JPS60256382A - Novel cosmid vector and production of gene bank using the same - Google Patents

Abstract

PURPOSE:A novel cosmid vector forming equimolar right and left arms with one cos (cohesive end site), respectively, is prepared by cleavage with a restriction enzyme and the vector is used as a fraction of 35-45Kbp to enable the high-efficiency formation of a gene bank of genome DNA. CONSTITUTION:The objective cosmid vector includes 2 coses of lambda-phage or lambdoid phage with the same direction of sequence. The vector is cleaved with 2 restriction enzymes to form right and left arms containing one cos, respectively and an external DNA fraction of various kinds having 35-45Kbp length is inserted between the two arms to form a tranducing phage. Then, the phage is cloned into Escherichea coli to prepare a gene bank of external DNA. An example of the novel vector is pDcosAp<r>/ori shown in the figure.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to genetic engineering technology, and more specifically to a novel cosmid vector created using genetic engineering technology and a method for producing a gene bank using the same.

The size of genes in mammals, including humans, is often over 20 kilobase pairs (hereinafter referred to as Kbp). Nowadays, it is widely used.
<Ifi-to? o-y(fZT@, 6DNA.

Because it is 20 Kbp in size, it is not possible to functionally clone an entire gene much larger than this.

lambda phage cohesive end 5ite
(hereinafter abbreviated as COS), ie, a cosmid vector, it is possible to clone DNA with a size of 35 to 45 Kbp. Among various vectors, cosmid vectors are the most capable of cloning DNA of the largest length, but they are difficult to prepare long genomic DNAs, have a high packing ground for vectors without inserts, and The use of cosmid vectors has not progressed due to obstacles such as improved efficiency. However, Ish-Horowicz & Burke used phosphatase to give vectors directionality, and succeeded in reducing the background value of the vector alone and increasing cloning efficiency.
Ish-Horowicz, & J. F. Burk
e, Nucleic Ac1ds Res, q9+28
89, (1981)).

The method for producing a gene bank of genomic DNA by the Ish-Horowicz & Burke method is as follows (for example, pJB-8t, which is one of the cosmid vectors currently widely used).
is 5.44 Kbp in size, and the restriction enzyme Ban HI
It is a cosmid vector that has been cut at one site each with , HindIII, and 5alI and has one COS of lambda phage. (2) pJB-8 is cut with Hind m to obtain linear DNA, and then the Hind m cut fragment is inactivated by treatment with phosphatase. Then, by cutting at 11am HI, two D
NA fragments are obtained, and large fragments containing coS, NA
m pieces (5,4 Kbp) are isolated by agarose gel electrophoresis. The obtained cos t-containing DNA fragment <
5.4 Kbp) corresponds to the left arm of the lambda phage. (2) Linear DNA is obtained by cutting pJB-8 with 9al I, and then the 5al I cut fragment is inactivated by treatment with phosphatase. and,
Two DNA fragments were obtained by cutting with BamHl, a small NA fragment containing coa (2,34 Kb
p) is isolated by Aga-4-Sugul electrophoresis. The obtained DNA fragment (2,34 Kbp) containing cos k corresponds to the right arm of lambda phage. ■For example, human genomic DNA is digested with restriction enzymes sau aA or Mb.
Partially cleaved at oI, D of size 35-45 Kbp
Fractionate the NA fragments. This 35-45 Kbp DNA fragment is inserted between the left and right arms prepared separately in ① and ② using a ligating enzyme (T4-DNA'J gauze) to prepare recombinant DNA. ■, recombinant DN
By converting A into transducing phage particles by 1nvitro packaging and then infecting E. i bacteria, a gene bank of human genomic DNA can be produced.

The above is the method for producing a genomic DNA gene bank using the Ish-Horowicz & Burke method.
There are three problems: First, pJB-8 is COW
There is only one cocoon, and the left and right arms must be prepared separately.Secondly, for the left and right arms, each
DNA fragments containing s must be separated by agarose gel electrophoresis. Third, the length of the left arm of pJB-8 is 5.4 Kbp, and the length of the right arm is 2.34 Kbp, and the length of the left arm and right arm are different, so it is 35 to 45 Kbp.
The probability of linking to the genomic DNA of p is also different, and the ratio of left and right arms to be mixed must be considered.

In order to solve these problems, the present inventors have two lambda phages 008 with the same directionality,
By cutting with two types of restriction enzymes, each COS
We created a new cosmid vector characterized by having equimolecular right and left arms of approximately equal length, each having one [36th Annual Meeting of the Japanese Society of Cell Biology, Abstracts of Presentations, 93]
．． 94 pages (1983)). As a result, we have completed a method for producing a foreign DNA gene bank using the cosmid vector of the present invention.

The cosmid vector of the present invention has two cos having the same directionality in the same molecule, and by cutting it with two types of restriction enzymes, the cosmid vector has almost equal length right arms each having one Co11. Since the same moles of the left arm and the left arm can be obtained, prepare the left leg and right arm separately. , 35-45 Kbp
Genome DNA VCT 4-DNA! It can be assumed that the degree of ligation by gauze is approximately equal, and if an equimolar mixture of the right and left arms cut with two types of restriction enzymes is directly ligated with 35 to 45 Kbp of genomic DNA, a recombinant DNA can be created. It can be expected that half of the DNA will be transformed into transducing phage particles. As described above, by using the vector of the present invention, a gene bank of genomic DNA can be constructed more easily and efficiently as 35 to 45 Kbp DNA fragments.

pDcos, one of the novel cosmid vectors of the present invention
Aprlori is shown in FIG. 1 [36th Annual Meeting of the Japanese Society of Cell Biology, Abstracts of Lectures, page 93 (1983)].

pDcos Aprlori is a co-directional cos derived from the phage vector Charon 4A.
2 and the neomycin resistance gene (neor) derived from Tn 903 [Oka, Sugisaki & Tak
anami, J, Mo1. Biol,,l 47.

217 (1981)), ampicillin resistance gene (Apr) and origin of replication (ori) derived from pBR-327
,,1゜(5oberon, Cavarrubias
& Bolivar, Gene, i287 (1980)
] Consists of. It is 4.7 Kbp thick and is cleaved at one site each with restriction enzymes pvu II, Bam HI, and Pst I, at two sites with restriction enzyme BstEniC, and at three sites with restriction enzyme Eco RI.

This vector has the following characteristics. ■There are two c'os with the same directionality. (2) Genomic DNA of 35 to 45 Kbp that has been limitedly digested with 5au3A or Mbo I can be cloned into the BamHI site. (2) The insert can be excised from the vector by cutting the Eco RI sites on both sides of the Bam HI site. (2) Cutting the vector with pvulI and treating it with phosphatase gives the vector directionality, and further cuts the BamHI site to create equimolar right and left arms. ■T 4-DN
A') Using gauze, 5aIA3A or Mb
o Genome D of 35-45 Kbp limitedly digested with I etc.
The right and left arms of the vector were ligated at the Bam HI site on both sides of the NA, and the recombinant DNA was injected in vitro.
It can be transformed into transducing phage particles by packaging and then infected into E. coli. (2) Since this transductant DNA contains Apr and ori derived from the vector, by selecting with ampicillin, only the large MJ bacteria having the recombinant DNA as a plasmid can be selectively kept alive. Furthermore, the method for producing a gene bank of genomic DNA (foreign DNA) using this vector is as follows (see Figure 2).

(2) By cleaving pDcos Apr/ori with pvu IF and Bam HI, a right arm and a left arm each having one COS and having approximately equal length are prepared. ■, Exogenous DNA is limitedly digested with BaIAaA Moshike Mb o I, etc., and 35-45 K
Prepare DNA fragments of bp size. ■, 35-45
A recombinant DNA is prepared by inserting the Kbp genomic DNA fragment between the left arm and right arm prepared in ① using [T4-DNA') gauze. ■, recombinant DNA i
n vitro packaging into transducing phage particles and infect E. coli. (2) By selecting the transduced ampicillin-resistant E. coli with ampicillin, only E. coli having the recombinant DNAff1 plasmid can be utilized.

In this way, a gene bank of genomic DNA can be created.

In addition, the present inventors have developed a cosmid vector, pDcos Tc, for the purpose of gene recovery from transformant cells.
r (Fig. 3), pDcos Tcrlori (Fig. 4)
(Figure) was successfully created (36th Annual Meeting of the Japanese Society of Cell Biology, Collection of Abstracts, p. 94 (1983)). Mammalian marker genes can be isolated using gene transfer into cultured mammalian cells.

At that time, there is a work to recover the DNA sequence that has been transferred into cultured cells from transformant cells into E. coli.
Novel cosmid vector pDcos encompassed by the present invention
Tcr and pDcos Tcrlori are useful vectors for this gene recovery. pDcosTcr
is the same directional 008 from Charon 4A
2, a tetracycline resistance gene (Tcr) derived from pBR-327, and an origin of replication (ori). pDcos TCrlori similarly cog
2, TCr and art derived from pBR-327, and the Sv-dihydrofolate reductase gene (SV-dhfr) as a spacer (Subramani, Mu
lligan & Berg, Mol.

Ce11. Biol, 1.854 (1981))
It consists of These vectors are pDe0 as previously described.
8Apr10ri (Fig. 1) and can be used to produce a gene bank of genomic DNA in the same way, but Sau 3A or Mb o I is added to the BglII site instead of the BamHI site.
The difference is that 35 to 45 Kbp genomic DNA that has been limitedly digested with, etc., can be cloned. pDcos Tc
Transducing phage particles made with r are plasmid or
Since it does not have i, it cannot multiply even if infected with E. coli.

The gene of interest can be recovered in E. coli as a tetracycline-resistant (Tcr) clone only when the ori is located near the gene of interest. On the other hand, pDcosT
Transducing phage particles made with crlori are or
It is possible to create a gene bank of genomic DNA as a Tc' clone by carrying the I and TCr markers. If there is a drug resistance marker such as AI)r near the gene of interest, only the drug resistance marker such as ampicillin resistance (Apr) can be selectively recovered in E. coli.

Furthermore, the present inventors have developed a cosmid vector, pDcosApr10ri/g, which has a selectable marker for mammalian cells and is intended for gene transfer into mammalian cells.
pt/'I'K (Figure 5) was created. pDcosA
pr10rt/gpt/TK contains two codirectional cos molecules derived from Charon 4A and pBR-327.
Apr and ori of origin.

psV2-dhfr-derived SV as a spacer
-dhfr gene, gpt gene C from pSV2 gpt, Richard.

Mulligan & Berg, Mo1. Ce1l,
Biol, 1, 449 (1981)), and pT
TKK gene derived from K-4 [l5hiura, M.
etal Mo1. Ge11. Biol, 2,607
(1982) ). This cosmid vector can be used to produce a gene bank of genomic DNA, similar to the above-mentioned pDcosAprlori. In other words, two cos having the same directionality, pv
By cutting with u II and Bam HI, cos
It is possible to create a right arm and a left arm with equal moles of 4, each having one molecule, and to create a recombinant DNA in which 35 to 45 Kbp of genomic DNA is inserted into the Bam HI site. And, this river recombinant DNA is in vitro
The recombinant DNA can be transformed into large phage particles by packaging and transferred to E. coli, and the recombinant DNA contains vector-derived Apr and ori.
Escherichia coli containing E. coli can be obtained as ampicillin-resistant clones by selecting with ampicillin.

Furthermore, since this recombinant plasmid carries the gpt gene and the TK gene, it can be used to culture mammals; By using TK) activity as a marker,
Determination of insertion of recombinant DNA derived from the vector is performed by culture a.
It has the advantage of being possible at the cellular level.

The present invention relates to such a novel cosmid vector and a method for producing a gene bank using the same. Examples of the COS used in the present invention include those derived from lambda phage or lambdoid phage. In particular, COS derived from lambda phage is preferred.

By cutting the vector of the present invention with two types of restriction enzymes, a right arm and a left arm each having one COS can be created.The combination of these two types of restriction enzymes is Pvu II and Bam:HI. , Pvu II and B
gl II, C1a I and Bam HI, C1a
Examples include Bgl I and Bgl II. Of course, other combinations can be considered, so the invention is not limited to these. Generally, in order to determine that a vector-derived DNA fragment has been inserted into the vector of the present invention, Selection is based on drug resistance. Therefore, it is preferable that a DNA fragment expressing drug resistance is also incorporated into the vector of the present invention. Drug resistance includes ampicillin resistance,
Tetracycline resistance, neomycin (kanamycin)
resistance or common antibiotic resistance such as chloramphenicol resistance. pDcos Aprlori
has ampicillin resistance genes and neomycin resistance genes, pDcosTcr and pDcosTcr10r.
i trif ) It has a rat icrin resistance gene. pDcosAp '10 r i/gpt
/TK has an ampicillin resistance gene.

Using the vector of the present invention, genomic DNA (foreign DNA)
) When producing a gene bank and transferring the gene (gene bank) into cultured mammalian cells, vector-derived D
Preferably, a DNA fragment expressing a specific physiological function is incorporated into the vector of the present invention in order to determine whether the NA fragment has been inserted. Examples of genes that express specific physiological functions include the thymidine kinase (TK) gene [WxglerpM-etzla, C
el l r 14+ 725 (1978), adenine phosphoribosyltransferase (APRT) gene [Wigler, M, 7et eLL, Pro,
Natl, Acad, Sci.

U, S, A, , 76.1373 (1979)),
Hyhoxanthin quanine phosphoribosyltransferase (HGPRT) gene (Graf, L, H, Jr.
,et41,,Somat,Ce1l,Genet
, .

and, 1031 (1979)), xanthine guani 1.
449 (1981)). Novel cosmid vector of the present invention pDcosApr10ri/gpt
/TK has a gpt gene and a TK gene.

Furthermore, it is preferable that the vector of the present invention contains a gene fraction having multiple functions derived from Co1EI.

Examples of foreign DNA used in the present invention include DNA derived from microorganisms such as bacteria, filamentous fungi, and yeast, as well as from various phages of higher animals and plants.

Furthermore, the relationship between the restriction enzymes used in the present invention (enzymes that have the function of selectively cleaving a specific base sequence site of a DNA fragment) and the DNA adhesive ends generated by restriction enzyme cleavage, and the complementary relationship thereof. An enzyme (ligase) used to join the sticky ends of certain other DNA fragments, or an enzyme that turns DNA ends into mutually complementary sticky ends in order to join DNA ends that are not complementary to each other, or Ligases that bind ligases are already well known, many enzymes are commercially available, and methods for using them have also been widely disclosed [Mo1ecu
lar Cloning, Co1d Spring H
arbor Laboratory, Maniatis,
T,,eta,1. , (1982)), so you can use them.

By using the vector of the present invention, it will become easier to analyze cancers, genetic diseases, etc. in mammals including humans at the molecular level. For example, a gene bank of genomic DNA derived from normal human cells is converted into pDco.
sAprlorif is created and genetically diseased cells are transformed (normalized) using this gene bank to obtain transformant cells. Genome D from transformant cells
Extract NA and add pDcosTcr to pDco
A gene bank of genomic DNA is created using sTc rlo r i and the gene of interest is recovered.

By examining the gene of interest from various aspects, human genetic diseases can be analyzed at the molecular level.

Furthermore, using the vector of the present invention, genomic DNA (foreign D
By creating a gene bank of NA), it is possible to efficiently discover specific genes that express useful substances. This specific gene includes DNA derived from microorganisms such as #1 bacteria, filamentous fungi, and yeast, higher animals and plants, and various phages.Specific useful substances include, for example, various hormones, enzymes, and even antibiotics. Examples include enzymes that synthesize substances and amino acids.

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example ID Creation and preparation of cosTcr (Figure 6) (a
t pAPcos (7) Creation and Preparation 1) CO derived from Charon 4A, a phage vector created
pH079 (H
ohn & CCo11in, Gene, 11,29
1 (1980)) 204 to the reaction mixture 200 Ill
(l OmM Tris-HCI (pH 7,6), 1
2 mNL NaC12, 10M DTT.

100μfiALI BSA (bovine disintegrated albumin)
So, 29.9! unit of restriction enzyme Bal I (N
After complete digestion by reacting with ew England Biolabs for 2 hours at 37°C, 500 μl of reaction fluid (150 mM NaCl, 6 mM Tris
-HCI (pH 7,9), 6 tnM NaCl2,6
mM DTT, 1004/Ill BSA), 4
1.1 unit of restriction enzyme BstEn (New En
grand Biolabs M) at 60° C. for 2 hours to complete digestion. After the reaction is complete, extract with an equal amount of water-saturated phenol, concentrate the h1 aqueous layer with secondary butanol, and add 0.1 times the volume of 3M alcohol 41 otoso + '7A.
and 2 times the volume of ethanol to obtain DNA as a precipitate, washed with 95% ethanol, dried with a water pump, and precipitated with 1 OmM Tria-HCI (pH 8).
, 0), o, im MEDTA (hereinafter abbreviated as 10t10.I
It was dissolved in (Miima)).

Next, this Bal I and BstE II digested DN
1 pi (~Lttl) of solution A was added to 25 μm of the reaction mixture (5 mm M Tris-HCI (pH 7,2),
10 m M M”y SO,.

0, 1 m, MDTT, 50 ttllAnlB S
A, 80 pM dcTP'.

80 9TP), 2 units of Klenow enzyme (Boehringer Mannheim) and 2 units of Klenow enzyme (manufactured by Boehringer Mannheim).
React at 2°C for 30 minutes to confirm that the two strands have mutually complementary strands.
Double-stranded DNA fragment (coheaive end) with both ends aligned (blunt end)
changed to

After the reaction, the reaction solution was heat-treated at 65°C for 10 minutes to remove Kl.
The enow enzyme was inactivated.

Then, 10μ7 of this solution (~0.4μg of DNA) was added to 50μ/(50mM Tris-HCI (pH
7,4), 10 m M MyC12, l OmMDT
TX 1mM spermidine, 1mM ATP, 10
0 pg/fil BS A), 15 units of T4
-DNA ligase (New England Biola
A DNA fragment (4,75
Kbp) was ring-closed. After the reaction was completed, phenol extraction was performed, and the DNA was precipitated with ethanol, and the precipitate was dissolved in 20 pl of 0.1 M Trim-HCI (pH 7,5) solution using a VC susceptor.

11) Transformation Transform 10μ of this DNA solution into E. coli HBIOI.
Etent cell 1001tlc logarithmically growing E. coli 1 (BJOlt - 0.1 M CaCl at 0-4℃
, after treatment with the solution for 15 minutes, the original culture solution was removed. 0. Resuspend in 1 M CvCA/14% glycerol solution, mix well (stored at -70 U) and incubate at 0 °C for 1
After incubating at 37°C for 5 minutes to infiltrate the DNA into E. coli, add L-gloss (1% Ba
ctotr-yptone, 0.5% yeast extract 0.
Add 27d of 5% NaCl, 0.1% glucose),
The culture was incubated with shaking at 37°C for 1 hour. 20μ of this bacterial cell Mm solution
A plate containing 7fnl of ampicillin and 20 μg of tetracycline) (a plate containing 1% agar in L-broth) was plated and incubated at 37°C for 24 to 30 minutes.
After culturing for 48 hours, ampicillin and tetracycline resistant clones were obtained.

111) Small-scale culture and small-scale DNA preparation Ap rIT
Cr's eyes are L-Pros 2 in the waste (Ap20
(containing μW) at 37°C overnight, propagate to tc0 culture, and add 1.5 at to an Eppendorf tube9.
After collecting bacteria using a desk centrifuge, extract the bacteria* (8
% sucrose, 0.5% TritonX-100,50
mMEDTA (pH 8,0), i.

m M Tris-HCI (pH 8,0)) 0.35
Resuspend at 1 m. Next, lysozyme solution 25μ7
(10mg/go, 10mM Tris-HCI (pH
After adding and mixing, the tube was placed in boiling water for 40 seconds. Then, the tube was immediately centrifuged at 4°C (21,00 rpm for 15 minutes) to obtain a supernatant, which was then added with wisteriase A solution (20w, y
/ml.

1 otlo, tE) Add 0.5 μl and heat at 37°C.
The mixture was kept warm for 1 hour to degrade the RNA. Thereafter, the protein was removed by phenol extraction, the DNA was precipitated with ethanol, and the precipitate was dissolved in 10 tlo and IE 20 till K to obtain a plasmid DNA solution.

iV) Characterization of plasmid DNA and 1μl or 2tt1 of this plasmid DNA solution
1, restriction enzymes EcoRI, BstE]I, BglII,
After Pvull digestion, 0.4-1.5% agarose gel electrophoresis (40mM Tris, 20mM
M sodium acetate, 2 m M EDTA (pH 8,
15) ; l 07? L.A.

It took about 12 to 24 hours). Decomposed DNA fragments can be detected by ethidium bromide staining and long-wavelength ultraviolet illumination. In this manner, the plasmid DNA possessed by each clone was characterized by constructing a restriction map of the plasmid DNA. And P? 1079 Ba1
l DNA fragment between the BstEII site and the BstEII site (1
, 68Kbp) Gold-deficient plasmid: pAPcos
(Fig. 6) was obtained.

■) Large-scale culture and preparation of plasmid DNA This E. coli was cultured in TYG-P medium (1% Bactory-pto
ne, 0.1% yeast extract, 0.2% glucose, 0
, 1% NH,C1,0,3%NaC1,0,01%
Na25o.

0.08% MgCl2-6H20, 1,52% Na2
f(PO, *12H20,0,3To KF(2PO4
) 17 Cultured with shaking at 37°C, and the absorbance at 600 nm (A6oo) reached 1.0 at 7'C. and cultured with shaking at 37°C for an additional 15 hours.

fft After the completion of the culture, centrifuge (6,000 rpm, 10
25 min) to collect the bacteria, and then collect the bacterial cells using 25 tisiosaccharide, 50mM '□Tris-HCI (pH 8,0).
To this, a lysozyme solution [5 m9/ml, 0.25 M Tris-
Add 3.6 ml of HCI (pH 8,0)), mix gently, and leave at 0°C for 5 minutes.
7.2 ml of DTA (pH 8,0) bath rfi was added, mixed gently, and incubated at 0° C. for 5 minutes.

After that, 2.7 ml of 1O% SDSg solution and 5M NaCl
Add 1 ml of solution B, mix gently, leave overnight at 0°C, and centrifuge the mixture at 4 m (20,00 rpm).
.

45 minutes) to remove the crude lysate (cleared lysate).
ate)~39ml was obtained. In this crude lysate, RNas
e Am liquid (20η/WLl, 1Ot10.IE)
After adding 20 μl and incubating at 37° C. for 1 hour to degrade the RNA, protein was removed by phenol extraction, and the mixture was subjected to gel filtration with Sepharose 2B. DNA was obtained from the void fraction containing Plasmid DNA by ethanol precipitation, and then the DNA was precipitated with cesium chloride-ethidium bromide solution (IMTris-HCI (pHs,
0 ) 0375 I, 0.25 MEDTA (pI(8
,0)O,a Oml, H,06,45mJ,CsC
17.5g.

EtBr solution (4 ta/mJ) (0.375 ml) was subjected to equilibrium density gradient centrifugation (60,000 rpm, 12 hours).

Then, the plasmid D is covalently closed and the transferable plasmid D
When NA (cce-DNA) is exposed to long-wavelength ultraviolet rays in a centrifuge tube, it appears as a band below the fluorescent bands of linear plasmid DNA and chromosomes. This ccc-D
The NA band was fractionated and extracted with an inproper tool saturated with 08C1 aqueous solution to increase endium bromide.
After removal, dialysis was performed against Lotlo and IE1.

Then, ccc-DNA was obtained from the dialyzed fluid as ethanol precipitation, and the precipitate was collected at 10t10. Dissolve pAPcos plasmid DNA in 2 ml of lE (0,41
8 da/hemp) was prepared.

bl Creation and preparation of pAPcos△ (EcoRI) Mix 10 μm of pAPcos in the reaction mixture fl 00 t
tlcl 00mM Tris-HCI (pH7,5)
, 50mM Nacl, 5mMMy01□, 10
0 μl Ad BSA) and 60.6 units of the restriction enzyme EcoRI (New England Biol
Abs M) at 37° C. for 2 hours to completely digest. After protein removal by phenol extraction, the DNA was precipitated with ethanol for 10t10. I E7μノ(
~lμVμl).

Next, add 1 μl of this EcoRI-digested DNA solution (
~LAjq) and 25 μl of the reaction mixture (50 mM Trl
s-HCI (pH 7,2), l Om MMyS04.
0.1 rnM DTT.

50 μi AI BSA, 80 μM dATP, 80
μM dCTP, 80μM dGTP, 80μMd
The cohesive end was purified by reacting with 2 units of Klenow enzyme at 22°C for 30 min.
Changed to unt end. Then, the reaction solution was heated to 65°C for 1
The Klenow enzyme was inactivated by heat treatment for 0 minutes.

Next, ioμl (~0.4μ, !i') of this DNA solution
Add 50 μl of the reaction mixture (50mM Tris-HCI (
pH7,4), 10 rn M M2CI□, i 0m
MDTT, 1mM spermidine, 1mM T P
After reaction with 15 units of T4-DNA ligase at 12°C for 17.5 hours at 100 µl/fnlBSA), cyclization was performed, followed by phenol extraction.
) NA was obtained as ethanol precipitate 1,) precipitate was 0.1
M Tris-HCI (pH 7,5) solution 20
It was dissolved in ttl.

Then, add 10 μl of this DNA solution to Escherichia coli (f(B10
) was transformed into AI)r and TCr clones. Next, we performed small-scale culture and small-scale DNA analysis of these clones.1) The characteristic participle of NA and the plasmid possessed by each clone, pAP
Plasmid lacking cog EC0RI site: pA
E. coli harboring PcosΔ(EcoRI) (Figure 6) was obtained.

After growing this E. coli in 1 liter of TYG-P medium, ace-DNA was isolated from the bacterial cells and pAPco
Plasmid DNA of sΔ(EcoRI) was prepared.

(cl pHPcos creation and preparation pAPcosΔ (EcoRI) 20 ttl was added to the reaction mixture at 200 pH (60 mM Nacl, 6 mM
is HCI (pH 8,0), 10 m-MMiC1
26mM DTT, 100μWBSA), 75
．． 8-L 2” restriction enzyme AvaI (New Eng
landBiolabs Inc.) for 2 hours at 37°C for complete digestion, and then add 500 μl of the reaction mixture (
60mMNac1.7mMT'ris 7HC1 (p
f(7,4), 7mMM@C12, 100μIIALl
BSA), 101 units of nd for restriction enzymes.
It was completely digested by reacting with llI (manufactured by New England Biolabs) at 37°C for 2 hours. After protein removal by phenol extraction, DNA was precipitated with ethanol, and the precipitate was extracted with 10 ml of ethanol. IB 14 μl (~
1μl! yltl).

Next, 1 μl (~1 μg) of the DNA solution digested with AvaI and Hlnd m was added to 25 μl of the reaction mixture. Then, the cohesive end was changed to a blunt end by reacting with 2 units of Klenow enzyme at 22°C for 30 minutes. Then, the reaction solution was heat-treated at 65°C for 10 minutes to
lenow enzyme was inactivated.

Next, add 10 μg (~0.4 μg) of this DNA solution to 50 μg of the reaction mixture. and reacted with 15 units of T4-- DNA ligase at 12°C for 17.5 hours to separate
ri, AI)' DNA fragment containing f (3,75 Kbp
) was ring-closed ( Hind [site was reconstructed and vol.

After that, phenol extraction was performed to extract I, -s, DNAt-
Obtained as ethanol precipitate, precipitate was 0.1 M Trim
-Dissolved in 20μ71C of HCI (pH 7,5) solution.

Then, add 10 μl of this DNA bath solution to Escherichia coli () (BIO
After transforming I), Apr KatsuT
c (tetracycline sensitive) clone was obtained. Next, the characteristics of the plasmid DNA of these clones were investigated, and pAPcosΔ(Ec.

Grasmid lacking the DNAfM piece (IKbp) between the AvaI site and HindIII site of R1):
I got Dai & Kan with pi (Pcos (Figure 6)).

After growing this Escherichia coli in t-TYG-P medium IJ, c c c-DNA was separated and purified from the bacterial cells and pHPc
Plasmid DNA of os was prepared.

(dl ”Deaf I TIcoa (7) Creation and preparation of PF
(Pcos 10 pg was added to the reaction mixture 100 Al(6
0mM NaCl, 6mM Tris-HCI (pH 7
,5) 6mMMyc12.6ynMDTT, l
00 tt Le fJ13SA), 1 of 51.2 units
2 at 37°C with the endogenous enzyme Pvu (manufactured by Takara).
Completely digested by reacting for an hour (bluntend)
). Then, after removing protein by phenol extraction, DNAt-ethanol precipitate was obtained, and the precipitate and Pi were 1Ot10, IE
7μl←1μg/1tll).

Next, add 0.4a7 of the DNA solution digested with this Pvul (
~0.4μg) and Bl? L II clinker [d (pC
-A-G-A-T-C-T-G)) 2pi,
20 μl of reaction mixture 50mM Tria-HCI (
pH7,4) l OmMM2c12. lo mock DTT
, 1mM spermidine, 1mM ATP, 100μg
~B5Al was reacted with 6 units of 'I'4-DNAII-ase at 22°C for 6 hours to ligate. Thereafter, phenol extraction was performed to obtain In and DNA as ethanol precipitates.

This DNA precipitate was mixed with a reaction mixture of 250μ/[60mM N
aC1, 10mM Tris-HCI (pH 7°4
) 10mM2Cl2, lOTrLMDT
T , l 00 a 11 /wLlB S A )
and 200 units of restriction enzyme B&L II (Ta
After digestion by reacting with KARA Co., Ltd. for 4 hours at 37°C, it was applied to a 0.8% preparative agarose gel for 10rn
Electrophoresis was performed for 12 hours at k. Decomposed DNA fragments become visible through ethidium bromide staining and long-wavelength ultraviolet illumination. DNA fragment consisting of COS, ori, and Apr (
After cutting out the gel containing 2,55 Kbp), 5 volumes of extract solution (0,5M NH40Ac, 1mM
EDTA (pH 7,5): water saturated phenol = 1:1
), the gel was crushed to elute the DNA. Then, the extracted fl was mixed with secondary butanol and 0.1 volume of 3M sodium acetate and 2 volumes of ethanol were added to precipitate the DNA.

This DNA precipitate was reacted with 0.20 μl of reaction mixture at 12°C for 17.5 hours with (i units of T4-DNA ligase) to generate a DNA fragment (2,55 Kbp) consisting of C08, ori, and Apr. Thereafter, phenol extraction was performed to obtain a DNAk ethanol precipitate, and the precipitate was dissolved in 20 μm of 0.1 M Tris-HCI (7,5) solution.

Then, add 10 μl of this DNA solution to Escherichia coli (HBIOI).
)'t) After formation, AprCr
It was a clone. Next, we investigated the characteristics of the plasmid DNA of these clones and determined that the plasmid DNA of ρIP008 had an 82L IT clinker inserted at the pvu[;yx<)':
Escherichia coli harboring p.

After increasing the concentration of this E. coli in TYG-P medium 17,
Separate and purify ccc-DNA from the bacterial cells, p) iBy, f
Plasmid DNA of l II cos was prepared.

(el pAPcos (BOIL II) was prepared and -Icono-pAPcoslOμg was added to the reaction mixture 1001
Digestion was performed by reacting with 60.6 units of EcoRI at 37° C. for 2 hours. Then, after removing protein by phenol extraction, the DNA was obtained as a total ethanol precipitate, and the precipitate was collected in 10 tons.
10.1E 7μ! (~lμνμ)).

Next, add 1μ of this EcoRI-digested DNA solution (
~lμg), 25μg of the reaction mixture! Then, 2 units of [l
Cohe
sive end t-blunt end K changed. Then, the reaction solution was heat-treated at 65°C for 10 minutes [1en
The ow enzyme was inactivated.

Next, this DNA solution lOμ) (0.4μg) and Bgl
L II clinker Cd (pC-A-G-A-T-C-
T-G)]2~ were ligated by reacting with 6 units of T4-DNAu gauze at 22°C for 6 hours using 20 μl of the reaction mixture.

Then, 10 μl of this reaction solution was added to 50 μl of the reaction mixture,
17. with 15 units of T4-DNA ligase at 12°C.
After reacting for 5 hours, co8 and ori, Apr, T
The cr caranal DNA fragment (4,75 Kbp) was circularized. Thereafter, phenol extraction was performed to obtain ADNA as ethanol precipitate, and the precipitate was dissolved in 0, 1 M Tris-HC.
I (pH 7,5) bath solution 20 ttl! IIC was dissolved.

10 μl of this DNA solution “t’ E. coli (HB, 1O
,l) After f transformation, Apr
A TCr clone was obtained. Next, the characteristics of the plasmid DNA of these clones were investigated, and pAPcoa
Plasmid with B21T1 linker at EcoRI site: pAPcos (Bflll II)
(Fig. 6) and obtained E. i bacteria.

After growing this Escherichia coli in 1 liter of TYG-P medium, ccc-DNA was isolated from the bacterial cells and prepared in I)A.
Grasmid DNAf of Pcoll (Bln),! I took off my clothes.

(fJ pDcos Ap'/Tcr (2B, ■) creation and aXapAPcoa (Bg-I II)
20tt& was digested by reacting with 200 μl of reaction mixture and 2 units of f202 units of BgII at 37°C for 2 hours, and then subjected to 0.8% AHA agarose gel electrophoresis to obtain a DNA@piece containing cos and Tcr (1,86 Kbp).
)'t-+ separation and purification, DNA was obtained as ethanol precipitate, and the precipitate was precipitated with 10 tlo, IE 5.5μ (~1μ
DI/DI).

On the other hand, pHBgl II cos 10 μ, yt-1 reaction mixture io.

Digestion was performed by reacting with 94 units of Byl II at 37°C for 2 hours. After removing protein with phenol extraction,
DNA was obtained by ethanol precipitation, and the precipitate was precipitated with 10 ml of ethanol.
1E 7μ! (~1 μi/ltl), pAPcoa (B, yj! u)
Digest 1 μl (~1 μl) of the DNA bath solution of the B21 IF fragment and 1 μl (~1 μl) of the DNA bath solution of the B21 IF fragment, digest the pHB, $1 II cos with B, 21 [1 μl (~1 μl)
1μy) and 6 units of T4-
The mixture was ligated by reacting with DNA ligase at 12° C. for 17.5 hours. Thereafter, phenol extraction was performed to obtain DNA as an ethanol precipitate, and the precipitate was precipitated with 0.1M Tris-HCI.
(pH 7,5) solution 20 ttl9 K was dissolved.

Next, add 10 μl of this DNA bath solution to the large intestine 11 (HBIol).
) was transformed, April KatsuTc
I got a clone. Next, the characteristics of the plasmid DNA of these clones were examined to find cDNAs with the same orientation.
Plasmid that carries 2 os: pDcoa Apr/
E. coli harboring 1rer (2Byl II) (Figure 6) was obtained.

After growing this E. coli in TYG-P medium 1/1, the bacterial cells were purified by ace-DNAt and TepDc
os ApriTcr(2B$J II)ノ:/2
Sumitomo DNA was prepared.

(Creation and preparation of gl pDcos ApriTcr p
Dcog Apr/Tc' (2Bgfl II) 1
0 pif: Limited digestion was carried out by reacting 100 μm of reaction solution a with 11.0 units of Bll II at 37° C. for 30 minutes. Next, the protein was removed by phenol extraction, and the DNA gold was obtained as an ethanol precipitate. Add this DNA precipitate to reaction mixture 1
After complete digestion with 21.8 units of Pvu II at 37°C for 2 hours + 1lij at 00μ, 0.8
% preparative agarose gel electrophoresis, two e0
f) NA fragment (containing 8 and ori, Apr, Tcr)
4,2K, bp) minutes! Purification was performed to obtain I) NA as a total ethanol precipitate, and the precipitate was purified by 10 t10. IE 6.5f
il (~Lttfi/Ill).

Next, 1 μg (~1 μg) of this DNA solution was reacted with 2 units of Klenow enzyme in 25 pl of the reaction mixture at 22°C for 30 minutes to bl the cohesive end.
Changed to untend. Then, the reaction solution was heated to 65°C for 10
The Klenow enzyme was inactivated by heat treatment for 1 minute.

Next, add this DNA solution lOμ/(0,4μI) and Pv
u II clinker (a (pc -C-A-a-C-T
-a-a)] 2 μg and 2 μg of the reaction mixture, 6
The mixture was ligated by reaction with Unit's T4-DNA ligase at 22°C for 6 hours.

Then, 10μ of this reaction solution was mixed with 50μ of the reaction mixture,
17. with 15 units of T4-DNA ligase at 12°C.
After reacting for 5 hours, two cogkUori, April,
Tar, d>6e DNA Al1r piece (4.2 Kbp) was circularly closed. After that, two phenol extractions were performed, and DN
A was obtained as ethanol precipitate, and the precipitate was diluted with 0.1 M Tr.
This DNA solution was dissolved in 20 fil of ia-HCI (pH 7,5) solution.
) was transformed to obtain Apri) h TQr clone. Then, we investigated the characteristics of the plasmid DNA of these clones and determined that pDcos Ap
PvuII length and B of riTcr (2Bll II)
DNA fragment between ll and II sites (0.2Kbp)
Plasmid lacking: pDcos Apri Tcr
(Fig. 6) was obtained.

After growing this E. coli in 1 liter of TYG-P medium, ace-DNA was separated and purified from the bacterial cells and pDC
To prepare plasmid DNA of os ApriTcr,
(hl pDcoa Apr/Tc'-XhoI" Creation and Preparation pDcos ApriTcrl 0pift
At 100 ttl of reaction mixture, 42.9 units of Ava
It was digested by reacting with I at 37°C for 2 hours. After protein removal by phenol extraction, the DNA was precipitated with ethanol, and a precipitate #10t10. lE 7μl (~lμg/μ
l).

Next, lμ of the D i'J A bath solution digested with this AvaI
1 (~1 μg) was reacted with 2 units of Klenow enzyme in 25 μ4 of the reaction mixture for 30 min at 22°C, and co
change hesive end to blunt end fc
, - and heat treated at 65°C for 10 minutes to form Kle.
-nov enzyme was inactivated.

This DNA solution (10 ttlco, 4 ttfi) and
XhoI linker (d (pC-C-T-C-G-A-
GG)) were ligated by reacting 20 μg of the reaction mixture with 6 units of T4-DNA ligase at 22° C. for 6 hours. After that, phenol extraction was performed and the DNA
was obtained as an ethanol precipitate.

This f) NA precipitation was added to 600 μl (150 μl) of the reaction mixture.
m M NaCl,6m M Tris-HCI (p
H7,9), 6mM MyC12, 6mM-DTT,
100μ1 ALI BSA) and 1000 units of XhoI (New England Biolabs
After digestion by reacting with 37°C for 4 hours, 0.
Two c.
DNA fragment containing os, ori, Apr, and Tcrk (4
, 2I' (bp) was separated and loaded, and DNA was obtained as ethanol precipitate.

This DNA precipitate was cyclized by reacting with 6 units of T4-DNA ligase at 12°C for 17° and 5 hours in a reaction mixture of 20μ, followed by phenol extraction.
f) NAe obtained as ethanol precipitation, precipitation tjtk O,
l M Trim-HCI (pH7,5) f&liquid 2
0/J.

Then, add 10 μl of this DNA bath solution to the InuM plane (HBIOI).
) after transformation, Apr and Tc
r clone was obtained. Next, we investigated the characteristics of the plasmid DNA of these clones and determined that pDaos Apr/'
Plasmid with Xh o I linker inserted into the AvaI site of l'cr: pDcos Apr/'f'c
E. coli containing "Xhol" (Figure 6) was collected (4).

After growing this Escherichia coli in TYG-P medium IA', QC (!-DNA) was isolated from the bacterial cells.
8Apr/'1rcr-XhOIa Nophrasmite DNA
was prepared.

(il pDcos Tcr creation and preparation pDcos
Apr/Tcr-XhoIal 0μji to reaction mixture 100IJ T6.2-=”l hOBsLEn&60
A limited crude reaction was carried out by reacting for CT°°6.5. After protein removal by phenol extraction, the DNA was precipitated with ethanol, and the precipitate was 10.1E 7μ] (~l/iμ
)).

Next, limited digestion with BstE I[*DNA solution l
The Klenow enzyme was inactivated by heating 25 μl of the reaction mixture at 65° C. for 10 minutes.

This DNA solution lOμ/(0.4μg) and PstI linker-[d (+)G-C-T-G-C-A-G-C)
] 2 μg and 6 units of T in a reaction mixture of 20 μg.
4-DNA! J gauze and ligated by reacting at 22°C for 6 hours.

Thereafter, phenol extraction was performed to obtain a DNA Atl-ethanol precipitate.

This DNA was added to the reaction mixture 400μ7 (50mM
(NH4) 2 SO2, 20m M Tris-HC
I (pH 7,5), 10 ml (M, yc12, l
00 μWBSA) Te, 600 units of PstI
(manufactured by New England Biolabs) at 37°C for 4 hours and digested, 0.8%
Applied to rjL4 agarose gel mass electrophoresis Mil+,
2 cos and ori, i'c'°k t tr
D N A M piece (~4Kbp) gold fraction kk, purified,
Precipitate the DNA with ethanol and use 4fcn Next, this DNAI! t# with 20 μl of reaction mixture, 6 units of T4-L)NA! 7 gauze and 17.5 at 12℃
After reacting for a period of time to achieve ring closure, phenol extraction was performed to obtain DNA as an ethanol precipitate, and the precipitate was 0.1
M Tris-HCI (p)f 7.5) Bath liquid 20
It was dissolved in μ/.

Then, 10μ of this DNA solution was added to Escherichia coli (HBIOI).
), a Tcr clone was obtained. Next, the plasmid DNA of these clones
Check the characteristic gold of A, pDcosApr/? cr-Xho
We obtained Escherichia coli containing the b77 plasmid: pD(!011 Ter (Fig. i316)) lacking the DNA fragment (~0.2 Kbp) between the B a LE (I site and the Pstl f site) of Ha. The strain was sent to the Laboratory of Imitation Biological Technology, Institute of Technology and Technology [Microtechnology Research Institute No. 7551 (1?ERM
P-7s 51) I was entrusted with the role of J.

After growing this large intestine in 1 liter of TYG-P medium, ccc-DNA was separated and purified from the bacterial cells to produce pDcos.
Tcr plasmid DNA' was prepared.

Example 2 Creation and preparation of pDcos Tcrlori (Figure 7) (Al Creation and preparation of pBR-327/XhoIp and mff
10μg of pBR-327 was added to the reaction mixture (100μ) for 24 hours.
Digestion was performed by reacting with 8 units of PstI at 37°C for 2 hours. Then, after removing protein with phenol extraction, DNA
h ethanol precipitate, and the precipitate was 10t10. IE
7 μl (~1 μg/μ!). Next, this p
Digested with stI* DNA solution 1.5 µm (~15
μI ) to 30 ttl (33 m M T
rim-aceta-to (pH 7,9), 66mMm
Potassium acid, 10mM MgCl□, 0.5mM, I) TT
, 1001ti/ME, BSA, 0.1mM d
ATP, 0. lmM dCTP, O, lmMd
GTP.

0, l ry5M dTTP) and 5 units of T
4- DNA polymerase (P, L company M) and 3
Incubate the cohesive
Changed the ending to a blunt end. Then, the reaction mixture was heat-treated at 65°C for 10 minutes to inactivate T4-DNA polymerase.

8 μJ (0.4 μy) of this DNA solution and XhOI linker [d (pC-C-T-C-G-A-G-G)
) and 6 units of T in 20 μl of the reaction mixture.
4-DNA! The mixture was ligated by reacting with J gauze at 22°C for 6 hours.

Thereafter, phenol extraction was performed to obtain DNA'fr as ethanol precipitate.

This DNA precipitate was digested by reacting it with 1000 units of XhoI in 600 μl of the reaction mixture at 37°C for 4 hours, and then subjected to O18 preparative agarose gel electrophoresis to separate and purify a 3.27 Kbp DNA fragment. , D.N.
A was obtained as a precipitate using ethanol.

Prepare 6 units of this DNA precipitate using 20 μl of the total reaction mixture.
After ring closure by reacting with '4-DNA ligase at 12°C for 17.5 hours, phenol extraction was performed to obtain DNA as ethanol precipitate, and the precipitate was precipitated with 0.1 M Tri8.
-Dissolved in 20 pi of HCI (pH 7,5) solution.

Then, add large & bacterial cells (HBlo) with 10 μl of this DNA bath.
Tc' clone was obtained after transformation. Next, we investigated the characteristics of the DNA of these clones and determined the Psε of ρBR-327.
Plasmid with KXhoI linker inserted:
E. coli harboring pBR-327.Xbolp (Fig. 7) was obtained.

After growing this E. coli in 1 liter of TYG-P medium, Jccc-DNA was isolated from the bacterial cells and pBR-3
7-Xholp plasmid DNA was prepared and ft.

(bl) Creation of pDcos Tcr (2ori) and PA-made pDcos 'rer20 fiI/, reaction mixture 500AA' ((150 mg Nacl, 6
is-HCI (pH 7,9).

67X M-MFCI z, 6 tn M, DTT
, 100 μg/” BSA) and 167 units of the restriction enzyme BamHI (New England Bio
labs) and 500-L=:7 to XhoI and 3
After complete digestion at 7°C for 2 hours, two COBs were subjected to electrophoresis on a 0.8 inch preparative agarose gel
DNA flT fragment (2-4 hbp) containing and ori
e minutes 4sHL Te, D N A 't' Engineering 1 'obtained as a nord precipitate, precipitation! i school 1Ot10.1E 9.
It was dissolved in 5 μl (~1 ttll/ltl!).

Meanwhile, pBR-327Xbolp204 was added to the reaction mixture at 50%
After complete digestion by reacting with 167 units of BamHI and 500 units of ,14K
bp) was separated and purified, DNA was obtained as ethanol precipitation, and the precipitate was washed with 10t10. IE 9.0μl (~lμg
/ μm.

Then, 1 μl of a DNA solution of BamF (I/Xho I fragment containing e03 and ori of pDcos Tcr) was added.
C~1. μg) and pBR-327-XhoIp or
1μ of DNA solution of BamHI/XhoI fragment containing i'x! (~1 μg) were ligated by reacting with 6 units of T4-DNA ligase in 2 μl of the reaction mixture at 12° C. for 17.5 hours. After that, complete phenol extraction,
L) NATh was obtained as ethanol precipitate, and the precipitate was 0.1
M Tris-HCI (p) (7,5) solution 20 μl
I made K-folding.

Next, Escherichia coli (Tomo 101) was transformed with 10 μl of this DNA solution to obtain a Tc clone. Next, the characteristics of the plasmid DNA of these clones were investigated, and two eoll and 21 ori, T
Plasmid with cr: pDcosTcr (2or
i) Escherichia coli having (Fig. 7) was obtained.

After growing this Escherichia coli in 1 liter of TYG-P medium, ace-DNA was separated and purified from the bacterial cells to produce pDcos.
Plasmid DNA of Tcr (2ori) was prepared.

(Cl psV2-dhfr Δ(B1■) Creation and Preparation psi2-dhfr (Subramani,
Mulligan & Berg.

Mo1. Ce11. Biol,, 1, 854 (1
981) 110 μg to 100 μg of reaction mixture! So, 48.
Digestion was performed by reacting with 5 units of Blf at 37°C for 2 hours.

After protein removal by phenol extraction, the DNA was precipitated with ethanol, and the precipitate was extracted with 1Ot10. lE 7 μl (~
1μνμl).

9 Next, add 1μ of this B@12II-digested DNA solution.
! (~1 μl) was reacted with 2 units of Klenow enzyme in 25 μl of the reaction mixture for 30 min at 22°C and co
Change hesive end to blunt end Ic. Then, the reaction solution was heat-treated at 65°C for 10 minutes to obtain Kle.
-nov enzyme was inactivated.

Next, add 10 μg (~0.4 μg) of this DNA solution to 50 μg of the reaction mixture. After reacting with 15 units of T4-DNA ligase at 12°C for 17.5 hours to achieve ring closure, phenol extraction was performed to obtain the DNA as an ethanol precipitate.
7.5) Dissolved in 20 μm of M solution.

Then, add 10 μl of this DNA solution to E. coli (HBIOI).
) was transformed, Apr clone t'' was obtained. Next, the characteristics of the plasmid DNA of these clones were investigated, and B) λ of pSV, 2 to dhfr was obtained.
■Plasmid lacking the site: psV2 dhfr△
(B2i II) (Fig. m7) was obtained.

After growing this Escherichia coli in 1 liter of TYG-P medium, ace-DNA was isolated and purified from the bacterial cells, and pSV2-
dhfrΔ(BgiII) plasmid DN ”A
t-prepared.

fdl Creation and preparation of pDcos Tc/ori 201tli of pDcos Tcr(2ori) was added to 500ttll of reaction mixture (60mM Nacl, 6mM
Tris-HCI (pH 7,5), 6 mM MyC
l, , 6 mM-DTT, 100 t4/Ill
BSA), 42.5 units of PvuII and 35
．． After complete digestion by reacting with 8 units of PstI at 37°C for 2 hours, the DNA AFr pieces containing two cog and ori, Tcrf: 3,l Kbp)'! r minute tlt
a'W L, Te, DNAt-obtained as ethanol precipitate,
Precipitate at 10. IE 9.5μl (~lμg/μ)
) was dissolved in

On the other hand, psv2-dhfrΔC5yJ2II)2oμ
38.8 units of P in 500 μl of the reaction mixture.
vu [[ and 32.7 units of PstI at 37°C.
After complete digestion by reaction for several hours, the DNA fragment (2,45 Kbp) containing SV-dhfr was separated and purified by 0.8% preparative agarose gel electrophoresis.
NA was obtained as ethanol precipitate, and the precipitate was 10t10, l
E was dissolved in 7 μl (~μfi/ltl).

and cos and ori of pDcos Tcr (2ori).

DNA lysis of Pvu II/PstI ffjr fragment containing Tc k wlpi (~1Ag) and psV2-dhf
SV of r△(By2 II ) -dhf P containing r
DNA of vuII/Ps t I mr piece?
l'i Lttl! (~lμg) and 20μg of the reaction mixture.
17 units of T4-DNA ligase at 12°C.
．． The mixture was reacted for 5 hours and ligated. The DNA was extracted with phenol, and the DNA was precipitated with ethanol.
．． 1 M Tri 5-HCI (pH 7,5) 16
It was dissolved in 20 # of liquid.

Next, add 10μ4 of this DNA bath solution to the large intestine (HBIO)
After transformation, a Tcr clone was obtained. Next, the plasmid DN of these sunsets
Check the characteristics of A and get 2111! coo and ori of d
, Tcr, SV-dhfr: p
E. coli harboring Dcos Tcr/art (Figure 4.7) was obtained. In addition, this strain was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology [Copyright Research Institute No. 7554 (FERMD-75)].
54) Deposited as J.

After growing this E. coli in TYG-P medium 11, ace-DNA was isolated and purified from the bacterial cells, and pD
Plasmid DNA of coa Tcrlori was prepared.

Example 3 Creation and preparation of pDcos Aprlori (at pBR-327XhoI)
10 .mu.l of R-327 was digested by reaction with 110 units of AvaI at 37.degree. C. for 2 hours in reaction mixture ioo.mu. After protein removal by phenol extraction, the DNA was precipitated with ethanol, and the precipitate was t1Ot10, IE 7μ (~1μ9/1tlj) Kmm.

Next, the DNA solution lμ! digested with this human vaI! (~l
μg) in 25 μl of the reaction mixture, 2 units of Klen
ow enzyme for 30 minutes at 22°C to
Changed the e end to a blunt end. and,
The reaction solution was heat-treated at 65° C. for 10 minutes to inactivate the Klanow enzyme.

10 μl (0.4 μg) of this DNA solution and XhoI
Linker-[d (PC-C-T-C-G-A-G-G
)) 2μg and 9 months, oh, yo 2゜ttllT6-=
Wow. The mixture was ligated by reacting with 14-9NA ligase at 22°C for 6 hours. After that, phenol extraction was performed and lx, pN
4 with A as ethanol precipitation.

This DNA precipitate was digested by reacting it with 100 units of XhoI at 37°C for 4 hours using 600 μl of the reaction mixture, and then subjected to gas phoresis on a 0.8% preparative agarose gel to obtain a 3°27 Kbp DNA fragment. After separation and purification, DN
A was obtained as an ethanol precipitate.

This DNA precipitate was mixed with 6 units of T4 in a reaction mixture of 20 μm.
- After reacting with DNA ligase at 1.2°C for 17.5 hours to achieve ring closure, perform phenol extraction. , DNA was obtained by ethanol precipitation, and the precipitate # was 0. I M Tris
-HCI (pH 7,5) was dissolved in 20 pl of bath solution.

Then, with 101 Lll of this DNA bath solution, E. coli (FiB
After transforming Ap r
A Tcr clone was obtained. Next, the characteristics of the plasmid DNA of these clones were investigated, and pBR-32
Insert the XhoI linker into the Ava I putite of 7.
tp5 SumiI': pBR-327・XhoIaD
A large intestine atresia was observed (Fig. ig8).

This large intestine was grown in 1 liter of kTYG-P medium.
Separate and purify ccc-DNA from the bacterial cells and create pBR.
-327・Xhola (D 7' lasmid DNA
'kt4H.

Add 20 μp of prepared pDcoa Tcrlori to the reaction mixture 20
106 units of EcoRI at 0 μi and 2 at 37°C.
After reacting for a time and digesting, add 500μ of the reaction mixture)
It was digested by reacting with 530 units of XhoI at 37°C for 2 hours. Thereafter, a DNA piece (~2.9 Kbp) containing two COS and SV-dhfr was separated and purified by 0.8 multi-preparation agarose gel electrophoresis, and the DNA was precipitated with ethanol.
．． On the other hand, pBR-3274hoIa20 pg k Ij
176 units of E(!) in 200 μl.
After digestion with ORI at 37°C for 2 hours, 880-L nit XhoI was added to 500 μl of the reaction mixture.
and digested at 37°C for 2 hours. Therefore, when subjected to 0.8% g agarose gel kick-electrophoresis, ori and Ap rk
MiyabD NAM piece (1,84Kbp) (%l in r minutes
lJL.

DNA'if obtained as ethanol precipitation, precipitate 10
t10. IE 8 tt cedar (~lμg/1tll) was added to mm.

And QOB and 5V of pDcoa Tcrlori
1 containing -dhfr (D of coRI/Xh o i fragment
N A mi shallow LtJl (~1t4) t, pB
R-327・XhoI" (D art and U Ap'
k Contains trEcoRI/XhoIte? One piece ON
A MW 1μZ (~ld) and 20μ of the reaction mixture
6 units of T4-DNA ligase and 1 unit of T4-DNA ligase at 12°C.
The mixture was reacted for 7.5 hours and ligated. Thereafter, phenol extraction was performed to obtain DNA as an ethanol precipitate, and the precipitate was dissolved in 011M Tris-HCI (pH 7,5) solution 2.
0 ttil K solution 11=1=.

Next, use 10 μl of this DNA solution to extract E. m. bacteria (HBlol).
After transformation, I obtained an Apr clone. Next, the plasmid DNA of these clones
Check the attachment of A and find two aos and ori, Apr
, S V -dhfrΔCB, glU): pDcos Apr10ri/dhfrΔ
(B7IU) (Fig. 8) was obtained.

After this large #T bacterium was grown in TYG-P medium, ccc-DNAtl was isolated and purified from the bacterial body, and pD
cos Apr/'ori/dhfrΔ(ByflI)
plasmid DNA was prepared.

(cl pSV2- dhfr Δ(BamHI/Bg
111) Creation and preparation of psV2-dhfr 10
ttll was digested by reacting with 100 pi 48° 5 units of B'II at 37°C for 2 hours.
In addition, 58.2 units of Bam were added to the reaction mixture at 250 μm.
Digestion was performed by reacting with HI for 2 hours. After protein removal with phenol extraction, the DNA was precipitated with ethanol, and the precipitate was collected in 1Ot10, IE 7μl (~1μVltll).
It was dissolved in

Next, digest with this BgIII and BamHI *oNh
1 μg of ml solution was reacted with 2 units of Klenow # in 25 μl of the reaction mixture for 30 minutes at 22°C.
let's oohesive end k blunt e
Changed to nd. Then, the reaction solution was heat treated at 65° C. for 10 minutes to inactivate the Klenow enzyme.

Then this f) NA solution lo μA (~o, tutg
) and 15 units of T4-DNA in 50 μl of the reaction mixture.
After reacting with ligase at 12°C for 17.5 hours to achieve ring closure, phenol extraction was performed, the DNA was precipitated with ethanol, and the precipitate was precipitated with 0.1M Tris-HCI.
(pH 7,5) solution was dissolved in 20 # of solution.

Then, with 10 μl of this DNA solution, E. coli (f(Blo)
After transforming t), Apr clone was obtained. Next, plasmid D of these clones
By examining the properties of NA, psV2-dhfr ByI
DNA fragment between II site and BamHr site (0
, 85Kbp): psV2
- dhfr Δ (BamHI/B, ?JII) (
Escherichia coli with g 8 1m was present.

After growing this E. coli in TYG-P coral 17,
Separate and purify the ccc-DNA from the bacterial cells to generate psV.
2-dhfrΔ(BamHI/f3/F II)
Plasmid :) DNA was prepared.

(dl pDcoa Apr10ri/dhfrΔ(B
am HI/B l 'II) and preparation of pDcos Apr10ri/dhfr Δ(B21
II) 20t4'r: 200 μl of reaction mixture, 2
．． Limited digestion was carried out by reacting with 9 units of pstI at 37°C for 30 minutes. Next, after removing protein with phenol extraction, DN
A was obtained as an ethanol precipitate.

This DNA precipitate was digested by reacting with 34.0 units of Pvu in 200 μl of the reaction mixture at 37° C. for 2 hours. Thereafter, the DNA fragment containing two cos, ori, and Apr was subjected to 0.8 inch preparative agarose gel electrophoresis (
2, aKbp) was purified by tm, the DNA was obtained as ethanol precipitate, and the precipitate was 1Ot10, IE 7ttlJ.
(~14/#) Seta understands Km.

On the other hand, pSV2-dhfrΔ(J3amHI/B
gllllI) 20ztll was reacted with 500 μl of the reaction mixture for 2 hours at 37°C with 46.8 units of Pv uII and 39.5 units of PstI, and then photocompletely digested. , 5V-dhfr f containing L) NA fragment (1,
61Kb)'i fraction was separated and the DNA was precipitated with ethanol. IE 5.5til
l (~1tti/1tlJ) K r? It's been a long time.

and % I) DCO8Apr10ri/dhfr △
(BpII) cos and ori, Pvu including Apr
II/PstI fragment DNA solution 1ttlC~Lttf
pSV2-dhfrΔ(BamHI/Bp
II) S V dhfr f sayPvulL/P
Ligation was performed by reacting 1 μl of a DNA solution containing the st I fragment (~1 μm aliquot with 2 μl of the reaction mixture) with 6 units of T4-DNA ligase at 12°C for 17.5 hours, followed by phenol extraction. ? XDNA was obtained by ethanol precipitation, and the precipitate was dissolved in 0.1 M Tris-HCI (pH 7,
5) Dissolve the solution in 20μljK.

Next, and t7) DNA liquid 10 Ill "'C big H!
Transformation L for Ijm(l(BIOl),
Apr clone fc was obtained from fc. Next, we investigated the characteristics of the plasmid DNA of these clones, and
cos and ori, Apr+ SV -dhfr
Plasmid with Δ(Bamt(I/B@]]II): pDcos Apr10ri/dhfrΔ(Bar
ru(I/Bfi'lI) (BS 8 figure) Dog waiting +yi: m*1↓Ita.

After growing this large 9J bacterium in TYG-P medium iz, ace-DNA was isolated and purified from the bacterium, and pD
cos Apr/ark/dhfrΔ (BamHI/
B) J! II) Plasmid DNA was prepared.

(el pi)cog Apr10ri/dhfr (
BamHI ) creation and toning pDcos Apr10ri/dhfr Δ(BamH
I/B,lp II) 10μ,! i', reaction mixture lOOμ! and 73.7 units of EcoRI and 37°C.
The mixture was reacted for 2 hours and digested.

After protein removal with phenol extraction, 1) NA was precipitated with ethanol, and the precipitate was collected at 10 t10. lE 7μl
(~lμνμIJ).

Next, digest this EC0RI and add 1 pi of fcDNA solution.
(~1 μl) to 25 μl of the reaction mixture! So, 2 units of K
React with lenow$ at 22℃ for 30 minutes
I changed it to eslve end f blunt end. Then, the reaction solution was heat-treated at 65°C for 10 minutes to obtain Kle.
The now enzyme was inactivated.

, (jD DNA bath solution 10''A' (0.4 μg> and B
am[(I linker-Cd (pC-G-G-A-T-C
-C-G)) 214 was reacted with 6 units of T4-DNA ligase in 20 μl of the reaction mixture at 22° C. for 6 hours to ligate.

Thereafter, phenol extraction was performed to obtain DNA' and ethanol precipitate.

This DNA precipitate was digested by reacting it with 600 μl of the reaction mixture and 100 units of BamHI at 37°C for 4 hours, and then subjected to 0.8% preparative agarose gel electrophoresis to separate and purify a 3.91 Kbp DNA fragment. DNA
was precipitated with ethanol.

Add 6 units of T4 to this DNA precipitate in 20 μl of the reaction mixture.
- After reacting with DNA ligase at 12°C for 17.5 hours to achieve ring closure, phenol extraction was performed and the DNA (il
-obtained as an ethanol precipitate, and the precipitate was precipitated with O, l M Tri
It was dissolved in 20 ttll of s-HCI (pH 7,5) R1 solution.

And this D N A m'1eL10 ttll
After transforming the large PIh box (HB, +01) t), I served the AI) r clone. Next, we investigated the characteristics of the plasmid DNA of these clones,
pDcos Apr10ri/dhfr △(BamH
Bam on the f:coRI site of I/B, yf n)
f (plasmid with an I linker inserted (in this case, B
ffcoRI site is reconfigured on both sides of amHT site): pDcoa Apr/ori/dhfr
(BamHI) (Figure 8) 1? I got E. coli with.

After growing this E. coli in 110 ml of TYG-P medium, ace-DNA was isolated and purified from the bacterial cells, and pD
cos Apr10ri/dhfr (BamHI)
The entire plasmid DNA was prepared.

Creation and preparation of IfJpsVO1o/neor pA04
3 (Oka, Sugisaki & Takanam
i, J, Mol.

Biol, l4u, 214 (1981)) 20μ
Ji'm mixture 200μ/(60mM NaC1,6
mW Tris-HCI (pH 8,0), l 0mM
M@C12, 6mMDTT, 100μ,! 150 units of the restriction enzyme Ava [(N
ew England Biolabs) and 37°C
After reaction and digestion for 2 hours with
f x t/-A/ obtained as precipitate, precipitate #t-10t
It was dissolved in 5 μl of 10JE (~1 μV~).

Add 2 μl (~2 μμ) of this DNA solution to 50 μl of the reaction mixture.
The cohesive end was bred by reacting with 4 units of Klenow enzyme at 22°C for 30 min at a still temperature.
Changed to l untend. After protein removal by phenol extraction, the DNA was precipitated with ethanol, and the precipitate 'c
10t10. lE 1.5ttll (~1pfl
Atll ) K lytic sasse fc.

On the other hand, psVOIO [xVX (Brain 5eed
, Nucleic Acids Bes,, 11.24
psV with a polylinker derived from 27 (1983)
o 1 (Tjan, R, et al., Proc.

Natl, Acad, Sci, U, S, A, (77,
6491 (1980) related plasmid 3toμg,
After reacting with 100 μl of the reaction mixture and digesting with 129 units of Bamf (I) at 37°C for 2 hours, phenol extraction was performed and the DNA was precipitated with ethanol. ltl
? ) was dissolved in

2 μl (~2 μg) of this DNA solution, 50 μl of the reaction mixture
Cohesive en
Changed to d f bluntend. Then, by phenol extraction, the protein d<, DNA was precipitated with ethanol, and the precipitate was 1Ot10.1g 1.5μI! , (~lμν
d) was bathed in camellia.

Next, pAO43 neo”r Miyamu DNA piece eKl
- now lμl of DNA solution treated with acetic acid (~lμgong, psVOlo'r: digested with Bamt(I, K
fcDNk solution i ttl (
~x4) and 6 units of T4-
The mixture was ligated by reacting with DNA ligase at 12° C. for 17.5 hours.

The DNA was extracted with a trowel and phenol was precipitated with ethanol, and the precipitate was extracted with phenol.
(C1(pf(7,5)# solution 20 tJl
fc.

Then, add 1υμl of this DNA solution to Escherichia coli (1 (Blo)
After the transformation of T), Apr.
rnr(n1eO reclones were obtained. Next, the physical properties of the plasmid DNA of these clones were investigated and psV
OIO's Bamt (neo”e inserted into the I site) 7 Sumito* psVOIo/neor (Figure 8) 5
) was obtained.

After growing the entire Escherichia coli in TYG-P medium, the bacterial cells were isolated and ccc-DNA #I was prepared.
Plasmid DNA of VO1O/neor was prepared.

(Creation and preparation of gl pBR-322/neor p
Reaction mixture of sVO1o/neor20 pg 200 μm
After digestion by reacting with 46.6 units of PstI at 37°C for 2 hours, 500 μl of the reaction mixture was added to 166
It was digested by reacting with unit E (!
Kbp) was subjected to subdivision purification, and the DNA was obtained as ethanol precipitation.
It was dissolved in water.

On the other hand, pBR-32220μI was digested by reacting with 37.2 units of PlstI in 200μl of the reaction mixture at 37°C for 2 hours, and then further digested with 500μl of the reaction mixture for 13
Digestion was performed by reacting with 2 units of EcoRI for 2 hours.

Then, 0.8% agaro-'geyv'di, air
Add 17 degrees to K@ and say "°'" and "1°'", (D
The NA fragment (3.6 Kbp) was separated and loaded, and the DNA was obtained as ethanol precipitate.
It was dissolved in microorganisms (~lμi/1tll).

and % psVO10/neOrtl-containing EcoRI/Pstl fragment l) NAM solution 1 ttl
J (~1, c+9) and 1 μl (~1 μs) of a DNA solution of the EcofjI/PstI fragment containing pRR-322 ori and TCr were mixed with 6 units of T4-DNA ligase in a 20μ reaction mixture at 12°C for 17 hours. , and ligated after reacting for 5 hours. After that, Phenol Tsukuda withdrawal, D
NA was obtained as ethanol precipitate, and precipitated di 'c O,l
M Tris-HCI (pH 7,5) solution 20μl
It was dissolved in %.

Meanwhile, add 10μ of this DNA solution to E. coli (dish 101).
-H Randomation, Kmr(n e
or) The Tc r and Ap S clones are 4. Next, the characteristics of the plasmid DNA of these clones were investigated, and E. coli containing grasmid pBR322/neor (Fig. 8) in which the neor fragment was inserted into pBR-322 was obtained.

After growing this Escherichia coli in 1/1 TYG-P medium, ace-DNA was prepared from the bacterial cells and pB
R-3227neor plasmid oNp, gold v* was made.

(hl pBR-322/neorΔ(EcoRI/B
a1I) Creation and preparation pBR-322/neor10ttl': After digestion by reacting with 40 units of Ba1I in 100 ttll of the reaction mixture at 37°C for 2 hours, further adding 250 ttl of the reaction mixture.
It was digested by reacting it with 120 units of 1i3coRI in μl at 37°C for 2 hours. Then, after removing protein with phenol extraction, the DNA was precipitated with ethanol and the precipitate was 1Ot1.
It was dissolved in 7μl of 0.1K (~lμVμl).

Next, digest with this Ba1I and E (!ORI and fc D
Add 1 μl (~1 μg) of NA # solution to 25 μl of reaction mixture.
Then, react with 2 units of Klenow enzyme at 22°C for 30 minutes to obtain a cohesive end f blunt.
Change the temperature to fc, and then react for 10 minutes at 65°C.
The Kle new enzyme was inactivated by heat treatment for 1 minute.

Next, add 10μl of this DNA bath solution (~o, 4.a, !i+
) to 50 μl of the reaction mixture, 15 units + 7)'
After ring closure by reacting with r4-DNA ligase at 12°C for 17.5 hours (864 EeoRI sites are created at this stage), phenol extraction was performed and DNA
A was obtained as ethanol precipitate, and precipitate #0.1 M Tr
Dissolved in 20μ7 of is-HCl (pf(7,5) solution.

Then, with 10 μl of this DNA solution, large intestine m<HBIOI
)'t) After running home, neor (K
mr) and the T c 8 and Ap8 clones.

Next, the characteristics of the plasmid DNA of these clones were investigated, and a DNA fragment (1,42K
bp): pBR-322/neo
Escherichia coli harboring rΔ(EcoRI/Ba1I) (Fig. 8) was obtained.

After growing this E. coli in 1 liter of TYG-P medium, ace-DNA was isolated and purified, and pBR-
Plasmid DNA of 322/neorΔ (EcoRI/Ba1I) was prepared using alS.

(Creation and preparation of pDcos Aprlori p
Dcos Apr10ri/dhfr (BamE(I
) 20t4k reaction di) 4. With 200 μl of the mixed solution.
Limited digestion was carried out by reacting with 1 unit of PstI at 37°C for 30 minutes. Next, protein and DNA were removed by phenol extraction.
was obtained as an ethanol precipitate.

This DDNA precipitate was digested by reacting with 49 units of Pvu[200 μA'C' of the reaction mixture at 37°C for 2 hours.

Thereafter, a DNA fragment (2.3 Kbp) containing two cos, ori, and Apr was separated by electrophoresis on a 0.8% agarose gel, and the DNA was precipitated with ethanol. .1E 8
td3 (~lμVμ)).

On the other hand, pBR-322/neorΔ(EcoRI/Ba
1I) 20 μg to 500 μg of reaction mixture! Then, 2 DNA fragments (1.9 Kbp) were separated and purified with 56 units of Pvu [and 47 units of PstI] at 37°C, and D
Obtained as NAt-ethanol precipitate, precipitate 'klOt10
．． 1E 8μ! (~1 μl).

and % pDcos Apr10ri/dhfr (
cos and art of Bam1(I), Aprf, Tomamu PvulI4) s t I DN Am of 19℃ piece
Solution til(~1μ,!i?) and pBR-322
/neorΔ (EcoRI/Ba1I) DNA solution of PvulI/Ps t I fragment containing neor (EcoRI/Ba1I)
~lμI) and 6 units of T4 in 2oμ of the reaction mixture.
- Ligation was performed by reacting with DNA ligase at 12°C for 17° for 5 hours. The sickle was extracted with phenol, -=DNA
was obtained as an ethanol precipitate, mf: 0.1 M
It was dissolved in 20μ of Tris-MCI (pH 7,5) solution.

Next [, Ko (1) D N A Bath liquid 10 tt/Cusp Nk
After transforming tM (HB, 101), I cloned Apr (Kyn). Next, I transformed the plasmid DNA of these clones!
p! Check the j properties and find two 0cos and ori, April
, noor: pDcos Apr
E. coli harboring lori (Fig. 1, 8) was obtained. This strain has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as F'ERMP-7553 J.

After growing E. coli in 1 liter of whole TYG-P medium, ccc-DNA was isolated and purified from the bacterial cells, and pD
cos Aprlori plasmid DNA'fi-X
Manufactured.

pSV2-yptc Richard, Mullig
an & berg.

Mo1. Ce11. Biol,,l, 449. (1
981) )lOPJ"j?, reaction mixture 100μ,
46 unit output manager JJI and 46 unit Bgl
Digestion was performed by reacting with llI at 37°C for 2 hours. and,
After removing protein with phenol extraction, the DNA was precipitated with ethanol, and the precipitate was 10t10.1E7μ! (~1μ microtll).

Next, digest with Hind[[ and Bglll and fcD
NA solution lμ! (~Lt4) into 25 μl of reaction mixture
Cohesive end blunt enzymatic reaction with Klenow enzyme at 22°C for 30 minutes
d i/I was shaking.

Then, the reaction solution was heat-treated at 65°C for 10 minutes to obtain Klen.
The ow enzyme was inactivated.

Next, Kono +) NA bath 110 μA (~0.4 μ, 9)
15 units of T4-υ in 50 μl of the 'c reaction mixture.
After ring closure by reacting with NA ligase at 12°C for 17.5 hours, the DNA was precipitated with ethanol without phenol extraction, and the precipitate was precipitated with 0.1 M Tris-HCl.
(pH 7,5) dissolved in solution M 201i11.

Then, with 10μ of this DNA solution, E. coli (t(BI)
OI) After k transformation, Apr clone was created. Next, we investigated the characteristics of plasmid DNk possessed by these clones and determined that the Hi
nd 111 site and Bgl [[1 between the site] N
Plasmid with A fragment (0.12Kbp): ps
V2-yptΔ(Hind[[/BgiII)
(Fig. 9) was obtained.

After growing this E. coli in 1 liter of TYG-P medium, ccc-DNA was isolated and purified from the bacterial cells, and psV2-
Plasmid 1) NA of yptΔ(Hi nd ifI /Bgl[) was prepared.

(bl Hirohe Nakayonie tΔ(HindilI/Bg
III)/Bam1 (Creation 1 of I) and preparation psV2-,yptΔ(Hindllf/BglI
I) 18.8- at 100 tJ of 104k reaction mixture
'-nit was digested by reacting with Pvu, [I at 37°C for 2 hours. Then, remove protein and DNA by phenol extraction.
The precipitate was obtained as an ethanol precipitate using Lotlo, IB.
It was dissolved in 7 μl (~1 μg/μ).

Next, 0.4 μl (~0.4 μg) of this DNA bath solution,
BamE(I linker-Cd(+)C-G-G-A-T
-C-C-G): 12 μl of the reaction mixture was reacted with 6 units of T4-DNAIJ gauze at 22° C. for 6 hours to ligate with 20 μl of the reaction mixture. Preliminary phenol extraction was performed to obtain DNA as an ethanol precipitate.

This DNA precipitate was digested by reacting with 100 μm of the reaction mixture and 1000 units of Bamt (I) at 37°C for 4 hours, and then digested with 0.8% preparative Aga "Coase Gel It Dd".
By J, 5. A DNA fragment with a length of 1 Kbp was separated and purified, and the DNA was obtained as an ethanol precipitate.

Add 6 units of this I) NA precipitate to 20 μl of the reaction mixture.
l'4-1) After reacting with NA ligase at 12°C for 17.5 hours to close the ring, perform phenol extraction,
DNA was obtained as ethanol precipitate, and the precipitate was 0.1 M
It was dissolved in 20 μl of Tris-HCI (pH 7,5) solution.

Then, with 10 μl of this DNA solution, E. coli (f(B 1
After transformation of 01), Apr clone was obtained. Next, we investigated the characteristics of the plasmid DNA of these clones and determined that pSv2-yptΔ(H
plasmid with a BamHI linker inserted into the pvuII site of indIII/BglII): pSV
2-ypt △(HindlTI/BglII)/B
E. coli containing amHI (Figure 9) was obtained.

After growing this Escherichia coli in TYG-P medium IJ, ccc-DNA was isolated and purified from the bacterial cells, and psV2-
yptΔ(Hi nd III /Bg I II )
/BamHI plasmid DNA was prepared.

(cl pDcos Apr10ri/pt (2B
Creation and preparation of ank(I) pSV2-ypt Δ(HindlII/Bg・1
TIl) /Bamf(I 20ti9 to reaction mixture 20
In 0 μl, 226 units of Bamf (I) and 2
After reaction and digestion for several hours, the DNA fragment containing ypt (2
, 2Kbp) was separated and purified, the DNA was obtained as ethanol precipitation, and the precipitate was washed at 10t, 10. lFE 8.5μl
(~1 μm diameter).

On the other hand, pDcos Apr10ri/dhfr (Ba
mE-II) 74 LOx& with 100μ of the reaction mixture
The mixture was digested by reacting with unit BamHI at 37°C for 2 hours. After protein removal by phenol extraction, the DNA was precipitated with ethanol, and the precipitate was extracted with 1Ot10. IE 7μ
l (~1 μg/μl).

And psV2-yptΔ(Hind IJI/B
Group g→/D of BamHI fragment containing 2pt of BamHI
1 µl of NA bath solution (~1 µl, 9) and pDcos
Apr10ri/dhfr (Bam [(I)
DNA digested with mHI [III (~Lltj
l ) and pDcos Apr10ri/dhfr (
BamHI) was digested with BamHI in a DNA bath solution lμ
1 (~1 μ!q) and gold were ligated by reacting with 6 units of T4-DNA ligase in 20 μl of the reaction mixture at 12° C. for 17.5 hours.

After that, phenol extraction was performed to obtain DNA:
, as ethanol precipitate, and precipitate as 0. I M Tr
It was dissolved in 20 μl of is-HCI (pH 7,5) solution.

Next, add E. coli (HBIOI) to 10 μl of this DNA solution.
After F transformation, Ap clone was obtained. Next, the plasmid DNA of these clones
By investigating the characteristics of pDcosApr10ri/dhfr
Plasmid with 2pt inserted into the BamHI site of:
pDcos Apr10ri/Ipt (2BamH
Obtain E. coli having I) (Figure 9).

After growing this Escherichia coli in 1 liter of TYG-P medium, the bacterial cells were separated and purified to produce pDcos.
Plasmid DNA of Apr10ri/@pt (2BamE(I) was prepared.

(dl Creation and preparation of pDcos Apr10ri/@pt pDcos Apr10ri/%pt (2Ba
mI (I) LOt4 was limitedly digested by reacting with 9.1 units of BamF (I) in 100 μl of the reaction mixture at 37°C for 30 minutes. After removing protein with phenol extraction, DNA was obtained as an ethanol precipitate. , the precipitate is 1
ot10. It was dissolved in 7 μl of IE (~Lafi/μl).

Next, perform limited digestion with this BamHI and add 1μ of fCDNA solution.
l (~1 μs) to 1 nit of Kl in 25 μl of the reaction mixture.
React with enow enzyme for 30 minutes at 22°C to
After changing the sive end to a blunt end,
0.8 Good for preparative agarose gel electrophoresis, 6,
Separate and purify the 3 Kbp long DNAfr piece to obtain DN
A was obtained as an ethanol precipitate.

This DNA precipitate was cyclized by reacting 20 μl of the reaction mixture with 6 units of T4-DNA ligase at 12°C for 17.5 hours, followed by phenol extraction to obtain the DNA as an ethanol precipitate. IMTris-H
CI (pH 7,5) Dissolved in 201 tll of R solution 1 t
0 Then, with 10 μl of this DNA solution, E. coli (1 (B 1
After transforming 01), an Ap clone was obtained. Next Dekokura's clone has Bunsumido DN
, check the special characteristics of A, I) J) (! 03Apr/
ori/ypt (2BamHI) C03 and ypt
BamF (IA tablasmid lacking the I site):
A canine bacterium carrying pDcosApr10ri4pL (Figure 9) was obtained.

This Escherichia coli was propagated in TYa-pr@ji11, and ccc-DNA was isolated and purified from the bacterial cells to prepare plasmid DNA of pDcoa Apr10ri/>pt.

Generation of tel pTK-4/BamHI and mMpT
K-4[1shiura, M, etal, Mo1. c.
ell Biol.

Z, 607 (1982)] tolt, 9 was reacted with 3.0 units of PvulI at 100 Ai at 37°C.
After reaction for 30 minutes and limited digestion, the DNA was subjected to 0.8% iGalose gel electrophoresis to separate and purify a DNA fragment with a length of 6.43 KbpO, and the DNA was precipitated with ethanol.
Precipitate 1Ot10.1E 3μ! (~lμI/ltl
) was dissolved in

Next, (7) DNA solution 0.4 pi (~o<tt
fi) and BamHI linker-Cd (pC-G-G
-A-T-C-C-G),)2μ,i? 20 μl of the reaction mixture was reacted with 6 units of T4-(DNA IJ gauze) at 22° C. for 6 hours to ligate.

Then, add 10 μl of this reaction solution to 5 μl of the reaction mixture.
17.5 units of T4-DNA ligase at 12°C.
The reaction was carried out for a period of time to achieve ring closure. After that, phenol extraction was performed to obtain DNA as an ethanol precipitate, and the precipitate was precipitated with O, l
M'['ris-HCI (pH 7,5) solution 20
It was dissolved in μe.

Next, add E. coli ()LB to this DNA solution at 10/'IJ.
lol) was transformed into L&C clones, Apr and TCr clones were obtained. Then, we investigated the characteristics of the plasmid DNA of these clones, and determined the origin of pTK-4.
Plasmid with Bam1 (I linker inserted at Pvu[i site: ρ■-4/BamHI) between ri and TK
(Fig. 9) was obtained.

After growing this E. coli in 1 liter of 'rYG-P medium, ace-DNA was isolated and purified from the bacterial cells, and pTK
-4/Bamf(I plasmid DNA was prepared.

rl pTK-4/BamHI/BamHI Creation and Preparation pTK-4/BamHI LOt4': Reaction mixture 100/All with 9 units of BamHI and 3
After limited digestion by reacting at 7°C for 30 minutes, phenol extraction was performed, and the DNA was precipitated with ethanol (- and (
! □4.

This ethanol precipitate was completely digested by reacting 100 μl of the reaction mixture with 15 units of PvuTl at 37°C for 2 hours, followed by phenol extraction to obtain DNA as an ethanol precipitate. IE 3μl (
~ lμy/μl).

Next, 1 μl of the DNA solution was subjected to limited digestion with BamHI and further digested with pvμ■! (~Iμg) was mixed with 2 units of Klenow enzyme in 25μl of the reaction mixture at 22°C for 3 hours.
Incubate for 0 minutes and turn the coheaive end blue.
After changing to nt end, ori and Ap r were subjected to 0.8% preparative agarose gel electrophoresis.

A DNA fragment (4,67 Kbp) containing TK was separated and purified, and the DNA was obtained as ethanol precipitate.

This DNA I! t# was reacted with 6 units of T4-DNA ligase in 20 μl of the reaction mixture at 12°C for 17.5 hours to achieve ring closure (in this case, the BamHI site was reconstituted), and then phenol extraction was performed. was precipitated with ethanol, and the precipitate was precipitated with 0.1 M Tr.
It was dissolved in 20μ of is-HCI (pH 7,5) solution.

And this DNA solution is 10μ! So, Escherichia coli (HB 1
ot) e) After the run home station A p
r and Ta' clones were obtained. Next, the percentage of plasmid DNA in these clones was examined, and pTK-
4/Grasmid lacking the DNA fragment (i, 76 Kbp) between the PvulI site of BamHI and the BamHI site (on Tcr): pTK-47BamH'J3
E. coli containing amHI (Figure 9) was obtained.

This E. coli was grown in TYG-P medium lJ! After growing in the microorganism, ace-DNAf is isolated and purified from the bacterial cells, and pTK
-4/BamHI/Bamf (I plasmid ONA
was prepared.

(gl pDcos Aprillort, now pt/'rK
Creation and preparation of (2Barr+f-H) pTK-4/13amHI/BamHI 20tt9
React ay, zo.

The DNA fragment containing TK (2Kbp
) was separated and purified to obtain DNA as a total ethanol precipitate, and the precipitate was 10.1E 6 μm (~1/i!/μ
l).

On the other hand, pDcos Apr10ri/ypt LOp9
91 units of BamHI in the reaction mixture LOOtt.
Digestion was performed by reacting with 37°C for 2 hours. After protein removal with phenol extraction, DNA'e was precipitated with ethanol (10 tlo, lE 7 μg/μg/μ).
It was dissolved in

And TK of pTK-4/BamHI/BamHI
1 μl (~1 μg) of DNA solution of BamHI fragment containing
and pDcos Apr10ri/ypt f Bam
1 μg (~1 μg) of the DNA solution digested with HI was ligated by reacting with 6 units of T4-DNA ligase in 20 μl of the reaction mixture at 12° C. for 17.5 hours. Thereafter, phenol extraction was performed to obtain l, -t, DNA as ethanol precipitate, and the precipitate was precipitated with 0.1 M Tris-HCI (pH
7,5) Dissolved in 20μ of solution.

Next, add 10 μl of this DNA solution to E. coli (f(Blot).
) was transformed, and then the AI)r clone was obtained. Next, plasmid D possessed by these clones
Examining the properties of NA, pDC08Ap rlo r i
/2 Plasmid with TK inserted into the BamHI site of pt: pDcos Apr10ri/ypt/T
E. coli harboring K(2BamHI) (Figure 9) was obtained.

After growing this E. coli in 1 liter of TYG-P medium, ccc-DNA was isolated and purified from the bacterial cells, and pD
cos Ap 10 rl/pt/TK (2Ba
Grasmid DNA of mHI) was prepared.

pDcos Apr10ri/ypt/TK (2Ba
mHI) 10 μl9 tl-reaction mix*100
in μlJ with 6.9 Ut of BamHI and 30 μl at 37°C.
Limited digestion was performed by reacting for minutes. After protein removal by phenol extraction, the DNA was precipitated with ethanol, and the precipitate was
0t10. It was dissolved in 7 μl of IE (~it4/pi).

Next, 1μl of this BamHI-digested DNA solution
(~ ■ μI) in 25 μl of the reaction mixture, 2 units of Kl
React with enow enzyme for 30 minutes at 22°C to
After changing the sive end to a blunt end,
0.8% Preparative Agarose Gel I4 Pneumophoresis, 8
, 3 Kbp length 1) NA fragment was completely isolated and purified to obtain DNA as ethanol precipitate.

Then, this DNA precipitate was reacted with 20 μl of the reaction mixture with 6 units of T4-DNA ligase at 12°C for 17.5 hours to achieve ring closure, and then phenol extraction was performed.
A was obtained as ethanol precipitate, and the precipitate was diluted with 0.1 M Tr.
It was dissolved in 20μ of is-HCI (pH 7,5) solution.

E. coli (HBIOI)'ff with 10 μl of this DNA solution.
: After transformation, Apr clone was obtained. Next, the plasmid DNA of these clones
A'l? Sex't-A, pDcos Apr10r
i/lpt/TK (2BamHI) TK and Ap
Ban...l site between r and plasmid lacking: pD
E. coli harboring COsApr10ri/1Ppt/TK (Figure 5.9) was obtained. This strain was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology [Feikoken Bacteria No. 7552 (FE
BMP-7552) J.

After this E. coli was grown in TYG-P medium 1/2, ace-DNA was isolated and purified from the bacterial cells, and the entire plasmid DNA was prepared into pDcos Apr10ri4pt.

Example 5 Mouse L cells (Ltk) transfected with a recombinant thymidine kinase (TK) gene of herpes simplex virus type 1 (H8V-1') having genomic DNA pBR-322 (lD[group & e) by pDcos Aprilori- ~[f(SV-ITK+))lt.

Cultured in Eagle MEM supplemented with 10% calf disintegration, confluent monolayers (10
0 mm-9 plates, ~IQ cells/grade) and PBS (137 mM, NaCl, 3
mM KCI, 8 mM Na2I (PO4+ 1 m
After washing twice with M KH2PO4), solubilizer (10
0A, 9/kl Proteinase 0.5%SD
S +150 m1VI NaCl, 10mM EDT
A, 10 mM Tris-HCI (pH 7,5)
) was added at 1.0 me per plate and heated at 65℃ for 5 to 3 hours.
The mixture was kept warm for 0 minutes and then mixed. This solubilized material is 50ml!
The mixture was transferred to a centrifuge tube and kept at 37° C. overnight, and 100 μg Al of Protinasek was further added thereto. After the reaction, it was gently extracted twice with phenol saturated with Tris buffer at 50 m m T.
ris-HCI (pH 8,0), 10 mM ED
Dialysis was performed four times against three volumes of dialysis buffer consisting of TA and 10mM NaCl. Dialysis fluid is 50 μgA
ll ωNa5e-1,000 roe RNase A was added and kept at 37° C. for 3 hours to degrade RNA.

After the reaction, the slightly aqueous heel layer was gently diluted twice with an equal amount of phenol and chloroform mixture to 10 mM Tris-HCI.
The purified JAM
50ml of DNA solution was obtained. 260 n of DNA solution
m and 280 nm 1 term “1f)l (OD) is 10
0, 667, 0.363 respectively (OD2g
o10D2jo'=0.544), and the concentration of DNA uo, 667X0.050 (μg/100)X
10 = 0.334 μg/μl.

(Preparation of genomic DNA of b135-45 Kbp 5.98y (21ng) of purified genomic DNA hemorrhoid fluid was added to 29.96ml of reaction mixture (50mM NaCl, 10mM
MTris-HCI (pH 7,5), 10 mM
Sau 3A (New Engl-and B
iolabs) for 1 hour at 37°C for limited digestion, and then 0.5 MEDTA (pH 8,0) solution was added to
．． 3 d and gently extracted twice with an equal volume of Tris buffer saturated phenol [7. After concentrating the aqueous layer to 10ml with secondary butanol, 3.0M sodium acetate and 20at of ethanol were added to obtain genomic DNA as an ethanol precipitate. The ethanol precipitate was dissolved in TB; 2rrtl.

Next, 300 μl of this DNA solution, 5-25%
Sucrose density gradient centrifugation (20mM Tris-HCI
(pH 7°6), 5 mM EDTA, Beck-r
7 SW28, 20.00Orpm, 9 hours, 20℃
) and fractionated every 30 cells. 20 μl of each fraction
．． 4 Measure the length by thiagarose gel electrophoresis,
After collecting 1IiII of DNA with a size of 5 to 45 Kbp, dialysis was performed three times against TE3A, and the dialyzed solution was extracted with phenol. obtained as a precipitate,
The precipitate was dissolved in TE1mlC to obtain a 35-45 Kbp genomic DNA solution. 0D260 and 0 of DNA solution
0.216 and 0.1 respectively when diluted 50 times in D28o
12 (OD28010D260=0.519), and the concentration of DNA is 0.216X O,050 (μ, 9/
l0C))X50 = 0.54ttl/1tll
I thought so.

Preparation of tel cosmid vector DNA pDcoa A
prlori 40 t4 at 1 volume at 800μ/reaction mixture.
Digestion was performed by reacting with 52 units of Pvu■ at 37°C for 2 hours. After that, perform phenol extraction. XDNA was obtained by ethanol precipitation, and the precipitate was precipitated at 10t10. IE10
It was dissolved in 0 μl.

95 μl of this Pvu[-digested DNA solution was added to 500 μl of the reaction mixture (50 mM Tris-HCI (
pH9,0).

1mM Mlc12, 0.1mM ZnC1, 1mM
suberdimine) and 6 units of phosphatase ((:
:Jfintestinal alkaline ph
osphatase, Boehringer.

q) Mannheim) for 15 minutes at 37°C.
After incubating for 15 minutes at 56°C, an additional 6 units of phosphatase were added and incubated at 37°C for 15 minutes, followed by 15 minutes at 56°C.
The cleavage fragment of Pvu■ was inactivated by reacting for 1 minute. Then, H2O400ttlJ, sTE (100mM
Tris-HCI (pH 8,0), IM NaCl,
l OmMEDTA) 100 ttl, 10% SDS
The mixture was treated with 50 ttll k and incubated at 68° C. for 15 minutes to inactivate the phosphatase. After protein removal by phenol extraction, the DNA was precipitated with ethanol, and the precipitate #1 was dissolved in 1Ot10 and 30μl of IF.

Next, this Pvu [30 μl of DNA solution digested with phosphatase] was added to the reaction mixture at 500 ttl at 152 μl. The mixture was digested by reacting with Nit's BamHI at 37°C for 2 hours. After removing protein with phenol extraction, D
NA was obtained as ethanol precipitate, and the precipitate was 10. I
A cosmid vector DNA solution was obtained by dissolving in 30 μl of E. 0D26o and 0D28oid of DNA solution
300 times diluted (-re('shio, 046, 0.
026 (OD28o10f)2ao=:Q, 565)
and the concentration of DNA is 0.046 x 0.050
(μ, 9/xOD) xaoo-o, 690μg/μ
It was l.

(35-45 Kbp genome solution 2 prepared with bl,
611tlJc 1.4μ, 9) and 0.86μl of the fc cosmid vector DNA solution prepared in (C) (0,
sc+μμ) was reacted with 10 μl of the reaction mixture and 5.9 units of T4-DNA ligase at 12° C. for 17.5 hours to ligate. After the reaction was completed, the reaction solution was stored at 4°C.

Escherichia coli BHB2690 was grown in NZY medium (11NZamin).
s, 0.5% yeast extract, 0.5% NaCl, 0, 2
%MgCl2・6H20, pH 7,5) After measuring the OD 600 of the overnight culture in 100 ml,
Bacteria were inoculated into 500 ml of NZY medium (in 27 flasks) kept at 32°C so that the initial 0D6o0 was 0.025, and shaking culture was started at 32°C. And 0D
When 6oo reached 0.3 (2 hours after the start of culture)
After 0 minutes), glophages were induced at 45°C every 15 minutes. Thereafter, the culture temperature was changed to 38-39°C and cultured with vigorous shaking for another 3 hours (induction was tested by adding a drop of chloroform to a small amount of the culture solution).
.

After culturing, 6. Collect bacteria by centrifuging at ooorpm for 10 minutes, and incubate the bacteria with 20mM Tri8-HCI.
(pH 8,0), 1 rnMEDTA, 5mM β-
3.6 ml of a mild solution consisting of mercaptoethanol K
suspended. Then, after performing ultrasonic treatment at 4℃ or below (maximum output for 5 seconds x 20 times), 20.000 rpm
Centrifuge for 10 minutes at m (4°C), and remove the supernatant liquid
7nl! I got it.

Next, the supernatant was mixed with 3FdK, etc. of the above buffer and packaging buffer (6mM Tris-HCI (p) (8
,0), 50mM suberdimine, 50?7LM putrescine, 20mMMfL□, 30mM ATP,
After adding 0.5ml of 30mM β-mercaptoethanol, 15μl each was dispensed into 1.5ml Ebbendorff tubes, immediately frozen in liquid nitrogen, and -70
Saved at 0.

E. coli 2688 was cultured overnight in 100 m of NZY medium, and after measuring 0D6oo, it was added to 500 m1 of NZY medium (in a 211 flask) kept at 32°C in advance so that the initial of) soo was 0.025. Inoculate the bacteria into LJc and start shaking culture at 32°C. And 0D
At the time of reaching 6oφ5O93 (2 hours from the start of culture)
After 0 minutes), prophage induction was performed at 45°C every 15 minutes.

Thereafter, the culture temperature was changed to 38-39° C., and the culture was continued with vigorous shaking for another 3 hours (induction was tested by adding a drop of chloroform to a small amount of the culture solution).

After completion of the culture, collect the bacteria by centrifuging at 6,000 rpm for 10 minutes, and transfer the bacterial cells to a sucrose solution (10% sucrose, so m M Tris-HCI (pH 8,0)).
'') After suspending in 3-3, 0.5 d each was placed in six Etzbendorf tubes, and lysozyme solution (2 mq/ml, 0.25 M Tris) was added to each tube.
-HCI (pH 8,0)) was added (4°C), mixed gently, and quickly frozen in liquid nitrogen.

Next, the tubes were placed on ice to completely thaw the extract, and then 25 μl of packaging buffer was added to each tube and mixed. And each extract together, 21,000
A supernatant was obtained by centrifuging at 0 rpm for 1 hour (4°C), and aliquots of lθμz of this were dispensed into 1.5 ph Espendol 7 tubes, and immediately frozen in liquid hydroxide at 1 to -70 ℃.
Stored at °C.

(fl in vitro packaging) Place the bacterial cell extracts of BHB2690 and BHB2688 stored at -70°C on ice, and thaw the BHB2688 first.
The extract was added to the extract of Bf (82690) and mixed gently.

When almost all the cells were melted, 2.5μ7 (0.5μ!?) of the recombinant DNA solution prepared in (d) was added, mixed well, and reacted at room temperature for 1 hour to give 1nvi tr.
o Packaging was performed.

After the reaction, add 8M solution (0,581NaC1,0,2%
M1S04-7 H2O, 50mM Tris-HCI
(pH 7,5) 0, 01% gelatin) lTnl! and chloroform-drops were added and mixed. Then, the Etsbendol 7 tube was placed in a desk centrifuge for 30 seconds to obtain a supernatant liquid as a transduced 7A particle solution.

□□□) Transduction and grating colon I HBI
OI-L-broth (added with 0.4% maltose)-
200 μl of the overnight culture was collected using a desk centrifuge, and the cells were resuspended in 100 μl of 10 mM Mp12 solution. Next, 100μ of this cell suspension was diluted 10 times with 7KSM solution, and 100μ of transducing phage particle solution was added thereto, kept warm at 37°C for 15 minutes for transduction, and then L-broth 1. Add IM and heat at 37℃ for 4 hours.
Shake and incubate for 5 minutes. Then, add 100 ml of this scoop M suspension.
μl Fampicillin 20 μg/rJ Gold Containing 100
After applying the mixture to L-plates of 1.0 mm in size and leaving the plates at 37° C. overnight, the resulting colonies were packed with a bamboo comb and grown in L-gross (containing 20 μg of ampicillin and 7 fnl). Also, the number of colonies per plate is 20-1001!
It was jt.

Ampicillin resistant colonies (transformants) 20
After culturing overnight in L-gloss (containing ampicillin 20 μj9/fnl) for 2 hours, 1.5 ml of each culture solution was placed in a 1.5-tube Etsubendol 7 tube and collected using a desk centrifuge. These bacterial cells were mixed with solution I (50mM glucose, 25mM Tria-HCI (pFI 8
．． 0), 4mHi lysozyme with 10mM EDT)
After resuspending the suspension at 100 µm, it was left at room temperature for 5 minutes. Next, solution n (0,2N NaoH.

Add 200 μl of 5DS), mix gently, and leave on ice for 5 minutes, then remove the solution! II C5M Potassium Acetate (
Add 1150 μl of pH 4,8) and mix gently.
Placed on ice for 5 minutes. Then, the Eppendorf tube was centrifuged on a desk to obtain a supernatant, and twice the volume of ethanol was added to this to obtain plasmid DNA as an ethanol precipitate.

After dissolving a small amount of 10.1EK plasmid DNA of each transformant, size marker DNA (Lambda DNA: 48 Kbp, Lambda D
NA) digested with Iindlff: 22.3K
bp.

When subjected to 0.4 thiagarose gel electrophoresis with ampicillin-resistant colonies 2
The presence of plasmid DNA was observed in all of the 0 samples with a size of 22.3 Kbp to 48 Kbp.

[Brief explanation of the drawing]

Figure 1 shows the new cosmid vector pDcos Aprlo.
Figure 2 shows a method for producing a genomic DNA gene bank using the novel cosmid vector shown in Figure 1, Figure 3 shows the new cosmid vector pDcos Tc'', and Figure 4 shows the new cosmid vector pDcos Tc. pDcos Tc
Figure 5 shows the new cosmid vector pDcos Apr10.
ri4yut/TK, Figure 6 shows how to create pDcos Tcr, Figure 7 shows how to create pDcoa Tcr/ori, Figure 8 shows how to create tri pDcos Apr/ori, and Figure 9 shows how to create pDcos Apr/ori. The figure is pDcos Apr10ri/ypt
/'I' shows how to create it. , 1' Procedural amendment filed November 26, 1980 Commissioner of the Japan Patent Office 1. Indication of the case 1982 Patent Application No. 112206 2. Name of the invention Novel cosmid hector and method for producing gene bank using the same 3 , Relationship with the amended person's case Patent applicant address 2-7-8 Nihonbashi Honmachi, Chuo-ku, Tokyo Name (5
07) Banyu Pharmaceutical Co., Ltd. 4, Agent Address: 406 Kita-Otsuka Building, 2-16-9 Kita-Otsuka, Toshima-ku, Tokyo 170 Telephone: (917) 1917 As per the voluntary attachment. (1) rBiol, 4th line from the top of page 12 of the specification,
. 1," is corrected to "rBiol,, Sat." (2) rdGTPJ in the 3rd line from the bottom on page 20 of the specification
Correct the statement rdGTP, 80μM ATP, 80μMTT to "suspend." (4) Line 9 from the bottom of page 24 of the Middle East specification: “In 1β”
The statement has been corrected to "in 1β (contains 20 pg/ml of Ap)". (5) Third line from the top of page 25 of the Middle East specification rO, 25J
Correct the statement to read ro, 25MJ. (6) The 5th line from the bottom of the same page says r1Mj, “IM
” he corrected. (7) In the specification box, page 28, line 8 from the bottom, line 7, 1 narrow” is corrected to rMJ. (8) Correct "rMJ" in the 9th line from the bottom of page 30 of the specification to rMJ. (9) On the 7th line from the bottom of the same page, replace rPvuJ with [P
Correct it as vuUJ. I Q (1) Second line from the top of page 31 of the specification rlom
Correct MJ to “lOmMJ.” (11) Box 32, line 10 from the top of the specification r (7,
5) Correct J to r(pH7,5)J. (12) Box 34, line 5 from the bottom of the statement r (BII
[)J is corrected to r(Bgl II)J. (13) 4th line from the bottom of the same page r (2B II)
Correct the word J to [(28gβ II)J. (14) 7th line from the bottom of page 43 of the specification r100p
g/m7! ,” it says r100μg/m7! Correct it with J. (15) 7th line from the bottom of page 51 in box ``cohe''
Correct sieJ to rcohesiveJ. (16) Mei 1III, page 55, line 10 from the top r
Correct B IIJ to rBg# Ill. (17) 8th line from the top of page 62 of the Middle East Specification “πVXB
rainJ and aru wo r (yrVX (BrainJ
I am corrected. (18) In the 9th line from the top of the same page, correct "r(1983)J" to "r(1983))J". (19) In the 11th line from the top of the same page, correct "r(1980)J" to "r(1980))j". (20) On the 122nd line from the top of the same page, correct the text "plasmid" to "1 plasmid)". (21) rpDcos on page 74 of the statement box, line 8 or line 6 from the bottom rpDcos... 1μ7! (~μg),”
Delete certain. (22) Box 82, line 5 from the top of the statement r10ri
/pt/TKJ and aru wo rlo r i/gp t/T
Correct KJ. (23) Box 84, line 9 from the bottom of the specification r3mMJ
(24) 7th line from the bottom of the same page rProteinas
"e" is corrected to [Proteinase KJ. (25) Box 85, line 6 from the bottom of the statement r/1.0
0) Correct J to r/1 0DJ. (26) In the 8th line from the top of page 91 of the specification, ``Homare j'' is corrected to ``Yo''. (27) Figures 2 and 9 of the box in the drawings of this application are corrected as shown in the attached sheet. that's all

Claims (1)

[Claims] A cosmid vector. (2) By having two COSs of lambda phage or lambdoid phage with the same directionality and cutting them with two types of restriction enzymes, a right arm and a left arm of approximately equal length each having one eoi9 are created. The cosmid vector according to claim 1, in which the cosmid vector can have equimolar amounts of . (3) The cosmid vector according to claim 1, which contains a gene sequence that exhibits drug resistance or a specific physiological function for determining the insertion of a gene fraction derived from the cosmid vector. (4) The cosmid vector according to claim 3, which has a gene sequence that expresses ampicillin resistance, tetracycline resistance, neomycin resistance, or chloramphenicol resistance as drug resistance. (5) As a specific physiological function, thymidine kinase (TK
) activity, adenine phosphoribosyltransferase (
APRT) activity, hypoxanthine guanine phosphoribosyltransferase (HG P RT) activity, xanthine guanine phosphoribosyltransferase (XG
4. The cosmid vector according to claim 3, which has a gene sequence for expressing PRT or gpt) activity, or dihydrofolate reductase (dhfr) activity. (6) The cosmid vector according to claim 1, which contains a gene fraction having multiple functions derived from CoLFA. COS obtained by cutting the cosmid vector described in claim 1 with two types of restriction enzymes.
35-45 K between the right arm and the left arm holding one each.
Inserting various foreign DNA fragments of bp length, in v
Foreign D
A method for producing a gene bank, characterized by producing a gene bank for NA. (8) The foreign DNA is derived from bacteria, filamentous fungi, microorganisms such as yeast, higher animals and plants, or various phages, etc.
The manufacturing method according to claim 7, which is A.