JP2599111B2 - Polydeoxyribonucleotides for expression of human gamma interferon-like polypeptide - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention
Gamma interferon-like polypeptide (hereinafter γ-I
FN).

The chemically synthesized DNA molecules disclosed herein have substantially the same amino acid sequence and composition as human gamma interferon and have the immunological or biological activity of human gamma interferon. It is characterized by a DNA sequence that encodes the polypeptide.

[0003]

2. Description of the Related Art Interferon induces antiviral activity in cells and is said to exhibit antitumor properties, and is classified into three groups, alpha, beta and gamma.

For γ-IFN, Messenger R
It has been reported that γ-IFN is produced by a genetic engineering technique using a gene produced by reverse transcriptase from NA (Nature 295 , 503, 198).
2) has a drawback that the codon suitable for the host cell cannot be selected because the gene sequence is defined by the messenger RNA.

[0005]

It is considered that the above-mentioned drawbacks can be overcome by chemically synthesizing the γ-IFN gene, and a chimeric polypeptide can be produced by adding a restriction enzyme cleavage site arbitrarily. However, since the gene is composed of several hundred base pairs, synthesis is not easy.

[0006]

Means for Solving the Problems The present inventors have solved this difficulty and succeeded in synthesizing the γ-IFN gene, and have pioneered a method for producing γ-IFN using this gene by a genetic engineering technique.

[0007] The present invention relates to a polydeoxyribonucleotide having the following nucleotide sequence: 1 Cys Tyr Cys Gln Asp Pro Tyr Val Lys TGC TAC TGC CAG GAC CCA TAC GTG AAG ACG ATG AGC TGC GTC GTC ATC GTC ATC GTC ATC tyr Phe GAA GCT GAA AAC CTG AAG AAA TAC TTC CTT CGA CTT TTG GAC TTC TTT ATG AAG 20 Asn Ala Gly His Ser Asp Val Ala Asp AAC GCT GGT CAT TCT GAC GTT GCT GAC TTG CGA CCA GTA AGA CTG CAA CGA CTG 30 Asn Gly Thr Leu Phe Leu Gly Ile Leu AAC GGT ACT CTG TTC CTG GGT ATC CTG TTG CCA TGA GAC AAG GAC CCA TAG GAC 40 Lys Asn Trp Lys Glu Glu Ser Asp Arg AAA AAC TGG AAA GAA GAA TCT GAC CGT TTT TTG ACC TTT CTT CTT AGA CTG GCA 50 Lys Ile Met Gln Ser Gln Ile Val Ser AAA ATC ATG CAG TCT CAG ATC GTT TCT TTT TAG TAC GTC AGA GTC TAG CAA AGA 60 Phe Tyr Phe Lys Leu Phe Lys Asn Phe TTC TAC TTC AAG CTG TTC AAA AAC TTC AAG ATG AAG TTC GAC AAG TTT TTG AAG 70 Lys Asp Asp Gln Ser Ile Gln Lys Ser AAG GAC GAC CAG TCT ATC CAG AAA TCT TTC CTG CTG GTC AGA TAG GTC TTT AGA 80 Val Glu Thr Ile Lys Glu Asp Met Asn GTT GAA ACT ATC AAG GAA GAC ATG AAC CAA CTT TGA TAG TTC CTT CTG TAC TTG 90 Val Lys Phe Phe Asn Ser Asn Lys Lys GTT AAG TTC TTC AAC TCT AAC AAG AAA CAA TTC AAG AAG TTG AGA TTG TTC TTT Lys Arg Asp Asp Phe Glu Lys Leu Thr AAG CGT GA GAC TTC GAA AAG CTT ACT TTC GCA CTG CTG AAG CTT TTC GAA TGA 100 Asn Tyr Ser Val Thr Asp Leu Asn Val AAC TAC TCT GTT ACT GAC CTT AAT GTA TTG ATG AGA CAA TGA CTG GAA TTA CAT 110 Gln Arg Lys Ala Ile His Glu Leu Ile CAG CGT AAA GCT ATC CAT GAA CTG ATC GTC GCA TTT CGA TAG GTA CTT GAC TAG 120 Gln Val Met Ala Glu Leu Ser Pro Ala CAG GTT ATG GCT GAA CTG TCC CCG GCT GTC CAA TAC CGA CTT GAC AGG GGC CGA 13 Ala Lys Thr Gly Lys Arg Lys Arg Ser GCT AAA ACT GGT AAG CGT AAA AGA TCT CGA TTT TGA CCA TTC GCA TTT TCT AGA 140 Gln Met Leu Phe Arg Gly Arg Arg Ala CAG ATG CTG TTC CGT GGT CGT CGT GCT GTC TAC GAC AAG The present invention relates to GCA CCA GCA GCA CGA 146 Ser Gln TCT CAG AGA GTC and a plasmid incorporating the same, and a strain belonging to Escherichia coli transformed with the plasmid.

A preferred example of the above polydeoxyribonucleotide has a sequence encoding the amino acid sequence of 1-146 shown in FIG. 2 and has a translation initiation code such as ATG at the 5'-terminal and a 3'-terminal Those having a translation termination code such as TAA are mentioned.

For convenience of genetic manipulation, it is preferable to add a nucleotide sequence which can be cleaved by a restriction enzyme to each end of the translation initiation and termination codes. 'A nucleotide sequence to be cleaved with SalI was added to the terminal side.

The above polydeoxyribonucleotide is
For example, they can be synthesized as follows. First, the 62 gene fragments (oligodeoxyribonucleotides) shown in FIG. 1 were synthesized using the modified triester method and the solid phase method known per se, and then these fragments were ligated using T4 ligase to complete the fragment. Let it be the γ-IFN gene (see FIG. 2). This synthetic gene contains a restriction enzyme cleavage site as shown in FIG. This is to facilitate the search for the active site structure and the construction of chimeric genes.

Next, the above gene is inserted into a vector plasmid. An example of the operation is as follows. First, a plasmid pKO70 in which the promoter region (9 g base pairs) of the lactose operon of Escherichia coli was inserted into the EcoRI and ClaI cleavage sites of pBR322.
3 and the synthetic gene having an EcoRI site at the 5 ′ end and a SalI site at the 3 ′ end shown in FIG. 2 inserted into the EcoRI and SalI cleavage sites of the plasmid to construct a plasmid pGIF (FIG. 9). reference).

If desired, a plasmid in which a gene other than the γ-IFN gene is inserted into the above-mentioned cleavage site is constructed, and a host cell transformed with this plasmid is cultured, whereby the product of the gene can be obtained under the control of the lactose promoter. Advantageously, it can be produced.

As a host cell, a strain belonging to Escherichia coli is used. Using the plasmid pGIF obtained as described above, a strain of the genus Escherichia is transformed. When Escherichia coli WA802 is used as a strain, a transformed strain WA802 / pGIF is obtained. The strain has ampicillin resistance (Amp ^r ) and tetracycline sensitivity (Tet ^s ). Can be more separated.

When this transformant is induced and cultured in a medium such as L-broth using isopropyl-β-D-thiogalactopyranoside, an interferon active substance is produced in the cells obtained.

In addition, Escherichia coli WA802 / p
GIF4 is deposited with the identification label of SBMG105 at the Institute of Microbial Industry, National Institute of Advanced Industrial Science and Technology, and has the accession number No. 6522 of the microbe lab.

[0016]

EXAMPLES The embodiments of the present invention will be described below in the form of examples. Escherichia coli is described as Escherichia coli.

EXAMPLE Chemical Synthesis of γ-IFN Gene Fragment The chemical synthesis of 62 oligodeoxyribonucleotides, designated F _{1 to} F ₆₂ , respectively, shown in FIG. Is Ken-ichi M
The solid-phase method reported by Iyoshi et al. [Ken-
Ichi Miyoshi et al (1980) N
ucl. Acids Res. 8 5507] was used.

The outline of the chemical synthesis of oligodeoxyribonucleotides according to this improved technique is as follows. a) First, as shown in FIG. 3, the procedure was started by immobilizing a 3 ′ terminal mononucleoside and a unit on an aminomethylated polystyrene resin. That is, a resin crosslinked with 1% divinylbenzene was used as the aminomethylated polystyrene resin shown in FIG. 3, and a resin in which two β-alanines were linked to this resin was synthesized. This resin has three types (B is thymine,
N-benzoylcytosine or N-isobutyrylguanine) to act on the 3 '
The terminal nucleoside was immobilized on the resin. A polystyrene resin was obtained.

An example of the method for synthesizing this resin is shown in FIG.
The case of isobutyrylguanine is as follows. Aminomethylated polystyrene.HCl (divinylbenzene 1%, 100-200 mesh, NH ₂ : 0.
63 mmole / g) 20 g of the resin was added to CH ₂ Cl ₂ 100
After swelling well with mL, Nt-BOC-β-alanine (1.34 g, 15.1 mmole) was added, and DCC was added.
(3.1 l, 15.1 mmole) for 3 hours at room temperature. After filtering the resin with a glass filter, CH
₂ Cl ₂ twice with 200 ml, DMF 150 ml three times,
Then wash three times with 100 ml of pyridine. Next, unreacted amino groups were acetylated by treating the resin with 100 ml of 25% acetic anhydride-pyridine for 1 hour. Removing the t-BOC group by treatment at room temperature for 30 minutes the following resin _{20% CF 3 COOH-CH 2} Cl 2 120ml, then the resin was washed with 5% triethylamine -CH ₂ Cl ₂ 200ml, further CH ₂ Cl _{2 The} resin was washed four times with 100 ml. By returning again repeated the same operation, the resin i.e. in Fig 3 2, beta-alanine to aminomethylated polystyrene resin was obtained two linked resin. 10 g of the resin obtained here (FIG. 3) was dissolved in 60 ml of DMF, and the compound of FIG. 3 (B is N-isobutyrylguanine)
11 mmole and 1 ml of triethylamine were added and room temperature 1
Shake for 2 hours. After the reaction, the resin was filtered off, washed with DMF and then with pyridine, and then treated with 150 ml of 10% phenyl isocyanatopyridine for 3 hours to protect the unreacted amino groups.
After washing with methanol and drying under reduced pressure in the presence of P ₂ O ₅ , the resin in FIG. 3 (B is N-isobutyrylguanine) was obtained.

A portion of the resin obtained here (FIG. 3, B is N-isobutyrylguanine) was J. Gait et al. [M. J. Gait et al, (1980) Nu
cl. Acids Res. 8 1081] Quantitative determination of the amount of 3 'end Nukureosaido immobilized on resin 1g was used was 0.12 mmole. Resin (B is thymine or N-benzoylcytosine) was also produced in the same manner as in the above synthesis method. The amount of the 3'-terminal nucleoside immobilized on 1 g of the resin was 0.126 mmole when B was thymine, and 0.125 mmole when B was N-benzoylcytosine. b) The three types of resins obtained here and the 3'-phosphoric diester dimer block shown in FIG. 4 (0 in FIG. 4n) and the 3'-phosphoric diester trimer block (1 in FIG. 4n).
6 consisting of F _{1 to} F ₆₂ having a fixed base sequence
Two oligodeoxyribonucleotides were synthesized. That is, a 10% trichloroacetic acid (hereinafter abbreviated as TCA) -CH
After removing the DMTr group by treating with Cl ₃ ,
3 'nucleoside unit 1 immobilized on resin
4 equivalents of 3 'phosphoric acid diester dimer (0 in FIG. 4n) or 3' phosphoric acid diester trimer (FIG.
n is 1) 3 equivalents were sequentially condensed using 20 to 50 equivalents of a condensing agent mesitylenesulfonyltetrazolide (MsTe). Excess 3 'phosphoric diester block and condensing agent
The reaction mixture can be easily removed by filtration with a glass filter.

Next, in order to protect the unreacted 5 'hydroxyl group, the resin is treated with 10% acetic anhydride-pyridine for 1 hour at room temperature, or the resin is treated with 10 ml of 10% acetic anhydride-pyridine in 10 ml.
The unreacted 5 'hydroxyl group was acetylated by treating with a solution containing 20 mg of dimethylaminopyridine (DMAP) at room temperature for 30 minutes. Next, the 5 ′ terminal DMTr group was removed by treating the resin with 10% TCA, and the following condensation reaction was performed. This operation was repeated until the desired length was obtained.

[0022] a follows To illustrate this series of operations for the synthesis of F ₁₆ fragments. Figure 3 200 mg of resin (B is N-isobutyrylguanine) was added to 10% TCA
The resin was treated with 20 ml of -CHCl ₃ for 30 seconds three times to obtain a resin from which the DMTr group had been removed. This resin has a 3 ′ phosphate dimer block, CA, (FIG. 4, n =
0, B ₁ is N- benzoyl cytosine, B ₃ after was repeated N- benzoyl adenine) 96Myumole was added pyridine 3ml azeotroped three times, Kusase's Ken pyridine 2 ml, 2 hours at room temperature using a condensing agent MsTe 1 mmole A condensation reaction was performed. After the reaction, excess 3 'phosphoric acid dimer and condensing agent can be easily removed by filtering the reaction mixture using a glass filter and thoroughly washing the resulting resin with pyridine. Next, in order to protect the unreacted 5 'hydroxyl group, the resin was suspended in 20 ml of 10% acetic anhydride-pyridine and shaken at room temperature for 1 hour. After the reaction, the resin is filtered off using a glass filter, and washed thoroughly with pyridine and then with CHCl ₃ . Next, the resin was added to 20% of 10% TCA-CHCl _3.
For 30 seconds for 3 times to remove the DMTr group from the resin. The resin was then washed well with CHCl ₃ followed by pyridine, and the block condensation was repeated in the same manner until the desired length was obtained. That is, in order to synthesize the oligodeoxyribonucleotide of F-16, the following 3 ′ phosphate dimer block, CC, TA, GA, T
C, and the condensation reaction is repeated to obtain F-1
A completely protected oligodeoxyribonucleotide resin having a base sequence of 6 was obtained. Similarly, the other fragment F
₁ to F ₁₅ and F ₁₇ each fragment to F ₆₂ also other changing the combination of the nucleotides were prepared using the same way as described above.

C) The oligodeoxyribonucleotide completely protected with a certain base sequence obtained in this manner is cleaved from the resin and completely deprotected. The resin is reacted with concentrated aqueous ammonia-pyridine (2: 1 v / v) for 55 minutes. Obtained by treating at room temperature for 10 hours followed by 80% acetic acid for 10 minutes at room temperature (method A). However, the fragment, F ₂ ,
_{F 3, F 5, F 7} ~F 10, F 22 ~F 24, F 26, F 29, F
_{30, F 33 ~F 42, F} 53 ~F 55, F 57 ~F 62, could not be obtained in this deprotection conditions. Therefore, these fragments were used for the method of Reese et al. [C. B. R
eese, et al, (1981), Nucl. Aci
ds Res. 9 , 4611] (Method B).

These deprotection reactions were carried out using fragment F
If Write down the ₁₆ and F ₂₆ are as follows.

Method A: 50 mg of fragment F ₁₆ resin
In a sealed tube with 1 ml of pyridine and 2 ml of concentrated ammonia water.
After shaking at 10 ° C. for 10 hours, the resin is separated by filtration, and the filtrate is concentrated under reduced pressure. Toluene is removed by adding toluene to the obtained residue and repeating azeotropic distillation. Next, 80% acetic acid 2 was added to the residue.
The resulting mixture was treated at room temperature for 10 minutes to obtain oligodeoxyribonucleotides completely deprotected.

[0026] Method B: resin 50mg of fragments F ₂₆
Was treated with 0.3 M 1,1,4,4, -tetramethylguanidinium 2-pyridinealdoximate dissolved in 3.5 ml of dioxane-water (7: 1 v / v) at 40 ° C. for 48 hours, Concentrated aqueous ammonia-pyridine (2: 1 v /
v) Treated with 3 ml in a sealed tube at 55 ° C. for 10 hours. Subsequently, treatment with 2 ml of 80% acetic acid at room temperature for 20 minutes gave oligodeoxyribonucleotides completely deprotected.

D) Separation and purification of the final product were performed using high performance liquid chromatography (HPLC). This HPLC uses an ALTEX model 110A liquid chromatograph, and uses a solvent A (20% CH ₃ CN-1m
MKH ₂ PO ₄ , pH 6.5) and solvent B (20% CH
Partisil 10 S using a linear concentration gradient method with ₃ CN-500m MKH ₂ PO ₄ , pH 6.5).
AX (Whatman) column (φ4.6 × 250m
The compound was separated and purified in m). Starting with solvent A, 1
A gradient was added by adding 3% solvent B every minute. Elution was performed at 58 ° C. at a flow rate of 2 ml per minute.
The target peak was collected, desalted by dialysis, and freeze-dried. The homogeneity of the thus obtained 62 completely deprotected oligodeoxyribonucleotides F _{1 to} F ₆₂ was determined by labeling the 5 ′ end with [γ- ³² P] ATP using T4 polynucleotide kinase. After that, it was checked by electrophoresis on a 20% polyacrylamide gel. The base sequence of each oligodeoxyribonucleotide is T4 After labeling the 5 'end with [γ- ³² P] ATP using polynucleotide kinase, the partially hydrolyzed product using nuclease was subjected to pH 3.5 electrophoresis using cellulose acetate and DEA.
It was confirmed by a two-dimensional mapping method obtained by performing homochromatography using E-cellulose [Bambar
a, J. et al. E. FIG. et al (1974) Nucl. Aci
des. Res. 1 881].

Ligase of γ-IFN gene fragment
® down chemically synthesized the 62 oligodeoxyribonucleotide fragments (1-62) and corresponding to the gamma-IFN consisting 450 base pairs according ligation plan shown in FIG. 5 gene (Figure 2) was ligated to. The details will be described below.

[A] to [F] blocks were synthesized. The fragment (1,1) at the 5 'end of each block
4,15,24,25,32,33,44,45,5
(4, 55, 62), 1 nmol of each of the remaining 50 fragments was dissolved in 22 μl of distilled water, and dissolved at 90 ° C. for 2 minutes.
After heating for 1 minute, it was immediately cooled to 0 ° C. 5μ in this aqueous solution
[Γ- ³² P] ATP of Ci (5100 Ci / mmol)
Was added, and the solution was treated with 10 mM 70 mM Tris-HCl (pH 7.5).
Adjust to be mM MgCl ₂ kinase buffer,
Add units of polynucleotide kinase and add total volume of 30μ
l. The 5 'end was labeled with ³² P by reacting at 37 ° C. for 15 minutes. Next, 4 nmol of ATP and 3 units of polynucleotide kinase were added in order to phosphorylate all 5 'ends, and the mixture was reacted at 37 ° C. for 45 minutes.
The reaction was stopped by heating at 5 ° C. for 5 minutes. Phosphorylated 2 to 13 above and unphosphorylated 1 and 1
4 (A block), phosphorylated 16-23 and non-phosphorylated 15 and 24 (B block), phosphorylated 26-31 and non-phosphorylated 25 and 32
(C block), phosphorylated 34 to 43 and unphosphorylated 33 and 44 (D block), phosphorylated 46 to 53 and unphosphorylated 45 and 54
(E block), 2.1 μg each of phosphorylated 56 to 61 and non-phosphorylated 55 and 62 (F block) were mixed, placed in a dialysis tube, and dialyzed against distilled water at 4 ° C. for 16 hours. Reaction ATP and kinase buffer components were removed. The dialyzed DN of each of the A to F blocks
The A solution was concentrated to dryness and 14.5 μl of ligase buffer (20 mM Tris-HCl, pH 7.6, 10 mM MgC
l ₂ ).

This solution was placed in a 40 μl glass capillary tube, which was sealed at both ends, then placed in a 16.5 mm test tube containing 20 ml of water, and heated at 95 ° C. for 2 minutes. Next, the whole test tube was left at room temperature for 2 hours, and the DNA fragment was annealed and then placed at 0 ° C. The DNA solution was taken out from the glass tube, and a ligase buffer solution containing 0.4 mM ATP and 10 mM DTT was added. 30 units of T4 DNA ligase was added thereto, and the total volume was 80 μl, and the mixture was reacted at 15 ° C. for 16 hours. A part of the reaction solution was taken out, subjected to 14% polyacrylamide gel electrophoresis containing 7 M urea, and examined for ligation by radioautograph. As a result, the maximum size of the ligation reaction product was equal to the size of the target product of 6 blocks from A to F, respectively. Next, the entire amount of the ligation reaction was separated by 14% polyacrylamide gel electrophoresis containing 7 M urea. After confirming the position of each of A to F by radioautograph, the gel piece of that part was cut out, put into a dialysis tube, and 9 mM
After filling with Tris boric acid (pH 8.3, containing 0.25 mM EDTA) buffer (elution buffer), binding both ends of the tube, the gel was electrophoresed in the elution buffer at a constant voltage of 200 V for 3 hours. The DNA in was eluted.
Next, the DNA was taken out of the dialysis tube and freeze-dried. Dry the product with 100 μl of 0.3 M sodium acetate (p
H7.0), add 2 volumes of ethanol, and add -80 ° C.
After 30 minutes, the DNA was recovered as a precipitate by centrifugation. 20 μl of DNA precipitates from blocks A to F
After dissolving in 1 ml of distilled water, 1 μl of the solution was
It was examined by polyacrylamide electrophoresis. As a result, FIG.
As shown in FIG. 0, D of each block from [A] to [F]
An almost uniform band corresponding to the size of NA was obtained.
The DNA solution of each of the blocks A to F was adjusted to have the composition of the kinase buffer, 2 nmol ATP and 6 units of polynucleotide kinase were added, and the mixture was reacted at 37 ° C. for 1 hour to phosphorylate the 5 ′ end of each block. [A],
[B], [C] 20 pmol of each DNA of each block were mixed, and adjusted so as to have a ligase buffer composition.
ATP and D to give M ATP, 10 mM DTT
Add TT to bring the total volume to 50 μl and add 8 units of T4DN
The reaction was performed at 15 ° C. for 16 hours using A ligase.
Similarly, 5 pmol of each of the DNAs of the blocks [D], [E], and [F] was mixed and ligated at 15 ° C. for 16 hours. After completion of the reaction, a part of each reaction product was taken, and the ligation was examined by 8% polyacrylamide gel electrophoresis containing 7 M urea.

As a result, as shown in FIG.
It was found that [I] and [II] blocks corresponding to the above were synthesized. Next, the entire amount of the reaction solution was separated by 8% polyacrylamide gel electrophoresis containing 7 M urea. After confirming the DNA of 225 base pairs by radioautograph, a gel piece of that portion was cut out, and the DNA was eluted by the above-mentioned electrophoretic elution method. After freeze-drying the DNA solution, the DNA solution was dissolved in 100 μl of a 0.3 M sodium acetate (pH 7.0) solution, and then 2.5 times the amount of ethanol was added to precipitate the DNA. To the obtained DNA, 0.5 nmol ATP and 3 units of polynucleotide kinase were added in the same manner as described above, and reacted at 37 ° C. for 1 hour. [I]
[II] 1 pmol of the DNA of each block was mixed and adjusted so as to have a ligase buffer composition, and 0.4 mM ATP,
Add ATP and DTT to 10 mM DTT,
The reaction was carried out at 15 ° C. for 16 hours using 5 units of T4 DNA ligase in a total volume of 20 μl. After completion of the reaction, a part of the reaction product was taken and examined for ligation by 6% polyacrylamide gel electrophoresis containing 7 M urea. FIG. 12 shows the result. In the figure, I and II indicate the separated and purified [I] and [II] blocks, respectively.

G shows the result of ligation of all the γ-IFN genes, and DN corresponds to 450 base pairs.
It turned out that A molecule was obtained.

Next, 1/10 amount of 3
DNA was recovered as a precipitate by adding M sodium acetate and adding 2.5 volumes of ethanol. 10 precipitates
0 μl of TA buffer (33 mM Tris acetic acid, pH 7.
9,66 mM potassium acetate, 10 mM magnesium acetate,
0.5 mM DTT) and 20 units of restriction enzyme E
coRI, 20 units of restriction enzyme SalI were added, and 37 ° C.
For 1 hour. DNA was recovered as a precipitate by adding 10 μl of 3M sodium acetate to the reaction solution and adding 2.5-fold amount of ethanol. DNA was dissolved in a solution containing 80% formamide, 10 mM NaOH, and 1 mM EDTA, and separated by 6% polyacrylamide gel electrophoresis containing 7 M urea. After confirming 450 base pairs by radioautograph, a gel piece of the portion was cut out, placed in a dialysis tube, and filled with an elution buffer,
After sealing, the DNA in the gel was eluted by electrophoresis in the same buffer at a constant voltage of 200 V for 3 hours. Next, the DNA solution was taken out of the dialysis tube and freeze-dried. The dried product was washed with 100 µl of 0.3 M sodium acetate (p.
H7.0), add 2.5 volumes of ethanol and dissolve in D
NA was recovered as a precipitate. About 0.05 pmol of γ-
The IFN gene was obtained. This DNA was dissolved in 10 μl of a kinase buffer, 2 units of polynucleotide kinase and 0.1 nmol ATP were added, and the mixture was reacted at 37 ° C. for 1 hour to obtain a DNA for transformation.

In order to express the chemically synthesized γ-IFN gene as a complete protein in Escherichia coli, γ-IFN was inserted downstream of the UV5 promoter operator region of the lactose operon of Escherichia coli.
An E. coli plasmid pKO703 into which the N gene can be inserted was constructed, and the γ-IFN gene was cloned into this pKO703 plasmid.

The construction of a series of plasmids used in this experiment will be described below in detail (see FIG. 9).

[0036] (1) pKO703 plasmid the Construction of plasmid PKO703 including UV5 promoter operator region of the Construction of E. coli lactose operon was performed by the following method.

(A) Cut restriction enzymes EcoRI and ClaI
Preparation of pBR322 plasmid filled with a cleavage site 1.5 μg of pBR322 DNA in 1 × T in 20 μl
A reaction solution (33 mM Tris acetate buffer pH 7.6, 66
Restriction enzyme EcoR in mM potassium acetate, 10 mM magnesium acetate and 0.5 mM dithiothreitol).
Cleavage was performed at 37 ° C. for 60 minutes using 5 units each of I and ClaI. The reaction solution was heated at 65 ° C. for 30 minutes to inactivate the enzyme.
A was obtained as a precipitate. The obtained DNA precipitate is subjected to T4D
After suspending in 40 μl of NA polymerase reaction solution,
After heating at 10 ° C. for 10 minutes, 5 μl each of 4 mM solution of dATP, dCTP, and dGTP and 5 μl of 100 mM β-mercaptoethanol were added, and the mixture was reacted at 37 ° C. for 60 minutes using 1 unit of T4 DNA polymerase. The reaction solution was heated at 65 ° C. for 30 minutes to inactivate the enzyme, followed by ethanol precipitation, and finally the DNA precipitate was dissolved in 15 μl of distilled water.

(B) Restriction site for restriction enzyme EcoRI
Meta LacUV5 promoter operating record over Tar region DNA
The following experiment was performed to separate the UV5 promoter operator region of the lactose operon. Plasmid p
KO601 is a plasmid having a lactose operon promoter operator region between two restriction enzyme EcoRI cleavage sites. This plasmid pKO601
80 μg was reacted at 37 ° C. for 60 minutes in a 1 × TA reaction solution in 500 μl using 150 units of a restriction enzyme EcoRI. The reaction solution was subjected to 10% polyacrylamide gel electrophoresis, and a 95 bp promoter operator region DN having a restriction enzyme EcoRI adhesion site cohesive end was used.
The fragment A was separated from the gel by electrophoresis (for the separation method, see the example of Japanese Patent Application No. 56-163303).

After ethanol precipitation, 40 μl of the DNA precipitate was
Dissolved in 40 μl of T4 DNA polymerase reaction solution of
After heating at 5 ° C for 30 minutes, it is cooled rapidly, and dATP, dTTP, dC
5 μl each from 4 mM solution of TP and dGTP and 1
After adding 5 μl of 00 mM β-mercaptoethanol,
37 ° C., 60 units using 10 units of T4 DNA polymerase.
Allowed to react for minutes. The reaction solution was heated at 65 ° C. for 30 minutes to inactivate the enzyme, and then subjected to ethanol precipitation.
The DNA was dissolved in 1 liter of distilled water.

(C) The process adjusted in (A) and (B)
Rasmid and lactose UV5 promoter operators
Ligation of region DNA and transformation of Escherichia coli (A) with 0.8 μg of restriction enzyme EcoRI and pBR322 DNA filled with ClaI cleavage site, and 0.8 μg of UV5 promoter operator region DNA of lactose operator prepared with (B), Ligation was performed at 16 ° C. for 20 hours using 2.5 units of T4 DNA ligase. (The ligation method is described in Japanese Patent Application No. 56-1633.
The method described in Example No. 03 was followed. )

After ligation, ethanol precipitation was performed and DN
A precipitate was dissolved in 10 μl of distilled water, and E. coli 12 · W was dissolved.
A802 was transformed. (The method of transformation was in accordance with the method described in Examples of Japanese Patent Application No. 56-163303.) The transformed strain was an M9S-Xgal medium [5 g of NaCl, NH ₄ containing 1 μg of 40 μg of ampicillin].
_{Cl1g, Na 2 HPO 4 5.94g,} KH 2 PO 4 3
g (pH 7.0) MgSO ₄ 0.2 g, casamino acid 2
g, glucose 5 g, 5-bromo-4-chloro-3-indoyl-β-D-galactopyranoside 400 mg, agar 15 g, water 1000 ml: hereinafter abbreviated as M9S-Xgal medium), and ampicillin-resistant and concentrated. Select 10 blue colonies, WA802 / pKO701-WA8
02 / pKO710.

Plasmid pBR32 of the restriction enzyme ClaI
2 is within the promoter region of the tetracycline resistance gene, and if another DNA fragment is inserted between the EcoRI-ClaI restriction sites of the pBR322 plasmid, E. coli harboring the plasmid becomes tetracycline sensitive. However, the lactose operon U
If the DNA fragment of the V5 promoter operator region is inserted in a desired orientation, the transformant having the plasmid can be expected to be tetracycline-insensitive.

The thus obtained WA802 / pKO701
~ WA802 / pKO710 strain with tetracycline 10
A single colony was formed on a normal nutrient agar medium containing μg / ml, and tetracycline sensitivity was examined. As a result, WA802 / pKO701 and WA802 / pKO70
3, WA802 / pKO706, WA802 / pKO7
09 were tetracycline insensitive. Therefore, plasmid DNA was isolated from these strains and 2 μg of DNA was isolated.
In a 20 μl 1 × TA reaction solution with the restriction enzyme EcoR
Reaction was carried out at 37 ° C. for 60 minutes using 5 units each of I and HpaII, and the orientation of the promoter operator region of the lactose operon was examined by 10% polyacrylamide gel electrophoresis. As a result, WA802 / pKO703, W
A802 / pKO709 had the promoter operator region inserted in the desired orientation.

This pKO703 plasmid was transformed into γ-IFN
Used for gene cloning. Plasmid pKO70
No. 3 has an EcoRI restriction enzyme recognition site at one position downstream in the direction of transcription from the promoter, and pB
Since the tetracycline resistance gene of R322 remains on the plasmid as it is, a gene having a cohesive end of a restriction enzyme, such as EcoRI and SalI or BamHl, which cleaves a tetracycline resistance structural gene, obtained chemically or from the plasmid, is ligated using a ligation reaction. hand,
It can be easily introduced into the plasmid pKO703, and the target clone can be easily selected as an ampicillin-resistant tetracycline-sensitive bacterium.

[0045] (2) limit the 15μg of Construction plasmid pKO703 of plasmid pGIF ₄ enzyme EcoR
30 μl of 100 mM using 10 units each of I and SalI
The reaction was performed at 37 ° C. for 60 minutes in a solution of 5 mM MgCl ₂ and 50 mM NaCl in a Tris-HCl buffer (pH 7.3). The reaction solution was heated at 65 ° C. for 30 minutes to inactivate the enzyme, and then subjected to 0.7% agarose gel electrophoresis to 3.8 kb.
Was separated from the gel by electrophoresis.
After ethanol precipitation, the DNA was dissolved in 4.5 μl of distilled water. Restriction enzyme E of pKO703 thus obtained
0.3 μg of the coRI and SalI restriction enzyme fragment DNA and 29 ng of the γ-IFN gene phosphorylated at the 5′-OH end obtained above were dissolved in 24 μl of a T4 DNA ligase reaction solution, heated at 65 ° C. for 10 minutes, and cooled on ice. 100 mM DTT
Add 3 μl and 3 μl of 4 mM ATP then add T4D
Ligation was performed at 16 ° C. for 20 hours using 3 units of NA ligase. A 2.5-fold amount of cold ethanol was added to the ligation reaction solution to convert the DNA into an ethanol precipitate. Then, 10 μl of distilled water was added to the obtained DNA precipitate, and E. coli W
A802 was transformed. The transformant was cultured on an M9S-Xgal medium containing 40 μg / ml of ampicillin, an ampicillin-resistant and dark blue colony was selected, and a single colony was formed on a normal nutrient agar medium containing 10 μg / ml of tetracycline, and the resulting mixture was sensitized to tetracycline sensitivity. Is selected and WA802 / pGIF1
It was named WA802 / pGIF5.

After separating the plasmid DNA, the restriction enzyme Ec
oRI and SalI, EcoRI and HindIII, and E
Plasmid DNA is cleaved with coRI and BglII, and the size of the DNA fragment is measured by agarose electrophoresis or polyacrylamide gel electrophoresis, and WA802 in which the desired γ-IFN gene has been cloned.
/ PGIF4, WA802 / pGIF5. In these pGIF4 and pGIF5 plasmids, Shine-D of the UV5 promoter region of the lactose operon was used.
γ 10 base pairs downstream from the algano DNA base sequence
-The start codon ATG of the IFN gene is located.

[0047]

[Table 3]

Determination of base sequence of γ-IFN gene 450 bp γ-IF obtained by digesting pGIF4 plasmid DNA with restriction enzymes EcoRI and SalI
The nucleotide sequence of the region corresponding to the N gene was determined according to the plan shown in FIG. The determination of the base sequence was performed using Maxam-Gi
lbert method (Proc. Nat. Acad. Sci.
USA. 74, 560-564, 1977). As a result, it was confirmed that all 450 base pairs were in the original design (FIG. 2).

(3) Human gamma interferon
And identification of E. coli-like polypeptide in E. coli E. coli WA802 / pGIF4 and WA802
In / pGIF5, the γ-IFN gene is inserted downstream of the promoter region of the lactose operon.
Therefore, γ-IFN is expected to be expressed under the control of the lactose operon. Therefore, E. coli is obtained by the method described in detail below. coliWA802 / pGIF4, WA80
The expression and fixation of γ-IFN in 2 / pGIF5 cells were performed. Strain E. E. coli WA802 / pGIF4 and W
A802 / pGIF5 was added to M9S-glycerol medium containing 40 μg / ml of ampicillin (NaCl 5 g, NH
_{4 Cl 1g, Na 2 HPO 4} 5.94g, KH 2 PO
_{4 3g, MgSO 4 · 7H 2} O0.2g, glycerol 2.5g, casamino acid 2g, water 1000 ml) 2.0 m
1 and inoculated at 37 ° C. overnight. This culture solution was inoculated into four 5 ml M9S-glycerol mediums each containing 40 μg / ml of ampicillin so that the absorbance OD 660 nm became 0.1, followed by culturing at 37 ° C.
When 60 nm reached 0.8, the lactose operon inducer isopropyl-β-D-thio-galactopyranoside (hereinafter abbreviated as IPTG) was added to four culture tubes so that the final concentration was 1 mM. 37 ° C,
Further culturing was performed for 2.5 hours. The remaining two were further cultured at 37 ° C. for 2.5 hours without adding IPTG. After 5 ml of these cultures were centrifuged at 3,000 rpm for 10 minutes, the cells were obtained as a precipitate. 1 × containing 1 mg / ml bovine serum albumin in the two cell pellets with and without IPTG
PBS (10 mM phosphate buffer pH 7.0, 150 mM
NaCl) was added, and the cells were sonicated. Also, 1 × PBS 0.5% containing 1 mg / ml egg white lysozyme was added to the two cell precipitates with and without IPTG.
After lysozyme treatment at 0 ° C. for 40 minutes, the mixture was sonicated. 0.5 ml of the sonicated cells was added for 25,000
Centrifugation was performed at 0 rpm for 30 minutes at 4 ° C., and the supernatant was used for measuring the activity of γ-IFN.

[0050]

[Table 2] Measurement of interferon activity 5 on Coaster plastic 96-well multiplate
The interferon samples were multipipetted into dilution series using Eagle's minimal medium containing 5% newborn calf serum. Next, 100 μl of FL cells (5 × 10
⁵ cells / ml) and cultured overnight at 37 ° C. under 5% CO ₂ . The culture supernatant was discarded, and 100 μl of Sindbis virus having a concentration 100 times higher than TCID ₅₀ was added to each well.
The cells were cultured overnight at 5 ° C. and 5% CO ₂ . After about 17 hours, the supernatant was discarded and stained with a 1% crystal purple solution containing 20% ethanol. The titer of the interferon was determined by setting the dilution of the interferon sample that showed half the difference in staining between the wells to which no virus was added and the wells to which only the virus was added without adding the interferon as the lab unit / ml. . This assay system is 7 to 25 times more sensitive than α-interferon with international standards.

Whether the interferon activity is indeed human gamma interferon activity depends on the anti-human
It was confirmed by an immunological technique using a gamma interferon antibody. In this case, 10 μl of anti-human gamma interferon antibody (having an activity of inactivating human gamma interferon activity of 2,000 units μl / ml) is added to a 50 μl interferon activity sample.
Was left at room temperature for 3 hours, followed by centrifugation (10,000 rotations, 5 minutes), and the supernatant was used as a sample.

[0052]

[Table 1] As shown in Table 1, the transformed E. coli obtained in the present invention was used. E. coli WA802 / pGIF4 and WA802
/ PGIF5 was clearly found to produce a human gamma interferon-like substance under the control of the lactose operon, indicating that non-transformed bacteria (WA802 / pKO7
No. 03) showed no human gamma interferon activity.

Ultracentrifuged supernatant of cell disruption of Escherichia coli showing interferon activity in the above example was subjected to Ultragel ACA54 (0.7 cm in diameter × 47 in height).
cm), and the molecular weight was estimated by gel permeation chromatography. Using a physiologically balanced salt solution (1 × PBS), fractionation was performed in 0.7 ml portions. As a standard protein, ribonuclease A (molecular weight 13,70
0), chymotrypsinogen A (molecular weight 25,000)
0), ovalbumin (molecular weight 43,000) was used. Interferon activity is determined by chymotrypsinogen A
And ovalbumin. Under this condition, the main peak of interferon activity has a molecular weight of about 30,000.
(See FIG. 13).

[Brief description of the drawings]

FIG. 1 shows the sequence of each of 62 oligodeoxyribonucleotides used for γ-IFN gene synthesis.

FIG. 2 shows the amino acid sequence of synthetic γ-IFN and the nucleotide sequence of the gene.

FIG. 3 shows an oligodeoxyribonucleotide synthesis reaction step.

FIG. 4 shows a synthesis reaction step of an oligodeoxyribonucleotide.

FIG. 5 shows an oligodeoxyribonucleotide ligation scheme.

FIG. 6 shows a scheme for determining the nucleotide sequence of a synthetic γ-IFN gene.

FIG. 7 is a restriction map of a synthetic γ-IFN gene, and the numbers in parentheses indicate distances expressed in base pairs with the EcoRI recognition cleavage site as a starting point.

FIG. 8 is a restriction map of plasmid pKO703, in which the numbers in parentheses indicate the distance expressed in base pairs from the EcoRI recognition cleavage site as a starting point.

FIG. 9 shows a construction step of a pGIF plasmid.

FIG. 10. Synthesized polydeoxyribonucleotide A,
B, C, D, E, F block and molecular weight marker M
(Φ × 174RF decomposed with HinfI) 14
2 shows mapping by% polyacrylamide gel electrophoresis.

11] [I] and [I] ligated according to FIG.
I] Each block and molecular weight marker M (φ × 174R
8 shows the mapping of F obtained by decomposing F with HpaII) by 8% polyacrylamide gel electrophoresis.

FIG. 12 shows the above [I] and [II] blocks and the composite γ
-IFN gene and molecular weight marker M (pBR322
3 shows the mapping of DNA (decomposed with HpaII) by 6% polyacrylamide gel electrophoresis.

FIG. 13 is a graph of the molecular weight of each fraction obtained by gel filtration of the supernatant of a centrifuged E. coli coli cell showing interferon activity.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical display location C12R 1:19) (C12P 21/02 C12R 1:19)

Claims (11)

(57) [Claims] 1. A polydeoxyribonucleotide having the following nucleotide sequence: 1 Cys Tyr Cys Gln Asp Pro Tyr Val Lys TGC TAC TGC CAG GAC CCA TAC GTG AAG ACG ATG ACG GTC CTG GGT ATG CAC TTC 10 Glu Ala Glu Asn Leu Lys Lys Tyr Phe GAA GCT GAA AAC CTG AAG AAA TAC TTC CTT CGA CTT TTG GAC TTC TTT ATG AAG 20 Asn Ala Gly His Ser Asp Val Ala Asp AAC GCT GGT CAT TCT GAC GTT GCT GAC TTG CGA CCA GTA AGA CTG CAA CGA CTG 30 Asn Gly Thr Leu Phe Leu Gly Ile Leu AAC GGT ACT CTG TTC CTG GGT A C CTG TTG CCA TGA GAC AAG GAC CCA TAG GAC 40 Lys Asn Trp Lys Glu Glu Ser Asp Arg AAA AAC TGG AAA GAA GAA TCT GAC CGT TTT TTG ACC TTT CTT CTT AGA CTG GCA 50 Lys Ile Met Gln Ser Gln Ile Val Ser AAA ATC ATG CAG TCT CAG ATC GTT TCT TTT TAG TAC GTC AGA GTC TAG CAA AGA 60 Phe Tyr Phe Lys Leu Phe Lys Asn Phe TTC TAC TTC AAG CTG TTC AAA AAC TTC AAG ATG AAG TTC GAC AAG TTT TTG AAG 70 Lys Asp Asp Gln Ser Il Gln Lys Ser AAG GAC GAC CAG TCT ATC CAG AAA TCT TTC CTG CTG GTC AGA TAG GTC TTT AGA 80 Val Glu Thr Ile Lys Glu Asp Met Asn GTT GAA ACT ATC AAG GAA GAC ATG AAC CAA CTT TGA TAG TTC CTT CTG TAC TTG 90 Val Lys Phe Phe Asn Ser Asn Lys Lys GTT AAG TTC TTC AAC TCT AAC AAG AAA CAA TTC AAG AAG TTG AGA TTG TTC TTT Lys Arg Asp Asp Phe Glu Lys Leu Thr AAG CGT GAC GAC TTC GAA AAG CTT ACT TTC GCA CTG CTG AAG C TT TTC GAA TGA 100 Asn Tyr Ser Val Thr Asp Leu Asn Val AAC TAC TCT GTT ACT GAC CTT AAT GTA TTG ATG AGA CAA TGA CTG GAA TTA CAT 110 Gln Arg Lys Ala Ile His Glu Leu Ile CAG CGT AAA GCT ATC CAT GAA CTG ATC GTC GCA TTT CGA TAG GTA CTT GAC TAG 120 Gln Val Met Ala Glu Leu Ser Pro Ala CAG GTT ATG GCT GAA CTG TCC CCG GCT GTC CAA TAC CGA CTT GAC AGG GGC CGA 130 Ala Lys Thr Gly Lys Arg Lys Arg Ser GCT AAA A CT GGT AAG CGT AAA AGA TCT CGA TTT TGA CCA TTC GCA TTT TCT AGA 140 Gln Met Leu Phe Arg Gly Arg Arg Ala CAG ATG CTG TTC CGT GGT CGT CGT GCT GTC TAC GAC AAG GCA CCA GCA GCA CGA 146 Ser Gln TCT CAG AGA GTC 2. The polydeoxyribonucleotide according to claim 1, which has a translation initiation code at the 5 'end and a translation termination code at the 3' end. 3. A nucleotide sequence which is cleaved by a restriction enzyme is added to both ends, and preferably, the 5 'end is EcoR
3. The polydeoxyribonucleotide according to claim 1, wherein the I and 3 'ends are cleaved with SalI. 4. A polydeoxyribonucleotide having the following nucleotide sequence: 1 Cys Tyr Cys Gln Asp Pro Tyr Val Lys TGC TAC TGC CAG GAC CCA TAC GTG AAG ACG ATG ACG TGA TCG TGC GTC GTC GTC ATC GTC ATC tyr Phe GAA GCT GAA AAC CTG AAG AAA TAC TTC CTT CGA CTT TTG GAC TTC TTT ATG AAG 20 Asn Ala Gly His Ser Asp Val Ala Asp AAC GCT GGT CAT TCT GAC GTT GCT GAC TTG CGA CCA GTA AGA CTG CAA CGA CTG 30 Asn Gly Thr Leu Phe Le Gly Ile Leu AAC GGT ACT CTG TTC CTG GGT ATC CTG TTG CCA TGA GAC AAG GAC CCA TAG GAC 40 Lys Asn Trp Lys Glu Glu Ser Asp Arg AAA AAC TGG AAA GAA GAA TCT GAC CGT TTT TTG ACC TTT CTT CTT AGA CTG GCA 50 Lys Ile Met Gln Ser Gln Ile Val Ser AAA ATC ATG CAG TCT CAG ATC GTT TCT TTT TAG TAC GTC AGA GTC TAG CAA AGA 60 Phe Tyr Phe Lys Leu Phe Lys Asn Phe TTC TAC TTC AAG CTG TTC AAA AAC TTC AAG ATG AAG TTC GA AAG TTT TTG AAG 70 Lys Asp Asp Gln Ser Ile Gln Lys Ser AAG GAC GAC CAG TCT ATC CAG AAA TCT TTC CTG CTG GTC AGA TAG GTC TTT AGA 80 Val Glu Thr Ile Lys Glu Asp Met Asn GTT GAA ACT ATC AAG GAA GAC ATG AAC CAA CTT TGA TAG TTC CTT CTG TAC TTG 90 Val Lys Phe Phe Asn Ser Asn Lys Lys GTT AAG TTC TTC AAC TCT AAC AAG AAA CAA TTC AAG AAG TTG AGA TTG TTC TTT Lys Arg Asp Asp Phe Glu Lys Leu Thr AAG CGT GAC GA C TTC GAA AAG CTT ACT TTC GCA CTG CTG AAG CTT TTC GAA TGA 100 Asn Tyr Ser Val Thr Asp Leu Asn Val AAC TAC TCT GTT ACT GAC CTT AAT GTA TTG ATG AGA CAA TGA CTG GAA TTA CAT 110 Gln Arg Lys Ala Ile His Glu Leu Ile CAG CGT AAA GCT ATC CAT GAA CTG ATC GTC GCA TTT CGA TAG GTA CTT GAC TAG 120 Gln Val Met Ala Glu Leu Ser Pro Ala CAG GTT ATG GCT GAA CTG TCC CCG GCT GTC CAA TAC CGA CTT GAC AGG GGC CGA 130 Al a Lys Thr Gly Lys Arg Lys Arg Ser GCT AAA ACT GGT AAG CGT AAA AGA TCT CGA TTT TGA CCA TTC GCA TTT TCT AGA 140 Gln Met Leu Phe Arg Gly Arg Arg Ala CAG ATG CTG TTC CGT GGT CGT CGT GCT GTC TAC GAC AAG A plasmid in which GCA CCA GCA GCA CGA 146 Ser Gln TCT CAG AGA GTC has been incorporated. 5. A polydeoxyribonucleotide having EcoRI at the 5 'end and a SalI cleavage site at the 3' end, and EcoRI and Sal of plasmid pKO703.
5. The plasmid of claim 4 integrated between the I cuts. 6. The plasmid according to claim 5, comprising pGIF. 7. The plasmid according to claim 6, comprising pGIF4. 8. A polydeoxyribonucleotide having the following nucleotide sequence: 1 Cys Tyr Cys Gln Asp Pro Tyr Val Lys TGC TAC TGC CAG GAC CCA TAC GTG AAG ACG ATG ACG TGC GTC GTC GTC GTC GTC GTC GTC GTC ATC tyr Phe GAA GCT GAA AAC CTG AAG AAA TAC TTC CTT CGA CTT TTG GAC TTC TTT ATG AAG 20 Asn Ala Gly His Ser Asp Val Ala Asp AAC GCT GGT CAT TCT GAC GTT GCT GAC TTG CGA CCA GTA AGA CTG CAA CGA CTG 30 Asn Gly Thr Leu Phe Le Gly Ile Leu AAC GGT ACT CTG TTC CTG GGT ATC CTG TTG CCA TGA GAC AAG GAC CCA TAG GAC 40 Lys Asn Trp Lys Glu Glu Ser Asp Arg AAA AAC TGG AAA GAA GAA TCT GAC CGT TTT TTG ACC TTT CTT CTT AGA CTG GCA 50 Lys Ile Met Gln Ser Gln Ile Val Ser AAA ATC ATG CAG TCT CAG ATC GTT TCT TTT TAG TAC GTC AGA GTC TAG CAA AGA 60 Phe Tyr Phe Lys Leu Phe Lys Asn Phe TTC TAC TTC AAG CTG TTC AAA AAC TTC AAG ATG AAG TTC GA AAG TTT TTG AAG 70 Lys Asp Asp Gln Ser Ile Gln Lys Ser AAG GAC GAC CAG TCT ATC CAG AAA TCT TTC CTG CTG GTC AGA TAG GTC TTT AGA 80 Val Glu Thr Ile Lys Glu Asp Met Asn GTT GAA ACT ATC AAG GAA GAC ATG AAC CAA CTT TGA TAG TTC CTT CTG TAC TTG 90 Val Lys Phe Phe Asn Ser Asn Lys Lys GTT AAG TTC TTC AAC TCT AAC AAG AAA CAA TTC AAG AAG TTG AGA TTG TTC TTT Lys Arg Asp Asp Phe Glu Lys Leu Thr AAG CGT GAC GA C TTC GAA AAG CTT ACT TTC GCA CTG CTG AAG CTT TTC GAA TGA 100 Asn Tyr Ser Val Thr Asp Leu Asn Val AAC TAC TCT GTT ACT GAC CTT AAT GTA TTG ATG AGA CAA TGA CTG GAA TTA CAT 110 Gln Arg Lys Ala Ile His Glu Leu Ile CAG CGT AAA GCT ATC CAT GAA CTG ATC GTC GCA TTT CGA TAG GTA CTT GAC TAG 120 Gln Val Met Ala Glu Leu Ser Pro Ala CAG GTT ATG GCT GAA CTG TCC CCG GCT GTC CAA TAC CGA CTT GAC AGG GGC CGA 130 Al a Lys Thr Gly Lys Arg Lys Arg Ser GCT AAA ACT GGT AAG CGT AAA AGA TCT CGA TTT TGA CCA TTC GCA TTT TCT AGA 140 Gln Met Leu Phe Arg Gly Arg Arg Ala CAG ATG CTG TTC CGT GGT CGT CGT GCT GTC TAC GAC AAG A strain belonging to Escherichia coli transformed with a plasmid into which GCA CCA GCA GCA CGA 146 Ser Gln TCT CAG AGA GTC has been incorporated. 9. The polydeoxy according to claim 8, wherein the plasmid is a plasmid.
EcoRI at the 5 'end of the siribonucleotide and 3' at the
A SalI cleavage site was added and plasmid pKO703 was added.
The plasmid integrated between the EcoRI and SalI cuts
9. The strain according to claim 8, which is a sumid. 10. Escherichia coli K12 WA80
The strain according to claim 9, comprising 2 / pGIF. 11. Escherichia coli K12 WA80
The strain of claim 10, comprising 2 / pGIF4.