JP3145454B2 - Cell activator for pearl culture - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a cell activator for pearl culture containing an artificially functional polypeptide.

[0002]

2. Description of the Related Art Cultured pearls are found in shells such as pearl oysters, white butterfly shells, black butterfly shells, ikecho shells, flatfish shells, crow shells, lobster shells, kawashinju shellfish, mabe, abalone, etc. It is produced by artificially inserting the core material of sections and pearls. In the culture of pearls, compatibility between the nucleus for cultivation and the mother oyster, activation of mantle cells and the like are important. Therefore, the compound described in JP-B-45-5555 is a cell activator for pearl culture. It is used as Platonin Neoxin [Japan Photosensitive Dye Laboratories]. Recently, collagen and fibronectin, which are bio-related substances, have been used in culture nuclei in order to increase the affinity between the mother mussel and the culture nucleus (Japanese Utility Model Application No. 53-183).
No. 98, JP-B-3-33290).

[0003]

The bio-related substance is:
It is less toxic and has less adverse effect on the mother of pearl culture. However, it is difficult to obtain large quantities of uniform preparations of these natural substances. The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a substance which is useful in pearl culture and can be supplied in a uniform and large amount, particularly a substance useful as a cell activator for pearl culture. It is in.

[0004]

SUMMARY OF THE INVENTION In general, the present invention relates to a mantle cell activator for pearl culture, and
Pearl culture cell seeds used for processing mantle mantle sections
An activator characterized by containing an artificial polypeptide having an Arg-Gly-Asp sequence in the molecule and having cell adhesion activity and cell activation activity .

The artificial polypeptide used in the present invention has an Arg-Gly-Asp sequence in its molecule.
In addition, the artificial polypeptide only needs to exhibit cell adhesion activity. The Arg-Gly-Asp sequence is an essential unit for cell adhesion [Nature 309].
Arg-Gly-, Vol. 30-33 (1984)].
Examples of natural polypeptides containing an Asp sequence in a molecule include fibronectin, thrombin, hydronectin, von Willeland factor, collagen, discodin I, etc. [Proceedings of the National Academy of Sciences of the
USA (Proc. Natl. Acad. Sci. USA) Vol. 81, No. 59
85-5988 (1984)]. The polypeptide used in the present invention includes, for example, Ar
An artificial polypeptide containing the g-Gly-Asp sequence can be prepared and used by protein engineering, chemical synthesis, or genetic engineering. For example, as an artificial polypeptide containing an Arg-Gly-Asp sequence in fibronectin, for example, a 30 amino acid residue polypeptide or a 108 amino acid residue polypeptide described in JP-A-59-501548 may be used. JP-A-2-97
No. 397, a polypeptide represented by SEQ ID NO: 1 in the sequence listing may be used. Other artificial polypeptides having the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing and having cell adhesion activity include, for example, JP-A-1-18009.
No. 00, No. 1-206998 and No. 2-152990, and these polypeptides may be used.

Further, it has an Arg-Gly-Asp sequence,
A chimeric polypeptide of a polypeptide having cell adhesion activity and a polypeptide having another physiological function may be prepared by genetic engineering and used, for example, containing the polypeptide represented by SEQ ID NO: 1 in the sequence listing. Japanese Patent Application Laid-Open Nos. 1-261398 and 2-31
No. 1498, No. 3-59000, No. 3-232898
Gazettes, Japanese Patent Application Nos. 2-80676 and 2-15962.
No. 4 and No. 3-117886.

Further, a polypeptide obtained by transferring a polypeptide containing an Arg-Gly-Asp sequence into a polypeptide having another physiological function by genetic engineering may be used. For example, JP-A-3-34996 , Japanese Patent Application No. 3-1
For example, a polypeptide to which an Arg-Gly-Asp sequence described in Japanese Patent No. 62521 is transferred can be used.

[0008] Further, Arg-like compounds described in JP-A-2-174797 and JP-A-3-173828 may be used.
A cyclic polypeptide containing a Gly-Asp sequence;
A chain polypeptide containing a repeating sequence of the rg-Gly-Asp sequence may be synthesized and used.

As described above, Arg used in the present invention is
Artificial polypeptides having the -Gly-Asp sequence can be prepared by various techniques. Whether or not they have cell adhesion activity may be measured, for example, according to the method of Luoslaty et al. [Methods in Enzymology ( M
ethods in Enzymology), Vol. 82, 803-831
P. (1981)]. That is, the prepared sample is dissolved in PBS or the like, and adsorbed on a microtiter plate.
After blocking with SA, pre-cultured B
Add HK or NRK cells and incubate at 37 ° C. By observing the spread of cells under a microscope,
The presence or absence of cell adhesion activity is determined, and a polypeptide having cell adhesion activity may be used. As described in each specification, these polypeptides can be prepared in large quantities and as a uniform preparation.

[0010] When these polypeptides are used as a cell activator for pearl culture, they may be formulated as needed using excipients, carriers and the like. Excipients, carriers and the like are selected from those which do not adversely affect the shellfish, and for example, water, alcohols or animal and vegetable oils such as soybean oil, olive oil and mineral oil, or synthetic oils are used as liquid carriers. As the solid carrier, sugars such as maltose and sucrose, amino acids, cellulose derivatives such as hydroxypropylcellulose, and organic acid salts such as magnesium stearate are used.

In the case of a liquid, the dissolving solution is, for example, a physiological saline solution,
Various buffer solutions, glucose, inositol, mannitol, saccharide solutions such as lactose, ethylene glycol,
Glycols such as polyethylene glycol are preferred. In addition, lyophilized preparations with activators such as sugars such as inositol, mannitol, lactose and sucrose, and amino acids such as phenylalanine, and suitable dissolution solutions at the time of use, for example, sterile water, physiological saline solution, glucose solution, electrolyte solution, amino acid solution, etc. Can also be administered after dissolving in a liquid. Although the content of the polypeptide used in the present invention in the preparation varies depending on the preparation, it is usually 0.1 to 100% by weight.
Preferably it is 1 to 98% by weight. For example, in the case of a liquid preparation, it is usually 0.0001 to 1% by weight, preferably 0.001 to 1% by weight.
It is desirable to include 0.1% by weight of the active ingredient.

The activator of the present invention is described, for example, in JP-A No. 2-9 / 1990.
No. 7397 can also be prepared by dissolving a lyophilized preparation of a 274 amino acid residue polypeptide having the amino acid sequence represented by SEQ ID NO: 1 in a PBS solution in distilled water with distilled water. For example, the polypeptide
A 0.001-0.1% (W / V) PBS solution may be prepared, and then the mantle section of the mother pearl culture shell may be processed. For example, an antibiotic may be added to the solution, and a coloring agent such as phenol red may be added. As a method for treating the slice, for example, immersion treatment in an aqueous solution, application treatment of the aqueous solution, or the like may be performed, and the slice may be activated with the polypeptide.

When cultivation is performed using mantle sections treated with the polypeptide, the ratio of the number of beaches to the number of treatments is higher than that of the control, and the productivity of pearls is improved. Also,
The ratio of the number of merchandise beads to the number of beached fish is higher than the control,
The commercialization rate is also improved.

[0014]

Next, the present invention will be described in detail with reference to examples, but the scope of the present invention is not limited to the examples.

Example 1 2 prepared by the method described in JP-A-2-97397
A freeze-dried product of a 74 amino acid residue polypeptide in PBS solution was dissolved in distilled water, and a 0.001% (W / V) solution (hereinafter referred to as solution A) and a 0.01% (W / V) solution (hereinafter referred to as solution A) were used. B) was prepared. A group of 1500 pearl oyster shells per group
The mantle section and the untreated section treated with the solution and the solution B were transferred together with nuclei, respectively, and cultured. Table 1 shows the current year's number of beach beads and the number of beads after removing lower and upper scraps.
In addition, the quality of the beads was evaluated on a three-point scale for the winding and the grain (3 in the table is excellent, 2 is good, and 1 is normal).

[0016]

[Table 1] Table 1 個数 Number of treatments Number of beaches Number of beads Volume A Liquid A 1500 849 626 3 2 Liquid B 1500 858 645 3 3 Unprocessed 1500 826 574 22 2 ──────────────────────────────────

As shown in Table 1, high quality pearls could be obtained with high productivity by using Liquid A and Liquid B.

Example 2 A mantle section and a nucleus treated with the solution A were transferred to 1840 mother shells according to Example 1, and a mantle section and a nucleus treated with the solution B were transferred to 1800 mother shells. Transplanted. Untreated sections and nuclei were transferred to 1806 mother mussels and cultured for the current year.
As shown in Table 2, high quality pearls could be efficiently obtained by using the solution A and the solution B.

[0019]

[Table 2] Table 2 個数 Number of treatments Number of beaches Number of beads Color A Liquid A 1840 1026 735 33 Liquid B 1800 1069 793 33 No treatment 1806 973 651 32

Example 3 A solution A and a B solution were prepared in the same manner as in Example 1, and commercially available platonin neoxin [manufactured by Nippon Kogaku Dye Laboratories, Inc.]
Was used as a control, and one tube of each of the above was completely dissolved in 40 ml of distilled water. According to Example 1, the mantle section and the nucleus treated with the solution A were placed at two places on each of the 1440 mother shells, and the mantle section and the nucleus treated with the solution B were placed at two places on each of the 1280 mother shells. And the mantle section and nucleus treated with a platenin / neoxin solution
Untreated mantle sections and nuclei were transferred to each of two sites of 440 mother shells, and further to each of two sites of 3040 mother shells, and were cultured for the current year. As shown in Table 3, solution A,
By using the liquid B, a good quality pearl could be obtained efficiently.

[0021]

[Table 3] Table 3 個数 Number of treatments Number of beaches Number of beads Winding color ────────────────────────────────── liquid A 1440 × 2 1621 1115 3 2 B liquid 1280 × 2 1626 1140 3 3 Platonin / neoxin solution 1440 × 2 1602 1098 3 2 No treatment 3040 × 2 3116 1920 22 2 ────────

Example 4 According to Example 3, the mantle section and nucleus treated with solution A
The mantle sections and nuclei treated with solution B were placed in each of two places of 80 mother mussels, and the mantle sections and nuclei treated with platonin / neoxin solution were placed in each of two places of 1280 mother shells.
The untreated mantle section and the nucleus were transferred to each of two places of 0 mother shells, and further to each of two places of 2760 mother shells, and the current year's cultivation was carried out. As shown in Table 4, high quality pearls could be efficiently obtained by using solution A and solution B.

[0023]

[Table 4] Table 4 個数 Number of treatments Number of beaches Number of beads Volume A 液 Liquid A 1280 × 2 1583 1112 23 B Liquid 1280 × 2 1574 1140 3 3 Platonin / neoxin solution 1920 × 2 1894 1236 23 No treatment 2720 × 2 3006 1789 22 2 ────────

The production rate (number of hamage / number of treatments × 100) and the commercialization rate (number of pearl beads / number of treatments × 100) when the solution A, the solution B, and the platonin / neoxin solution were used and in the case of no treatment, respectively. ) And average quality are summarized in Table 5 below.

[0025]

[Table 5] Table 5 ────────────────────────────────── Treatment Total treatment Total Sogohama Total product production Ratio Commercialization Volume Color Number Qty. Number of beads Ratio ────────────────────────────────── Solution A 8780 5079 3588 57.8 40.9 2.8 2.5 Solution B 8420 5127 3718 60.9 44.2 3.0 3.0 Platonin / neoxin solution 6720 3496 2334 52.0 34.7 2.5 2.5 No treatment 14826 7921 4934 53.4 33.3 2.3 2.0 処理────────────────

As shown in Table 5, by using the activator of the present invention, both the production rate and the commercialization rate are increased, and pearls of good quality can be obtained.

[0027]

Industrial Applicability According to the present invention, it is possible to industrially provide a cell activator for pearl culture having high safety and uniform quality, and by using the activator, pearls of good quality can be efficiently produced. Can be produced.

[Sequence list]

 SEQ ID NO: 1 Sequence length: 274 Sequence type: amino acid Number of chains: single-stranded Topology: linear Sequence type: peptide Sequence: Pro Thr Asp Leu Arg Phe Thr Asn Ile Gly Pro Asp Thr Met Arg 1 5 10 15 Val Thr Trp Ala Pro Pro Pro Ser Ile Asp Leu Thr Asn Phe Leu 20 25 30 Val Arg Tyr Ser Pro Val Lys Asn Glu Glu Asp Val Ala Glu Leu 35 40 45 Ser Ile Ser Pro Ser Asp Asn Ala Val Val Leu Thr Asn Leu Leu 50 55 60 Pro Gly Thr Glu Tyr Val Val Ser Val Ser Ser Val Tyr Glu Gln 65 70 75 His Glu Ser Thr Pro Leu Arg Gly Arg Gln Lys Thr Gly Leu Asp 80 85 90 Ser Pro Thr Gly Ile Asp Phe Ser Asp Ile Thr Ala Asn Ser Phe 95 100 105 Thr Val His Trp Ile Ala Pro Arg Ala Thr Ile Thr Gly Tyr Arg 110 115 120 Ile Arg His His Pro Glu His Phe Ser Gly Arg Pro Arg Glu Asp 125 130 135 Arg Val Pro His Ser Arg Asn Ser Ile Thr Leu Thr Asn Leu Thr 140 145 150 Pro Gly Thr Glu Tyr Val Val Ser Ile Val Ala Leu Asn Gly Arg 155 160 165 Glu Glu Ser Pro Leu Leu Ile Gly Gln Gln Ser Thr Val Ser Asp 170 175180 Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr Ser Leu 185 190 195 Leu Ile Ser Trp Asp Ala Pro Ala Val Thr Val Arg Tyr Tyr Arg 200 205 210 Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 215 220 225 Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu Lys 230 235 240 Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Gly Arg 245 250 255 Gly Asp Ser Pro Ala Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg 260 265 270 Thr Glu Ile Asp

────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Ikunoyuki Kato 3-4-1, Seta, Otsu City, Shiga Prefecture Inside Takara Shuzo Co., Ltd. Central Research Laboratory (56) References JP-A-1-148135 (JP, A Michael D.). et al. , Proc. Natl. Acad. Sc i. USA. , Vol. 81, p. 5985-5988 (1984) (58) Fields investigated (Int. Cl. ⁷ , DB name) A01K 61/00 BIOSIS (DIALOG)

Claims (3)

(57) [Claims] 1. A method for treating a mantle section of a pearl culture mother mussel.
Pearl culture cell activator used
It has -gly-Asp sequence and cell adhesion activity and cell vehicles
A mantle cell activator for pearl culture, comprising an artificial polypeptide having an active activity . 2. A method comprising the sequence represented by SEQ ID NO: 1 in the sequence listing.
The mantle cell activator for pearl culture according to claim 1, wherein the activator comprises an artificial polypeptide . (3) The person whose sequence is represented by SEQ ID NO: 1 in the sequence listing
2. The method according to claim 1, which comprises a polypeptide of the present invention.
The mantle cell activator for pearl culture according to the above.