JPS62228280A - Novel cosmid vector, derived from lambda phage and having replication function and production of genetic bank using said cosmid vector - Google Patents

Abstract

PURPOSE:To readily and efficiently produce a genetic bank of an extraneous DNA using a novel cosmid vector, by preparing the novel cosmid vector characterized by having replication function derived from lambda phage employing a genetic manipulation technique. CONSTITUTION:A cosmid vector having >=2 COS of lambda phages or lambdoid phage with the same orientation property in the same molecule and replication function derived from the lambda phage is cleaved with 2 kinds of restriction enzymes to give the right arm and left arm of almost the same length each having at least one COS and various kinds of extraneous DNA fragments having 35-45kb base pairs are inserted between the arms to convert the arms into tranducing phage particles by packaging. The resultant particles are then cloned in Escherichia coli to produce a genetic bank of the extraneous DNA. Since the number of copies of recombinant DNA per cell in a host is large and almost constant, the quality and stability of the genetic bank are high.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to J4 gene manipulation technology, and more specifically, a novel cosmid vector with a replication function derived from lambda phage created using gene manipulation technology and a gene using the same. This relates to the method of manufacturing banks.

BACKGROUND OF THE INVENTION Many genes of mammals, including humans, are larger than 20 kilobase pairs (hereinafter referred to as kbp). Currently, the size of DNA that can be cloned into the widely used lambda vector is 20 kbp, so it is not possible to clone an entire gene larger than this in a robust manner.

lambda phage cohesive encl 5it
If you use a vector having a cosmid vector (hereinafter abbreviated as COS), you can clone an NA with a size of 35 to 45 kbp. Among various vectors, cosmid vectors are capable of cloning DNA of the largest length, but they are difficult to prepare, and the background of only vectors without inserts is high. Furthermore, the use of cosmid vectors has not progressed due to the lack of efficiency. However, Ish-trorowicz gBt
Jrke (Ishhorowitz and Bulke) devised a way to give vectors directionality using phosphatase, and succeeded in reducing the background value of the vector alone and increasing the efficiency of cloning (
D, Ish-11orowicz & J, F, B
Urke.

(Isshuhorowitz and Bulke) NuCI
eic Ac1ds Res, (Nucreitsk Acids Research), 9.2889 (1981))
.

Problems that Zadeaki is trying to solveIsb-11 Genomic DNA analysis using the Orowicz & Burke method
The method for producing a gene bank is as follows (taking pJB8, one of the currently widely used cosmid vectors, as an example). pJB8 is 5.44 kbp
The major enzyme (-1 restriction enzyme BamHI, Hind[l
, 5allc- is a cosmid vector that has been cleaved at one site each and has 1 t111 of lambda phage CO3. '(Dl) Cut JB8 with 11-1dII [117
The DNA is made into a linear DNA by iv treatment, and the cut fragment of trindlI is inactivated by treatment with phosphatase. Then, two DNA fragments are obtained by cleaving with Bam111, and a large NA fragment (5.4 kbl) containing cos is isolated by agarose gel electrophoresis. The obtained cos-containing DNA [ffi fragment (
5.4 kbl)) corresponds to the left arm of the lambda phage.

(2) pJ88 is made into a linear DNA by cutting with 5all, and then the 5all fragment is inactivated by treatment with phosphatase. And BamHl
Two DNA fragments are obtained by cutting with c
A DNA fragment (2,34 kbp) containing os is isolated by agarose gel electrophoresis. The obtained cos-containing DNA fragment (2,34 kbp) corresponds to the right arm of lambda phage. ■For example, human genomic DNA can be partially cleaved with restriction enzyme Sau 3A or ML+OI.
J rlJi L/, 35-45kl) p size D
Fractionate one piece of NA. This DNA fragment having a size of 35 to 45 kbp is inserted between the left arm and the right arm, which were separately made by UA using ■ and ■, using a ligating enzyme (T4 DNA ligase) to prepare recombinant DNA. ■ By converting the recombinant DNA into transducing phage particles by in VitrO packaging and then infecting E. coli.
A gene bank of genomic DNA can be created.

That's it for Isb-Ilorowicz & Burke.
This is a method for producing in-gene bank genomic DNA using the (Ishuborowitz and Bulke) method, but there are the following four problems. First, pJB8 has only one COS, and the left and right arms must be prepared separately.

Second, for the left arm and the right arm, D containing cos, respectively.
The NA fragments must be separated by agarose gel electrophoresis. Thirdly, the length of the left arm of ρJB8 is 5.4 kb
p, the length of the right arm is 2.34 kbp, and the length of the left arm and right arm are different, so the genomic DNA is 35 to 45 kbp.
The probability of connecting to the left and right arms is also different, and the ratio of mixing left and right arms must be considered. Fourth, the origin of the replication function of 0JBB is p8R322 (Bol 1var, F, ).

(Ho1,1 Bar F) et al, Gene
(Gene), L95 (1977)), p.
The copy number per cell of BR322 in the host is ~20, with the copy number per cell decreasing further in recombinant plasmids containing foreign DNA as large as ~40 kbp.

Varies depending on the structure of the incorporated foreign DNA (
P, F, R, Little & S, Il, Cross
(PFR Re1~le & Nis H Cross), Proc, Natl, Acad,
Sci, U, S, A, (Proceedings
of the National Academy of Sciences United State of America IJ Power), male,
3159 (1985)). The fact that the copy number of the recombinant plasmid per cell varies depending on the structure of the foreign DNA does not result in instability of the gene bank constructed using the cosmid vector. Furthermore, if the copy number of the recombinant plasmid containing the gene to be cloned is low, it may not be possible to detect clones carrying the desired DNA by colony hybridization, or the expression of the gene may not be detected due to low expression. There is a problem that says it cannot be done.

In order to solve these problems, the inventors of the present invention discovered that two or more lambda phage cos having the same directionality were cut with two types of restriction enzymes. by c
A new cosmid vector was created, which is characterized by having equimolar right and left arms having at least one os and having approximately the same quality, and having lambda phage-derived replication ability.

Then, foreign DNA using the cosmid vector of the present invention
This led to the completion of a method for producing a gene bank.

■ The cosmid vector of the present invention has two or more COSs with the same directionality in the same molecule, and by cutting with two types of restriction enzymes, each cosmid vector has a length of at least one cos. Since the right arm and the left arm can be produced in approximately equal moles, there is no need to prepare the left arm and right arm separately, and there is no need to separate them by agarose gel electrophoresis. Furthermore, since the lengths of the right and left arms are approximately equal, it can be assumed that the 1σ is approximately equal enough to be ligated to 35 to 45 kbp of genomic DNA using T4 DNA ligase. If an equimolar mixture of right and left arms is ligated as is with genomic DNA of 35 to 45 kbp, it can be expected that half of the recombinant DNA will be transformed into transducing Fur9 particles. Since the recombinant plasmid has a replication function derived from lambda phage, the number of copies per cell of the recombinant plasmid is expected to be higher than that of a recombinant plasmid with a replication function derived from DBR322, and it is expected to remain almost constant regardless of the structure of the foreign DNA. (P, F, R, L
ittle & S, 11. Cross (PFR Re1~le and Nis H Cross),
proc, Na mouth, Acad, Sci, U,
S, 8, (Broadcastings of the National Academy of Sciences United
Stay 1-of America), Village, 3159 (1985
) ).

The novel cosmid vector of the present invention, pTcO3λori
/Al)'/neo'/Cnl'/Cnl' is shown in FIG.

pT CO3λOri / AI)' /near /
Cm'/TK is the cosmid vector Lori
Replication function [λOri (P, O)] derived from c. and neomycin resistance road gene neo') (P, F, R, L
ittle & S, 11. Cross (P.F.R. Little and Niss H. Cross).

Proc, Natl, Acad, Sci, U,
S.A. (Proceedings of the National Academy of Sciences United State of America), 82.3159 (1)
985) ), Sv-dihydrofolate reductase gene (SV-dMr) (Subr
amani, Hulliuan & BerO (subsima 1, mulligan and barb), tool,
Ce11. Biol, (Molecular and
Cellular Hight Rossi), 1.854 (198
1)), pTK20 (3SVE/SV 010-derived nothymicin'Ft-t'31 gene (TK) rCbarl
es N, Coile & George) 1.5an
tanoelo, (Charles en Cole and George Emlintanzi x Mouth) Mo1.
Ce11. Biol, (Molecular and
Cellular Biology), 3.267 (1983
)], co-directional cos derived from the cocosmid vector pRH(-44) or pLH(+129)
3 pieces ()1iWa & +atsubara (Miwa Anto Matsuhara), Gana (Gene), 20
．． 267 (1982)], IINT-31 Yamaki's chloramphenicol resistance gene (cm') rTomiz
awa & Itot+ (Tomizawa & Ito), Proc, Natl, Acad, Sci
, U.S.A. (Proceedings of the National Academy of Sciences United States of America), Shin, 6096
(1981) 3, pBR327 Yamaki Ap'
[5ol) eron, Cavarrubias &
Bolivar, Gene, 9.28
7 (1980). In addition, pr cos which is a starting material in the production of the cosmid vector of the present invention
How to obtain Apr/Ori/cmr, please refer to JP-A-6
In Publication l-132187q, pSV2-dMrΔ
(Bol II) can be obtained from Japanese Patent Application Publication No. 60-25638.
This is disclosed in Publication No. 2.

This vector has the following characteristics. ■There are three COSs with the same direction. ■Bam1ll Sai 1
Genomic DNA of 35 to 45 kbp that has been limitedly digested with Sau 3A or Mbol can be cloned into ~.

■By cleaving the vector with C1al and treating it with phosphatase, the vector has directionality, and
By cutting the am1ll site, equimolar right and left arms are created. ■ Using T4 DNA ligase, Sau
3A or 1 (35-4 limitedly digested with boI, etc.)
The right and left arms of the vector were ligated to both sides of the 5 kbp genomic DNA at the Bam1ll site, and the recombinant D
The NA can be converted into transducing phage particles by in VitrO packaging and then infected into E. coli. ■This transductant DNA contains vector-derived neo', Ap', Cm', λori
(P, O), so by selecting with neokanamycin (or kanamycin), ampicillin, or chloramphenicol, it is possible to selectively keep alive only E. coli cells that have the recombinant DNA as a plasmid. ■Also, since this recombinant DNA has a TI gene, when the gene is transferred into cultured cells of lI+li mammals, by using TK activity as a marker, the recombinant DNA derived from the vector can be transferred. can be determined at the level of cultured cells. ■Furthermore, this recombinant DN
Since A has a replication function derived from a lambda phage, it has the characteristic that the number of copies of recombinant DNA per cell in the host is relatively large and remains almost constant regardless of the structure of the foreign DNA.

A novel cosmid vector of the present invention, pT cosλori
/Ap'/cm' is shown in FIG. I)TC
To O3, Iori/8p' cmr is a cosmid vector and is derived from ori c? ! 2 g function λ
ori, dihydrofolate reductase gene (dMr) as a spacer, cosmidvec'l-theal1)
Rn(-44) is also L/<1. t pLIl(+12
9) Three cos molecules with the same orientation of origin, pNT-3
Chloramphenicol resistance gene derived from 1 (cm')
, and Apr derived from pBrt327.

This vector has the following characteristics. ■There are three COSs with the same direction. ■3 that was limited to Sau 3A or Hbol on the Bam1ll site
Genomic DNA of 5-45 kbp can be cloned.

■By cleaving the vector with C1al and treating it with phosphatase, the vector has directionality, and
By cutting the am1ll site, Kasamoru's right and left arms are created. ■ Using T4 DNA ligase, Sau
35-45 limited digested with 38 or Mbol etc.
kl) The right and left arms of the vector can be ligated at the Bam1ll sites on both sides of the genomic DNA of p, and the recombinant DNA can be converted into transducing Far2 particles by in vitro packaging and then infected into E. coli. ■This transductant DNA contains vector-derived Apr, cmr, and λ ori, and by selecting with ampicillin or chloramphenicol, only E. coli having the recombinant DNA as a plasmid is selectively kept alive. I can do it. ■Furthermore,
Since this recombinant DNA has a replication function derived from a lambda phage, the number of copies of the recombinant DNA per cell in the host is relatively large and remains almost constant regardless of the structure of the foreign DNA. There is. Novel cosmid vector of the present invention 1) D CO3Apl' /λ
The ori is shown in FIG. pD cos Ap' /λ
o r t Ha, the replication function λ ori derived from the cosmid vector Loric, the dihydrofolate reductase term gene (dllrr) as a spacer, two identically oriented cos and p8R327 from the phage vector Charon 4A Yamaki. It consists of the next April.

In addition, DD CO3AD't'ori/dMr(
How to obtain BamHI> is from JP-A-60-256382.
In addition, pBR327/Bgl II Ccos
How to obtain 8t+cn9k is JP-A-61-132187
It is disclosed in the publication No.

This vector has the following characteristics. ■There are two COSs with the same direction. ■Bamt (I site is limited to 3 digested with Sau 3A or Mbol, etc.)
Genomic DNA of 5 to 45 kbp can be cloned.

■By cleaving the vector with C1al and treating it with phosphatase, the vector has directionality, and
By cutting the amHI site, equimolar right and left arms are created. ■ Using T4 DNA ligase, Sau
35 ~ limitedly digested with 3A or Mbo I, etc.
The right and left arms of the vector were ligated at the Bamf (I site) on both sides of the 45 kbp genomic DNA, and the recombinant DNA
transduced by in VitOrO packaging]? - can be converted into diparticles and then infected with E. coli. (2) This transductant DNA contains the vector-derived 80'1λ ori, so by selecting with ampicillin, only E. coli having the recombinant DNA as a plasmid can be selectively kept alive. ■
Furthermore, since this recombinant DNA has a replication function derived from Lambtuff 1-di, the number of copies of the recombinant DNA per cell in the host is relatively large and remains almost constant regardless of the structure of the foreign DNA. It has characteristics.

In addition, genomic DNA (foreign DNA) using this vector
The production method for gene banks is as follows (
(See Figure 2). ■pT CO3λori / Ap'
/ne.

'/Cm'/T is made into a linear DNA by cleaving with CIaI, and then the CIaI cut fragment is inactivated by treatment with phosphatase. By cutting with BamHI, a right arm and a left arm having approximately equal length and having one and two cos molecules, respectively, are prepared.

(2) Exogenous DNA is limitedly digested with Sau 3A or )lboI to prepare a DNA fragment having a size of 35 to 45kl). (1) A genomic DNA fragment of 35 to 45 kbl) is inserted between the left and right arms prepared in (2) using 74 DNA ligase to prepare recombinant DNA. ■The recombinant DNA is transformed into transducing phage particles by in vitro packaging and infected into Escherichia coli.

■Transduced kanamycin resistance, ampicillin resistance,
Alternatively, by selecting chloramphenicol-resistant E. coli with kanamycin, ampicillin, or chloramphenicol, only E. coli carrying recombinant DNA as a plasmid can be kept alive. In this way, a gene bank of genomic DNA can be produced.

This recombinant DNA can also be converted into transducing phage particles by in VitrO packaging and transferred to E. coli (in which the lambda phage has been lysogenized), and the E. coli containing the recombinant DNA can be treated with kanamycin, ampicillin or It can be obtained by selection with chloramphenicol. Next, a gene bank can be produced by inducing a transformant (E. coli) and converting the recombinant DNA having two cos into transducing phage particles by in vivo pancasing.

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

By cutting the vector of the present invention with two types of restriction enzymes, the right arm and the
ji arm is formed, and the combination of these two types of restriction enzymes is Pvu II and 8aI old.

Pvu fl and BgIII, C1al and Bam1l
Examples include C1aI, BolII, and the like. Of course, other combinations can be considered, so the invention is not limited to these.

In order to determine that a vector-derived DNA fragment has been inserted into the vector of the present invention, selection based on drug resistance is generally performed. Therefore, it is preferable that a DNA fragment expressing drug resistance is also incorporated into the vector of the present invention. &5 drug resistance includes common antibiotic resistance such as ampicillin resistance, tetracycline resistance, neomycin (kanamycin) resistance, or chloramphenicol resistance. pT CO3λOri /
Ap'/neo'/cm'/T has an ampicillin resistance gene, a neomycin resistance gene, and a chloramphenicol 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 cells of mammals, 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) a gene (Wigler, M., et al.
al,, Ce1l (cell) 14, 725 (197
8) ), adenine phosphoribosyltransferase (APRT)
Acad, Sci.

U.S.A. (Proceedings of the National Academy of Sciences United State of America), 76, 1373
(1979)), hypoxanthine guanine phosphoribosyltransferase (HGPRT) gene (Gr
af, L, H, Jr.
GRA) et al, Somat, C
e11. Genet, ('J Matheixel Geneteix), 5.1031 (1979)), xanthine guanine phosphoribosyltransferase (
XGPRT or gpt) gene (Berg, P.
(Barb P'), et al, Mo1.
Ce11. Biol, (Molecular and
Cellular Biology).

1.449 (1981)). pT
CO3λOri / AO' / neo' / cm'
/TK 4;i:dhfriden! Has a trochanter Ki Butsuko.

Furthermore, the replication ability of the cosmid vector of the present invention is derived from lambda phage, and lambda phage DNA,
It can be prepared from ml Sumid vector LOrlC, 7Fuge vector, plasmid vector λdv, etc.

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

In addition, the restriction enzyme used in the present invention (enzyme that has the ability to selectively cleave a specific base sequence site of a DNA fragment) or the DNA adhesive end generated by restriction enzyme cleavage, and the complementary relationship therewith. 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,
Alternatively, ligases that bind these are already well known, many enzymes are commercially available, and methods using them have been widely disclosed (Molecul
ar Cloning, Co1d Spring H
arbor Laboratory (Molecular
Cloning (Cold Spring Harbor Laboratory) 1 Maniati S, T., et al. (1982)), so they can be used.

Using the vector of the present invention, genomic DNA (foreign DNA)
) By creating a gene bank, it is possible to efficiently discover specific genes that express useful substances. This specific gene includes DNA derived from bacteria, filamentous fungi, yeast, microorganisms, higher animals and plants, and various phages.Specific useful substances include various hormones, enzymes, and even antibiotics. and amino acid synthesis enzymes.

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

Example 1. ' r r
r) Created Loric 10m Reaction Mixture 100 Recommended (6m)! T
ris-HCI (pH 7,5), 6m) (hcI2.
6mM DTT.

100 restriction enzymes/d BSA), 60 units of restriction enzyme S
ac II (New Enaland Biolab
(manufactured by S. Co., Ltd.) at 37°C for 2 hours for digestion. After protein removal by phenol extraction, 1 q of DNA was precipitated with ethanol, and the precipitate was dissolved in 10 t/0.1E 7 m (~1 t/JJ1.).

Next, 1.5 m of the DNA solution digested with this sac
(~1.5#) to 30d (33m) of reaction mixture
s-acetate (1) 87.9), 66mM
Potassium acetate.

10mM MgCl2. 0.5m)l DTT,
100m/mBS8, 0.1m dATP
, 0.1. , M dCTP, 0.1d
4 Cohesive
Changed d to blunt end. Then, add the reaction solution to 6
T4-DNA polymerase was inactivated by heat treatment at 5°C for 10 minutes.

This DNA solution 8Ai(0,4JJ!I), XhO
I linker (d(1) C-C-T-C-G-A-G-G
)) 2N, reaction mixture 20 recommended (50n+HTr
is-HCI (pH 7°4), 10mt(H(lcI
2, 10mM DTT, 1mM spermidine, 1
mM A1P, 100i/IdBS8), 6 units of T4 DNA ligase (New England
(manufactured by Biolabs) at 22°C for 6 hours. Thereafter, phenol extraction was performed to obtain DNA as ethanol precipitate. This DNA precipitate was mixed with reaction mixture 600J11 (150mM NaCl, 6mM Tr
is-HCI (pH 7,9).

61118) + (lcI2, 6mM DTT,
After digestion by reacting with 1000 units of restriction enzyme Xhol (manufactured by Takara) at 37°C for 4 hours using 100 JR/d BSA), the mixture was applied to a 0.8% preparative agarose gel and electrophoresed at 10 mA for 12 hours. (
40n+HTris, 20n+H sodium acetate,
2mM EDTA (1)88.15)). The degraded DNA fragments become visible through ethidium bromide staining and long-wavelength ultraviolet illumination. λori (0,P), ne
After cutting out the gel containing the DNA fragment (6.3 kbp) consisting of
The gel was ground to elute the DNA in the presence of NHa08c, 1mM EDTA (pH 7,5): water-saturated phenol = 1:1). After concentrating the eluate with secondary butanol, 0.1 volumes of 3M sodium acetate and 2 volumes of ethanol were added to obtain DNA as a precipitate. This DNA precipitate was cyclized by reacting with 6 units of T4 DNA ligase at 12°C for 17.5 hours in a reaction mixture of 20%, followed by phenol extraction.
was obtained as an ethanol precipitate, and the precipitate was 0. IHTris-
It was dissolved in HCI (pt17.5) solution 20J11.

■) Transfer 10 parts of this DNA solution to Escherichia coli 11B 101.
patent cell 100J11 (logarithmic growth phase E. coli 118101) at 0 to 4°C at 0.1f (CaCl2
After treatment with the solution for 15 minutes, 0.1) 1 cac12 /14X glycerol of 1710 volumes of the original culture solution was added.
Resuspend in solution and store at -70 °C), mix well and store at 0 °C.
for 10 minutes at
Bactotryl) tone, 0.5% yeast extract, 0.5% NaCl, 0.1x glucose) 2-
was added and cultured with shaking at 37°C for 1 rf.

This bacterial cell suspension was treated with 2075/d kanamycin (K
m) containing L-plate (1'g agar in L-broth)
Kanamycin-resistant clones were obtained by inoculating the cells on the same plate and culturing at 37° C. for 24 to 48 hours.

111),
(neo') clone in L-broth 2d (
III 20113/rail) at 37° C. overnight with shaking. Culture solution 1.5mi! was placed in an Eppendorf tube, subjected to desktop centrifugation, and after Togoku, the bacterial cells were extracted with an extract [8% sucrose, 0.5X Tr.
iton X-100, 50n14EDTA (pH 8,
0), 10mM Tris-HCI (pH 8,
0) resuspended in 0135ml. Next, lysozyme solution 25JJ1 (10mff/m(, 10mM Tr
is-HCI(1)H8,0)) and mixed,
The tube was placed in FIIjm water for 40 seconds. and,
Immediately centrifuge the tube at 4°C (21,000
rpm for 15 minutes) to obtain a supernatant, and add f
? NaSe A solution <20RI/rnll, 10t/
0. IE) 0.51 Jfl was added and kept at 37°C for 1 hour to degrade RNA. After that, the protein was removed by phenol extraction, and the DNA was precipitated with ethanol. Dissolve plasmid D in IE 20
An NA solution was obtained.

IV) Preparation of restriction map of plasmid mini-DNA and incubate this plasmid DNA solution 1JIi or 2d with restriction enzymes Eco R1, Bam11I, Nrul.

Digested with BstE I [etc., 0.4-1.5%
Agarose gel electrophoresis (40+nHTris, 20
mH sodium acetate, 2+nHEDTA (pH 8,1
5); 10 mA for 12 to 24 hours]. Decomposed DNA fragments can be detected by ethidium bromide staining and long-wavelength ultraviolet illumination. The plasmid DNA possessed by each clone was analyzed by creating a series of restriction n-base maps of the plasmid DNA. And L.O.
E. coli containing the plasmid Loric/X (Fig. 3) in which an XhOI linker was inserted into the Sac II site of riC was obtained.

v > 7, Hi7rasmi)' DNA (7): JLjJ This E. coli was cultured in TYG-P medium (1% BaCtOtrypt
one, 0.1% yeast extract, l, 0.2X g/l/D-su, 0.1% N84CI, 0.
3% NaC1,0,011%Na2SO4,0,08
% H(lc12 ・6H20, 1,52"X Na2
HP04 ・12)+20, 0.3X KH2
PO4) <20 tll/ml Km in 1 bottle), 3
The culture was incubated overnight at 7°C with shaking. After completing the culture, centrifuge (6
, ooorpm, io minutes) to collect bacteria, and then the bacterial cells were incubated with 25X sucrose, 501118 rris-
Resuspended in isotonic buffer 18K consisting of tlcl (p118.0). To this, lysozyme solution C5m9/rd,
0.25HTris-tlcI (pH8,0))
3.6- was added, mixed gently, and kept at 0°C for 5 minutes, then 0.258 EDTA (pH 8,0) solution 7.
2 ml was added, mixed gently and kept at 0°C for 5 minutes.

Thereafter, 10% SO8 solution 2.1- and 51-NaCI solution 8.1 were added and mixed gently, left at 0°C overnight, and then centrifuged at tl tJ (20,000 rpm,
45 minutes)
ate) ~39m! I got l. Add RN to this crude lysate.
ase A solution (2h+y/d, 10t10°IE)
After adding 20% of the protein and incubating at 37°C for 1 hour to degrade the RNA, protein was removed by phenol extraction, and the mixture was further subjected to gel filtration with Sephas 28. plasmid DNA
DNA was obtained from the void fraction containing ethanol as precipitated with ethanol, and then the DNA was precipitated with cesium chloride-ethidium bromide solution (18Tris-tlcI(1)) f 8.0
) 0.375d, 0.25HEDTA(p)
I 8.0>0.30m, R206,45me,
CsCl7. FJ7, EjBr solution (4 (Q/m
Equilibrium density gradient centrifugation (60,
OOOrpm, 12:00 f2U). Then, the covalently closed circular plasmid DNA
(ccc-DNA) appears as a band under the fluorescent band of linear plasmid DNA and chromosomes under long-wavelength ultraviolet irradiation in a centrifuge tube. This ccc-
The DNA band was fractionated and extracted with isopropanol saturated with CsCI aqueous solution to remove endium bromide, and then extracted with 10t10. Dialysis was performed against IE 11. Then, ccc-DNA was recovered from the dialysis fluid as an ethanol precipitate, and the precipitate was washed with 10 ml of ethanol. IE
I dissolved it in 2d.

A ric/X plasmid DNA solution was prepared.

(b) LOriC/X1l(7): I' and:
J”IHLoric/X 10 ml reaction mixture 10011
i(100+nHNaC1,6mHTris-11cI
(ptl 7.4), 6+nHMoC12°5mMD
100R/d BSA), 50 units of the restriction enzyme NruI (New England Bi
(manufactured by Olabs) at 37°C for 2 hours to digest (bluntend). After protein removal by phenol extraction, the DNA was precipitated with ethanol, and the precipitate was
0t10.1E7d (~1119/di>).

0.4 d (0°4 埒) of this DNA solution and old ndmu
jy -(d(DC-A-A-G-C-T-T-G)
) Add 2#t to 6 units of T4 with 20d of reaction mixture.
The mixture was ligated by reacting with DNA ligase at 22°C for 6 hours.

This DNA solution was mixed with a reaction mixture of 60 kg and 18 units of T.
After reacting with 4 DNA ligase at 12°C for 17.5 hours to cyclize r11, phenol extraction was performed.1. D.N.
A was obtained as an ethanol precipitate, and the precipitate was 0. IHTris
-f(CI (pH 7,5) solution).

Then, with 10 d of this DNA solution, Escherichia coli (HBlol)
After transformation, an mr clone was obtained. Next, the plasmid DNA of these clones
Examining the characteristics of A, Loric/X's Nr old sasite f
lind[[Blamid Loric with linker inserted
/Xll (Figure 3) was obtained.

After growing this E. coli in TYG-P medium 11, ccc-DNA was separated and purified from the bacterial cells and Lori
c/XH plasmid DNA was prepared in vA.

(C) 206SVE/5VOIO/old ndI [
[)Created and prepared pTK206sVE/5VO1010
, qwo reaction mixture 10100d (60NaCl, 6m
HTris-IIcI (pfl 7.5), 6mHN
aCl2.6mHDTT, 100I! ! I/Recommendation B
SA) r, restriction enzyme PvuI [(T
akara) at 37° C. for 2 hours to digest (bluntend). After protein removal with phenol extraction, the DNA was precipitated with ethanol, and the precipitate was extracted with 10 tons of
10.1E7d (~111!I/recommendation).

This DNA solution 0.4d (0.4N> and 1lind
■Linker-“(d (pc-A-A-G-C-cho-1-cho-
G))) and 6 units of T in 20 recommended reaction mixtures, 4
The mixture was ligated by reacting with DNA ligase at 22°C for 6 hours.

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

This DNA precipitate was digested by reacting with 1000 units of lindl[[ at 37°C for 4 hours in a reaction mixture of 600 μl, and then subjected to 0.8x preparative agarose gel electrophoresis to determine the amount of DNA containing Ap', ori, and T. The DNA fragment (4.3 kbp) was separated and purified, and the DNA was obtained as ethanol precipitate.

This DNA precipitate was mixed with 20 Jji of reaction mixture to produce 6 units of T4 DNA! After ring-closing by reaction between J Garcet and 12°C T-17,5rR, phenol extraction was performed and the DNA
was obtained as an ethanol precipitate, and the precipitate was 0. IHTris-
It was dissolved in 20ml of HcI (1) H7,5) solution.

Then, with this DNA solution 10111, E. coli 01B10
After transforming 1), Apr clone was obtained. Next, we examined the plasmid DNA of these clones and added 206SVE/5VOtO (7
) Escherichia coli carrying the plasmid pTK206sVE/5VO10/old ndII [(Figure 3) in which the old ndlI[jJ linker was inserted into the PvulI site was obtained.

After growing this Escherichia coli in TYG-P medium 11, ccc-DNA was isolated and purified from the bacterial cells, and T pTK
20(3SVE/5VO10/flindI [[(7)
Plasmid DNA was prepared.

(d) Preparation of LoriC/Xll/TK and 10 g of prepared Loric/XH in a reaction mixture of 200 units and 40 units of old ndII [and 50 units of restriction enzyme Bam].
After complete digestion by reaction with HI (manufactured by Takara) at 37°C for 2 hours, λori (0,P) was subjected to 0.8% preparative agarose gel electrophoresis.

DNA fragment (6.3 kbp) containing neor 81 minutes
TR'IJ, the DNA was precipitated with ethanol,
10.1E7.0j11 (~11I!l/
JJi).

On the other hand, pTK206sVE/5VOIO/ll1ndI
[[10tt, q, 200ml of reaction mixture, 60 units of old ndlI [and 60 units of Bam] and 37°C.
After complete digestion for 2 hours, the DNA containing TK was subjected to 0.8x preparative agarose gel electrophoresis.
The fragment (2.0 kbp) was separated and purified, the DNA was obtained as ethanol precipitation, and the precipitate was collected at 10 t/0. IE 3.
5d (~1~/Ji1).

And λori (0,P) of Loric/Xll
, It ind m / Bam including neo'
1 l I fragment DNA solution 1111 (1 tg) and pTK
A DNA solution 1J11 (~1R) of 1lindI/BamHI fragment containing 20GsVE/5VOIO/1lindln of TK was ligated by reacting with 6 units of T4 DNA ligase at 12°C for 17.5 hours in a reaction mixture. Thereafter, phenol extraction was performed to obtain DNA as an ethanol precipitate. IHTris-H
It was dissolved in CI (pH 7,5) solution 20A1.

Next, E. coli 1B101) was transformed with this DNA solution, and 1q of neo' clones were obtained. Next, the plasmid DNA of these clones
Examine A, λori (0, P), neo'
, Plasmid with TK: Loric/XH/TK
E. coli having (Fig. 3 and Fig. 4) was obtained.

After growing this E. coli in TYG-P medium 11, ccc-DNA was isolated and purified from the bacterial cells.
Plasmid DNA of c/XH/TK was prepared.

(e) pT cos 8D' / ori / c
Creation and preparation of m'-Xhol pT CO3Ap'/Ori/CIO' 10
IJ! I to 100% of the reaction mixture (60mM NaCl,
10m) f Tris-HCI (pH 7,4), 10
lac12.10mM DTT, 100
tqy/BiaoBSA), 60 units of restriction enzyme Bo
The mixture was digested by reacting with lII (manufactured by Takara) at 37°C for 2 hours. After removing protein with phenol extraction, DN
A was obtained as ethanol precipitate, and the precipitate was 10t10, IE
It was dissolved in 7J11 (~1/recommendation). Next, this h
The DNA solution 1A1 (~1R) digested with lII was added to the reaction mixture 250ml (50mM Tris-HCI (pH 7,
2), 10mM Mg5o, 0.1mHD
TT, 50/dBSA, 80+nHdATP,
80as dCTP, 80m dCT
P.

80IIj4dTTP), 2 units of leno
Enzyme (Boehringer)
Cohesive DNA fragments containing mutually complementary strands were reacted at 22°C for 30 minutes with
d) is a double-stranded DNA ArIfT piece with both ends shaved (blue
nt end) LC modification t;:.

After the reaction was completed, the reaction solution was heat-treated at 65°C for 10 minutes to obtain TKI.
The enowr11f element was inactivated.

This DNA solution 1oJ11 (0,4u!I) and Xho
I linker-[d(pC-C-T-C-G-A-G-G)
] 60 units of T4 DNA in 20 d of reaction solution
The mixture was ligated by reacting with ligase at 22°C for 6 hours. Thereafter, phenol extraction was performed to obtain DNA as ethanol precipitate. Add this DNA precipitate to the reaction mixture at 600°C or 1000°C.
Unit (7) kbp) was separated and purified to obtain DNA as an ethanol precipitate.

This DNA precipitate was mixed with 6 units of T4 in 20 d of reaction mixture.
After reacting with 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, and the precipitate was dissolved in 0.1M di-ris-tlcI.
(pH 7,5) solution 20JJ1.

And, with this DNA solution 10 recommendations, 181 large FmJ bacteria ()
After transformation of 01), Apr clone was obtained. Next, the plasmid DNA of these clones was examined and pTCO3AD'/Ori/
Plasmid with an XhoI linker inserted into the hlII site of Cm' pT C03Ap' / ori /
E. coli containing cm'-XhoI (Figure 4) was obtained.

After growing this E. coli in TYG-P medium 11, ccc-DNA was isolated and purified from Kokutai, and pT
cos Ap' / ori / cm' - Xhol
plasmid DNA was prepared.

psV2-dMrΔ(Ba1l[) 1hywo reaction U
Liquid 101001i1 (20KCI, 10n+HTri
s-HCI (pH 7,4).

10mHhcIz, 1mHDTT, 100I
J! 20 to 7 g/m BSA) of the restriction enzyme HpaI.
(manufactured by New EnalandBiO1abS) and 3
After reacting at 7°C for 2 hours, the reaction mixture was further reacted with 60 units of Ba111HI at 31°C for 2 hours at 400 d for digestion. After removing protein with phenol extraction, D
NA was obtained as ethanol precipitate, and the precipitate was washed with 10t10. I
It was dissolved in E7 recommended (~IR/ld). Next, this H
gar.

DNA B solution 111I digested with BamHI! (~
1~) in 25d of reaction mixture, 2 units of Kleno
React with wlff element for 30 minutes at 22°C to
After changing ive end to b+unt end, 0
．． Ap' was subjected to 8% preparative agarose gel electrophoresis.
, ori and dMr (4,82k
bp) was separated and purified to obtain DNA as ethanol precipitate.

Then, this DNA precipitate was reacted with 15 units of T4 DNA ligase at 12°C for 17.5 hours in a reaction mixture of 5°C to obtain a DNA fragment consisting of Apr, ori, and 1fr.
4,82 kbp) was closed. After that, phenol extraction was performed to obtain the DNA as an ethanol precipitate, and the precipitate was
．． IHTris-HCI (1) 117.5) solution 20d
It was dissolved in

After transposition of E. coli (HB 101) with this DNA solution, Apr clone was obtained.

Next, we investigated the characteristics of the plasmid DNA of these clones and found that 1) SV2-dMrΔ(hlI[) Bam
Plasmid pSV2-dhrrΔ(Bglff)Δ lacking a piece of DNA between the old site and HpaIcy1-
(8iml(r/HpaI) (Fig. 4)) was obtained.

After growing this E. coli in TYG-P medium 11, ccc-DNA was isolated and purified from the bacterial cells and oSV2
- dMrΔ(hlπ)Δ(Bam[/ )lpar)
plasmid DNA was prepared.

1) SV2-dhfrΔ(B(7111)Δ(Bam
Old/1lpal) 10R, 1ooy of reaction mixture, 2
Digestion was performed by reacting with 5 units of Pvu II at 37°C for 2 hours (blunt end). After protein removal by phenol extraction, the DNA was precipitated with ethanol, and the precipitate was extracted with 10 t10. It was dissolved in IE 71J1 (~1 g/ml).

This DNA solution 0.4J11 (0,4i) and CIaI
Linker-[d(pC-A-T-C-G-A-T-G)]
2 to 2 were ligated by reacting with 6 units of T4 DNA ligase at 22° C. for 6 hours in 20 m of the reaction mixture. after that,
Phenol extraction was performed, and the DNA was precipitated with ethanol. This DNA precipitate was added to the reaction mixture 600JJiT-1
After digestion with 000 units of C1al at 37°C for 4 hours, o, ax:X7! A DNA fragment (4°a2kbp) containing Apr, Ori, and dMr was purified by agarose gel electrophoresis (manufactured by J.
NA was obtained as ethanol precipitate. This DNA precipitate was cyclized by reacting with 6 units of 74-DN'A ligase at 12°C for 17.5 hours in a reaction mixture of 20%, followed by phenol extraction to obtain DNA as an ethanol precipitate. It was dissolved in 20ml of IHTris-11cI (pH 7,5) solution.

Then, with 10 parts of this DNA solution, E. coli (!lB101
) was transformed, and an Apr clone was obtained. Next, plasmid D possessed by these clones
By investigating the characteristics of NA, psV2-dhfrΔ(B(I
III) Pvu f of Δ(BamHI/Hpal)
Plasmid p with a C1al linker inserted into the l site
sV2-dMrΔ(BalI[)Δ(BamHl/
HpaI) -C1aI (Figure 4) was obtained.

This E. coli was transferred to TYG-P medium 1j! After growing in the culture medium, the ccc-DNA was separated and purified from the Kokutai.1)
SV2-dhfrΔ(hlI[)Δ(Bam old/H
plasmid DNA of paI)・C1al was prepared.

(h) pT cos Ap' / ori /cm
' / dhfr creation and 肚 pT cos Ap' / ori / cmr -X
hol 10°C, reaction mixture 200m, 80 units of Eco old and 25 units of C1al at 37°C.
After reaction and digestion for a period of time, 3 cos, Apr,
DNA fragment containing ori and c n+ r (3,5k
bl)) was separated and purified to obtain DNA as an ethanol precipitate, and the precipitate was dissolved in 10t10.1E7 (~1R/resolution).

On the other hand, pSV2-dMrΔ(hlII)Δ(Baml
ll/HpaI) -ClaI 10ts was completely digested by reacting the reaction mixture with 200 units of Eco R1 and 201 units of CIaI at 37°C for 2 hours, and then subjected to 0.8XUA agarose gel electrophoresis. , DNA fragment containing dMr (2,52kbp)
;f: Purify the DNA by 1 q and precipitate with ethanol. , IE 3.5j11 (~
1R/recommended).

and pT cos AEI'/ori/c
3 cos of rnr-Xhol, AI)',
DNA solution of Eco RI/C1aI fragment containing ori and cm'i&! (~1 埒) and psV2- dMr
Δ(BallI)Δ(Bamtll/upal)
A DNA solution of Eco R1/C1al fragment containing the dhfr of -C1al 11i1 (~1 tg) was mixed with 6 units of T4 DNA ligase at 2° C. for 11 min at 12° C.
The mixture was reacted for 5 hours and ligated. Thereafter, phenol extraction was performed to obtain DNA as an ethanol precipitate. I
It was dissolved in 20% of HTris-HCI (DH7°5) solution.

Next, use this DNA solution to extract Escherichia coli (Ha 101).
After transformation, an Aor clone was obtained. Next, the plasmid DNA of these clones
Examine the properties of A and find the three cos, Ap', o
Plasmid with ri and cmr: pT cos
Ap' / ori / cm' / dMr (4th
We obtained Escherichia coli with (Fig.).

After growing this E. coli in TYG-P medium 11, ccc-DNA was isolated and purified from the bacterial cells, and pT
cos 8p' / ori / cmr / dhf
Plasmid DNA of r was prepared.

(i) Construction of pBR327・5alI and preparation of pBR
32710R'li:, reaction a'tt 100Jt11
00Jt1(60,6mHTris-11cI(pH8
,0), 10mHHoc+2.

6 Aval (DTT, 100JJ!g/d BSA), 55 units of restriction enzyme Aval (New Engl.
and Biolabs) for 2 hours at 37°C for digestion, followed by further reaction with 90 units of Eco R1 at 37°C for 2 hours at 200 d of reaction mixture for digestion. After protein removal with phenol extraction, the DNA was precipitated with ethanol, and the precipitate was 10t10.1E7m
(~1 m/d).

Next, this AVa I and Eco R1 digested DN
Solution A IIJ1 (~1 tg) was reacted with 2 units of KlenowiW at 25°C for 30 minutes at 22°C to change the cohesive end to the old unend.

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

This DNA solution 10J11 (0,4N> and 5ail linker [d(DG-G-T-C-G-A-C-C)12
6 units of T4 ligase and 22
The mixtures were ligated by reacting at ℃ for 6 hours. Thereafter, phenol extraction was performed to obtain DNA as ethanol precipitate.

This DNA precipitate was mixed with a reaction mixture of 600. cffl (150
mM NaCl, 6mHTris-HCI (pH 7,9
), 6 mH) lOcIz.

6mHDTT, 100Jj! J/d BSA) at 1
000 units of 5all (New England
Biolabs) was digested at 37°C for 4 hours, and then subjected to 0.8x preparative agarose gel electrophoresis to obtain a DNA fragment containing Apr and ori (2,94
kbl)) was separated and purified to obtain DNA as ethanol precipitate.

This DNA precipitate was mixed with 6 units of T4 in a reaction mixture of 20
After ring closure by reacting with DNA ligase at 12°C for 17.5 hours, phenol extraction was performed to obtain the DNA as an ethanol precipitate, and the precipitate was reduced to 0.5%. IHTris-HCI (1
)H7,5) Solution 2QI11 was dissolved.

Then, with 10 d of this DNA solution, E. coli (H8101)
After transformation, /pr clone was obtained. Next, the plasmid DNA of these clones
By examining the characteristics of A, we obtained Escherichia coli containing plasmid pBR327.5all (Fig. 4), which lacks the DNA fragment (0.33 kbp) between the Eco RI site and AVal site of pBR327 and inserts a 5alI linker between them. .

After growing this E. coli in TYG-P medium 11, ccc-DNA was isolated and purified from the bacterial cells, and pBR
Plasmid DNA of 327-Sa II was prepared.

(j) pBR327-5alI/BamHI (7)
Creation and preparation 1) BR327-5all 10JJ!
1 of the reaction mixture (75 mH NaCl, 10 m
HTris-HCI (pH 8,0), 10m) 1
hc12. 5mHDTT, 100. a/d BS
A), 24 units of restriction enzyme AhaIII (Ne
w Enaland Biolabs) and 3 at 37°C.
Limited digestion was performed by reacting for 0 minutes (bltlnt end
). After protein removal by phenol extraction, the DNA was precipitated with ethanol, and the precipitate was extracted with 10 t10. IE7J!
1 (~1113/ld).

This DNA solution 0.4d (0,4,s) and BamH
I linker-[d(1)C-G-G-A-Tm〇-C-G
)I2# in a reaction mixture of 20JIi and 6 units of T4.
The mixture was ligated by reacting with DNA ligase at 22°C for 6 hours.

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

This DNA precipitate was mixed with a reaction mixture of 600 units and 1000 units.
The DNA fragment (2,94 kl) containing Apr and ori was digested by reacting with Bamtll of - at 37°C for 4 hours, and then subjected to 0.8x preparative agarose gel electrophoresis.
I)) was separated and purified to obtain DNA as an ethanol precipitate.

This DNA precipitate was mixed with 6 units of T4 in a reaction mixture of 20
After ring closure by reacting with DNA ligase at 12°C for 17.5 hours, phenol extraction was performed, the DNA was precipitated with ethanol (19), and the precipitate was purified with 0.1H ris-HCI (19).
DH7,5) solution 20I11.

Then, with this DNA solution 10111, Escherichia coli (HBlo
After transformation of 1), Apr clone was obtained. Next, the plasmid DNA of these clones was examined and pBR327-3all Apr and or
Add a BamtlI linker to the Aham site between i.
Plasmid pBR327-Sal I/
E. coli containing BamH (Figure 4) was obtained.

After growing this E. coli in TYG-P medium 11, ccc-DNA was isolated and purified from the bacterial cells, and pBR
327-Sal I/BamH 5; Z, Mit:
DNA was prepared.

pT cos Ap' / ori / cmr /
dMr 10IJ! I to the reaction mixture 200. In fl,
50 units of BamHI and 120 units of 5al
After digestion by reacting with 0.8X
Three Cs were subjected to FJI agarose gel electrophoresis.
O3, AI)', Ori, cIIl' and hd
One piece of hrr-containing DNA (6,12 kbD) was separated and purified, and the DNA was precipitated with ethanol.
10t10. It was dissolved in IE 7.0tti (~11J!I/recommended).

On the other hand, pBR327-5all/BamHI 10tt
s to the reaction mixture 200 Ij 1 te 100 t (7)
BamHt and 240L Ni) - 5all and 37℃
After complete digestion by reaction for 2 hours, DNA containing Apr was subjected to agarose gel electrophoresis for O, aX preparation.
The A fragment (1.1 kbp) was separated and purified to obtain DNA as ethanol precipitate. IE 3.
5 recommendations (~111!l/J11').

And pchocos Ap' / ori / cm
' / dMr's three cos, Ap', or
BamHI/5al containing i, cm' and dMr
I fragment DNA solution 1j11 (-1 埒) and pB113
Bam containing Apr of 27-5alI/Bam1ll
A DNA solution of the HI/5alI fragment (~1N) was ligated by reacting with 6 units of T4 DNA ligase at 12°C for 17.5 hours in a reaction mixture of 20 parts. Thereafter, phenol extraction was performed to obtain DNA as an ethanol precipitate, and the precipitate was dissolved in 0.1HTris-HCI (pH 7,5) solution 20/71J2.

Next, use this DNA solution to prepare E. coli (JI8101).
After transformation, Apr clone was obtained. Next, the plasmid DNA of these clones
Examine the properties of A and find 3 cos, 2 AI)'
, ori , Cmr and dMr plasmid pr cos Apr 10ri/ cml”Ap'
/ cm' / dMr (Figure 4)
II I got bacteria.

After growing this E. coli in TYG-P medium 11, ccc-DNA was isolated and purified from the bacterial cells, and pT
cos Ap' / ori / Ap' / cm'
/dMr plasmid DNA was prepared.

pT cos Apr / ori / Ap' /
cm'/dMrloR in a reaction mixture of 200 mL, 4
0 units of BamHI and 200 units of XhoI
After digestion at 37°C for 2 hours, 0.8%
Three COSs were subjected to preparative agarose gel electrophoresis.
.

DN including Apr, ori, cm' and dMr
The ATh piece (5.4 kbp) was separated and purified to obtain DNA as ethanol precipitate. iε5.
It was dissolved in Od (~1 binding/recommendation).

On the other hand, add Loric/XH/TK 10R to reaction mixture 2.
After digestion with 30 units of Bam and 180 units of XhoI at 37°C for 2 hours, the
．． 8% preparative agarose gel electrophoresis, λOr
A DNA fragment (6,1 kbp) containing i (0, P), neo' and TK was separated and purified, and the DNA was precipitated with ethanol. IE 4. Od(~
It was dissolved in 1 katana/sei).

And pT cos Ap' / ori / Ap
' / cmr/dllll'r 3 CO3, Ap'
, Bam1ll /Xh containing Cm' and dhfr
oI fragment DNA solution 1JI1. (~IJJ!J) and
λori (0,P) for Lor ic/XH/T
, neo', Bam1ll /X including TK
DNA solution 1d (~1~) of the hol fragment was ligated by reacting with 6 units of T4 DNA ligase at 12°C for 17.5 hours in a reaction mixture of 20. Thereafter, phenol extraction was performed to obtain DNA as an ethanol precipitate. IM Tris-HCI (pH 7,5) solution 20d
It was dissolved in

Next, add this DNA solution 10. E. coli (11810
After transforming 1), a neor clone was obtained. Next, the characteristics of the plasmid DNA of these clones were investigated, and the three COS, λori
(0,P), neo', Ding K.

9 plasmids with Apr, Cm' and dMr COSλori/Ap'/neor/cmr
/TK (7J Figures 1 and 4) was obtained. This strain has been approved by the Institute of Microbiology and Industrial Technology, Agency of Industrial Science and Technology as "Feikoken Bacterial Serial No. 8537 (FErl) IP-8573".
) Deposited as J.

This E. coli was grown in 1 J of TYG-P medium! After growing in the microorganism, ccc-DNA was isolated and purified from the bacterial cells, and pT
cO3λori / Ap' / neo' / cm'
/T plasmid DNA was prepared.

Example 2 TCO8λOri/A'/neo
Mouse L cells (Ltk-(H3V-ITK)) transfected with a recombinant herpes simplex virus type 1 (ll5V-1) thymidine kinase (TK) gene having the base sequence '/Cm'/pBR322.
C. Cultured in Eagle 14EH supplemented with 10% calf serum, C0nr+uent monolayers (
9 100 mm plates, ~107Ce11S/plate) and PBS (137 mM NaCl, 3
mMKCl, 8mM Na5iflPOa,
After washing twice with 1mM KH2PO4), solubilizer (100R/rrdl proteinase)
0.5X SDS, 150mM NaCl.

10mM EDTA, 10mM Tris-HC
I (pH 7,5)) per plate 1. Od added, 6
It was kept warm at 5°C for 15 to 30 minutes to solubilize. This solubilized material was transferred to a 50 rd volume centrifuge tube and further incubated at 37°C overnight.
0i/d was added. After the reaction, it was gently extracted twice with phenol saturated with Tris buffer, and the aqueous layer was extracted with 50 m
M Tris-HCI (pH 8,0), 10mt
Dialysis was performed four times against dialysis buffer 31 consisting of 4 EDTA and 110 In NaCl. The dialysis fluid contains 0Nase-f'ree RNase at a rate of 50~/m
A was added and kept at 37°C for 3 hours to degrade RNA.

After the reaction, it was gently extracted twice with an equilateral phenol:chloroform mixture, and the aqueous layer was extracted with 10mM Tris-HCI.
(pH 8,0), 1 m) Dialysis was performed 9 times against an aqueous solution consisting of 1 ED (Din E) 31 to obtain 50 ml of purified DNA solution. 260nm and 280nm of DNA solution
The absorbance (OD>) is 0.66 for each 10-fold dilution.
7, 0.363 (00z8o/OD 2aO=0.
544) and the concentration of DNA is 0.667x O,0
50 (sword/l0D) X10=0.334*/recommendation.

(b) Preparation of 35-45 kbp genomic DNA 5.98 ml of the purified genomic DNA solution was added to 29.96 ml of the reaction mixture (50 mM NaCl
, 10mHTris-IICI(1)H7,5),
10mM M(JCI2, 100#/d BSA
) and 15.61 nits of Sau 3A (New En
After limited digestion by reacting with Grand Biolabs) at 37°C for 1 hour, 0.5HEDTA (pH 8
, 0) solution was added for 1.3 d and gently extracted twice with equilateral Tris buffer saturated phenol. After concentrating the aqueous layer with secondary butanol to 10d,
Genomic DNA was obtained as an ethanol precipitate by adding 1.1 ml of M sodium acetate and 20 d of ethanol, washed with 70% ethanol and dried with a water pump, and the ethanol precipitate was dissolved in TE 2 m.

, Next, (7) DNA solution 300. ccg wo5~2
5X sucrose density gradient centrifugation (20m) 4 Tris-
HCI (pH 7,6).

5n+HEDTA, 5W28; 20.
00 Orpm, 9 hours, 20°C] and fractionated every 30 drops.

Each fraction 20J11 was subjected to 0.4x agarose gel electrophoresis to measure the length, and DNA with a size of 35 to 45 kbp was detected.
After collecting the fractions, dialysis was performed three times against TE 3f. After the dialyzed fluid was extracted with phenol, the aqueous layer was concentrated with secondary butanol, DNA was precipitated with ethanol, and the precipitate was dissolved in TE 1d to obtain 35-45k
A bp genomic DNA solution was obtained. OD2 of DNA solution
9G and OD 280 are each 0.21 at 50 times dilution.
6゜0, 112 (OD 280/OD 26+1
= 0.51'9), and the concentration of DNA is 0.216
X O,050 (#/10D) x50 = 0.54 埒/J11.

(c) Preparation of Cosmiζ vector DNA pT CO3λori / Ap' / neo' /
cm'/TK 40 埒 in 800J11 reaction mixture
Digestion was performed by reacting with 0 units of C1aI at 31°C for 2 hours. After that, phenol extraction was carried out (1) NA was precipitated with ethanol, and IL precipitation was performed for 10 t10. iE 1
00m t, knee H solved.

The DNA solution 95A1 digested with C1al was mixed with 500 Jli (50 m) of the reaction mixture using Tris-HCI (p
H9,0), 1mM MgCl2. 0.1mM
ZnCl2.1mM spermine), 6 units of phosphatase (Calfintestinal alk)
aline phosphotase, Bochri
(manufactured by Ha1111110im) and 31°C
After incubation for 15 minutes at 56°C, an additional 6 units of small sphatase were added, followed by 15 minutes at 37°C, followed by 15 minutes at 56°C.
The C1al cut fragment was inactivated by reacting at 6°C for 15 minutes.

After that, H2O400J11. STE (100m
HTris-HCl (pH 8,0), 1) INa
Cl, 10mM EDTA) 100JJi.

10% SDS 50d was added and kept 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 was extracted with 10 t10. Dissolved in IE 30111.

Next, the DNA solution digested with this C1al and inactivated with phosphatase was prepared using 30. J with 500 recommended reaction mixtures, 1
Digestion was performed by reacting with 52 units of fsamHI at 37°C for 2 hours. After removing protein with phenol extraction, DN
A was precipitated with ethanol and the IL precipitate was precipitated with 10t10. IE
The cosmid vector DNA solution was 1 rJ by dissolving the cosmid vector DNA in a diluted solution. (0.70 埒/d).

(d) 35-45 kbp genome solution 2 prepared in (b) between 35-45 kbp Tsum DNA and cosmid vector DNA.
I JJl (1,4IJ3) and cosmid vector DNA solution prepared in (C) 0.84 d (0,59#)
and 5.9 units of T4 DNA in 10 μl of the reaction mixture.
The mixture was ligated by reacting with ligase at 12° C. for 17.5 hours.

After the reaction was completed, the reaction solution was stored at 4°C.

Escherichia coli BHB 2690 was grown in NZY medium (IX NZ a
Mine.

0.5x yeast extract, 0.5x NaCl, 0.
2χ +gC12・6H20, pH7.5) 10
After measuring the 00@ of the overnight culture at 0,
500 m of NZY medium kept at 2℃ (2,!!
The bacteria were inoculated into a flask (in a flask) so that the initial 0D61Xl was 0.025, and shaking culture was started at 32°C. Then, when OD@ reached 0.3 (2 hours and 40 minutes after the start of culture), 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 the completion of the culture, the bacteria were collected by centrifugation at 6,000 rpm for 10 minutes, and the cells were diluted with 20mM Tris-HCI.
(pH 8,0), 1mHEDTA, 5mHβ-
It was suspended in 3.6 d of buffer consisting of mercaptoethanol. Then, after performing ultrasonic treatment at 4℃ or below (maximum output for 5 seconds x 20 times), 20.000 rpm Te1
The supernatant liquid was centrifuged for 0 molecules and 1 minute at 4° C. to obtain a supernatant of ~3 ml.

Next, the above M1 is isothermal to the supernatant liquid 3-! ! solution and packaging buffer (8 mHTrisJICl (pHa, o)
, 50n+H suberdimine, 50mM Bretocin, 20mM MOCI2, 30mM ATP,
After adding 0.5 m of 30 mM β-mercaptoethanol, the mixture was dispensed into 1.5 m Eppendorf tubes in 15 d portions, immediately frozen in liquid nitrogen, and stored at -70°C.

ff) After measuring the 0DaO of the overnight culture of E. coli 2688 in NZY medium 100, the first 0DaO was added to NZY medium 500d (in a 2J flask) kept at 32°C in advance. Inoculate the bacteria so that the value is 0.025,
A shaking culture was started at 32°C. And 0Dauo is 0
．． 3 (2 hours and 40 minutes after the start of culture),
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 culturing, collect the bacteria by centrifuging at 6.0OOrpa+ for 10 minutes, and collect the bacteria in a sucrose solution (10% sucrose, 50mM Tris-HCI (pH 8,0)).
After suspending in 0.5 d of water, 0.5 d of each was placed in 6 Etzpendorf tubes, and lysozyme solution <2 m9/rnll and 0.25HTris-HCI (1
)H8,0)) 25JJi was added (4°C), mixed gently, and quickly frozen in liquid nitrogen.

Next, the tubes were placed on ice to thaw the extract, and then 25 d of packaging buffer was added to each tube and mixed. Then, each extract was combined to give 21, O
A supernatant was obtained by centrifuging at OOrpm for 1 hour (4°C), and aliquots of 10 portions were dispensed into 165D Eraspendle tubes, immediately frozen in liquid nitrogen, and incubated at -70°C.
Saved with.

(r“) in VitrO packaging BHB 2690 and BIIB 2688 stored at -70°C
Place the bacterial cell extract on ice and thaw it first.BHB
Add the extract of 2 (388) to the extract of BIIB 2690 and mix gently.
2.5 m of recombinant DNA solution prepared in (d) (0
,5iI! 1) was added and mixed well, and then reacted at air temperature for 1 hour to perform in VitrO packaging.

After the reaction is completed, add 8M solution (0.58X NaCl).

0.2%HQSOa 7H20, 50mM Tr
is-11cI (Dl17.5) 0.01% gelatin]1rI11 and a drop of chloroform were added and mixed. The Etzbendorf tube was then placed in a desk centrifuge for 30 seconds to obtain a supernatant as a transduced phage particle solution.

(g) MLjLE and platein E. coli He
101 L-broth (added with 0.4x maltose) was cultured overnight in 200J11 using a desk centrifuge to collect bacteria, and the cells were resuspended in 100mM MOCI2 solution 100JIi. Next, add 8M to this national suspension 100Jji.
Transducing phage particle solution diluted 10 times with solution 100
After adding L-broth 1.1- and incubating at 37°C for 15 minutes for transduction, add L-broth 1.1- and incubating at 37°C for 45 minutes.
Incubate with shaking for 1 minute. Then, 100% of this bacterial cell FJ suspension was added.
100m containing JIi 20 more kanamycin/d
Plates 1 to 1 were left overnight at 37°C, and the resulting colonies were picked up using bamboo sticks and grown in L-broth (containing 20 grammes/d of kanamycin). Moreover, the number of colonies per plate was 20 to 100.

Kanamycin resistant colony (transformer I-) 2
0 pieces in L-broth (containing 20R/rdl of kanamycin)
After culturing overnight at 2d, 1.5I of each culture solution was added to 1.5I.
The cells were placed in an I11 Eppendorf tube and centrifuged in a tabletop centrifuge to collect bacteria. These Kokutai were dissolved in solution I (50mM
Glucose, 25mM Tris-HCI (pHa,
o), 10mM EDTA, 10mg/-lysozyme) After resuspending in 100d, it was left at room temperature for 5 minutes. Next, the solution [(0,2N NaOH, 1% 5O3
) 200J11, mix gently, leave on ice for 5 minutes, and add solution I[[[5M potassium acetate (p
H4,8) ] 1501!1 was added, mixed gently, and placed on ice for 5 minutes. The Eppendorf tube was then subjected to a desk centrifuge to obtain a supernatant, and twice the volume of ethanol was added to this to obtain plasmid DNA as an ethanol precipitate.

After dissolving the plasmid DNA of each transforman 1- in a small amount of 10t10.1E, size marker DNA (lambda DNA: 48 kbp) was added.

Lambda DNA digested with lindI: 22.
3kbl), 9.5kbE)) toIMni,
0.4X; #j When subjected to low gel electrophoresis,
Of the 20 kanamycin-resistant colonies, all 22
．． The presence of plasmid DNA was observed at a size of 3 to 48 kbl).

pT cosλori / AD'/neo' /T
K 10R was added to the reaction mixture at 100%. u[50mM NaCl
, 10mM Tris-HCI (r1M8.0), 1
0mM MnCl2, 51118 00 tons,
100 #/dBSA], 50 L of 27 restriction enzymes Nbel (New England B101a
bS) for 37 minutes for digestion. Furthermore, the reaction mixture 200. 20 units + 7 in cf)
Digestion was performed by reacting with BamllI at 37°C for 2 hours.

After protein removal with phenol extraction, the DNA was precipitated with ethanol, and the precipitate was precipitated with 10t10°IE7R (~1J
J3/J11).

Next, the DNA digested with BamHI and Nhel
React 1 component (~1~) of the reaction mixture with 2 units of KlenOW enzyme at 22°C for 30 minutes to obtain CO2.
After changing to 11eSiVe end@b+unt end, it was subjected to 0.8% preparative agarose gel electrophoresis, and 3cos, dhfr, Ap', λori,
A DNA fragment (8.3 kbp) containing cmr was separated and purified to obtain DNA as ethanol precipitate.

Then, this ethanol precipitate was mixed with 15 units of T4 DNA ligase in reaction mixture 50111 at 12°C for 17 minutes.
After 5 hours of reaction, 3cos, dMr, Δp', λo
DNA fragment (8.3kbl) containing ri, cmr)
was closed. After that, phenol extraction was performed to extract the DNA.
was obtained as an ethanol precipitate, and the precipitate was 0. IHTris-
Dissolved in 20d of HCI (pif 7.5) solution.

Next, with 10 JJi of this DNA solution, Escherichia coli (HBlol
), an Apr clone was obtained. Next, the plasmid DNA of these clones
Examine A and find three COS and Ap' 1dhfr, λ
Plasmid with ori 1cm': pT cosλ
E. coli having ori/Ap'/cm' (Fig. 5.7) was obtained. This strain has been approved by the Institute of Microbiology, Agency of Industrial Science and Technology as “FERMP-8947” (FERMP-89).
47) Deposited as J. TY this E. coli
After growing in G-P medium 11, ccc was obtained from the bacterial cells.
- Separate and purify DNA to pr cos 2 or
i/Ap'/cmr plasmid DNA was prepared.

pOcos AD' 10ri/dMr(BamHI
) 10 Immediately, 50 units of Bol with 100 recommended reaction mixtures
Digestion was performed by reacting with 1I at 37°C for 2 hours. After protein removal by phenol extraction, the DNA was precipitated with ethanol, and the precipitate was extracted with 10 t10. IE 711i (~111!
I/Recommendation).

On the other hand, pBR327/Bat II cos(HCN)
10 scraps in 200d of reaction mixture, 70 units of Bi)1
After completely digesting by reacting with ■ at 37℃ for 2 hours,
．． Ap' was subjected to 8x preparative agarose gel electrophoresis.
Containing DNA fragment (1,1kbp) molecule fl1%
L, r, DNA was obtained by ethanol precipitation, and the precipitate was
t10.1E It was dissolved in 3 recommendations (~1 ton/recommendation).

Then, po CO3Ap' 10ri/dhfr(B
amtlI) digested with Bolff (1 recommendation)
~1~) and pBR327/Ba1IICcos(II
cN) (7) AI) BolI [fragment DNA
1 m (~1#) of the solution was ligated by reacting with 6 units of T4 DNA ligase at 12°C for 17.5 hours in a reaction mixture of 20 parts. Thereafter, phenol extraction was performed, the DNA was precipitated with ethanol, and the precipitate was dissolved in 0.1) I Tris.
-HCI (1) 7.5) solution was dissolved in 20 recommended solutions.

Next, Escherichia coli (HBIOI) was transformed with this DNA solution to obtain an Apr clone. Next, the plasmid DNA of these clones was examined and pD cos Ap'10ri/dMr (Bam
One piece of Ap' DNA is inserted into the B(llI[site of 1ll), and the Toughhrasmid: pDCO8Apr10ri/Ap
Escherichia coli harboring r (Fig. 8) was obtained.

After growing this Escherichia coli in TYG-P medium 11, ccc-DNA was isolated and purified from the bacterial cells and pDC
Plasmid DNA of os Ap' 10ri/Apr was prepared.

(b) D cosAp'10ri/An' /C
Creation and preparation of IaI pDCO3AI)' / Ori
/Ap' 10 #Te reaction U solution 100Jli was reacted with 10 units of Pvu ■ at 37℃ for 2 hours to digest (
blunt end). After protein removal with phenol extraction, the DNA was precipitated with ethanol, and the precipitate was extracted with 10 tons of
10.1E 7 m (~1 m/d) was dissolved.

This DNA solution 0.4. d(0,4JJ3) and CIa
I linker-[d(1)C-A-T-C-G-A-T-G
)] 2u! g was ligated by reaction with 6 units of T4 DNA ligase at 22° C. for 6 hours in a reaction mixture of 20 d. Thereafter, phenol extraction was performed to obtain DNA as ethanol precipitate. This DNA precipitate was digested by reacting with 600 units of reaction mixture and 1000 units of CIaI at 37°C for 4 hours, and then subjected to 0.8% preparative agarose gel electrophoresis to obtain 2cos, 2Ap', ori,
A DNA fragment (5.0 kbp) containing dhfr was separated and purified, and the DNA was obtained as ethanol precipitate. This D
6 units of T4 DNA with 20 recommended NA precipitated reaction mixtures.
After reacting with ligase at 12°C for 17.5 hours to achieve ring closure, phenol extraction was performed to obtain DNA as an ethanol precipitate. IM Tris-HCI(1)1
17.5) Dissolved in solution 20d.

And, with this DNA solution 10 recommendations, Escherichia coli (HB 01)
After transformation, a clone was obtained.

Next, we investigated the characteristics of the plasmid DNA of these clones and found a plasmid in which a C1al linker was inserted into the Pvu II site of pD cosAp' 10ri/Apr: pD C03Ap'101'i/Ap
'/C1aI (Figure 8) was obtained.

After growing this E. coli in TYG-P medium 11, ccc-DNA was isolated and purified from the bacterial cells and pDC
Plasmid DNA of os Ap'10ri/Ap'/C1al was purified.

(C) I) Creation and preparation of D cosA r /λori pD CO3Ap'/λori/Ap' /C1
20tts was digested by reacting it with 40 units of Bam and 100 units of Xhol at 37°C for 2 hours in a 200ml reaction mixture. Then 0.8x preparative agarose gel electrophoresis 1. : Kake T, 2cos, Ap'
, 197 pieces of DNA containing dMr (3.2kb
l)) Minutes (M) DNA was precipitated with ethanol, and the precipitate was precipitated with 10 ml of ethanol. IE 10 recommendations (~1u!
I/Recommendation).

On the other hand, pdCO3λori/Ap'/Cmr 20
11! I to reaction mixture 200/ljj! After complete digestion by reacting with 40 units of BamHI and 100 units of Xbol at 37°C for 2 hours, the DNA fragment (2,93 kbl) containing λori was separated by electrophoresis on a 0.8% preparative agarose gel. After purification, DNA was obtained as ethanol precipitate, and the precipitate was 10t10.1E5d(
~IR/recommended).

And po cos At)' 10ri/Apr
/C1aI 2 cos 5Apr, dhfr
DNA solution 1A1 of BamHI/XhoI fragment containing
~1~) and pT cosλori/Apr/cm
DN of Ba) IHI/Xhol fragment containing λori of '
A solution 1d (~1~) was ligated by reacting 20 units of the reaction mixture with 6 units of T4 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 0. IHTris-H
It was dissolved in CI (pH 7.5) solution.

Next, add this DNA solution 10. d for E. coli (HB 101
) was subjected to 1-transformation, and an Apr clone was obtained. Next, plasmid D of these clones
Examine the NA and find the two COS and Apr, λori,
Plasmid with dMr: pDcosAp'/
One strain of E. coli harboring λori (Figure 6.8). This strain has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as FERHP-8946 J.

After growing this E. coli in TYG-P medium 11, ccc-DNA was isolated and purified from the bacterial cells, and pD
Plasmid DNA of cos Ap'/λori was prepared.

Effects of the Invention If the vector of the present invention is used, a DN of 35 to 45 kbp can be obtained.
As an A fragment, a correspondent bank of genomic DNA can be produced more easily and efficiently. Furthermore, the number of copies of recombinant DNA per cell in the host is large, and the number of copies remains almost constant regardless of the structure of the foreign DNA.
The quality and stability of the gene bank created using the cosmid vector of the present invention is increased, and furthermore, since the copy number has been low until now, the target DNA can be
The use of this vector brings advantages such as the fact that it is now possible to detect clones with this vector or to detect the expression of the gene due to low gene expression.

[Brief explanation of drawings]

Figure 1 shows the new cosmid vector pT cosλori
/hep'/neo'/cm' /DingK, Figure 2 shows a method for producing a genomic DNA gene bank using the novel cosmid vector of Figure 1, and Figures 3 and 4 show pr. cosλori / At)
'/neo' / cm' /rK creation method is shown. Figure 5 shows the new cosmid vector pD CO3λ01'l
/All' / Cl1l' shows L/", m6 diagram shows the new cosmid vector pT cos Apl"/λori
shows. Patent Applicant Municipal Mulberry Co., Ltd. Ope 01 - Item 0 o < 0 Nibezu Procedural Amendment (Voluntary) October 17, 1985

Claims (9)

[Claims] (1) A cosmid vector characterized by having two or more cos of lambda phage or lambdoid phage having the same directionality and having a replication function derived from lambda phage. (2) A right arm and a left arm each having at least one cos can be created by cutting two or more cos of lambda phage or lambdoid phage with the same directionality with two types of restriction enzymes. Claim 1 that can be made in equimolar amounts and has a replication function derived from lambda phage.
Cosmid vectors described in section. (3) The cosmid vector according to claim 1 or 2, wherein the replication function derived from a lambda phage is derived from a lambda phage, a phage vector, or a λdv plasmid. (4) The cosmid vector according to claim 1, which contains a gene sequence that expresses drug resistance or a specific physiological function for determining insertion of a gene fraction derived from the cosmid vector. (5) The cosmid vector according to claim 1, which has a gene sequence that expresses ampicillin resistance, tetracycline resistance, neomycin resistance, or chloramphenicol resistance as drug resistance. (6) As a specific physiological function, thymidine kinase (TK
) activity, adenine phosphoribosyltransferase (
APRT) activity, hypoxanthine guanine phosphoribosyltransferase (HGPRT) activity, xanthine guanine phosphoribosyltransferase (XGPR
2. The cosmid vector according to claim 1, which has a gene sequence for expressing T or gpt) activity or dihydrofolate reductase (dhfr) activity. (7) The cosmid vector according to claim 1, which contains a marker DNA sequence useful for recovering a cosmid vector-derived gene fraction from transformant cells. (8) between the right arm and the left arm, which have at least one cos obtained by cutting the cosmid vector described in claim 1 with two types of restriction enzymes;
Inserting various foreign DNA fragments of kbp length, in
A method for producing a gene bank, comprising producing a gene bank of foreign DNA by in vitro packaging into transducing phage particles and then cloning into E. coli. (9) Foreign DNA is DNA derived from bacteria, filamentous fungi, microorganisms such as yeast, higher animals and plants, or various phages, etc.
The manufacturing method according to claim 8.