JP3718734B2 - 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

【００７７】
【配合表】
配列番号：４
配列の長さ：1326
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：アコヤ貝（Pinctada fucata ）
細胞の種類：外套膜上皮細胞
配列の特徴
特徴を表す記号：CDS
存在位置：38..1039
特徴を決定した方法：Ｐ

【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 cell) of Japanese pearl oyster (Pincta fucata), a vector containing the cDNA, a host cell transformed with the vector, and a product produced thereby The present invention relates to a polypeptide to be produced, a method for producing the same, a DNA encoding the polypeptide, an antibody to the polypeptide, and a cosmetic containing the polypeptide or a degradation peptide thereof.
[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]
It also aims to develop new raw materials for cosmetics or a new method for sorting pieces in pearl culture.
[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 identifying the function of genes from genes is also remarkable.
[0007]
Therefore, the present inventors 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, the base sequence was determined, and the 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 sequence and amino acid sequence registered in the amino acid sequence database (EMBL-GDB Rel.41) and protein database (SWISS-PROT Rel.30) were investigated. 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. Further, the pearl oyster shell has structural differences of three layers, which are called the shell layer, the ridge column layer, and the nacre 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 drugs for some time and is still used in some areas. It has also been used as an active ingredient in cosmetics for a long time.
[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. Pearls emerge from the pearl oyster gonad along with a piece that is a piece of pearl oyster mantle in about 1-3 years.
[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.
[0015]
(Claim 1) A cDNA encoding the nucleotide sequence of SEQ ID NO: 1.
[0016]
(Claim 2) A cDNA encoding the nucleotide sequence of SEQ ID NO: 2.
[0017]
(Claim 3) A cloning vector having the cDNA according to claim 1 or 2.
[0018]
(Claim 4) DNA comprising the nucleotide sequence represented by SEQ ID NO: 1 .
[0019]
(Claim 5) A DNA comprising the nucleotide sequence represented by SEQ ID NO: 2 .
[0020]
(Claim 6) An expression vector containing the cDNA according to claim 1 or 2.
[0021]
(Claim 7) form transformants who hold the expression vector of claim 6.
[0022]
(Claim 8) A polypeptide produced by the transformant according to claim 7 and encoded by the cDNA according to claim 1 or 2 .
[0023]
(Claim 9) The polypeptide according to the above item consisting of the amino acid sequence of SEQ ID NO: 3.
[0024]
(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.
[0025]
(11) An antibody prepared by using the polypeptide of claim 8 or 9 as an antigen.
[0026]
DETAILED DESCRIPTION OF THE INVENTION
In the above, the DNA having the base sequence represented by SEQ ID NO: 1 (or 2) (Claims 4 and 5) is generally 90% or more, for example, 95%, 98% or 99% of DNA. It means that the DNA has a base sequence represented by 1 (or 2). In addition, these homologous fragments mean at least 10 bases, preferably at least 15 bases, for example, 20, 25, 30 or 40 bases, and such fragments are also included in the DNA of the present invention (similarly Effect).
[0027]
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. Means a polypeptide having the amino acid sequence encoded by the DNA having the base sequence represented by SEQ ID NO: 1. Also, the homologous 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 Are homologous more than 95%, and such homologues are hereinafter described as polypeptides of the present invention (similar effects are obtained).
[0028]
The fragment that selectively hybridizes to the DNA having the base sequence represented by SEQ ID NO: 1 or 2 generally has 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 fragments are hereinafter described as DNA of the present invention (similar effects can be obtained). ).
[0029]
Furthermore, the present invention includes a cloning vector 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 production of RNA corresponding to DNA and transformation of host cells.
[0030]
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.
[0031]
Furthermore, the present invention includes a method for producing a polypeptide comprising 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.
[0032]
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.
[0033]
In addition, 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 this polypeptide between individuals, Can be compared.
[0034]
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 conventional methods such as inoculating a host animal such as a rat or rabbit with the polypeptide of the present invention and recovering serum.
[0035]
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.
[0036]
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.
[0037]
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.
[0038]
The DNA of the present invention represented by SEQ ID NO: 2 is an embodiment of the DNA represented by SEQ ID NO: 1 and represents a natural sequence.
[0039]
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) Synthesize 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 (production 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.
[0040]
As an explanation, step (I) is described in the method of Chomczynski, p. Et al. [Anal. Biochem., 162, 156 (1987), using pearl oyster mantle as a material. ] Is performed according to the above.
[0041]
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). Phage vectors referred to here include phage vectors (Phagemid) that can be converted into plasmids by infecting helper phages. included.
[0042]
Step (V) is performed according to the method described in Molecular Cloning [Sambrook, J., Frisch, EF, and Maniatis, T. Cold Spring Harbor Laboratory Press, (1989)].
[0043]
The sequencing of the step (VI) is performed by the Maxam-Gilbert method or the dideoxy terminator method.
[0044]
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 signal peptide of the highly hydrophobic region at its N-terminus), and (C) the DNA is full-length or almost full-length It is necessary to confirm that there is.
[0045]
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.
[0046]
The method for obtaining the polypeptide of the present invention includes (1) a method for isolation and purification from a living body or cultured cells. (2) A method for peptide synthesis, or (3) a method for production using a gene recombination technique, and the like. Among them, the method described in (3) is preferred industrially.
[0047]
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.
[0048]
For example, when expressed 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.) An expression vector is prepared by inserting into a vector that functions in E. coli (for example, pBR322, pUC18, pUC19, etc.). 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.).
[0049]
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.
[0050]
Next, E. coli transformed with this expression vector is cultured in an appropriate medium, and the desired polypeptide can be obtained from the cells. In addition, 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.
[0051]
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.
[0052]
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.
[0053]
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.
[0054]
【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.
[0055]
Example 1 (Separation and purification of mRNA)
Mantle epithelial cells of pearl oysters (Pinctada fucata) (the oyster oysters are readily available at home and abroad today. These mantle epithelial cells cannot withstand cryopreservation, and other preservation methods are also available. However, since the storage conditions (medium, etc.) have not yet been found, it is not possible to survive in the separated state (as a cell) at present. The center has refused to accept this cell.Acceptance of the acceptance rejection certificate has been obtained (indication for identification: P. fucata-M01) and the acceptance of the pearl oyster itself has not been accepted. , Using the method of Chomczynski, P. et al. Anal. Biochem., 162, 156 (1987). ] To isolate total RNA.
[0056]
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, from this, using oligo (dT) latex particles (sold by TAKARA), mRNA was separated, and 2.5 μg of Poly (A) ⁺ RNA was purified and recovered.
[0057]
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)].
[0058]
That is, single-stranded DNA (first strand) was synthesized from 2.5 μg of Poly (A) ⁺ RNA separated and purified in Example 1 using 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.
[0059]
The above cDNA synthesis step was performed using a time saver cDNA synthesis kit (Time Saver cDNA Synthesis kit, sold by Pharmacia).
[0060]
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, a cDNA library having 5.5 × 10 ⁵ pfu of independent clones was obtained.
[0061]
Example 3 (Cloning)
From the cDNA library obtained in Example 2, a density of about 10,000 pfu / plate was prepared on a 10 cm × 14 cm LB plate. On the other hand, a 20-mer synthetic nucleotide was prepared by requested synthesis (sold by Biologica) based on the data already obtained by the present inventors (with reference to the amino acid sequence obtained from the CNBr-degrading peptide of the pearl oyster nacre layer insoluble protein). This was labeled with an isotope using [γ- ³² P] adenosine triphosphate and T4 polynucleotide kinase to prepare a labeled probe. Next, screening was performed by plaque hybridization according to the method described in Molecular Cloning [Sambrook, J., Fritsch, EF, and Maniatis, T. Cold Spring Harbor Laboratory Press, (1989)], and a number of positive clones were obtained.
[0062]
Example 4 (Clone analysis)
Furthermore, the DNA of the obtained phage clone was cleaved with the restriction enzyme EcoRI, and Southern plot hybridization was performed according to a conventional method using one of them (clone Cl-6) as a probe. As a result, 8 clones out of 12 clones were the same clone.
[0063]
Further, this clone Cl-6 was subcloned into the EcoRI site of the pUC118 plasmid vector, and the plasmid DNA was separated and purified by a conventional method (cloning vector).
[0064]
Therefore, Northern blot analysis of the obtained Cl-6 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 Cl-6 cDNA insert was hybridized as a probe, a band that was specific only to the mantle and expressed in a large amount at a position of about 1500b was observed. Although the insert size of the Cl-6 clone was about 1300 bp, it was confirmed that the Cl-6 clone was a substantially full-length cDNA in consideration of the result of the Southern hybridization.
[0065]
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. In addition, in order to read the inside of the cDNA, a dilation clone was prepared according to a conventional method. DNA sequencing was performed using a Bucabest sequencing kit (BcaBEST Dideoxy Sequencing Kit, sold by TAKARA) based on the dideoxy terminator method of Sanger, F. et al.
[0066]
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.
[0067]
Example 6 (Preparation of transformed bacteria)
The pUC118 plasmid vector into which the Cl-6 protein cDNA was subcloned in Example 4 was cleaved with EcoRI to prepare a Cl-6 protein cDNA, which was incorporated into the expression plasmid pGEX-4T-3 (sold by Pharmacia). . This expression vector was introduced into E. coli DH5 to obtain a transformed bacterium. This transformed bacterium has been deposited at the Patent Deposit Center, National Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (identification: E. coli MK001, accession number: FERM P-15316).
[0068]
Example 7 (Production of polypeptide)
The transformant obtained in Example 6 was cultured in 1 liter of ampicillin-containing L-broth until the absorbance reached 0.6, then IPTG was added to 0.5 mM, and the culture was further continued for 3 hours. .
[0069]
Next, the cells were collected, sonicated, and the supernatant from which the crushed cells had been removed was mixed with glutathione sepharose (sold by Pharmacia). After washing the glutathione sepharose with a phosphate buffer, 5 mM Cl-6 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.
[0070]
Example 8 (Production of antibodies)
The Cl-6 protein purified in Example 7 was inoculated subcutaneously in rabbits three times every two weeks with complete Freund's adjuvant, and the serum was collected.
[0071]
When this serum was tested using a Western blotting method, it showed a certain reactivity to Cl-6 protein even at a dilution of about 1000, confirming that it could be used as an antibody against Cl-6 protein. .
[0072]
【The invention's effect】
According to the present invention, unknown useful polypeptides can be efficiently mass-produced. This polypeptide can be used as a cosmetic raw material.
[0073]
In addition, it becomes possible to develop a new method for sorting pieces in pearl culture.
[0074]
[Composition table]
SEQ ID NO: 1
Sequence length: 1002
Sequence type: Number of nucleic acid strands: Double-stranded topology: Linear organism name: Pinctada fucata
Cell type: mantle epithelial cell array
ATGAAGCCCT TTGTCACACT CGCAAGCTTG ATCGTCTTAA TTGCTTCGGC TTCTGCCGAC 60
GGATATGATG ACTATAAGAA ATACGGAAGT GTCGGTTATG GACCTGGCAT CAGCCTTGGA 120
GGTGGTGGTT TAGGTGGCGG AGGAGGCATT ATCTCGGTTG GAGGCGGAGG AGGCCTCGGA 180
GGAGGCCTCG GAGGAGGCTT GGGTTGTGGT CTCGTAGGTG TCGGAGGCGG TGGACTTATC 240
GGAGGAGGGT TTGGCCCCGG TAGAGTGTCT GGTACCATCA ACGCAGGAGG TGGAGTTTTC 300
GCAAGCGGAT CCGGATTGGG AGGACTCTCA CCAGCTGGAA GAGGTGCAGC ACAAGGTGCA 360
GCCACTCTAA GCGCTCTCCA AATTGCTTCC GGAAGACCAG GCAGAGTCTC AGGTGTATCC 420
GTCGGAACTG GAGGAGGTCG TGCTGTTGTT TCCGGAAGTG CTACTCCAGT TGGAGGCTTT 480
GGTGTTCCCT ATGGAGGTTA CGGATACAAT TACGGTGTCC CAAGTTATGG AGTTGGCCTC 540
CCAAGCTACG GAGTCAGCCT TCCAAGTTAC GGAGTAGGTT TGGGAGGAGG TTATGGTGGC 600
TATGGATACG GACTAGACCT TGCCAGCTTC CAAGGTTCTA CCTACGGAAA TCTTGCAACT 660
GGACAAATTA ATACCGCAGT GGTAGCATTC CATATGGCGG TTCTCTTGGA ATCTATGGAA 720
GCGGAATCGG ATACGGAGGT GGATACGGAG GATATGGACT CGGAGGAGGA TATGGAATCG 780
GAGGAGGATA CGGAATCGGA GGAGGATACG GAATCGGAGG AGGATACGGA ATCGGAGGAG 840
GATATGGAAT CGGAGGAGGA TATGGACTCG GAGTCCTCGG TGGTGGATCA AGTCTCTATG 900
GTGTATCCCA ATCACTTTAC GGGGGACGTG CTGTTCTGTC AGGTCAGGCT TCAGGAGCTG 960
GAGTTCCCAG CTTTGGAAGC ATTAGTTTCG GTGGTTTTGG AG 1002
[0075]
[Composition table]
SEQ ID NO: 2
Sequence length: 1326
Sequence type: Number of nucleic acid strands: Double-stranded topology: Linear sequence type: cDNA to mRNA
Origin organism name: Akoya oyster (Pinctada fucata)
Cell type: Mantle epithelial cell Sequence characteristics: mRNA
Location: 1..1326
Method for determining characteristics: E
Array
AACATCTCCA GATAAAGAAC GGTACCCATC CACAATCATG AAGCCCTTTG TCACACTCGC 60
AAGCTTGATC GTCTTAATTG CTTCGGCTTC TGCCGACGGA TATGATGACT ATAAGAAATA 120
CGGAAGTGTC GGTTATGGAC CTGGCATCAG CCTTGGAGGT GGTGGTTTAG GTGGCGGAGG 180
AGGCATTATC TCGGTTGGAG GCGGAGGAGG CCTCGGAGGA GGCCTCGGAG GAGGCTTGGG 240
TTGTGGTCTC GTAGGTGTCG GAGGCGGTGG ACTTATCGGA GGAGGGTTTG GCCCCGGTAG 300
AGTGTCTGGT ACCATCAACG CAGGAGGTGG AGTTTTCGCA AGCGGATCCG GATTGGGAGG 360
ACTCTCACCA GCTGGAAGAG GTGCAGCACA AGGTGCAGCC ACTCTAAGCG CTCTCCAAAT 420
TGCTTCCGGA AGACCAGGCA GAGTCTCAGG TGTATCCGTC GGAACTGGAG GAGGTCGTGC 480
TGTTGTTTCC GGAAGTGCTA CTCCAGTTGG AGGCTTTGGT GTTCCCTATG GAGGTTACGG 540
ATACAATTAC GGTGTCCCAA GTTATGGAGT TGGCCTCCCA AGCTACGGAG TCAGCCTTCC 600
AAGTTACGGA GTAGGTTTGG GAGGAGGTTA TGGTGGCTAT GGATACGGAC TAGACCTTGC 660
CAGCTTCCAA GGTTCTACCT ACGGAAATCT TGCAACTGGA CAAATTAATA CCGCAGTGGT 720
AGCATTCCAT ATGGCGGTTC TCTTGGAATC TATGGAAGCG GAATCGGATA CGGAGGTGGA 780
TACGGAGGAT ATGGACTCGG AGGAGGATAT GGAATCGGAG GAGGATACGG AATCGGAGGA 840
GGATACGGAA TCGGAGGAGG ATACGGAATC GGAGGAGGAT ATGGAATCGG AGGAGGATAT 900
GGACTCGGAG TCCTCGGTGG TGGATCAAGT CTCTATGGTG TATCCCAATC ACTTTACGGG 960
GGACGTGCTG TTCTGTCAGG TCAGGCTTCA GGAGCTGGAG TTCCCAGCTT TGGAAGCATT 1020
AGTTTCGGTG GTTTTGGAGT AGGTAGTCCA TACAGTATTT ATGGAGGTGG ATATCCAATT 1080
GGAATTGGAG GCGGCGGCGG TGGTATCATC GGCGGAGGTG GTATCATCGG CGGAGGTGGT 1140
ATAGGAGGCG GTATCATCGG CGGAGGTGGT ATCGGACCAA TCGGCGGTGG AATCATTAGA 1200
AAGAAGAAGT ATTAAGCTGA TGTTTGATGT ATGGGAATTG CCAAATGGAC TCACCTTTTT 1260
CCTGGATATA CCTTTTTCAT GGATGTTTGA TATTTAATTT TTTGCAATAA ATGAGCATAT 1320
ACAAAA 1326
[0076]
[Composition table]
SEQ ID NO: 3
Sequence length: 334
Sequence type: Amino acid Topology: Linear sequence type: Protein origin organism: Pinctada fucata
Cell type: Characteristic features of mantle epithelial cell sequence: peptide
Location: 1..334
Method for determining characteristics: E

[0077]
[Composition table]
SEQ ID NO: 4
Sequence length: 1326
Sequence type: Nucleic acid Topology: Linear sequence type: cDNA to mRNA
Origin organism name: Akoya oyster (Pinctada fucata)
Cell type: Characteristic features of mantle epithelial cell sequence: CDS
Location: 38..1039
Method for determining characteristics: P

[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 that have a 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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