JP3713568B2 - Novel polypeptide gene cDNA, vector containing the cDNA, host cell transformed with the vector, polypeptide produced thereby, method for producing the same, DNA encoding the polypeptide, and antibody of the polypeptide - Google Patents

Description

【００７８】
【配列表】
配列番号：４
配列の長さ：３３３１
配列の型：核酸
トポロジー：直鎖状
配列の種類：ｃＤＮＡ to ｍＲＮＡ
起源
生物名：アコヤガイ(Pinctada Fucata)
細胞の種類：外套膜上皮細胞
配列の特徴
特徴を表わす記号：ＣＤＳ
存在位置：５０．．２２６３
特徴を決定した方法：Ｐ

TAAATTATTT GACATTATCC GCCATTTGAC TCATTTTTCT TAGTTCTCTA TGTTTTATAC 2323
TTCACCTTAG ATTGTTTTAG TTTGATTGAA TAAATTATGT TTTCGATATA AATTTTTTTT 2383
AAATTAAATT AAACTTTATT AGTTGACCTG TAAACTTTTT CATGGAGTTA TAATCTAAGG 2443
AACAAAAAAC ATACATAATA TGTTCAGTAT TGTGGTAAAG CACCTGTACC GCAAACACAA 2503
TCACCTCTAT ACATGTATAC AAAATCAGTA ATGCTGACAA AATCTTCTAC ACTCTCACCT 2563
ACACACTCGC ACACAGTCCT CTTACATACA CAGCACTATA ATATCCTGAA CATGAAGTTT 2623
GTGTTGATAA AAAGTTCAGA AAAATCTCCC CTACATCACC TGATCTTTCA CTGAAAATTT 2683
ACGACAAGTA TTGAAAATAG CAGAAAGAAA ACGGGAAATT GAGAAGTTTT CTATAAAAAA 2743
CAATCGGAAC AATGACTGGA ATGACAAGGA TGAAAATAAT GATAACTTAC ATTAATTAAG 2803
GCCCCAATAA TCTCTCTATT TTCAAACTTT TTTTTCAAAT GTTCTCTCTA ACTCACTTGC 2863
ATCTATGTGG AAATTCACAT ACTATACTAA ATTACCACAA GTATCAAGGT TTCACAACCT 2923
CTCATGCCTT CATGGCAGAC CATGCTGGGT ATTTGTCTAA CAATGCCTCA TAAATACATA 2983
AAACTAACTA ACAAAATAGG TCAGTCTGTA ACAAATTATT AATGCACCAT TATTGCATTT 3043
TCTAAAACAA AGCATACACT GGATATTGGC AGACAAAATG TTGTTATTGG ATACCTTTCC 3103
ATTCTATCTA GACACTTGCT TTCCACAAGT CATCATAAAT AAATCCCCCC TATCCCAAAT 3163
GTCAATGGAA TGCCCCAACC CTTCCCCCAT AATTTTAAAA CCTAGAATAA ATTAAAACAT 3223
CTATAGTTCG TCATGATCAT CTTTCTTATC ATCCTCTTCT TCTTCCTCCT CCTCCTTCTT 3283
CTTCTTCCTC CTCCTCAGGT TCTTGGCTGC CTGCTCCTTC CTTGCCAA 3331
【図面の簡単な説明】
【図１】本発明のクローニングベクターの一実施例を示す構成図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a cDNA of a novel polypeptide gene produced by mantle tissue (mantle epithelial cells) of Japanese pearl oyster (Pincta fucata), a vector containing the cDNA, a host cell transformed with the vector, Relates to a polypeptide produced by the method, a method for producing the same, a DNA encoding the polypeptide, an antibody of the polypeptide, and a cosmetic containing the polypeptide or a degradation peptide.
[0002]
[Object of invention]
Various factors related to the maintenance, proliferation and growth of cells are produced from the cells. For example, from the pearl oyster mantle epithelial cells, factors relating to shell formation, factors relating to biological defense, and many other factors are produced. The present inventors paid attention to this point, and intensively studied to find a novel factor (polypeptide) produced by certain types of cells (for example, pearl oyster mantle epithelial cells).
[0003]
Another object is to develop a new raw material for cosmetics or a new method for selecting pieces in pearl culture (a method for selecting pieces containing a large amount of the polypeptide).
[0004]
[Prior art and problems to be solved by the invention]
Conventionally, when obtaining a specific polypeptide or DNA encoding the same, the target polypeptide is confirmed in tissue or cell culture medium, and then the gene is cloned through isolation and purification of the polypeptide. Or the method of expression cloning of genes using their biological activity as an indicator.
[0005]
However, there is very little information on factors produced by the pearl oyster mantle epithelial cells, and many of them show insolubility, which makes it difficult to isolate, purify, and confirm biological activity.
[0006]
On the other hand, cDNA production technology and sequencing technology have rapidly developed, and it has become possible to rapidly sequence a large amount of cDNA. In addition, the development of reverse genetics methods for fixing the function of a gene from a gene is also remarkable.
[0007]
Therefore, the present inventors have decided to find a novel polypeptide using these methods. That is, mRNA was isolated from pearl oyster mantle epithelial cells and the like, cDNA was obtained using this as a starting material, its nucleotide sequence was determined, and amino acid sequence was deduced. As a result, the inventors succeeded in finding a DNA encoding a novel polypeptide and completed the present invention.
[0008]
The known nucleotide and amino acid sequences registered in the nucleic acid sequence database (EMBL-GDB Rel. 41) and protein database (SWISS-PROT Rel. 30) were examined. It was. Therefore, it was confirmed that the DNA and polypeptide of the present invention are completely novel.
[0009]
Here, the polypeptide secreted by the pearl oyster mantle epithelial cells will be described. The pearl oyster covers its outer surface with a strong shell to protect itself from foreign enemies. It is known that this shell responsible for a kind of biological defense is made by mantle epithelial cells. The shell is mainly composed of calcium carbonate crystals and polypeptides. Furthermore, the pearl oyster shell has a structural difference consisting of three layers, which are called the shell layer, the ridge pillar layer, and the pearl layer from the outside. This structural difference is believed to be due to differences in polypeptides.
[0010]
The only difference in the polypeptide constituting each layer, known to date, is the difference in the amino acid composition. This is because most polypeptides are insoluble in water and most other solvents, and this is a factor that delays the analysis of these polypeptides.
[0011]
Among these polypeptides, the polypeptide constituting the nacre has been used as various medicines such as cold medicines, stomach medicines, etc., and is still used in some areas. In addition, it has been used as an active cosmetic ingredient from the past to the present.
[0012]
In addition, the nacre on the shell structure is considered to be equivalent to the pearl that is widely loved by people as a gem. A pearl is created in about 1 to 3 years by transplanting a round core made of various shells into a pearl oyster gonad along with a piece of tissue of the pearl oyster mantle.
[0013]
From this, it can be seen that these polypeptides play a very important role for the colors and windings that greatly affect the value of pearls as gems.
[0014]
[Means for Solving the Problems]
The present invention is as follows. That is,
(Claim 1) A cDNA encoding the nucleotide sequence of SEQ ID NO: 1.
[0015]
(Claim 2) A cDNA encoding the nucleotide sequence of SEQ ID NO: 2.
[0016]
(Claim 3) A cloning vector containing the cDNA of claim 1 or 2.
[0017]
  (Claim 4) The nucleotide sequence represented by SEQ ID NO: 1Consist ofDNA.
[0018]
  (Claim 5) The nucleotide sequence represented by SEQ ID NO: 2Consist ofDNA.
[0019]
(Claim 6) An expression vector containing the cDNA according to claim 1 or 2.
[0020]
  (Claim 7) The expression vector of claim 6 is retained.FormTransformant.
[0021]
  (Claim 8) Produced by the transformant of claim 7., Encoded by the cDNA of claim 1 or claim 2Polypeptide.
[0022]
  (Claim 9) The amino acid sequence of SEQ ID NO: 3Consist ofThe polypeptide of claim 8.
[0023]
(Claim 10) A method for producing a polypeptide, comprising culturing the transformant according to claim 7 under conditions for expressing the polypeptide according to claim 8 or 9.
[0024]
(11) An antibody prepared by using the polypeptide according to claim 8 or 9 as an antigen.
[0025]
DETAILED DESCRIPTION OF THE INVENTION
In the above, DNA having the base sequence represented by SEQ ID NO: 1 (or 2) (Claims 4 and 5) generally means that 90% or more, for example, 95%, 98% or 99% of DNA is SEQ ID NO: 1 ( Or it means that it is DNA which has a base sequence shown by 2). The homologous fragment means a portion of at least 10 bases, preferably at least 15 bases, such as 20, 25, 30 or 40 bases, and such fragments are also included in the DNA of the present invention (similar effects) Is obtained).
[0026]
The polypeptide encoded by the DNA having the base sequence represented by SEQ ID NO: 1 and the polypeptide having the amino acid sequence represented by SEQ ID NO: 3 are generally 90% or more, for example, 95, 98 or 99% of the polypeptide, It means a polypeptide having an amino acid sequence encoded by a DNA having the base sequence represented by SEQ ID NO: 1. Their homologue fragments are generally at least 20, preferably at least 30, for example 40, 60 or 100 consecutive amino acid regions, at least 70%, preferably at least 80 or 90%, more preferably 95%. These are homologous, and such homologues are hereinafter described as polypeptides of the present invention (similar effects can be obtained).
[0027]
The DNA that selectively hybridizes to the DNA having the base sequence shown in SEQ ID NO: 1 or 2 is generally at least 20, preferably at least 30, for example, 40, 60, or 100 consecutive base sequence regions. And at least 70%, preferably at least 80 or 90%, more preferably 95% or more homologous, and such DNA is hereinafter described as the DNA of the present invention (similar effects can be obtained). ).
[0028]
Furthermore, the present invention includes a cloning or expression vector comprising the DNA of the present invention. Examples of the vector include a plasmid, virus, or phage vector comprising an ori region and, if necessary, a promoter for expression of the DNA, a promoter control factor, and the like. The vector may contain one or more selective marker genes, such as an ampicillin resistance gene. The vector can be used in vitro, for example, for the production of RNA corresponding to DNA and transformation of host cells.
[0029]
Furthermore, the present invention also includes a host cell transformed with a vector for replicating or expressing the DNA of the present invention, which comprises DNA having the nucleotide sequence represented by SEQ ID NO: 1 or 2, or an open reading frame thereof. included. Examples of the cell include bacteria, yeast, insects, insect cells and mammalian cells (non-human when introduced directly into a living body).
[0030]
Further, the present invention includes a method for producing the polypeptide of the present invention, which comprises culturing the host cell of the present invention under conditions for expressing the polypeptide of the present invention. Culturing is preferably performed under conditions where the polypeptide of the present invention is expressed and produced from a host cell.
[0031]
Antisense RNA can also be produced by inserting the DNA of the present invention into the antisense region of the vector as described above. Such antisense RNA can also be used to control the level of a polypeptide of the invention in a cell.
[0032]
Further, the DNA of the present invention, its homologue or fragment can be used as a probe for Northern analysis or as a primer for PCR (Polymerase Chain Reaction), so that the expression level of the polypeptide between each solid, Can be compared.
[0033]
The present invention also includes monoclonal or polyclonal antibodies of the polypeptides of the present invention. The present invention further includes a method for producing a monoclonal or polyclonal antibody of the polypeptide of the present invention. A monoclonal antibody can be produced by a conventional hybridoma technique using the polypeptide of the present invention or a fragment thereof as an antigen. Polyclonal antibodies can be produced by a conventional method such as inoculating a host animal such as a rat or rabbit (non-human) with the polypeptide of the present invention and collecting the serum.
[0034]
As the polypeptide of the present invention, in addition to the polypeptide having the amino acid sequence shown by SEQ ID NO: 3, a part thereof is deleted, a part thereof is substituted with another amino acid (for example, amino acids having similar physical properties) And those having other amino acids added or inserted in part thereof are also included.
[0035]
As is well known, 1 to 6 codons encoding one amino acid are known. Therefore, the base sequence of DNA can be changed without changing the amino acid sequence of the polypeptide.
[0036]
The DNA of the present invention represented by SEQ ID NO: 1 includes all the base sequence groups encoding the polypeptide represented by SEQ ID NO: 3. By changing the base sequence, production of the polypeptide may be improved.
[0037]
The DNA of the present invention represented by SEQ ID NO: 2 is one embodiment of the DNA represented by SEQ ID NO: 1 and represents a natural sequence.
[0038]
The DNA having the base sequence represented by SEQ ID NO: 2 is produced according to the following method. That is,
(I) isolating mRNA from the pearl oyster mantle tissue producing the polypeptide of the present invention,
(II) synthesizing single-stranded DNA (first strand) and then double-stranded DNA (second strand) from the mRNA (synthesis of cDNA);
(III) incorporating the cDNA into an appropriate phage vector;
(IV) After packaging, infect host cells with the resulting recombinant infectious phage (preparation of cDNA library),
(V) screening the obtained cDNA library with a specific probe;
(VI) The obtained clone can be prepared by sequencing its full length.
[0039]
In addition, step (I) is described in the method of Chomczynski, P. et al. [Anal. Biochem., 162, 156 (1987), using the pearl oyster mantle as a material. ] Is performed according to.
[0040]
Steps (II), (III) and (IV) are steps for preparing a cDNA library and are described in the modified Gubler & Hoffman method [Gene., 25, 263 (1983). ] Is performed according to. There are many known phage vectors used in the step (III) (for example, λgt10, λgt11). The phage vectors referred to here are phage vectors that can be converted into plasmids by infecting helper phages (Phagemid). Is also included.
[0041]
The step (V) is performed according to the method described in Molecular Cloning [Sambrook, J, Fritsch, E.F., and Maniatis, T. Cold Spring Harbor Laboratory Press, (1989)].
[0042]
The sequencing of the step (VI) is performed by the Maxam-Gilbert method or the dideoxy terminator method.
[0043]
The DNA thus obtained (A) converts its base sequence into an amino acid sequence in a possible reading frame, (B) a hydrophobic profile is prepared from the obtained amino acid sequence, and an initiation codon (ATG) ) Immediately after (the secreted protein has a high hydrophobic region signal peptide at its N-terminus), and (C) the DNA is full-length or almost full-length. It is necessary to confirm that there is.
[0044]
During the above confirmation operation, (C) is performed by Northern analysis. Once the nucleotide sequences shown in SEQ ID NOs: 1 and 2 are confirmed, the DNA sequence is then synthesized by chemical synthesis, by PCR (Polymerase Chain Reaction) method, or by hybridizing a fragment of the nucleotide sequence as a probe. The inventive DNA can be obtained. Furthermore, a necessary amount of the DNA of the present invention can be obtained by introducing a vector DNA containing the DNA of the present invention into an appropriate host and propagating it.
[0045]
Examples of the method for obtaining the polypeptide of the present invention include (1) a method for isolation and purification from living organisms or cultured cells, (2) a method for peptide synthesis, or (3) a method for production using genetic recombination techniques. However, industrially, the method described in (3) is preferable.
[0046]
Examples of an expression system (host-vector system) for producing a polypeptide using a gene recombination technique include expression systems for bacteria, yeast, insects, insect cells, and mammalian cells.
[0047]
For example, when expressing in Escherichia coli, a DNA having the base sequence represented by SEQ ID NO: 1 is connected downstream of a suitable promoter (eg, trp promoter, lac promoter, λPL promoter, T7 promoter, etc.). Then, it is inserted into a vector that functions in E. coli (for example, pBR322, pUC18, pUC19, etc.) to prepare an expression vector. Alternatively, it can also be prepared by connecting the DNA represented by SEQ ID NO: 1 to a commercially available expression vector (for example, pKK223-3: Pharmacia, pET vector: Stratagene, pTV vector: Takara, etc.).
[0048]
Examples of E. coli include E. coli. coli JM109, E. coli. coli HB101, E. coli. E. coli DH5 strain can be used, but is not limited thereto.
[0049]
Next, E. coli transformed with this expression vector is cultured in an appropriate medium, and the desired polypeptide can be obtained from the cells. Further, if a bacterial signal peptide (for example, a pelB signal peptide) is used, the target polypeptide can be secreted into the periplasm. Furthermore, it can be produced as a fusion protein with other polypeptides to obtain the desired polypeptide.
[0050]
For expression in insects or insect cells, for example, a transfer vector is prepared by inserting the DNA represented by SEQ ID NO: 1 or 2 into an appropriate vector. Next, a recombinant virus is produced by co-transfecting with a vector having a promoter of baculovirus (Autographa california NPV). The obtained recombinant virus is introduced into an appropriate insect cell (for example, Sf9, Sf21, or Tn5 cell) or the body of an insect (for example, silkworm) to produce the desired polypeptide.
[0051]
When expressed in mammalian cells, for example, the DNA represented by SEQ ID NO: 1 or 2 in an appropriate vector (for example, a retrovirus vector, papilloma virus vector, vaccinia virus vector, SV40 vector, etc.) An expression vector is prepared by inserting it downstream of an appropriate promoter (for example, SV40 promoter, LTR promoter, metallothionein promoter, etc.). Next, an appropriate mammalian cell (eg, monkey COS cell, Chinese hamster CHO cell, mouse L cell, etc.) is transformed with the obtained expression vector, and the transformant is cultured in an appropriate medium. The desired polypeptide is produced in the cell. The polypeptide obtained as described above can be isolated and purified by a general biochemical method.
[0052]
The polypeptide of the present invention can be applied to cosmetics. That is, the polypeptide of the present invention can be mixed with other commonly used components to make a cosmetic composition. In addition, for example, the PCR method makes it possible to develop a new method for selecting pieces in pearl culture (a method for selecting pieces containing a large amount of the polypeptide).
[0053]
【Example】
The present invention will be described more specifically with reference to the following examples, but these examples do not limit the scope of the present invention.
[0054]
Example 1 (Separation and purification of mRNA)
Mantle epithelial cells of the pearl oyster (Pinctada fucata) [The pearl oyster is readily available at home and abroad today. These mantle epithelial cells cannot withstand cryopreservation, and other storage methods and storage conditions (such as culture medium) have not yet been found. As)) cannot survive. For this reason, the acceptance of this cell was rejected by the Patent Microorganism Depositary Center of the Biotechnology Institute of Industrial Technology. Acknowledgment certificate rejected (displayed for identification: P. fucata-M01). Neither the pearl oyster himself was accepted. As described above, Chomczynski, P. et al. [Anal. Biochem., 162, 156 (1987)] uses about 3 g of the above-mentioned pearl oyster (Pinctada fucata) which is stored by the applicant himself and can be transferred. ] To isolate total RNA.
[0055]
That is, the tissue was homogenized in 4M GTC solution (4M guanidine thiocyanate, 25 mM sodium citrate, 0.5% sarkosyl, 0.1M 2-mercaptoethanol), phenol / chloroform extraction was performed twice, isopropanol was added, and centrifugation was performed. (10,000 × g, 10 minutes) and about 200 μg of total RNA was recovered during precipitation. Furthermore, using oligo (dT) latex particles (sold by TAKARA), mRNA was separated and 2.5 μg of Poly (A)⁺RNA was purified and recovered.
[0056]
Example 2 (Preparation of cDNA library)
The cDNA library was prepared by a modification of the Gubler & Hoffman method [described in Gene., 25, 263 (1983)].
[0057]
That is, Poly (A) separated and purified in Example 1⁺Single-stranded DNA (first strand) was synthesized from 2.5 μg of RNA by reverse transcriptase using a random hexamer primer and an oligo (dT) primer. Subsequently, after synthesizing double-stranded DNA (second strand), unreacted primers and RNA were removed by gel filtration column chromatography, and EcoRI adapter was ligated. Again, the unreacted adapter was removed by gel filtration column chromatography, and the cDNA fraction was recovered.
[0058]
The above cDNA synthesis step was performed using a time saver cDNA synthesis kit (Time Saver cDNA Synthesis kit, sold by Pharmacia).
[0059]
After ligation of the cDNA prepared above and the λgt10 phage vector, packaging was performed using lambdain (In vitro Packaging Kit LAMBDA IN, sold by Nippon Gene), and the titer was confirmed by a conventional method. As a result, the number of independent clones was 5.5 × 10.^FiveA pfu cDNA library was obtained.
[0060]
Example 3 (Cloning)
From the cDNA library obtained in Example 2, a LB plate of 10 cm × 14 cm was prepared to have a density of about 10,000 pfu / plate. On the other hand, based on the data already obtained by the present inventors, a 20mer synthetic nucleotide was prepared by a requested synthesis (sold by Biologica), and this was converted into [λ-³²Isotope labeling using P] adenosine triphosphate and T4 polynucleotide kinase produced a labeled probe. Next, screening by plaque hybridization was performed according to the method described in Molecular Cloning [Sambrook, J., Fritsch, E.F., and Maniatis, T. Cold Spring Harbor Laboratory Press, (1989)], and a number of positive clones were obtained.
[0061]
Example 4 (Clone analysis)
Further, the DNA of the obtained phage clone was cleaved with the restriction enzyme EcoRI, and Southern blot hybridization was performed according to a conventional method using one of them as a probe. As a result, 8 clones out of 12 clones were the same clone. Of the 4 non-identical clones, 3 clones showed the same size, while the remaining 1 clone (clone C2-5) showed a larger size.
[0062]
Furthermore, the cDNA library was screened using this clone C2-5 as a probe to obtain three positive clones. Each of these clones was subcloned into the EcoRI site of the pUC118 plasmid vector, and the plasmid DNA was separated and purified by a conventional method.
[0063]
Therefore, Northern blot analysis of the obtained clone C2-5 was performed. That is, total RNA extracted from each pearl oyster tissue was subjected to agarose gel electrophoresis and then blotted onto a nylon membrane. When the C2-5 cDNA insert was hybridized as a probe, a band specific to the mantle only and expressed at a position of about 3500b was observed.
[0064]
Example 5 (Sequencing)
Since the plasmid has the structure shown in FIG. 1, sequencing was performed using an M13 forward primer and a reverse primer. Thus, the nucleotide sequence can be read from the 5 ′ side and 3 ′ side of the cloned cDNA. Moreover, in order to read the inside of cDNA, the dilation clone was produced according to the conventional method. DNA sequencing was performed using a Bucabest sequencing kit (BcaBEST Dideoxy Sequencing Kit, sold by TAKARA) based on the dideoxy terminator method such as Sanger, F.
[0065]
The sequence data thus obtained was edited into a continuous sequence using the GENETYX DNA sequence linking program, and the nucleotide sequence shown in SEQ ID NO: 2 was obtained. An open reading frame was determined from this cDNA full-length sequence data, and further translated into amino acids to obtain the sequence shown in SEQ ID NO: 3.
The formulation table (Formulation No. 4) shows the entire base sequence of cDNA and the primary amino acid sequence of the protein encoded thereby.
[0066]
Example 6 (Construction of full-length cDNA)
Based on the sequence results of Example 5, cDNA of full-length C2-5 protein was constructed in the pUC118 vector from two positive clones using the SacI cleavage site.
[0067]
Escherichia coli (E. coli JM109 strain) into which the cloning vector thus constructed has been introduced has been deposited internationally at the Patent Deposit Center of the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (identification: E. coli MK002, consignment). Number: FERM BP-5937, (transferred from domestic deposit [FERM P-15687] to international deposit under the Budapest Treaty)).
[0068]
Example 7 (Production of transformed bacteria)
The pUC118 plasmid vector into which the C2-5 protein cDNA was subcloned in Example 6 was cleaved with EcoRI and SalI to prepare a C2-5 protein cDNA, which was expressed by the expression plasmid pGEX-5X-1 (sold by Pharmacia). ). This expression vector was introduced into Escherichia coli JM109 to obtain a transformed bacterium.
[0069]
Example 8 (Production of polypeptide)
After culturing the transformant obtained in Example 7 in 1 liter of ampicillin-containing L-broth until the absorbance reached 0.6, IPTG was added to 0.5 mM, and the culture was further continued for 7 hours. It was.
[0070]
Next, after collecting the bacterial cells, sonication was performed, and the supernatant from which the disrupted bacterial cells were removed was mixed with glutathione sepharose (sold by Pharmacia). After washing the glutathione sepharose with a phosphate shock solution, 5 mM The C2-5 protein was released with a phosphate buffer containing glutathione. After dialysis of this protein with a phosphate buffer, a part thereof was analyzed by SDS-polyacrylamide gel. As a result, a GST fusion protein having a purity of 90% or more was synthesized.
[0071]
Example 9 (Production of antibody)
The C2-5 protein purified in Example 8 was subcutaneously inoculated into rabbits three times every two weeks together with complete Freund's adjuvant, and then the serum was collected.
[0072]
When this serum was tested using the Western blotting method, it was confirmed that it could be used as an antibody against the C2-5 protein even at about 1,000-fold dilution.
[0073]
【The invention's effect】
According to the present invention, unknown useful polypeptides can be efficiently mass-produced. This polypeptide can be used as a raw material for cosmetics.
[0074]
In addition, it becomes possible to develop a new method for sorting pieces in pearl culture.
[0075]
[Sequence Listing]
SEQ ID NO: 1
Sequence length: 2214
Sequence type: Nucleic acid
Number of strands: double strand
Topology: Linear
origin
Organism name: Pinctada Fucata
Cell type: mantle epithelial cells
Array
ATGAAGTTAC TGGTGGTTCT CACCACCCTC GTGGGCTTTA GCTCAGCACT AAGTTTCGGT 60
TGTAATTACA GACCAGTATT AGGCTTCAAT TCACAGTATA TGCTGGGAGG ACTAAGACTT 120
TTCTGTATGC CTGCCATGGT TTATGATCCA TGGGCATGTG GTTGCGTTTC GGCATGGAGC 180
AGTGCAGGTC TTTACGGTGT CGGAGGGGGC GGAGGCGCCT GGGGAGCTGG CGGTGCTGGA 240
GGAGCCGACG GCGGACGCGG CGGCGGCGGT GGAGATTGGG AATATGACTA TGATGACGAC 300
AGCGATGACG ATGATGAATG GGACTGGGAT GATGACGGTG GAATGGGAGC TGGCGCCGGA 360
GGTGGTGCTG GTGGTGGTGC CGGAGGTGGT GCTGGTGCTG GTGCTGGAGC AGGCGCAGGA 420
GCAGGAGCAG GTGCTGGACT CGGACTTGGA TTGGGCGGAG GTCTCGGAGG TGGACTTGGC 480
GGACTTGGAG GTCTTGGCGG ACTTGGCGGT GGAGACGATT TATTTGATTT AGATTTCGAT 540
GATCTTGGTG CAGCTCTTGC CCTCGGTGGA GCTGGTGGAG CTGGAGGTGC TGCTGCTGCT 600
GCTGCAGCTG CCGCTGCTGC CGCCGGGGGT GGAGTTGGTG GAGCTGCTGC CGCAGCCGCA 660
GCCGCTGCTG CCGCTGCAGG AGGAGGCGCA GGTAGACTTG GAGGAGCTGC TGCTGCAGCC 720
GCAGCCGCTG CTGCCGCTGC AGGAGGCGCA GGTGGACTTG GAGGACTCGG TGGCGGACTT 780
GGAGGACTCG GTGGCGGACT TGGAGGCCTC GGAGGTCTTG GTGGCCTCGG AGGATATGGA 840
GGATCTGCTG CTGCCGCTGC TGCTGCTGCC GCCGCTGCTG CCGGAGGTGG AGGACTCGGT 900
GGTGTTGGTT TCTACGGTGG ACGAGGAGGT AGACGCGGTC GAGGAAGAGG AGGCCGCAGA 960
CGTGCTGCTG CTGCCGCTGC TGCAGCTGCC GCCGCAGCCG CTGGTGGTGG CGGAGGAGGT 1020
GGAGGTGGTG GAGGAGGAGG CGGAGGCGCT GGTGCTGCCG CTGCCGCTGC AGCCGCTGCT 1080
GCATCTGCTT CAGCTTCTAG ACAAATGAGT GGTATAAGGG ACGCATTAGG AGACATTAAA 1140
GACCTTCTCA GGAGTAATGG AGCCTCTGCA AAAGCCTCTG CTAAAGCATC AGCAGTAGCA 1200
AGCACAAAAT CTCAAATTGA CGATTTGAAG GATGTCTTAA AGGATCTTGC AGGTCTATTG 1260
AAAAGCTCAG CATCTGCTTC AGCATCTGCA TCTGCATCAG CTTCAGCTGG AGGTGGAGGC 1320
GGTGGTGGTA ACGGAGGTGG TAACGGAGGA GGAGGCGGCG GTGGAGCTGG AGCTCTAGCT 1380
GCTGCTCTCG CTGCTGCAGG AGCCGGAGGT GGACTTGGAG GTGGAGGCGG AGGCGGAGCT 1440
TTAGCCGCTG CACTAGCTGC TGCTGGTGCA GGTGGAGGAG GTTTTGGTGG ACTTGGAGGA 1500
CTAGGCGGTC TTGGTGGGGG ATCTGCCGCA GCTGCTGCAG CCGCTGCCGC TGCTGCATCA 1560
GGTGGTGGAG GAAGAGCACT TAGAAGGGCT TTGAGAAGAC AAATGCGTGG AGGTGGATCC 1620
GCTGCTGCCG CTGCTGCTGC TGCTGCAGCT GCTGCTGGAG GTGGATGGGG AGGTGGAATG 1680
GGTGGAGGAT TCGGAGTAGG TCTCGGTGGA GGATTCGGAG GAGGATTTGG TGGTGGATCA 1740
TCAGCAGCAG CTGCTGCCGC TGCTGCAGCC GCCGCTGGAT TTGGTGGAGG TGGACGAAGA 1800
GGTAGAGGTA GAGGACGTGG AGGCGATGGC GACGGTAACG GAGCTAGTGC TGTAGCTGCA 1860
GCCGCCGCCG CTGCTGCTGC TGCTGGAGGA TCTGCTGCTG ATGTTGCCGC TGCCGCTGCT 1920
GCAGCCGCAG CTATGTACGG TGACGGTGCT GATGGACCTG ATTTCGATAA TGGATTCGGT 1980
GGTGGAAACG GAAATGGAGG TGGCGGATCT GGTGGTGGCG GATCCGGCGG AGGTGGATCC 2040
GGTGGCGGAT CTGGAGGTGG CGGTGGATCT GGTGGATCAG GCGGTGGCGG CGGATCTGGT 2100
GGTTCAGGCG GTGGCGGATC AGGCGGCGGT GGAAACAATG GATGGGGAAA TAACGGCAAC 2160
AATAAATATG ACGATGATGA CTGTGATGAA TATGGTAACC CTATTAGAAG GGGG 2214
[0076]
[Sequence Listing]
SEQ ID NO: 2
Sequence length: 3331
Sequence type: Nucleic acid
Number of strands: double strand
Topology: Linear
Sequence type: cDNA to mRNA
origin
Organism name: Pinctada Fucata
Cell type: mantle epithelial cells
Sequence characteristics: mRNA
Location: 1. . 3331
Method for determining characteristics: E
Array
TCGGATCTCC CCACACAACA TAGATAGAGG ATATCCGCCT GGGTTCACAA TGAAGTTACT 60
GGTGGTTCTC ACCACCCTCG TGGGCTTTAG CTCAGCACTA AGTTTCGGTT GTAATTACAG 120
ACCAGTATTA GGCTTCAATT CACAGTATAT GCTGGGAGGA CTAAGACTTT TCTGTATGCC 180
TGCCATGGTT TATGATCCAT GGGCATGTGG TTGCGTTTCG GCATGGAGCA GTGCAGGTCT 240
TTACGGTGTC GGAGGGGGCG GAGGCGCCTG GGGAGCTGGC GGTGCTGGAG GAGCCGACGG 300
CGGACGCGGC GGCGGCGGTG GAGATTGGGA ATATGACTAT GATGACGACA GCGATGACGA 360
TGATGAATGG GACTGGGATG ATGACGGTGG AATGGGAGCT GGCGCCGGAG GTGGTGCTGG 420
TGGTGGTGCC GGAGGTGGTG CTGGTGCTGG TGCTGGAGCA GGCGCAGGAG CAGGAGCAGG 480
TGCTGGACTC GGACTTGGAT TGGGCGGAGG TCTCGGAGGT GGACTTGGCG GACTTGGAGG 540
TCTTGGCGGA CTTGGCGGTG GAGACGATTT ATTTGATTTA GATTTCGATG ATCTTGGTGC 600
AGCTCTTGCC CTCGGTGGAG CTGGTGGAGC TGGAGGTGCT GCTGCTGCTG CTGCAGCTGC 660
CGCTGCTGCC GCCGGGGGTG GAGTTGGTGG AGCTGCTGCC GCAGCCGCAG CCGCTGCTGC 720
CGCTGCAGGA GGAGGCGCAG GTAGACTTGG AGGAGCTGCT GCTGCAGCCG CAGCCGCTGC 780
TGCCGCTGCA GGAGGCGCAG GTGGACTTGG AGGACTCGGT GGCGGACTTG GAGGACTCGG 840
TGGCGGACTT GGAGGCCTCG GAGGTCTTGG TGGCCTCGGA GGATATGGAG GATCTGCTGC 900
TGCCGCTGCT GCTGCTGCCG CCGCTGCTGC CGGAGGTGGA GGACTCGGTG GTGTTGGTTT 960
CTACGGTGGA CGAGGAGGTA GACGCGGTCG AGGAAGAGGA GGCCGCAGAC GTGCTGCTGC 1020
TGCCGCTGCT GCAGCTGCCG CCGCAGCCGC TGGTGGTGGC GGAGGAGGTG GAGGTGGTGG 1080
AGGAGGAGGC GGAGGCGCTG GTGCTGCCGC TGCCGCTGCA GCCGCTGCTG CATCTGCTTC 1140
AGCTTCTAGA CAAATGAGTG GTATAAGGGA CGCATTAGGA GACATTAAAG ACCTTCTCAG 1200
GAGTAATGGA GCCTCTGCAA AAGCCTCTGC TAAAGCATCA GCAGTAGCAA GCACAAAATC 1260
TCAAATTGAC GATTTGAAGG ATGTCTTAAA GGATCTTGCA GGTCTATTGA AAAGCTCAGC 1320
ATCTGCTTCA GCATCTGCAT CTGCATCAGC TTCAGCTGGA GGTGGAGGCG GTGGTGGTAA 1380
CGGAGGTGGT AACGGAGGAG GAGGCGGCGG TGGAGCTGGA GCTCTAGCTG CTGCTCTCGC 1440
TGCTGCAGGA GCCGGAGGTG GACTTGGAGG TGGAGGCGGA GGCGGAGCTT TAGCCGCTGC 1500
ACTAGCTGCT GCTGGTGCAG GTGGAGGAGG TTTTGGTGGA CTTGGAGGAC TAGGCGGTCT 1560
TGGTGGGGGA TCTGCCGCAG CTGCTGCAGC CGCTGCCGCT GCTGCATCAG GTGGTGGAGG 1620
AAGAGCACTT AGAAGGGCTT TGAGAAGACA AATGCGTGGA GGTGGATCCG CTGCTGCCGC 1680
TGCTGCTGCT GCTGCAGCTG CTGCTGGAGG TGGATGGGGA GGTGGAATGG GTGGAGGATT 1740
CGGAGTAGGT CTCGGTGGAG GATTCGGAGG AGGATTTGGT GGTGGATCAT CAGCAGCAGC 1800
TGCTGCCGCT GCTGCAGCCG CCGCTGGATT TGGTGGAGGT GGACGAAGAG GTAGAGGTAG 1860
AGGACGTGGA GGCGATGGCG ACGGTAACGG AGCTAGTGCT GTAGCTGCAG CCGCCGCCGC 1920
TGCTGCTGCT GCTGGAGGAT CTGCTGCTGA TGTTGCCGCT GCCGCTGCTG CAGCCGCAGC 1980
TATGTACGGT GACGGTGCTG ATGGACCTGA TTTCGATAAT GGATTCGGTG GTGGAAACGG 2040
AAATGGAGGT GGCGGATCTG GTGGTGGCGG ATCCGGCGGA GGTGGATCCG GTGGCGGATC 2100
TGGAGGTGGC GGTGGATCTG GTGGATCAGG CGGTGGCGGC GGATCTGGTG GTTCAGGCGG 2160
TGGCGGATCA GGCGGCGGTG GAAACAATGG ATGGGGAAAT AACGGCAACA ATAAATATGA 2220
CGATGATGAC TGTGATGAAT ATGGTAACCC TATTAGAAGG GGGTAAATTA TTTGACATTA 2280
TCCGCCATTT GACTCATTTT TCTTAGTTCT CTATGTTTTA TACTTCACCT TAGATTGTTT 2340
TAGTTTGATT GAATAAATTA TGTTTTCGAT ATAAATTTTT TTTAAATTAA ATTAAACTTT 2400
ATTAGTTGAC CTGTAAACTT TTTCATGGAG TTATAATCTA AGGAACAAAA AACATACATA 2460
ATATGTTCAG TATTGTGGTA AAGCACCTGT ACCGCAAACA CAATCACCTC TATACATGTA 2520
TACAAAATCA GTAATGCTGA CAAAATCTTC TACACTCTCA CCTACACACT CGCACACAGT 2580
CCTCTTACAT ACACAGCACT ATAATATCCT GAACATGAAG TTTGTGTTGA TAAAAAGTTC 2640
AGAAAAATCT CCCCTACATC ACCTGATCTT TCACTGAAAA TTTACGACAA GTATTGAAAA 2700
TAGCAGAAAG AAAACGGGAA ATTGAGAAGT TTTCTATAAA AAACAATCGG AACAATGACT 2760
GGAATGACAA GGATGAAAAT AATGATAACT TACATTAATT AAGGCCCCAA TAATCTCTCT 2820
ATTTTCAAAC TTTTTTTTCA AATGTTCTCT CTAACTCACT TGCATCTATG TGGAAATTCA 2880
CATACTATAC TAAATTACCA CAAGTATCAA GGTTTCACAA CCTCTCATGC CTTCATGGCA 2940
GACCATGCTG GGTATTTGTC TAACAATGCC TCATAAATAC ATAAAACTAA CTAACAAAAT 3000
AGGTCAGTCT GTAACAAATT ATTAATGCAC CATTATTGCA TTTTCTAAAA CAAAGCATAC 3060
ACTGGATATT GGCAGACAAA ATGTTGTTAT TGGATACCTT TCCATTCTAT CTAGACACTT 3120
GCTTTCCACA AGTCATCATA AATAAATCCC CCCTATCCCA AATGTCAATG GAATGCCCCA 3180
ACCCTTCCCC CATAATTTTA AAACCTAGAA TAAATTAAAA CATCTATAGT TCGTCATGAT 3240
CATCTTTCTT ATCATCCTCT TCTTCTTCCT CCTCCTCCTT CTTCTTCTTC CTCCTCCTCA 3300
GGTTCTTGGC TGCCTGCTCC TTCCTTGCCA A 3331
[0077]
[Sequence Listing]
SEQ ID NO: 3
Sequence length: 738
Sequence type: amino acid
Topology: Linear
Sequence type: protein
origin
Organism name: Pinctada Fukata
Cell type: mantle epithelial cells
Sequence features
Symbols representing features: putide
Location: 1. . 738
Method for determining characteristics: E

[0078]
[Sequence Listing]
SEQ ID NO: 4
Sequence length: 3331
Sequence type: Nucleic acid
Topology: Linear
Sequence type: cDNA to mRNA
origin
Organism name: Pinctada Fucata
Cell type: mantle epithelial cells
Sequence features
Symbols representing features: CDS
Location: 50. . 2263
Method for determining characteristics: P

  TAAATTATTT GACATTATCC GCCATTTGAC TCATTTTTCT TAGTTCTCTA TGTTTTATAC 2323
TTCACCTTAG ATTGTTTTAG TTTGATTGAA TAAATTATGT TTTCGATATA AATTTTTTTT 2383
AAATTAAATT AAACTTTATT AGTTGACCTG TAAACTTTTT CATGGAGTTA TAATCTAAGG 2443
AACAAAAAAC ATACATAATA TGTTCAGTAT TGTGGTAAAG CACCTGTACC GCAAACACAA 2503
TCACCTCTAT ACATGTATAC AAAATCAGTA ATGCTGACAA AATCTTCTAC ACTCTCACCT 2563
ACACACTCGC ACACAGTCCT CTTACATACA CAGCACTATA ATATCCTGAA CATGAAGTTT 2623
GTGTTGATAA AAAGTTCAGA AAAATCTCCC CTACATCACC TGATCTTTCA CTGAAAATTT 2683
ACGACAAGTA TTGAAAATAG CAGAAAGAAA ACGGGAAATT GAGAAGTTTT CTATAAAAAA 2743
CAATCGGAAC AATGACTGGA ATGACAAGGA TGAAAATAAT GATAACTTAC ATTAATTAAG 2803
GCCCCAATAA TCTCTCTATT TTCAAACTTT TTTTTCAAAT GTTCTCTCTA ACTCACTTGC 2863
ATCTATGTGG AAATTCACAT ACTATACTAA ATTACCACAA GTATCAAGGT TTCACAACCT 2923
CTCATGCCTT CATGGCAGAC CATGCTGGGT ATTTGTCTAA CAATGCCTCA TAAATACATA 2983
AAACTAACTA ACAAAATAGG TCAGTCTGTA ACAAATTATT AATGCACCAT TATTGCATTT 3043
TCTAAAACAA AGCATACACT GGATATTGGC AGACAAAATG TTGTTATTGG ATACCTTTCC 3103
ATTCTATCTA GACACTTGCT TTCCACAAGT CATCATAAAT AAATCCCCCC TATCCCAAAT 3163
GTCAATGGAA TGCCCCAACC CTTCCCCCAT AATTTTAAAA CCTAGAATAA ATTAAAACAT 3223
CTATAGTTCG TCATGATCAT CTTTCTTATC ATCCTCTTCT TCTTCCTCCT CCTCCTTCTT 3283
CTTCTTCCTC CTCCTCAGGT TCTTGGCTGC CTGCTCCTTC CTTGCCAA 3331
[Brief description of the drawings]
FIG. 1 is a block diagram showing an example of a cloning vector of the present invention.

Claims (11)

 A cDNA encoding the nucleotide sequence of SEQ ID NO: 1.  A cDNA encoding the nucleotide sequence of SEQ ID NO: 2.  A cloning vector containing the cDNA according to claim 1 or 2. DNA consisting of the base sequence shown in SEQ ID NO: 1 . A DNA consisting of the base sequence represented by SEQ ID NO: 2 .  An expression vector containing the cDNA according to claim 1 or 2. Form transformants who hold the expression vector of claim 6 wherein. A polypeptide produced by the transformant according to claim 7 and encoded by the cDNA according to claim 1 or 2 . The polypeptide according to claim 8, which consists of the amino acid sequence of SEQ ID NO: 3.  A method for producing a polypeptide, comprising culturing the transformant according to claim 7 under conditions for expressing the polypeptide according to claim 8 or 9.  An antibody prepared by using the polypeptide according to claim 8 or 9 as an antigen.

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