JPS6192573A - Dna fragment - Google Patents

Abstract

PURPOSE:A DNA fragment that has a codon sequence corresponding to cardiodilatin-like polypeptide. CONSTITUTION:A DNA fragment that is given in the formula (X is CUC, CTC, GGG or CGG). The DNA fragment or a plasmid containing the same is used to produce cardiodilatin-like peptide, which can be used as a medicine for vasodilation and hypotensor.

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention relates to a DNA fragment containing a base sequence encoding a cardiodeilatin-like peptide.

Cardiodilatin
)teeth.

This peptide has a molecular weight of approximately 7300, is derived from a precursor protein in the atrium-derived protein, and is known to have a muscle relaxing effect.

<Prior art> It is known that the sequence of 30 amino acids on the N-terminal side of cardiodeilatin derived from pigs is as follows (
Anatomy and Embryology 168, 307-3/3
．． /9za3). Aen-Pro-Val-Tyr-
Gly-8er-Val-8or-Aen-Ala-A
ep-Leu-Met-Asp-Phe-Lys-As
n-Leu-Leu-Aep-Hls-Leu-Glu
-Asp-Lys-Met-Pro-Leu-Glu-
Aep Although this precursor protein itself has been investigated at the protein level, it has not yet been elucidated at the DNA level.

In the process of investigation to obtain a cDNA fragment encoding the precursor protein of Cardionatrin, which is an atrium-derived peptide consisting of 21 amino acids and has Na diuretic effects, the present inventors
I) The DNA sequence encoding diodeiratin is present together with the NA sequence encoding cardionatrin.
It was revealed that it exists on DNA fragments (r]
ature.

310, 23'84).

Furthermore, the present inventor conducted studies and found that D
The present invention was achieved by identifying the NA fragment.

<Configuration of the Invention> The gist of the present invention is a DNA7 fragment containing a base sequence encoding a cardiodeilatin-like peptide. Cardiodeiratin-like peptide refers to cardiodeilatin itself and cardiodeilatin analogs, which have similar physiological activity to cardiodeilatin. An example of a base sequence encoding a cardiodeiratin-like peptide is shown in the following formula (■).

Furthermore, as an example of a DNA fragment containing a base sequence encoding a cardiodeiratin-like peptide, a translation initiation codon, 3 Examples include a DNA fragment represented by the following formula (II) with a translation stop codon added to the 'terminus.

The present invention will be explained in detail below.

First, an example of a method for preparing a DNA fragment containing a DNA sequence encoding a cardiodilatin-like peptide according to the present invention will be described.

Human heart lrM tFfr pieces are homogenized with guadinylthionanate and total RNA is isolated by OsCl equilibrium density gradient ultracentrifugation (Chirgwin et al. 7ri).

Next, this was purified by oligo(dT) cellulose column chromatography in a conventional manner to obtain poly(A)-containing RNA.
(mRNA raw material).

From this mRNA raw material, the method of Okayama and Berg Parg (
MOLLOLAR and CELLULAR BIO
A cDNA library is obtained using the molecular and cell biology method (2, 1ti-170, 12). That is, a vector primer and an oligo(aG) tail linker are obtained using a hybrid plasmid of pBRJ22 and 8v. This vector primer and the above mRNA are coexisted with reverse transcriptase to act on cD.
NA is synthesized, then digested with the restriction enzyme Flind I, and then cyclized using the linker described above. After that, m
The RNA portion is replaced with DNA to obtain a cDNA fragment-containing plasmid.

Next, using this, Escherichia coli (Esche.
richia coli) etc. to be ampicillin resistant to obtain a cDNA library.

Then, cardioonatrine (5er-Lθu-Arg
DNA encoding Met-Asp-Arg-E-Gly
The following nucleotides complementary to the sequence were synthesized as probes.

Using this clone, the above-mentioned cDNA library is screened to select a clone that hybridizes with the same probe. cDNA obtained from the clone
Fragments were generated using the Maxam-Gilbert method ((Metho
ds in 11CnZ7mnlOg7) Methods in Enzymology-Jj, ≠ buta zto, i r, r
Determine the base sequence by O).

The cDNA fragment is not necessarily required to have a certain nucleotide sequence and number of nucleotide residues, and if the substance encoded by DljA is a cardiodeiratin-like substance having the same physiological activity as cardiodeilatin, A nucleotide sequence may be a nucleotide sequence in which part of the nucleotide sequence has been substituted or deleted, or nucleotides have been added.

Furthermore, the cDNA fragments are not limited to those derived from humans, but are derived from other higher animals, such as cows, pigs,
It may be derived from horses, mice, rats, etc.

By adding a methionine linker, a linker having a liposome binding site, and a termination linker to the DNA fragment thus obtained, an NA fragment capable of expressing the desired cardiodeilatin-like peptide can be obtained.

The DNA fragment of the present invention represented by the above formula (II) is characterized by having a start codon at the j' end and a stop codon at the 3' end. When a cardiodeilatin-like peptide is produced by translation using the DNA fragment of the present invention as a template by introducing the DNA fragment of the present invention into the cloning site of an expression vector having an expression control region such as a promoter upstream of the cloning site, non-fusion This is advantageous because protein can be obtained.

Next, a part of the present invention in which a plasmid suitable for expression is constructed using this DNA fragment will be further explained with reference to the drawings. That is, plasmid pHANF
48 (Nature 310, 2J, A Buta '74
30/ containing carteiodeilatin obtained from
After combining the bp fragment with methionine phosphoryl, it was digested with the restriction enzyme Bind I to obtain j 33' bp fragment.
Get fragment. This is combined with a linker having a liposome binding sequence (RBEI) shown in FIG. 1, and digested with restriction enzymes Ba and mHI to obtain a 3 A j bp fragment shown in FIG. This is plasmid pUC! J' (
plasmid pUO (purchased from LBiochemicals) was introduced into the former site of P.
c(1g (Fig. 3)). Then, this pU Ccd I
was partially digested with the restriction enzyme Ayal and a termination ((Term) TGA TAG0TATO) linker was inserted to obtain the plasmid pUcdff term (Fig. 4).
Digesting J4'0 (purchased from P,L Biochemicals) with EcqRI and Ban old, approximately 370
bp fragment, while the above plasmid plod
I term was digested with Apal and BamHI,
Obtain x ao' bp fragment. On the other hand, pHAN
Obtained a large fragment of F4'Jr's Katsutaka after plastic I digestion. These three are ligated and large pI & bacteria are transformed to obtain the desired grasmid phcD (Figure t).

<Effects of the Invention> The DNA7 fragment according to the present invention and the plasmid containing it are effective for producing cardiodeilatin-like peptides.

The obtained peptides are useful as vasodilators and antihypertensive agents.

<Example> Hereinafter, the present invention will be further explained in detail with reference to Examples.

Example 1 <Preparation of raw material DNA fragment> (1) Human heart fragments were crushed with liquid nitrogen, and then a guanidinium thiocyanate aqueous solution was added and homogenized. The obtained homogenate was processed using the method of Chirgwin et al. (Biochemistry 18, 5294-5299, 1979).
Total RNA was isolated by cesium chloride equilibrium density gradient ultracentrifugation according to. Next, this was purified by oligo(dT) cellulose column chromatography in a conventional manner to isolate poly(A)-containing RNA, which was used as an mRNA raw material.

(2) On the other hand, the Okayama and Berg Park methods (Molec
ularand Ce1lular Biolop;y
MoVKie2and Cellular Biology 2,
/41-/70. pBRJ J,
Vector primers and oligo(dG) tail linkers were obtained using the BVIAOF)/S hybrid plasmids.

That is, a hybrid plasmid of pBRJJJ and 5V4 (0 (map units 0,7/-0, IrA)≠00μ?) was digested with the restriction enzyme Kpnl at 37°C for 1 hour in a buffer containing bovine serum albumin. .

Next, the DNA was recovered by ethanol precipitation using a conventional method, dissolved in a buffer containing dTTP, terminal deoxynucleotidyl transferase was added, and the reaction was carried out at 37°C for 30 minutes.
After about 10 dT details were added to the digestion site, the DNA was recovered by ethanol precipitation. Next, this D
NA was digested with the restriction enzyme Hpal in a buffer containing bovine serum albumin (37°C, 5 hours). The larger DNA7 fragment was purified by agarose gel electrophoresis and purified using the glass powder method (Vogelstein et al., Pro.
c. Natl. Acad, Sci, U, S, A.

Vogelstein Proceedings of the National Academy of! Ien Utsu 61-11-Nisei 76
, 615-619, 1979). Thereafter, this DNAQ was applied to an oligo(dA) cellulose column at 0° C., eluted with water, and recovered with ethanol to obtain a vector primer having an oligo(dT) tail.

On the other hand, 100 μ2 of a hybrid plasmid of pBR,J 22 and 5v4co (map unit 0./li ~ 0.3 J) was digested with the restriction enzyme Sittl in a buffer containing bovine serum albumin ( 37°C, 1.5 hours). Then, the DNA was collected, dissolved in a buffer containing dGTP, terminal deoxynucleotidyl transferase was added, and the mixture was reacted at 37°C for 20 minutes.
Approximately 10~/! dG detail was added. Collected I)
NA was digested with restriction enzyme H1n4 M in a buffer containing bovine serum albumin (37°C, 7 hours) and then subjected to agarose gel (/, t%) electrophoresis to detect small oligo(dG) tail linker-DNA. Extract.

Recovered.

(3) Okayama and Berg Park method (Molecul
arand Cellular Biolocrv Molecular and Cell 2- Biology 2, 16
1-170, 1482), a cDNA library was obtained.

That is, Tris-RC2 (pH 1, 3), Hg0l
z.

KCl, dithiothreitol, dATP.

Add 30 μt of the mRNA obtained in (1) above to an aqueous solution containing dTTP, dGTP and (up)acTP and (2) above.
Vector primer i obtained in

μt was added and reacted at 37° C. for 20 minutes in the presence of reverse transcriptase to synthesize plasmid-cDNA:mRNA, which was precipitated with ethanol and collected in the form of a pellet. This pellet was dissolved in a buffer containing OQ O'l, dithiothreitol, poly(A), [arp]dCTP, and terminal deoxynucleotidyl transferase, and reacted at 37°C for IO minutes to dOM
All residues from IO to /j of P were added.

Then, the recovered oligo(aO) tail plasmid-c
DNA: Dissolve the mRNA-containing pellet in a buffer containing bovine serum albumin and digest with restriction enzyme H1ndl for 3
After digestion at 7°C for 7 hours and ethanol precipitation, Hlnd
I-digested oligo(ao) Qi/l/(!DNA:mRN
A plasmid was recovered.

This was dissolved in a buffer containing the oligo(aG) tail linker DNA obtained in (2) above, incubated at tj°C for 2 minutes, further maintained at λ°C for 30 minutes, and cooled to 0°C.

Then, Escherichia coli (E, C011) DNA ligase was added to β-NAD in the presence of cotin adenine dinucleotide), and the mixture was incubated overnight.

Then dATP% a'r'rp-, -ao'rP.

dCTP, β-MAD, RE. E. coli DNA ligase, K., colt DNA polymerase and E. coli DNA ligase. col
iRNaF3e H was added and the mixture was incubated at 12°C.
After incubating for 1 hour and then 7 hours at room temperature, the reaction was stopped and the desired cDNA fragment-containing plasmid was obtained.

Next, using this plasmid, E. coli (
Fi, coli) HB/0/ was transformed.

(4) On the other hand, M et -A of cardionatrin
Two groups of nucleotides complementary to the DNA sequence encoding sp-Arg-I1e-G1y were synthesized.

Next, a cDNA library is screened using these oligos ■ and ■ as probes, and the μo and ooo transformants are hybridized with the same probes.
Two clones were selected. From these 72 clones, cDNA fragments were extracted, a restriction enzyme map was created, and based on this, the Maxam-Gilbert method (Meth
ods in Knz7m010g7methods in
Enzai Sevenology 62, ≠ Buta! r60. /ri rO
) was used to determine the base sequence of the fragment (pHA
NF4'ri.

(Nature 310, 23, 699 '84).

The DNA sequence of Naori Rudiodeiratin is that its molecule f
g7. The number of amino acids estimated from s00 is 72-7j
It was estimated from the position of arginine, which is easily processed in vivo.

The above human-derived cardiodeiratin/ is the above-mentioned pig-derived cardiodeiratin (AnatOm7 and
Embryology Anatomy and Encyclopedia 16za, 307-313, 1rJ), in the N-terminal (l) 1130 amino acid sequence,
≠Differences in places.

<Preparation of plasmid ph(!d)> (1) Digest pHANF≠t with S!■ and SIP I to obtain a 30/bp fragment containing cardiodeiratin. (Ir°C, /≠ waiting time, then restriction enzyme H1
Digest with nd i at 37° C. for 2 hours) to obtain a 333 bp fragment. This fragment was ligated with a linker having the liposome binding sequence shown in Figure 2 using T≠DNA ligase at tr°C for an i≠ waiting time, and then digested with restriction enzymes at 37°C for 1 hour). A 36 j bp fragment shown in 2 was obtained.

On the other hand, plasmid pU (!f was digested with restriction enzyme BamHI at 37° C. for 2 hours) and the above 3 A j bp fragment was introduced into this BamH1 site.

Next, E. coli JM10! Transform X tthl
Select the white colony on top and add plasmid pUOcdJ'
(Figure 3) was obtained.

Partially digest the above plasmid with restriction enzyme Ayal.
7°C, 30 minutes), the linker described above containing a stop codon (
Termination linker) is ligated in the presence of T≠DNA polymerase to create a plasmid pUo cd r ter
m was obtained (Figure ≠).

Furthermore, plasmid pDRj with Tac promoter
Restriction enzyme Mr. F1. ! , digested with μ History H1 37
°C, 2 h), yielding an approximately 370 bp fragment (Fig.
). On the other hand, the above plasmid pU Ccd I term
was digested with Apal and BamHI (37°C for 2 hours) to obtain the 2&Obp fragment (Figure j).

On the other hand, pHAN F-Hirot was digested with Rikita■ and Shisai-R1,
A large fragment containing the ampicillin resistance gene was obtained (Figure.

The three 7-ragment fragments obtained in this way were ligated using T-Hiro DNA ligase (f℃, /Hiro time), the resulting plasmid was used to transform Escherichia coli JM10, and the transformed strain was transformed into plasmid phCD (Fig. 6 ) was obtained.

<Production of cardiodeiratin-like peptide> When E. coli, TMios, transformed with plasmid phOD was cultured in MterOA medium, a molecular weight of about 7. ! Production of 00 carteiodeiratin-like peptide was confirmed.

[Brief explanation of drawings]

Figures /, ≠ and j are schematic diagrams of examples of the plasmid surplus process in the present invention, and FIG.
Figure 3 shows the restriction map of plasmid pU Ccd I; Figure t shows the restriction map of plasmid phOD; Figure 7 shows the restriction map of plasmid phOD;
The base sequence of the DIJA portion of the plasmid containing the jj-sequence encoding the cardiodeilatin-like peptide is shown. Partner Sankyen Kasei Kogyo Co., Ltd. Representative Patent attorney Hase - Nika/Meishu 2 3b5bp□ Mi 3 &mHI BamHI Fig. 4 ↓ Recombinant/Cρθferm @ 5 Fig. b

Claims (2)

[Claims] (1) A DNA fragment containing a base sequence encoding a cardiodeilatin-like peptide. (2) The DNA fragment according to claim 1, wherein the base sequence encoding a cardiodeiratin-like peptide is represented by the following formula (I). [There is a base sequence] (I) (In the above formula, X indicates [There is a base sequence] or [There is a base sequence]) (3) DNA containing a base sequence encoding a cardiodeilatin-like peptide The DNA fragment according to claim 1, wherein the fragment is represented by the following formula (II). [There is a base sequence] (II) (In the above formula, X indicates [There is a base sequence] or [There is a base sequence])