JPH09154584A - Cdna of new polypeptide gene, vector containing the same cdna, host cell transformed with the same vector, polypeptide produced thereby, its production, dna capable of coding the same polypeptide and antibody against the same polypeptide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new cDNA, capable of coding a polypeptide produced by a mantle tissue of Pinctada fucata martensii Dunker, having a specific base sequence and capable of providing a polypeptide useful for development of a new raw material for cosmetics or a new method, etc., for selecting a piece in pearl culture. SOLUTION: This new cDNA is capable of coding a polypeptide produced by a mantle epithelial tissue of Pinctada fucata martensii Dunker and used for genetic engineering production, etc., of a new polypeptide useful for developing a new raw material for cosmetics or development, etc., of a new method for selecting a piece in pearl culture. The new cDNA is obtained by isolating an mRNA from a mantle epithelial tissue of Pinctada fucata martensii Dunker, synthesizing a cDNA with a reverse transcription enzyme using the mRNA as a template, preparing a cDNA library therewith according to a conventional method, then screening the resultant cDNA library with a synthetic nucleotide capable of coding a part of an insoluble protein of a pearl layer of the Pinctada fucata martensii Dunker as a probe and recovering the cDNA from a positive clone.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel polypeptide gene c produced by the mantle tissue (mantle epithelial cells) of Japanese pearl oyster (Pinctada fucata).
DNA, a vector containing the cDNA, a host cell transformed with the vector, a polypeptide produced thereby, a method for producing the same, a DNA encoding the polypeptide, an antibody of the polypeptide, and the polypeptide or It relates to a cosmetic product containing the degraded peptide.

[Object of the Invention] Various factors related to the maintenance, proliferation, growth and the like are produced from cells. For example, pearl oyster mantle epithelial cells produce many factors such as factors related to shell formation and biological defense. The present inventors have paid attention to this point and have conducted extensive studies to find out a novel factor (polypeptide) produced by a certain type of cell (for example, pearl oyster mantle epithelial cell).

Another object is to develop a new raw material for cosmetics or a new method for selecting pieces in pearl culture.

[0004]

2. Description of the Related Art Conventionally, when obtaining a specific polypeptide or a DNA encoding the same, a desired polypeptide is confirmed in a tissue or cell culture, A method of isolating and purifying a polypeptide followed by cloning of a gene or a method of expressing and cloning a gene using its biological activity as an index has been generally used.

However, information on factors produced by pearl oyster mantle epithelial cells is extremely small, and many of them show insolubility, which makes isolation, purification and confirmation of biological activity difficult.

[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. Further, the development of various methods of Reverse Genetics for identifying the function of a gene from the gene is remarkable.

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 nucleotide sequence was determined, and the amino acid sequence was deduced. As a result, they succeeded in finding a DNA encoding a novel polypeptide, and completed the present invention.

Amino acid sequence database (EMBL-GDB Re
l.41), protein database (SWISS-PROT Rel.30)
A known nucleotide sequence and amino acid sequence registered in the above were investigated, but none of them were the same, and none of them were highly homologous. Therefore, it was confirmed that the DNA and polypeptide of the present invention are completely novel.

Here, the polypeptide secreted by the pearl oyster mantle epithelial cells will be described. The pearl oysters cover their outer surfaces with strong shells and protect themselves from external enemies. It is known that this shell, which is a kind of host defense, is made by mantle epithelial cells. Shells are mainly composed of calcium carbonate crystals and polypeptides. Furthermore, the shells of the pearl oyster have structural differences consisting of three layers, and they are called the crust layer, ridge layer, and nacre layer from the outside. This structural difference is thought to be due to the difference in polypeptide.

[0010] The only difference between the polypeptides constituting each layer, which has been known so far, is the difference in the amino acid composition. This is because most of the polypeptides are insoluble in water and most other solvents, which is a factor that delays the analysis of these polypeptides.

Among these polypeptides, the polypeptide constituting the nacre has been used as various drugs for a long time, and is still used in some regions even now. It has also been used as an active ingredient in cosmetics for a long time.

The nacre on the shell structure is considered to be equivalent to a pearl widely used as jewelry by people. A pearl is transplanted into a pearl oyster gonad together with a round nucleus made of shells of other species together with a piece, which is a tissue piece of the pearl oyster mantle, and emerges in about 1 to 3 years.

This suggests that these polypeptides play a very important role in the color and volume that greatly affect the value of pearls as gemstones.

[0014]

The present invention is as follows.

(Claim 1) A cDNA encoding the nucleotide sequence of SEQ ID NO: 1.

(Claim 2) A cDNA encoding the nucleotide sequence of SEQ ID NO: 2.

(Claim 3) The cDNA of claim 1 or 2.
Cloning vector with A.

(Claim 4) A DNA having the nucleotide sequence represented by SEQ ID NO: 1, or a fragment which selectively hybridizes to the sequence.

(Claim 5) A DNA having the nucleotide sequence represented by SEQ ID NO: 2 or a fragment which selectively hybridizes to the sequence.

(Claim 6) The cDNA of claim 1 or 2.
An expression vector containing A.

(Claim 7) A transformant carrying the expression vector according to claim 6, such as bacteria, yeast, insects, insect cells or mammalian cells.

(Claim 8) A polypeptide produced by the transformant according to claim 7.

(Claim 9) The polypeptide according to the above item having the amino acid sequence of SEQ ID NO: 3.

(Claim 10) A method for producing a polypeptide, which comprises culturing the transformant according to claim 7 under conditions for expressing the polypeptide according to claim 8 or 9.

(Claim 11) An antibody prepared by using the polypeptide according to claim 8 or 9 as an antigen.

[0026]

BEST MODE FOR CARRYING OUT THE INVENTION In the above, a DNA having the nucleotide sequence shown in SEQ ID NO: 1 (or 2) (claim 4,
5) is generally 90% or more of DNA, for example 95
%, 98% or 99% means that the DNA has the nucleotide sequence shown by SEQ ID NO: 1 (or 2).
The homologue fragment means at least 10 bases, preferably at least 15 bases, for example, 20, 25, 30 or 40 base portions, and such fragments are also included in the DNA of the present invention (similar The effect is obtained).

D having the base sequence shown in SEQ ID NO: 1
The polypeptide encoded by NA and the polypeptide having the amino acid sequence represented by SEQ ID NO: 3 are generally 90% or more of the polypeptide, for example, 95, 98 or 99% have the nucleotide sequence represented by SEQ ID NO: 1. Have DN
It means that the polypeptide has an amino acid sequence encoded by A. In addition, a fragment of a homologue thereof is generally at least 20, preferably at least 30, for example, a continuous amino acid region of 40, 60 or 100, and at least 70%, preferably at least 80 or 90%, more preferably Is 95% or more homologous, and such a homologue is hereinafter described as a polypeptide of the present invention (similar operational effects can be obtained).

The fragment which selectively hybridizes to the DNA having the nucleotide sequence represented by SEQ ID NO: 1 or 2 is generally at least 20, preferably at least 30, for example, 40, 60 or 100 contiguous fragments. At least 70%, preferably at least 80 or 90%, and more preferably 95% or more homologous in the nucleotide sequence region, and such a fragment is described as the DNA of the present invention (similar action effect). Is obtained).

Further, 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 expressing the above DNA, a promoter regulatory factor, and the like. The vector may include one or more selectable marker genes, eg, ampicillin resistance gene. The vector can be used in vitro, for example, for producing RNA corresponding to DNA and for transforming a host cell.

Further, in the present invention, SEQ ID NO: 1 or 2
Or a host cell transformed with a vector for replicating or expressing the DNA of the present invention, which contains a DNA having the nucleotide sequence of or a DNA having an open reading frame thereof. Examples of cells include bacteria, yeast,
Insects, insect cells or mammalian cells are mentioned.

Further, the present invention also includes a method for producing the polypeptide, which comprises culturing the host cell of the present invention under the conditions for expressing the polypeptide of the present invention.
The cultivation is preferably carried out under conditions in which the polypeptide of the present invention is expressed and produced from host cells.

The DNA of the present invention is inserted into the antisense region of the above vector to obtain antisense RN.
A can also be produced. Such antisense R
NA can also be used to control the level of a polypeptide of the invention in a cell.

Further, the DNA of the present invention, a homologue or a fragment thereof, is used as a probe for Northern analysis or PCR (Polymerase Chain React).
ion) as a primer, it becomes possible to compare the expression level of the present polypeptide among other individuals and other factors.

The present invention also includes monoclonal or polyclonal antibodies of the polypeptides of the present invention. Furthermore, the present invention includes a method for producing a monoclonal or polyclonal antibody of the polypeptide of the present invention. Monoclonal antibodies can be produced by ordinary hybridoma technology using the polypeptides of the present invention or fragments thereof as antigens. The polyclonal antibody can be produced by a usual method such as inoculating a host animal such as rat or rabbit with the polypeptide of the present invention and collecting serum.

As the polypeptide of the present invention, in addition to the polypeptide having the amino acid sequence shown in SEQ ID NO: 3, a part thereof is deleted, and a part thereof is replaced with another amino acid (for example, in the physical properties). (Substituted with similar amino acids), and those in which other amino acids are added or inserted in part thereof are also included.

As is well known, 1 to 6 types of codons encoding one amino acid are known. Thus, DN without changing the amino acid sequence of the polypeptide.
The base sequence of A can be changed.

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 nucleotide sequence, production of the polypeptide may be improved.

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 native sequence.

D having the nucleotide sequence shown in SEQ ID NO: 2
The preparation of NA is performed according to the following method. That is, (I) mRNA is isolated from pearl oyster mantle tissue producing the polypeptide of the present invention, and (II) the mRNA is isolated.
To single-stranded DNA (first strand), then 2
Synthesized double-stranded DNA (second strand) (cDNA
(III), (III) incorporating the cDNA into an appropriate phage vector, (IV) packaging, and then infecting a host cell with the obtained recombinant infectious phage (cDN
Preparation of A library), (V) The obtained cDNA library was screened with a specific probe, and (VI)
The obtained clone can be prepared by sequencing the full length thereof.

In addition, in the step (I), the method of Chomczynski, p. Et al. [Ana
l.Biochem., 162, 156 (1987). ].

The steps (II), (III) and (IV) are steps for preparing a cDNA library and were modified.
Gubler & Hoffman method [Gene., 25, 263 (1983). ]. Many phage vectors are known to be used in the step (III) (for example, λgt1
0, λgt11), the phage vector mentioned here also includes a phage vector (Phagemid) that can be converted into a plasmid by infecting a helper phage.

The step (V) is carried out by Molecular Cloning [Sa
mbrook, J., Frisch, EF, and Maniatis, T.Cold Spring H
arbor Laboratory Press, (1989)].

Sequencing of the step (VI) is carried out by Maxam-Gilbert method or dideoxy
It is performed by the terminator method.

The DNA thus obtained is (A)
The base sequence is converted into an amino acid sequence in a possible reading frame, (B) a hydrophobicity profile is prepared from the obtained amino acid sequence, and immediately after the initiation codon (ATG) (the secretory protein has its N It is necessary to confirm the presence of the highly hydrophobic region (having a signal peptide of the highly hydrophobic region at the end), and further (C) confirm that the DNA is full length or substantially full length.

During the above confirmation operation, (C) is a Northern (No)
rthern) analysis. Once the nucleotide sequences shown in SEQ ID NOs: 1 and 2 have been determined, they are then chemically synthesized or by PCR (Polymerase Chain React).
ion) method or by hybridizing with a fragment of the nucleotide sequence as a probe,
NA can be obtained. Furthermore, a required 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 growing the vector.

The method of obtaining the polypeptide of the present invention includes (1) a method of isolating and purifying from a living body or cultured cells. Examples of the method include (2) peptide synthesis method, (3) method using gene recombination technology, and the like. Industrially, the method described in (3) is preferable.

Examples of the expression system (host-vector system) for producing a polypeptide using the gene recombination technique include expression systems of bacteria, yeast, insects, insect cells and mammalian cells.

For example, Escherichia coli (Escherichia)
E. coli), the DNA having the nucleotide sequence represented by SEQ ID NO: 1 is used as a suitable promoter (eg, trp promoter, lac promoter, λPL).
Promoter, T7 promoter, etc.)
Vectors that function in E. coli (eg, pBR322,
pUC18, pUC19, etc.) to produce an expression vector. Alternatively, commercially available expression vectors (eg,
pKK223-3: Pharmacia, pET vector: Stra
It can also be prepared by connecting the DNA shown in SEQ ID NO: 1 to a tagene, pTV vector: Takara, etc.).

Examples of Escherichia coli include E. coli. coli
JM109, E.I. coli HB101, E. coli. col
i DH5 strain and the like can be used, but the present invention is not limited thereto.

Next, Escherichia 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 (eg, pelB signal peptide) is used, the target polypeptide can be secreted into the periplasm. Furthermore, the desired polypeptide can be obtained by producing it as a fusion protein with another polypeptide.

When expressed in insects or insect cells, for example, DN shown in SEQ ID NO: 1 or 2 is used.
A is inserted into an appropriate vector to prepare a transfer vector. Next, baculovirus (Autographa cal
ifornia NPV) and a vector having a promoter are co-transfected to produce a recombinant virus. The resulting recombinant virus is introduced into an appropriate insect cell (eg, Sf9, Sf21, or Tn5 cell) or an insect body (eg, silkworm) to produce a desired polypeptide.

When expressed in mammalian cells, for example, the DNA represented by SEQ ID NO: 1 or 2 is a suitable vector (eg, retrovirus vector, papillomavirus vector, vaccinia virus vector, SV40 system vector, etc.). An expression vector is prepared by inserting it into the downstream of an appropriate promoter (for example, SV40 promoter, LTR promoter, metallothionein promoter, etc.). Next, a suitable mammalian cell (for example, monkey COS cell, Chinese hamster CHO cell, mouse L cell, etc.) is transformed with the obtained expression vector, and the transformant is cultured in a suitable 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.

The polypeptide of the present invention can be applied to cosmetics. That is, the polypeptide of the present invention can be mixed with other components usually used to prepare a cosmetic composition.

[0054]

The present invention will be described in more detail with reference to the following Examples, which do not limit the scope of the present invention.

Example 1 (Separation and Purification of mRNA ) Mantle epithelial cells of pearl oyster (Pinctada fucata) (The pearl oysters are readily available today in Japan and overseas. The mantle epithelial cells are frozen. Since it cannot withstand storage, and other storage methods and storage conditions (medium, etc.) have not been found yet, it cannot currently survive in a separated state (as cells). The deposit of this cell was rejected by the Patent Microorganism Depositary Center of the Institute of Biotechnology, Institute of Biotechnology. A certificate of refusal of deposit has been obtained (indication for identification: P.fucata-M01). The above-mentioned pearl oyster (Pinctadafucata) is stored by the applicant himself and is transferable. Approximately 3 g of
omczynski, P. et al. [Anal. Biochem., 162, 156 (1987)]
Described in. ], The total RNA was separated.

That is, 4M GTC solution (4M guanidine thiocyanate, 25 mM sodium citrate,
The tissue was homogenized in 0.5% sarcosyl, 0.1M 2-mercaptoethanol, extracted twice with phenol / chloroform, added with isopropanol, centrifuged (10,000 xg, 10 minutes), and allowed to precipitate during precipitation. 200 μg of total RNA was recovered. Further, from this, mRNA was separated using oligo (dT) latex particles (sold by TAKARA), and 2.5 μg of Poly (A) ⁺ RNA was purified and recovered.

Example 2 (Preparation of cDNA Library ) The cDNA library was prepared by the Gubler & Hoffman method [Gene.,
25, 263 (1983)].

That is, Pol separated and purified in Example 1
Single-stranded DNA (first strand) was synthesized from 2.5 μg of y (A) ⁺ RNA by a reverse transcriptase using a random hexamer primer and an oligo (dT) primer. Then, after synthesizing double-stranded DNA (second strand), unreacted primers and RNA were removed by gel filtration column chromatography, and E
Ligation of the coRI adapter was performed. again,
The unreacted adapter was removed by gel filtration column chromatography to recover the cDNA fraction.

The above-mentioned cDNA synthesis steps are carried out by the Time Saver cDNA Synthesis Kit
s kit, sold by Pharmacia).

After ligating the cDNA prepared above with the λgt10 phage vector, lambda (In vit)
Ro Packaging Kit LAMBDA IN (sold by Nippon Gene Co., Ltd.) was used for packaging, 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.

Example 3 (Cloning) From the cDNA library obtained in Example 2, 10 cm ×
A 14 cm LB plate was prepared at a density of about 10,000 pfu / plate. On the other hand, based on the data that the present inventors have already obtained, 2 by the requested synthesis (sold by Biologica)
To prepare a synthetic oligonucleotide of 0Mer (the amino acid sequence obtained from CNBr degradation peptides oyster pearl layer insoluble protein that was produced in the reference), this [.gamma. ³²
P] Adenosine triphosphate and T4 polynucleotide kinase were used for isotope labeling to prepare a labeled probe. Next, Molecular Cloning [Sambrook, J., Fritsch,
EF, and Maniatis, T. Cold Spring Harbor Laboratory
Press, (1989)], and a large number of positive clones were obtained by screening by plaque hybridization.

Example 4 (Analysis of clones) The DNA of the obtained phage clone was subjected to restriction enzyme E.
cut with coRI and one of them (clone Cl-6)
Southern blot hybridization was carried out according to a conventional method using as a probe. As a result, 8 out of 12 clones
The clone was the same clone.

Further, this clone Cl-6 was cloned into pUC11
8 The plasmid DNA was subcloned into the EcoRI site, and the plasmid DNA was separated and purified by a conventional method (cloning vector).

Then, Northern blot analysis of the obtained Cl-6 was carried out. That is, total RNA extracted from each pearl oyster tissue was subjected to agarose gel electrophoresis and then blotted on a nylon membrane. Cl-6 cDN
When hybridized with the A insert as a probe, a band which was specific only to the mantle and was expressed in a very large amount at a position of about 1500b was observed. The size of the Cl-6 clone insert was approximately 1300 bp.
Considering the results of Southern hybridization, C
It was confirmed that the 1-6 clone was a substantially full-length cDNA.

Example 5 (sequencing) Since the plasmid has the structure shown in FIG. 1, M
Sequencing was performed using a 13-system forward primer and a reverse primer. This allows the nucleotide sequence to be read from the 5 'and 3' sides of the cloned cDNA. Also, cd
In order to read the inside of NA, a dilation clone was prepared by a conventional method. DNA sequencing is Sanger,
BcaBEST Dideoxy Sease Kit based on dideoxy terminator method such as F.
quencing Kit, sold by TAKARA).

The sequence data thus obtained was edited into a continuous sequence using the DNA sequence ligation program of GENETYX to obtain the nucleotide sequence shown in SEQ ID NO: 2. The 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 recipe (Formulation No. 4) shows the entire nucleotide sequence of the cDNA and the primary amino acid sequence of the protein encoded thereby.

Example 6 (Preparation of transformant) The pUC118 plasmid vector obtained by subcloning the cDNA of Cl-6 protein in Example 4 was digested with EcoRI to prepare cDNA of Cl-6 protein, which was used as expression plasmid pGEX. -4T-3 (sold by Pharmacia)
Incorporated in. This expression vector was introduced into Escherichia coli DH5 to obtain a transformant. This transformant has been deposited at the Patent Depositary Center, Institute of Biotechnology, Institute of Industrial Science and Technology (identification label: E. coli MK001, deposit number:
FERM P-15316).

Example 7 (Production of Polypeptide) The transformant obtained in Example 6 was cultured in 1 liter of L-broth containing ampicillin until the absorbance reached 0.6, and IPTG was adjusted to 0.5 mM. And culturing was continued for another 3 hours.

Then, the cells were collected, sonicated, and the supernatant obtained by removing the disrupted cells was mixed with glutathione sepharose (sold by Pharmacia), and the glutathione sepharose was washed with a phosphate buffer. Cl-6 protein was released with a phosphate buffer containing 5 mM glutathione. After dialyzing this protein against phosphate buffer, part of it is SD
As a result of analysis by S-polyacrylamide gel, purity 9
0% or more of the GST fusion protein was synthesized.

Example 8 (Preparation of Antibody) The Cl-6 protein purified in Example 7 was subcutaneously inoculated into a rabbit with complete Freund's adjuvant three times every two weeks, and the serum was collected.

When this serum was tested using the Western blotting method, Cl-
Since it showed a reliable reactivity to 6 proteins, Cl-
It was confirmed that it can be used as an antibody against 6 proteins.

[0072]

According to the present invention, unknown useful polypeptides can be efficiently mass-produced. This polypeptide can be used as a raw material for cosmetics.

Further, it becomes possible to develop a new method for selecting pieces in pearl culture.

[0074]

[Recipe]

 SEQ ID NO: 1 Sequence length: 1002 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear origin Organism name: Pinctada fucata Cell type: Mantle epithelial cell Sequence 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 GACTAGACCACT TGCCAGCTTCCAGAGTTCTACT GCAGT 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]

[Recipe]

 SEQ ID NO: 2 Sequence length: 1326 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin organism name: Pearl oyster (Pinctada fucata) Cell type: Mantle features of epithelial cells sequence: mRNA present position: 1..1326 method to determine the characteristics: E SEQ 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 TGGC CTCCCA 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]

[Formulation list] SEQ ID NO: 3 Sequence length: 334 Sequence type: Amino acid Topology: Linear Sequence type: Protein Origin organism name: Pearl oyster (Pinctada fucata) Cell type: Mantle epithelial cell Sequence characteristics Characteristic symbol: peptide Location: 1..334 Method of determining feature: E sequence Met Lys Pro Phe Val Thr Leu Ala Ser Leu Ile Val Leu Ile Ala Ser 1 5 10 15 Ala Ser Ala Asp Gly Tyr Asp Asp Asp Tyr Lys Lys Tyr Gly Ser Val Gly 20 25 30 Tyr Gly Pro Gly Ile Ser Leu Gly Gly Gly Gly Leu Gly Gly Gly Gly 35 40 45 Gly Ile Ile Ser Val Gly Gly Gly Gly Gly Leu Gly Gly Gly Leu Gly 50 55 60 Gly Gly Leu Gly Cys Gly Leu Val Gly Val Gly Gly Gly Gly Leu Ile 65 70 75 80 Gly Gly Phe Gly Pro Gly Arg Val Ser Gly Thr Ile Asn Ala Gly 85 90 95 Gly Gly Val Phe Ala Ser Gly Ser Gly Leu Gly Gly Leu Ser Pro Ala 100 105 110 Gly Arg Gly Ala Ala Gln Gly Ala Ala Thr Leu Ser Ala Leu Gln Ile 115 120 125 Ala Ser Gly Arg Pro Gly Arg Val Ser Gly Val Ser Val Gl y Thr Gly 130 135 140 Gly Gly Arg Ala Val Val Ser Gly Ser Ala Thr Pro Val Gly Gly Phe 145 150 155 160 Gly Val Pro Tyr Gly Gly Tyr Gly Tyr Asn Tyr Gly Val Pro Ser Tyr 165 170 175 Gly Val Gly Leu Pro Ser Tyr Gly Val Ser Leu Pro Ser Tyr Gly Val 180 185 190 Gly Leu Gly Gly Gly Tyr Gly Gly Tyr Gly Tyr Gly Leu Asp Leu Ala 195 200 205 Ser Phe Gln Gly Ser Thr Tyr Gly Asn Leu Ala Thr Gly Gln Ile Asn 210 215 220 Thr Ala Val Val Ala Phe His Met Ala Val Leu Leu Glu Ser Met Glu 225 230 235 240 Ala Glu Ser Asp Thr Glu Val Asp Thr Glu Asp Met Asp Ser Glu Glu 245 250 255 Asp Met Glu Ser Glu Glu Asp Thr Glu Ser Glu Glu Asp Thr Glu Ser 260 265 270 Glu Glu Asp Thr Glu Ser Glu Glu Asp Met Glu Ser Glu Glu Asp Met 275 280 285 Asp Ser Glu Ser Ser Val Val Asp Gln Val Ser Met Val Tyr Pro Asn 290 295 300 His Phe Thr Gly Asp Val Leu Phe Cys Gln Val Arg Leu Gln Glu Leu 305 310 315 320 Glu Phe Pro Ala Leu Glu Ala Leu Val Ser Val Val Leu Glu 325 330

[0077]

[Formulation list] SEQ ID NO: 4 Sequence length: 1326 Sequence type: Nucleic acid Topology: Linear Sequence type: cDNA to mRNA Origin Biological name: Pearl oyster (Pinctada fucata) Cell type: Mantle epithelial cell Sequence Features of the symbol: CDS Location: 38..1039 Method of determining the feature: P sequence AACATCTCCA GATAAAGAAC GGTACCCATC CACAATC 37 ATG AAG CCC TTT GTC ACA CTC GCA AGC TTG ATC GTC TTA ATT GCT TCG 85 Met Lys Pro Phe Val Thr Leu Ala Ser Leu Ile Val Leu Ile Ala Ser 1 5 10 15 GCT TCT GCC GAC GGA TAT GAT GAC TAT AAG AAA TAC GGA AGT GTC GGT 133 Ala Ser Ala Asp Gly Tyr Asp Asp Tyr Lys Lys Tyr Gly Ser Val Gly 20 25 30 TAT GGA CCT GGC ATC AGC CTT GGA GGT GGT GGT TTA GGT GGC GGA GGA 181 Tyr Gly Pro Gly Ile Ser Leu Gly Gly Gly Gly Leu Gly Gly Gly Gly 35 40 45 GGC ATT ATC TCG GTT GGA GGC GGA GGA GGC CTC GGA GGA GGC CTC GGA 229 Gly Ile Ile Ser Val Gly Gly Gly Gly Gly Leu Gly Gly Gly Leu Gly 50 55 60 GGA GGC TTG GGT TGT GGT CTC GTA GGT GTC GGA GGC GG T GGA CTT ATC 277 Gly Gly Leu Gly Cys Gly Leu Val Gly Val Gly Gly Gly Gly Leu Ile 65 70 75 80 GGA GGA GGG TTT GGC CCC GGT AGA GTG TCT GGT ACC ATC AAC GCA GGA 325 Gly Gly Gly Phe Gly Pro Gly Arg Val Ser Gly Thr Ile Asn Ala Gly 85 90 95 GGT GGA GTT TTC GCA AGC GGA TCC GGA TTG GGA GGA CTC TCA CCA GCT 373 Gly Gly Val Phe Ala Ser Gly Ser Gly Leu Gly Gly Leu Ser Pro Ala 100 105 110 GGA AGA GGT GCA GCA CAA GGT GCA GCC ACT CTA AGC GCT CTC CAA ATT 421 Gly Arg Gly Ala Ala Gln Gly Ala Ala Thr Leu Ser Ala Leu Gln Ile 115 120 125 GCT TCC GGA AGA CCA GGC AGA GTC TCA GGT GTA TCC GTC GGA ACT GGA 469 Ala Ser Gly Arg Pro Gly Arg Val Ser Gly Val Ser Val Gly Thr Gly 130 135 140 GGA GGT CGT GCT GTT GTT TCC GGA AGT GCT ACT CCA GTT GGA GGC TTT 517 Gly Gly Arg Ala Val Val Ser Gly Ser Ala Thr Pro Val Gly Gly Phe 145 150 155 160 GGT GTT CCC TAT GGA GGT TAC GGA TAC AAT TAC GGT GTC CCA AGT TAT 565 Gly Val Pro Tyr Gly Gly Tyr Gly Tyr Asn Tyr Gly Val Pro Ser Tyr 165 170 175 GGA GTT GGC CTC CCA AGC TAC GGA GTC AGC C TT CCA AGT TAC GGA GTA 613 Gly Val Gly Leu Pro Ser Tyr Gly Val Ser Leu Pro Ser Tyr Gly Val 180 185 190 GGT TTG GGA GGA GGT TAT GGT GGC TAT GGA TAC GGA CTA GAC CTT GCC 661 Gly Leu Gly Gly Gly Tyr Gly Gly Tyr Gly Tyr Gly Leu Asp Leu Ala 195 200 205 AGC TTC CAA GGT TCT ACC TAC GGA AAT CTT GCA ACT GGA CAA ATT AAT 709 Ser Phe Gln Gly Ser Thr Tyr Gly Asn Leu Ala Thr Gly Gln Ile Asn 210 215 220 ACC GCA GTG GTA GCA TTC CAT ATG GCG GTT CTC TTG GAA TCT ATG GAA 757 Thr Ala Val Val Ala Phe His Met Ala Val Leu Leu Glu Ser Met Glu 225 230 235 240 GCG GAA TCG GAT ACG GAG GTG GAT ACG GAG GAT ATG GAC TCG GAG GAG 805 Ala Glu Ser Asp Thr Glu Val Asp Thr Glu Asp Met Asp Ser Glu Glu 245 250 255 GAT ATG GAA TCG GAG GAG GAT ACG GAA TCG GAG GAG GAT ACG GAA TCG 853 Asp Met Glu Ser Glu Glu Asp Thr Glu Ser Glu Glu Asp Thr Glu Ser 260 265 270 GAG GAG GAT ACG GAA TCG GAG GAG GAT ATG GAA TCG GAG GAG GAT ATG 901 Glu Glu Asp Thr Glu Ser Glu Glu Asp Met Glu Ser Glu Glu Asp Met 275 280 285 GAC TCG GAG TCC TCG GTG GTG G AT CAA GTC TCT ATG GTG TAT CCC AAT 949 Asp Ser Glu Ser Ser Val Val Asp Gln Val Ser Met Val Tyr Pro Asn 290 295 300 CAC TTT ACG GGG GAC GTG CTG TTC TGT CAG GTC AGG CTT CAG GAG CTG 997 His Phe Thr Gly Asp Val Leu Phe Cys Gln Val Arg Leu Gln Glu Leu GAG TTC CCA GCT TTG GAA GCA TTA GTT TCG GTG GTT TTG GAG 1039 Glu Phe Pro Ala Leu Glu Ala Leu Val Ser Val Val Leu Glu 325 330 TAGGTAGTCGA ATACAGTATT TATGGAGGTGGAATTC GTGGTATCAT CGGCGGAGGT GGTATCATCG GCGGAGGTGG TATAGGAGGC GGTATCATCG 1159 GCGGAGGTGG TATCGGACCA ATCGGCGGTG GAATCATTAG AAAGAAGAAGGATTATTAAGCTG 1219 ATGTTTGATG TATGGGAATT GCCAAATGGATCATGGA CTCACCTTTT TCCTGGATAT ACCTTTATTATT CACTTTTTTATT 1279

[Brief description of the drawings]

FIG. 1 is a block diagram showing one embodiment of the cloning vector of the present invention.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI Technical display location C12N 1/21 C12N 1/21 5/10 C12P 21/02 C C12P 21/02 21/08 21 / 08 7823-4B C12Q 1/68 A C12Q 1/68 G01N 33/53 D G01N 33/53 33/531 A 33/531 A61K 7/00 J // A61K 7/00 K C12N 5/00 B (C12N 15 / 09 ZNA C12R 1:91) (C12P 21/02 C12R 1:19) (54) Title of the Invention cDNA of novel polypeptide gene, vector containing said cDNA, host cell transformed with said vector, Polypeptide produced thereby, method for producing the same, DNA encoding the polypeptide, and antibody of the polypeptide

Claims (11)

[Claims] 1. cD encoding the nucleotide sequence of SEQ ID NO: 1.
NA. 2. cD encoding the nucleotide sequence of SEQ ID NO: 2.
NA. 3. A cloning vector containing the cDNA according to claim 1 or 2. 4. A DNA having the nucleotide sequence represented by SEQ ID NO: 1, or a fragment that selectively hybridizes to the sequence. 5. A DNA having the nucleotide sequence represented by SEQ ID NO: 2, or a fragment that selectively hybridizes to the sequence. 6. An expression vector containing the cDNA according to claim 1 or 2. 7. An expression vector according to claim 6,
Transformants such as bacteria, yeasts, insects, insect cells or mammalian cells. 8. A polypeptide produced by the transformant according to claim 7. 9. The polypeptide according to claim 8, which has the amino acid sequence of SEQ ID NO: 3. 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. 11. An antibody prepared by using the polypeptide according to claim 8 or 9 as an antigen.