JP2004143165A - Periodontium regenerant, method for extracting active ingredient of the same, and plasmid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a periodontium regenerant capable of effectively recovering the periodontium which is removed when a periodontal disease, or the like, is treated. <P>SOLUTION: This periodontium regenerant contains a sheath protein derived from a mammal as an active ingredient, wherein the sheath protein derived from a pig and having an amino acid sequence of VPAFPRQPGTPGVASLSLETMRQLGSLQGLNMLSQYSRFGFGKSFNSLWMHGLLPPHSSFQWMRPREHETQQYEYSLPVHPPPLPSQPSLQPQQPGQKPFLQPTVVTSIQNPVQKGVPQPPIYQGHPPLQQVEGPMVQQQVAPSEKPPEAELPGLDFADPQDPSMFPIAR is exemplified. The active ingredient is contained in an amount of 0.0001-3 mg per 100 mg of the periodontium regenerant. The sheath protein associated with amelogenin or enamelin may be contained as the active ingredient, and in this case, the active ingredient is contained in an amount of 0.0003-10 mg per 100 mg of the regenerant. <P>COPYRIGHT: (C)2004,JPO

Description

The present invention relates to a periodontal tissue regenerating agent capable of effectively recovering periodontal tissue, particularly cementum, periodontal ligament removed by treatment of periodontal disease, etc., and an effective component extraction method thereof, and further its effectiveness The present invention relates to a plasmid that can be used for production of components.

Plaque and calculus generated by the bacteria and their products in the mouth adhering to the teeth cause tooth decay and periodontal disease. Periodontal disease affects the periodontal ligament and cementum, and it has been known that alveolar bone is lost and teeth fall out as it progresses. About 80% of adults in Japan suffer from periodontal disease. It is said to have been affected.
Periodontal disease is treated by removing the causative plaque, calculus, and defective granulation to regenerate and recover new periodontal tissue, but remove plaque, calculus, and defective granulation. However, it is difficult to regenerate the removed periodontal tissue even though it is possible to prevent the progression of periodontal disease.

Now, the present inventors have succeeded in separating sheathin, which is an enamel protein consisting of 421 amino acids, from the juvenile enamel, and have also revealed the gene sequence (J Dent. Res. 76 (2): 648-657).
In addition, as a result of investigations on the portion of this sheathrin that once plays a role in enamel formation, for example, in the case of pigs, the amino acid sequence of the 27th to 196th stages is included in the 421 amino acid sequence constituting the sheathrin. It was discovered that an enamel protein having a molecular weight of 13 to 17 kDa has the activity, and this was named sheath protein.

By the way, it has recently been clarified that these enamel proteins involved in the formation of enamel are also involved in the regeneration of periodontal tissues, and young enamel was prepared from porcine permanent tooth germs. Preparations obtained by solubilizing with 5M acetic acid solution, dialyzing quickly, freeze-drying as acetic acid soluble fraction and further processing regenerate periodontal tissue lost due to treatment of periodontal disease, etc. It has been developed as a drug that can be used (trade name “EMDOGAIN” manufactured by Seikagaku Corporation). The main component of this drug is amelogenin, which is used for the purpose of regenerating periodontal tissue after periodontal disease treatment, but contains multiple proteins other than those that act on periodontal tissue regeneration. In addition, there are problems such as the possibility of causing side effects depending on the individual used. Further, there is a problem that the content of the active ingredient contained per unit dose of emdogain is small, and a sufficient amount effective for the regeneration of periodontal tissue cannot be applied to the treatment site.
J. et al. Dent. Res. 76 (2): 648-657

The present invention exhibits a periodontal tissue regeneration action quickly and with only a small amount of application, and does not contain extra protein in addition to the enamel protein directly involved in periodontal tissue regeneration as an active ingredient. Therefore, it is an object of the present invention to provide a periodontal tissue regenerating agent that hardly causes side effects due to this, a method for extracting the active ingredient, and a plasmid that can be used for producing the active ingredient.

As a result of diligent research to solve the above problems, the inventors of the present invention not only have an effect on enamel formation on a sheath protein composed of a specific number of steps in the amino acid sequence constituting sheathrin. The inventors have found that there is an excellent periodontal tissue regeneration action, and have completed the present invention.
That is, the present invention is, periodontal tissue regeneration agent characterized by containing the sheath proteins from mammalian as active ingredient, a porcine, by containing a sheath protein having the amino acid sequence of VPAFPRQPGTPGVASLSLETMRQLGSLQGLNMLSQYSRFGFGKSFNSLWMHGLLPPHSSFQWMRPREHETQQYEYSLPVHPPPLPSQPSLQPQQPGQKPFLQPTVVTSIQNPVQKGVPQPPIYQGHPPLQQVEGPMVQQQVAPSEKPPEAELPGLDFADPQDPSMFPIAR as an active ingredient Periodontal tissue regenerative agent characterized by human origin, VPFFPQQSGTPGMASLSLETMRQLGSLQRLNTLSQYSRYGFGKSFNSLWMHGLLPPHSSLPWMRPREHETQQYEYSLPVHPPPLPSQPSLKPQQPGLKPFLQSAAATTNQATALKEALQPPIHLGHLPLQ EGELPLVQQQVAPSDKPPKPELPGVDFADPQGPSLPGMDFPDPQGPSLPGLDFADPQGSTIFQIAR contains a sheath protein having an amino acid sequence as an active ingredient, and it is preferable to contain 0.0001 to 5 mg of the active ingredient in 100 mg of the regenerant.
In the present invention, the above-described sheath protein may be one in which amelogenin and enamelin are associated, and the active ingredient in this case is preferably contained in 0.0003 to 10 mg in 100 mg of periodontal tissue regenerating agent. . Furthermore, the gist of the present invention is a periodontal tissue regenerating agent comprising a peptide derived from a sheath protein.

In addition, the present invention comprises (1) preparing a young enamel from a mammalian tooth germ, (2) adding a neutral buffer thereto, and homogenizing at a low temperature to obtain a neutral insoluble fraction. (3) Alkaline buffer solution is added to this and homogenized at a low temperature to obtain an alkali-soluble fraction. (4) The alkali-soluble fraction is concentrated with an ultrafiltration membrane and (5) subjected to chromatography. Periodontal tissue regenerating agent characterized by fractionating eluted fraction, (6) performing ion exchange on this fraction, and fractionating first eluted fraction (1) Prepare young enamel from mammalian tooth germs, (2) Add alkaline buffer to this and stir extract, centrifuge to remove precipitates, (3) Ultrafiltration of the obtained sample The active ingredient extraction method of the periodontal tissue regeneration agent characterized by performing an operation that is desalted also as its gist, also as its gist the plasmid characterized by having a DNA encoding a sheath protein.

In the present invention, the periodontal tissue means gums, cementum, periodontal ligament, gingiva, alveolar bone and the like. Therefore, the periodontal tissue regenerating agent of the present invention has the purpose of regenerating those tissues, but is particularly excellent mainly in the regeneration action of cementum and periodontal ligament, and other periodontal tissues are secondary. Have a regenerative action.
The periodontal tissue regenerative agent of the present invention comprises a portion having an action of regenerating periodontal tissue, ie, sheath protein, as an active ingredient in sheathrin which is an enamel protein derived from juvenile enamel prepared from a mammalian tooth germ. Is. Specifically, in the case of pigs, enamel protein having a molecular weight 13~17kDa having an amino acid sequence consisting VPAFPRQPGTPGVASLSLETMRQLGSLQGLNMLSQYSRFGFGKSFNSLWMHGLLPPHSSFQWMRPREHETQQYEYSLPVHPPPLPSQPSLQPQQPGQKPFLQPTVVTSIQNPVQKGVPQPPIYQGHPPLQQVEGPMVQQQVAPSEKPPEAELPGLDFADPQDPSMFPIAR is 27-196 stage of the 421 amino acid sequence constituting the sheathlin is sheath proteins, in humans, VPFFPQQSGTPGMASLSLETMRQLGSLQRLNTLSQYSRYGFGKSFNSLWMHGLLPPHSSLPWMRPREHETQQYEYSLPVHPPPLPSQPSLKPQQ which is the 27th to 222nd stage of the 447 amino acid sequence constituting the sheathlin PGLKPFLQSAAATTNQATALKEALQPPIHLGHLPLQEGELPLVQQQVAPSDKPPKPELPGVDFADPQGPSLPGMDFPDPQGPSLPGLDFADPQGSTIFQIAR enamel protein having a molecular weight of 13 to 19 kDa is a sheath protein.
In addition, although it is a fact that sheath protein contains the part required for periodontal tissue reproduction | regeneration efficiently compared with sheathlin etc., for example, it contains parts other than the part directly related to periodontal tissue reproduction | regeneration. There is a possibility that not all the amino acid sequences are necessary. That is, the amino acid sequence constituting the sheath protein may include a site with a stronger periodontal tissue regeneration action, and by using such a site, that is, a peptide derived from the sheath protein, the amino acid sequence can be more efficiently used. In some cases, periodontal tissue regeneration can be obtained.

The sheath protein contains 0.0001 to 5 mg, preferably 0.001 to 3 mg, more preferably about 0.01 to 1 mg of the periodontal tissue as an active ingredient in 100 mg of the periodontal tissue regenerating agent of the present invention. It is possible to sufficiently exhibit the regenerating action.
In addition, the sheath protein that is an active ingredient of the periodontal tissue regenerating agent of the present invention may be associated with enamel proteins such as amelogenin and enamelin, and it is better to use a sheath protein in which these enamel proteins are associated. Peripheral tissue regeneration may be exerted. In the case of using a complex in which sheath protein is associated with amelogenin or enamelin, the content of the active ingredient composed of the complex is 0.0003 to 10 mg, preferably 0.003 to 100 mg of the periodontal tissue regenerating agent of the present invention. 003 to 5 mg, more preferably about 0.03 to 3 mg.
If the amount of the active ingredient is less than the above, the periodontal tissue regeneration action may not be sufficiently obtained. If the amount is more than the above, the effect is saturated, but the cost becomes high and economically disadvantageous. Become.

The sheath protein that is an active ingredient of the periodontal tissue regenerating agent of the present invention may be prepared by extraction from a mammalian tooth germ, or prepared as a recombinant protein by using a plasmid having a DNA encoding the sheath protein. You may do it. Examples of suitable mammals include pigs, cows, monkeys, rats, horses, humans and the like, and among them, pigs, humans and the like can be preferably used. In any of the above methods, since preparation of sheath protein requires a tooth germ and / or erupted tooth, it is preferable to use an individual at an early stage for both, specifically, for example, in the case of a pig, after birth It is preferable to use an individual of about 5 to 7 months.

In the present invention, there are two sheath protein extraction methods. The first method is (1) preparing a young enamel from a mammalian tooth germ, (2) adding a neutral buffer to this, and homogenizing at a low temperature to obtain a neutral insoluble fraction. 3) Add alkaline buffer to this and homogenize at low temperature to obtain an alkali-soluble fraction. (4) Concentrate the alkali-soluble fraction with an ultrafiltration membrane, (5) Chromatography, elution first (6) This is a method in which ion exchange is performed on this fraction and the fraction that elutes first is collected, which may not be suitable for mass production. The degree of purification of the extracted sheath protein is high and the extraction effect is also high.
In this extraction method, when it is desired to further increase the degree of purification of the sheath protein, gel filtration may be performed in the guanidine solution while suppressing the association of other enamel proteins, if necessary.

To prepare juvenile enamel in (1), remove the tooth germ from the jawbone of a young mammal, remove the pulp and wash it, and then the cheese-like hard part, that is, the juvenile enamel in the substrate formation stage What is necessary is just to scrape only the surface layer part from the quality. The reason why only the surface layer portion is separated is that there is a neonatal juvenile enamel (that is, a juvenile enamel that has just been made) in this portion, and a strong periodontal tissue regeneration action exists in this portion. The tooth germ can be used for both permanent teeth and deciduous teeth. At this time, it is advisable to remove unnecessary tooth debris. The neutral buffer used in (2) is added in an amount of 5 to 50 ml, preferably 10 to 30 ml per gram of juvenile enamel, homogenized at a low temperature, that is, 0 to 8 ° C., preferably 2 to 5 ° C., and centrifuged. By repeating the operation of separating and removing the supernatant 3 to 4 times, the crude sheath protein from which proteases and the like have been removed remains as an insoluble matter, that is, a neutral insoluble fraction. In addition, when repeating this operation, it is better to be careful not to raise the temperature of the sample too much. The alkaline buffer used in (3) is added to the obtained neutral insoluble fraction in an amount of 5 to 50 ml, preferably 10 to 30 ml, and homogenized at a low temperature, that is, 0 to 8 ° C., preferably 2 to 5 ° C. Then, an alkali-soluble fraction can be obtained by centrifugation.

In the above-described sheath protein extraction method, the neutral buffer used in (2) is a buffer having a pH of 6 to 8, preferably 7.0 to 7.6, more preferably 7.2 to 7.4. Specifically, phosphate buffer, Tris-HCl buffer, Sorensen buffer and the like can be used, but phosphate buffer is preferably used, and 10 to 100 mM, preferably 40 to It is preferable to use about 60 mM. The alkaline buffer used in (3) refers to a buffer having a pH of 9 to 13, preferably 10 to 11, more preferably 10.5 to 10.8. Specifically, a carbonate buffer, an ammonia solution, etc. However, it is preferable to use a weak salt buffer such as a carbonate buffer, and it is preferable to use a solution of about 10 to 100 mM, preferably about 40 to 60 mM. The reason why the steps (2) and (3) are performed at this temperature is to increase the extraction efficiency of the sheath protein. If the extraction operation is performed at a temperature higher than this, the extraction efficiency is extremely reduced. May end up. As the ultrafiltration membrane used in (4), those having a molecular weight of about 1 to 3 kDa, specifically YM-1 or Ultracell PLAC (manufactured by Millipore) may be used. (5) As chromatography, gel filtration may be performed using a column such as Sephadex G-100, Sephadex G-100 Superfine or Sepharose 6B (Amersham Pharmacia Biotech), or TSKgel G3000PW (Tosoh Corporation). High-performance liquid chromatography may be performed using a column such as that manufactured by the same company, but preferably gel filtration using Sephadex G-100 Superfine is performed, which is the same as the buffer used in (3). It may be used after equilibration with a buffer solution. Ion exchange in (6) is performed in Whatman Q (trade name “EXPRESS-ION EXCHANGER Q” manufactured by Whatman) in 5-8 M urea (pH adjusted to around 7.4 with a buffer solution). A cellulose ion exchanger is used. Of the above alkali-soluble fraction, a maximum of about 15% by weight is eluted as this fraction, ie, sheath protein.

In order to further increase the purity of the sheath protein obtained as described above, it is 2 to 8 M, preferably 3.5 to 4.5 M in guanidine solution, or 2 to 10 M, preferably 6 if necessary. Gel filtration chromatography may be performed in ~ 8M urea. Examples of the column used include Cellulofine GCL-2000 (manufactured by Chisso Corporation), TSKgel G3000 (manufactured by Tosoh Corporation), TSKgel G4000PW (manufactured by Tosoh Corporation), and the like. Further, this step is a step for suppressing the association of amelogenin or enamelin with sheath protein, and the above solvent may be used instead of protein extraction and gel filtration of the first fraction.
Further, when it is desired to obtain the sheath protein in the absence of an aggregate, in addition to the above steps, for example, RESOURCE RPC (Pharmacia Biotech) is used as a carrier, and in reverse phase in a trifluoroacetic acid-acetonitrile solution. Chromatography may be performed.
In the above step (3), a small amount of the crude sheath protein is adhered to what remains as crystals without shifting to the alkali-soluble fraction side. Therefore, acetic acid can be added to the crystals to elute the crude sheath protein and remove it. Thereafter, the steps (4) to (6) above may be performed to collect the sheath protein.

The second method is (1) preparing juvenile enamel from mammalian tooth germs, (2) adding alkaline buffer solution to this, stirring and extracting, centrifuging to remove precipitates, and under low temperature This is a method in which ammonium sulfate fractionation is performed, and (3) the obtained sample is desalted with an ultrafiltration membrane, and the extraction and purification effect of the sheath protein is lower than that of the first method, but mass production is necessary. It is effective when.
To prepare juvenile enamel in (1) in this method, fractionation may be carried out in the same manner as in (1) in the first method. All of them may be used not only on the surface layer but also in the hard part, that is, juvenile enamel at the substrate formation stage. The tooth germ can be used for both permanent teeth and deciduous teeth. The juvenile enamel can be separated by removing the pulp of the extracted tooth germ and washing it, and then scraping it out or putting it into a buffer solution and stirring vigorously with a stirrer. At this time, it is advisable to remove unnecessary tooth debris.

After preparing the juvenile enamel in (1), you may proceed immediately to the step (2), but if necessary, the step (2) in the first method, ie, neutral buffer The step of removing protease and the like may be performed.
In the second method, the alkaline buffer and, if necessary, the neutral buffer may be the same as in the first method, but the neutral buffer is preferably 10 to 50 ml per gram of juvenile enamel. 20-40 ml, alkaline buffer is 10-50 ml per gram of juvenile enamel, or 10 grams per gram of precipitate obtained when step (2) in the first method is performed, preferably 20 Use ~ 40 ml. Extraction in this method does not require the use of a homogenizer, and a buffer solution is added in each step of step (2) in the first method and step (2) of this method, as necessary. Stirring may be performed overnight, that is, for about 16 to 24 hours.

Subsequent ammonium sulfate fractionation is performed at a low temperature, that is, at 0 to 8 ° C., preferably at 2 to 5 ° C., and specifically in ice water or the like. Specifically, the ammonium sulfate fractionation is performed as follows. First, ammonium sulfate is added so as to be 5 to 10% by weight, preferably 6 to 7% by weight of the saturated ammonium sulfate, and the resulting precipitate is removed. Next, ammonium sulfate is added to 40 to 50% by weight, preferably 45 to 46% by weight, and the resulting precipitate is collected. Ammonium sulfate is desalted by dissolving it in about 10 to 50 mM acetic acid and using an ultrafiltration membrane similar to that used in the first method. Moreover, you may freeze-dry the sample obtained as needed.
In the present extraction method, when it is desired to further increase the degree of purification of the sheath protein, the following operation (5) in the first method may be performed in addition to the above operation as necessary.

The sheath protein obtained by any of the above methods may be obtained as a complex associated with amelogenin or enamelin, but even if this complex is used as an active ingredient of the periodontal tissue regenerating agent of the present invention, no matter what. There is no hindrance.
In addition, when formulating the sheath protein prepared by extraction, in order to inactivate the protease contained in the obtained sheath protein as necessary after extraction and to prevent infection of the individual virus, etc., sterilization It is preferable to carry out the treatment. Specifically, for example, E-type virus possessed by pigs may be sterilized by heating at about 80 ° C. for about 30 minutes, and other mammals may also be sterilized according to the respective conditions. However, it is generally good to perform sterilization for about 10 to 60 minutes at about 60 to 100 ° C. If the sterilization treatment is performed at a higher temperature for a longer time than necessary, the sheath protein as an active ingredient may be denatured and deactivated. In some cases, the regenerative action is improved.
The sheath protein extracted in this way is an active ingredient of the periodontal tissue regenerating agent of the present invention, and of course has an action related to enamel formation, but has an excellent periodontal tissue regenerating action. Can be used as a therapeutic agent for the purpose of regenerating the removed periodontal tissue after treating mainly dental diseases such as periodontal disease, and used for treatment of gingival retraction by brushing or treatment It can be used as a therapeutic agent aimed at regenerating periodontal tissue by dealing with root exposure by dentures, etc., and can also be used as a biomaterial constituting an implant surface.

The sheath protein extracted as described above may be used as it is or dissolved in pure water or the like, but it can be safely put in the mouth and has a high viscosity PGA ( When it is used by dissolving in propylene glycol alginate) or hyaluronic acid, it is held at the site to be coated and is easily fixed. In addition to the sheath protein, which is an active ingredient, in order to make it easy to use for the above-mentioned applications, proliferation such as anti-inflammatory agents, bulking agents, fragrances, cell growth growth factor (TGF) and bone morphogenetic factor (BMP) It is good also as a periodontal tissue regeneration agent by adding a factor.
Moreover, it can be expected that the periodontal tissue regeneration action can be obtained more effectively by combining the treatment using the periodontal tissue regeneration agent of the present invention and the treatment such as GTR method.

Moreover, the sheath protein which is an active ingredient of the periodontal tissue regenerating agent of the present invention includes the amino acid sequence of human sheathlin (underlined portion is the sheath protein) (the sheath protein is the 27th to 222nd steps), and the base sequence ( The DNA encoding the sheath protein is 154 to 741), and Table 2 (pig sheathin (underlined is the sheath protein) amino acid sequence (sheath protein is 27 to 196), and the base sequence (sheath protein is The encoded DNA has been cloned at the gene level as in the 167th to 676th steps)), and can be produced in large quantities by genetic engineering or culture engineering. That is, by transforming bacteria such as Escherichia coli and fungi such as yeast, or animal cells such as mammals and insects with plasmids having DNA encoding sheath protein, and culturing the obtained cells under appropriate conditions, A large amount of sheath protein can be prepared as a recombinant protein.

[Table 1]
10 20 30 40 50 60
gtcctgaaaaaaattttaatcttcttttcttagaactatcttggttggcatcatcaggcc

70 80 90 100 110 120
ctgagggcacagtgcatgtcagcatctaagattccacttttcaaaatgaaggacctgata
M S A S K I P L F K M K D L I

130 140 150 160 170 180
ctgatcctatgcctcctggaaatgagttttgca gtgccgttctttcctcagcaatctgga
L I L C L L E M S F A V P F F P Q Q S G

190 200 210 220 230 240
acaccgggtatggctagtttgagccttgagacaatgagacagttgggaagtctgcagaga
T P G M A S L S L E T M R Q L G S L Q R

250 260 270 280 290 300
ttaaacacactttctcagtattctagatacggctttggaaaatcatttaattctttgtgg
L N T L S Q Y S R Y G F G K S F N S L W

310 320 330 340 350 360
atgcacggtctcctcccaccacattcctctcttccatggatgaggccaagagaacatgaa
M H G L L P P H S S L P W M R P R E H E

370 380 390 400 410 420
actcaacagtatgaatattctttgcctgtgcatcccccacctctcccatcacagccatcc
T Q Q Y E Y S L P V H P P P L P S Q P S

430 440 450 460 470 480
ttgaagcctcaacagccaggactgaaaccttttctccagtctgctgctgcaaccaccaac
L K P Q Q P G L K P F L Q S A A A T T N

490 500 510 520 530 540
caggccacagcactgaaagaagcacttcagcctccaattcacctgggacatctgcccttg
Q A T A L K E A L Q P P I H L G H L P L

550 560 570 580 590 600
caggaaggagaactgcctctggttcagcagcaggtggcaccatcagataagccaccaaag
Q E G E L P L V Q Q Q V A P S D K P P K

610 620 630 640 650 660
cctgagctcccaggagtagattttgctgatccacaaggtccatcactcccaggaatggat
P E L P G V D F A D P Q G P S L P G M D

670 680 690 700 710 720
tttcctgatccacaaggtccatcactcccaggattggattttgctgatccacaaggttca
F P D P Q G P S L P G L D F A D P Q G S

730 740 750 760 770 780
acaattttccaaatagcccgt ttgatttctcacggaccaatgccacaaaataaacaatct
T I F Q I A R L I S H G P M P Q N K Q S

790 800 810 820 830 840
ccactttatccaggaatgttgtacgtgccttttggagcaaatcaattgaatgcccctgcc
P L Y P G M L Y V P F G A N Q L N A P A

850 860 870 880 890 900
agacttggcatcatgagttcagaagaagtggcaggcgggagagaagacccaatggcctat
R L G I M S S E E V A G G R E D P M A Y

910 920 930 940 950 960
ggagccatgtttccaggatttggaggcatgaggcccggctttgagggaatgccccacaac
G A M F P G F G G M R P G F E G M P H N

970 980 990 1000 1010 1020
ccagctatgggcggtgacttcactctggaatttgactccccagtggctgccaccaaaggc
P A M G G D F T L E F D S P V A A T K G

1030 1040 1050 1060 1070 1080
cctgagaacgaagaaggaggtgcacaaggctcccctatgccggaggccaacccagacaat
P E N E E G G A Q G S P M P E A N P D N

1090 1100 1110 1120 1130 1140
ctagaaaacccagctttccttacagagctagaacctgctccccacgcagggctccttgct
L E N P A F L T E L E P A P H A G L L A

1150 1160 1170 1180 1190 1200
ctccctaaggatgacattcccggcctgccaaggagcccttcagggaagatgaagggactc
L P K D D I P G L P R S P S G K M K G L

1210 1220 1230 1240 1250 1260
cccagcgtcaccccagcagctgctgacccactgatgacccctgaattagctgatgtttat
P S V T P A A A D P L M T P E L A D V Y

1270 1280 1290 1300 1310 1320
aggacctacgatgctgacatgaccacatccgtggatttccaggaagaagcaaccatggat
R T Y D A D M T T S V D F Q E E A T M D

1330 1340 1350 1360 1370 1380
accacgatggccccaaactctctgcaaacatccatgccaggaaacaaagcccaggagccc
T T M A P N S L Q T S M P G N K A Q E P

1390 1400 1410 1420 1430 1440
gagatgatgcatgacgcatggcatttccaagagccctgacagctctaagatattagctac
E M M H D A W H F Q E P *

1450 1460 1470 1480 1490 1500
tttctgtatgcacaagcttcccagctttgtccccacagtgtacctttttgctaaaacact

1510 1520 1530 1540 1550 1560
tattacccttctgcagcaaaggcattaaaagcgctaagcatatattaataaatgcaagtg

1570 1580 1590 1600 1610 1620
gctagaaatagtgtaggtccccttcttgctttcaatatcttgttgaaataaaatgtgtca

1630 1640 1650
attgtctctgtgaaaaaaaaaaaaaaaaa

[Table 2]
10 20 30 40 50 60
gtcccaggctacagtccagaaaaaaaatttaatcttcttttcttagagctatcttggttg

70 80 90 100 110 120
gcatcaccaggccctgagagcactgtgcatgccagcattgaagattccacttttcaaaat
M P A L K I P L F K M

130 140 150 160 170 180
gaaggacatggtactgatcctgtgcctcctgaaaatgagttctgca gtgccggcatttcc
K D M V L I L C L L K M S S A V P A F P

190 200 210 220 230 240
tcggcaacctggaaccccaggtgtggctagtttgagccttgagacaatgagacagttggg
R Q P G T P G V A S L S L E T M R Q L G

250 260 270 280 290 300
aagtttgcagggactaaacatgctttctcagtattcaagatttggctttgggaaatcatt
S L Q G L N M L S Q Y S R F G F G K S F

310 320 330 340 350 360
taattctctgtggatgcatggtctccttccaccacactcctccttccaatggatgagacc
N S L W M H G L L P P H S S F Q W M R P

370 380 390 400 410 420
aagggaacatgaaactcaacagtatgaatattctttgcctgtgcaccccccacctctccc
R E H E T Q Q Y E Y S L P V H P P P L P

430 440 450 460 470 480
atcgcagccatccttgcagcctcaacagccgggacagaagcctttcctccagcccactgt
S Q P S L Q P Q Q P G Q K P F L Q P T V

490 500 510 520 530 540
cgtaacctccatccagaacccagtccagaagggagtacctcagcctccaatttaccaggg
V T S I Q N P V Q K G V P Q P P I Y Q G

550 560 570 580 590 600
acatccgcccttgcagcaagtagaggggccaatggttcagcagcaggtggcaccatcaga
H P P L Q Q V E G P M V Q Q Q V A P S E

610 620 630 640 650 660
aaagccaccagaggccgagctaccaggactggattttgctgatccacaagatccatcgat
K P P E A E L P G L D F A D P Q D P S M

670 680 690 700 710 720
gtttccaatagcccgt ttgatatctcagggaccagtgccacaagataagccatctccact
F P I A R L I S Q G P V P Q D K P S P L

730 740 750 760 770 780
ttatccaggaatgttttacatgtcctacggagcaaatcaattgaactctcctgccagact
Y P G M F Y M S Y G A N Q L N S P A R L

790 800 810 820 830 840
tggcatcctgagctcagaagaaatggcgggaggaagaggaggccccttggcctatggagc
G I L S S E E M A G G R G G P L A Y G A

850 860 870 880 890 900
catgttcccaggatttggaggcatgaggcccaaccttggagggatgccccccaactcagc
M F P G F G G M R P N L G G M P P N S A

910 920 930 940 950 960
caagggtggggactttactctggagtttgactccccagctgctggaaccaaaggccccga
K G G D F T L E F D S P A A G T K G P E

970 980 990 1000 1010 1020
gaagggagaaggaggtgcagaaggctctccagtggcggaggccaacacagctgatcctga
K G E G G A E G S P V A E A N T A D P E

1030 1040 1050 1060 1070 1080
aagcccagctctcttttcagaggtagcatctggtgtcctcggagggcttcttgctaatcc
S P A L F S E V A S G V L G G L L A N P

1090 1100 1110 1120 1130 1140
aaagggcaaaattcccaacctggcaagaggccctgcagggcggagcaggggaccccccgg
K G K I P N L A R G P A G R S R G P P G

1150 1160 1170 1180 1190 1200
ggtgaccccagcagatgctgacccactgatgacccccggattagctgatgcttatgagac
V T P A D A D P L M T P G L A D A Y E T

1210 1220 1230 1240 1250 1260
ctacggtgctgatgagaccacaactctgggtctccaggaagaaatgaccatggattccac
Y G A D E T T T L G L Q E E M T M D S T

1270 1280 1290 1300 1310 1320
agcgaccccatacagtgagcatacatcaatgccaggcaacaaagcccagcagccccagat
A T P Y S E H T S M P G N K A Q Q P Q I

1330 1340 1350 1360 1370 1380
taagcgtgatgcgtggcgtttccaagagccctgagagccttgaaatatcggctactttct
K R D A W R F Q E P *

1390 1400 1410 1420 1430 1440
gtatgcacaagctccccagctttgtccccatagtatatctttttgcaaaaacacttatta

1450 1460 1470 1480 1490 1500
cccttctgcagcaaaggcattaaaagcgctaagcacatattaataaatacaagtggctag

1510 1520 1530 1540 1550 1560
aaatagtataggtcctcttcttgctttcattctcttattgaaataaaatgtgtcacctgt

1570 1580 1590 1600 1610 1620
cactgtgatttagaaacacttttaatgacatgagagcaagtctaagggtctctgcattca

1630 1640 1650 1660 1670 1680
aaaatcagtttcttagctgtcttggcattatgaatttctcttactagcatgacactgtta

1690 1700 1710 1720 1730 1740
tatccaggaaatgtgacactgctttggaaattttactctaagcaaaggcacatattctta

1750 1760 1770 1780 1790 1800
gaattataagttatttcactcaaacgttattaaacagggtttggtggacaatccctgact

1810 1820 1830 1840 1850 1860
ggtattactgggtttggtatattggattttaaattctcatttgtagagtattttatttaa

1870 1880 1890 1900 1910
tctactaaaaacatttagcctattttaaataaagaatttttctcactg

A plasmid having DNA encoding a sheath protein can be prepared in the following manner. First, mammalian tooth germs and / or erupted teeth are prepared, and a cell group containing enamel blasts and / or odontoblasts in the matrix-forming stage or mature stage is prepared. Tooth germs and / or erupted teeth may be permanent teeth or deciduous teeth. Total RNA is extracted from the obtained cell group by a conventional method to prepare cDNA thereof. Using appropriate primers, DNA encoding sheath protein is amplified by PCR, etc., and the target gene is prepared by PCR using appropriate primers to which restriction enzyme cleavage sites have been added. The target gene obtained above is cleaved at a cleavage site using a restriction enzyme, and then ligated to obtain a target plasmid, that is, a plasmid having DNA encoding a sheath protein. Specifically, for example, in the case of humans, it is a plasmid having the base sequence of 154 to 741 in the base sequences described in Table 1, and in the case of pigs, it is 167 of the base sequences described in Table 2. A plasmid having a base sequence of ˜676th stage.
The DNA encoding the above-mentioned sheath protein may include DNA encoding a portion other than the portion directly related to periodontal tissue regeneration, and the entire base sequence may not always be necessary. That is, the DNA encoding the above-mentioned sheath protein may include a region having a stronger periodontal tissue regeneration action, that is, a DNA encoding a peptide derived from the sheath protein. As described above, a plasmid can be prepared in the same manner as described above. In some cases, a recombinant protein can be obtained more efficiently by using such a plasmid.

As the vector to be used, a pET vector or the like is used when the host is Escherichia coli or the like, a baker's yeast or the like, a Pichia expression vector or the like, or a pcDNA3 vector or the like in the case of cultured mammalian cells.
As the host, animal cells such as Escherichia coli, baker's yeast, and human-derived 293 strain cells can be used.
A large amount of sheath protein can be extracted from the culture solution by introducing the plasmid obtained as described above into a host cell and performing appropriate culture.

The periodontal tissue regenerative agent of the present invention exhibits a periodontal tissue regeneration action quickly and excellently only by applying a small amount, and in addition to the enamel protein directly involved in periodontal tissue regeneration as an active ingredient. Since it does not contain extra protein, it also has effects such as almost no side effects.
Moreover, if the active ingredient extraction method or plasmid of the present invention is used, a large amount of sheath protein can be obtained.

Example 1
Permanent tooth germs of incisors were removed from the mandible of 6 months old pigs, and the surrounding soft tissues and pulp were removed. After washing this with physiological saline and wiping off the water, the surface of the cheese-like hardness portion (ie, the neonatal juvenile enamel during the substrate formation stage) was scraped about 60 μm. Although the yield varies depending on the maturity of the tooth germ, about 150 mg of neonatal juvenile enamel was obtained from the incisors for 50 pigs. 20 ml of 50 mM phosphate buffer (pH 7.0) per gram is added to this newborn juvenile enamel and stirred for a total of 30 seconds at 6000 rpm at 4 ° C using a homogenizer (manufactured by Polytron). Since the temperature rose during the extraction, the mixture was extracted by occasionally stopping stirring or using an ice-water bath, and centrifuged to remove the supernatant. This operation was repeated three times. 20 ml of 50 mM carbonate buffer (pH 10.8) per gram was added to the obtained insoluble matter, homogenized at 2 ° C., and centrifuged to obtain 35 mg of an alkali-soluble fraction. The alkali-soluble fraction is concentrated as it is using an ultrafiltration membrane YM-1, and fractionated by gel filtration using a Sephadex G-100 column equilibrated with 50 mM carbonate buffer (pH 10.8). The fraction eluted first was collected. This fraction was further ion-exchanged with Whatman Q cellulose ion exchanger in 6 M urea (50 mM Tris buffer, pH 7.4), and the first eluted fraction was collected to obtain a sheath protein. 4.5 mg of a complex in which small amounts of amelogenin and enamelin were associated was extracted (of which 0.5 to 1.5 mg of sheath protein).

Example 2
Permanent tooth germs were removed from the mandible of the pig about 6 months old, and the surrounding soft tissues and pulp were removed. After washing this with physiological saline and wiping off the moisture, the hard portion for cheese (that is, the young enamel during the substrate formation stage) was shaved and collected. About 120 mg of juvenile enamel was obtained from the incisor for one pig. To the collected sample, 30 ml of 50 mM phosphate buffer (pH 7 to 7.4) per gram was added and stirred at 4 ° C. overnight. The precipitate was collected by centrifugation, and the same volume of 50 mM carbonate buffer (pH 10.8) was added to the precipitate and stirred overnight. The precipitate was removed by centrifugation, and the following ammonium sulfate fractionation was performed in ice water. First, ammonium sulfate is added to 6% by weight of saturated ammonium sulfate, and the resulting precipitate is removed. Next, ammonium sulfate is added to 45% by weight, and the resulting precipitate is collected. The collected precipitate was dissolved with 50 mM acetic acid to remove ammonium sulfate, desalted with ultrafiltration membrane YM-1, and freeze-dried to obtain a complex in which amelogenin and enamelin were associated with 3.5 mg of sheath protein.

Example 3
Permanent tooth germs of incisors were removed from the mandible of cows about 12 months old, and the surrounding soft tissues and pulp were removed. After washing this with physiological saline and wiping off the water, the surface of the cheese-like hardness portion (ie, the neonatal juvenile enamel during the substrate formation stage) was scraped about 60 μm. Although the yield varied depending on the degree of maturity of the tooth germ, about 25 mg of neonatal juvenile enamel was obtained from the incisors of 20 cattle. 20 ml of 50 mM phosphate buffer (pH 7.0) per gram is added to this newborn juvenile enamel and stirred for a total of 30 seconds at 6000 rpm at 4 ° C using a homogenizer (manufactured by Polytron). Since the temperature rose during the extraction, the mixture was extracted by occasionally stopping stirring or using an ice-water bath, and centrifuged to remove the supernatant. This operation was repeated three times. 20 ml of 50 mM carbonate buffer (pH 10.8) per gram was added to the obtained insoluble matter, homogenized at 2 ° C., and centrifuged to obtain 6 mg of an alkali-soluble fraction. The alkali-soluble fraction is concentrated using an ultrafiltration membrane YM-1 as it is, and is subjected to high performance liquid chromatography using a column of TSKgel G3000PW (manufactured by Tosoh Corporation) equilibrated with 50 mM carbonate buffer (pH 10.8). Fractionation by gel filtration was performed by chromatography, and the fraction eluted first was collected. This fraction was further subjected to gel filtration by high performance liquid chromatography using a column of TSKgel G4000PW (manufactured by Tosoh Corporation) in 4M guanidine solution (50 mM Tris buffer, pH 7.4), and the fraction eluted last. About 0.2 mg of sheath protein containing amelogenin and enamelin was extracted (of which 0.05 to 0.1 mg of sheath protein).

Example 4
Periodontal tissue regenerating agent 1 was prepared by dissolving 0.09 mg of the sheath protein extracted in Example 1 in 100 mg of 6% PGA solution. In addition, 0.5 mg of the sheath protein lyophilized product extracted in Example 2 was dissolved in 100 mg of 6% PGA solution to prepare periodontal tissue regenerating agent 2.
The periodontal ligament in contact with the incisor on the labial side of the dog was peeled off, and the alveolar bone, periodontal ligament and cementum were scraped off and removed. Here, the periodontal tissue regenerating agents 1 and 2 prepared above were applied, and the state of the gingiva after 56 days was observed with an optical microscope (× 10). The micrographs are shown in FIGS. 1 and 2, respectively.
As a control, the state after 56 days of the periodontal tissue to which nothing was applied is shown in FIG. 3, and the state after 56 days of the periodontal tissue to which Emdogain 30 mg / ml was applied is shown in FIG.
As is clear from FIGS. 1 to 4, the periodontal tissue regenerated with sheath protein (FIGS. 1 and 2) is 2 to 8 times as much as the regenerated amount (FIG. 4). 1, 2, and 4, the portion indicated by the arrow indicates the regenerated portion), and that the sheath protein has an extremely excellent effect in the regeneration of periodontal tissue Understand.

Example 5
The surrounding soft tissue and pulp were removed from the human first premolars (erupted teeth) that were removed for treatment. After washing this with physiological saline and wiping off the moisture, the teeth were split along the vertical axis to remove the pulp, and the odontoblasts remaining on the surface of the dentin were scraped to obtain a cell group sample. Total RNA was extracted from this sample by a conventional method, and their cDNAs were prepared.
DNA encoding the sheath protein (underlined portion in Table 1) was amplified by the PCR method using the following primers.

Sense primer 5'- gtgccgttctttcctcagcaa -3 '
Antisense primer 5'- atgggctatttggaaaattgt -3 '

The amplified DNA has a restriction enzyme (HindIII) cleavage site at the 5 ′ end, a KOZAK sequence necessary for mRNA synthesis, a methionine (M) codon, and a restriction enzyme (EcoRI) cleavage site at the 3 ′ end. , And a stop codon were added by the PCR method using the following primers to obtain the target gene.

Base sequence of primer used for addition

M
5'- aagcttccaccatggtgccgttctttcctcagcaa -3 '
HindIII KOZAK

5'- gaattctcaatgggctatttggaaaattgt-3 '
EcoRI stop

The obtained target gene and pcDNA3 vector are cleaved with restriction enzymes (EcoRI and HindIII), joined with ligase and incorporated into the pcDNA3 vector to obtain a plasmid having DNA (human) encoding a sheath protein. Obtained.

Example 6
An incisor permanent tooth germ was removed from the mandible of 6 months old pig, the surface of the soft tissue surrounding the tooth germ was removed, and a cell group containing the deep enamel blasts was prepared. Total RNA was extracted from the cell group containing the enamel blasts by a conventional method, and their cDNAs were prepared.
DNA encoding the sheath protein (underlined portion in Table 1) was amplified by the PCR method using the following primers.

Sense primer 5'- gtgccggcatttcctcggcaa -3 '
Antisense primer 5'- acgggctattggaaacatcga -3 '

For the amplified DNA, a restriction enzyme (HindIII) cleavage site and a restriction enzyme (EcoRI) cleavage site were added to both ends of the 5 ′ end and 3 ′ end by PCR using the following primers. I got the gene.

Base sequence of primer used for addition

5'- ccgaattcgtgccggcatttcctcggcaa -3 '
EcoRI
5'- caagcttcacgggctattggaaacatcga -3 '
HindIII

The obtained gene of interest and pET vector are cleaved with restriction enzymes (EcoRI and HindIII), joined with ligase, and the gene of interest is incorporated into the pET vector, so that a plasmid having DNA (pig) encoding a sheath protein is obtained. Obtained.

Since the periodontal tissue regenerating agent of the present invention has an excellent periodontal tissue regeneration action, the treatment aimed at regenerating the removed periodontal tissue after mainly treating dental diseases such as periodontal disease It can be used as a medicine, and can also be used as a therapeutic drug for the purpose of regenerating periodontal tissue to cope with gingival recession due to brushing and tooth root exposure due to dentures used during treatment. It can also be used as a biomaterial constituting the surface.
Moreover, it can be expected that the periodontal tissue regeneration action can be obtained more effectively by combining the treatment using the periodontal tissue regeneration agent of the present invention and the treatment such as GTR method.

The microscope picture which showed the mode of the periodontal tissue which apply | coated the regeneration agent using the sheath protein obtained in Example 1. FIG. The microscope picture which showed the mode of the periodontal tissue which apply | coated the regeneration agent using the sheath protein obtained in Example 2. FIG. A photomicrograph showing the periodontal tissue with nothing applied. A photomicrograph showing the state of periodontal tissue coated with emdogain.

Claims (10)

Periodontal tissue regenerative agent characterized by containing a sheath protein derived from mammal as an active ingredient. A porcine, periodontal tissue regeneration agent characterized by containing the sheath protein having the amino acid sequence of VPAFPRQPGTPGVASLSLETMRQLGSLQGLNMLSQYSRFGFGKSFNSLWMHGLLPPHSSFQWMRPREHETQQYEYSLPVHPPPLPSQPSLQPQQPGQKPFLQPTVVTSIQNPVQKGVPQPPIYQGHPPLQQVEGPMVQQQVAPSEKPPEAELPGLDFADPQDPSMFPIAR as an active ingredient. Human origin and VPFFPQQSGTPGMASLSLETMRQLGSLQRLNTLSQYSRYGFGKSFNSLWMHGLLPPHSSLPWMRPREHETQQYEYSLPVHPPPLPSQPSLKPQQPGLKPFLQSAAATTNQATALKEALQPPIHLGHLPLQEGELPLVQQGPS The periodontal tissue regenerative agent according to any one of claims 1 to 3, comprising 0.0001 to 5 mg of an active ingredient in 100 mg of the periodontal tissue regenerative agent. The periodontal tissue regenerating agent according to any one of claims 1 to 3, wherein the sheath protein is an association of amelogenin and enamelin. 6. The periodontal tissue regenerative agent according to claim 5, comprising 0.0003 to 10 mg of an active ingredient in 100 mg of the periodontal tissue regenerative agent. A periodontal tissue regenerating agent comprising the sheath protein-derived peptide according to any one of claims 1 to 3 as an active ingredient. (1) Prepare juvenile enamel from mammalian tooth germ, (2) Add neutral buffer to this and homogenize at low temperature to obtain neutral insoluble fraction, (3) Alkaline buffer Add the liquid and homogenize at low temperature to obtain an alkali-soluble fraction. (4) Concentrate the alkali-soluble fraction with an ultrafiltration membrane and (5) Chromatography. (6) A method for extracting an active ingredient of a periodontal tissue regenerating agent, comprising performing an operation of performing ion exchange on this fraction and fractionating a fraction that elutes first. (1) Prepare juvenile enamel from mammalian tooth germs, (2) Add alkaline buffer to this and extract with stirring, centrifuge to remove precipitates, and perform ammonium sulfate fractionation at low temperature. (3) A method for extracting an active ingredient of a periodontal tissue regenerating agent, which comprises performing an operation of desalting the obtained sample with an ultrafiltration membrane. A plasmid characterized by having DNA encoding a sheath protein.

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