JPH0436184A - Human b-fgf acceptor gene - Google Patents

Abstract

NEW MATERIAL:A human b-FGF acceptor DNA base sequence containing a recombinant DNA, a chromosome DNA or cDNA base sequence which code a polypeptide selected from (a) a polypeptide having an amino acid sequence shown by the formula, (b) a mature polypeptide prepared by removing a signal peptide comprising about 20 amino acids from the first Met approximately to the 20th Thr from the amino acid sequence, (c) a polypeptide wherein Met is added just before to the first amino acid of the amino acid sequence and (d) a variant polypeptide observed to have variance such as deficiency, addition or replacement of amino acid in (a) the polypeptide, (b) the polypeptide and (c) the polypeptide. USE:Preparation of recombinant human b-FGF acceptor. PREPARATION:An oligonucleotide corresponding to FLG sequence is synthesized, human placental cDNA library is identified by using the oligonucleotide as a probe, a cDNA to code the whole human b-FGF acceptor is obtained, the whole base sequence is determined and the whole amino acid sequence of human b-FGF acceptor is elucidated.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to human basic fibroblast growth factor (human basic fibroblast growth factor).
basic-Fibloblast Growth
Concerning novel DNA containing a DNA base sequence that coats a cell surface receptor for human b-FGF receptor (hereinafter abbreviated as human b-FGF), and microorganisms and animal cells transformed with the novel DNA. It is related to.

The human b-FGF receptor coated by the DNA sequence of the present invention is useful for screening research or drug design research for agonists and antagonists for human b-FGF useful for healing burns and wounds.
The base sequence is determined by using recombinant DNA technology.
Recombinant human b-F using microorganisms or animal cells as hosts
It enables the production of GF receptors and is industrially useful.2 In addition, it has recently been revealed that the expression level of human b-FGF receptors in the brain is high.
FGFi body DNA sequence is associated with aging/dementia and human b-FG
This is expected to be a means of elucidating the relationship with F receptors.

The DNA sequence of the present invention reveals the main body of the human b-FGF receptor for the first time, and the expression level of the human b-FGF receptor in the human body can be determined using the sequence of the present invention or similar mutant sequences. Since it can be measured, it can be used as a material for manufacturing test drugs for diseases caused by the human b-FGF receptor, and it can also be used as a material for manufacturing therapeutic drugs for diseases caused by the human b-FGF receptor, so it is an industrial item. It is very useful.

(Prior Art) In general, b-FGF is known as a substance that exhibits various biological properties such as angiogenesis, cell division, and cell differentiation, and is used as a drug for healing burns, wounds, and angiogenesis. Research is underway to confirm its usefulness as a biotechnology (Biotechnology, 6; 25-30.
1988.1vid″7: 793-798.1989
, Experimental Medicine 8(3) 46-48.1990).

Additionally, b-FGF binds to specific cell surface receptors, namely b-FGF receptors, and activates b-FGF receptors to transmit biological responses. The b-FGF receptor was identified as a protein that binds to 125-labeled b-FGF (Annu, Rev.
Biochem.

Flash: 575-609.1989. In this document, b-F
GF is classified and named as HBGF-2).

cDNA for b-FGF receptor is chicken cDNA
The peptide encoded by the cDNA that was first isolated from the library had an amino acid sequence that matched the amino acid sequence of the tryptic digest of chicken b-FGF receptor, proving that it was the b-FGF receptor gene. (Science 245: 57-60.1989
).

On the other hand, cDNA for human FLG (fms-1ike gene).

human integumentary cell-derived cells (endotherial ce
ll) It was isolated from a cDNA library by screening at low stringency using the v-fms oncogene as a probe (Oncogen
e 3; 9-15° 1988). Human FLG cDNA
The polypeptide encoded by the chicken b-FGF receptor cD was not full-length and was incomplete, lacking the N-terminus.
It was revealed that there is 93% homology at the amino acid level with the amino acid sequence of the polypeptide coated by NA.

In addition, in recent years, a mouse b-FGF-like receptor cDNA sequence has been discovered, and when compared with the sequence of the chicken b-FGF receptor, it has been reported that there is 91% homology at the amino acid level (Proc, Natl, Acad. , Sc
i, USA.

87:1596-1600.1990).

Therefore, the DNA encoding the b-FGF receptor of the present invention
Chicken b-FGF receptor c as a sequence related to the sequence
DNA, mouse b-FGF-like receptor cDNA and human F
There is a DNA sequence for each of the LG cDNAs. Regarding the human-derived sequences targeted by the present invention, human FLG cDNA D
Although the NA sequence is a related sequence, it lacks the amino acid sequence near the N-terminus and cannot be said to be a sequence that reveals the human b-FGF receptor body.

(Problems to be solved by the invention) The body of the human b-FGF receptor can be clarified,
Using the recombinant DNA or cDNA base sequence encoding it, recombinant b-FGF is produced using genetic recombination technology.
If the receptor can be produced, it will become possible to carry out screening research or drug design research for agonists and antagonists for the human b-FGF receptor, which is currently impossible due to the unavailability of the human b-FGF receptor material. Furthermore, by using the human b-FGF receptor, it becomes possible to research diagnostic or therapeutic agents for receptor-mediated diseases that cause aging and dementia in humans.

However, as mentioned above, the main body of the human b-FC;F receptor has not been clarified, so the present inventors cloned the human b-FGF receptor cDNA based on the homology and cloned the human b-FGF receptor. We conducted intensive research to identify the human b-FGF receptor encoded by the human b-FGF receptor.

(Means for solving the problem) The present inventors synthesized an oligonucleotide corresponding to the sequence of FLG, used it as a probe, and used it as a probe for human placenta cDNA.
A library was searched, a cDNA encoding the full length of the human b-FGF receptor was obtained, the entire base sequence was determined, and the entire amino acid sequence of the human b-FGF receptor was elucidated, thereby completing the present invention.

Specific examples of the present invention will be explained in detail below.

The material for cloning human b-FGF receptor cDNA may be obtained from human tissue or human cultured cells expressing human b-FGF receptor. Human tissues that express the human b-FGF receptor include the placenta, lung, liver, and brain, and cultured human cells include various types of fibroblasts, human epithelial cells, and neuroplasmodoma cells. 1. The present inventors were able to obtain good results using easily available placenta.

The production of cDNA and the production of a cDNA library using λ phage may be carried out by any known method as described in the experimental book.

In addition, cDNA libraries using plasmids also have λ
Although it is less efficient than phage, it can still be used. A method that uses plasmids and increases efficiency is the Okayama-Barb method (Mo1. Ce11. Biol.

2, 161-170.1982). A commercially available cDNA library can be easily used. The inventors have obtained good results using commercially available human placenta cDNA. The experimental book referred to here is, for example, Molecular Cloning, edited by Maniatis, T., et al.
1 Laboratory Manual, 1989,
Eds, Sambrook, J, Fr1tsc.
h, E, F., Maniatis, T., Co.
1d Spring Harbor Laborato
ry Press.

The oligonucleotide probe used for human b-FGF receptor cDNA cloning was the known chicken b-F
GF receptor cDNA (Science, 245: 5
7-6L 1989), mouse b-FGF receptor-like cD
NA (Proc, Natl, Acad.

Sci, USA, 87: 1596-1600.1
990) or human FLG cDNA (Oncoge
ne, 3: 9-15.198g), and synthesize it as an oligonucleotide having a length of preferably 15 bases or more based on the 10 base base length, and obtain it by the NA method. For example, human FLG cDNA D
Select the vicinity of the 5'-end of the NA sequence, synthesize a 20 base long oligonucleotide using a commercially available DNA synthesizer, purify it using a predetermined purification method, label it with an isotope, for example, and use it as a probe for target gene detection. It is efficient when used as <
Screening of cDNA libraries can be performed.

Chicken b-FGF receptor cDNA, mouse b-FGF
Probes can also be prepared by labeling part or the entire length of receptor-like cDNA or human FLG cDNA with, for example, an isotope.

The DNA used as a probe is labeled with an isotope to enable highly sensitive screening.

Isotope vsmization can be done by a terminal labeling method using Plγ-ATP and T4 polynucleotide, but other labeling methods such as nick translation and primer extension methods may also be used. The method of converting isotopes to IjIWt can be used, although the sensitivity is slightly lowered.

c required for human b-FGF receptor cDNA cloning
A series of molecular biological experiments such as DNA production, hybridization screening, recombinant DNA production, and DNA base sequencing can be carried out using, for example, the aforementioned Maniat.
It may be carried out according to the known method described in the experimental book edited by IS, T, etc.

cDNA of human b-FGF receptor elucidated by the present inventors
The base sequence is shown in Figure 1 for uncoated #l of the DNA double strands.
(RNA-1ike). The base sequence shown in Figure 1 is the 5'-untranslated region (228 base pairs long),
Human b-FGF receptor coat region (2193 base pairs long)
and 3'-untranslated region (907 base pairs long).

The amino acid sequence of the human b-FGF receptor coated on the human b-FGF receptor cDNA of the present invention (Fig. 1) is
Amino acids are shown in the figure using three-letter abbreviations. The amino acid sequence (731 amino acids) shown in FIG. 2 is shown divided into a hypothesized signal peptide and a hypothesized mature human b-FGF receptor. Furthermore, the hypothesized transmembrane site is underlined.

The basis for the fact that the polypeptide shown in Figure 2 is the human b-FGF receptor is the previously identified chicken b-FGF receptor cD.
N^(Science, 245: 57-60.19
89), the DNA of the present invention has a length of 267 base pairs (
At the amino acid level, apart from the deletion of 89 amino acids)
3% homology and mouse b-FGF
-like receptor cDNA (Proc.

Natl, Acad, Sci, USA, 87.
1596-1600.1990) Short @ (Short
This is because compared to S>, which has a deletion of 267 bases in length, a surprisingly high homology of 98% at the amino acid level is observed despite species differences such as human and chicken or human and mouse. Functionally, the extracellular immunoglobulin-like domain, transmembrane domain, and tyrosine kinase domain reported in chicken and mouse are found in the human b-FGF receptor of the present invention.

The amino acid sequence of the human b-FGF receptor of the present invention (Fig. 2; 731 amino acids) is based on the previously reported human FLG (O
ncogene, 3: 9-15.1988; 62
3 amino acids), the N-terminal side is 10
The human b-FGF receptor of the present invention was determined to be a substance consisting of a novel amino acid sequence different from human FLG because it is 8 amino acids long and there is a difference of 6 amino acids at two locations in the 623 amino acids on the C-terminal side. It was done.

The mature human b-FGF receptor as used in the present invention is a polypeptide obtained by removing the signal peptide. The N-terminal amino acid of the mature human b-FGF receptor is originally human b-F.
Determination becomes possible after the GF receptor is isolated and purified from humans, but human b-FGF receptor decontaminants are not easily obtained, and even if obtained, chicken b-FG
A report that the N-terminal amino acid of the F receptor was blocked and could not be determined (Science+ 245:57-6
0.1989), it is not easy to correctly determine the N-terminal amino acid even in the case of the human b-FGF receptor. .

Therefore, in the present invention, the reported chicken b-FG
Comparison of the amino acid sequence of the hypothesized signal peptide of the F receptor, the hypothesized signal peptide of the mouse b-FGF-like receptor, and the amino acid sequence near the N-terminus of the human b-FGF receptor of the present invention and human b-FG
The signal peptide of the F receptor was tentatively set from the 1st Met to the 200th Thr, but this has not been confirmed, and this signal peptide contains a signal at around 20 amino acids from the 1st Met to around the 200th Thr. It can be considered that a peptide is formed.

The term "mutant polypeptide in which mutations such as deletion, addition, or substitution of amino acids are observed" as used in the present invention refers to mutated polypeptides that are mutated without impairing the function of the human b-FGF receptor of the present invention.Discovered in nature A mutant polypeptide that has been modified or artificially modified.

Recombinant DNA encoding the polypeptide of the present invention
, chromosomal DNA, or cDNA base sequence" means a cDNA encoding a human b-FGF receptor of interest.

It is characterized by containing chromosomal DNA containing introns and exons, recombinant DNA obtained by removing introns from the same chromosomal DNA and ligating exons, or recombinant DNA obtained by ligating oligonucleotides artificially prepared by the synthetic NA method. This is the DNA base sequence.

When using the method of artificially creating oligonucleotides using the synthetic NA method and ligating them to obtain the desired DNA base sequence, it is possible to obtain the desired DNA base sequence without changing the amino acid sequence due to the degeneracy of the genetic code. It can be changed. The present invention includes all base sequences based on the degeneracy of the gene coat of the gene related to the polypeptide of interest.

Recombinant DNA, chromosomal DNA or cDNA that coats a mutant polypeptide may be chromosomal DNA or cDNA that coats a mutant human b-FGF receptor that has variations at the amino acid level due to intraspecies or allelic variations that occur in nature; Chromosome or cD that coats a mutant human b-FGF receptor with a mutation that can be created artificially using the point mutation method
NA, and mutant human b-FG produced by synthetic NA method.
Obtained as a recombinant DNA that coats the F receptor.

Even if there are no mutations at the amino acid level, chromosomal DNA and cDNA isolated from nature can be converted into DNA without changing the amino acid sequence due to the degeneracy of the gene coat.
Examples of base sequence variations are often observed, and 5′
Since the -untranslated region and the 3'-untranslated region are not involved in defining the amino acid sequence of a polypeptide, the DNA base sequence is easily mutated, and therefore mutations in these base sequences are included in the present invention.

The mouse b-FGF-like receptor cDNA has a long
Two isomers have been reported: for+s) and short form (Proc, Natl.
, Acad, Sci, IJSA, 87.159
6-1600.1990).

Chicken b-FGF receptor cDNA (Science
, 245: 57-60.1989) is mouse b-F
It encoded a polypeptide corresponding to the long chain of a GF-like receptor. The two isomers, long-chain and short-chain, are an example of the dopamine D2 receptor (EMBOJ, 8: 4025-4).
034.1989), Insulin Receptor Examples (Pro
c, Natl, Acad, Sci, USA,
86: 114-118.1989).

Although the human b-FGF receptor cDNA of the present invention encodes a polypeptide corresponding to a short chain, it can be expected that cDNA corresponding to a long chain exists as in mouse or chicken.

The DNA base sequence of the present invention is inserted into an appropriate expression vector to obtain a transformant that uses a microorganism or cultured animal cell as a host, and the transformant produces a polypeptide exhibiting human b-FGF receptor activity. It can be used as a deterrent.

When producing mature human b-FGF receptor in the cells of microorganisms such as Escherichia coli, Bacillus subtilis, or yeast, Me
t-added polypeptide (Met-human b-FGF
It is best to express it as a receptor). After MetLt expression, high intracellular and extracellular expression can be removed in some cases.

If the aim is to produce a completely human b-FGF receptor, it is preferable to use a full-length sequence including a signal peptide and to express it in a nucleated organism, particularly in cultured animal cells, as a host. The obtained transformed cells expressing the human b-FGF receptor are treated with an agonist for human b-FGF k,
It can be used for antagonist screening research or drug design research.

Fibroblasts are the best animal cultured cells to use as hosts, but mouse cells, which are easily available, and mouse 3T cells are most suitable.
3. Mouse C127, mouse myeloma.

CJIO, C) Good results are expected with IL, He1a or human myeloma cells.

As the expression vector, it is preferable to use a known HECTOR developed for each host.

The present invention will be explained in more detail with reference to Examples below, but these are merely examples of the present invention.

Example (1) Preparation of probe Human FLG (Oncogene, 3: 9-15.1
988) Amino w! 4-10 and amino acids 1g-28
Two 20 base long oligonucleotides, namely fig-4 (20mar) and f
ig-18 (20mer) was synthesized.

The base sequence of fig-4 is 5'-TAACGGACCTT
GTAGCCTCC-3', the base sequence of flg-18 is 5'-TCAGAGGGGCACCACAGAGTC-3
'Met.

Synthetic oligonucleotides are produced using a fully automatic D method based on the solid phase method.
It was produced using an NA synthesizer. As a fully automatic DNA synthesizer, Applied Biosystems Model 381A was used. Nucleotides, carriers with immobilized 3'-nucleotides, solutions, and reagents were used according to the instructions of the company. After the prescribed coupling reaction was completed and the 5'-end protecting group was removed with trichloroacetic acid, the oligonucleotide carrier was left in concentrated ammonia at room temperature for 1 hour to release the oligonucleotide from the carrier. Next, in order to release the protecting groups on the nucleic acid and phosphoric acid, the reaction solution containing the nucleic acid was heated with concentrated ammonia solution in a sealed vial at 55°C for 14 to 24 hours.
Incubated for hours. Each oligonucleotide, from which the carrier and protecting group had been removed and released, was dried in vacuo.

60 μg of each dried synthetic oligonucleotide.

Electrophoresis buffer (10mM Tris-borate, pH
7, 6, 1mM EDTA, 7M urea, 0.1% bromophenyl blue, and 2% sucrose) and fractionated by 7M urea-8% polyacrylamide gel electrophoresis. After fractionation by electrophoresis, the length of each synthetic oligonucleotide was examined using the UV shadow method.
A gel section containing an oligonucleotide of the desired length was cut out, each oligonucleotide of the desired length was electrically extracted, and then purified by ion exchange chromatography.

1 Mg of each purified oligonucleotide was dissolved in 22 μQ of the reaction solution specified by the supplier, and 15 units of T4 polynucleotide kinase [Takara Shuzo] and 3 μQ of (3Z p
) y-^TP (Amersham Company; )5,0QO
Ci/meal) was added and incubated at 37°C for 1 hour, firstly incubated at 65°C for 5 minutes, and cooled to 0°C. Labeled oligonucleotides were purified by ion exchange chromatography. The specific activities of the obtained probes were 6 x 10'cpm/μg and 4 x lO'cp, respectively.
m/pg.

(2) Screening of human placenta cDNA library A human placenta cDNA library (C1o
ntechLab, Inc.) from E. coli
When we diluted and plated Y1090 plates until plaques appeared from 10' to 10' per plate with a diameter of 90 mm, we found that 10' appeared on approximately 50 plates.
Approximately 6 plaques appeared.

Approximately 10× of plaques that appeared were screened for candidate recombinant phages using the plaque hybridization method described below.

The plaque that appeared was transferred to a nylon filter, and the transferred nylon filter was treated with alkali (0,5
N (shake 3 times for 5 minutes in NaOH solution) to neutralize
．． Washed with 5M Tris-H (4, pH 7,5 solution, shaken once for 5 minutes), 2X SSC (0.3M sodium chloride, 0.03M sodium citrate) for 5 minutes, and finally once with 90% ethanol. , washed twice with 95% ethanol for 3 minutes each time, and air-dried. The filter contains prehybridization solution (50+aM Na-PO
4, pH7,0,2+*M EDTA, 5XSSC,
0.1% SDS, 0.5% non-fat dried
m1lk, 100. (contains ug/mfl boiled sonicated salmon sperm DNA) at 55°C in 100a+Q
Prehybridization was performed after incubation for 2 hours.

To the filter after prehybridization,
Labeled probe fig-4 (3X 107 counts/min)
60 mQ of a solution with the same composition as the prehybridization solution containing
was added and incubated at 55°C for 16 hours to perform hybridization.

The filters were then washed four times for 10 minutes at room temperature with 5XSSC-0,1% SOS solution and washed once at 45°C with a solution of the same composition.
It was washed for 0 minutes and air-dried.

The air-dried filter was exposed to X-ray film at -70°C for 12 hours using an intensifying screen.

As a result of autoradiography, 6 plaques reacted with the probe.

The plaques of these six candidate phages were numbered #1 to #6, and each phage was diluted and plated on E. coli Y1090 plates, so that about 10" to 103 plaques appeared on each plate. Labeled probe f1g-4 or fi
When probes were examined by plaque hybridization using G-18, it was confirmed that all of 11 to 16 reacted with both probes and contained the desired sequences.

Six candidate phages were diluted and plated on E. coli Y1090 plates and isolated as single plaques by repeated plaque hybridization.

For recombinant phages #1 to #6, phage DNA
When we prepared a large amount of phage and determined the length of the inserted cDNA using the cloning site εcoRI, recombinant phage #1 was the longest.

The inserted DNA of Il was EcoRI and consisted of fragments of about 600 base pairs and about 2,000 base pairs. Both D
After recloning each NA-cleaved J4 into M13mp18 phage and preparing each recombinant phage DNA, the nucleotide sequence was examined using synthetic primers while walking using the dideoxy method, and the entire nucleotide sequence was determined.6 As a result, 11 recombinants were found. The inserted cDNA of the phage is 3,328 base pairs long,
It was found to consist of a 228 base pair length of the '-untranslated region, a length of 2193 base pairs of the human b-FGF receptor coding region, and a length of 907 base pairs of the 3'-untranslated region (
Figure 1). An EcoRI fragment of about 600 bases and about 2.0
A 00 base long EcoRI fragment was inserted into the EcoRI site of plasmid pUc19 to obtain plasmid pFGFR1 containing the full length human b-FGF receptor gene.

A restriction enzyme cleavage map was created from the elucidated sequence (3rd
Although not shown in the figure, BamHI-Hin
d■, XbaI, XhoI, Pvul, 5acI,
5alI%Ba11. C1aI and NruI sites were absent.

[Brief explanation of the drawing]

Figure 1 shows the base sequence of the human b-FGF receptor cDNA as the non-coding strand (RNA-1ike) of the DNA double strands.
It was shown in The number on the right indicates the number of the rightmost base, which indicates the 5'-untranslated region (228 bases long), the human b-FGF receptor coding region (2193 bases long) or the 3'-untranslated region (907 bases long).
The boundaries of base length) are shown in the figure. FIG. 2 shows the amino acid sequence of the human b-FGF receptor encoded by the human b-FGF receptor cDNA using three-letter amino acid abbreviations. The postulated position of the signal peptide and the postulated mature human b-FGF receptor region are shown in the figure, and the postulated transmembrane domain is underlined. The number on the right side indicates the number of the amino acid on the right side. FIG. 3 shows the restriction enzyme cleavage site of human b-FGF receptor cDNA. Numbers are base numbers, coding region is A
The start codon is indicated by TG and the stop codon is indicated by TGA. Agent Patent Attorney 1) Parent Male Diagram

Claims (3)

[Claims] (1) Recombinant DNA, chromosomal DNA or cDNA encoding a polypeptide selected from the following groups:
A human b-FGF receptor DNA base sequence comprising the base sequence. (a) A polypeptide consisting of the amino acid sequence shown in FIG. (b) The first Met from the amino acid sequence shown in (a)
A mature polypeptide obtained by removing a signal peptide consisting of around 20 amino acids from to around the 20th Thr. (c) A polypeptide obtained by adding Met immediately before the first amino acid of the amino acid sequence shown in (b). (d) A mutant polypeptide in which mutations such as deletion, addition, or substitution of amino acids are observed in (a), (b), or (c). (2) D containing the DNA base sequence of claim 1
Microorganisms transformed with NA. (3) D containing the DNA base sequence of claim 1
Animal cells transformed with NA.