JP3243531B2 - Method for screening functional proteins - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel screening method for a functional protein, and more particularly to a novel screening method for a G protein-coupled receptor.

[0002]

2. Description of the Related Art G protein-coupled receptors are coupled to intracellular GTP-binding proteins when they bind to extracellular signal transducers (ligands) to activate intracellular calcium dynamics and activate protein kinases. Furthermore, it is known to cause various intracellular metabolic changes. Therefore, G protein-coupled receptors are important for both basic and higher-order functions of living organisms, and controlling the activity of these receptors is important for treating diseases related to nerves, immunity, blood pressure, and metabolism. It is valid. In addition, genomic information obtained from recent genomic analysis research has revealed that there are many unidentified G protein-coupled receptors, and elucidation of their functions will provide new therapeutic and diagnostic agents. It is expected.

[0003] As a screening method for G protein-coupled receptor cDNA or genomic DNA, (1) an oligonucleotide deduced from a partial amino acid sequence of a purified receptor protein is synthesized and prepared from a tissue or cultured cells as a probe. Method for searching for a cDNA or genomic DNA library (Nature 323 (6087): 411 (1986)),
(2) A method for searching a cDNA or genomic DNA library by gentle hybridization using a cDNA or genomic DNA fragment of a similar receptor or a synthetic DNA as a probe (Science 237: 527 (1987)), (3) Oligo dT Alternatively, cDNA is synthesized from RNA prepared from tissue or cultured cells using a primer containing a random oligonucleotide. A method for searching for a similar receptor cDNA from this cDNA library by expression cloning (Science 241: 558)
(1988)), and (4) oligonucleotides were synthesized from two or more well-conserved positions in the amino acid sequence of a related G protein-coupled receptor, and these were used as primers.
Methods such as a method of searching for a similar receptor cDNA by performing PCR using a cDNA or a genomic DNA prepared from a tissue or a cultured cell as a template (Science 244: 569 (1989)) are already known.

It has also been shown that a chimeric receptor in which the C-terminal region of a G protein-coupled receptor is replaced with a related receptor has essentially no effect on ligand binding or signal transduction (FEBS lett. 241: 119 (1988)). However, there is no known method for screening a G protein-coupled receptor cDNA using a chimeric G protein-coupled receptor cDNA library.

[0005]

The progress of genome research has made it possible to obtain enormous amounts of sequence information. However, since the original function of the sequence has not been analyzed, even if the sequence is known, There is a so-called “orphan receptor” that is unknown. At present, the sequences of many G protein-coupled receptors are already known, and it is difficult to find further unknown receptors using the conventionally known screening methods for G protein-coupled receptor cDNA / DNA. It has become to. However, in order to further analyze the function of the living body and to develop a new drug for diagnosis, treatment and prevention of various diseases, it is necessary to develop a screening method that is not dependent on a conventionally known sequence and thereby to use a novel G protein coupling. The discovery and study of type receptors is desired. Accordingly, the present invention provides a novel method for isolating a G protein-coupled receptor cDNA by identifying a cDNA that expresses receptor activity from a chimeric G protein-coupled receptor cDNA library.

Furthermore, the novel screening method obtained by the present invention is not limited to G protein-coupled receptors, but also to other biological functions (neural functions, immune functions, endocrine functions,
It has been clarified that the present invention can be applied to screening of proteins having a circulatory function) and functional proteins having a chemical reaction catalytic function. That is, the present invention also provides a novel screening method for, for example, an ion channel, a transporter, a growth factor, a hydrolase, a synthase, an oxidase / reductase, a protein inhibitor, a bioactive peptide, and a peptide poison. It is. Accordingly, the present invention provides a novel method for isolating a functional protein cDNA by identifying a cDNA that expresses a functional protein having activity from a functional protein chimeric cDNA library.

[0007]

Means for Solving the Problems The present inventors selected a C-terminal region which does not significantly affect the function among the regions conserved between functional proteins, and used an antisense strand oligonucleotide primer. The primer was used to synthesize a primer-extended cDNA library using tissue or cell poly (A) + RNA as a template. Further, this cDNA was ligated so that the translation frame coincided with the cDNA encoding the C-terminal region of a known homologous functional protein.

The resulting chimeric protein cDNA
The library is a collection of various functional protein cDNAs enriched, and by incorporating these into an expression vector so that they can be expressed, chimeras with different origins from the N-terminal side and the C-terminal side containing the functional region The function can be expressed in the protein. That is, a functional protein cDNA can be efficiently isolated by expression screening of this chimeric protein cDNA library using its activity as an index.

That is, the present invention provides the following (1) to (9)
I will provide a. (1) A method for producing a cDNA library comprising a cDNA encoding a functional protein having a functional region and having a region between the functional region and the carboxyl terminus and conserved between homologous functional proteins, A method for preparing a chimeric cDNA library, comprising the following steps (a) to (d): (A) synthesizing an antisense oligonucleotide complementary to the sequence of a region located between the functional region and the carboxyl terminus of the functional protein and conserved between proteins having the same type of function as the functional protein, (b) tissue Alternatively, poly (A) ⁺ RNA is prepared from cultured cells. (C) Using the above poly (A) ⁺ RNA as a template and the above oligonucleotide as a primer, cDNA containing an amino terminal and a functional region is obtained by reverse transcription or PCR. (D) including the carboxyl-terminal side of the conserved region of the known homologous functional protein synthesized
The protein is inserted into an expression vector together with the cDNA so that the protein can be expressed in a state where the protein translation frames are linked together.

(2) A method for preparing a cDNA library containing a cDNA encoding a functional protein having a functional region and having a region located between the functional region and the carboxyl terminus and conserved between functional proteins of the same kind. A method for preparing a chimeric cDNA library, comprising the following steps (a) to (d). (A) synthesizing an antisense oligonucleotide complementary to the sequence of a region located between the functional region and the carboxyl terminus of the functional protein and conserved between proteins having the same type of function as the functional protein, (b-1) ) Preparing poly (A) ⁺ RNA from tissue or cultured cells, (b-2) subjecting the poly (A) ⁺ RNA to normalization or subtraction, (c) (b-2) treatment Using the subsequent poly (A) ⁺ RNA as a template and the above oligonucleotide as a primer, reverse transcription or PC
Synthesizing a cDNA containing an amino terminus and a functional region by R; and (d) including the carboxyl-terminal side from the conserved region of the synthesized known homologous functional protein.
The protein is inserted into an expression vector together with the cDNA so that the protein can be expressed in a state where the protein translation frames are linked together.

(3) A functional protein chimeric cDNA library prepared by the method according to (1) or (2). (4) A function having a functional region, which comprises the following steps (a) to (f), and having a region between the functional region and the carboxyl terminus and conserved between homofunctional proteins. A method for screening a sex protein. (A) synthesizing an antisense oligonucleotide complementary to the sequence of a region located between the functional region and the carboxyl terminus of the functional protein and conserved between proteins having the same type of function as the functional protein, (b) tissue Or preparing poly (A) ⁺ RNA from cultured cells; (c) using the above poly (A) ⁺ RNA as a template and the above oligonucleotide as a primer to synthesize cDNA containing amino terminus and functional region by reverse transcription or PCR (D) the cDNA comprises a carboxyl-terminal side from the conserved region of the synthesized known homofunctional protein.
(e) transcription and translation in vivo or in vitro to express the chimeric protein; and (f) expression. The function of the chimeric protein is screened.

(5) A region having a functional region, which is characterized by including the following steps (a) to (f), and which is located between the functional region and the carboxyl terminus and conserved between homofunctional proteins: A method for screening a functional protein having (A) synthesizing an antisense oligonucleotide complementary to the sequence of a region located between the functional region and the carboxyl terminus of the functional protein and conserved between proteins having the same type of function as the functional protein, (b-1) ) Preparing poly (A) ⁺ RNA from tissue or cultured cells, (b-2) subjecting the poly (A) ⁺ RNA to averaging or subtraction treatment, (c) poly (A) ⁺ RNA after (b-2) treatment (A) Reverse transcription or PC using ⁺ RNA as template and the above oligonucleotide as primer
Synthesizing a cDNA containing an amino terminus and a functional region by R; and (d) including the carboxyl-terminal side from the conserved region of the synthesized known homologous functional protein.
(e) transcription and translation in vivo or in vitro to express the chimeric protein; and (f) expression. The function of the chimeric protein is screened.

(6) The method for producing a chimeric cDNA library according to the above (1) or (2), wherein the functional protein is a G protein-coupled receptor. (7) The chimeric cDNA according to (3), wherein the functional protein is a G protein-coupled receptor.
Library. (8) The screening method according to the above (4) or (5), wherein the functional protein is a G protein-coupled receptor.

(9) The screening method according to (8), wherein the conserved region is in the seventh transmembrane region of a G protein-coupled receptor. (10) The screening method according to (8), wherein the amino acid sequence of the conserved region is SEQ ID NO: 1. (11) The screening method according to (8), wherein the sequence of the primer is SEQ ID NO: 13.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, "functional protein" includes G protein-coupled receptor and other biological functions (neural function, immune function, Endocrine function, cardiovascular function)
Examples include proteins and functional proteins having a chemical reaction catalytic function. Specifically, for example, an ion channel,
Transporters, growth factors, hydrolases, synthases,
Oxidation / reduction enzymes, proteinaceous inhibitors, bioactive peptides, peptide-type toxins and the like. The size of the functional protein to which the present invention can be applied is preferably such that the amino acid length is up to about 1,200 and the cDNA is about 3.5 kb at the maximum.

In the present invention, the term “functional region” refers to a region that directly exerts its function in the protein molecule. For example, in the case of a G protein-coupled receptor, the ligand binding region and the coupling with the G protein The specificity determining region corresponds to an ion selection filter in the case of an ion channel, and the region including an active center in the case of an enzyme.

"Same functions" refers to those having the same or similar functions even if their origins (species of animals, plants, etc., types of tissues, etc.) differ, for example, differences in ligands, ions, etc. The effect on these, if any, is the same. In the art, proteins having the same kind of functions as described above may be referred to as "family". For example, in the case of G protein-coupled receptors, G protein-coupled receptor families including those having different ligands Are collectively referred to as In the present invention, all proteins belonging to these families are defined as having the same type of function. In the present invention, “poly (A) ⁺ RNA” is RNA in which a plurality of adenines are present at the 3 ′ end of RNA, and mainly means mRNA.

Hereinafter, the present invention will be described as an example of a functional protein, particularly a G protein-coupled receptor. First, as step (a), an antisense oligonucleotide complementary to the sequence of the region between the functional region of the functional protein and the carboxy terminus and conserved between proteins having the same type of function as the functional protein is synthesized. .

The G protein-coupled receptor is known to have seven transmembrane regions I to VII as shown in FIG. 1 and is also called a seven-transmembrane receptor. Also,
Many of them are said to have a ligand binding region from the N-terminus to the III transmembrane region and a conjugate specificity determining region for G protein between V and VI transmembrane regions (Protein Nucleic Acid Enzyme 42: 275 ( 1997) and 42: 343 (1997)). Therefore, the sequence of the region conserved between the functional region and the protein having the function of the G protein-coupled receptor between the carboxy terminus and the conserved region located on the C-terminal side from the VI transmembrane region was determined as the sequence of the antisense oligo. Design and synthesize nucleotides. Specifically, G
Any region can be used as long as it is a region conserved between protein-coupled receptor subtypes. However, in order to obtain a novel receptor that has not been known, it is preferable to select a region near the VII transmembrane region. Preferred as an amino acid sequence conserved in this region in the present invention is NP (I / L / F)
(I / L / V) Y (T / A / P) (SEQ ID NO: 1). An antisense nucleotide sequence that can be suitably used as a primer is the sequence GT (A
/ G) TA (G / A) ATIA (T / A) NGG (A / G) TT. Here, parentheses indicate the mixture of bases shown, N indicates A,
A mixture of G, C, and T indicates that I is synthesized by adding inosine.

Another embodiment of the present invention is a K ⁺ channel family having six transmembrane membranes. The amino acid sequence of this family is, for example, (Neurosci. 12: 538 (1992))
And a pore forming portion of an ion channel is assumed as a functional region. Therefore, in the case of this protein, the oligonucleotide containing the antisense nucleotide sequence TAICCIAINGTNGTCAT (SEQ ID NO: 4) is derived from the amino acid sequence MTT (V / L) GY (SEQ ID NO: 3) contained in the P region known as a conserved region. Primers can be used.

Still another embodiment of the present invention includes an inward rectifier K ⁺ channel family. The amino acid sequence of this family can be found, for example, in (J. Biol. Chem. 270: 5691).
(1995)), and assumes a pore-forming portion of an ion channel as a functional region. Therefore, in the case of this protein, the amino acid sequence TIG (F / Y) G (SEQ ID NO: 5) contained in the H5 region known as a conserved region is used to convert the antisense nucleotide sequence CC (A / G) (A / T ) AICC (A
Oligonucleotide primers containing (/ G / T) ATNGT (SEQ ID NO: 6) can be used.

In still another embodiment of the present invention, Kunitz
/ BPTI serine protease inhibitor family. The amino acid sequence of this family is, for example,
Protein database; P05067, Q06481, P36992, P00978,
And P10646, and an inhibitory reaction site is assumed as a functional region. Therefore, in the case of this protein, the amino acid sequence YGGC (G / L / M) G
(SEQ ID NO: 7) shows the antisense nucleotide sequence CCI
Oligonucleotide primers containing AI (A / G) CAICCNCC (G / A) TA (SEQ ID NO: 8) can be used.

In still another embodiment of the present invention, Na ⁺ / P
O ₄ Shin porter family, and the like. The amino acid sequence of this family can be found, for example, in "The transporter factsbo
ok ", (Griffith, J., Sansom, C., Academic Press, 1
998), and a transmembrane region is assumed as a functional region. Therefore, in the case of this protein, the amino acid sequence (E / D) (I / K) Q (D /
From P) W (SEQ ID NO: 9), an oligonucleotide primer containing an antisense nucleotide sequence (CCA (G / A) TC (T / C) TGIATNTC (SEQ ID NO: 10) can be used.

Still another embodiment of the present invention includes a nerve growth factor family. The amino acid sequence of this family is known, for example, by Protein Nucleic Acid Enzyme 36: 1211 (1991). In the case of this family, since the peptide is relatively small, the whole peptide has bioactivity. Therefore, the setting position of the primer is almost at the C-terminal, and the amino acid sequence WR (F / W) IRI
(SEQ ID NO: 11), an antisense nucleotide sequence
Oligonucleotide primers containing ATICI (A / G / T) ATI (A / C) ANC (G / T) CCA (SEQ ID NO: 12) can be used.

When an oligonucleotide containing the above antisense nucleotide sequence is used as a primer in the present invention, the nucleotide length is preferably at least 23 mer. At this time, S
It is preferable to add a restriction enzyme cleavage site (B) such as peI or NotI, and to add a protection sequence for promoting annealing of the DNA terminus and increasing the efficiency of digestion by the restriction enzyme. In addition, it is preferable to design the primer so that the degree of degeneracy is 100 or less so that the annealing efficiency with the target functional protein does not become too low.

In the step (b), the tissue or cell poly
(A) ⁺ RNA is prepared by a conventional method. For example, TRIZOL (LifeT
ech Oriental Co., Ltd.) to obtain all RNs from tissues such as brain, heart, kidney, etc. or their localized sites, or cells.
After extracting A, use Poly (A) ⁺ R with OligoTex (Takara Shuzo) ^.
Purify NA.

At this stage, the extracted RNA contains many of those encoding structural proteins, metabolic enzymes, etc., and if the process proceeds to the next cDNA synthesis stage, the target protein will be buried, and the screening itself Is possible, but unnecessary cDNAs are amplified at the same time, resulting in low screening efficiency. Therefore,
In order to selectively remove those having a high RNA content and increase the relative ratio of RNA encoding the target protein, poly (A) ⁺ RNA averaging (DNA Res. 1:91 (1992)
4)) or subtraction ("Molecular Cloning 2nd
ed. ").

Next, in step (c), primer extension using the above oligonucleotide as a primer is carried out.
The cDNA synthesis reaction may be performed using a reverse transcriptase or a PCR reaction according to a conventional method, and is not particularly limited. For example, Revertra Ace (manufactured by Toyobo Co., Ltd.) is used for reverse transcriptase.
And DNA synthase (Amersham-Pharmacia). Alternatively, SMART II oligonucleotides (Cl
ontech) and PCR using the above primers.

The synthesized cDNA is separated by, for example, 1% agarose gel electrophoresis, and the DNA is extracted by cutting out the neighborhood of the size expected from homology. After attaching a primer adapter or linker for restriction enzyme (A), digest with restriction enzymes (A) and (B), and cDNA library fragment (AB)
(See flowcharts in FIGS. 2 and 3). On the other hand, in the next step (d), a cDNA containing the carboxy terminal side of a known functional protein is synthesized by a conventional method, and based on this, a restriction enzyme of the above-mentioned primer (point mutation introduction method or PCR method) is used. B) Create a restriction enzyme (B) cleavage site corresponding to the cleavage site.

Further, this cDNA is expressed in an expression vector (for example,
It is cloned into pcDNA3 (Invitrogen) or pSD64vector). Expression vectors include Escherichia coli, Bacillus subtilis, and yeast-derived plasmids, bacteriophages such as λ phage, retroviruses, vaccinia viruses,
Animal viruses such as baculovirus can be used and are appropriately selected depending on the host in which the functional protein is expressed. For example, when expressed in Xenopus,
When expressing D64 in cultured animal cells, it is preferable to use a vector such as pcDNA3. Also, depending on the host
The expression vector is preferably ligated downstream of the promoter so that the expression is performed under the control of a suitable promoter such as the SP6 promoter or the CMV promoter. Furthermore, if necessary, a regulatory sequence such as a signal sequence and an enhancer may be contained, and these techniques are known in the art. As the host, for example, bacteria such as genus Escherichia and Bacillus, yeast, insects, animal cells and the like, any of which are known in the art can be used, but for the purpose of analyzing the physiological function of the expressed protein, It is preferable to use animal cells such as Xenopus oocytes and cultured cells. Digest with restriction enzymes (A) and (B)
A DNA fragment (BA) containing the C-terminal cDNA of the functional protein and the vector is obtained. (See FIGS. 2 and 3)

The cDNA library fragment (AB) obtained as described above, the C-terminal cDNA of the functional protein and the DNA fragment (BA) containing the vector were ligated to synthesize in the above step (b). A cDNA containing an amino terminus and a functional region, and a cDNA containing a carboxy terminus from the above-mentioned conserved region of a known functional protein are incorporated into an expression vector so that they can be expressed in a state in which protein translation frames are linked together. A chimeric protein cDNA library is created.

Alternatively, in the step (d), first,
The cDNA containing the amino terminus and the functional region synthesized in the step (b) was ligated from the conserved region of the known functional protein to the cDNA containing the carboxy terminus with the protein translation frame, and then ligated. Chimeric cDNA
May be incorporated in an expression vector so that it can be expressed. The chimeric protein is expressed by transforming cultured cells from these cDNAs, or the cDNA is transcribed and translated in vitro to express the chimeric protein, and the function of the expressed chimeric protein is screened.
The specific method is known in the art.

For example, in the case of a G protein-coupled receptor, as a function screening method, the activity of the receptor can be measured in a Xenopus oocyte expression system or the like. Measurement of receptor activity is targeted
Depending on the type of G protein-coupled receptor, a test substance (eg, a fractionated tissue extract, a low molecular compound, a synthetic peptide, etc.) is added to a receptor expression system such as a cultured cell or an oocyte, It can be performed by observing the fluctuation of intracellular calcium ion, pH change and intracellular potential change accompanying metabolic fluctuation.

In another embodiment, in the case of the six-transmembrane type K ⁺ channel family and the inward rectifier K ⁺ channel family, the function screening thereof is carried out, for example, by measuring the intracellular current by membrane voltage fixation of oocytes and the like. There is a measurement method (for example, Nature 362: 127 (1993)). Furthermore, in the case of the Kunitz / BPTI-type serine protease inhibitor family, screening can be performed, for example, by measuring trypsin inhibitory activity using a fluorescent substrate.

Further, in the case of the Na ⁺ / PO ₄ symporter family, Na ⁺ -dependent phosphate transport is measured in an expression system such as an oocyte (J. Biol. Chem. 265: 12331 (1990)). ).
Furthermore, in the case of the nerve growth factor family, the ability to differentiate into nerve cells is observed using neural differentiated cells such as PC12 cells.

[0036]

EXAMPLES The present invention will be described in detail with reference to the following examples, which do not limit the scope of the present invention. Unless otherwise specified, details of the genetic engineering technique are described in “Molecular Cloning 2nd ed.” (Sambroo
k, J., Fritsch, EF, Maniatis, T. Cold Spring Har
vor Laboratory Press, 1989).

The serotonin receptor-related cDNA library
Preparation and Functional Screening Example 1 Synthetic primers common to serotonin receptors
Production of 10 known serotonin (5HT) receptor subtypes, 5-HT1B (M89478), 5-HT1C (M81778), 5-HT1D (M899)
55), 5-HT1E (M91467), 5-HT2 (X57830), 5-HT2B (X7730
7), 5-HT4B (Y12505), 5-HT4 (Y08756), 5-HT6 (L4114
The amino acid sequences predicted from the nucleotide sequences of 7) and 5-HT7 (L21195) were compared. Here, () is the search system provided by Japan DNA Data Bank, DDBJ / GenBank / EMBL Da
Indicates the Accession Number registered in tabase. Among the putative seventh transmembrane regions having particularly high homology among these sequences,
Select the region at the C-terminal side, base sequence corresponding to the amino acid sequence (SEQ ID NO: 1) and codon usage (codon us
age), considering the following antisense strand 5HT
Receptor degenerate primers were synthesized.

5'-GTTG ACTAGT GT (A / G) TA (G / A) ATIA (T / A) NG
G (A / G) TT-3 ′ (SEQ ID NO: 13) where the parentheses indicate a mixture of the bases indicated therein, and N indicates A, G, C,
A mixture of T, I indicates that synthesis is performed with inosine. At the 5 'end, a SpeI recognition sequence (underlined) and its protection sequence were introduced for the convenience of the subsequent library construction.

Example 2 Poly (A) + RNA from rat cerebrum
Fraction preparation, averaging, and cDNA synthesis Total RNA was extracted from rat cerebrum using TRIZOL (LifeTech Oriental), and Oligotex-dT30 (Takara Shuzo)
Was used to prepare poly (A) + RNA. Synthesis of single-stranded cDNA on Oligotex-dT30 (Takara Shuzo) and averaging of poly (A) + RNA using the same are described in the literature (DNA Res. 1:91 (1992).
The method described in 4)) was followed. Here, single-stranded DNA was synthesized on latex using 200 μg rat cerebral poly (A) + RNA and 25 mg Oligotex-dT30. In the subsequent averaging step, 20 μg rat cerebral poly (A) + RNA was annealed with the single-stranded cDNA synthesized on the latex for 15 minutes at 55 ° C., and the centrifuged supernatant was subjected to a second annealing. In the primer extension reaction, the fourth centrifugation supernatant obtained by the same operation was collected by ethanol precipitation and used as a template.
CDNA was synthesized from 1 nmol of the primer of SEQ ID NO: 13 using a cDNA synthesis kit (Stratagene). A BamHI adapter (Takara Shuzo) was added thereto, phosphorylated with polynucleotide kinase, digested with SpeI, and subjected to 1% agarose gel electrophoresis to extract DNA of about 1-2 kb. (QIAquick Gel Extraction Kit; Qiagen)

Example 3 C-terminal of serotonin 2C receptor
Amplification of cDNA to be Encoded and Construction into Vector C-terminal cDNA of human 5HT2c receptor was amplified by PCR from its known base sequence. That is, the human 5HT2c receptor
Upstream primer containing SpeI recognition site (ACTAGT) designed based on cDNA base sequence (M81778) 5'-GGTGTATAC aCTagT CAACAAAA-3 '(SEQ ID NO: 14) and 3'
Using the downstream primer 5'-GCT gGTACC GTAGGAAAAGACTG-3 '(SEQ ID NO: 15) containing the KpnI recognition site (GGTACC) in the untranslated region (herein lowercase letters introduce artificial modifications to the human 5HT2c receptor cDNA base sequence) This creates a recognition site for SpeI and KpnI underlined, respectively), and performed PCR using human brain-derived cDNA (Clontech) as a template. The composition of the reaction solution was 200 pM each of synthetic DNA primers (SEQ ID NOs: 14 and 15), 1 ng of template cDNA, 0.2 mM dNTPs, 0.025
Use unit / μl Taq DNA polymerase (ExTaq, Takara Shuzo) and the buffer attached to the enzyme.
μl. The temperature cycle is a thermal cycler (Perk
in-Elmer), 94 ° C for 2 minutes, 94 ° C for 1 minute, 58 ° C
A cycle of 1 minute and 72 ° C. for 1 minute was repeated 30 times. Confirmation of the PCR product was performed by 1.2% agarose electrophoresis and ethidium bromide staining. See "Molecular Cloning
2nd ed. "(Sambrook, J., Fritsch, EF, Maniatis, T.
Author, Cold Spring Harvor Laboratory Press, 1989). As a result, the specifically amplified DNA product of about 300 bp was digested with SpeI and KpnI, and pSD64TF
(BH) vector (FIG. 4; pSP64T (Nucleic Acids Res. 1)
2: 7057 (1984), a vector modified by adding a multicloning site and the 5 ′ and 3 ′ untranslated regions of the frog β-globin gene to increase expression efficiency in Xenopus oocytes. Supplied by Dr. T. Snutch. ) S
It was inserted into the peI and KpnI sites using Ligation High (Toyobo) (pSD64TF-5HT2cR C-term: FIG. 5). Plasmid from transformed Escherichia coli XL1Blue (Stratagene)
DNA was prepared (Wizard, Promega). The nucleotide sequence of the inserted DNA is BigDye Terminator Cycle Sequencing FS
Reaction using Ready Reaction Kit (PE Biosystems) and SP6 promoter primer (Promega)
Decoding was performed using a fluorescence automatic sequencer (ABI Sequencer 377). The obtained base sequence information is from Lasergene (DNASTAR)
The inserted DNA fragment is analyzed using a known base sequence (M
81778) was not different from the corresponding part.

Example 4 Serotonin receptor-related protein
The BamHI-SpeI fragment (1-2 kb) of the cDNA extended from the 5HT receptor degenerate primer obtained in Example 2 of the construction site of the chimeric cDNA library was constructed using the plasmid pSD64TF- obtained in Example 3. 5HT2cR C-term Ba
It was inserted into the mHI and SpeI sites using Ligation High (Toyobo). As a result, the 5HT receptor-related cDNA extended from the 5HT receptor common primer is translated into the translation frame (translation frame).
frame) is matched, and it is linked to the C-terminal cDNA of 5HT receptor 2C. Using this cDNA, Escherichia coli XL1-Blue strain
(Stratagene) was transformed to construct a rat cerebral 5HT receptor-related protein chimeric cDNA library.

Example 5 Functional Tan Responding to Serotonin
Expression screening of protein cDNA The cDNA library prepared in Example 4 was spread on an agar medium, and about 10,000 colonies were subdivided into 96 pools (about 100 colonies / pool). Plasmid DNA for each pool
And then cap analog (Pharmacia)
And cRNA synthesis using SP6 RNA polymerase (MEGA
Script SP6 Kit; Ambion).

The cRNA pool was microinjected into Xenopus oocytes, and after 2-3 days, the current response induced by serotonin was measured. (These electrophysiological assays are described in Science 241: 558 (1988).) Positive pools that respond to serotonin were further subdivided and repeated in a similar manner until a single clone was obtained. At least three serotonin-reactive clones were obtained. The nucleotide sequences of these clones were determined and analyzed according to the method described in Example 3. As a result of a homology search with the database, cDNAs corresponding to rat 5HT receptor subtypes 2A (M30705), 2B (X66842), and 2C (M21410) were isolated. That is, searching from the chimeric cDNA library according to the present invention using the physiological activity as an index,
It became clear that it was possible to obtain the related protein cDNA.

G protein-coupled receptor chimeric cDNA library
Preparation of the library and orphan G protein-coupled receptor c
DNA isolation Example 6 G protein-coupled receptor chimeric cDNA live
Preparation of Rally Poly (A) + RNA was prepared from mouse brain in the same manner as in Example 1, and SMART was prepared using 0.5 μg mouse brain poly (A) + RNA as a template.
CDNA was synthesized using a PCR cDNA synthesis kit (Clontech). However, the primer used in Example 1 (SEQ ID NO: 13) was used as the downstream primer in both the primary strand extension and cDNA amplification reactions.

(1) The amplified cDNA was blunt-ended with T4 DNA polymerase (Blunting Kit; Takara Shuzo Co., Ltd.), followed by the addition and phosphorylation of a BamHI adapter (Takara Shuzo Co., Ltd.) by the same steps as in Example 2. After digestion with, DNA of about 1-2 kb was fractionated and extracted by 1% agarose gel electrophoresis and ethidium bromide staining. (QIAquick Gel
(Extraction Kit; Qiagen) (2) As in Example 4, C-terminal cD of human 5HT2c receptor
Bam of vector pSD64TF-5HT2cR C-term (Figure 5) with NA
The BamHI-SpeI cDNA fragment (1-2 kb) isolated in (1) was inserted into the HI and SpeI sites using Ligation High (Toyobo) and transformed into E. coli XL1-Blue strain (Stratagene) to obtain a mouse. A brain-derived receptor chimeric cDNA library was constructed.

Example 7 Orphan G Protein Conjugated Type
Isolation of Receptor cDNA The cDNA library prepared in Example 6 was transferred to an agar medium (LB,
75 μg / ml ampicillin), and 36 growing colonies were randomly picked up, and plasmid DNA was prepared from each (Wizard, Promega). The nucleotide sequence of the inserted DNA was determined in the same manner as in Example 3. As a result of a homology search based on this nucleotide sequence, it was found that one obtained cDNA encodes a part of an orphan G protein-coupled receptor whose ligand in vivo is unknown (FIG. 6).

Example 8 Orphan G Tan by RACE Method
Acquisition of C-terminal cDNA of protein-coupled receptor Mouse brain poly (A) + RNA prepared by the same process as in Example 2
Using 2.7 μg as a template, 162 ng of random nonamer pd (N)
CDNA was synthesized using a cDNA synthesis kit (BRL) with 9 (Takara Shuzo) as a primer. After adding BamHI linker (Takara Shuzo) and digesting with BamHI, the vector pBlues
It was cloned into the BamHI site of cript II KS- (Stratagene). Escherichia coli XL1-Blue (Stratag
ene) and a mouse brain cDNA library was prepared. This cDNA library was amplified on an agar medium, and the plasmid was recovered from a 1/30 volume of the E. coli suspension.
DNA was prepared (Wizard, Promega) and used as a template for RACE. The sense strand primer is the orphan G protein-coupled receptor cDNA identified in Example 7.
The following S1 and S2 were used based on the nucleotide sequence of S1 5'-TGCCACAGAGCAGTACCTC-3 '(SEQ ID NO: 16) S2 5'-CGTGAAGATGCTGGTGCTTG-3' (SEQ ID NO: 17)

As the antisense strand primer, T7 (T7 promoter primer; Nippon Gene) in the vector pBluescript II KS- was used. The composition of the reaction solution is
S1 and T7 200 pM each, template cDNA 150 ng, 0.2 mM dNTPs,
0.025 unit / μl Taq DNA polymerase (ExTaq; Takara Shuzo) and a buffer attached to the enzyme were used, and the total reaction volume was 20 μl. The thermal cycle was performed using a thermal cycler (Perkin-Elmer) at 94 ° C for 2 minutes and at 94 ° C for 1 minute.
After 5 cycles of 1 minute, 58 ° C for 1 minute, and 72 ° C for 1 minute, 2 µl was added to the second round using nested primers S2 and T7.
PCR was performed. That is, S2 and T7 each 200 pM, the first P
PCR was performed under the same conditions as for CR, except that the temperature cycle was performed 25 times. 1 μl of the reaction solution was subcloned into the pCR2.1-TOPO vector according to the prescription of TOPO TA Cloning Kit (Invitrogen). This was introduced into E. coli TOP10 (Invitrogen), and the resulting transformant was used in Example 3
The sequence of the inserted cDNA fragment was determined by the same method as described above. However, here, T7 promoter primer and M13
Reverse primer (Invitrogen) was used for the sequencing reaction. As a result, a cDNA overlapping with the orphan G protein-coupled receptor cDNA isolated in Experimental Example 7 and encoding the C-terminal portion was obtained. That is,
The cDNA base sequence containing the entire coding region of the orphan G protein-coupled receptor and the predicted primary structure of the receptor protein were clarified (FIG. 6, SEQ ID NO: 18).
And 19). This is almost the same as the known nucleotide sequence (M80481), and two nucleotide sequence substitutions and accompanying amino acid substitutions were observed (double line in FIG. 6; T → C at nucleotide number 323 and G at nucleotide number 475). → A). The underlined portion in FIG. 6 indicates the position where the 5HT receptor degenerate primer (SEQ ID NO: 13) is presumed to have annealed. The arrows in FIG. 6 indicate the positions of the S1 and S2 primers.

[0049]

[Sequence List] SEQUENCE LISTING <110> Director-General of Agency of Industrial Science and Technology <120> The screening method of functional proteins <130> 11900312 <160> 19 <170> PatentIn Ver. 2.0 <210> 1 <211> 6 <212> PRT <213> Artificial Sequence <220><223> Description of Artificial Sequence: conserved amino acid sequence in the seventh transmembrane segment of 5HT receptors <220><221> variation <222> 3 <223> Xaa = Ile , Leu or Phe <220><221> variation <222> 4 <223> Xaa = Ile, Leu or Val <220><221> variation <222> 6 <223> Xaa = Thr, Ala or Pro <400> 1 Asn Pro Xaa Xaa Tyr Xaa 1 5 <210> 2 <211> 17 <212> DNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: synthetic primer deduced from S eq ID No: 1 <220><221> variation <222> 9 <223> i <220><221> variation <222> 12 <223> N = A, C, G or T <400> 2 gtrtaratna wnggrtt 17 <210> 3 <211> 6 <212> PRT <213> Artificial Sequence <220><223> Description of Artificial Sequence: conserved amino acid sequence in the voltage gated K ⁺ channels <220><221> variation <222> 4 <223> Xaa = Val or Leu <400> 3 Met Thr Thr Xaa Gly Tyr 1 5 <210> 4 <211> 17 <212> DNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: synthetic primer deduced from Seq ID No: 3 <220><221> variation <222> 3,6,8 <223> i <220><221> variation <222> 9, 12 <223> N = A, C, G or T <400> 4 tanccnanng tngtcat 17 <210> 5 <211> 5 <212> PRT <213> Artificial Sequence <220><223> Description of Artificial Sequence: conserved amino acid sequence in the inward rectifier K ⁺ channels <220><221> variation <222> 4 <223> Xaa = Phe or Tyr <400> 5 Thr Ile Gly Xaa Gly 1 5 <210> 6 <211> 14 <212> DNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: synthetic primer deduced from Seq ID No: 5 <220><221> variation <222> 6 <223> i <220><221> variation <222 > 12 <223> N = A, C, G or T <400> 6 ccrwanccda tngt 14 <210> 7 <211> 6 <212> PRT <213> Artificial Sequence <220><223> Description of Artificial Sequence: conserved amino acid sequence i n the serine protease inhibitors <220><221> variation <222> 5 <223> Xaa = Gly, Leu or Met <400> 7 Tyr Gly Gly Cys Xaa Gly 1 5 <210> 8 <211> 17 <212> DNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: synthetic primer deduced from Seq ID No: 7 <220><221> variation <222> 3,5,9 <223> i <220><221> variation <222> 12 <223> N = A, C, G or T <400> 8 ccnanrcanc cnccrta 17 <210> 9 <211> 5 <212> PRT <213> Artificial Sequence <220><223> Description of Artificial Sequence: conserved amino acid sequence in the Na ⁺ / PO ₄ synporters <220><221> variation <222> 1 <223> Xaa = Glu or Asp <220><221> variation <222> 2 <223> Xaa = Ile or Lys <220><221> variation <222> 4 <223> Xaa = Asp or Pro <400> 9 Xaa Xaa Gln Xaa Trp 1 5 <210> 10 <211> 15 <212> DNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: synthetic primer deduced from Seq ID No: 9 <220><221> variation <222> 10 <223> i <220><221> variation <222> 13 <223> N = A, C, G or T <400> 10 ccartcytgn atntc 15 <210> 11 <211> 6 <212> PRT <213> Artificial Sequence <220><223> Description of Artificial Sequence: conserved amino acid sequence in the nerve growth factors <220><221> variation <222> 3 <223> Xaa = Phe or Trp <400> 11 Trp Arg Xaa Ile Arg Ile 1 5 <210> 12 <211> 17 <212> DNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: synthetic primer deduced from Seq ID No : 11 <220><221> variation <222> 3,5,9 <223> i <220><221> variation <222> 12 <223> N = A, C, G or T <400> 12 atncndatnm anckcca 17 <210> 13 <211> 27 <212> DNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: degenerated primer for 5HT rece ptors <220><221> variation <222> 19 <223> i <220><221> variation <222> 22 <223> N = A, C, G or T <400> 13 gttgactagt gtrtaratna wnggrtt 27 <210> 14 <211> 23 <212> DNA <213> Artificial Sequence <220 ><223> Description of Artificial Sequence: upstream primer to amplify the C-terminal portion of 5HT2c receptor <400> 14 ggtgtataca ctagtcaaca aaa 23 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: downstream primer to amplify t he C-terminal portion of 5HT2c receptor <400> 15 gctggtaccg taggaaaaga ctg 23 <210> 16 <211> 19 <212> DNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: sense primer for RACE method <400> 16 tgccacagag cagtacctc 19 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: sense primer for RACE method <400> 17 cgtgaagatg ctggtgcttg 20 <210> 18 <211> 1272 <212> DNA <213> mus musculus <220><221> CDS <222> ( 1) .. (1272) <400> 18 atg aag gtt cct cct gtc ctg ctt ctc ttt ctt ctg tcc tca gtg cga 48 Met Lys Val Pro Pro Val Leu Leu Leu Phe Leu Leu Ser Ser Val Arg 1 5 10 15 gct act gag caa ccg cag gtc gtc act gag cat ccc agc atg gag gca 96 Ala Thr Glu Gln Pro Gln Val Val Thr Glu His Pro Ser Met Glu Ala 20 25 30 gcc ctg acc ggg ccc aac gcc tcc tcg cac ttc tgg gcc aac tac act 144 Ala Leu Thr Gly Pro Asn Ala Ser Ser His Phe Trp Ala Asn Tyr Thr 35 40 45 ttc tct gac tgg cag aac ttc gtg ggc agg aga cgt tat ggg gcc gag 192 Phe Ser Asp Trp Gln Asn Phe Val Gly Arg Arg Arg Tyr Gly Ala Glu 50 55 60 tcc cag aac ccc acg gtg aaa gca ctg ctc atc gtg gcc tac tca ttc 240 Ser Gln Asn Pro Thr Val Lys Ala Leu Leu Ile Val Ala Tyr Ser Phe 65 70 75 80 acc atc gtc ttc tcg ctc ttc ggt aat gtc ctg gtc tgt cat gtc atg Thr Ile Val Phe Ser Leu Phe Gly Asn Val Leu Val Cys His Val Ile 85 90 95 ttc aag aac cag cgc atg cac tcg gcc acc agc ccc ttc att gtc aac 336 Phe Lys Asn Gln Arg Met His Ser Ala Thr Ser Pro Phe Ile Val Asn 100 105 110 ctg gca gtg gcg gac atc atg atc aca ttg ctc aac acg ccc ttc act 384 Leu Ala Val Ala Asp Ile Met Ile Thr Leu Leu Asn Thr Pro Phe Thr 115 120 125 ttg gtc cgc ttt gtg aac agc aca tgg gtg ttt ggg aag ggc atg tgt 432 Leu Val Arg Phe Val Asn Ser Thr Trp Val Phe Gly Lys Gly Met Cys 130 135 140 cat gtc agt cgc ttt gct cag tac tgt tct cta cat gtc tca aca ctg 480 His Val Ser Arg Phe Ala Gln Tyr Cys Ser Leu His Val Ser Thr Leu 145 150 155 160 act ctg aca gct atc gca gtg gac cgc cac cag gtc atc atg cat cca 528 Thr Leu Thr Ala Ile Ala Val Asp Arg His Gln Val Ile Met His Pro 165 170 175 ctg aag cct cgg atc tcc atc acc aag ggt gtc ata tat att gct gtc 576 Leu Lys Pro Arg Ile Ser Ile Thr Lys Gly Val Ile Tyr Ile Ala Val 180 185 190 atc tgg gtc atg gct acc ttc ttc tct ctg cca cat gcc atc tgc cag 624 Ile Trp Val Met Ala Thr Phe Phe Ser Leu Pro His Ala Ile Cys Gln 195 200 205 aaa ctg ttt acc ttc aag tac agt gag gac att gtg cgc tcc ctc tgc 672 Lys Leu Phe Thr Phe Lys Tyr Ser Glu Asp Ile Val Arg Ser Leu Cys 210 215 220 ctg ccg gac ttc ccg gag cca gct gac ctc ttc tgg aag tat ctg gac 720 Leu Pro Asp Phe Pro Glu Pro Ala Asp Leu Phe Trp Lys Tyr Leu Asp 225 230 235 240 ctg gcc acc ttc atc ctg cta ctt cca ctc ttc att atc tca 768 Leu Ala Thr Phe Ile Leu Leu Tyr Leu Leu Pro Leu Phe Ile Ile Ser 245 250 255 gtg gcc tat gct cgt gtg gcc aag aag ctg tgg ctc tgt aac acc att 816 Val Ala Tyr Alar Val Ala Lys Lys Leu Trp Leu Cys Asn Thr Ile 260 265 270 ggc gac gtg acc aca gag cag tac ctc gcc ctg cga cgc aag aag aag 864 Gly Asp Val Thr Thr Glu Gln Tyr Leu Ala Leu Arg Arg Lys Lys Lys 275 280 280 acc acc gtg aag atg ctg gtg ctt gtg gta gtc ctc ttt gcc ctc tgc 912 Thr Thr Val Lys Met Leu Val Leu Val Val Val Leu Phe Ala Leu Cys 290 295 300 tgg ttc cct ctc aac tgc tat gtc act cct gc gcc atc 960 Trp Phe Pro Leu Asn Cys Tyr Val Leu Leu Leu Ser Ser Lys Ala Ile 305 310 315 320 cac acc aac aat gcc ctc tac ttt gcc ttc cac tgg ttt gcc atg agc 1008 His Thr Asn Asn Ala Leu Tyr Phe Ala Phe His Trp Phe Ala Met Ser 325 330 335 agt act tgt tat aac ccc ttc atc tac tgc tgg ctc aat gag aac ttt 1056 Ser Thr Cys Tyr Asn Pro Phe Ile Tyr Cys Trp Leu Asn Glu Asn Phe 340 345 350 350 agg gtt gag ctt aag gca ttg ctg agc atg tgc caa agg cca ccc aag 1104 Arg Val Glu Leu Lys Ala Leu Leu Ser Met Cys Gln Arg Pro Pro Lys 355 360 365 ccg cag gaa gac agg cta ccc tcc cca gtt cct tcc ttc agg gtg gca 115 Gln Glu Asp Arg Leu Pro Ser Pro Val Pro Ser Phe Arg Val Ala 370 375 380 tgg aca gag aag agc cat ggt cgg agg gct cca cta cct aat cac cac 1200 Trp Thr Glu Lys Ser His Gly Arg Arg Ala Pro Leu Pro Asn His His 385 390 395 400 ttg ccc tct tcc cag atc cag tct ggg aag aca gat ctg tca tct gtg 1248 Leu Pro Ser Ser Gln Ile Gln Ser Gly Lys Thr Asp Leu Ser Ser Val 405 410 415 gaa ccc gtt gtg gcc atg agt tag 1272 Glu Pro Val Val Ala Met Ser 420 <210> 19 <211> 423 <212> PRT <213> mus musculus <400> 19 Met Lys Val Pro Pro Val Leu Leu Leu Phe Leu Leu Ser Ser Val Arg 1 5 10 15 Ala Thr Glu Gln Pro Gln Val Val Thr Glu His Pro Ser Met Glu Ala 20 25 30 Ala Leu Thr Gly Pro Asn Ala Ser Ser His Phe Trp Ala Asn Tyr Thr 35 40 45 Phe Ser Asp Trp Gln Asn Phe Val Gly Arg Arg Arg Tyr Gly Ala Glu 50 55 60 Ser Gln Asn Pro Thr Val Lys Ala Leu Leu Ile Val Ala Tyr Ser Phe 65 70 75 80 Thr Ile Val Phe Ser Leu Phe Gly Asn Val Leu Val Cys His Val Ile 85 90 95 Phe Lys Asn Gln Arg Met His Ser Ala Thr Ser Pro Phe Ile Val Asn 10 0 105 110 Leu Ala Val Ala Asp Ile Met Ile Thr Leu Leu Asn Thr Pro Phe Thr 115 120 125 Leu Val Arg Phe Val Asn Ser Thr Trp Val Phe Gly Lys Gly Met Cys 130 135 140 His Val Ser Arg Phe Ala Gln Tyr Cys Ser Leu His Val Ser Thr Leu 145 150 155 160 Thr Leu Thr Ala Ile Ala Val Asp Arg His Gln Val Ile Met His Pro 165 170 175 Leu Lys Pro Arg Ile Ser Ile Thr Lys Gly Val Ile Tyr Ile Ala Val 180 185 190 Ile Trp Val Met Ala Thr Phe Phe Ser Leu Pro His Ala Ile Cys Gln 195 200 205 Lys Leu Phe Thr Phe Lys Tyr Ser Glu Asp Ile Val Arg Ser Leu Cys 210 215 220 Leu Pro Asp Phe Pro Glu Pro Ala Asp Leu Phe Trp Lys Tyr Leu Asp 225 230 235 240 Leu Ala Thr Phe Ile Leu Leu Tyr Leu Leu Pro Leu Phe Ile Ile Ser 245 250 255 Val Ala Tyr Ala Arg Val Ala Lys Lys Leu Trp Leu Cys Asn Thr Ile 260 265 270 Gly Asp Val Thr Thr Glu Gln Tyr Leu Ala Leu Arg Arg Lys Lys Lys 275 280 285 Thr Thr Val Lys Met Leu Val Leu Val Val Leu Phe Ala Leu Cys 290 295 300 Trp Phe Pro Leu Asn Cys Tyr Val Leu Leu Leu Ser Ser Lys Ala Ile 305 31 0 315 320 His Thr Asn Asn Ala Leu Tyr Phe Ala Phe His Trp Phe Ala Met Ser 325 330 335 Ser Thr Cys Tyr Asn Pro Phe Ile Tyr Cys Trp Leu Asn Glu Asn Phe 340 345 350 Arg Val Glu Leu Lys Ala Leu Leu Ser Met Cys Gln Arg Pro Pro Lys 355 360 365 Pro Gln Glu Asp Arg Leu Pro Ser Pro Val Pro Ser Phe Arg Val Ala 370 375 380 Trp Thr Glu Lys Ser His Gly Arg Arg Ala Pro Leu Pro Asn His His 385 390 395 400 Leu Pro Ser Ser Gln Ile Gln Ser Gly Lys Thr Asp Leu Ser Ser Val 405 410 415 Glu Pro Val Val Ala Met Ser 420

[0050]

[Sequence List Free Text] SEQ ID NO: 1: 5HT receptor V
Conserved amino acid sequence in II transmembrane region Location 3: Xaa = Ile, Leu or Phe Location 4: Xaa = Ile, Leu or Val Location 6: Xaa = Thr, Ala or Pro SEQ ID NO: 2 Derived from SEQ ID NO: 1 Synthetic primers to be present Position 9: Inosine Position 12: N = A, C, G or T SEQ ID NO: 3: Conserved amino acid sequence in 6-transmembrane K ⁺ channel Position 4: Xaa = Val or Leu SEQ ID NO: 4 : Synthetic primer derived from SEQ ID NO: 3 Location 3, 6, 8: Inosine Location 9, 12: N = A, C, G or T SEQ ID 5: Conserved amino acid sequence in inward rectifier K ⁺ channel 4: Xaa = Phe or Tyr SEQ ID NO: 6: Synthetic primer derived from SEQ ID NO: 5 Position 6: Inosine Position 12: N = A, C, G or T SEQ ID NO 7: Conserved amino acid sequence in serine protease inhibitor Position 5: Xaa = Gly, Leu or Met SEQ ID NO: 8: Synthetic primers present position derived from column 7 3, 5, 9: Inosine existing position 12: N = A, C, G or T SEQ ID NO 9: Na ⁺ / PO ₄ stored in symporter amino acid sequence present position 1: Xaa = Glu or Asp Location 2: Xaa = Ile or Lys Location 4: Xaa = Asp or Pro SEQ ID 10: Synthetic primer derived from SEQ ID 9 Location 10: Inosine Location 13: N = A, C , G or T SEQ ID NO: 11: Conserved amino acid sequence in nerve growth factor Location 3: Xaa = Phe or Trp SEQ ID 12: Synthetic primer derived from SEQ ID NO: Location 3, 5, 9: Inosine Location 12: N = A, C, G or T SEQ ID NO: 13: Degenerate primer for 5HT receptor Location 19: Inosine Location 22: N = A, C, G or T SEQ ID NO: 14: C- of 5HT2c receptor Upstream primer for amplifying the terminal sequence SEQ ID NO: 15: Accepting 5HT2c SEQ ID NO: 16: a sense primer for the RACE method SEQ ID NO: 17: a sense primer for the RACE method

[Brief description of the drawings]

1 shows a schematic diagram of a three-dimensional structure of the 5HT _2C R as an example of a G protein-coupled receptors having seven transmembrane domains. In the figure, N indicates the N-terminus, C indicates the C-terminus, and I to VII indicate transmembrane regions, respectively, indicating that the protein is a chimeric protein having different origins from the black line and the gray line.

FIG. 2 is a conceptual diagram showing steps (a) to (c) of the method for producing a cDNA library of the present invention.

FIG. 3 is a conceptual diagram showing step (d) of the method for producing a cDNA library of the present invention.

FIG. 4 shows a structural diagram of a plasmid vector pSD64TF (BH).

FIG. 5 shows a structural diagram of a plasmid vector pSD64TF-5HT2cR C-term.

FIG. 6 shows an estimated amino acid sequence of a base sequence of an orphan G protein-coupled receptor cDNA. In the figure, the double line indicates the substitution site with the known sequence, and the thick line indicates the assumed annealing position of the 5HT receptor degenerate primer. Arrows indicate the positions of S1 and S2 primers used for RACE.

──────────────────────────────────────────────────の Continuation of the front page (51) Int.Cl. ⁷ identification code FI G01N 33/566 C12N 15/00 ZNAA (58) Field surveyed (Int.Cl. ⁷ , DB name) C12N 15/00-15 / 90 C12Q 1/68 BIOSIS (DIALOG) MEDLINE WPI (DIALOG)

Claims (11)

(57) [Claims] 1. A functional protein having a functional region and having a region between the functional region and the carboxyl terminus and conserved between homologous functional proteins.
A method for preparing a cDNA library containing a cDNA, comprising the following steps (a) to (d):
How to make NA library. (A) synthesizing an antisense oligonucleotide complementary to the sequence of a region located between the functional region and the carboxyl terminus of the functional protein and conserved between proteins having the same type of function as the functional protein, (b) tissue Alternatively, poly (A) ⁺ RNA is prepared from cultured cells. (C) Using the above poly (A) ⁺ RNA as a template and the above oligonucleotide as a primer, cDNA containing an amino terminal and a functional region is obtained by reverse transcription or PCR. (D) including the carboxyl-terminal side of the conserved region of the known homologous functional protein synthesized
The protein is inserted into an expression vector together with the cDNA so that the protein can be expressed in a state where the protein translation frames are linked together. 2. Encoding a functional protein having a functional region and having a region between the functional region and the carboxyl terminus and conserved between homologous functional proteins.
A method for preparing a cDNA library containing a cDNA, comprising the following steps (a) to (d):
How to make NA library. (A) synthesizing an antisense oligonucleotide complementary to the sequence of a region located between the functional region and the carboxyl terminus of the functional protein and conserved between proteins having the same type of function as the functional protein, (b-1) ) Preparing poly (A) ⁺ RNA from tissue or cultured cells, (b-2) subjecting the poly (A) ⁺ RNA to normalization or subtraction, (c) (b-2) treatment Using the subsequent poly (A) ⁺ RNA as a template and the above oligonucleotide as a primer, reverse transcription or PC
Synthesizing a cDNA containing an amino terminus and a functional region by R; and (d) including the carboxyl-terminal side from the conserved region of the synthesized known homologous functional protein.
The protein is inserted into an expression vector together with the cDNA so that the protein can be expressed in a state where the protein translation frames are linked together. 3. A functional protein chimeric cDNA library prepared by the method according to claim 1 or 2. 4. A method comprising the steps of: (a) to (f), comprising a functional region and a region between the functional region and the carboxyl terminus and conserved between homofunctional proteins. A method for screening a functional protein having (A) synthesizing an antisense oligonucleotide complementary to the sequence of a region located between the functional region and the carboxyl terminus of the functional protein and conserved between proteins having the same type of function as the functional protein, (b) tissue Or preparing poly (A) ⁺ RNA from cultured cells; (c) using the above poly (A) ⁺ RNA as a template and the above oligonucleotide as a primer to synthesize cDNA containing amino terminus and functional region by reverse transcription or PCR (D) the cDNA comprises a carboxyl-terminal side from the conserved region of the synthesized known homofunctional protein.
(e) transcription and translation in vivo or in vitro to express the chimeric protein; and (f) expression. The function of the chimeric protein is screened. 5. A method having a functional region, comprising the following steps (a) to (f), wherein a region which is located between the functional region and the carboxyl terminus and conserved between homofunctional proteins is provided: A method for screening a functional protein having (A) synthesizing an antisense oligonucleotide complementary to the sequence of a region located between the functional region and the carboxyl terminus of the functional protein and conserved between proteins having the same type of function as the functional protein, (b-1) ) Preparing poly (A) ⁺ RNA from tissue or cultured cells, (b-2) subjecting the poly (A) ⁺ RNA to averaging or subtraction treatment, (c) poly (A) ⁺ RNA after (b-2) treatment (A) Reverse transcription or PC using ⁺ RNA as template and the above oligonucleotide as primer
Synthesizing a cDNA containing an amino terminus and a functional region by R; and (d) including the carboxyl-terminal side from the conserved region of the synthesized known homologous functional protein.
(e) transcription and translation in vivo or in vitro to express the chimeric protein; and (f) expression. The function of the chimeric protein is screened. 6. The method for producing a chimeric cDNA library according to claim 1, wherein the functional protein is a G protein-coupled receptor. 7. The chimera according to claim 3, wherein the functional protein is a G protein-coupled receptor.
cDNA library. 8. The screening method according to claim 4, wherein the functional protein is a G protein-coupled receptor. 9. The method according to claim 9, wherein the conserved region is in a seventh transmembrane region of a G protein-coupled receptor.
The screening method according to claim 8. 10. The screening method according to claim 8, wherein the amino acid sequence of the conserved region is SEQ ID NO: 1. 11. The sequence of the above-mentioned primer is SEQ ID NO: 1.
9. The screening method according to claim 8, wherein the method is 3.