JP2949351B2 - Plasmid (p) NI10 and gene recombination method using it as a vector - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a novel plasmid useful as a vector for gene recombination using Pseudomonas sp. As a host and a gene recombination method using the same as a vector. In particular, the present invention relates to a novel plasmid pNI10 having a molecular weight of about 3.7 Kb and characterized by the restriction enzyme cleavage map shown in FIG. 1, and a gene recombination method using the same as a vector.

At any restriction enzyme site of the plasmid pNI10 of the present invention,
Gene DNA encoding the target protein is ligated and introduced into Gram-negative bacteria such as Pseudomonas bacteria and Escherichia coli, whereby the target peptide hormones and various enzymes are added to the inside or outside of these bacteria. Etc. can be mass-produced.

[Prior art] Conventionally, gene recombination experiments have been widely studied mainly in a system using Escherichia coli as a host, and have produced great results such as mass production of insulin, interferon, human growth hormone, various enzymes, etc. in Escherichia coli. . In addition to Escherichia coli, host / vector systems have been developed for yeast, Bacillus subtilis, actinomycetes, and the like, and ways of application are being studied.

Pseudomonas bacteria have a wide range of habitats, can use various organic compounds as a single carbon source, and can utilize chemically synthesized compounds. Development has become a more effective tool for breeding industrially useful microorganisms.

[Problems to be solved]

Although some plasmids have been reported from Pseudomonas sp., They all have a molecular weight of 30 Kb or more and involve various operational difficulties. Attempts have been made, but nothing has yet been achieved.

Also, in gene recombination experiments of Pseudomonas genus bacteria, examples of using a broad host range plasmid that can be used as a multicopy plasmid in Gram-negative bacteria such as RSF1010 are known, but this plasmid is also 8.9 Kb. And the transformation efficiency is not very high.

Therefore, development of a small vector that can insert a foreign gene of any length, can transform a Pseudomonas genus bacterium with high efficiency, and can easily detect the gene has been desired.

[Method to solve the problem]

Under such circumstances, the present inventors have intensively studied to develop a host and a vector suitable for Pseudomonas bacteria, and finally use the new strain Pseudomonas flavida IF-4 strain as a vector from a new strain of Pseudomonas genus Pseudomonas flavida. The present inventors have found a plasmid pNI10 suitable for the present invention and completed the present invention.

Since the plasmid pNI10 of the present invention can be retained in Escherichia coli, it can be used as a shuttle vector for Escherichia coli-Pseudomonas sp.

Also, as is clear from FIG.
It has cleavage sites at specific and limited positions by restriction enzymes such as RI, Hinc II, Pvu II, Sca I, and Sma I. This is advantageous in that, when using pNI10 as a vector, the site of introduction of the target gene to be inserted can be significantly selected.

In order for a plasmid DNA to be a vector, it is essential that the plasmid has an autonomous growth ability in a host and a selection marker (a marker indicating that the plasmid is present in the host). In general, Pseudomonas bacteria are often sensitive to kanamycin (hereinafter abbreviated as Km) and streptomycin (hereinafter abbreviated as Sm), and if such drug resistance is imparted, the presence of a vector plasmid can be easily identified. be able to. We found that pUC-4K was present at the restriction enzyme cleavage site of pNI10.
[Vieria, J. and Messing, J. Gene, 19, 259 (1982)]
Km resistance gene and RSF1010 (Scherzinger, E. et al., Pr.
oc. Natl. Acad. Sci. USA, 81, 654 (1984)]
A resistance gene was introduced, and a plasmid vector derived from pNI10 having a selection marker was completed.

The plasmid pNI10 of the present invention has a high copy number in a cell, and therefore, when a foreign gene is recombined, a large amount of gene amplification can be expected. It is also possible to insert a foreign gene of 20 Kb or more.

Furthermore, the use of the plasmid pNI10 of the present invention as a vector can significantly improve the breeding of useful Pseudomonas bacteria that can utilize a large number of compounds.

Plasmid pNI10 is obtained from the strain IF-4 newly isolated from the soil by the present inventors. The bacteriological properties of this strain are as follows.

I. Cell morphology: Short bacilli 0.7-1.1 × 1.0 with 1-2 polar flagella
Mobility: Yes Gram stain: Negative Spore formation: No II. Growth state in each medium (1) Standard agar plate culture Colonies are circular, the entire periphery is round, the surface is smooth, transparent, and bright.

(2) Standard agar slant culture Linear, smooth surface, buttery, iridescent, moderately growing.

(3) Standard liquid culture There is a slight powdery precipitate. No film formation.

(4) Standard gelatin puncture culture Liquefaction.

(5) Litmus milk alkaline III. Physiological properties Catalase: Positive Cytochrome oxidase: Negative GC content: 63% Pigment generation: yellow (tryptosoy agar medium, water-insoluble dye), yellow (KingB medium, fluorescent water-soluble dye) O -F test: oxidized acid production: glucose + xylose + mannitol-lactose-sucrose-maltose-salicin-fructose + development: 5 ° C + 41 ° C-Mac Conkey + SS + Nac + CVT + 6% NaCl + DL-α-HB-polybetahydroxybutyric acid accumulation + nitrate reduction-gas from nitrite-egg yolk reaction-arginine dihydrolase + lysine decarboxylase-ornithine carboxylase-acylamidase-Tween80 degradation-gelatin degradation + casein degradation-starch degradation − DNA degradation − Esculin degradation − Urea degradation − Cellulose degradation − Chitin Decomposition-Agar decomposition-Production of 2-ketogluconic acid from gluconic acid + VP.MR reaction-Indole and hydrogen sulfide reaction-Formation of dioxyacetone-Utilization of citric acid + TSI-/-ONPG-Formation of mucoid-Litmus milk Alkaline phenylalanine reaction-Growth temperature (° C) 5-37 Growth pH 5.0-9.2
y's Manual of Determinative Bacteriology) Eighth Edition,
Cowan Stale Manual (STCowan et al.:197
4 Manual for the Identification of Medical Bacteri
a), CDC manual (REWeaver and DGHollis: 1983
Gran-Negative Organisms: An approach to Identifica
The strain was identified as Pseudomonas by a search using Action Centers for Disease Control.

However, the species was determined to be none of the conventional species because it is a new bacterium not described in each manual and has a novel plasmid pNI10. Since this strain is a particularly shiny yellow fungus, it was named Pseudomonas flavida IF-4 strain. This strain is manufactured by B.I.K. 10865 (FERM P-10865)
And deposited at the Institute of Microbial Industry and Technology, National Institute of Advanced Industrial Science and Technology.

Example 1 (1) Pseudomonas flavida IF-4 (FERM P-1086)
5) Isolation of pNI10 DNA from the cultured cells A PY medium (5 g of peptone, 5 g of yeast extract, water 1, pH 7.0) was inoculated with Pseudomonas flavida IF-4 (FERM P-10865) strain, and incubated at 30 ° C. overnight. And shake culture. 1 ml of the main culture was inoculated into 100 ml of the same fresh medium and cultured at 30 ° C. for 16 hours.

The culture is collected by centrifugation under cooling and centrifuged, and SET buffer (20%
Sucrose, 50 mM EDTA, 50 mM Tris-HCl (pH 8.0)] 20
Suspend in ml. 2.25 ml of lysozyme (10 mg / ml, Boehringer Mannheim) and pronase (10 mg / m
l, 250μl RNase (5mg / ml, Boehringer Mannheim product) 250μ, add lightly,
Leave on ice for 5 minutes. Next, 45 ml of an alkaline SDS solution (1% SDS, 0.2N NaOH) is added and stirred up and down in a centrifuge tube. After cooling on ice for 10 minutes, 32 ml of 3M sodium acetate solution (pH 4.8) is added, mixed, and left standing in ice water for 1 hour. After cooling and centrifugation at 19,000 rpm for 15 minutes, the supernatant was separated. Further, the same volume of isopropanol as the supernatant was added, and the mixture was cooled on ice for 2 hours, and then centrifuged at 8000 rpm for 10 minutes.
After centrifugation, the precipitate was washed with a 70% ethanol solution. The precipitate was dried in a desiccator under reduced pressure using a vacuum pump, and then 8 ml of TE buffer (10 mM Tris-HCl, 1 mM EDTA pH 8.0)
To give a crude DNA sample. 0.56 ml of 10 mg / ml ethidium bromide and 8.48 g of cesium chloride were added thereto, and
The plasmid DNA was separated by ultracentrifugation at 16 ° C for 17 hours at rpm. The separated plasmid DNA was treated with isopropanol saturated with saturated cesium chloride water to remove ethidium bromide, and dialyzed against TE buffer to give plasmid DN.
A got. Next, the DNA was subjected to electrophoresis using 0.7% agarose gel and TBE buffer (59 mM Tris-borate, 2 mM EDTA, pH 8.0). Lambda phage D as a DNA molecular weight marker
When the HindIII digest of NA was used, the mobility of the circular plasmid of pNI10 was the same as that of the linear DNA fragment of about 2.3 Kb. This portion is cut out from the agarose gel, the gel is dissolved with an 8 M sodium perchlorate solution, and the solution is subjected to suction filtration with a Whatman 10FC filter to adsorb the plasmid DNA on the filter. 6M perchloric acid solution,
After washing the filter with 100% ethanol, remove the filter
Plasmid DNA was recovered by immersion in E buffer. By the above operation, a highly pure pNI10 plasmid was prepared.

(2) Measurement of molecular weight of pNI10 plasmid The molecular weight of pNI10 was determined by 0.7% agarose gel electrophoresis of a fragment obtained by cutting pNI10 with the restriction enzyme PstI. As a molecular weight marker at this time, λ phage DNA
Of Hind III and BstE II were used. From this, the molecular weight of pNI10 was calculated to be 3.7 Kb.

(3) Preparation of pNI10 restriction enzyme map The purified pNI10 plasmid DNA was cut with various restriction enzymes. After cleavage, the plasmid DN is prepared in the same manner as in (2).
The molecular weight of the restriction fragment of A was determined by 1% agarose gel electrophoresis. Table 1 shows the number of cleavages for various restriction enzymes.

A restriction enzyme cleavage map as shown in FIG. 1 was prepared from the restriction enzyme cleavage patterns in Table 2.

(4) Measurement of copy number of pNI10 Pseudomonas flavida IF-4 (FERM P-10865)
The strain was cultured by the method described in the above (2) to obtain bacterial cells. From the cells, a method known to Saito and Miura [Biochimica et Biophysica Acta, 72
Vol., Pp. 619-629 (1963)]. Next, about 10 μg of the DNA was completely digested with the restriction enzyme EcoRI, and a part thereof was subjected to agarose gel electrophoresis by the method described in the above (2). The separation pattern in agarose gel electrophoresis was photographed, and the ratio of the peak of the pNI10-derived band to the peak of the other DNA portion was measured with a densitometer. Assuming that the total amount of DNA per cell of Pseudomonas bacteria was 4 × 10 ⁹ daltons, the copy number of pNI10 per cell was calculated to be 30-40 / cell.

(5) Construction of plasmids pNI105, pNI106, pNI107 and pNI111 a) A known plasmid pUC-4K [Vieria, J. and Messing, J., Gen.
e, 19 vol, 259 (1982)] with restriction enzyme Hinc II processing T ₄ DNA fragment containing the Km ^r gene caused approximately 1250bp by
Ligated using DNA ligase, and transformed Pseudomonas flavida IF-42 strain obtained by removing all of the circular plasmids of Pseudomonas flavida IF-4 by the method described in the following section, and then a PY medium containing 25 μg / ml of Km. The Km ^r transformant was selected. From this transformant to obtain a plasmid pNI105 having Km ^r marker.

b) In the same manner as in a), the restriction enzyme Sma I of pNI10 was used.
The cleavage site was ligated the DNA fragment containing the Km ^r gene 1250bp caused by the restriction enzyme Hinc II processing pUC-4K,
Pseudomonas Furabida IF-42 strain was transformed to obtain a plasmid pNI106 having from Km ^r selectable marker this.

c) The restriction enzyme Hinc II cleavage site of pNI106 was replaced with the restriction enzyme Bam
By changing to the HI cleavage site, pNI107 was obtained.

 The construction procedure of pNI107 is as follows.

First, pNI106 is digested with the restriction enzyme Hinc II. This DNA
Fragment commercial phosphorylated BamH I linker (Takara Shuzo Co., Ltd., having a CGGATCCG sequence) was ligated with T ₄ DNA ligase. Then, by the action of BamH I, the both ends and cohesive ends, was circular DNA again using T ₄ DNA ligase. Using this, Pseudomonas flavida IF-4 strain (FERM-P
10865), thereby obtaining pNI107.

d) pNI107 is partially digested with the restriction enzyme ClaI, and the DNA fragment cut at one site is separated by agarose gel electrophoresis.
to recover. The resulting plasmid DNA was blunt-ended using a DNA polymerase, and the restriction enzyme Hinc II was removed from RSF1010.
-About 1.9 Kb of D containing the Sm resistance gene excised by Pvu II
It was ligated with NA fragments and T ₄ DNA ligase. Pseudomonas putida (Pseudomonas
putida) IF3 strain was transformed and an Sm resistant strain was isolated. Sm
A plasmid was prepared from the resistant transformant and its structure was examined. As shown in FIG. 2, the plasmid pNI107 having the Sm resistance gene inserted at the Cla I site in the Km resistance gene of pNI107 was obtained.
111 was obtained. FIG. 2 shows the outline of each vector.

(6) Transformation of Pseudomonas sp. And other Gram-negative bacteria Transformation of Pseudomonas sp.
gdasarian et al., Gene, 16, 237 (1981)].

PY medium is inoculated with a conserved seed of Pseudomonas sp.
Shaking culture was performed at 30 ° C. for 15 hours. Collect 1 ml of this culture,
Next, 2.5 ml of transformation buffer I (1
0 mM MOPS, 10 mM RbCl, 100 mM MgCl ₂ , pH 7.0) were added to suspend the bacteria. After standing at 0 ° C. for 30 minutes, the cells were collected by centrifugation, and 0.3 ml of the same buffer solution I was added thereto and resuspended to obtain competent cells. 25μ DNA sample in competent cell solution
Was added and left at 0 ° C. for 45 minutes. Next, after heat treatment at 40 ° C. for 5 minutes, 1.7 ml of PY medium is added, and after shaking culture at 30 ° C. for 3 hours, 0.15 ml of culture solution is applied to the surface of any selective medium to select the desired transformant. did.

(7) Transformation efficiency The transformation efficiency of some Pseudomonas bacteria was measured according to the method of (6). Transformation efficiency is 1
How many transformants per μg of pNI105 plasmid DNA (Km
(Resistant strain) was obtained. Table 2 shows the results.

When each of pNI106, pNI107 and pNI111 (Sm resistant) vectors was used, almost the same results as in Table 2 were obtained.

(8) Search for host bacterium of pNI105 It was examined whether the plasmid pNI105 replicated and expressed in Escherichia coli as a gram-negative bacterium other than Pseudomonas sp. Transformation methods are known methods [Kushner,
SR, Genetic Engineering, page 17, Elsevier (1978)] to prepare competent cells.

As a result, it was found that the pNI105 vector was stably retained in the Escherichia coli C-600 strain. In particular, the transformation efficiency of the E. coli C600 strain was 2 × 10 ⁵
With a very high ratio of cells / μg DNA, pNI105 was sufficiently usable as a shuttle vector between Pseudomonas bacteria and Escherichia coli.

In addition, each of the plasmids pNI106, pNI107, and pNI111 had the same results as pNI105.

As described above, the pNI10-based plasmid is useful as a shuttle vector between Pseudomonas bacteria and Escherichia coli.

(9) Length of insertable DNA The following method was used to determine how large a foreign DNA can be inserted into pNI107.

First, foreign DNA was prepared from Pseudomonas putida according to the method described in (4) above. Next, the DNA was partially digested with a restriction enzyme EcoRI to obtain a foreign DNA sample.

The other pNI107DNA was cleaved with EcoR I, the vector DNA and the foreign DNA was ligated with T ₄ ligase to the IF3 strain Pseudomonas putida in the method of the preceding paragraph (6) was transformed. Km
50 resistant strains were arbitrarily selected, and plasmid DNA was isolated from the resulting transformant. Plasmid DNA obtained from each strain was cut with a restriction enzyme EcoRI to cut out the inserted foreign DNA. The length of this DNA was determined by agarose gel electrophoresis according to the method described in the above section (2). As a result, it was revealed that DNAs of each length were inserted into the vector and that the largest DNA had a length of 20 Kb or more.

〔The invention's effect〕

The plasmid pNI1 obtained according to the present invention as described above
Numeral 0 is a small vector, to which a gene DNA encoding a target protein can be ligated to an arbitrary restriction enzyme site. Therefore, by introducing this into various bacteria,
Proteins such as a target peptide hormone and various enzymes can be mass-produced inside or outside the cells of these bacteria.

[Brief description of the drawings]

FIG. 1 shows a restriction map of plasmid pNI10 of the present invention. FIG. 2 shows a restriction map of each plasmid derived from the plasmid pNI10 of the present invention, in which a) shows the restriction of plasmid pNI105, b) shows the restriction of plasmid PNI106, c) shows the restriction of plasmid pNI107, and d) shows the restriction of plasmid pNI111. Each enzyme map is shown. Also,

Claims (3)

(57) [Claims] The present invention relates to the following: 1. A molecular weight of about 3.7 Kb, having an enzymatic cleavage map shown below, and restriction enzymes BamHI, SacI and Sal.
Plasmid pNI10 from Pseudomonas flavida not cleaved by I. 2. A plasmid obtained by adding a kanamycin resistance or streptomycin resistance selection marker to the plasmid pNI10 according to claim 1. 3. A gene recombination method using the pNI10 or the pNI10-derived plasmid according to claim 1 as a vector.