JPS62181784A - Mass-production of insecticidal protein by expression of insecticidal protein gene in escherichia coli - Google Patents

Abstract

PURPOSE:To mass-produce an insecticidal protein, by forming an expression plasmid, containing an insecticidal protein of Bacillus thuringiensis aizawai IPL strain and capable of expressing the strain in microbial cells. CONSTITUTION:An expression vector, e.g., expression vector pUC18 holding Lac promoter, expression vector pKK223-4 holding Tac promoter which is a powerful promoter for Escherichia coli and terminator for rrbB ribosome RNA, etc., is linked to an insecticidal protein gene of Bacillus thuringiensis aizawai IPL strain to form an expression vector holding the insecticidal protein gene. The resultant expression vector is then introduced into a host microorganism, e.g. Escherichia coli JM103 strain, etc., to give a transformant microorganism, which is then cultivated in a culture medium to mass-produce the aimed insecticidal protein.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention contains an insecticidal protein gene of Bacillus thuringiensis aizawai IPL strain that exhibits strong insecticidal activity against lepidopteran insects, diamondback moth and Spodoptera spp. Expression plasmids that are expressed in microorganisms such as Bacillus thuringiensis,
The present invention relates to a microorganism that produces an insecticidal protein of Aizawai IPL strain, and a method for producing an insecticidal protein, which is characterized by culturing the microorganism.

Prior Art Various strains of Bacillus thuringiensis form 1-2 μm crystals made of insecticidal proteins during the sporulation stage, and insects that ingest these crystal proteins stop their feeding activity.
It is known to cause death after intestinal rupture. Bacillus thuringiensis strains are classified into 29 subspecies based on flagellar antigen, esterase activity, etc., and each strain exhibits specific and different insecticidal activities.

The present inventors have developed a known bacterium, Bacillus thuringiensis aizawai, which exhibits strong insecticidal activity against the diamondback moth and Spodoptera spp., which are pests of vegetables among lepidopteran insects.
With the aim of utilizing the insecticidal protein produced by the L strain as an insecticide, we conducted research and completed the present invention.

Means for Solving the Problem The present inventors cloned the gene for the insecticidal protein produced by Bacillus thuringiensis aizawai IPL strain, which exhibits strong insecticidal activity against diamondback moth and Spodoptera spp., and then cloned the structural gene portion of the gene. The entire sequence of 3465 bases was determined and the primary structure of the insecticidal protein was elucidated. Furthermore, by connecting the gene for this insecticidal protein to various expression vectors and introducing it into microorganisms, a microorganism that produces the insecticidal protein is produced, and by culturing this microorganism, Bacillus thuringiensis aizawai IPL We have completed a manufacturing method for producing large amounts of insecticidal protein from strains.

Bacillus thuringiensis aizawa rP of the present invention
The insecticidal protein gene of the L strain is specified by the base sequence and amino acid sequence shown in FIG.

A plasmid containing the insecticidal protein gene of the present invention can be obtained by constructing a gene library from the plasmid DNA of Bacillus thuringiensis aizawai IPL strain and using an appropriate probe, for example, the insecticidal protein gene of the present invention shown in FIG. Synthetic oligonucleotides created based on the base sequence of Bacillus thuringiensis, a related strain in Japan
I (by screening this library using a synthetic oligonucleotide prepared from the known base sequence of the insecticidal protein gene of the D-1 Guibel strain as a probe and isolating a plasmid containing the insecticidal protein gene of the present invention. In this case, the production of a recombinant plasmid containing the gene of the present invention can be advantageously carried out by selecting and using a strain exhibiting high insecticidal activity from the Bacillus thuringiensis Aizawai IPL strain. can.

As is well known, for many amino acids, there are multiple gene DNA base sequences encoding them.
The base sequence is not uniquely determined and there are many possibilities. In the case of the gene encoding the amino acid sequence of the insecticidal protein of Bacillus thuringiensis aizawai IPL strain, which was revealed by the present inventors, the DNA base sequence is numerous in addition to the DNA base sequence of the natural gene. However, the genetic DNA of the present invention has a natural D
It is not limited to only NA base sequences, but also includes other DNA base sequences encoding the amino acid sequences of insecticidal proteins revealed by the present invention.

Furthermore, with genetic recombination technology, mutations can be made artificially in specific parts of the basic DNA in a way that does not change the basic characteristics of what the DNA encodes or improves its characteristics. can be caused. Genetic DNA having a natural base sequence or a genetic DNA having a base sequence different from that of nature provided by the present invention
Similarly, A can be artificially inserted, deleted, or replaced to have characteristics equivalent to or improved from the natural gene, and the present invention also includes such mutant genes. It is something that

Bacillus thuringiensis aizawai I of the present invention
The insecticidal protein gene of the PL strain is expressed in a suitable expression vector, for example, the expression vector pUc carrying the Lac promoter.
18 (Pharmacia), an expression vector pKK223- containing the Tac promoter, which is a strong E. coli promoter, and the rrbB liposomal RNA terminator.
4. Expression vector p carrying the 'l'rp promoter
An expression vector that produces the insecticidal protein of Bacillus thuringiensis Aizawai IPL strain in microorganisms such as Escherichia coli by connecting to DR720 (Pharmacia), inducible expression vector pPL-Lambda (Pharmacia), etc. can be constructed.

The expression vector carrying the insecticidal protein gene of the present invention is used in Escherichia coli [for example, Escherichia coli JM103 strain (Pharmacia)].
By introducing the microorganism into a host microorganism such as the following, it is possible to obtain a microorganism that produces insecticidal proteins within its body.

The transformed microorganism produced in this way is placed in an appropriate medium,
It is possible to mass-produce insecticidal proteins by culturing under these conditions. In this case, the production of insecticidal proteins can be advantageously achieved by adding an inducer such as isopropylthiogalactoside at the early stage of culture.

The insecticidal protein can be isolated after culturing, for example, by crushing the bacterial cells with ultrasound and centrifuging them.
This can be done by easily concentrating and collecting the a aggregate.

In addition to the E. coli host-vector system, Bacillus subtilis,
Host-vector systems such as yeast, Shutomonas, and actinocontae can also be used, and insecticidal proteins can be mass-produced by taking advantage of the characteristics of each host-vector system.

The present invention will be explained in more detail with reference to Examples below. The present invention is not limited only to the following examples,
Modifications common to the art of the invention may be made.

Reference example Isolation of insecticidal protein genes Selection of Bacillus thuringiensis Aizawai IPLNaT strain showing high insecticidal activity against the Lepidoptera insects Diamondback moth and Spodoptera spp. Step 1 Purification of Bacillus thuringiensis Aizawai IPL strain Aizawai IPL strain [U Nis Department of Agriculture Research Service (U, S, D
equipment of agriculture
PY plate medium (10 g of tryptone (Sigma), 5 g of NaCl (Hanka Kagaku), 5 g of yeast extract (Sigma))
g, and 12 g of agar (Sigma) to make 11,
Purified bacterial strain 32 obtained by purification on the prepared agar medium)
Plasmid analysis was performed for each individual. l of each strain
After culturing for 24 hours in Qml of PY liquid medium (PY plate medium with agar removed), the bacteria were centrifuged (20,000
x g for 30 minutes) and Btrnboi
m and Doly (Nucl'etc Ac1ds R
Plasmid DNA was prepared according to the method of es・'L 1513-1523, et al., and plasmid analysis was performed by 0.8% agarose gel electrophoresis. As a result, the 32 purified strains analyzed did not show the same plasmid pattern, but could be classified into at least nine different plasmid patterns; 4. OMd, 4.8Md, 5.4M
d, 8.5Md, 12Md, 15Md, 17M
d, 21Md, 37Md, 40Md, 50Md,
The strain in which 12 52Md plasmid DNA bands were observed was named Aizawai IPLNaT strain.

Step 2 Selection and classification of Bacillus thuringiensis aizawai I P LN17 strains by insecticidal test
Each of the two purified Aizawai IPL strains was cultured on two PY plate media (15c+a diameter) at 30°C for 7 days, and after confirming sporulation and crystal formation using a phase-contrast i-microscope, the bacteria were harvested and subjected to freeze-thawing. After repeating this three times, the suspension was suspended in 2 ml of distilled water and subjected to ultrasonic crushing treatment to prepare a crystal Qi liquid. Stock solution of the obtained preparation solution, 10 times diluted solution,
After soaking 1 mJ of each 100-fold diluted solution into artificial feed, 10 4th instar Spodoptera were fed.
The number of dead animals was observed after 3 days. Similar tests were conducted on the diamondback moth using 3rd instar larvae. Purified strain Aizawai I
Crystal suspension of PLN17 strain (2.2
When 1 ml of 10' spores (containing 10' spores) was given to diamondback moths, l
10 out of 0 died, and when the crystal suspension (containing 2.6 x 10'' spores per ml) was fed to Spodoptera spp., 10 out of 10 died.

Therefore, it was revealed that the Aizawai IPLNIT strain exhibits high insecticidal activity against both pests.

Step 1 Preparation of synthetic NA probe Base sequence of insecticidal protein gene of Bacillus thuringiensis Christakii strain HD-IDipe1 (Whiteley et al.
J. Biol. Chem. 260.6264-72
(1985)) was synthesized with reference to NA probe (5
'-CAC 88 Heto CCAGCACCGGG-3')
was manufactured. Applied Biosystems' DNA
Synthesis After a DNA oligomer was synthesized using a synthetic demo model 380A, 1 ml of 27% ammonium hydroxide was added to a DNA 17i collection vial, heated at 55° C. for 4 hours, and dried in a concentrator. After drying, 100 μl of 0. OIM
Triethylamine-acetic acid (TEAA) (pH 7.2
), applied to a reverse phase HPLC column C18, and eluted with a solvent system of acetonitrile-0.1 MTEAA (pH 7.2). The absorption peak fraction of 260 no+ was collected, and after drying, 100 μl of 80% acetic acid prepared with acetonitrile was added and left for 15 minutes. After 15 minutes, when it becomes pale orange, dry it and add 0.01M
TEAA (p[I7.2) 100 /j l was added, and further 100 μl of ethyl acetate was added and mixed.

The ethyl acetate phase was discarded, 100 μl of diethyl ether was added, and the same operation was performed twice to dry up.

0, dissolved in OIM TEAA (all 7.2),
The 260rv absorption peak fraction was collected again by HPLC, and after drying, 10mM) lithium-hydrochloric acid (pH 7,5) +
Dissolved in 1 mM EDTA (TE) solution. Next, the prepared synthetic NA probe was labeled with stp.

5 μl of synthetic NA probe (approx.
? I Tris-HCl (pH 7,6), 0.1 MM
g Cl g 0.1M 2-mercaptoethanol) was added, and then distilled water was added to bring the total volume to 10
After reaction at 37° C. for 1 hour, chloroform:phenol (1:1) was added and mixed, and the supernatant was collected. The separated supernatant was applied to a DE-52 column (Whatman, 0.5 ml bed size) equilibrated with TEII street solution (pH 7,5), and 3 ml of TEII street solution (p
After washing with H7,5), 0.7M Na C1-TE
Elution was performed with loose cellulose solution (pH 7.5), a radioactive fraction was collected, and a 3tp-labeled synthetic NA probe was prepared.

Step 2 Plasmid DNA of Aizawai IPL strain 7
Preparation of A library Cells of Aizawai [PIllT strain] cultured in 200 m It of PY liquid medium were cultured in 30 m J! G buffer (10%
The cells were washed with glycerol, 1mM EDTA, 50mM) lis-hydrochloric acid (all 8.0), suspended in 3ml of lysozyme (5μ/mj!), and reacted at 30°C for 2 hours. Next, add an alkaline solution (0,2NN a OH
.

Add 16 ml of 1% sodium dodecyl sulfate (SDS), mix, and leave at room temperature for 10 minutes.
12ml of 3M sodium acetate (ptl 4.8)
The mixture was then left at 4° C. for 1.5 hours. Centrifugal operation (14,0
The supernatant was separated by 0 Orpm for 20 minutes, 79mj7 of cold ethanol was added, and the mixture was left to stand at -20°C for 1 hour, followed by ethanol precipitation to recover DNA.

After drying, suspend in 5 ml of 0.1M sodium acetate,
After treatment with phenol equilibrated with E buffer (pH 7.5) was performed twice, the supernatant was collected, 2 volumes of cold ethanol was added, and ethanol precipitation was performed again.

Furthermore, after drying, the plasmid DNA was suspended in 6 ml of TEi solution (pH 7.5) and purified by cesium chloride equilibrium density gradient centrifugation according to a conventional method.

Next, 5 μg of plasmid DNA was cut with 10 units of the restriction enzyme BamHI, and an equal volume of phenol:chloroform (1:1) was added and mixed. The supernatant was removed, and the DNA was recovered by ethanol precipitation and dried. After that, 20μl
of TE buffer (pH 7.5). Also, 2 μg
puca vector plasmid (Pharmacia)
After digestion with Unit's restriction enzyme 13 am HI, the DNA was recovered in the same manner and added to 20 μl of TE buffer (pH
7,5), 5 μl of E. coli alkaline phosphatase (Takara Shuzo, 2.0 units), 50 μl of 0.1 M
After adding 1-lis-hydrochloric acid buffer (pH 8.0) and 15 μl of distilled water, the mixture was incubated at 60° C. for 1 hour and subjected to alkaline forsterase treatment. After the reaction, phenol-chloroform treatment was performed twice, and the supernatant was collected.
Collect DNA by ethanol precipitation and add 20 μl of TE.
It was suspended in a buffer solution (pH 7.5).

Next, add 15μl of BamHI cut plasmid DNAΔ solution and L5ul of I3amHI3μpUC8 hector DN.
A? Yong? T4DNA'J of night kongo 1 barley, l, t+A
Gauze (Takara Shuzo, 0.1 unit), 7.5 μl of 0
．． 1M dithiothreitol (Gyudon Kagaku), 7.5 μl
10mM adenosine triphosphate (Gyudon Kagaku), 25μl
A 3-fold concentrated reaction mixture (200mM Tris-Salt H (pH
7,6) 20 mM g Cj! z) and 5μβ of distilled water to bring the total volume to 75μl, and incubate at 14°C for 6 hours.
Incubated for hours. Obtained gauze reaction solution 10
μl, the method of Skott et al. (Cell Engineering, Main, 616-6
26.1983) 100 μl of E. coli DHI
strain (ATCC33849), incubated at 0°C for 15 minutes, heat-treated at 42°C for 40 seconds, and added to 0.9ml of broth liquid medium (tryptone 10g, yeast extract 5g, NaCl).
Add 5 g of glucose (Gyudon Kagaku) Ig and bring the total volume to 11 with distilled water), incubate for 1 hour at 37 °C, and add L-Pross plate medium (L-Pross liquid medium) containing ampicillin at a final concentration of 50 μg/ml. (with agar added to give a concentration of 1.2%).

Step 3 Isolation of insecticidal protein gene clones by colony hybridization The colonies spread on the plate were replicated onto two nitrocellulose filters, and ampicillin (50 Mg
/ml) and chloramphenicol (600μg/ml
) and incubate overnight. 2.5ml of 0.5N N per filter
a Add OH, treat for 5 minutes, air dry, then add an equal volume of 1M
Tris-hydrochloric acid (pH 17.5) was added and left for 5 minutes. After air drying, add an additional 2.5 ml of IM) lithium-hydrochloric acid (pH
7,5) - treated with 1,5M Na C12 for 5 minutes,
After air drying, it was treated under vacuum at 80°C for 3 hours. 0.1% of 10mj+ per 4 filters 5DS-4xSS
C (SSC indicates 0.15M NaCl-0,015M sodium citrate (pH 7,5)) and 60
After processing for 15 minutes at °C, wipe off the colonies on the filter.
6 ml of prehybrid solution (4XSSC, 1Qx Denhart, 1100II/ml Salmon Testis 1
Soak in Hon@' (DNA (Sigma); however, 10x Denhart is a solution containing 0.2% Ficoll, 0.2%, polyvinylpyrrolidone, and 062% BSA), and
It was treated at ℃ for 3 hours. Next, the filter was placed in a hybrid solution in which the 32P-labeled synthetic NA probe prepared in step 1 was added to the above solution, and incubated overnight at 56°C. After hybridization, 4XS at 56°C.
After washing with SC-0, 1% SDS solution four times for 15 minutes, the filter was dried, placed on X-ray film, and subjected to autoradiography. Colonies giving a positive signal were picked from the master plate, and similar colony hybridization was performed again to isolate a positive clone E. coli DHI/pAB6.

Plasmid pAB6 was isolated from the isolated positive clone E. coli DHI/pAB6 according to the method of Birnboim and Doly et al. Plasmid pstl, B
glII, pvu ■, EcoRI, Yaham, K
The insert DNA was cut using pnl and C1al and analyzed by 0.7% agarose electrophoresis.
A 22.4-kb restriction enzyme map was created (upper part of Figure 3), and a Southern hybridization method using the synthetic NA probe described above (J, Mo1. Bi
In order to identify the insecticidal protein gene region in the insert DNA and create a detailed restriction enzyme map of that region, we used various restriction enzymes (lower part of Figure 3). After subcloning the cut DNA fragment into vector pUC8,
Plasmid DNA containing DNA fragments was prepared by the method of is+ and Doly et al.

The obtained DNA was suspended in 18 μl of TE (pf 17.5), 2 μl of 2N NaOH was added, the mixture was left at room temperature for 5 minutes, 8 μl of 7.5 M ammonium acetate was added, and 1
00 μl of cold ethanol was added to perform ethanol precipitation.

Centrifugation was performed at 12.00 rpm for 5 minutes, and the precipitate was collected and dried to yield 0.5 pn+ol/5 m j! It was dissolved in distilled water so that 5 μl of plasmid D prepared twice
NA (5 pmo I), 1.5 μβ of 10 times the concentration of Klenow buffer (Takara Shuzo), and 1 μl of primer DNA.
A (PL Biochemical Co., Ltd.), add 4.5 μl of distilled water to make a total volume of 12 μl, and after heating at 60°C for 15 minutes,
It was left at room temperature for 20 minutes. Add 2 μl of C4- to this reaction solution.
"r') ATP (400 Ci/vwol, Amar-Jam Japan) and 1 μl of Klenow fragment enzyme (Takara Shuzo, 2 units) were added and mixed.
．． Add 2 μl each of 4 types of 2 μp dNTP + ddN
It was added to a TP mixture (Takara Shuzo) and left at 42°C for 20 minutes. 1 μl of Chase solution (Takara Shuzo) was added to each, and the mixture was left at 42° C. for 20 minutes. Finally, 6 μl of 9
5% formamide dye (containing 0.1% bromophenol blue and 0.1% xylene cyatool) was added.

A 6% acrylamide/urea gel was prepared according to a conventional method,
Apply 2 μl of the above reaction solution and heat at 1700V for 6 hours.
Electrophoresis was performed. After drying the gel, it was placed between X-ray films and the base sequence was read after intense light exposure.

The insecticidal protein gene of the Bacillus thuringiensis Aizawai IPLIkT strain, whose elucidated base sequence is shown in Figure 2, begins with the start codon ATG and ends with the stop codon TA.
It has a coding region of 3465 bases ending with A, and 1
It encoded 155 amino acids.

Example Presentation Step 1: Preparation of Aham DNA Fragment Approximately 10 μg of recombinant plasmid pAB6 containing the insecticidal protein gene
Add about 30 units of the restriction enzyme Ahan to the
ttl Aham reaction solution (10mM Tris-HCl (pH
7,5), 60mM NaC1, 7mM MgCl1z,
3 in 10mM EDTA, 1mM dithiothreitol]
After reacting for 1 hour at 7°C, the reaction solution was applied to a 0.8% low melting point agarose gel (manufactured by Bethesda Research Laboratory) containing 0.1 μg/ml ethidium bromide, and agarose electrophoresis was performed. . After electrophoresis, the gel portion corresponding to the 3.5 Kb Ahal[l DNA fragment was cut out under an ultraviolet lamp, placed in a test tube, and heated at 65° C. for 5 minutes. Add twice the volume of TE11 buffer (lomM) to the melted gel.
) Lis-hydrochloric acid (pH 8°0), 0.5mM EDTA
), add phenol saturated with TE buffer,
Phenol extraction was performed. 10.000 rp-5
After centrifuging for a minute and separating the upper layer, 1/40! 4M of it
Add NaCl and 2 times the volume of ethanol and heat to -80°C.
After precipitating the DNA with ethanol for 10 minutes, the DNA was centrifuged at 10°00 rpm for 10 minutes.
was collected and suspended in 10 μl of distilled water.

Step 2: Preparation of vector Add 1 unit of restriction enzyme HincII (Takara Shuzo) to 1 μg of expression vector pUc18 (Pharmacia), add 20 pl of If inc fl reaction solution (10 m
M) Lis-hydrochloric acid (pH 7,4), 100mM Na Cj
! , 7 mM Mg C12, 10 mM dithiothreitol] at 37° C. for 1 hour. After the reaction, an equal volume of phenol/chloroform (l:1) mixture was added to the reaction solution, mixed, and after centrifugation at 10,000 rpm for 5 minutes, the supernatant was collected. Next, add twice the amount of cold ethanol,
After standing at -80℃ for 15 minutes, 10.000 rpv
The DNA was centrifuged for 10 minutes at A, and the DNA was collected and suspended in 10 μl of distilled water.

AhanlDN prepared in step 1 and step 2
1 μg each of A fragment and expression vector pUc18
Mix together, add 7.2 units of T4 DNA ligase (Takara Shuzo), and add 45μ! Glue gauze reaction solution (66mM
Tris-hydrochloric acid (pH 7,6).

The reaction was carried out at 16°C for 2 hours in 6.6mM MgClz, 10mM dithiothreitol, 1°0mM ATP). Thereafter, the reaction solution was transformed into Escherichia coli JM103 (Pharmacia) according to the method of Cohen et al.

The ampicillin-resistant colonies that appeared were cultured and Birn
Plasmid DNA was prepared according to the method of Boim et al.

Add 3 units of restriction enzyme Hindm to about 1 μg of plasmid DNA, and add Hindm [[reaction solution (
10mM) Lis-HCl (pl! 7.5), 60 m
M NaCl -10m M M g Cl z
1 mM 2-mercaptoethanol, 100 μg7
ml bovine blood (serum albumin) for 1 hour at 37°C and analyzed by agarose electrophoresis. From the results of agarose electrophoresis, the expression plasmid in which the insecticidal protein gene was connected in the forward direction to the Lac promoter of the expression vector was A
H8, and the expression plasmid connected in the opposite direction is called pAH
7 (see Figure 1) - Step 1: Ps tr - containing the insecticidal protein gene
Preparation of BamHI fragment Approximately 5 μg of expression plasmid pA containing the insecticidal protein gene
Approximately 20 units of restriction enzymes were added to H8! and about 2
Add 0 units of restriction enzyme BamHI and add 50ttll.
Pst1 reaction solution (10mM Tris-HCl (1) H7,
5), 50mM NaCj! , 10 m M M g C
l t , 1 m M 2-mercaptoethanol, 1
00 μg/m 12 bovine serum albumin] at 37°C.
After reacting for 1 hour, phenol saturated with 30 μl of TE loose solution was added to perform phenol extraction. 10.00
Centrifuge at 0 rpts for 5 minutes, separate the upper layer, and then divide into 1/40
4mM NaCl and 2 times the amount of ethanol were added, and the mixture was left at -80°C for 10 minutes.

After centrifugation at 10,000 rpm for 10 minutes, the DNA was collected, dried, and suspended in 20 μl of distilled water.

Step 2: Preparation of vector Add 0.1 unit of restriction enzyme BamHI to about 5 μg of expression vector pKK223-3 (manufactured by Pharmacia).
(Takara Shuzo) and BamHI reaction solution (10mMl-
Lis-hydrochloric acid (p) I8. O) ,? mMMgClz
, 100mM Nacj! , 2mM 2-/lucaptoethanol, o.i.% bovine serum silbumin] at 37°C.
The reaction took place for 15 minutes. The reaction solution was applied to a 0.8% low melting point agarose gel containing 0.1 μg/ml of ethidium bromide, and electrophoresis was performed.

After electrophoresis, a DNA molecule (4.6 Kb), which is estimated to have been cleaved at only one of the two 3 μm HI recognition sites held by pKK223-3, was cut out under an ultraviolet lamp, placed in a test tube, and incubated at 65°C. Heated for 5 minutes. Melt the gel, perform phenol extraction, and perform ethanol precipitation to extract DNA.
was collected and suspended in 20 μl of distilled water. - Prepared D
Four deoxyribonucleotides and 5 units of E. coli DNA polymerase I were added to the NA solution at a final concentration of 3 mM.
Add large fragment, 60μl)(indl
[[Reaction solution [10mM Tris-HCl (pH 7.5),
60mM Nacl, 10mM MgCj2z, 1m
M2-mercaptoethanol, 100 μg/m I
After reaction at 25° C. for 2 hours in bovine serum albumin], phenol extraction and ethanol precipitation were performed, and the DNA was recovered and suspended in 20 μl of distilled water. Approximately 1 μg of recovered D
Add 3 units of T4 DNA ligase (Takara Shuzo) to NA, and add 45 μl of glue gauze reaction solution (66 mM Tris-HCl (pH 7,6), 6.6 mM MgCf!z, 1
0mM dithiothreitol, l.

The reaction was carried out at 16° C. for 2 hours in OmMATP). after that,
The reaction solution was transformed into large intestine seedling JM103 strain according to the method of Cohen et al. The ampicillin-resistant colonies that appeared were cultured, and plasmid DNA was prepared according to the method of Birnboim et al. About 1 μg of plasmid DNA, 3
Unit's restriction enzyme Dandoko HI was added, and the mixture was reacted for 1 hour at 37°C in a 11mHI reaction solution, and analyzed by agarose electrophoresis. Among the plasmids analyzed, pKK223-3
A plasmid in which the BamHI site of the plasmid's multiple cloning site was conserved and the other BamH1 sites were deleted was selected and named pKK223-4. Next, about 5 μg of pKK223-4 DNA was added with 10 units of restriction enzyme Pstl and 10 units of restriction enzyme B.
amHI was added and 50 μl of PsLT reaction solution (10 mM
Tris-hydrochloric acid (pH 7,5), 50 mM Na
After reaction at 37°C for 1 hour in Cl, 10mM MgC1z + 1mM 2-mercaptoethanol, 100μg/m 1 bovine serum albumin], phenol extraction and ethanol precipitation were performed, and the DNA was collected and suspended in 20μl of distilled water. did.

Step 3: Construction of expression plasmid pTB1 Step l
Then, mix 1 gg each of the Pstl-BamHI cut DNA fragment prepared in step 2 and vector pkk223-4, add 7.2 units of T4 DNA ligase (Takara Shuzo), and add 45 μl of glue gauze reaction solution (66
mMl-Lis-HCl (pH 7,6), 6.6mM
g(1!z, 10mM dithiothreitol, 1
The reaction was carried out at 16° C. for 2 hours in OmMATP). The reaction solution was transformed into E. coli strain JM103 according to the method of Cohen et al. Cultivate the ampicillin-resistant colonies that appear,
Plasmid DNA was prepared. Approximately 1 μg of plasmid D
Add 3 units of restriction enzyme BamHr to NA and add 30
React in ttl BamHI reaction solution at 37°C for 1 hour,
Analyzed by agarose electrophoresis. Based on the results of agarose electrophoresis, a plasmid in which an insecticidal protein gene was connected downstream of the Tac promoter of the expression vector was selected, and this was designated as the expression plasmid pTB1 (see FIG. 1).

3. Each expression plasmid pAH? , pAH8 and pTBl were introduced into E. coli strain JM103 according to the method of Cohen et al.

The obtained E. coli recombinant JMI 03/pAH7゜JM
Identification and analysis of the insecticidal proteins of B. thurigensis, B. thurigensis, and Aizawai IPL strains produced by I03/pAH8 and JM103/pTB1 were performed as follows.

Each E. coli strain is cultured overnight in L-Pros liquid medium (10 g of tryptone (Difco), 5 g of NaCl', 5 g of yeast extract (Difco) dissolved in 11 parts of distilled water). An overnight culture of 1
Transferred to 0 ml of broth liquid medium, cultured at 37°C, and
When the value of D660nn+ reaches 0.1, the final concentration is 2m
Add M isopropylthiogalactoside. Furthermore, culture was continued at 37°C, and after 20 hours, 0.3 m
j! The bacteria were collected by centrifugation (3,00 rpm, 15 minutes), and 50 μl of sample buffer (62,
5mM Tris-HCl (pH 8,0), 2% (w/w)
Sodium dodecyl sulfate, 5% (v/v) 2-
Mercaptoethanol, 10% (w/v) glycerol, 0. After suspending in O1% bromophenol blue], 10
Heat treatment was performed at 0°C for 5 minutes, followed by the method of Laemn+li et al.
According to Nature 227.680-685),
5DS-polyacrylamide electrophoresis. After electrophoresis, the gel was stained with Coomassie Brilliant Blue, degassed and dried, and then fixed on a lip paper. As a result, an insecticidal protein band for the molecule ff1125 was detected in E. coli JM103 strain containing the expression plasmid, and this protein band showed specific cross-reactivity with anti-insecticidal protein antibody (IgG).

When each protein band on the gel was measured with a densitometer, E. coli JM103/pAH7, JM103/pA
H8 and JMI 03/pTB 1 produced insecticidal proteins of 1%, 8%, and 14% based on total bacterial protein, respectively (see Figure 4). Therefore, these E. coli recombinants are
Efficiently Bacillus thuringiensis aizawai I
It was confirmed that the PL strain produced insecticidal protein.

4. Preparation of insecticidal protein produced by Enterobacteriaceae. The cells were transferred to L Pross medium and cultured at 37°C, and when the OD660nm value reached 0.1, isopropylthiogalactoside was added at a final concentration of 2mM. Furthermore, culture was continued and 5 ml of the culture solution was taken out.
．． Centrifuge at 00 rpm for 15 minutes to collect bacteria, -8
It was frozen at 0°C. Next, after melting at room temperature and repeating this operation three times, it was suspended in 2 ml of TE buffer (10 mM), Lis-HCl (pH 7,5), and 1 mM EDTA).
Ultrasonication was performed three times for 30 seconds. Next, this crude extract was centrifuged at 7,000 rpm for 10 minutes, and the precipitate was collected. When this precipitated fraction was suspended in a sample buffer for electrophoresis and analyzed by 5DS-PAGE, approximately 80% of the proteins contained in this precipitated fraction were insecticidal proteins. Therefore, it has been revealed that insecticidal proteins can be easily and efficiently prepared by using the above preparation method. It has been confirmed that this preparation method is effective for large amounts of culture fluid.

[Brief explanation of drawings]

Figure 1 shows the expression plasmid pAH? , pAII8, and pTBl. black, vertical line,
Each box containing a horizontal line, white color and dot is
Insecticidal protein gene, Lac promoter, liposome R
NA terminator-1Tac promoter and 1ac
Showing the Z gene. ATG and TAA are the start and stop codons of the insecticidal protein gene, respectively. Ah, Kp. pv, Bm, Hc, Ps are restriction enzymes AhaI[I, MotoI, PvuIF, BamHI, Hin
c ■, and Pstl are shown. Figure 2 shows 3465 structural gene parts of insecticidal protein genes.
The entire base sequence consisting of bases is shown. The upper row shows the base sequence, and the lower row shows the amino acid sequence deduced from it. In the base sequence, the region from base number 1 to base number 3465 is the region that codes for the structural gene of the insecticidal protein gene. Figure 3 shows the insert DNA (22
, 4kb) showing the restriction enzyme map of Aizawai IPL.
The lower part of the figure shows the insecticidal protein gene region of the strain in a large frame.
Detailed restriction enzyme sites in the insecticidal protein gene region are shown. FIG. 4 shows the results of quantifying the expressed insecticidal protein using a densitometer. (1), (2), (31 are JM103/pAH?, JM103/pAI(8), respectively.
The insecticidal protein in the bacterial cell crude extract of JMI O3/pTB 1 was measured. The peak indicated by the arrow corresponds to the insecticidal protein band. 5. Written amendment to procedures subject to amendment (voluntary) April 15, 1986

Claims (12)

[Claims] (1) Bacillus thuringiensis aizawai IPL
An expression plasmid that contains the insecticidal protein gene of the strain and expresses it within the microorganism. (2) The plasmid according to claim 1, wherein the gene is specified by the amino acid sequence shown in FIG. (3) The plasmid according to claim 1, wherein the gene is specified by the base sequence shown in FIG. (4) The expression plasmid according to claim 1 named pAH7, pAH8 or pTB1 (5) Bacillus thuringiensis aizawai IPL
A microorganism that produces an insecticidal protein by being transformed with an expression plasmid that contains the insecticidal protein gene of the strain and expresses it within the microbial cell. (6) The microorganism according to claim 5, wherein the gene is specified by the amino acid sequence shown in FIG. (7) A microorganism according to claim 5, wherein the gene is specified by the base sequence shown in FIG. (8) The microorganism according to claim 5, which carries an expression plasmid named pAH7, pAH8, or pTB1. (9) Bacillus thuringiensis aizawai IPL
A method for producing an insecticidal protein, which comprises culturing a microorganism that is transformed with an expression plasmid that contains the insecticidal protein gene of a strain and expresses it within the microbial cell body, and that produces the insecticidal protein. (10) The production method according to claim 9, wherein the gene is specified by the amino acid sequence shown in FIG. (11) The manufacturing method according to claim 9, characterized in that the gene is specified by the base sequence shown in FIG. (12) The production method according to claim 9, which comprises culturing a microorganism carrying an expression plasmid named pAH7, pAH8, or pTB1.