. for 17.5 hours for ligation. Then, phenol extraction was performed to obtain DNA as ethanol precipitate,
The precipitate was dissolved in 0.1 M Tris-HCl (pH 7.5) solution 2
It was dissolved in 0 μl.

Next, after transforming E. coli (HB101) with 10 μl of this DNA solution, c
m ^r and Ap ^r clones were obtained. Then, the characteristics of the plasmid DNA possessed by these clones were examined, and pHC-
79 ClaI site plasmid was inserted the cm ^r of: pHC-79 / cm ^r was obtained E. coli with (Figure 5). After this E. coli was grown in 1 liter of TYG-P medium, ccc-DNA was separated and purified from the cells to obtain pHC-
79 / cm ^r of plasmid DNA was prepared.

(F) pHC-79 / cm ^r Δ (Hind
Preparation and preparation of III) 10 μg of pHC-79 / cm ^{r was} added to 100 μl of the reaction mixture.
It was digested by reacting with 35 units of Hindlll at 37 ° C for 2 hours. After deproteinization with phenol extraction, D
NA was obtained as an ethanol precipitate and the precipitate was 10 t / 0.1
It was dissolved in E7 μl (˜1 μg / μl). Next, 1 μl (~ 1 μg) of this HindIII-digested DNA solution
Was reacted with 2 units of Klenow enzyme for 30 minutes at 22 ° C. in 25 μl of the reaction mixture, and then cohesive
The end was changed to blunt end. Then, the reaction solution was heat-treated at 65 ° C. for 10 minutes to inactivate the Klenow enzyme. Next, 10 μl of this DNA solution (up to 0.4 μm)
g) in 15 μl of T4-D with 50 μl of reaction mixture
Cyclization was performed by reacting with NA ligase at 12 ° C. for 17.5 hours. Then, phenol extraction was performed to obtain DNA as an ethanol precipitate. The precipitate was extracted with 0.1 M Tris-HCl.
It was dissolved in 20 μl of (pH 7.5) solution.

Next, after transforming E. coli (HB101) with 10 μl of this DNA solution, Ap
^r and cm ^r clones were obtained. Then, the characteristics of the plasmid DNA possessed by these clones were examined, and pHC-7
A plasmid lacking the HindIII site at 9 / cm ^r :
Escherichia coli having pHC-79 / cm ^r Δ (HindIII) (Fig. 5) was obtained. This E. coli was grown in 1 liter of TYG-P medium, and ccc-DNA was separated and purified from the cells to prepare a plasmid DNA of pHC-79 / cm ^r Δ (HindIII).

(G) Preparation and Preparation of pUC-8 / cm ^r pHC-79 / cm ^r Δ (HindlII) 20 μg
Reaction mixture 200 μl [50 mM (NH ₄ ) ₂ SO ₄ , 2
0 mM Tris-HCl (pH 7.5), 10 mM Mg
Cl ₂ , 100 μg / ml BSA], 22 units of the restriction enzyme PstI (New England Biol).
Abs) and reacted at 37 ° C. for 2 hours for complete digestion. Then, the reaction mixture was further reacted with BamHI (80 units) at a temperature of 37 ° C. for 2 hours for complete digestion, and then subjected to 0.8% preparative agarose gel electrophoresis to give a DNA fragment containing cm ^r (1.9 Kbp). Is separated and purified to obtain DNA as an ethanol precipitate.
It was dissolved in 0t / 0.1E4.0 µl (~ 1 µg / µl).

On the other hand, pUC-8 [JFV VIEIRA and Geochime Measing, Gene (Je
ferry Vieira & Joachim Me
ssing, Gene), 19 , 259 (1982)].
20 μg was added to 200 μl of the reaction mixture to prepare 60 units of P
It was reacted with stI at 37 ° C. for 2 hours for complete digestion. Then, the reaction mixture was further reacted with BamHI of 215 units at 37 ° C. for 2 hours in 500 μl of the reaction mixture to completely digest it, and then subjected to 0.8% preparative agarose gel electrophoresis to obtain a DNA fragment containing Ap ^r and ori (2. 7Kbp)
Was separated and purified to obtain DNA as an ethanol precipitate, and the precipitate was 10 t / 0.1E14.0 μl (∼1 μg / μl)
Dissolved in.

PHC-79 / cm ^r Δ (Hin
DN of the Pst / BamHI fragment containing the cm ^r of dIII)
A solution 1 μl (up to 1 μg) and Ap ^r , o of pUC-8
1μ DNA solution of PstI / BamHI fragment containing ri
1 (.about.1 .mu.g) was ligated by reacting 20 .mu.l of the reaction mixture with 6 units of T4-DNA ligase at 12.degree. C. for 17.5 hours. Then, phenol extraction was performed to obtain DNA as an ethanol precipitate, and the precipitate was extracted with 0.1 M Tris-H.
It was dissolved in 20 μl of Cl (pH 7.5) solution. next,
After transforming E. coli (HB101) with 10 μl of this DNA solution, cm ^r and Ap ^r clones were obtained. Then, the plasmid D possessed by these clones
By investigation of the characteristics of the NA, plasmid having Ap ^r, ori and cm ^r: obtain a E. coli with pUC-8 / cm ^r (Fig. 5). After this E. coli was grown in 1 liter of TYG-P medium, ccc-DNA was separated and purified from the cells,
Plasmid DNA of pUC-8 / cm ^r was prepared.

(H) pUC-8 / cm _r Δ (EcoRI
/ PstI) preparation and preparation 20 μg of pUC-8 / cm ^{r in} 200 μl of reaction mixture,
It was reacted with 60 units of EcoRI at 37 ° C. for 30 minutes for limited digestion. Then, deproteinize with phenol extraction,
DNA was obtained as an ethanol precipitate. This DNA precipitate was mixed with 35 units of PstI and 3 with 200 μl of the reaction mixture.
The reaction was carried out at 7 ° C for 2 hours for complete digestion. After deproteinization by phenol extraction, DNA was obtained as an ethanol precipitate, and the precipitate was collected at 10 t / 0.1E10 μl (up to 1 μg / μm).
l).

Next, 1.5 μl of this DNA solution (up to 1.
5 μg) in 30 μl of the reaction mixture [33 mM Tris-
acetate (pH 7.9), 66 mM potassium acetate, 1
0 mM MgCl ₂ , 0.5 mM DTT, 100 μg / m
1BSA, 0.1 mM dATP, 0.1 mM dCTP,
0.1 mM dGTP, 0.1 mM dTTP], and 5 units of T4-DNA polymerase (manufactured by PL) and 3
Allow to react for 15 minutes at 7 ° C, then cohesive end
Was changed to blunt end and then subjected to 0.8% preparative agarose gel electrophoresis to give Ap ^r , ori and cm.
A DNA fragment containing ^r (3.85 Kbp) was separated and purified to obtain DNA as an ethanol precipitate.

Then, this DNA precipitate was mixed with 50 μl of the reaction mixture.
15 units of T4-DNA ligase at 1 ° C and 1 at 12 ° C
The reaction was carried out for 7.5 hours for ring closure. Then, phenol extraction was performed to obtain DNA as an ethanol precipitate, and the precipitate was dissolved in 20 μl of 0.1 M Tris-HCl (pH 7.5) solution.
Dissolved in. Next, after transforming E. coli (HB101) with 10 μl of this DNA solution, A.
p ^r and cm ^r clones were obtained. Then, the characteristics of the plasmid DNA possessed by these clones were examined, and pUC-
Plasmid lacking the DNA fragment (0.75 Kbp) between the EcoRI and PstI sites of 8 / cm ^r : p
UC-8 / cm ^r Δ (EcoRI / PstI) (Fig. 5)
E. coli having This E. coli was transformed into TYG-P medium 1
After the cells were grown in lcc, ccc-DNA was separated and purified from the cells, and (pUC-8 / cm ^r Δ (EcoRI / Ps
The plasmid DNA of tI) was prepared.

(I) pUC-8 / cm ^r Δ (EcoRI
/ PstI) Δ (BamHI) Preparation and Preparation pUC-8 / cm ^r Δ (EcoRI / PstI) 10μ
g of BamHI of 150 units in 100 μl of the reaction mixture
And digested at 37 ° C. for 2 hours. After deproteinization by phenol extraction, DNA was obtained as ethanol precipitate,
The precipitate was dissolved in 10 t / 0.1E7 μl (˜1 μg / μl). Next, 1 μl (to 1 μg) of this BamHI-digested DNA solution was reacted with 2 units of Klenow enzyme at 22 ° C. for 30 minutes with 25 μl of the reaction mixture,
Cohesive end was changed to blunt end. Then, heat the reaction solution at 65 ° C. for 10 minutes to obtain Kl.
The enow enzyme was inactivated.

Then, 10 μl of this reaction solution was mixed with 50 μl of the reaction mixture and 15 units of T4-DNA ligase.
The ring was closed by reacting at 17.5 hours for 1 hour. Then, phenol extraction was performed to obtain DNA as ethanol precipitate,
The precipitate was dissolved in 0.1 M Tris-HCl (pH 7.5) solution 2
It was dissolved in 0 μl. Next, after transforming E. coli (HB101) with 10 μl of this DNA solution, Ap ^r and cm ^r clones were obtained. Then, by examining the characteristics of the plasmid DNA possessed by these clones, p
Bam of UC-8 / cm ^r Δ (EcoRI / PstI)
Plasmid lacking HI site: pUC-8 / cm ^r Δ
E. coli with (EcoRI / Pst) Δ (BamHI) (FIG. 5) were obtained. After this E. coli was grown in 1 liter of TYG-P medium, ccc-DNA was separated and purified from the cells, and pUC-8 / cm ^r Δ (EcoRI / Pst) Δ
(BamHI) plasmid DNA was prepared.

(J) pUC-8 / cm ^r Δ (EcoRI
/ PstI) Δ (BamHI) / HindIII Preparation and Adjustment pUC-8 / cm ^r Δ (EcoRI / PstI) Δ (B
amHI) 10 μg reaction mixture 100 μl [20 mMK
Cl, 6 mM Tris-HCl (pH 8.0), 6 mM
MgCl ₂ , 6 mM DTT, 100 μg / ml BSA]
With 125 units of restriction enzyme SmaI (New Eng
(manufactured by Land Biolabs) and completely digested at 37 ° C. (blunt end). After deproteinization by phenol extraction, DNA was obtained as an ethanol precipitate, and the precipitate was dissolved in 10 t / 0.1E7 μl (up to 1 μg / μl).

Next, 0.4 μl of this DNA solution (.about.0.
4 μg) and HindIII linker [d (pC-A-A-
G-C-T-T-G)] 2 μg and 20 μl of reaction mixture
Then, it was ligated by reacting with 6-unit T4-DNA ligase at 22 ° C. for 6 hours. And 10 μl of this reaction solution
Was cyclized by reacting with 15 units of T4-DNA ligase in 50 μl of the reaction mixture at 12 ° C. for 17.5 hours.
Then, phenol extraction was performed to obtain DNA as an ethanol precipitate. The precipitate was extracted with 0.1 M Tris-HCl (pH
7.5) Dissolved in 20 μl of solution.

Next, after transforming E. coli (HB101) with 10 μl of this DNA solution, Ap
^r and cm ^r clones were obtained. Then, the characteristics of the plasmid DNA possessed by these clones were examined, and pUC-8
/ Cm ^r Δ (EcoRI / PstI) Δ (BamHI)
The SmaI site in the plasmid was inserted the HindIII ^{linker: pUC-8 / cm r Δ} (EcoRI / Ps
E. coli with tI) Δ (BamHI) / HindIII (FIGS. 4 and 5) were obtained. After this E. coli was grown in 1 liter of TYG-P medium, ccc-DNA was separated and purified from the cells and pUC-8 / cm ^r Δ (EcoRI / P
stI) Δ (BamHI) / HindIII plasmid DNA was prepared.

(K) Preparation and Preparation of pDcos / cm ^r 10 μg of pDcos was reacted with 100 μl of the reaction mixture with 60 units of HindIII at 37 ° C. for 2 hours for digestion. After deproteinization by phenol extraction, DNA was obtained as an ethanol precipitate, and the precipitate was 10t / 0.1E7 μl.
(˜1 μg / μl). On the other hand, pUC-8 /
cm ^r Δ (EcoRI / PstI) Δ (BamHI) /
20 μg of HindIII was added to 200 μl of the reaction mixture to give 250
After reacting with Unit's HindIII at 37 ° C. for 2 hours for complete digestion, it was subjected to 0.8% preparative agarose gel electrophoresis to give a DNA fragment containing cm ^r (1.15 Kb).
p) was separated and purified to obtain DNA as an ethanol precipitate.
l).

Then, 1 μl (~ 1 μg) of a DNA solution obtained by digesting pDcos with HindIII and pUC-8 / cm
^r Δ (EcoRI / PstI) Δ (BamHI) / Hi
1 μl (∼1 μg) of a HindIII fragment DNA solution containing cm ^r of ndIII was ligated by reacting 20 μl of the reaction mixture with 6 units of T4-DNA ligase at 12 ° C. for 17.5 hours. Then, perform phenol extraction and DN
A was obtained as an ethanol precipitate and the precipitate was extracted with 0.1 M Tris.
It was dissolved in 20 μl of -HCl (pH 7.5) solution. Next, after transforming E. coli (HB101) with 10 μl of this DNA solution, Ap ^r and cm ^r clones were obtained. Then, the characteristics of the plasmid DNAs possessed by these clones were examined, and two cos and Ap ^r , o
A plasmid with ri, cm ^r : pDcos / cm ^r
E. coli having (Fig. 4) was obtained. This E. coli was designated as TYG-
After being grown in 1 L of P medium, ccc-DN was extracted from the cells.
A was separated and purified to obtain pDcos / cm ^r of plasmid D
NA was prepared.

(L) Preparation and Preparation of pHCcos 10 μg of pRH (−44) was added to 100 μl of the reaction mixture, and 2
After complete digestion with 3 units of PvuII at 37 ° C for 2 hours, 250 μl of reaction mixture was further reacted with 70 units of BamHI at 37 ° C for 2 hours to complete digestion, and then 0.8% for preparation. A DNA fragment (2.48 Kbp) containing cos, Ap ^r , and ori was separated and purified by agarose gel electrophoresis to obtain DNA as an ethanol precipitate, and the precipitate was 10 t / 0.1E 4.0 μl.
(~ 1 μg / μl). Next, 1 μl (~ 1 μg) of this DNA solution was reacted with 25 μl of the reaction mixture with 2 units of Klenow enzyme at 22 ° C. for 30 minutes, and c
Replaced ohseive end with blunt end. Then, heat the reaction solution at 65 ° C. for 10 minutes to obtain Kl.
The enow enzyme was inactivated.

10 μl (0.4 μg) of this DNA solution and ClaI linker [d (pC-A-T-C-G-A-T]
-G)] 2 µg was ligated with 20 µl of the reaction mixture by reacting with 6 units of T4-DNA ligase at 22 ° C for 6 hours. Then, 10 μl of this reaction solution is added to the reaction mixture 50
This was cyclized by reacting 15 μl of T4-DNA ligase with 12 μl at 12 ° C. for 17.5 hours. After that, phenol extraction was performed to obtain DNA as an ethanol precipitate, and the precipitate was dissolved in 0.1 M Tris-HCl (pH 7.5) solution 20 μm.
dissolved in l.

Next, after transforming E. coli (HB101) with 10 μl of this DNA solution, Ap
^{An r} clone was obtained. Then, the characteristics of the plasmid DNA of these clones were examined, and the pRH (-44) P
Escherichia coli having a plasmid: pHCcos (FIG. 6) in which a DNA fragment (1.72 Kbp) between the vuII site and the BamHI site was deleted and a ClaI linker was inserted at that position was obtained. This Escherichia coli was grown in 1 liter of TYG-P medium, and ccc-DNA was separated and purified from the cells to prepare pHCcos plasmid DNA.

(M) Preparation and Preparation of pBglIICcos 10 μg of pHCcos was reacted with 100 μl of the reaction mixture with 100 units of HindIII at 37 ° C. for 2 hours for digestion. After deproteinization by phenol extraction, DNA was obtained as an ethanol precipitate, and the precipitate was washed with 10t / 0.1E7μ.
1 (~ 1 µg / µl). Next, this Hin
1 μl (~ 1 μg) of the DNA solution digested with dIII was added to 25 μl of the reaction mixture and 2 units of Klenow enzyme and 22
After reacting at 0 ° C. for 30 minutes, the cohesive end was changed to the blunt end. Then, the reaction solution is heated to 65 ° C.
The Klenow enzyme was inactivated by heat treatment for 10 minutes.

10 μl (0.4 μg) of this DNA solution
And a BglII linker [d (pC-A-G-A-T-C
-TG)] 2 μg was ligated by reacting with 20 μl of the reaction mixture with 6 units of T4-DNA ligase at 22 ° C. for 6 hours. Then, 10 μl of this reaction solution was mixed with 50 μl of the reaction mixture to prepare 15 units of T4-DNA ligase.
The ring was closed by reacting at 17.5 hours for 1 hour. Then, phenol extraction was performed to obtain DNA as ethanol precipitate,
The precipitate was dissolved in 0.1 M Tris-HCl (pH 7.5) solution 2
It was dissolved in 0 μl.

Next, after transforming E. coli (HB101) with 10 μl of this DNA solution, Ap
^{An r} clone was obtained. Then, the characteristics of the plasmid DNA possessed by these clones were investigated, and Hin of pHCcos was examined.
Escherichia coli having a plasmid pBglIICcos (FIG. 6) having a BglII linker inserted at the dIII site was obtained.
After this E. coli was grown in 1 liter of TYG-P medium,
Ccc-DNA was isolated and purified from the bacterial cells to obtain pBglIIC.
Cos plasmid DNA was prepared.

(N) Preparation and Preparation of pBR-327BamHI ^a 10 μg of pBR-327 was added to 100 μl of the reaction mixture [6
0 mM NaCl, 6 mM Tris-HCl (pH 8.
0) 10 mM MgCl ₂ , 6 mM DTT, 100 μg
/ MlBSA] and reacted with 55 units of the restriction enzyme AvaI at 37 ° C. for 2 hours for digestion. After deproteinization by phenol extraction, DNA was obtained as ethanol precipitate,
The precipitate was dissolved in 10 t / 0.1E7 μl (˜1 μg / μl). Next, this DNA solution 1 digested with AvaI
μl (up to 1 μg) was reacted with 25 μl of the reaction mixture with 2 units of Klenow enzyme at 22 ° C. for 30 minutes, and c
Replaced ohseive end with blunt end. Then, heat the reaction solution at 65 ° C. for 10 minutes to obtain Kle.
The now enzyme was inactivated.

10 μl (0.4 μg) of this DNA solution
And a BamHI linker [d (pC-G-G-A-T-
C-C-G)] 2 μg were ligated by reacting with 20 μl of the reaction mixture with 6 units of T4-DNA ligase at 22 ° C. for 6 hours. Then, phenol extraction was performed to obtain DNA as an ethanol precipitate. This DNA precipitate was mixed with 600 µl of the reaction mixture and 1,000 units of BamHI and 37 ° C.
After 4 hours of reaction and digestion, it was subjected to 0.8% preparative agarose gel electrophoresis to obtain D containing Ap ^r and ori.
The NA fragment (2.22 Kbp) was separated and purified to obtain DNA as an ethanol precipitate.

This DNA precipitate was treated with 20 μl of the reaction mixture to give 6
After reaction with unit T4-DNA ligase at 12 ° C. for 17.5 hours for cyclization, phenol extraction was performed, and D
NA was obtained as an ethanol precipitate and the precipitate was 0.1 M Tri
It was dissolved in 20 μl of s-HCl (pH 7.5) solution.
Then, with 10 μl of this DNA solution, E. coli (HB10
After transforming 1), an Ap ^r clone was obtained. Then, the characteristics of the plasmid DNA possessed by these clones were examined to find a DNA fragment (1.05 Kb) between the BamHI site and the AvaI site of pBR327.
p) was deleted and ^a BamHI linker was inserted in its place to obtain Escherichia coli harboring ^a plasmid: pBR-327BamHI ^a (FIG. 6). This E. coli was mixed with 1 liter of TYG-P medium.
After grown in medium, plasmid DNA of pBR-327BamHI ^a the ccc-DNA from the cells was separated and purified
Was prepared.

(O) pBR-327 / BglIICcos
Preparation and Preparation of pBglIICcos (20 μg) in a reaction mixture (200 μl)
After reacting with 65 units of PstI at 37 ° C. for 2 hours for complete digestion, 500 μl of the reaction mixture was further reacted with 77 units of ClaI for 2 hours at 37 ° C. for complete digestion and then 0.8% preparative agarose. The cos-containing DNA fragment (0.94 Kbp) was separated and purified by gel electrophoresis to obtain a DNA ethanol precipitate, and the precipitate was dissolved in 10 t / 0.1E 5.0 μl (˜1 μg / μl).

On the other hand, pBR-327BamHI ^a 20 μ
g of the reaction mixture was reacted with 70 units of PstI at 37 ° C. for 2 hours for complete digestion, and then 500 μl of the reaction mixture was added to 85 units of ClaI.
After 2 hours of reaction at ℃ for complete digestion, 0.8% preparative agarose gel electrophoresis was performed and D containing ori was added.
The NA fragment (1.45 Kbp) was separated and purified to obtain DNA as an ethanol precipitate, which was precipitated with 10 t / 0.1E9.
It was dissolved in 0 μl (˜1 μg / μl).

Then, 1 μl of DNA solution of Pst / ClaI fragment containing cos of pBglIICcos (1 μl
g) and P containing pBR-327BamHI ^a ori
DNA solution of stI / ClaI fragment 1 μl (~ 1 μg)
Were ligated by reacting 20 μl of the reaction mixture with 6 units of T4-DNA ligase at 12 ° C. for 17.5 hours. Then, phenol extraction was performed to obtain a DNA ethanol precipitate, and the precipitate was 0.1 M Tris-HCl (pH 7.
5) Dissolved in 20 μl of solution. Next, after transforming E. coli (HB101) with 10 μl of this DNA solution, an Ap ^r clone was obtained. Then, the characteristics of plasmid DNA possessed by these clones were examined, and
Plasmid containing s and Ap ^r , ori: pBR-3
E. coli with 27 / BglIICcos (FIGS. 4 and 6) was obtained. After this E. coli was grown in 1 liter of TYG-P medium, ccc-DNA was separated and purified from the cells to obtain pB.
R-327 / BglIICcos plasmid DNA was prepared.

[0076] ^{(p) pTcosAp r / ori /} cm r
Preparation and Preparation of pDcos / cm ^r 20 μg in a reaction mixture 200 μl,
After reacting with 37 units of ClaI at 37 ° C for 2 hours for complete digestion, the reaction mixture was added with 500 µl of 11
After reacting with 0 unit of BamHI at 37 ° C for 2 hours to completely digest it, it was subjected to 0.8% preparative agarose gel electrophoresis to separate and purify a DNA fragment (1.5 Kbp) containing two cos and cm ^r. Then, the DNA was obtained as an ethanol precipitate, and this precipitate was collected at 10 t / 0.1E4.0 μm.
1 (~ 1 µg / µl).

On the other hand, pBR-327BglIICcos2
0 μg was added to 200 μl of the reaction mixture to obtain 80 units of Cla.
After digestion with I for 2 hours at 37 ℃, the reaction mixture was further added with 500 µl of 240 units of BamH.
After reacting with I at 37 ° C for 2 hours to completely digest,
Apply 0.8% preparative agarose gel electrophoresis to
DNA fragment containing s, Ap ^r , and ori (2.0 Kb
p) was separated and purified to obtain DNA as an ethanol precipitate, which was precipitated with 10 t / 0.1E12 μl (∼1 μg / μl).
). Then, 1 μl (to 1 μg) DNA solution of ClaI / BamHI fragment containing two cos and cm ^r of pDcos / cm ^r and pBR-327 / B
glIICcos of cos and Ap ^r, C, including the ori
DNA solution of laI / BamHI fragment 1 μl (up to 1 μl
g) and 20 μl of the reaction mixture with 6 units of T4-DN
Ligation was performed by reacting with A ligase at 12 ° C. for 17.5 hours. Then, phenol extraction was performed to obtain DNA as an ethanol precipitate. The precipitate was extracted with 0.1M Tris-HCl (p
H7.5) solution was dissolved in 20 μl.

Next, after transforming E. coli (HB101) with 10 μl of this DNA solution, A.
p ^r and cm ^r clones were obtained. Then, the characteristics of the plasmid DNAs possessed by these clones were examined, and the three co
plasmid containing s and Ap ^r , ori, cm ^r : p
TcosAp ^r / ori / cm ^r was obtained E. coli with (Figure 4). After this E. coli was grown in 1 liter of TYG-P medium, ccc-DNA was separated and purified from the cells and p.
The TcosAp ^r / ori / cm ^r plasmid DNA was prepared.

(Q) Preparation and Preparation of pSV010 / neo pAO43 [Oka, Sugisaki and Takanami, Journal of Morequiller Biology (OKa,
Sugisaki & Takanami, J. et al. Mo
1, Biol), 147 , 214 (1981)] 20μ
g to 200 μl of reaction mixture [60 mM NaCl, 6 mM
Tris-HCl (pH 8.0), 10 mM MgC
1 ₂ , 6 mM DTT, 100 μg / ml BSA], 1
50 units of restriction enzyme AvaII (New Engla
nd Biolabs) at 37 ° C. for 2 hours for digestion, and then subjected to 0.8% preparative agarose gel electrophoresis to give a DNA fragment containing neo ^r (1.24 Kb).
p) is separated and purified to obtain DNA as an ethanol precipitate, and the precipitate is 10t / 0.1E 5 µl (∼1 µg / µl)
Dissolved in.

2 .mu.l (.about.2 .mu.g) of this DNA solution was mixed with 50 .mu.l of the reaction mixture and 4 units of Klenow enzyme and 2
Allow to react at 2 ° C for 30 minutes, cohesive end
Was changed to blunt end. After deproteinization by phenol extraction, DNA was obtained as an ethanol precipitate, and the precipitate was dissolved in 10 t / 0.1E1.5 μl (˜μg / μl). On the other hand, pSV010 {πVX [Bryan Seed, Nyukraykku Acids, Research (Brain
seed, Nucleic Acids Res), 1
1 , 2427 (1983)] having a polylinker derived from pSV01 [Jiang, Earl et al., Proceeding]
Of National Academy Sciences You
S.A. (Tjan, R, et al, Proc, N
atl, Acad, Sci. U. S. A. ) 77 , 64
91 (1980)] and 10 .mu.g of the similar plasmid with BamHI of 129 units in 37 .mu.l of the reaction mixture.
After reacting at ℃ for 2 hours for digestion, phenol extraction was performed to obtain DNA as ethanol precipitate, and the precipitate was extracted with 10 t.
/0.1E7 µl (~ 1 Mg / µl).

2 .mu.l (.about.2 .mu.g) of this DNA solution was added to 50 .mu.l of the reaction mixture and 4 units of Klenow enzyme and 22
After reacting at 0 ° C. for 30 minutes, the cohesive end was changed to the blunt end. After deproteinization by phenol extraction, DNA was obtained as an ethanol precipitate, and the precipitate was dissolved in 10 t / 0.1E1.5 µl (~ 1 µg / µl). Next, 1 μl of a DNA solution obtained by treating the DNA fragment containing neo ^r of pAO43 with Klenow enzyme (~ 1
μg) and pSV010 were digested with BamHI and Kle
1 μl (˜1 μg) of the DNA solution treated with now enzyme was ligated by reacting 20 μl of the reaction solution with 6 units of T4-DNA ligase at 12 ° C. for 17.5 hours. Then, phenol extraction was performed to obtain DNA as an ethanol precipitate. The precipitate was extracted with 0.1 M Tris-HCl (pH
7.5) Dissolved in 20 μl of solution.

Then, E. coli (HB101) was transformed with 10 μl of this DNA solution, and then A
p ^r and obtained the Km ^r (neo ^r) clone. Then
By investigation of the characteristics of plasmid DNA possessed by these clones, the plasmid was inserted the neo ^r into the BamHI site of pSV010: pSV010 / neo ^r (FIG. 4) to obtain Escherichia coli with. This E. coli was mixed with 1 liter of TYG-P medium.
After grown in medium, and plasmid DNA was prepared for pSV010 / neo ^r a ccc-DNA from the cells was separated and purified.

[0083] ^{(r) pTcosAp r / ori /} cm r
/ Neo ^r creation and preparation pTcosAp ^r / ori / cm ^r 10μg reaction mixture 100 [mu] l, 3 with EcoRI and 37 ° C. for 27 Yunitsuto
The reaction was allowed to proceed for 0 minutes for limited digestion. After deproteinization by phenol extraction, DNA was obtained as an ethanol precipitate, and the precipitate was dissolved in 10 t / 0.1E7 μl (up to 1 μg / μl). On the other hand, in pSV010 / neo ^r 20μg reaction mixture 200 [mu] l, 325 Yunitsuto of EcoRI and 37 ° C.
After complete digestion by reacting for 2 hours, 0.8% preparative agarose gel electrophoresis was performed and D containing neo ^r was added.
The NA fragment (1.3 Kbp) was separated and purified to obtain a DNA ethanol precipitate, and the precipitate was 10 t / 0.1E5.0 μ.
1 (~ 1 µg / µl).

[0084] and, pTcosAp ^r / ori / cm
DNA was limited digested ^r with EcoRI solution 1μl (~1
and μg), E, including the neo ^r of pSV010 / neo ^r
1 μl (˜1 μg) of the DNA solution of the coRI fragment was ligated by reacting 20 μl of the reaction mixture with 6 units of T4-DNA ligase at 12 ° C. for 17.5 hours. Then, phenol extraction was performed to obtain DNA as an ethanol precipitate, and the precipitate was dissolved in 20 μl of 0.1 M Tris-HCl (pH 7.5) solution.

Next, after transforming E. coli (HB101) with 10 μl of this DNA solution, A.
p ^r, to obtain a cm ^r and Km ^r (neo ^r) clone.
Then, by examining the characteristics of plasmid DNA possessed by these clones, pTcosAp ^r / ori / cm ^r of Ec
Plasmid with neo ^r inserted in the ori site: pT
^{cosAp r / ori / cm r /} neo r ( 1, 4) to obtain Escherichia coli with. In addition, this strain was designated by the Institute of Life Science and Technology, Institute of Industrial Science and Technology, under the name of "Micromachine Research No. 7888 (F
ERM P-7888) ". After this E. coli was grown in 1 liter of TYG-P medium, ccc-DNA was separated and purified from the cells to obtain pTcosApr ^.
/ Ori / cm plasmid DNA of ^r / neo ^r was prepared.

[0086] Example 2 pTcosAp ^r / ori /
Creating and preparation TK / cm ^r / neo ^r (FIG. 7) (a) pTK-4 / BamHI creation and preparation pTK-4 [Ishiura, M. etc., Morekiyura and Cellular Biology (Ishiura, M., e
t al, Mol. Cell Biol), 2 , 607
(1982)] 10 μg with 100 μl of the reaction mixture, 3.
After reacting with 0 unit of PvuII at 37 ° C. for 30 minutes for limited digestion, it was subjected to 0.8% preparative agarose gel electrophoresis to separate and purify a DNA fragment having a length of 6.43 Kbp, and the DNA was subjected to ethanol precipitation. Get 1 precipitate
It was dissolved in 0t / 0.1E3 µl (~ 1 µg / µl).

Next, 0.4 μl of this DNA solution (.about.0.
4 μg) and a BamHI linker [d (pC-GG-
A-T-C-C-G)] 2 μg and 20 μl of reaction mixture
And ligated by reacting with 6 unit T4-DNA ligase at 22 ° C. for 6 hours. Then, 10 μl of this reaction solution
The reaction mixture was reacted with 15 units of T4-DNA ligase in 50 µl at 12 ° C for 17.5 hours for cyclization. Then, phenol extraction was performed to obtain DNA as an ethanol precipitate, which was precipitated with 0.1 M Tris-HCl (pH 7.
5) Dissolved in 20 μl of solution.

Next, after transforming E. coli (HB101) with 10 μl of this DNA solution, A.
p ^r and Tc ^r clones were obtained. Then, the characteristics of the plasmid DNA possessed by these clones were examined, and pTK-
BamHI at the PvuII site between the 4 ori and TK
Plasmid with inserted linker: pTK-4 / Bam
E. coli with HI (Fig. 7) was obtained. This E. coli is TY
After being grown in 1 liter of G-P medium, ccc-
The DNA was separated and purified to prepare pTK-4 / BamHI plasmid DNA.

(B) pTK-4 / BamHI / BamH
Preparation and Preparation of I 10 μg of pTK-4 / BamHI in 100 μl of reaction mixture
After reacting with BamHI (9 units) at 37 ° C. for 30 minutes for limited digestion, phenol extraction was performed to obtain DNA as an ethanol precipitate. This ethanol precipitate was mixed with 100 μl of the reaction mixture and 15 units of PvuII at 37 ° C. for 2 times.
After reacting for a time to complete digestion, phenol extraction was performed to obtain a DNA ethanol precipitate.
It was dissolved in 0.1E3 µl (~ 1 µg / µl).

Next, 1 μl (up to 1 μg) of a DNA solution was digested with BamHI and then digested with PvuII.
Was reacted with 2 units of Klenow enzyme in 25 μl of the reaction mixture at 22 ° C. for 30 minutes to obtain cohesive e
After changing nd to blunt end, 0.8% preparative agarose gel electrophoresis was performed to obtain ori and A
p ^r, was separated and purified the DNA fragment (4.67Kbp) containing TK, which give DNA as ethanol precipitation. This D
The NA precipitate was treated with 20 μl of the reaction mixture to prepare 6 units of T4-DN.
After the reaction with A ligase at 12 ° C. for 17.5 hours for cyclization and cyclization (in this case, the BamHI site is reconstituted), phenol extraction was performed to obtain DNA as an ethanol precipitate, and the precipitate was 0.1 M Tris-HCl. (PH 7.
5) The solution was dissolved in 20 μl.

After transforming E. coli (HB101) with 10 μl of this DNA solution,
Ap ^r and Tc ^s clones were obtained. Then, the characteristics of the plasmid DNA possessed by these clones were examined, and pTK
-4 / DNA fragment between the BamHI PvuII site and BamHI site (on Tc ^r) (1.76Kbp) the lack plasmid: pTK-4 / BamHI / BamHI
E. coli having (Fig. 7) was obtained. This E. coli was designated as TYG-
After being grown in 1 L of P medium, ccc-DN was extracted from the cells.
A is separated and purified to obtain pTK-4 / BamHI / BanH
I plasmid DNA was prepared.

[0092] ^{(c) pTcosAp r / ori /} TK /
cm ^r / neo creation of ^r (2BamHI) and preparation ^{pTcosAp r / ori / cm r /} neo r 10μg
The reaction mixture was mixed with 100 μl of BamHI (60 units) and 3
The reaction was carried out at 7 ° C for 2 hours for digestion. After deproteinization by phenol extraction, DNA was obtained as an ethanol precipitate, and the precipitate was dissolved in 10 t / 0.1E7 μl (up to 1 μg / μl). On the other hand, pTK-4 / BamHI / BamHI
20 μg was added to 200 μl of the reaction mixture to prepare 240 units of Ba.
After reacting with mHI at 37 ° C for 2 hours to completely digest, it was subjected to 0.8% preparative agarose gel electrophoresis,
A DNA fragment containing TK (2.0 Kbp) was separated and purified to obtain DNA as an ethanol precipitate.
It was dissolved in 0.1E 6.0 µl (~ 1 µg / µl).

[0093] and, pTcosAp ^r / ori / cm
DNA solution 1μl the ^r / neo ^r was digested with BamHI
(~ 1 μg) and pTK-4 / BamHI / BamHI
DNA solution of BamHI fragment containing TK of
μg) with 20 μl of the reaction mixture, and 6 units of T4-D
Ligation was performed by reacting with NA ligase at 12 ° C. for 17.5 hours. Then, phenol extraction was performed to obtain DNA as an ethanol precipitate. The precipitate was extracted with 0.1M Tris-HCl (p
H7.5) solution was dissolved in 20 μl.

Next, 10 μl of this DNA solution was added to E. coli.
After transforming (HB101), A
p^r, Cm^rAnd Km^r(Neo^r) A clone was obtained.
Then, the plasmid DNA of these clones was characterized.
Sex, pTcosAp ^r/ Ori / cm^r/ Ne
o^rWith TK inserted at the BamHI site of
Do: pTcosAp^r/ Ori / TK / cm^r/ Neo
^rE. coli with (2BamHI) (Fig. 7) was obtained. this
E. coli was grown in 1 liter of TYG-P medium and
Ccc-DNA is isolated and purified from the body and pTcosAp
^r/ Ori / TK / cm^r/ Neo^r(2 BamHI)
Was prepared.

[0095] ^{(d) pTcosAp r / ori /} TK /
cm ^r / neo ^r of creation and preparation ^{pTcosAp r / ori / TK / cm} r / neo
^r (2BamHI) 10 μg was added to 100 μl of the reaction mixture,
It was reacted with 14 units of BamHI at 37 ° C. for 30 minutes for limited digestion. And after deproteinizing with phenol extraction,
DNA was obtained as an ethanol precipitate, which was precipitated with 10 t / 0.
It was dissolved in 1E7 µl (~ 1 µg / µl). Next, 1 μl of the DNA solution limited to digestion with BamHI (up to 1 μm)
g) with 25 μl of the reaction mixture, 2 units of Kleno
React with w enzyme for 30 minutes at 22 ℃, cohesiv
0.8 after changing e end to blunt end
% Preparative agarose gel electrophoresis to
DNA fragment (6.93 Kbp) containing s and Ap ^r , ori, TK, cm ^r , neo ^r was isolated and purified to obtain DNA.
Was obtained as an ethanol precipitate.

This DNA precipitate was treated with 20 μl of the reaction mixture to give 6
After reaction with unit T4-DNA ligase at 12 ° C. for 17.5 hours to cyclize, followed by phenol extraction,
The DNA was obtained as an ethanol precipitate and the precipitate was extracted with 0.1 MTr.
It was dissolved in 20 μl of is-HCl (pH 7.5) solution. Then, with 10 μl of this DNA solution, E. coli (HB
101) and then Ap ^r ,
cm ^r and obtained the Km ^r (neo ^r) clone. Then, by examining the characteristics of plasmid DNA possessed by these ^{clones, pTcosAp r / ori / TK /} cm r / n
B between cm ^r of eo ^r (2 BamHI) and TK
The plasmid lacking the amHI site: pTcosAp ^{r
/ Ori / TK / cm r /} neo r ( 3, 7) to give E. coli with. In addition, this strain was designated as "Microindustrial Research Institute No. 7887 (FERM
P-7887) ". The E. coli, after grown in TYG-P medium 1l, was separated and purified ccc-DNA from the cells, pTcosAp ^r / o
Plasmid DNA of ^{ri / TK / cm r / neo} r was prepared.

[0097] Example 3 pTcosAp ^r / ori /
production of gene bank of cm ^r / neo ^r by genomic DNA (exogenous DNA) (Fig. 2) (a) with a nucleotide sequence of the prepared pBR-322 genomic DNA of high molecular weight, herpes thin pre poke virus type 1 (HSV-1 ) Thymidine kinase (T
K) mouse L cells (Ltk
^- [HSV-1TK ⁺ ]) was cultured in Eagle MEM supplemented with 10% fetal calf serum to obtain fluent mo
Nolayers (100 mm-9 plates, -10
⁷ cells / plate) and, PBS (137mMN
aCl, 3 mM KCl, 8 mM Na ₂ HPO ₄ , 1 mM
After washing twice with KH ₂ PO ₄ ), the solubilizing agent [100 μ
g / ml Proteinase K, 0.5% SDS,
150 mM NaCl, 10 mM EDTA, 10 mM Tr
is-HCl (pH 7.5)] at 1.0 per plate
ml was added, and the mixture was kept warm at 65 ° C. for 15 to 30 minutes for solubilization.

The solubilized product was transferred to a centrifuge tube having a volume of 50 ml and further incubated at 37 ° C. overnight, and in the middle of the process, Proteina was used.
seK was further added at 100 μg / ml. After the reaction, gently add 2 volumes of phenol saturated with an equal volume of Tris buffer.
Once, the aqueous layer is extracted with 50 mM Tris-HCl (pH 8.
0) Dialysis was carried out 4 times against 3 l of dialysis buffer consisting of 10 mM EDTA and 10 mM NaCl. The dialysis fluid is
50 μg / ml DNase-free RNaseA
Was added and the mixture was kept warm at 37 ° C. for 3 hours to decompose RNA.
After the reaction, the mixture was gently extracted twice with an equal volume of phenol: chloroform, and the aqueous layer was washed with 10 mM Tris-HCl (p
H8.0) Aqueous solution (TE) 3 consisting of 1 mM EDTA
It was dialyzed 9 times against l to obtain 50 ml of purified DNA solution. The absorbance (OD) of the DNA solution at 260 nm and 280 nm was 0.667 and 0.3 at 10-fold dilution, respectively.
63 (OD ₂₈₀ / OD ₂₆₀ = 0.544) and DN
The concentration of A is 0.667 × 0.050 (μg / l OD) ×
10 = 0.334 μg / μl.

(B) Preparation of 35-45 Kbp Genomic DNA Purified genomic DNA solution (5.98 ml, 2 mg) was added to
Reaction mixture 29.96 ml [50 mM NaCl, 10 mM
Tris-HCl (pH 7.5), 10 mM MgC
l ₂ , 100 μg / ml BSA] was reacted with Sau3A (manufactured by New England Biolabs) of 15.6 unit for 1 hour at 37 ° C. for limited digestion,
1.3 ml of 0.5 M EDTA (pH 8.0) solution was added, and the mixture was gently extracted twice with an equal volume of Tris buffer saturated phenol. Then, after concentrating the aqueous layer to 10 ml with secondary butanol, 1.1 ml of 3.0 M sodium acetate and 20 ml of ethanol were added to obtain genomic DNA as an ethanol precipitate, which was washed with 70% ethanol and dried with a water pump. Perform ethanol precipitation
It was dissolved in 2 ml of E.

Next, 300 μl of this DNA solution was added to 5-2.
5% sucrose density gradient centrifugation [20 mM Tris-HCl
(PH 7.6), 5 mM EDTA, Beckmann SW2
8; 20,000 rpm, 9 hours, 20 ° C.] 3
It fractionated every 0 drops. 20 μl of each fraction was subjected to 0.4% agarose gel electrophoresis and the length was measured.
After collecting a bp-sized DNA fraction, it was dialyzed against TE3l three times. After the dialysis solution was extracted with phenol, the aqueous layer was concentrated with secondary butanol to give DN.
A was obtained as an ethanol precipitate, and the precipitate was dissolved in TE1 ml to obtain a genomic DNA solution of 35 to 45 Kbp.
The OD ₂₆₀ and OD ₂₈₀ of the DNA solution were 0.216 and 0.112 (OD ₂₈₀ / OD ₂₆₀ =
0.519), and the concentration of DNA is 0.216 × 0.
050 (μg / l OD) × 50 = 0.54 μg / μl.

[0102] (c) Preparation of cosmid vector ^{DNA pTcosAp r / ori / cm r} / neo r 40μg
The reaction mixture was mixed with 80 μl of ClaI and 400 μl of the reaction mixture.
The reaction was carried out at 0 ° C for 2 hours for digestion. Then, phenol extraction was performed to obtain DNA as an ethanol precipitate.
It was dissolved in 100 μl of 0t / 0.1E. This ClaI
95μl of DNA solution digested with
[50 mM Tris-HCl (pH 9.0), 1 mMM
gCl ₂ , 0.1 mM ZnCl ₂ , 1 mM sperdimine], and 6 units of phosphatase (Calf in
testal alkaline phospha
Tase, Boehringer Mannheim) and after reacting at 37 ° C. for 15 minutes and at 56 ° C. for 15 minutes, 6 units of phosphatase were further added, and 37
The ClaI fragment was inactivated by reacting at 15 ° C for 15 minutes.

Then, 400 μl of H ₂ O and STE [10
0 mM Tris-HCl (pH 8.0), 1 M NaC
1, 10 mM EDTA] 100 μl, 10% SDS50
μl was added and the mixture was incubated at 68 ° C. for 15 minutes to inactivate the phosphatase. And after deproteinizing with phenol extraction,
DNA was obtained as an ethanol precipitate, which was precipitated with 10 t / 0.
It was dissolved in 30 μl of 1E. Next, 30 μl of this DNA solution digested with ClaI and inactivated with phosphatase
With 500 μl of reaction mixture, BamH of 176 units
It was digested by reacting with I at 37 ° C. for 2 hours. After deproteinization by phenol extraction, the DNA was obtained as an ethanol precipitate, and the precipitate was dissolved in 10 t / 0.1E30 μl to obtain a cosmid vector DNA solution (0.69 μg / μl).
).

(D) Ligation of 35-45 Kbp genomic DNA with cosmid vector DNA 2.6-35 Kbp genomic solution prepared in (b) 2.6
1 μl (1.4 μg) and 0.86 μl (0.59 μg) of the cosmid vector DNA prepared in (c) were reacted with 10 μl of the reaction mixture with 5.9 unit T4-DNA ligase at 12 ° C. for 17.5 hours. Connected. After the reaction was completed, the reaction solution was stored at 4 ° C.

(E) Preparation of packaging extract (1) Preparation of cell extract obtained by sonicating BHB2690 E. coli BHB2690 was diluted with NZY medium (1% NZamin).
e, 0.5% yeast extract, 0.5% NaCl, 0.2%
After measuring the OD ₆₀₀ of an overnight culture solution in 100 ml of MgCl ₂ · 6H ₂ O, pH 7.5, 500 ml of NZY medium preheated to 32 ° C (in a 2 l flask)
Bacteria were inoculated so that the initial OD ₆₀₀ was 0.025, and shaking culture was started at 32 ° C. And OD ₆₀₀
However, when it reached 0.3 (2 hours and 40 minutes after the start of culture), induction of profage was carried out at 45 ° C. for 15 minutes. After that, the culture temperature was changed to 38 to 39 ° C., and further 3
The culture was vigorously shaken for a period of time (induction was examined by adding one drop of chloroform to a small amount of the culture medium).

After completion of the culture, the cells were collected by centrifuging at 6,000 rpm for 10 minutes to collect the cells at 20 mM Tri.
s-HCl (pH 8.0), 1 mM EDTA, 5 mM β
-Suspension in 3.6 ml of a buffer consisting of mercaptoethanol. Then, after performing ultrasonic treatment (maximum output for 5 seconds × 20 times) at 4 ° C. or less, 20,000 rpm
Centrifuge for 10 minutes at 4 ° C (up to 3m)
1 was obtained. Next, in 3 ml of the supernatant, an equal amount of the above-mentioned buffer and packaging buffer [6 mM Tris-HCl (pH
8.0), 50 mM spermidine, 50 mM putrescine, 20 mM MgCl ₂ , 30 mM ATP, 30 mM β
-Mercaptoethanol] 0.5 ml and then 1
5 μl of each was dispensed into 1.5 ml of Eppendorf tube, immediately frozen in liquid nitrogen, and stored at −70 ° C.

(2) Preparation of bacterial cell extract obtained from BHB2688 by freezing and thawing Escherichia coli 2688 was cultured overnight in 100 ml of NZY medium, the OD _{600 of} which was measured, and then preliminarily kept at 32 ° C. 500 ml of plated NZY medium (in a 2 l flask)
Bacteria were inoculated so that the initial OD ₆₀₀ was 0.025, and shaking culture was started at 32 ° C. And OD ₆₀₀
When it reached 0.3 (2 hours and 40 minutes after the start of culture), induction of profage was carried out at 45 ° C. for 15 minutes. After that, the culture temperature was changed to 38 to 39 ° C., and further 3
The culture was vigorously shaken for a period of time (induction was examined by adding one drop of chloroform to a small amount of the culture medium).

After completion of the culture, the cells were collected by centrifuging at 6,000 rpm for 10 minutes to collect the cells, and the sucrose solution [1
0% sucrose, 50 mM Tris-HCl (pH 8.
0)] After being suspended in 3 ml, 0.5 ml of each of Eppendorf tubes was placed in 6 tubes, and a lysozyme solution [2 mg / ml, 0.25 MTris-H was added to each tube.
Cl (pH 8.0)] 25 μl was added (4 ° C.), mixed gently and quickly frozen in liquid nitrogen. Next, thaw the tube on ice to thaw the extract,
25 μl of packaging buffer was added to each tube and mixed. And each extract is put together to 21,000
A supernatant was obtained by centrifuging at rpm for 1 hour (4 ° C.), and 10 μl of the supernatant was dispensed into 1.5 ml of Eppendorf tube and immediately frozen in liquid nitrogen.
Stored at ° C.

(F) In vitro packaging BHB2690 and BHB26 stored at -70 ° C.
BH that 88 cell extract is put on ice and thawed first
The B2688 extract was added to the BHB2690 extract and gently mixed. When almost everything melted,
2.5 μl of the recombinant DNA solution prepared in (d) (0.
5 μg) was added and mixed well, and the mixture was allowed to react at room temperature for 1 hour for in vitro packaging. After completion of the reaction, SM solution [0.58% NaCl, 0.2% Mg
_{SO 4 · 7H 2 O, 50mMTris} -HCl (pH
7.5) 0.01% Gelatin] 1 ml and 1 drop of chloroform were added and mixed. Then, the Eppendorf tube was put on a desk centrifuge for 30 seconds to obtain a supernatant as a transduction-charge particle solution.

(G) Transduction and plating 200 μl of an overnight culture of Escherichia coli HB101 in L-broth (with 0.4% maltose added) was collected on a desk centrifuge to collect 100 cells of 10 mM MgCl ₂ solution.
Resuspended in μl. Next, 100 μl of this bacterial cell suspension was diluted 10-fold with SM solution to give a solution 1 for the transduction phage particles.
After adding 00 μl and incubating at 37 ° C. for 15 minutes for transduction, 1.1 ml of L-broth was further added, and 37
The cells were shaken at 45 ° C for 45 minutes. Then, 100 μl of this cell suspension was applied to a 100 mm L-plate containing 20 μg / ml of ampicillin, and the plate was kept at 37 ° C.
After overnight, attach the resulting colony with a bamboo stick and
-Grown in broth (containing 20 μg / ml ampicillin). The number of colonies per plate is 20-10
It was 0.

(H) Cultivation of transformant and preparation and characterization of plasmid DNA carried by the transformant.
After culturing overnight in 2 ml (containing μg / ml), each culture solution was placed in 1.5 ml of Eppendorf tube and centrifuged on a desk to collect the cells. Add these cells to solution I [50 mM
Glucose, 25 mM Tris-HCl (pH 8.
0) 10 mM EDTA, 4 mg / ml lysozyme) 1
After resuspending in 00 μl, the mixture was placed at room temperature for 5 minutes. Next, Solution II (0.2N NaOH, 1% SDS) 200μ
l, gently mixed and kept on ice for 5 minutes, and then solution III [5M potassium acetate (pH 4.8)] 150 μl
And gently mixed and placed on ice for 5 minutes. Then, Eppendorf tube was subjected to a desk centrifuge to obtain a supernatant liquid, and 2 volumes of ethanol was added thereto to obtain a plasmid DNA as an ethanol precipitate.

Plasmid D of each transformant
NA was dissolved in a small amount of 10 t / 0.1E and then size marker DNA (lambda DNA: 48 Kbp, lambda DNA digested with HindIII: 22.3 Kb
p, 9.5 Kbp) and subjected to 0.4% agarose gel electrophoresis to give ampicillin resistant colonies 2
The presence of plasmid DNA was observed at a size of 22.3 Kbp to 48 Kbp among all 0.
At this stage, the recombinant DNA exists as a plasmid, and the gene bank of the foreign DNA is the plasmid DN.
Manufactured as A.

[0112] (i) strain Ramudafuaji to transduction and in vivo Patsukejingu E. coli HB101 is lysogenic LN900 ^{[HB101 (λCI ts, Tc r)} : 30
At 37 ° C, lambda quartage is lysogenized, but at 37 ° C, lambda quartet shifts to the lysis cycle. It is also tetracycline resistant. ] Was cultured overnight at 30 ° C. in L-broth (0.4% maltose added). 200 μl of the culture solution was put on a desk centrifuge to collect the cells, and the cells were resuspended in 100 μl of a 10 mM MgCl ₂ solution.

Next, to 100 μl of this cell suspension,
After adding 100 μl of the transfection phage particle solution prepared in (f) (10-fold dilution with SM solution) and incubating at 30 ° C. for 15 minutes for transduction, L-broth 1.1 was further added.
ml was added, and the mixture was shake-cultured at 30 ° C. for 45 minutes. After that, ampicillin and chloramphenicol were added so that the final concentration was 20 μg / ml, and the total volume was 2 ml.
(L-broth: 0.4% maltose added) was cultured overnight at 30 ° C.

Next, 25 μl of this culture solution was added to L-broth (maltose 0.4
%, Ampicillin (20 μg / ml, chloramphenicol 20 μg / ml) was inoculated into the mixture, and shaking culture was started at 30 ° C. Then, when the OD ₆₀₀ reached 0.3 (3 hours after the start of culture), the mixture was allowed to stand at 40 ° C. for 10 minutes for induction, and then the culture temperature was changed to 37 ° C. and shaken for 30 minutes. Cultured. Then, to 100 μl of this culture solution, 9.9 ml of L-broth (0.4% maltose added) which had been kept warm at 37 ° C. was added, and after further shaking culture at 37 ° C. for 2 hours, several drops of chloroform were added. In addition, mixed.

Next, this solution was centrifuged (4,500 r).
pm, 10 min) to obtain the supernatant as a solution of transduction fare particles. In addition, 50 μl of this transfection phage particle solution was incubated with E. coli L at 30 ° C. overnight.
After adding 1 ml of N900 culture solution and incubating at 30 ° C. for 5 minutes for transduction, 50 μl of this bacterial cell suspension was applied to L-plate (containing 20 μg / μl of ampicillin), and the plate was heated to 30 ° C. I left it overnight at
1,000 to 3,000 colonies were observed per plate. At this stage, the recombinant DNA is present as transduction phage particles and the gene bank of foreign DNA is produced as transduction phage particles.

[Brief description of drawings]

FIG. 1 is a new cosmid vector pTcosAp ^r / or
shows the i / cm ^r / neo ^r.

FIG. 2 Genome D using the novel cosmid vector of FIG.
1 shows a method for producing a NA gene bank.

FIG. 3 is a new cosmid vector pTcosAp ^r / or
i ^r / TK / cm ^r / ne o ^r .

[Figure 4] ^{pTcosAp r / ori / cm r /} neo r
It shows how to create.

[Figure 5] subsequent to FIG. 4 pTcosAp ^r / ori / cm ^r
/ How to create a neo ^r shows a.

[6] subsequent to FIG. 5 pTcosAp ^r / ori / cm ^r
/ How to create a neo ^r shows a.

[7] ^{pTcosAp r / ori / TK / cm} r / n
This shows how to create eo ^r .

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Yoshio Okada 2-22-28 Hanmachi, Minoh City, Osaka Prefecture

Claims (2)

[Claims] 1. At least three cos of lambda or lambdoid having the same orientation and having at least one or two cos obtained by cutting with two kinds of restriction enzymes. Various foreign DNA fragments with a length of 35 to 45 Kbp were inserted between the right arm and the left arm, which were transformed into transduced sphere particles by in vitro packaging, and then cloned into Escherichia coli lysogenized by lambda phage. Then, by induction, the recombinant DNA is transformed into transduced phagy particles by in vivo packaging to induce the foreign D
A method for producing a gene bank using a novel cosmid vector, which comprises producing an NA gene bank. 2. The method for producing a gene bank according to claim 1, wherein the exogenous DNA is a DNA derived from a microorganism such as a bacterium, a filamentous fungus, a yeast, a higher animal or a plant, various types of phages and the like.