JPS60199385A - Plasmid vector ptp20-10 - Google Patents

Abstract

PURPOSE:To provide plasmid pTP20-10 by which the host bacterium can get the resistance to ampicillin, tetracyclin and trimethoprim, when it is introduced into the host. CONSTITUTION:The plasmid vector has about 5.6 kilobase, is cleaved in one site, respectively by restriction enzymes, Ava I , BamH I , Bal I , EcoR I , EcoRV, HindIII, Sal I , Pvu I , PvuII and Pst I and has markers resistant to Ap, Tc and Tp and the cleavage map as shown in the figure. The pTP20-10 is introduced into Ecoli K12C600 strain and kept stably.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a plasmid pTP20-10 which, when introduced into a host bacterium, allows the host bacterium to acquire resistance to ampicillin, tetracycline and trimethoprim.
(hereinafter abbreviated as p'rp 20-10).

In recent years, with the development of molecular biology and genetic engineering, methods have been developed to produce useful substances using recombinant DNA techniques, such as introducing foreign DNA into bacteria such as Escherichia coli and expressing the genes. Recently, research and development has been carried out to commercialize extremely useful substances such as interferon, insulin, and growth hormone using this genetic recombination method.

Generally, what is called a plasmid vector is used as a means for introducing the above-mentioned heterologous DNA and expressing the gene. When a plasmid vector is introduced into a host, it must have a gene that gives the host resistance to drugs, etc., so that it can be easily distinguished from a host that has not been introduced, that is, a genetic marker (hereinafter abbreviated as a marker). be. For example, plasmid vector pBR322
pACYC184 has tetracycline (hereinafter abbreviated as Tc) resistance and ampicillin (hereinafter abbreviated as Tc) resistance, and pACYC184 has chloramphenicol resistance and tumor resistance as markers.

As a method for inserting a heterologous gene into a plasmid vector, the plasmid vector is cut with an appropriate restriction enzyme, and the heterologous DNA having the same terminal structure as that obtained by cutting with the restriction enzyme used for this cut (generally, the plasmid vector is (using a DNA fragment obtained by cutting with the same restriction enzyme used to cut the DNA)
A method of ligating and re-linking using an enzyme called IJ gauze is commonly performed. The recombinant plasmid created in this way is introduced into a host, and the host bacteria into which the plasmid of interest has been introduced is grown to produce the protein encoded by the inserted foreign DNA. However, in the process of creating a recombinant plasmid, only the plasmid vector was reselected, and the heterologous DNA
It may be difficult to find a host with a plasmid in which A has been inserted. To prevent this, use a different DN.
A plasmid vector is constructed such that when A is inserted into a specific site of a plasmid vector, a specific marker is inactivated.

For example, the Tc resistance marker is inactivated by inserting heterologous DNA into the restriction enzyme BamHI cleavage site in the plasmid vector pBR322 described above. Such a site is called an insertion deactivation site. If a foreign DNA is inserted into the 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 addition, as the site for inserting foreign DNA, a site that is cut by a specific restriction enzyme is used.
It is necessary that there is only one cleavage site per plasmid vector.

Recently, various restriction enzymes have been developed, so it is desirable to construct a plasmid so that as many restriction enzymes as possible can be used. It is desirable to construct a plasmid vector having such a site.

Furthermore, since expression of the gene in the inserted DNA is expected, it is desirable to construct a plasmid vector that can also efficiently express the gene.

In view of these circumstances, the present inventors have developed a plasmid vector that has many types of markers and has many types of insertion sites for heterologous DNA (that is, has a large number of restriction enzymes that cut the plasmid vector at only one site), and As a result of intensive research to obtain a plasmid vector that efficiently expresses the gene in the inserted DNA, the present inventors have already constructed a plasmid vector pTP30-5 (Japanese Unexamined Patent Application Publication No. 1983-1973).
Described in Publication No. 46100. (hereinafter abbreviated as pTP30-5), Tc resistance, Ap resistance, and trimethoprim (produced T
Although the plasmid pTP6-1 has three types of resistance markers, it has the disadvantage of having two or more restriction enzyme 3al I and pst I sites each.
0 (described in the patent application 1987-973.Hereafter p'rp6-1
0), the expression efficiency of the dihydrophyllase reductase (hereinafter abbreviated as DHFR) gene, which is a Tp resistance marker, is very high, and the host carrying pTP6-10 is
Although FR is produced to account for approximately 9.3% of the total soluble protein, we focused on the drawback that it only has two markers, Ap resistance and Tp resistance, and has the advantages of both.
After conducting various studies to construct a plasmid vector that eliminates the drawbacks, we created the plasmid vector pBR322ΔEco (
Described in JP-A-57-192400. Below pBR3
We have devised a method for producing a recombinant plasmid in which a part of p'rp 6-10 (abbreviated as 22ΔEco) is combined with a part of p'rp 6-10, and by producing a recombinant plasmid according to the production method, the present invention has been carried out. It was completed.

p'l'p20-10 of the present invention has a molecular weight of about 5.6 kilobase pairs, and contains restriction enzymes AvaI, BamHI, Bal
I, EcoRI, EcoRV, H4ndIII
, 5alI, PvuI, PvuII, and Pst
■Ap resistance, T
This is a plasmid vector having c resistance and Tp resistance markers and the restriction enzyme cleavage map shown in FIG. Additionally, a host harboring pTP20-10 has approximately 1.
By producing 87j-nit/rn9 protein DHFR, it is possible to produce DHFR up to about 4696 of the total soluble protein.

The method for creating pTP20-10 of the present invention is as described in the Examples below, but pTP20-10 is a completely new plasmid vector. And pTP20-10
was introduced into the E. coli K12 C600 strain and kept stable, and the E. coli containing pTP20-10
The E.coli K12C600 strain has been deposited at FERMP- as FERMP-.

FIG. 1 shows a restriction enzyme cleavage map of pTP20-10 (approximately 5.6 kilobase pairs) of the present invention. A, Bm, B
+, RI-RV, H2, H3, S, Pvl, Pv2 and P are restriction enzymes Ava L BamHL Ba
tI, EcoRI.

EcoRI, EcoRV, Hinc II, Hin
dIII, 5alI, PvuI.

The cleavage sites of Pvu n and Pst I are shown, tet,
amp and fol are Tc resistance genes, Ap
It means a resistance gene and one resistance gene, and the arrow indicates the direction of expression.

pTP20-10 of the present invention has seven insertion deactivation sites. Restriction enzyme site BamHI, EcoRV
- HindII and Sat I are Tc-resistant insertion inactivation sites, restriction enzyme sites pvu I and Pst I are Ap-resistant insertion inactivation sites, and restriction enzyme site E
coRI is an insertion inactivation site for Japanese resistance.

pTP20-10 of the present invention has b with increased expression efficiency.
Resistance genes exist. D, which is the product of the Tp resistance gene.
The production amount of HFR is determined by the p value already created by the present inventors.
E. coli K 1206 containing TP30-5
This is approximately 10 times the production amount of DHFR strain 00, which is 0.215 units/~ protein. Utilizing this property, it is expected that by inserting a heterologous gene into the restriction enzyme EcoRI site present on the T9 resistance gene in p'rP20-10, the inserted gene will be efficiently expressed. In other words, it is expected that it can be used as a "plasmid vector for material production."

Next, examples of the present invention will be shown. In addition, isolation of plasmids from bacterial cells in Examples was performed by Tanaka and Weis.
blum's method [T, Tanaka, B, Wei
sblum; J, Bacteriology.

121.354 (1975)) and also plasmid D
NA was introduced into the host using the method of Novgard et al. (M,
VNovgarc%, Kee+n, J, J, Mon
ahan: Qene, 3. 279 (1979))
I followed.

Implemented IPJ 1 Production of p'rP20-10.

pBR322ΔEco (described in JP-A-57-192400) About 5 trg was added to 400 al of reaction solution I.
(7mM Tris-FIce, pH7.4, 7mM
MgCl2. The DNA cleavage reaction was performed with 10 units of the restriction enzyme pst I in 7 rrM 2-mercaptoethanol, 60 mM NaCl) for 37°C], followed by 8 ml of IM Tris HCl, pstI.
H8,0,4μm I M Mg CI2.5μm I
M CaCl2. 8/11 5M NaCl and 1
! , +0.25 M ethylenediaminetetraacetic acid, pl-
I8 (hereinafter abbreviated as EDTA) was added and maintained at 25°C. Two units of exonuclease BAL 31 were added to this reaction solution, and the reaction was allowed to proceed for 4 minutes. For the reaction, after adding 400 μl of water-saturated phenol and stirring,
The mixture was centrifuged to separate into an aqueous layer and a phenol layer, and the aqueous layer was taken and dialyzed against 50 mM Tris-HCI, pH 7.4.

Next, approximately 5 μg of p'rp 6-10 (described in Japanese Patent Application No. 1987-9735) was added to DNA in a 400/I+ reaction solution using 1.10 units of restriction enzyme Pvun at 37°C for 1 hour.
After performing the A-cleavage reaction, 8 μm IMTris HCI
, pH 8, 0, 4 μm I M Mg CI 2.5 μm
m IM CaClz, 8 sl 5M NaCl and 1μ
m EDTA was added and kept at 25°C. Add 2 units of exonuclease BAL31 to this reaction solution,
The reaction was allowed to proceed for 10 minutes. For the reaction, 400 μm of water-saturated phenol was added and stirred, followed by centrifugation to separate the aqueous layer and the phenol layer, and the aqueous layer was taken and dialyzed against 50 mM Tris-HCI, pH 7.4. A 50 μm sample was taken from the solution obtained in this way, and the above-mentioned p
507I+ was taken from the dialyzed solution obtained by treating BR322ΔEco, mixed together, 300/11 ethanol was added to this, and the DNA contained was precipitated by leaving it at -20°C overnight. . The DNA precipitate was collected by centrifugation, and after discarding the ethanol solution, the DNA was dried in a vacuum desiccator.

The DNA thus obtained was mixed with a 20μm ligase reaction solution (66mM Tris HCI, pH 7,4,7
mM MgC12, 10mM dithiothreitol, 5mM
ATP).

This includes 5 units of T4DNA! Add I gauze, 16
The ligation reaction was carried out for 12 hours at °C. This ligation mixture is
E, cot i K according to the method of Norgard et al.
12 was introduced into C600 strain. This treated bacterial body,
50 μg/lnl ampicillin sodium, 10/J
g/lnl of tetracycline hydrochloride and 50 μg/ln
Spread on a nutrient agar medium containing 3 liters of trimethoprim and
Six colonies could be obtained by leaving at 7°C for 24 hours. Select one colony from among these, prepare a plasmid from the bacterial cells according to Tanaka and Weisblum's method 1, and transfer this to p'
It was named rp20-10.

p'rp 20-10 again E, coli K 12
When introduced into C600 strain, it was possible to transform the product into Tc-resistant, Hep-resistant, and Tp-resistant at a frequency of about 105/μg plasmid.

Example 2 Structure of pTP20-10.

In order to determine the restriction enzyme cleavage map of p'rp20-10 obtained in Example 1, cleavage with the restriction enzymes shown in the table was performed, and this was analyzed by agarose gel electrophoresis. Using the DNA fragments obtained by HindlII cleavage as molecular size markers, the molecular sizes of the DNA fragments obtained by each cleavage were determined, and the results shown in the table below were obtained.

AvaI 5.6 Bam HI 5.6 Bat I 5.6 Eco RI 5.6 EcoRV 5.6 Hinc II 3.2+1.3+1.1Hind m
5.6 Sal I 5.6 Pvu I 5.6 Pvun 5.6 Pst I 5.6 Eco R1+ Ava I 3.3 +2.3Eco
RI + Bam HI 4.4 + 1.2Eco
RI + Bal I 3.3 + 2.3Eco
RI + Eco RV 4.6 +1.0Eco R
I+Hincn 3.2 + 1.3+0.9+062
Eco RI + HindIIl 4.7 + 0.9
Eco RI + Sal I 4.1 + 1.5E
co RI + Pvu I 4.5 +1.1Eco
RI + Pvun 2.9+2.7EcoRI +P
st I 4.4+1.2I (ind III 10Av
a I 4.2 + 1.4 Hind Ill + B
amHI 5.3 +0.3Hind m +Bal
I 4.2 +1.4HindI[I→-Eco RV
5.5 + 0.1 Hind Ill + Hinc
n 3.2+ 1.1 +0.7+0.6HindII
I-to 5alI 5. O+0.6 Hind III
+P vu I 3.6 +2.0HindI[10Flu II 3.6+2.0HindIII+ Rt
I 3.5+2.1PvuII + AvaI 5.
0+0.6Pvul[+ BamHI 3.9+ 1.
7Pvun + Bal I 5.1 +0.5Pvu
II + EcoRV 3.7+1.9PvuIr −
Hinc If 1.8 + 1.4 + 1.3
+ 1.1Pvun + 5alI 4.2+1.4
Pvul [+ Pvul 4. O+1.6Pvun+
PsiI 4.1+1.5 From the results shown in this table, the restriction enzyme cleavage map shown in FIG. 1 was determined.

Example 3 E. coli K12 containing pTP 20-10
DHFRHF property of C600 strain.

E, co! containing p T P 20-10! i
K12 C600 strain (hereinafter abbreviated as p'rp 20-10 strain) was cultured in 40 g of ST-Ap medium C2 g/l glucose, Ig/l 2 HPO4, 5 g/l yeast extract, 5 g/l of polypeptone and 50 W/l ampicillin sodium) aerobically at 37° C. until the number of turret units reached 120. The bacterial cells were collected by centrifugation, washed with 50 mM potassium phosphate buffer (pH 7.0) (hereinafter abbreviated as KPB), and suspended in 2 cups of KPB (then sonicated for 2 minutes and rotated at 20,000 rpm). Centrifugation was performed for 1 hour, and the resulting supernatant was measured for DHFR activity and protein concentration.The DHFR activity value was 13.8 units/ml at 37°C. The protein concentration of the supernatant used was 7.38 m9/ml. When the specific activity was calculated from this, a value of 1.87 units/ta protein was obtained. As the present inventors have already clarified (described in Japanese Patent Application No. 58-9735), the specific activity of completely purified DHFR is 407 units).
/mr protein, the above results indicate that pTP20
The results show that approximately 4.6% of the soluble protein in the sonicated centrifugation supernatant obtained from the -10 strain is DHFR. Similar measurements were performed on E
, coli K]2 C600 strain, the specific activity of DHFR was 0,010 units/da protein, indicating that approximately 0,025% of the soluble protein in this strain was DHFR.

[Brief explanation of drawings]

Figure 1 is a restriction enzyme cleavage map of p'rp20-10, and the symbols in the figure represent restriction enzymes and each resistance marker gene, A, Bm, Bl, RI, RV, H2, H3, S.
, Pvl, Pv2 and P are restriction enzymes AvaI, respectively.
. BamHI, Bol I, EcoRI, Eco
RV, HinclI, Hindl[, Satl5
PvuI, Pvun and PstI, tet
, amp and fol are Tc resistance gene, A
p resistance gene and Tp resistance gene are shown. Also,
The units of numbers indicate kilobase pairs.

Claims (2)

[Claims] (1) Plasmid vector pTP20i that has a molecular weight of 5.6 kilobase pairs, can confer ampicillin resistance, tetracycline resistance, and trimethoprim resistance to the host, and has the restriction enzyme cleavage map shown in Figure 1.
0° (2) Escherichia coli K12 C60 containing plasmid vector pTP20-10
0 shares.