JPS5846100A - Plasmid vector having three kinds of marker and its preparation - Google Patents

Abstract

PURPOSE:To plasmid vector having tetracycline-resistant (TC-resistant), amplicillin-resistant (Ap-resistant) and trimethoprim-resistant (Tp-resistant) markers, containing scission sites of various kinds of restricted enzymes, and capable of loading extrinsic DNA fraction. CONSTITUTION:A plasmid vector having Tc-resistant gene, Ap-resistant gene and Tp-resistant gene as markers. The plasmid can be prepared as follows: A plasmid vector pBR 322 having Tc-resistant and Ap-resistant markers is treated with a restricted enzume EcoRI to break the sites other than the insertion inactivated site, and then treated with enzyme nuclease SI to obtain straight-chain DNA having smooth end. Separately, the DNA fraction containing dihydrofolate reductase gene obtained from Escherichia coli K12 strain is shortened, and connected and cyclized with the above straight-chain DNA to obtain mixed plasmid. The plasmid is introduced into Escherichia coli K12 strain and the strain is cultured to obtain plasmid vector pTP30-5 having Tc-resistant, Ap-resistant and Tp-resistant markers and containing 6.3 kilo base pairs.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a plasmid vector having a gene for imparting tetracycline resistance, ampicillin resistance, and trimethoprim resistance to a host as a genetic marker, and a method for producing the same.

In recent years, with the development of genetic engineering, genetic recombination methods,
For example, methods of producing useful substances by introducing foreign DNA into bacteria such as Escherichia coli and expressing their genes are gaining attention.Recently, this gene recombination method has been used to produce interferon, insulin,
Research and development is underway to commercialize highly useful substances such as human growth hormone and glutathione.

A circular DNA generally called a plasmid is used as a means to introduce the above-mentioned heterologous DNA and express the gene.
Among these plasmids, a plasmid that has been modified to have the role of transferring foreign genes into host cells and increasing them therein is a plasmid vector. When introduced into a host, it is necessary to have a gene, that is, a genetic marker (hereinafter abbreviated as a marker), that confers resistance to a drug or the like on the host so that it can be distinguished from a host to which it has not been introduced. For example, plasmid vector pBR322 is tetracycline resistant (T
pMB 9 confers Tc resistance and colicin EI immunity, pACYo 184 confers chloramphenicol resistance and Tc resistance to the host, respectively. I have it as a marker.

By the way, one method for inserting a heterologous gene into a plasmid vector is to cut the plasmid vector with an appropriate restriction enzyme, connect linear heterologous DNA to both ends of the linear DNA, and circularize it again. , is commonly practiced. When inserting this heterologous DNA, the heterologous DNA is inactivated by inactivating the plasmid vector marker.
The success or failure of NA insertion can be examined. For example, restriction enzyme BamH in plasmid vector pBR322
By introducing heterologous DNA into the cleavage site of Tc
Resistance markers are inactivated. Such a site is called an insertion deactivation site, and if a foreign DNA is inserted into this insertion deactivation site, one marker will be lost. Therefore, it is desirable for a plasmid vector to have as many types of markers as possible.

In view of these circumstances, the present inventors have conducted intensive research to obtain plasmid vectors having various markers, and have found that plasmid vector pBR322 has two types of markers: Tc resistance and Ap resistance. , dihydrofolate reductase is strongly inhibited by trimethoprim, but trimethoprim resistance (hereinafter abbreviated as 'rp resistance) can be acquired by amplifying the gene in cells. By inserting DNA containing the dihydrofolate reductase gene into plasmid vector pBR322, Tc resistance and A
It was discovered that a plasmid vector having three types of markers, p-resistant and Tp-resistant, could be obtained, and the present invention was completed based on this finding.

That is, the present invention uses a Tc resistance gene as a marker.

The present invention provides a plasmid vector having an Ap resistance gene and an 'rp resistance gene, and a method for producing a plasmid vector having the above three types of markers by inserting DNA containing a dihydrofolate reductase gene into plasmid vector pBR322.

Plasmid vector pBR322'4° used in the present invention has a molecular weight of 2.6XI C1' Daltons and has two markers, Tc resistance and Ap resistance, and restriction enzymes Hind nLBamHI and 5all on the Tc resistance gene.
The insertion deactivation site recognized by A,
It has insertion inactivation sites recognized by restriction enzymes Pst I and Pvu I on the p-resistant gene,
It is the most widely used vector among all the vectors developed to date.

By the way, trimethoprim is a strong inhibitor of dihydrofolate reductase, for example in bacteria such as Escherichia coli.
Trimethoprim inhibits dihydrofolate reducing enzyme l''-, thereby stopping the supply of its product, tetrahydrofolate, which is essential for amino acid and nucleic acid biosynthesis reactions.For this reason, trimethoprim is used as an antibacterial agent.

For the above reasons, in order to impart resistance to trimethoprim to the host, the dihydrofolate reductase gene is incorporated into a plasmid vector and inserted into the host, increasing the content of this gene in the cell and increasing the dihydrofolate reductase gene. All you have to do is produce it in large quantities.

Therefore, the host of the present invention has Tc resistance, Ap resistance and Tc resistance.
To obtain a plasmid vector for imparting p resistance, insert a DNA containing the dihydrofolate reductase gene into the restriction enzyme cleavage site other than the insertion inactivation site of the plasmid vector pBR322, which has two markers of Tc resistance and Ap resistance.
Just insert A.

In addition, the molecular weight of dihydrofolate reductase (which is known to be about 20,000 Daltons) is thought to be less than a base pair, which makes it advantageous to use the Tp resistance gene as a marker. However, until now, a plasmid vector using the 'rp resistance gene as a marker has not yet been developed.

Next, embodiments of the present invention will be described.

First, add 1 to E. coli to plasmid vector pBR322.
A DNA fragment of approximately 9200 base pairs containing the dihydrofolate reductase gene obtained from the two strains was treated with the restriction enzyme Bam.
Insert using HT and T4 DNA IJ gauze to obtain a hybrid plasmid. Since the cleavage site of pB, R322 with the restriction enzyme BamHT is the insertion inactivation site on the Tc resistance gene, the resulting hybrid plasmid confers 'rp resistance and Ap resistance to the host. This hybrid plasmid was introduced into 12 E. coli strains, and the cells were cultured using a nutrient agar medium containing ampicillin sodium and trimethoprim. [referred to as)]. This plasmid has a gene that confers Ap resistance and 'rp resistance to the host, and also contains the restriction enzyme E as shown in Figure 1.
coRIHindjll, BamHI, 5alI
, Pstl: iBstEI[,5stH1XhoI
It has a part that is cut by .

Next, the above plasmid pTPI was cut at two places with the restriction enzyme BamHI, and then the enzyme Nuclease BAL31, which acts specifically on double-stranded DNA and degrades nucleic acids from both ends, was applied to transform the linear DNA containing the Tp resistance gene. Get a fragment of. Both ends of this linear DNA are blunt ends.

On the other hand, plasmid vector pBR322 is treated with restriction enzyme EcoRI to cut sites other than the insertion disqualification site to make linear DNA, and then nuclease ST, an enzyme that specifically acts on single-stranded DNA, is treated to make linear DNA. The single-stranded portions protruding from both ends of the DNA are deleted to create blunt ends. At this time, the EcoRI cleavage site is deleted.

The linear DNA thus obtained and the p'r
Linear DN containing the 'rp resistance gene obtained from p
A fragment is blunt-end ligated using T4 DNA IJ gauze and circularized to obtain a hybrid plasmid. This hybrid plasmid was introduced into 12 E. coli strains, and the cells were cultured using a nutrient agar medium containing ampicillin sodium, tetracycline hydrochloride, and trimethoprim. 6
．． A plasmid with a size of 3 kilobase pairs is selected to obtain the desired plasmid vector (referred to as pTP30-5).

Plasmid vector pTP30 thus obtained
-5 has 'rc resistance, Ap resistance and 'rp resistance to the host.
The restriction enzyme Eco
RI, former ndlll, BamHr 1Sal I 1
It has a site that is cleaved by PstI and BstEII.

The plasmid vector of the present invention can also be obtained by the following method.

That is, plasmid vector pBR322 is treated with restriction enzyme EcoRI to cleave sites other than the insertion devitrification site to obtain linear DNA, and then treated with enzyme nuclease SI.
to remove the single-stranded parts protruding from both ends to create blunt ends. On the other hand, a DNA fragment of approximately 9,200 base pairs containing the dihydrofolate reductase gene obtained from 12 strains of Bacillus Daenobu is shortened using the enzyme nuclease BAL31. Both ends of this shortened DNA are blunt ends, and this and the above-mentioned pBR322 are
A linear DNA with blunt ends obtained by the action of oRI and nuclease sr is ligated with blunt ends using T 4 DNA ligase, and circularized to obtain a hybrid plasmid.

This hybrid plasmid was introduced into 12 E. coli strains, and the cells were cultured using a nutrient agar medium containing ampicillin sodium, tetracycline hydrochloride, and trimethoprim.
A plasmid is isolated from the grown bacterial cells and screened for molecular size to obtain a plasmid with a size of about 6.3 kilobase pairs as a vector. This plasmid vector provides host with Tc resistance, Ap resistance and 'r resistance.
Provides p resistance.

The plasmid vector of the present invention has three types of markers that can easily identify host cells containing it: Tc resistance,
Because it has markers for Ap resistance and 'rp resistance, and cleavage sites for various restriction enzymes, foreign DNA
It is an extremely excellent vector that has a large ability to load the A fragment, has a small molecular weight, and does not bring in unnecessary genetic information.

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

In addition, in the examples, isolation of plasmid from bacterial cells was carried out using T.
anaka and Weisblum's method (T, Ta
Naka.

B, Weis'blum; J, Bacteriol
ogy, 121, 354 t (1975)). “Example 5aito and Miura's method (H, 5aito,
K, Miura Biochim, Biophys,
Acta, 72+ 619 (1963), ).
Approximately 1μ2 of DNA containing the dihydrofolate reductase gene obtained from the strain and approximately 1μ of plus vector pB and R'322 were added to a 75μt reaction solution (7mM Tris-HCI p
Digestion was performed using 1 to 5 units of BamH1 gold in H7,4,7mM MgC12t (7mM 2-mercaptoethanol, 60mM Mail) at 7°C for 1 hour. After further inactivating BamHI by keeping it at 65°C for 5 minutes, it was kept in water and added with 15μt of 50mM MgO1.
2+ 15 μl of 0.1 M dithiothreitol (hereinafter abbreviated as DTT), 15 μt of 5mM ATP, 7 μt of l
The BamHl-digested DNA mixture was ligated by adding M Tris-Mol pH 7, 4, 23 μt of water and 1 to 2 units of T4 DNA ligase and reacting at 4° C. for 18 hours. This ligation mixture was
The method of rd et al. (M. V. Norgard, Keem
+J, J, Monahan; Gene, 3, 27
9, (1978))
It was introduced into IA coli K12 C600 strain. The treated bacterial cells were mixed with 20μy/mt ampicillin sodium and 2μy/mt ampicillin sodium. The cells were plated on a nutrient agar medium containing /mL trimethoprim to obtain growing bacterial cells (Ap-resistant and Tp-resistant bacteria). A plasmid with a size of approximately 13.6 kilobase pairs was isolated from this bacterial cell. This plasmid is called plasmid pTPI (hereinafter abbreviated as pTPl).

pTPl restriction enzyme FlicoR1, Hindl,
BamHl rSal l, Pst l, Bs
The cutting turns by tEll, 5stIl, and Xhol are shown in FIG.

This pTPI was added to Escherichia coli K
12 When introduced into 0600 strain, it transforms into Ap- and Tp-resistant bacteria, and the Tc-resistant gene of pBR322 is B
%pT because it is cleaved by amHI and pBR322 confers Ap and Tc resistance to the host.
It is concluded that the Tp resistance of PI was obtained by inserting a fragment of DNA obtained from Fischer-ichia coli K 12 strain cut with BamHl into the BamHI cleavage site of pBR322. Approximately 5μr was added to 4oopt of reaction solution (7mMT
ris-HCI pH7,4,7m,M MgCl
2.7mM 2-memercaptoethanol,
5-10 units of Bam in 60mM Na1l)
After incubating at 37°C for 1 hour using HI, 8pt
I M Tris-HC! 1' pH 8, 4 μt j
'M MgO12, 5 pL I M CaCl2.8μ
t of 5 M NaCl and 1 μt of 0.25 MEDTA
was added and kept at 25°C. 5. Add 2 units of 1 μt nuclease BAL31 to this reaction solution. '10. '1
5, 20. After 25+30 minutes, 50 μt of each was taken, 50 μt of water-saturated phenol was added thereto, and the mixture was stirred for 5 minutes, and then the aqueous layer was collected. All the aqueous layers thus obtained were combined and added to 50 mM Tris-HOI pH 7,
Dialysis was carried out at 4. The dialyzed DNA mixture is used for the treatment described below. Meanwhile, approximately 1 μ2 of pBR322 was added to 100 μt of reaction solution (7
mM Tris-HCI pH 7,4, 7mM MgC
l2, 7mM 2-mercaptoethanol, 60 m
, MNa1l), 1 to 5 units of KcoRl
After digestion at 37°C for 1 hour using 100 Pl of 0.2M sodium acetate buffer pH 5.0 containing 24mM ZnS○4 and 60mM Na01, which had been kept at 18°C, 1. 100 in the next step
Unit nuclease S1 was allowed to act for 1 hour, and
After removing the single-stranded portions at both ends resulting from the digestion of oR'1, 2 oopt of water-saturated phenol was added to stop the digestion by nuclease S1, and the mixture was incubated for an additional 5 min at 1 p.p.
After mixing, centrifuge to separate the aqueous layer and phenol layer, separate the aqueous layer and add 50mM Tris-HOI pH.
7,>;'f, I underwent dialysis. 40 pt of the liquid thus obtained and 1. After digesting the aforementioned pTPl f: with BamHl, it was mixed with 50 μt of the solution obtained by treatment with nuclease BAL31, and further 15 pt of 50 mM MgO
12, 15μt 0.1MDTT, 15/ljt 5m
M ATP , 7pt I MTris-) (C1p
H7, 4, 8 μt of water and 10-20 units of T4D
Add NA ligase and react at 4℃ for 18 hours to mix D.
NA was ligated.

This ligation mixture was prepared according to the method of Norgard et al.
scherichia coli K I 20600
It was incorporated into the stock. The treated bacterial cells were sown on a nutrient agar medium containing 20 μy/mt Nanpi/Phosphate sodium, 10 μf 7 ml tetracycline hydrochloride, and 2 μy/mt Methoprim, and the growing bacterial cells (Ap-resistant, TC-resistant, and 'rp-resistant bacteria ) were obtained. When plasmids were isolated from these bacterial cells, they ranged in size from 13.6 kilobase pairs to 6.3 kilobase pairs. Among these plasmids, a plasmid with the smallest molecular weight of 6.3 kilobase pairs was selected as a vector and is referred to as plasmid vector pTP30-5.

pTP30-5 restriction enzymes KcoR1, Hindl
, BamHl, 5all, Pstl,
The cleavage map by BstK[l is shown in FIG.

This pTP30-5 was transformed into Escherichia cor.
i When introduced into the K12C600 strain, it became resistant to three types of drugs: ampicillin, tetracycline, and trimethoprim.

In addition, a heterologous D was added to the EcoRl cleavage site of pTP30-5.
The plasmid with NA inserted was transformed into Escherichia
When introduced into the coli K12 C600 strain, the resistance to trimethoprim was lost.

Escheri introduced with pTPl or pTP30-5
The amount of dihydrofolate reductase obtained from Chiacoli K 120600 strain was different from that of E.
The amount was about 10 times that obtained from Scheri-chia coli K120600 strain. Therefore, Tp resistance is thought to be due to increased intracellular production of dihydrofolate reductase, which releases or alleviates the inhibition of this enzyme by trimethoprim.

[Brief explanation of the drawing]

Figure 1 is a restriction enzyme cleavage map of pTPl, and Figure 2 is a restriction enzyme cleavage map of pTP30-5. The symbols in the figure represent restriction enzymes, E is KCORIIH3 is Hi
nd l, B is BamHl, Sa'l is 5al
l, BstFil is BstFli[, Pθt is Pstl
, E18tll indicates 5stl and xho) indicates Xhol. Furthermore, the unit of numbers is kilobases, and the internal arc represents DNA derived from plasmid vector pBR322. Patent applicant Makoto Ishizaka, Director of the Agency of Industrial Science and Technology - Designated agent Futoshi Hama, Institute of Textile and Polymer Materials, Agency of Industrial Science and Technology
Showa 1st figure 2nd figure

Claims (1)

[Scope of Claims] 1. A plasmid vector having a tetracycline resistance gene, an ampicillin resistance gene, and a trimethoprim resistance gene as genetic markers. 2. A method for producing a plasmid vector having a tetracycline resistance gene, an ampicillin resistance gene, and a trimethoprim resistance gene, which comprises inserting DNA containing a dihydrofolate reductase gene into plasmid vector pBR322.