JP2512436B2 - Tropoelastin cDNA - Google Patents

Description

TECHNICAL FIELD The present invention relates to a cDNA fragment encoding the precursor protein tropoelastin of elastin, and more particularly to a cDNA fragment encoding tropoelastin of chicken.

[Problems to be Solved by Prior Art and Invention]

Elastin (mature elastin) is a component of connective tissue of animals, like collagen and acidic mucopolysaccharides,
It is present in elastic organs such as lungs, aorta, ligaments, and skin. Since these elastins are high molecular weight proteins having a complicated cross-linking structure and having a molecular weight of several hundred thousand or more, they are insoluble in water and are difficult to extract and purify. In general, elastin is extracted and separated by a method in which elastin is separated as a decomposed product by extracting the tissue under severe conditions such as treatment with a strong alkali or high temperature. As a method of obtaining elastin with a relatively high molecular weight, Pa
rtridge et al.'s thermal oxalic acid treatment method (Partridge et al., Biochem.
J.61,11,1955) is known, and α is used as a water-soluble protein.
-Elastin [molecular weight about 70 KDa (kilodalton)] and β
-Elastin [molecular weight about 10 KDa or less] has been obtained. Recently, studies on the physiological action of elastin using α-elastin have been conducted, and it has been revealed that α-elastin has a function of inhibiting the aggregation of platelets that causes arteriosclerosis.

Tropoelastin is the precursor to the elastin protein. It is a water-soluble protein immediately after being translated from messenger RNA. Unlike mature elastin, it does not contain desmosine, isodesmosine, lysinonorleucine as residues, nor does it form a cross-link with them. Separation of tropoelastin from living organisms is very difficult and a method of separating from tissues of juveniles fed a copper-deficient diet to inhibit cross-linking enzymes (Smith; DW et al., Biochem. Biophys. Res.
Commum. 31, 309, 1968), but the amount of tropoelastin that can be isolated is very small. Therefore, research on the molecular structure of tropoelastin has not progressed easily, and only the molecular weight and amino acid composition have been clarified, and the amino acid sequence has not been clarified yet.

It is an object of the present invention to elucidate the structure of tropoelastin at the DNA level and provide a cDNA fragment necessary for producing tropoelastin using an expression vector.

[Means for solving problems]

The above object can be achieved by a cDNA fragment encoding tropoelastin according to the present invention.

 Hereinafter, the present invention will be described in more detail.

First, an example of a method for producing a cDNA fragment according to the present invention will be shown.

In chickens, tissues that produce a large amount of elastin (hereinafter sometimes referred to as positive tissues), for example, the aorta of chickens is homogenized with guanidyl thiocyanate to disrupt the cells, and RNA fractionation is performed by cesium chloride equilibrium density gradient ultracentrifugation. obtain. Then oligo dT
From the RNA fraction, RNA having poly A (poly A ⁺ RN) was analyzed by affinity chromatography loaded with cellulose.
A) is isolated.

Using this poly A ⁺ RNA as a template, a known method, preferably the method of Okayama-Berg (Mol.Cell.Biol.2,161,198).
A cDNA library is obtained by 2). The Okayama-Berg method is carried out as follows. That is, the poly A portion of poly A ⁺ RNA is adsorbed on the poly T portion of the Okayama-Berg vector and reacted with reverse transcriptase to synthesize cDNA. After adding oligo dC to the cDNA less than 3'with terminal deoxynucleotidyl transferase, the restriction enzyme HindII
Cut the vector DNA with I. After ligating the oligo dG linker and circularizing the vector, the RNA portion is replaced with DNA by DNA polymerase to obtain a cDNA-containing plasmid. Using these plasmids, the calcium chloride method (Strik, P.
, J. Bacteriol. 138, 1033, 1979) and the like to transform E. coli. By selecting an ampicillin-resistant strain on a plate medium containing ampicillin, a plasmid recipient bacterium is obtained.

On the other hand, the above-mentioned positive tissue, that is, a tissue that produces a large amount of elastin, and a tissue that does not produce a lot of elastin (hereinafter sometimes referred to as a negative tissue), such as chicken skin, are prepared, and poly A ⁺ Isolate RNA. Labeling 5'OH of RNA with ³² P using polynucleotide kinase and γ- ³² PATP,
This is used as a probe.

Next, colony hybridization method (Hanahan,
D. et al., Gene, 10, 63, 1980), there is complementarity with a probe derived from a positive tissue from the above-mentioned cDNA library,
Moreover, colonies not complementary to the probe derived from the minus tissue are selected. Plasmid DNA was obtained from the thus selected colonies and the dideoxynucleotide method (Messin
g, J.Methods in Enzymology101,20,1983)
Determine the base sequence. The amino acid sequence is deduced from the nucleotide sequence, it is confirmed that the amino acid sequence peculiar to elastin exists in the amino acid sequence, and the obtained cDNA clone is confirmed to be the cDNA encoding tropoelastin.

The cDNA fragment according to the present invention is a cDNA fragment encoding tropoelastin, and encodes tropoelastin as an amino acid sequence corresponding to the nucleotide sequence. An example thereof is shown in FIG. 1, but the cDNA fragment according to the present invention does not necessarily have a constant number of bases.

By ligating the cDNA fragment according to the present invention downstream of an appropriate promoter, tropoelastin can be produced by translation using E. coli, Bacillus subtilis, yeast, animal cells and the like with the cDNA fragment as a template.

The produced tropoelastin can be used as a platelet aggregation inhibitor or the like for preventing and improving arteriosclerosis.

〔Example〕

 Next, the present invention will be described in more detail with reference to examples.

Example 1 (1) The aorta around the heart was separated from 50 15-day-old eggs of chicken, disrupted with liquid nitrogen, and then an aqueous guanidinium thiocyanate solution was added to homogenize. The homogenate obtained was subjected to the method of Chirgwin et al. (Chirgwin et al., Biochem.
RNA was isolated by cesium chloride equilibrium density gradient ultracentrifugation according to (18,5294,1979). Poly A ⁺ RNA having poly A was isolated from the RNA fraction by affinity chromatography carrying oligo dT cellulose (Pharmacia, type 7).

(2) A cDNA library was prepared by a cDNA cloning system (Amersham) using a commercially available Okayama-Berg vector.

That is, Tris-HCl buffer (pH 8.3), magnesium chloride, potassium chloride, dithiothreitol, dATP, dGT
To the aqueous solution containing P, dTTP and α- ³² PdCTP, about 10 μg of the poly A ⁺ RNA obtained in (1) above and about 5 μg of Okayama-Berg vector primer were added, and a reverse transcriptase (Seikagaku Corporation) was added. Reverse transcription reaction was carried out at 37 ° C. for 30 minutes in the presence of. The vector plasmid containing the synthesized cDNA was recovered by ethanol precipitation. After drying this precipitate, it was dissolved in an aqueous solution containing Tris-HCl buffer (pH 6.9), sodium cacodylate, cobalt chloride, dithiothreitol, bovine serum albumin and α- ³² PdCTP, and the terminal deoxynucleotid derived from calf thymus was used. Add diltransferase and incubate at 37 ° C for 30 minutes, about 15-30
The residue oligocytosine was added to the cDNA. The obtained cDNA
After digesting the contained vector with the restriction enzyme HindIII, the oligocytosine-added cDNA-containing vector was recovered by ethanol precipitation.

The oligocytosine-added cDNA-containing vector was dissolved in a commercially available buffer solution containing oligo dG tail linker DNA (containing Tris-HCl pH8.0, magnesium chloride, EDTA, NAD, ammonium sulfate), E. coli DNA ligase was added, and the mixture was incubated at 16 ° C.
And reacted for 15 hours. After that, dATP, dCTP, dGTP, dTTP, NA
D, E. coli DNA ligase, E. coli DNA polymerase, and E. coli RNase H were added and reacted at room temperature for 1 hour to obtain a cDNA-containing plasmid.

Calcium chloride method (Mand
E. coli, M15, J. Mol. Biol. 53, 159, 1970).
The strain was transformed to obtain an ampicillin-resistant strain that grows in an LB medium containing 30 μg / ml of ampicillin.

(3) Poly A ⁺ RNA was isolated from the skin tissue of 15-day-old chicken eggs in the same manner as in (1) above. This poly A ⁺ RNA and the poly ⁺ RNA obtained in the above (1) were treated with Escherichia coli alkaline phosphatase (25 ° C. for 1 hour) in a pH 8.0 Tris-hydrochloric acid buffer solution to give a 5′-terminal phosphate. After removing the enzyme and inactivating the enzyme, T4 polynucleotide kinase and γ- ³² P · ATP were added, and phosphorylation was performed at 37 ° C for 1 hour, and ³² P-labeled poly A ⁺ RNA (probe) was added. Obtained.

(4) Using the probe obtained in (3) above, the transformed strain obtained in (2) above was subjected to colony high according to the method of Hanahan et al. (Hanahan D. et al., Gene, 10, 63, 1980). Hybridization was performed. When colonies showing complementarity to the aortic probe and not complementary to the skin probe were selected, 6 clones out of 1000 were obtained. When pramide DNA was extracted from these clones and a restriction enzyme map was created with the restriction enzyme P _VU II, three clones were
Common DNA fragments were found. CD with this common DNA fragment
Regarding NA, dideoxynucleotide enzymatic method using M13mp11 phage (Messing, J. Method in Enzymology, Vo
DNA base sequence was determined according to I.101, P20, Academic Press, 1983). The nucleotide sequence of the cDNA thus determined and the amino acid sequence predicted from it are shown in FIG. In the amino acid sequence shown in FIG. 1, a pentapeptide forming a β-sheet structure known as an amino acid sequence peculiar to elastin (Val-Pro-Gly-Val-Gly; underlined in FIG. 1 is shown. ) And crosslink forming peripheral sequences (indicated by a box in FIG. 1) were present. Therefore, the obtained cD
It has been shown that NA is a tropoelastin cDNA.

[Brief description of drawings]

FIG. 1 shows the nucleotide sequence of the cDNA fragment according to the present invention and the amino acid sequence expected therefrom.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Ichiro Tokimitsu 7-23-616 Shin-Ogawamachi, Shinjuku-ku, Tokyo (72) Inventor Masahiro Tajima 1050 Shinba-cho, Kohoku-ku, Yokohama-shi, Kanagawa Shiseido Research Institute Co., Ltd. (72 ) Inventor Toshio Fukasawa 2-8-2 Maehara Nishi, Funabashi City, Chiba (56) References Biochemical and Biophysical Research Communications, Vol. 103, No. 3 (1981) P. 880-885 “Applied Molecular Genetics” edited by Masahiko Kizumi and Tadaro Horiuchi, Kodansha (April 20, 1986), pages 85-89

Claims (1)

(57) [Claims] 1. The following nucleotide sequence: (In the formula, X does not exist or the following base sequence: ), Which encodes tropoelastin.