JPS61139390A - Novel dna to code human sod and bacterium having same - Google Patents

Abstract

PURPOSE:To mass produce human derived SOD, by transforming Escherichia coli with a plasmid integrated with complementary DNA by the use of mRNA from human placenta, cultivating Escherichia coli to give a mold, and isolating DNA from the mold. CONSTITUTION:mRNA is separated from human placenta, and a plasmid integrated with complementary DNA is obtained by Okayama-Berg method, etc. by using this mRNA. Escherichia coli is transformed with the plasmid, Escherichia coli is cultivated to give a mold, from which plasmid (DNA) containing the aimed base arrangement is isolated. Then, optionally DNA containing the aimed base arrangement is taken out, and connected to the downstream of a promoter of a vector, to give a vector plasmid containing the base arrangement.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention is directed to human superoxide dismutase (5up
Eroxide DisrnuLase is hereinafter referred to as 80D. ) and a microorganism transformed with the DNA.

[Prior art]

SOD in 1939, M;1nn and Kci l in
K, j: This is the first discovered enzyme metal oxygen in which two molecules of a polypeptide with a molecular weight of approximately 16,000 are associated with active oxygen mutagenic effects.

SOD is an inflammation that is thought to occur due to tissue damage due to superoquinde ion generated from oxygen molecules in the body, such as osteoarthritis 5 and chronic rheumatoid arthritis. It is attracting attention as a therapeutic agent.

and J abusch et al. [Biochem, i9
．． 2310-2316 (1980)] determined the amino acid sequence of SOD isolated and purified from red blood cells.

Also, Sherman et al. CProc, Natl, A.
cad, sci.

80, 5465-5469 (1983) ) ld
5v3Q thin Dl (51m1an V to human fibroblasts)
mRNA derived from 7h cells transformed with Irus 80
Clone 5OD DNA using A, and
The base sequence was determined.

[Problems to be solved by the present invention]

Since human-derived SOD is difficult to obtain in large quantities, pharmacological effects have been tested in animal studies and clinical settings.
However, there is currently a need for the development of technology that can easily mass-produce human-derived SOD.

[Means for solving problems]

The present inventors aimed to develop a technology to mass-produce human-derived 5ol using genetic engineering technology, and as a result of various studies, isolated mRNA encoding SOD derived from human placenta, and used this to produce human c DN encoding placental SOD
Human placenta SOD is synthesized by synthesizing A, and transforming the recombinant into which this has been incorporated into a microorganism to express the gene.
The present invention was completed by discovering that it is possible to produce a polypeptide.

The DNA of the present invention encodes human SOD using the following formula % formula %
This is DNA containing the base sequence shown in parentheses.

The base sequence of formula CI) in the present invention is a known J abu
The amino acid sequence of SOD reported by Sch et al.
The amino acids encoded are different at some locations.

Furthermore, it differs from the SOD reported by Sherman et al. in the codon at position 54.

As long as the DNA of the present invention contains the base sequence of the above formula (I),
It may be in the form of single-stranded DNA, double-stranded DNA, or plasmid.

The main abbreviations used in this specification and drawings are as follows.

DNA Deoxy/Liponucleic acid A Adenine T Thymine G Guani/ C7 4 No F? , NA ribonucleic acid dG deoxy/guanine dT deoxy/chimidi/ dATP deoquine adenosine triphosphate dGrP
Deoxy/guano-7-triphosphate dCTP Deoxytintidine triphosphate ATP Azonone triphosphate EDTA Ethylenediamine/tetraacetic acid oty Gly/No-Ala Alanine Val Vali 7 Leu Roy7-11e Inoloinone Ser Seri/Thr Threonine Cys Instein C1u Glutamate Asp Aspartate Lys Lysine Arg Arginine His Histidi/Phe Phenylalanine Trp Trigutophan Pro Proline Asn Asparagine Gln Glutamine TTP Tris Thymidine Triphosphate Tris Hydrochloride The novel DNA of the present invention can be used to transform hosts such as microorganisms and animal cells, and SOD used to produce active polypeptides.

In the novel DNA of the present invention, it is preferable to express the gene of the present invention in animal cells etc. when the (5') end of formula (1) contains the base sequence of the following formula.

The DNA of the present invention can be produced as follows.

(a) Isolation of mI (and NA) from human placenta (b) The m
Using RNA, for example, the Okayama-Berg method (Mol
, Ce1l, 13io1, 2 161-170
(1982)], etc., to obtain a plasmid incorporating complementary DNA, transform Escherichia coli with this plasmid, and extract the desired base from the bacterial cells obtained by culturing this Escherichia coli. Plasmid (DNA) containing the sequence!
V'1. tMIU Sur.

(e) Next, if desired, 1) NA containing the desired base sequence of 2 (1) can be excised from this plasmid. .

(f) By ligating this excised DNA downstream of the promoter of the vector, a vector plasmid containing the base sequence of formula (1) can be obtained.

In the above method, instead of the Okayama-Berg method, the synthesis of complementary DNA from mRNA is carried out in accordance with the conventional method, first using reverse transcriptase to convert it into single-stranded complementary DNA, then converting it into double-stranded DNA, and then converting it into vector. It can also be incorporated into a plasmid.

To produce SOD-like protein using the DNA of the present invention,
It can be obtained, for example, by transforming Escherichia coli with a vector plasmid containing the base of formula (1) above, culturing it, and isolating the protein from the cultured cells.

The Tac promoter, PL promoter, etc. can be suitably used as a promoter when producing SOD-like protein in E. coli.

The method for producing the DNA of the present invention and its expression will be explained below with reference to Examples.

Example (1) m11. from human placenta. NA separation and SODmR
Identification of NA: Approximately 300 g of fresh placenta, taken within 1 hour from the birth of the newborn, was washed with phosphate saline (1"BS solution rL) and guanidine/
・Thionanate method (Chirgwins: Bio
chem, 18, 5294-5299 (1979)
) total cytoplasmic RNA was extracted. This extracted total of 11. NA was added to a high salt buffer (Tris, 0.
5M NaCl (pH 7.4)K was dissolved and passed through an oligo(dT) Celllocus (manufactured by Pharmacia) column to obtain polyA RNA.
After adsorbing (mRNA), a low salt buffer 't
It was eluted with L (Tris, NaCl-free, pH 7.4) and precipitated with ethanol. 1.7 mg of +nL (NA was obtained from 150 mg of total RNA. The precipitate was
Dissolve in μl of sterile water, heat at 80°C for 2 minutes, cool quickly, and separate in order of molecular weight by 5-20φ/=1 sugar density gradient centrifugation.

Actually using Hitachi RPS 40T rotor, 35K
, rpm, and centrifuged at 0°C for 17 hours.

Next, a part of each separated surface (Q, 5m1).

Translation was performed using a rabbit reticulocyte lysate (manufactured by Ama/Yam) system, and the synthesized protein was analyzed using an immunological method (Engineering/Zyme immunoassay method) (J, Pharm, D).
yn, 5394-402 (1982)). In this way, S is added to the 10-12S fraction of mRNA.
OD mRNA (D presence was observed.

(2) mRNA annealing and cDNA synthesis:
Using the fraction obtained in (1), the method of Okayama-Berg (
Mo1. Ce11. Biol, , 'l, 161
-1700.3mM dithiothreitol (DTT), 8
mMMgC12, 40 μg/ml actinomycin D,
- 2mM each of dATP, dCTP, dGTP,
TTP, 3oμci (a-32P]dCTP (6
00 Ci/mmol) (manufactured by NEN), 280 units of glue bonuclease inhibitor (manufactured by Wayo Pure Chemical Industries, Ltd.), and 2.8 μg of plasmid Shira 1mer [Escherichia coli plasmid p8V7186 (using Pharma/A!
Approximately 6 T-tails synthesized according to the Okayama-Berg method
Prepare 101 liters of a solution containing 1 of 0 bases of Bly Juku-] and keep it at 37°C.

Then 10mMTris (p! [8), l r++Ml
10□1 solution containing flDTA and 3 pg of mRNA
was prepared, heated at 65°C for 5 minutes, immediately transferred to 37°C, mixed with 10 μm of the above solution, and heated for an additional 5 minutes. Subsequently, 5 units of reverse transferase (manufactured by Life Science) were added, and the mixture was heated at 37°C for 20 minutes. 2 μm of 250 mM FJDTA (pH 8,0) and 1 μm
After stopping the reaction by adding a 10% SDS solution of
After ethanol/chloroform extraction and ethanol precipitation, proceed to the next step (3) Synthesis of a plasmid containing the base sequence of formula (r) of the present invention (i''C) : The precipitate obtained in (2) was mixed with 140mM sodium cacodylate-30mM Tris (pH 6,8), 1mM
CoCl2, 0.1mM DTT, 1mM dCT
P and 50 μCI [α-32P] were dissolved in a solution containing dcTP, heated at 37°C for 2 to 3 minutes, and then 18 units of terminal deoxynucleotidyl transferase (manufactured by Pharma/A) was added to bring the total volume to 15 μl. . After heating at 37°C for 3 minutes, post-treatment similar to reverse re-reaction was performed to obtain an ethanol precipitate.

Next, the precipitate was mixed with 50mM NaC+, 50mM Tris (
pH 8.0), 10 mM Mgcl 2.100.
, Uno serum albumino (BSA), and 12 units of Hindu (manufactured by Novo/Gene) and heated at 37° C. for 2 to 4 hours.

After phenol/chloroform extraction and ethanol precipitation, this was mixed with 10 μl of 10 mM Tris (pH 7,3), 1
It was dissolved in a solution containing mM gDTA, and 3 μl of ethanol was added to make the total diameter 13 μm. This melt tL
O-04 pmal of oligo(dG) linker [E. coli plasmid pSV1932 (Pharma/
d synthesized according to the Okayama-Berg method using
o-tailing linker of about 12 bases], lOm
The MTris solution was heated at 65°C for 5 minutes and then at 42°C for ;t() minutes and then kept at O0C. Add to this 20mM Tris (
pH7,5), 4mM Mgcl2, IO+t
+M ammonium sulfate, 0.1 M KCI, 504g
/inl BSA, 0.1 mM β-nicotine agodoateno//dinucleotide (NA, D) and 0.6 μg
A concentrated solution containing Escherichia coli DNA ligase (Pharma/A) was added to make the final concentration solution 1007 zl, and 12
Warm at °C overnight. Then dA containing 20mM each
TP, dCTP, dOTP and TTP were set to 0.
4 al, 1 μM 1.5mM β-NAD, E. coli DN
0.4μg of A ligase, Escherichia coli DNA polymerase ■
0.3 □g of E. coli ribonuclease H and 1 unit of Escherichia coli ribonuclease H (total 10 μm) were added, and the mixture was further heated at 12° C. for 1 hour and at 25° C. for 1 hour.

(4) Transformation into large tooth: χ1776 (ATCC31244) was used as E. coli. Competent cells were developed by ManiAtis et al. [M
o1ecular Cloning+cold spr
ing harbor labor
ry, 254-255 (1982)) and dispensed in 0.2 ml portions.

Five tubes of 20 μl of the DNA solution were transformed, and Bactodrypton 10 g/I and yeast extract 5 g/L were transformed.
, Diaminopimelic acid 0.01%, Thymidine 0.00%
Approximately 30,000 coneys were obtained on a 1.5% agar medium containing 4% and 50 μg/ml of Ampias/(Ap).

(5) Colony high fritization = 1 no = Transfer about 10,000 of the obtained colonies onto an agar medium of the same composition (512 pieces/14 x 10 cm plate; 1 set of 2 plates, 1 plate) was stored as a master plate) and cultured until it grew to a diameter of approximately 3 mm. Whatman 5410 paper was slowly placed on this, and the paper was brought into close contact with the CoroTem medium, and cultured day and night. Fixation of DNA to the opening paper was performed as follows.

After culturing, the mouth paper was treated with 0.5 M NaOH twice for 5 minutes, and then treated with 0.5 M Tris (pH 7,4) f medium a.
K Modoshi, 2X8SC (pl-17) (IXSSC
: 0.15M NaCl, 0.015M sodium citrate) treatment, lightly washed with 95% ethanol aqueous solution, and then air-dried. (A) t7 nucleotide as probe: AA (TorC) 'l”T, (TorC)
GA (AorG) CA (AcirO) AA (
AorG) CIA (7) 32 types (B) 14 Nuku L/Ochito: OA ('l”orc) CA (T
orC) TO(TorC) AT(T, CorA
) Twenty-four types of AT were each chemically synthesized by the triester method and used in the hybridization described below.

(B) Greyhybridization/3-size paper 6 X IT (I X S[]T: 0.1
5M N a Cl, 0.015M Tris (pH 7,
5), 1 mM EDTA), 0.5% Nonidesoto P
40 (manufactured by Gyudon Kagaku Co., Ltd.) and 100 μg/ml of denatured E. coli DNA (E. coli DNA manufactured by Pharmacia Co., Ltd., boiled for 5 minutes and then rapidly cooled) was heated at 55° C. for 12 hours.

Next, instead of the denatured E. coli DNA, 100 μg/ml of yeast tR,NA (manufactured by BRL) and (,32P) A were used.
0.2 ng/ml of a globe labeled at the 5' position (32P) using TP acid (5000 Ci/mmol manufactured by NEN) and polynucleotide kinase (NEBt[)]
Hypolymerization was carried out at 29°C for 12 hours using

(c) The plasmid containing the base sequence of formula (1) was isolated and washed by (A) 5 minutes at 39°C and (B) 2 minutes at 290°C.
6X SSC for 0 min followed by 10 min treatment at room temperature.
Each step was repeated three times using the solution. After air drying the opening paper,
Autoradiography was performed using X-ray film (Kodasoku XA IL5), one colony positive for both (A) and (B) was selected, the plasmid was extracted from the bacterial body, and the plasmid was transformed into prlS 3237.
It was named.

(6) Confirmation of restriction map and nucleotide sequence of cDNA encoding human SOD: T)NA encoding human SOD can be excised with Pstj and Pvul (both manufactured by NEB, 1.), and Ava If, Tag l,
5tu(,, Hinf ■, 5au3AI and Al
When a restriction map was created using UL (both manufactured by Nino Bon Gene), it was confirmed that p[I8:(237) has the structure shown in Figure 1. Approximately 780 bp DNA inserted into this plasmid The fact that encodes SOD is demonstrated by the Maxam-Gilbert method (P
roc, Natl, Acad, Sci, ,
74°560-564 (1977)] and]M13-Dideoxy7 Method Proc, Natl, A
cad, sci, , 74゜5463-5467
(1977)] was used to determine the base sequence.

Figure 2 shows the base sequence. Downstream from the start codon ATG shown here, the codon ACA corresponding to the 54th Thr is the result of Sherman et al.
Unlike G.

Furthermore, Sherman et al. have not reported anything upstream from ATG. The inventors further upstream 130b
p (only a portion of which is shown in Figure 2), and almost full-length cDNA could be obtained.

(7) Construction of expression vector Hae II closest to the lactose promoter on E. coli plasmid pUc13 (manufactured by Pharmacia)
After cutting the site, exonuclease Bat 31 (N
T, DNA
Plasmid pliUc, 13 was prepared by recirculating with ligase (manufactured by Takara Shuzo Co., Ltd.) (this plasmid has lost its function as a lactose promoter).

Next, insert Hincl [Trp at the cleavage site] of this plasmid.
A. Plasmid pΔ(, ICT 13) into which a terminator (manufactured by Pharmacia) was inserted, Tac promoter or PL promoter (both manufactured by Pharmacia) was inserted, and 5OD DNA was further ligated to create an SOD expression vector as described below. It was constructed.

(8) Preparation of DNA encoding SOD The pH 83237 obtained in (c) of (5) above was digested with Pvu■,
Xba l licker (manufactured by NEB), DNA
The plasmid was ligated with ligase to create an Xba 1 site and named pH8X3237. pH8X323
7 was digested with r'sL l and exonuclease B
It was sequentially digested with al31. Furthermore, the ends of the T and Xba
(both manufactured by Ninobon Gene), approximately 630 to 700 bp of DNA was recovered using a 2 to 16% gradient polyacrylamide gel.

Insert @TacTa top and SOD Df'JA into Eco RI (manufactured by Novon Gene) and Bam HI
The 121 bp fragment containing the Tac promoter was recovered on polyacrylamide gel, and pΔUCT 13 noEc
o Approximately 3Kb obtained by inserting between RI and BamHI
The plasmid was named pTacT (Figure 3). p
Tacl-BamHI-Xba 1 (7) (8)
The resulting plasmid was inserted into Escherichia coli DHI (ATCC338
49). The base sequences of the various plasmids obtained were determined, and plasmids with a distance of 8 to 13 nucleotides from the SD sequence (AGGA) to the start codon ATG were named pTacsOD8-13 (Figure 3).
.

Preparation of plasmid with pPL bucomotor and SOD DNA inserted - mock 2. .

Tanju ÷→→ tl) When using the Pl-promoter containing the SD sequence, use the plasmid p having the PL7' promoter containing the SD sequence.
Hpa I and Sau
q61 (both manufactured by Nirapon Gene), and the approximately 480 bp PL promoter-containing DNA fragment was collected using polyacrylamide gel. The fragment was further partially digested with DNA polymerase Flenow fragment (NE) (inf 1) (manufactured by Nippon Gene).
After EcoRI digestion (manufactured by Company B), 350 bP DNA downstream of EcoRI was recovered using polyacrylamide gel.

Separately, pTac-SOD 8-13 was treated with BamHI.

The BamHI site was treated with DNA polymerase Flenow fragment or exonuclease SI to make the fcDNA blunt, and fcDNA was prepared and ligated with the approximately 350 bp DNA. Add this to Xba I and Ec.

A DNA fragment of approximately IKb was recovered by digestion with RI. The resulting IKb DNA fragment contains P
t, promoter, and the SOD coding sequence starting from human TG, which was converted into EcoRI of pΔUCT 13.
and Xba i. These plasmids were named pPL208-218 (Figure 5).

F8) Surprise of SOD-like polypeptide: l] Enno 20
Is 8-213 a temperature-sensitive repressor? E. coli N483 with Il-
A single colony obtained by transforming A600 (Pharmacia Shamaru) was cultured at 28-30°C in a medium
= 37°C to 42° when it becomes 0.1 to 0.3
The temperature is raised to 0.4°C and the culture is further cultured for several hours to produce SOD-like polypeptide in the culture. This production was confirmed by the Immunozo 7A/I/G method of reacting with SOD antibody.

In addition, pTac 5OD8-13 is a single colony obtained by transforming E. coli with 1itai and cultured at 37°C in an appropriate medium, and when it reaches an A60 of 0.1 to 0.3, inopropyl- β-Thiogolactobirano/de (manufactured by 7Gumman Co., Ltd.) was added to a final concentration of about 1mM, and cultured at 37°C for several hours to increase the SO content in the culture.
A D-like polypeptide can be produced.

[Brief explanation of the drawing]

Figure 1 shows the restriction map for pH 83237. Figure 2 shows the base sequence and SOD amino acid sequence of SOD cANA. Figure 3 shows pΔUCT 13 , pTacl and p
It is a block diagram of Tac5OD8-13. Figure 4 shows pΔUCT 13 and pPL80D 208
It is a block diagram of 213. In the figure, the loco is the SOD code sequence region, the mouth is the SD sequence region,
pus is the terminator region, and guco is the promoter region. Additionally, Ap is characterized by ampyridine resistance.

Claims (1)

[Claims] 1) A new DNA containing the base sequence represented by the formula [there is a base sequence] that codes for human superoxide dismutase (SOD) (2) The formula that encodes human SOD [there is a base sequence] A microorganism transformed with DNA containing the base sequence represented by