KR920005972B1 - Gene for superoxide dismutase - Google Patents

Abstract

A superoxide dismutase (Cu, Zn-SOD) is prepd. by (a) cloning cDNA of superoxide dismutase (SOD) to obtain the natural DNA sequence and (b) chemical synthesizing and mutating the obtd. DNA to obtain the SOD having Nco I-, Xho I-, Sac II- and Sal I restriction site. A primer 1 contg. Nco I restriction site is composed of 5'-ATCCATGGCGACGAAGGCCGTG-3'. A primer 2 contg. Sal I restriction site is composed of 5'-AAGTCGACTTATTGGGCGATCCCAATTACA3'. A primer 3 contg. Xho I restriction site is composed of 5'-ACCGTGTTTT CTCGAGAGAGGATTAAAGTG-3'.

Description

Superoxide dismutase (Cu, Zn-SOD) gene and preparation method thereof

1 shows the cDNA base sequence of the superoxide disproportionase.

Figure 2a, 2b and 2c is a clone search process containing a superoxide dismutase (SOD) gene from the cDNA library of superoxide dismutase according to the present invention and restriction enzymes NcoI, XhoI, SacII in the superoxide dismutase , SpeI and Sal I site insertion process is shown.

Figure 3 shows the nucleotide sequence of the superoxide disproportionase gene modified for the purpose of gene design according to the present invention and for the mass expression of genes in yeast.

The present invention relates to a superoxide disproportionase (Cu, Zn-SOD) gene and a method for preparing the same, wherein cDNA cloning of superoxide disproportionase (SOD) and a large amount of superoxide disproportionase (SOD) in yeast The present invention relates to a superoxide disproportionase (SOD) gene, which has been redesigned for ease of expression and genetic manipulation, and a method for preparing the same.

Superoxides produced by biological reactions in vivo have their strong reactivity, causing mutations in nucleic acids that can lead to cancer or tissue destruction. Superoxide Dismutase (SOD) acts to protect the living body by converting these superoxides into oxygen and hydrogen peroxide under a catalyst. There are three kinds of superoxide disproportionases in the eukaryotic cell stream, and the cloned by the present invention is cDNA of Cu, Zn-SOD in the stromal.

Human Cu, Zn-SOD is the same non-covalent bond of two polyfabtides of approximately 19,000 Daltons consisting of 153 amino acids (Liema-Hurwitz, J. et al., Bioche m. Int. 3,107 (1981)) It is known that the protein can be usefully used for the treatment of various human diseases including rheumatism.

Conventionally, by using a superoxide disproportionase (SOD) extracted from bovine as a therapeutic agent, there was a problem that the antibody is generated or low activity, there is a disadvantage that the price is expensive due to the problem of mass production.

Therefore, the present inventors have tried to solve the above problems, first, in order to mass-produce the superoxide disproportionase (Cu, Zn-SOD) according to the genetic engineering method, the existing natural state superoxide disproportionase ( CDNA of SOD) was cloned to obtain a natural base sequence, and then supernatant containing restriction enzymes NcoI, XhoI, SacII, SpeI and Sal I recognition sites for ease of genetic manipulation using oligonucleotide chemical synthesis and local mutation methods. Oxide disproportion and enzyme (SOD) genes were prepared.

That is, the present invention obtains the natural nucleotide sequence of the superoxide disproportionase (SOD) from the cDNA library of human liver cells and then the Nco I recognition site at the amino terminus of the gene, at the carboxy terminus. Superoxide dismutase (SOD) genes designed for easy genetic modification by making a superoxide dismutase (SOD) gene having a Sal I recognition site and restriction enzymes XhoI, SacII and SpeI recognition sites in between Characterized in the manufacturing method.

The present invention will be described in detail as follows.

Known superoxide dismutase (SOD) to search for SOD cDNA clones in human hepatocyte cDNA libraries (Clontech Laboratories, Inc., 4055 Fabian Way, Palo Alto, CA 94303, USA, Catalog No. HL1001b) A synthetic probe was prepared by the method of Suggs et al. (PNAS, 78.6613 (1981)) using 9 amino acids of and then plaque hybridization method (Benton, WD & Davis, RW, Science, 196,180 (1977)). Superoxide dismutase (SOD) cDNA clones were selected.

Selected cDNA clones were identified by Southern blotting (Southern Blotting: Southern EM, J. Mo l. Biol. 98,503 (1975)), Sanger Diodeoxy Method (PNAS, 74,5463 (1977)). ) Confirmed the base sequence. The confirmed nucleotide sequence is shown in FIG.

In order to directly obtain a superoxide dismutase (SOD) gene having a restriction enzyme NcoI recognition site at the amino terminus and a SalI recognition site at the carboxy terminus from a lambda phage containing the SOD cDNA. A single stranded 27-mer primer 1 (5'-ATCCATGGCGACGAAGGCCGTG-3 ') having a recognition site and a single stranded 30-mer primer 2 (5'-AAGTCGACTTATTGGGCGATCCCAATTACA-3') having a SalI recognition site were synthesized SOD-hexane fragments with NcoI and SalI restriction enzyme recognition sites were amplified by Polymerase Chain Reaction (PCR) using lambda DNA containing SOD cDNA as a template (Figure 2 a). .

This nucleic acid fragment was linked to the 5'-terminus by a T-4 polynucleotide kinase, and then inserted into M13mp18 digested with SmaI to obtain a template for local mutation.

30-mer primer 3 (5'-ACCGTGTTTTCTCGAGAGAGAGGATTAAAGTG-3 ') containing restriction enzyme XhoI recognition site, 33-mer primer 4 (5'-CACACCATCTTTGTCCGCGGTCACATTACCCAA-3') and restriction enzyme SpeI with restriction enzyme SpeII recognition site XhoI, 270 base pairs, located at about 200 base pairs away from the amino acid terminal of the superoxide disproportionase (SOD) gene by local mutation method using 27-mer primer 5 (5'-TTCATGGACCACTAGTGTGCGGCCAAT-3 ') having a recognition site. SacII and SpeI recognition sites were made at about 350 base pairs away (Figure 2b).

As shown in FIG. 2, the process of preparing cDNA library, SOD-gene synthesis having restriction enzyme NcoI, SalI recognition site, and insertion of restriction enzyme XhoI, SacII, SpeI site into SOD gene is shown in FIG. The designed superoxide dismutase (SOD) gene base sequence and the anoic acid sequence with are shown in FIG.

Applicant named such microorganism (M13mp18-SOD) containing superoxide disproportionase (SOD) gene as Escherichia coli LUCK-SOD-lE, which was deposited on June 29, 1990 with the Korean spawn association. It was deposited with KFCC-10699.

Hereinafter, the present invention will be described in more detail with reference to Examples.

Example 1

Search for Superoxide Dismutase (SOD) from Human Hepatocyte cDNA Library

In order to screen phage lambdas with superoxide disproportionase (SOD) genes in commercial human hepatocyte cDNA libraries, we used plaque hybridization (Benton WD & Davis, RW, Science 196,180 (1977)). Was carried out. At this time, the probe is a gene synthesizer (Applied Biosystem model 380B, USA) and 9 peptides (Asp-Val-Ser-Ile-Glu-Asp) between the 96th and 104th amino acid disulfase (SOD) amino acids. Based on -Ser-Val-Ile) a mixed sequence oligonucleotide probe 27-mer (5'-GATGT (G / Y) TCTATCGAAGATGT (G / T) ATC-3 ') was synthesized. Phage Clarke, first screened, was screened second and third, and phages containing SOD genes were purely selected. The phages thus obtained were cultured, DNA was extracted, digested with restriction enzyme EcoRI, electrophoresis on 1% agarose gel, and confirmed by Southern blotting (Southem, E., J. Mol. Biol. 98,503 (1975)). , EcoRI fragment was isolated and cloned into M13mp18 to confirm the DNA sequence of the superoxide dismutase (SOD) gene by Sanger dideoxy method (PNAS 74,5463 (1977)) (Figure 1).

Example 2

Synthesis of Superoxide Dismutase (SOD) Gene with Restriction Enzymes NcoI and SalI Recognition Sites

To facilitate cloning of the superoxide dismutase (SOD) gene into a yeast expression vector, the amino terminus of the gene was designed to be Nco I recognition site and the carboxy terminus of SalI recognition site.

Oligonucleotide synthesizer (DNA Synthesizer, Applied Biosystem Inc., USA) is a single stranded 27-mer with primer 1 (5'-ATCCATGGCGACGAAGGCCGTG-3 ') containing Nco I recognition sites and a single stranded 30-mer with SalI recognition sites Primer 2 of (5'-AAGTCGACTTATTGGGCGATCCCAATTACA-3 ') was synthesized.

The product was dried by taking 1.0 OD ₂₆₀ (33 μg / ml), dissolved in 200 μl of distilled water, and used for the next PCR reaction. 10μg of phage nucleic acid having a SOD-gene obtained in Example 1, 10μl of 10 × Taq plymerase buffer solution (100mM Tris-HCl, pH8.3, 500mM KCl, 15ml of MgCl ₂ , 0.1% (W / Gelatin, 10 μl of dNTP'S mixed solution: (including 1.25 mM of dGTP, dATP, dTTP, dCTO), 5 μl of primer 1, 5 μl of primer 2, 68.5 μl of distilled water, 0.5 μl of Ampli Taq DNA polymerase (Perkin) (Elmer Cetus, USA) and mix well.

50 μl of mineral oil was added to prevent evaporation of the solution (Saiki R.K, et al., Science 239,487 (1988)).

The temperature circulator (DNA Thermocycler, Perkin-Elmer Cetus, USA) was used.The temperature cycle program was repeated 25 times at 94 ° C for 30 seconds: 50 ° C for 30 seconds: 72 ° C for 1 minute and finally at 10 ° C for 72 ° C. It was further reacted for a minute.

The amplified superoxide dismutase (SOD) gene nucleic acid (about 470 base pairs) was purified by l% agarose gel electrophoresis extraction method and used for the reaction (FIG. 2 a).

Mix equal amounts of M13mp18 RF-DNA digested with Sma I restriction enzyme and nucleic acid fragments of superoxide disproportionase (SOD) obtained above, and 2μ1 of 10 × ligase buffer solution (500mM Tris-HCI, pH 7.6, 100mM MgCl Distilled water was added to ₂ , 20 mM DTT, 10 mM ATP, 50 μg / ml BSA), 2 μl 10 mM ATP, 1 μl T4 DNA ligase (New England Biolabs, USA) to give a final volume of 20 μl.

M13mp18-SOD clones were selected by conjugation of RF-DNA obtained from white plaques with EcoRI, HindIII in a transformed Escherichia coli agar plate at 14 ° C. for one day.

Example 3

Insertion of restriction enzymes Xho I, Sal I and SpeI sites into the superoxide dismutase (SOD) gene and sequence identification

M13mp18-SOD vector DNA containing superoxide dismutase (SOD) DNA obtained in Example 2 was obtained from E. coli CJ236 (Bio-Rad's Mutagene in Vitro Mutagenesis Kit., Bio-Rad Lab, 1000 Alfred Nobel Dr., Hercules , CA 94547, USA), and transformed and cultured to obtain a single-stranded U-DNA of phage containing Uridine.

Meanwhile, 30-mer primer 3 (5'-ACCGTGTTTTCTCGAGAGAGGATTAAAGTG-3 ') including the Xho I recognition site at the 67,68th amino acid position, and 33-mer primer 4 having the SacII recognition site at the 88,89th amino acid position 5'-CACACCATCTTTGTCCGCGGTCACATTGCCCAA-3 ') and 27-mer primer 5 (5'-TTCATGGACCACTAGTGTGCGGCCAAT-3') having the SPE I recognition site at the 118th and 119th amino acid positions were synthesized using a DNA synthesizer, and the absorbance at 260 nm was 0.1. The resulting amount was dried, and phosphorylated at 37 ° C. for 30 minutes under kinase reaction conditions (50 mM Tris-HCl, pH 8.0.10 mM MgCl ₂ , 10 mM DTT, 1 mM ATP, 10 Units polynucleotide kinase).

Using the single-strand u-DNA (M13mp18-SOD) obtained as a template, the primers phosphorylated under annealing conditions (Annealing: 20mMTris-HCl, pH 7.5, 2mM MgCl ₂ , 50mM NaCl) were mixed to 10μl, and then the water was heated to 70 ° C. Put in and let cool for about 1 hour. 1 unit of T4 DNA polymerase was added thereto, followed by 500 μM of dNTP, lmM of ATP, 8 mM of MgCl ₂ , 3 mM of Tris-HCl (pH 7.4), 2 mM of DTT, and 50 mM of NaCl at 90 ° C. at 90 ° C. The reaction was carried out for a minute (Fig. 2b). This was transformed into JM 01 (ATCC 33876) and the nucleotide sequence was confirmed by Sanger's Dideoxy DNA Sequencing method.

The identified superoxide disproportionase (SOD) gene nucleotide sequence is shown in FIG.

As described above, the superoxide disproportionase (SOD) gene of the present invention is a gene that can be usefully used for mass production of superoxide disproportionase (SOD) according to genetic engineering methods, and the superoxide produced thereby Oxide disproportionase (SOD) is not a problem of producing antibodies or low activity when using a small oxide disproportionase (SOD) prepared in the conventional method as a therapeutic agent, as well as to make a safe product for human use, It will be possible to mass-produce by genetic reconstruction method, so that it can be used not only for the treatment of inflammation, but also for other applications such as the treatment of other heart diseases and respiratory diseases.

Claims (15)

Cloning the cDNA of natural superoxide dismutase (Cu, Zn-SOD) to obtain the natural sequence, and then restriction enzyme Nco I, to facilitate genetic manipulation using oligonucleotide chemical synthesis and local mutation methods. The superoxide dismutase (SOD) gene of the following nucleotide sequence which has Xho I, SacII SpeI, and Sal I recognition sites.

2. The superoxide dismutase (SOD) gene of claim 1, wherein the restriction enzyme NcoI recognition site is configured to have a gene at the amino terminus. The superoxide dismutase (SOD) gene of claim 1, wherein the restriction enzyme Sal I recognition site is configured to have at the carboxy terminus of the gene. 2. The superoxide dismutase (SOD) gene of claim 1, wherein the restriction enzymes Xho I, SacII and Spe I are configured to be in the middle of the gene. The superoxide dismutase (SOD) gene according to claim 1 or 2, wherein the primer 1 of the single-strand 27mer including the Nco I recognition site is composed of 5'-ATCCATGGCGACGAAGGCCGTG-3 '. The superoxide dismutase (SOD) gene according to claim 1 or 2, wherein the primer 2 of the single strand 30mer including Sal I recognition site is composed of 5'-AAGTCGACTTATTGGGCGATCCCAATTACA-3 '. The superoxide dismutase (SOD) according to claim 1 or 4, wherein the primer 3 of 30mer including the Xho I recognition site at the 67th and 68th amino acid positions is composed of 5'-ACCGTGTTTTCTCGAGAGAGAGGATTAAAGTG-3 '. gene. The superoxide dismutase (SOD) gene of claim 1 or 4, wherein the primer 4 of 33mer having the SacII recognition site at the 88,89th amino acid position is composed of 5'-CACACCATCTTTGTCCGCGGTCACATTGCCCAA-3 '. The superoxide dismutase (SOD) gene according to claim 1 or 4, wherein the primer 5 of 27mer having the Spe I recognition site at the 118,119th amino acid position is composed of 5'-TTCATGGAUUAUTAGTGTGCGGCAAT-3 '. The natural sequence of the superoxide disproportionase (SOD) was obtained from the cDNA library of human hepatocytes, and then the Nco I cognitive site at the amino terminus, and the carboxy terminus, from the lambda phage containing the superoxide dismutase (SOD) cDNA. In order to directly obtain a superoxide dismutase (SOD) gene having a Sal I recognition site, a single strand 27-mer primer 1 having a Nco I recognition site and a single strand 30-mer primer 2 having a Sal I recognition site were used. SOD-nucleic acid fragments having Nco I and Sal I restriction enzyme recognition sites were obtained through the synthesis and reaction of polymerase chains based on lambda DNA containing superoxide disproportionase (SOD) cDNA. Phosphorylated 5'-terminus with T4 polynucleotide kinase, and then inserted into M13mp18 cleaved with Sma1 to obtain a template for local mutation, which was then identified as restriction enzyme Xho I. Prepared to have each recognition site by local mutation method using primer 3 of 30-mer and primer 4 of 33-mer having restriction enzyme SacII recognition site and primer 5 of 27-mer having Spe I recognition site including the above Method for producing a superoxide disenzyme (SOD) gene, characterized in that. The method of claim 10, wherein primer 1 is composed of 5′-ATCCATGGCGACGAAGGCCGTG-3 ′. The method of claim 10, wherein primer 2 is composed of 5′-AAGTCGACTTATTGGGCGATCCCAATTACA-3 ′. The method of claim 10, wherein primer 3 is composed of 5′-ACCGTGTTTTCTCGAGAGAGGATTAAAGTG-3 ′. The method of claim 10, wherein primer 4 is composed of 5′-CACACCATCTTTGTCCGCGGTCACATTGCCCAA-3 ′. The method of claim 10, wherein the primer 5 is composed of 5′-TTCATGGAUUAUTAGTGTGCGGCCAAT-3 ′.

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