U.S. patent application number 09/878262 was filed with the patent office on 2002-03-14 for antibody recognizing human 25-hydroxyvitamin d3-1a-hydroxylase.
Invention is credited to Anazawa, Hideharu, Hayashi, Matsuhiko, Ishimura, Yuzuru, Monkawa, Toshiaki, Saruta, Takao, Shimada, Hiroko, Shinki, Toshimasa, Suda, Tatsuo, Sugimoto, Seiji, Suzuki, Hiromichi, Wakino, Shu, Yoshida, Tadashi.
Application Number | 20020031798 09/878262 |
Document ID | / |
Family ID | 26503073 |
Filed Date | 2002-03-14 |
United States Patent
Application |
20020031798 |
Kind Code |
A1 |
Anazawa, Hideharu ; et
al. |
March 14, 2002 |
Antibody recognizing human 25-hydroxyvitamin D3-1a-hydroxylase
Abstract
The present invention relates to a polypeptide having
25-hydroxyvitamin D.sub.3-1.alpha.-hydroxylase activity, being
useful for the prevention, diagnosis and therapeutic treatment of
adult diseases such as osteoporosis induced by the decrease of
active type vitamin D.sub.3 and catalyzing the final stage of
vitamin D.sub.3 activation; and the gene encoding the polypeptide.
In accordance with the present invention, the following can be
provided; a polypeptide having 25-hydroxyvitamin
D.sub.3-1.alpha.-hydroxylase activity, DNA encoding the
polypeptide, a recombinant DNA prepared by inserting the DNA in a
vector, a transformant carrying the recombinant DNA, a method for
preparing 25-hydroxyvitamin D.sub.3-1.alpha.-hydroxylas- e by using
the transformant, a method for preparing 1.alpha.,
25-dihydroxyvitamin D.sub.3 comprising using the polypeptide having
25-hydroxyvitamin D.sub.3-1.alpha.-hydroxylase activity, and an
antibody recognizing the polypeptide.
Inventors: |
Anazawa, Hideharu; (Tokyo,
JP) ; Shimada, Hiroko; (Tokyo, JP) ; Sugimoto,
Seiji; (Tokyo, JP) ; Suda, Tatsuo; (Tokyo,
JP) ; Shinki, Toshimasa; (Tokyo, JP) ; Saruta,
Takao; (Tokyo, JP) ; Ishimura, Yuzuru; (Tokyo,
JP) ; Hayashi, Matsuhiko; (Tokyo, JP) ;
Wakino, Shu; (Tokyo, JP) ; Monkawa, Toshiaki;
(Tokyo, JP) ; Yoshida, Tadashi; (Tokyo, JP)
; Suzuki, Hiromichi; (Tokyo, JP) |
Correspondence
Address: |
FITZPATRICK CELLA HARPER & SCINTO
30 ROCKEFELLER PLAZA
NEW YORK
NY
10112
US
|
Family ID: |
26503073 |
Appl. No.: |
09/878262 |
Filed: |
June 12, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09878262 |
Jun 12, 2001 |
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09111730 |
Jul 8, 1998 |
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6274359 |
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Current U.S.
Class: |
435/58 ; 435/189;
435/252.3; 435/7.92; 536/23.2 |
Current CPC
Class: |
A01K 2217/05 20130101;
A61K 38/00 20130101; C12N 9/0071 20130101; A61K 39/00 20130101 |
Class at
Publication: |
435/58 ; 435/189;
435/252.3; 536/23.2; 435/7.92 |
International
Class: |
C12P 033/06; G01N
033/53; G01N 033/537; G01N 033/543; C07H 021/04; C12N 009/02 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 10, 1997 |
JP |
185399/97 |
Nov 25, 1997 |
JP |
322651/97 |
Claims
What is claimed is:
1. A polypeptide comprising an amino acid sequence selected from
the amino acid sequences represented by SEQ ID NOS. 1 and 2, or a
polypeptide comprising an amino acid sequence in which amino acids
residues are deleted, substituted or added in the amino acid
sequence of said polypeptide, and having 25-hydroxyvitamin
D.sub.3-1.alpha.-hydroxylase activity.
2. A DNA encoding a polypeptide according to claim 1 or a DNA which
hybridizes with said DNA under stringent conditions.
3. The DNA according to claim 2, wherein the DNA is DNA comprising
a nucleotide sequence selected from nucleotide sequences
represented by SEQ ID NOS. 3 and 4.
4. A recombinant DNA prepared by inserting the DNA according to
claim 2 or 3 into a vector.
5. A transformant carrying a recombinant DNA according to claim
4.
6. A method for producing 25-hydroxyvitamin
D.sub.3-1.alpha.-hydroxylase, comprising: cultivating the
transformant according to claim 5 in a medium to produce
25-hydroxyvitamin D.sub.3- 1.alpha.-hydroxylase in the culture; and
recovering said 25-hydroxyvitamin D.sub.3-1.alpha.-hydroxyla- se
from the resulting culture.
7. A method for producing 1.alpha., 25-dihydroxyvitamin D.sub.3,
comprising: putting the polypeptide according to claim 1 and
25-hydroxyvitamin D.sub.3 in an aqueous medium to produce 1.alpha.,
25-dihydroxyvitamin D.sub.3 in the aqueous medium; and recovering
said 1.alpha., 25-dihydroxyvitamin D.sub.3 from the aqueous
medium.
8. An antibody recognizing the polypeptide according to claim
1.
9. A method for immunologically detecting a polypeptide having
25-hydroxyvitamin D.sub.3-1.alpha.-hydroxylase activity, using the
antibody according to claim 8.
10. An immuno-histological staining method comprising using an
antibody according to claim 8.
11. An immuno-histological staining agent containing an antibody
according to claim 8.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to a polypeptide having
25-hydroxyvitamin D.sub.3-1.alpha.-hydroxylase activity, DNA
encoding the polypeptide, a recombinant DNA prepared by inserting
the DNA in a vector, a transformant carrying the recombinant DNA, a
method for preparing 25-hydroxyvitamin D.sub.3-1.alpha.-hydroxylase
by using the transformant, a method for preparing 1.alpha.,
25-dihydroxyvitamin D.sub.3 by using the polypeptide having
25-hydroxyvitamin D.sub.3-1.alpha.-hydroxylase activity and to an
antibody recognizing the polypeptide.
[0003] 2. Prior Art
[0004] Active type vitamin D.sub.3 has been known as a hormone
having various biological actions such as the action of controlling
calcium metabolism, the induction of cellular differentiation, and
immunomodulation.
[0005] It has been known that active type vitamin D.sub.3 is
generated from vitamin D.sub.3 having no biological actions through
the metabolism in biological organisms.
[0006] As one of the action mechanisms of active type vitamin
D.sub.3, an action mechanism through cytoplasmic receptors have
been known.
[0007] It has been known that active type vitamin D.sub.3 is
essentially 1.alpha., 25-dihydroxyvitamin D.sub.3 wherein the
positions 1.alpha. and 25 have been hydroxylated. As to the
metabolic pathway for the activation, it has been known that
vitamin D.sub.3 is firstly modified into 25-hydroxyvitamin D.sub.3
by introducing a hydroxyl group into the position 25 and the
position 1.alpha. of the resulting 25-hydroxyvitamin D.sub.3 is
hydroxylated to form 1.alpha., 25-dihydroxyvitamin D.sub.3 [All of
vitamin D.sub.3, edited by Etsuro Ogata, Tateo Suda, and Yosuke
Ogura, Kodansha Scientific, Co. (1993)].
[0008] As 25-hydroxylase gene which functions to introduce a
hydroxyl group into the position 25, a gene derived from rat liver
has been cloned (Japanese Published Unexamined Patent Application
No.2324893/1991). Furthermore, the gene of the hydroxylase of the
position 24 of vitamin D.sub.3 has been cloned [Japanese Published
Unexamined Patent Application No.207196/1992].
[0009] As an enzyme to hydroxylate the position 1.alpha. of vitamin
D.sub.3, human CYP27 has been reported [Proc. Natl. Acad. Sci.,
USA, 91, 10014 (1994)], but the activity of the enzyme to
hydroxylate the position 1.alpha. is a secondary activity, so the
activity is very weak, which is not an essential activity.
Additionally, the activity is not inducible.
[0010] It has been known that 25-hydroxyvitamin
D.sub.3-1.alpha.-hydroxyla- se activity is induced in the kidneys
of rats and chickens fed with vitamin D.sub.3 deficient diet
[Gerontology, 42 (Supplement 1), 67-77 (1996)].
[0011] Up to now, no report has been presented yet in any of animal
species, concerning the isolation of any enzyme polypeptide
catalyzing the final stage of vitamin D.sub.3 activation to
hydroxylate the most significant position 1.alpha., or the
isolation of a gene encoding the polypeptide.
[0012] As a method for producing 1.alpha., 25-dihydroxyvitamin
D.sub.3, a method comprising the use of kidney homogenates or
mitochondria fractions of animals such as chicken has been known
[Nature, 230, 228 (1971); J. Biol. Chem., 247, 7528 (1972);
Biochemistry, 25, 5512 (1986) ], but the method requires a vast
amount of animal kidney or liver and demands laborious works to
prepare them, so the method is insufficient and is not practical.
It has been found a microorganism having activity to directly
induce hydroxyl groups into the positions 1.alpha. and 25 (Japanese
Published Examined Patent Application No.64678/1992), but the
activity is very weak and substrate specificity is low, so it is
difficult to separate the product and byproducts.
SUMMARY OF THE INVENTION
[0013] An object of the present invention is to provide a
polypeptide having 25-hydroxyvitamin D.sub.3-1.alpha.-hydroxylase
activity and a gene encoding the polypeptide. 25-hydroxyvitamin
D.sub.3-1.alpha.-hydroxylase catalyzes the final stage of vitamin
D.sub.3 activation, and is useful for prevention, diagnosis and
therapeutic treatment of diseases such as osteoporosis induced by
the decrease of active type vitamin D.sub.3.
[0014] The present invention relates to a polypeptide having
25-hydroxyvitamin D.sub.3-1.alpha.-hydroxylase activity, DNA
encoding the polypeptide, a recombinant DNA prepared by inserting
the DNA in a vector, a transformant carrying the recombinant DNA, a
method for producing 25-hydroxyvitamin D.sub.3-1.alpha.-hydroxylase
by using the transformant, a method for producing 1.alpha.,
25-dihydroxyvitamin D.sub.3 by using the polypeptide having
25-hydroxyvitamin D.sub.3-1.alpha.-hydroxylase activity and an
antibody recognizing the polypeptide.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 is an HPLC chart of the identified vitamin D.sub.3
metabolites in cells to which pcMD3R or pcDNA3 is introduced,
wherein "A" shows the results of the identification of vitamin
D.sub.3 metabolites in the cells to which pcMD3R is introduced; and
"B" represents the results of vitamin D.sub.3 metabolites in the
cells to which pcDNA3 is introduced, wherein (1) represents
25-hydroxyvitamin D.sub.3; (2) represents 24, 25-dihydroxyvitamin
D.sub.3; (3) represents 10-oxo-19-nor-25-hydroxyvitamin D.sub.3 and
(4) represents .alpha., 25-dihydroxyvitamin D.sub.3.
DETAILED DESCRIPTION OF THE INVENTION
[0016] The present invention will now be described in detail.
[0017] As the polypeptide of the present invention, any polypeptide
having 25-hydroxyvitamin D.sub.3-1.alpha.-hydroxylase activity may
be used, for example including a polypeptide having an amino acid
sequence selected from amino acid sequences represented by SEQ ID
NOS. 1 and 2, or having an amino acid sequence in which one or more
amino acid residues are deleted, substituted or added in the amino
acid sequence of a polypeptide, the amino acid sequence being
selected from amino acid sequences represented by SEQ ID NOS. 1 and
2, and having 25-hydroxyvitamin D.sub.3-1.alpha.-hydroxylase
activity.
[0018] The polypeptide having an amino acid sequence in which one
or more amino acid residues are deleted, substituted or added in
the amino acid sequence, the amino acid sequence being selected
from amino acid sequences represented by SEQ ID NOS. 1 and 2, and
having 25-hydroxyvitamin D.sub.3-1.alpha.-hydroxylase activity may
be prepared according to the method described in Nucleic Acids
Research, 10, 6487 (1982); Proc. Natl. Acad. Sci., USA, 79, 6409
(1982); Proc. Natl. Acad. Sci., USA, 81, 5662 (1984); Science, 224,
1431 (1984); PCT WO85/00817 (1985); Nature, 316, 601 (1985); Gene,
34, 315 (1985); Nucleic Acids Research, 13, 4431 (1985); Current
Protocols in Molecular Biology, Chapter 8. Mutagenesis of Cloned
DNA, John Wiley & Sons, Inc. (1989) and the like.
[0019] DNA of the present invention includes DNA encoding the
polypeptide of the present invention, for example, DNA encoding the
polypeptide having an amino acid sequence selected from amino acid
sequences represented by SEQ ID NOS. 1 and 2, DNA encoding the
polypeptide having an amino acid sequence in which one or more
amino acid residues are deleted, substituted or added in the amino
acid sequence selected from amino acid sequences represented by SEQ
ID NOS. 1 and 2, and having 25-hydroxyvitamin
D.sub.3-1.alpha.-hydroxylase activity, DNA comprising a nucleotide
sequence selected from SEQ ID NOS. 3 and 4, or DNA hybridizable
with these DNAs under stringent conditions.
[0020] In the present application, "DNA hybridizable under
stringent conditions" means DNA recovered by using the DNA encoding
the polypeptide having 25-hydroxyvitamin
D.sub.3-1.alpha.-hydroxylase activity as a probe through colony
hybridization, plaque hybridization or Southern blot hybridization
or the like, specific example of which includes DNA identified by
hybridization in the presence of 0.7 to 1.0 M NaCl at 65.degree. C.
by using a filter on which a DNA prepared from colonies or plaques
is immobilized and then rinsing the filter at a condition of
65.degree. C. by using 0.1 to 2.times.SSC solutions (the
composition of 1.times.SSC solution is as follows; 150 mM NaCl and
15 mM sodium citrate).
[0021] The hybridization can be carried out according to the method
described in Molecular Cloning, A Laboratory Manual, 2-nd edition,
Sambrook, Fritsch & Maniatis, eds., Cold Spring Harbor
Laboratory Press (1989) (referred to as "Molecular Cloning, 2-nd
editions" hereinafter), Current Protocols in Molecular Biology,
Supplement 1 to 34, DNA Cloning 1: Core Techniques, A Practical
Approach, Second Edition, Oxford University (1995) or the like.
Hybridizable DNA includes for example DNA having homology of 60% or
more, preferably 80% or more, more preferably 95% or more to the
nucleotide sequence of the DNA encoding the polypeptide having an
amino acid sequence selected from amino acid sequences represented
by SEQ ID NOS. 1 and 2.
[0022] The antibody of the present invention includes antibodies
recognizing the polypeptide described above.
MODE FOR CARRYING OUT THE INVENTION
[0023] The present invention will now be described in detail
below.
[0024] 1) Preparation of cDNA library from mRNA derived from rat
kidney
[0025] From tissues, for example kidney of a rat fed with vitamin
D.sub.3 deficient diet to induce 25-hydroxyvitamin
D.sub.3-1.alpha.-hydroxylase activity, mRNA [sometimes referred to
as poly(A).sup.+RNA] is prepared.
[0026] Method for preparing such mRNA includes a method comprising
preparing the whole RNA from the rat tissues and preparing then
mRNA as poly(A) .sup.+RNA by using the oligo (dT) immobilized
cellulose column method [Molecular Cloning, 2-nd edition]; a method
comprising directly preparing mRNA from rat tissues by using kits
such as Fast Track mRNA Isolation kit manufactured by Invitrogen,
Co, and Quick Prep mRNA Purification Kit, manufactured by
Pharmacia, Co. and the like.
[0027] Method for preparing the whole RNA includes thiocyanate
guanidine-trifluoroacetic acid cesium method [Methods in Enzymol.,
154, 3 (1987)], AGPC method [Experimental Medicine, 9, 1937 (1991)]
and the like.
[0028] The whole RNA and mRNA may be prepared from rat tissues with
no induction of 25-hydroxyvitamin D.sub.3-1.alpha.-hydroxylase
activity by similar method described above.
[0029] By using the mRNA prepared above, a cDNA library is prepared
by a conventional method.
[0030] Method for preparing the cDNA library includes for example a
method for preparing a cDNA library, comprising synthesizing cDNA
from the mRNA derived from the kidney resected from a rat with
25-hydroxyvitamin D.sub.3-1.alpha.-hydroxylase activity induced, by
using ZAP-cDNA synthesis kit manufactured by Stratagene, Co., cDNA
Synthesis System manufactured by GIBCO BRL, Co. and the like,
ligating then an adapter with a digestible site with an appropriate
restriction enzyme, digesting a cloning vector .lambda. ZAP II with
the restriction enzyme, and inserting the cDNA into the digested
site of the cloning vector.
[0031] As the cloning vector to prepare the cDNA library, any
cloning vector capable of autonomously replicating in Escherichia
coli K12 may be used.
[0032] The cloning vector includes for example phage vector,
plasmid vector and the like, preferably including .lambda. ZAP II
described above, in addition to pUC18, pBluescript (Stratagene,
Co.) and the like.
[0033] As a host microorganism, any microorganism of species
Escherichia coli may be used, preferably including Escherichia coli
XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1,
Escherichia coli MC1000 and the like.
[0034] 2) Selection of an amino acid sequence characteristic to
vitamin D.sub.3 hydroxylase
[0035] Screening a region with the amino acid sequence present in
common with both the hydroxylase of the position 25 of rat vitamin
D.sub.3 [Japanese Published Unexamined Patent Application No.
232493/1991] and the hydroxylase of the position 24 thereof
(Japanese Published Unexamined Patent Application No. 207196/1992),
the amino acid sequence present in the region is selected as the
amino acid sequence characteristic to the hydroxylase of vitamin
D.sub.3.
[0036] The region with the amino acid sequence includes for example
adrenodoxin binding region (referred to as "Region A" hereinafter),
heme binding region (referred to as "Region H" hereinafter) and the
like.
[0037] 3) Amplification of a partial fragment of DNA encoding
25-hydroxyvitamin D.sub.3-1.alpha.-hydroxylase
[0038] Based on the amino acid sequence of the region selected in
2) above and with reference to the codons of rat, a sense primer
and an antisense primer are designed and prepared, which are
appropriate for the amplification of the DNA encoding
25-hydroxyvitamin D.sub.3-1.alpha.-hydroxylase by polymerase chain
reaction (referred to as "PCR" hereinafter).
[0039] Such primers include DNA comprising a nucleotide sequence
selected from nucleotide sequences represented by SEQ ID NOS. 7, 8
and 9.
[0040] Using the mRNA recovered in 1), first strand DNA is
synthesized by reverse transcriptase reaction. DNA synthesis may be
carried out using a cDNA synthetic kit manufactured by Stratagene,
Co.
[0041] Using the first strand DNA as a template and utilizing the
sense primers and antisense primers as prepared above, RT (reverse
transcription)-PCR is carried out to amplify a DNA region
containing a part of DNA encoding 25-hydroxyvitamin
D.sub.3-1.alpha.-hydroxylase.
[0042] Using the RT-PCR amplified fragments and 3' RACE system kit
manufactured by BRL, Co., PCR amplification is carried out between
the RT-PCR amplified fragment and the 3-terminal poly(A) structure
to recover a longer PCR amplified fragment additionally containing
the noncoding region on 3' side.
[0043] More specifically, a PCR amplified fragment containing the
3' noncoding region can be recovered by synthesizing cDNA using the
mRNA recovered in 1) and the oligo dT/AUAP primer in the 3' RACE
system kit manufactured by BRL, CO. and conducting PCR
amplification using the DNA as a template and using the AUAP primer
in the 3' RACE system kit manufactured by BRL and the RT-PCR
amplified fragment.
[0044] Using 5' RACE method in the same manner, a PCR amplified
fragment containing the 5' region can be recovered.
[0045] It can be confirmed that the amplified DNA fragment is a
partial fragment of the DNA encoding 25-hydroxyvitamin
D.sub.3-1.alpha.-hydroxyla- se by the following method.
[0046] Poly(A) .sup.+RNAs derived from a rat induced with
25-hydroxyvitamin D.sub.3-1.alpha.-hydroxylase activity and a
non-induced rat are individually subject to agarose
electrophoresis, and the poly(A) .sup.+RNAs electrophoresed are
then individually transferred onto each membrane filter in a
conventional manner.
[0047] Using these membrane filters, Northern hybridization is
carried out using the amplified DNA fragment as a probe.
[0048] By confirming that the amplified DNA fragment is
hybridizable only when using the membrane filter prepared from the
poly(A) .sup.+RNA derived from the rat induced with the activity,
it is revealed that the DNA fragment is a partial fragment of the
DNA encoding 25-hydroxyvitamin D.sub.3-1.alpha.-hydroxylase.
[0049] The amplified DNA fragment is then inserted into a plasmid,
and the resulting plasmid can be used for nucleotide sequencing and
the assay of expression specificity.
[0050] The method for inserting the fragment into a plasmid
includes a method for inserting the fragment into a plasmid,
comprising extracting the amplified DNA fragment from the agarose
using a DNA purification kit (manufactured by Bio Rad Co.) and
ligating the fragment with a vector pCRII (manufactured by
Invitrogen, Co.).
[0051] 4) Selection of a clone carrying DNA encoding
25-hydroxyvitamin D.sub.3-1.alpha.-hydroxylase
[0052] A cDNA library is screened by labeling the amplified DNA
fragment and subjecting the resulting fragment to colony- or plaque
hybridization in a conventional manner.
[0053] The labeling of the amplified DNA fragment can be carried
out using for example DIG labeling kit (#1 175 033, manufactured by
Boehringer Mannheim, Co.). More specifically, a DIG-labeled
amplified DNA fragment can be recovered by PCR using the amplified
DNA fragment as a template and utilizing the kit.
[0054] The plaque hybridization method includes for example the
following method.
[0055] The cDNA library (phage) prepared in 1) above is spread on
an agar culture medium and cultivated to a final concentration of
10,000 to 20,000 plaques per one petri dish.
[0056] Hybond N.sup.+ membrane (manufactured by Amersham, Co.) is
placed on the petri dish with plaques formed thereon to transfer
the plaque DNA onto the membrane.
[0057] The transfer membrane is subject to alkali treatment
(comprising for example immersing the membrane in 1.5 M NaCl, 0.5 M
NaOH solution) and SDS treatment (comprising for example immersing
in 2.times.SSC, 0.1% SDS solution), rinsing and drying, and the
resulting membrane is used for hybridization as a blotted membrane
with the plaque DNA immobilized thereon.
[0058] The blotted membrane is immersed in a hybridization solution
[5.times.SSC, 0.1% Sarkosyl, 0.02% SDS, 1% blocking reagent for
hybridization (manufactured by Boehringer Mannheim, Co.)] for 5
hours, and the labeled amplified DNA fragment which has been
subjected to thermal treatment is added thereto for
hybridization.
[0059] After hybridization, the membrane is subject to rinsing [for
example, rinsing twice in 2.times.SSC and 0.1% SDS at room
temperature for 5 minutes, and rinsing twice in 0.1.times.SSC and
0.1% SDS at 60.degree. C. for 15 minutes] and blocking [for
example, blocking in 1.times.blocking solution (manufactured
Boehringer Mannheim Co.), 0.1 M maleic acid, 0.15 M NaCl, pH 7.5],
and thereafter, the labeled amplified DNA is detected by a variable
method, depending on the labeling mode of the labeled amplified DNA
fragment, whereby an objective clone can be selected.
[0060] When a DNA fragment labeled with DIG is used, for example,
reaction with anti-DIG antibody labeled with AP and subsequent
alkali treatment [for example, immersing in 0.1 M Tris-HCl (pH
9.5), 0.1 M NaCl and 50 mM MgCl.sub.2 solution] are carried out,
and a plaque hybridized with the probe is screened on an X-ray film
using a DIG luminescence detection kit (#1 363 514, manufactured by
Boehringer Mannheim, Co.) to select a clone containing DNA encoding
25-hydroxyvitamin D.sub.3-1.alpha.-hydroxylase.
[0061] 5) Recovery of DNA encoding 25-hydroxyvitamin
D.sub.3-1.alpha.-hydroxylase
[0062] From the clone recovered by the screening procedure
described above in 4), DNA is isolated in a conventional manner to
recover DNA encoding 25-hydroxyvitamin
D.sub.3-1.alpha.-hydroxylase.
[0063] DNA nucleotide sequencing can be done by common nucleotide
sequencing methods, for example, the dideoxy method by Sanger et.
al. [Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)] or by sequencing
by using a nucleotide sequencer such as 373A.DNA sequencer
[manufactured by Perkin Elmer, Co.].
[0064] As the gene sequence of 25-hydroxyvitamin
D.sub.3-1.alpha.-hydroxyl- ase thus determined includes DNA
comprising the sequence represented by SEQ ID NO. 3 or 5.
[0065] Based on the DNA sequence thus determined by the method, an
objective DNA may be prepared by chemical synthesis with a DNA
sequencer. Such DNA sequencer includes a DNA sequencer based on the
thiophosphite method, manufactured by Shimadzu, and a DNA sequencer
Model 1392 based on the phosphoramidits method, manufactured by
Perkin Elmer, Co.
[0066] The rat-derived 25-hydroxyvitamin
D.sub.3-1.alpha.-hydroxylase gene as recovered above can be used to
recover 25-hydroxyvitamin D.sub.3-1.alpha.-hydroxylase gene derived
from other animals, for example, humans, by the following
method.
[0067] The DNA encoding 25-hydroxyvitamin
D.sub.3-1.alpha.-hydroxylase as recovered above is labeled with
.alpha.-.sup.32P-dCTP by using for example Megaprime DNA labeling
kit (manufactured by Amersham Co.). In the same manner as for the
method described above in 1), a cDNA library is prepared from
objective animal tissues, for example human kidney.
[0068] The cDNA library is screened by colony- or plaque
hybridization using the labeled DNA fragment described in 4) above
as a probe.
[0069] From the clone recovered through the screening, the
objective DNA is isolated by the method as described in 5) above,
and the nucleotide sequence is determined.
[0070] The nucleotide sequence having high homology to the
nucleotide sequence of the gene of rat 25-hydroxyvitamin
D.sub.3-1.alpha.-hydroxylas- e is defined as DNA encoding
25-hydroxyvitamin D.sub.3-1.alpha.-hydroxylas- e derived from the
objective animal.
[0071] The gene includes for example human kidney-derived DNA
comprising the sequence represented by SEQ ID NO. 4 or 6.
[0072] 6) Production of 25-hydroxyvitamin
D.sub.3-1.alpha.-hydroxylase polypeptide
[0073] To express the DNA encoding 25-hydroxyvitamin
D.sub.3-1.alpha.-hydroxylase as recovered in 5) above in a host
cell, the methods described in Molecular Cloning, 2-nd edition and
Current Protocols in Molecular Biology, Supplement 1 to 34 and the
like may be used.
[0074] More specifically, DNA recovered in 5) is modified into DNA
fragments with appropriate lengths so that the DNA encoding
25-hydroxyvitamin D.sub.3-1.alpha.-hydroxylase might be contained
therein, by using restriction enzymes or DNases, which are then
inserted into the downstream of a promoter in an expression vector,
and then, the expression vector with the DNA inserted therein is
introduced into a host cell appropriate for the expression
vector.
[0075] Any host cell capable of expressing the objective gene may
be used, including for example bacteria, yeast, animal cells and
insect cells.
[0076] As the expression vector, a vector, which is autonomously
replicable in the host cell or possibly inserted into the
chromosome and contains a promoter at the site on which the gene of
25-hydroxyvitamin D.sub.3-1.alpha.-hydroxylase can be transcribed,
may be used.
[0077] When procaryotic cells such as bacteria are used as such
host cells, it is preferable that an expression vector of
25-hydroxyvitamin D.sub.3-1.alpha.-hydroxylase gene is autonomously
replicable in the procaryotic cells and the vector is composed of a
promoter, a ribosome binding sequence, DNA encoding
25-hydroxyvitamin D.sub.3-1.alpha.-hydroxy- lase and a
transcription termination sequence. A gene regulating the promoter
may be contained in the vector.
[0078] Such expression vector includes for example pBTrp2, pBTacl,
pBTac2 (all commercially available from Boehringer Mannheim, Co.),
pKK2-2 (manufactured by Pharmacia, Co.) pSE280 (manufactured by
Invitrogen, Co.), pGEMEX-1 (manufactured by Promega, Co.), pQE-8
(manufactured by QIAGEN, Co.), pKYP10 (Japanese Published
Unexamined Patent Application No. 110600/1983), pKYP200 [Agric.
Biol. Chem., 48, 669 (1984)], pLSA1 [Agric, Biol. Chem., 53, 277
(1989)], pGEL1 [Proc. Natl. Acad. Sci., USA, 82, 4306 (1985)],
pBluescript (STRATAGENE, Co.), pTrs30 (FERM BP-5407), pTrs32 (FERM
BP-5408), pGHA2 (FERM BP-400), pGKA2 (FERM BP-6798), pTerm2
(Japanese Published Unexamined Patent Application No. 22979/1991,
U.S. Pat. Nos. 4,686,191, 4,939,094, 5,160,735), pKK233-2
(manufactured by Pharmacia, Co.), PGEX (manufactured by Pharmacia,
Co.), pET system (manufactured by Novagen, Co.) pSupex, pUB110,
pTP5, and pC194 and the like.
[0079] Any promoter which can be expressed in host cells such as
Escherichia coli may be used, including for example promoters
derived from Escherichia coli and phages, for example trp promoter
(Ptrp), lac promoter (Plac), P.sub.L promoter, P.sub.R promoter,
PletI promoter, and P.sub.SE promoter; SPO1 promoter, SPO2
promoter, penP promoter and the like. Additionally, artificially
designed and modified promoters, such as a promoter of two Ptrp's
in series (Ptrp.times.2) and tac promoter may be used.
[0080] Any ribosome binding sequence may be used, as long as the
sequence may be expressed in host cells such as Escherichia coli.
Preferably, a plasmid wherein the distance between the
Shine-Dalgarno sequence and the initiation codon is adjusted to an
appropriate distance (for example, 6 to 18 nucleotides) may be
used.
[0081] To express 25-hydroxyvitamin D.sub.3-1.alpha.-hydroxylase
gene of the present invention, a transcription termination sequence
is not necessarily required, but preferably, a transcription
termination sequence is arranged immediately below the structural
gene.
[0082] Examples of the host cell include microorganisms belonging
to the genus Escherichia, Serratia, Bacillus, Brevibacterium,
Corynebacterium, Microbacterium, Pseudomonas, and the like.
Specific examples include Escherichia coli XL1-Blue, Escherichia
coli XL2-Blue, Escherichia coli DH1, Escherichia coli MC1000,
Escherichia coli KY3276, Escherichia coli W1485, Escherichia coli
JM109, Escherichia coli HB101, Escherichia coli No.49, Escherichia
coli W3110, Escherichia coli NY49, Seratia ficaria, Seratia
fonticola, Seratia liquefaciens, Seratia marcescens, Bacillus
subtilis, Bacillus amyloliguefaciens, Brevibacterium ammoniagenes,
Brevibacterium immariophilum ATCC 14068, Brevibacterium
saccharolyticum ATCC 14066, Corynebacterium glutamicum ATCC 13032,
Corynebacterium glutamicum ATCC 14067, Corynebacterium glutamicum
ATCC 13869, Corynebacterium acetoacidophilum ATCC 13870,
Microbacterium ammoniaphilum ATCC 15354, Pseudomonas sp. D-0110 and
the like.
[0083] As the method for introducing the recombinant vectors, any
method for introducing DNA into the host cells may be used,
including for example a method comprising the use of calcium ion
[Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], protoplast method
(Japanese Published Unexamined Patent Application No.
2483942/1988), and methods described in Gene, 17, 107 (1982) and
Molecular & General Genetics, 168, 111 (1979).
[0084] In case of using yeast bacterial strains as host cells,
expression vectors for example YEp13 (ATCC 37115), YEp24 (ATCC
37051), YCp50 (ATCC 37419), pHS19, and pHS15 may be used.
[0085] As the promoter, any promoter which can be expressed in
yeast bacterial strains may be used. For example, promoters such as
PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal 1
promoter, gal 10 promoter, heat shock protein promoter, MF.alpha.1
promoter, CUP 1 promoter and the like may be listed.
[0086] Host cells used include for example Saccharomyces cerevisae,
Shizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon
pullulans, and Schwanniomyces alluvius.
[0087] As the method for introducing the recombinant vectors, any
method for introducing DNA into yeast cells may be used, for
example, electroporation method [Methods. Enzymol., 194, 182
(1990)], spheroplast method [Proc. Natl. Acad. Sci. USA, 84, 1929
(1978)], lithium acetate method [Journal of Bacteriology, 153, 163
(1983)], a method described in Proc. Natl. Acad. Sci. USA, 75, 1929
(1978) and the like.
[0088] In case of using animal cells as a host, the expression
vector includes for example pcDNAI, pcDM8 (commercially available
from Funakoshi, Co.), pAGE107 (Japanese Published Unexamined Patent
Application No. 22979/1991; Cytotechnology, 3, 133 (1990)), pAS3-3
(Japanese Published Unexamined Patent Application No. 227075/1990),
pCDM8 [Nature, 329, 840 (1987)], pcDNAI/Amp (manufactured by
Invitrogen, Co.), pREP4 (manufactured by Invitrogen, Co.), pAGE103
[J. Biochem., 101, 1307 (1987)], and pAGE210.
[0089] As a promoter, any promoter which can be expressed in animal
cells may be used, including for example a promoter of IE
(immediate early) gene of cytomegalovirus (human CMV), an early
promoter of SV40 or a promoter of metallothionein, a promoter
retrovirus, a heat shock promoter and an SR.alpha. promoter.
Additionally, the enhancer of the IE gene of human CMV may be used
in combination with such promoter.
[0090] Examples of the host cell include Namalwa cell, monkey cos
cell, Chinese hamster CHO cell, HST5637 (Japanese Published
Unexamined Patent Application No.299/1988) and the like.
[0091] As the method for introducing recombinant vector into animal
cells, any method for introducing DNA into animal cells may be
used, e.g., electroporation method [Cytotechnology, 3, 133 (1990)],
calcium phosphate method [Japanese Published Unexamined Patent
Application No. 227075/1990], lipofection method [Proc. Natl. Acad.
Sci., USA, 84, 7413 (1987)] and the method described in Virology,
52, 456 (1973)]. The preparation of a transformant and cultivation
of the transformant may be carried out according to the method
described in Japanese Published Unexamined Patent Application No.
227075/1990 or Japanese Published Unexamined Patent Application No.
257891/1990.
[0092] In case of using insect cells as a host, the protein may be
expressed according to the methods described in Baculovirus
Expression Vectors, A Laboratory Manual, W. H. Freeman and Company,
New York, 1992; Current Protocols in Molecular Biology, Supplement
1-38(1987-1997); Bio/Technology, 6, 47 (1988) and the like.
[0093] More specifically, the protein can be expressed by
co-introduction of the transfer vector containing interest gene and
helper DNA fragment of baculovirus into an insect cell to recover a
recombinant virus in the supernatant of the culture of the insect
cell and infecting an insect cell with the recombinant virus.
[0094] The transfer vector for gene introduction to be used in the
method includes for example pVL1392, pVL1393, pBlueBacIII (all
manufactured by Invitrogen, Co.) and the like.
[0095] As the helper DNA fragment of baculovirus, for example,
Autographa californica nuclear polyhedrosis virus, which is a virus
infecting insects of the family Barathra, may be used.
[0096] As such insect cell, Spodoptera frugiperda oocytes Sf9 and
Sf21 [Baculovirus Expression Vectors, A Laboratory Manual, W. H.
Freeman and Company, New York, 1992], Trichoplusia ni oocytes High
5 (manufactured by Invitrogen Co.) and the like, may be used.
[0097] The method for co-introducing the above-described transfer
vector containing interest gene and the helper DNA fragment of
baculovirus into insect cells to prepare the recombinant virus
includes for example calcium phosphate method (Japanese Published
Unexamined Patent Application No. 227075/1990), lipofection method
[Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)], and the like.
[0098] As the expression method of the gene, secretory production
and expression of fused protein may be carried out according to the
method described in Molecular Cloning, 2-nd edition and the like,
in addition to direct expression.
[0099] When the gene is expressed in yeast, animal cells or insect
cells, a glycosylated protein can be obtained.
[0100] The transformant thus obtained is cultivated in a culture
medium to form polypeptide of the present invention in the culture,
and the formed polypeptide is recovered from the culture, whereby
the polypeptide of the present invention can be produced. The
transformant of the present invention is cultivated in a culture
medium according to a conventional method for use in cultivating
hosts.
[0101] As the culture medium to cultivate a transformant recovered
by using procaryotic organisms such as Escherichia coli or
eucaryotic organisms such as yeast, any natural culture medium or
any synthetic culture medium may be used, so long as it contains
carbon sources, nitrogen sources, inorganic salts and the like
which can be assimilated by the organisms.
[0102] Any carbon source which can be assimilated by the organisms
may be used, including carbohydrates such as glucose, fructose,
sucrose, molasses containing them, starch and starch hydrolysates;
organic acids such as acetic acid and propionic acid; alcohols such
as ethanol and propanol.
[0103] As such nitrogen sources, ammonia; ammonium salts of
inorganic acids or organic salts, such as ammonium chloride,
ammonium sulfate, ammonium acetate, and ammonium phosphate; other
nitrogen containing compounds; peptone; meat extract; yeast
extract; corn steep liquor; casein hydrolysates; soy bean meal;
soybean meal hydrolysates; various fermentation products, and
digested products thereof, may be used.
[0104] As the inorganic substances, potassium dihydrogen phosphate,
dipotassium hydrogen phosphate, magnesium phosphate, magnesium
sulfate, sodium chloride, ferrous sulfate, manganese sulfate,
copper sulfate, calcium carbonate and the like, may be used.
[0105] Cultivation is generally carried out under aerobic
conditions, for example, by shaking culture or spinner culture
under aeration. The cultivation is carried out at 15 to 40.degree.
C. for 16 hours to seven days at pH 3.0 to 9.0. The pH is adjusted
with an inorganic or organic acid, an alkali solution, urea,
calcium carbonate, ammonia and the like.
[0106] During cultivation, antibiotics such as ampicillin and
tetracycline may be added to the culture medium, if necessary.
[0107] For cultivating microorganisms transformed with an
expression vector prepared using an inducible promoter, an inducer
may be added to the culture medium, if necessary. For cultivating
microorganisms transformed with an expression vector prepared using
lac promoter, for example, isopropyl-.beta.-D-thiogalactopyranoside
may be added to the medium; for cultivating microorganisms
transformed with an expression vector prepared using trp promoter,
for example, indole acrylic acid may be added to the medium.
[0108] As the culture medium for cultivating a transformant
recovered by using animal cells as the hosts, RPMI 1640 culture
medium [The Journal of the American Medical Association, 199, 519
(1967)], Eagle's MEM culture medium [Science, 122, 501 (1952)],
Dulbecco's modified MEM culture medium [Virology, 8, 396 (1959)],
DMEM culture medium (manufactured by GIBCO BRL, Co.), 199 culture
medium [Proceedings of the Society for the Biological Medicine, 73,
1 (1950)] for conventional use or culture media prepared by adding
fetal calf serum and the like to these culture media, may be
used.
[0109] Generally, cultivation is carried out in the presence of 5%
CO.sub.2 at pH 6 to 8 at 30 to 40.degree. C. for 1 to 7 days.
[0110] During cultivation, if necessary, antibiotics such as
kanamycin and penicillin may be added to the culture medium.
[0111] As the culture medium to cultivate transformants recovered
using insect cells as the hosts, culture medium for general use,
such as TNM-FH culture medium [manufactured by Pharmingen, Co.],
Sf-900 II SFM culture medium [manufactured by Life Technologies,
Co.], ExCell 400, ExCell 405 [both manufactured by JRH Biosciences,
Co.], Grace's Insect Medium [Grace, T. C. C. , Nature, 195, 788
(1962)] and the like, may be used.
[0112] Cultivation is carried out at pH 6 to 7 at 25 to 30.degree.
C. for 1 to 5 days.
[0113] During cultivation, if necessary, antibiotics such as
gentamycin may be added to the culture medium.
[0114] To isolate and purify the polypeptide expressed by the
method described above from the culture of the transformant,
conventional isolation and purification methods of enzymes may be
used.
[0115] When the polypeptide of the present invention is expressed
in cells at its dissolved state, a purified sample of the
polypeptide is obtained as follows. The cells are recovered through
centrifugation after the cultivation, suspended in an aqueous
buffer, and disrupted by means of ultrasonic oscillator, French
Press, Manton Gaulin homogenizer, Dynomill and the like, to recover
a cell-free extract. From the supernatant recovered by the
centrifugation of the cell-free extract, a purified sample can be
recovered by conventional isolation and purification methods of
enzymes, singly or in combination, such as solvent extraction
method, salting out methods with ammonium sulfate, etc., desalting
method, precipitation methods with organic solvents, anion exchange
chromatography by means of resins such as diethylaminoethyl
(DEAE)-Sepharose, DIAION HPA-75 (manufactured by Mitsubishi
Chemical Corporation); cation exchange chromatography by means of
resins such as S-Sepharose FF (manufactured by Pharmacia, Co.);
hydrophobic chromatography using resins such as butyl Sepharose and
phenyl Sepharose; gel filtration methods using molecular sieves;
affinity chromatography method; chromato-focusing method;
electrophoresis methods such as isoelectric focusing; and the
like.
[0116] When the polypeptide is expressed in cells in the form of an
inclusion body, a purified sample of the polypeptide is obtained as
follows. The cells are similarly recovered, disrupted, and
centrifuged to recover a precipitation fraction, from which the
polypeptide is recovered according to a conventional method, and
the inclusion body of the polypeptide is solubilized with a
polypeptide denaturant. The solubilized solution is diluted or
dialyzed in a dilute solution at such an extent that the resulting
solution does not contain any polypeptide denaturant or the
polypeptide is not any more denatured at the concentration of the
polypeptide denaturant, to renature the polypeptide into a normal
steric configuration, from which a purified sample can be recovered
according to the same isolation and purification method as
described above.
[0117] In case that the polypeptide of the present invention or
derivatives thereof such as a sugar modified product thereof are
secreted extracellularly, the polypeptide or the derivatives
thereof can be recovered from the culture supernatant. More
specifically, the culture is treated by the method as described
above, such as centrifugation, to recover a soluble fraction, and
from the fraction, a purified sample is recovered using the
isolation and purification method as described above.
[0118] Additionally, the polypeptide expressed by the above method
may be prepared by chemical synthetic methods such as Fmoc method
(fluorenylmethyloxycarbonyl method), tBoc method
(t-butyloxycarbonyl method) and the like. Alternatively, the
polypeptide can be prepared by utilizing peptide synthesizers
commercially available from Sowa Trade (manufactured by Advanced
chemTech, Co., USA), Perkin-Elmer Japan (manufactured by
Perkin-Elmer, Co., USA), Pharmacia Biotech (manufactured by
PharmaciaBiotech, Co., Sweden), Aroka (manufactured by Protein
Technology Instrument, Co., USA), KURABO (manufactured by
Synthecell-Vega, Co., USA), Japan PerSeptive Limited (manufactured
by PerSeptive, Co., USA), Shimadzu, Co. and the like.
[0119] 7) Production of 1.alpha., 25-dihydroxyvitamin D.sub.3
[0120] The polypeptide having 25-hydroxyvitamin
D.sub.3-1.alpha.-hydroxyla- se and 25-hydroxyvitamin D.sub.3 are
put in an aqueous medium to form 1.alpha., 25-dihydroxyvitamin
D.sub.3 in the aqueous medium, and the formed 1.alpha.,
25-dihydroxyvitamin D.sub.3 is recovered from the aqueous medium.
Thus, 1.alpha., 25-dihydroxyvitamin D.sub.3 can be produced.
[0121] As a polypeptide having 25-hydroxyvitamin
D.sub.3-1.alpha.-hydroxyl- ase activity, the polypeptide purified
by the method described above in 6) and the microbial culture
obtained by the method described above in 6) or a treated product
of the culture obtained by treating the culture in various ways and
the like, may be used.
[0122] Examples of the treated product of the culture broth include
a concentrated product of the culture, a dried product of the
culture, a culture supernatant obtained by centrifuging the
culture, a concentrated product of the culture supernatant, an
enzyme preparation obtained from the culture supernatant, cells
(including microbial cells) obtained by centrifuging the culture, a
dried product of the cells, a freeze-dried product of the cells, a
surfactant-treated product of the cells, an ultrasonic-treated
product of the cells, a mechanically disrupted product of the
cells, a solvent-treated product of the cells, an enzyme-treated
product of the cells, a protein fraction of the cells (fractions
having 25-hydroxyvitamin D.sub.3-1.alpha.-hydroxylase activity), an
immobilized product of the cells and an enzyme preparation obtained
by extraction from the cells.
[0123] The concentration of the polypeptide having
25-hydroxyvitamin D.sub.3-1.alpha.-hydroxylase activity is 0.01 to
50 g/l, preferably 0.05 to 10 g/l, as wet cells.
[0124] The aqueous medium includes water, buffers such as phosphate
salts, carbonate salts, acetate salts, borate salts, citrate salts,
and Tris; and aqueous solutions containing organic solvents such as
alcohols such as methanol and ethanol; esters such as ethyl
acetate; ketones such as acetone; amides such as acetoamide. If
necessary, surfactants such as Triton X-100 (manufactured by
Nakarai Tesque, Co.) and Nonion HS204 (manufactured by Nippon Oils
and Fats Co.), or organic solvents such as toluene and xylene may
be added at about 0.1 to 20 g/l.
[0125] The concentration of 25-hydroxyvitamin D.sub.3 is 0.01 to 50
g/l, preferably 0.01 to 10 g/l.
[0126] 1.alpha., 25-dihydroxyvitamin D.sub.3 can be produced by
adding polypeptide having 25-hydroxyvitamin
D.sub.3-1.alpha.-hydroxylase activity and 25-hydroxyvitamin
D.sub.3. The reaction is carried out at 15 to 80.degree. C.,
preferably 20 to 40.degree. C., at pH 3 to 11, preferably pH 4 to
9, for 5 minutes to 96 hours.
[0127] 8) Preparation of an antibody recognizing 25-hydroxyvitamin
D.sub.3-1.alpha.-hydroxylase
[0128] A purified product of the whole length or a partial fragment
of the protein obtained by the method described in the above in 6)
or a peptide having a partial amino acid sequence of the protein of
the present invention is used as the antigen. The antigen is
administered to animal by subcutaneous, intravenous or
intraperitoneal injection together with an appropriate adjuvant
(for example, complete Freund's adjuvant, aluminum hydroxide gel,
pertussis vaccine, or the like).
[0129] Examples of the animals used include rabbits, goats, 3- to
20-weak-old rats, mice, hamsters and the like.
[0130] Preferable dosage of antigen is 50 to 100 .mu.g per
animal.
[0131] When a peptide is used as the antigen, it is preferred to
use the peptide as the antigen after binding it covalently to a
carrier protein, such as keyhole limpet haemocyanin, bovine
thyroglobulin or the like. The peptide used as the antigen can be
synthesized using a peptide synthesizer.
[0132] Administration of the antigen is carried out 3 to 10 times
at one- to two-week intervals after the first administration. A
blood sample is recovered from the fundus of the eye 3 to 7 days
after each administration, and the serum is tested, for example, by
enzyme immunoassay (Enzyme-linked Immunosorbent Assay (ELISA),
published by Igaku Shoin (1976); Antibodies--A Laboratory Manual,
Cold Spring Harbor Laboratory (1988)) as to whether it is reactive
with the antigen used for immunization. A non-human mammal whose
serums shows a sufficient antibody titer against the antigen used
for immunization is submitted for use as the supply source of serum
or antibody producing cells.
[0133] A polyclonal antibody can be prepared by isolating and
purifying it from the serum.
[0134] A monoclonal antibody can be prepared by preparing a
hybridoma through fusion of the antibody producing cells with
myeloma cells of anon-humanmammal and culturing the hybridoma, or
administering the hybridoma to an animal to induce ascites tumor in
the animal, and then isolating and purifying it from the culture
medium or ascitic fluid.
[0135] Examples of the antibody producing cells include spleen
cells, lymph nodes and antibody producing cells in peripheral
blood. Particularly, spleen cells are preferred.
[0136] Examples of the myeloma cells include cell lines derived
from mouse, such as P3-X63Ag8-U1 (P3-U1) cell line [Current Topics
in Microbiology and Immunology, 18, 1-7 (1978)], P3-NS1/1-Ag41
(NS-1) cell line [European J. Immunology, 6, 511-519 (1976)],
SP2/O-Ag14 (SP-2) cell line [Nature, 276, 269-270 (1978)],
P3-X63-Ag8653 (653) cell line [J. Immunology, 123, 1548-1550
(1979)], P3-X63-Ag8 (X63) cell line [Nature, 256, 495-497 (1975)]
and the like, which are 8-azaguanine-resistant mouse (BALB/c)
myeloma cell lines.
[0137] Hybridoma cells can be prepared in the following manner.
[0138] Antibody producing cells and myeloma cells are fused,
suspended in HAT medium (normal medium supplemented with
hypoxanthine, thymidine and aminopterin) and then cultured for 7 to
14 days. After the culturing, a portion of the culture supernatant
is sampled and tested, for example, by enzyme immunoassay to select
those which can react with the antigen but not with protein which
does not contain the antigen. Thereafter, cloning is carried out by
limiting dilution analysis, and a hybridoma which shows stable and
high antibody titer by enzyme immunoassay is selected as monoclonal
antibody producing hybridoma cells.
[0139] With regard to the method for the isolation and purification
of the polyclonal antibody or monoclonal antibody, centrifugation,
ammonium sulfate precipitation, caprylic acid precipitation, or
chromatography using a DEAE-Sepharose column, an anion exchange
column, a protein A or G column, a gel filtration column and the
like may be employed alone or as a combination thereof.
[0140] 9) Utilization of the polypeptide and the DNA encoding the
polypeptide of the present invention and the antibody recognizing
the polypeptide of the present invention
[0141] (1) The polypeptide of the present invention can be utilized
for producing 1.alpha., 25-dihydroxyvitamin D.sub.3 as active type
vitamin D.sub.3.
[0142] (2) The whole length or partial fragments of the polypeptide
of the present invention can be utilized as an antigen against the
antibody recognizing 25-hydroxyvitamin
D.sub.3-1.alpha.-hydroxylase.
[0143] (3) By administering the whole length of the
25-hydroxyvitamin D.sub.3-1.alpha.-hydroxylase or partial fragments
thereof having the activity into biological organisms, diseases due
to the decrease of the enzyme protein, such as osteoporosis, can be
treated therapeutically.
[0144] (4) By using the DNA of the present invention, the mRNA of
25-hydroxyvitamin D.sub.3-1.alpha.-hydroxylase gene can be detected
by Northern hybridization method (Molecular Cloning, 2-nd edition),
PCR method [PCR Protocols, Academic Press (1990)], and RT-PCR
method and the like.
[0145] The diagnostic method for assaying the expression level of
the mRNA of the gene of 25-hydroxyvitamin
D.sub.3-1.alpha.-hydroxylase by utilizing the detection method, is
useful for suppressing the onset of adult diseases such as
osteoporosis induced by the decrease of active type vitamin D.sub.3
and is also effective for early diagnosis of genetic diseases due
to congenital deficiency of the 25-hydroxyvitamin
D.sub.3-1.alpha.-hydroxylase gene.
[0146] According to Northern hybridization method, the expression
level of mRNA is assayed on the basis of the label of a probe
hybridized, for example, on the basis of the radioactivity in case
of labeling with for example .sup.32P or the fluorescence in case
of fluorescent labeling. The expression level of mRNA is assayed,
on the basis of the fluorescence of a DNA specific fluorescent dye,
for example ethidium bromide and Cyber Green 1 which is used for
staining amplified fragments.
[0147] (5) The DNA of the present invention is inserted into virus
vectors such as retrovirus and adenovirus and other vectors, and
the resulting DNA can be used for therapeutic treatment according
to gene therapy.
[0148] (6) By using the anti-25-hydroxyvitamin
D.sub.3-1.alpha.-hydroxylas- e antibody of the present invention,
25-hydroxyvitamin D.sub.3-1.alpha.-hydroxylase can be detected and
assayed in samples of blood, some organs, cells and the like.
Specifically preferable methods therefor include ELISA method by
using microtiter plates, fluorescent antibody methods, Western blot
method and the like; additionally, immuno-histological staining by
using pathological sections may also be utilized. Thus, the
antibody of the present invention is useful for the diagnosis of
diseases such as osteoporosis, due to the decrease of the
expression of vitamin D.sub.3-1.alpha.-hydroxylase, the diagnosis
of the onset thereof and early prediction of the possibility of the
onset thereof and the like. Similarly, the antibody is also useful
as a laboratory reagent for research works for the protein.
[0149] (7) By using the antibody of the present invention,
polypeptides having 25-hydroxyvitamin D.sub.3-1.alpha.-hydroxylase
activity are immuno-histologically stained, and thus, an
immuno-histological staining agent containing the antibody can be
provided.
[0150] (8) By using the DNA of the present invention and through
the hybridization thereof with the genome DNA, the DNA in the
promoter region of the gene can be cloned. By using DNA fragments
in the promoter region, molecules involved in the regulation of the
expression of the gene can be screened and analyzed.
[0151] The present invention will now be described in detail in the
following examples. When kits were used in individual procedures,
experiments were progressed according to the protocols attached to
the kits, unless otherwise stated specifically. Fundamental genetic
manipulation techniques were according to Molecular Cloning, 2-nd
edition.
EXAMPLES
Example 1
[0152] Preparation of Kidney from Rats Fed with Vitamin D.sub.3
Deficient Diet
[0153] Immediately after weaning, four male SD rats were given
vitamin D.sub.3 deficient diet for 3 weeks (age 6 weeks).
[0154] DIET 11 [Suda, et. al., J. Nutrition, 100, 1049 (1970);
commercially available as Purified diet for Rat from Teklad Co,
Madison, Wis., USA] was used as the vitamin D.sub.3 deficient diet.
The diet was vitamin D deficient and low calcium diet at a calcium
content of 0.03% and a phosphate content of 0.6%.
[0155] Deionized water was used for supplementing the rats with
water.
[0156] 48 hours prior to sacrifice, 1.alpha., 25-dihydroxyvitamin
D.sub.3 (manufactured by Calviochem, Co., Calif., USA) was
intravenously injected at 1 .mu.g/rat into the rats.
[0157] After the designed dieting term was terminated, the rats
were anesthetized with ether. From the abdominal aortas of the
rats, blood was drawn out, and then, the rats were sacrificed to
death by phlebotomy and immediately thereafter, the rats were
autopsied to resect the kidneys.
[0158] The kidneys were rinsed in PBS [containing NaCl (8 g), KCl
(0.2 g), NaH.sub..2PO.sub.412H.sub.2O (2.9 g) and KH.sub.2PO.sub.4
(0.2 g) per one liter), and the resulting kidneys were frozen in
liquid nitrogen.
[0159] As a control group, rats were given normal diet (Rat diet
containing calcium (0.5 g), phosphate (0.6 g) and vitamin D.sub.3
(200 IU) per 100 g) in a similar fashion, and then, the kidneys
were prepared by the same method as described above. The resulting
kidneys were used as kidneys from rats with no activity
induction.
Example 2
[0160] Preparation of mRNA from Rat Kidneys
[0161] The kidneys prepared from the rats fed with the vitamin
D.sub.3 deficient diet and the kidneys derived from the rats fed
with normal diet, weighed 0.78 g and 0.94 g, respectively, were
rinsed in PBS and were then frozen in liquid nitrogen. The frozen
kidneys can be stored at -80.degree. C.
[0162] The frozen kidneys were cut into pieces in liquid nitrogen
with a wearing blender, until the tissues were hashed into sand
size form. Then, the liquid nitrogen was evaporated.
[0163] The sand-like tissues were homogenized in ice cooling with a
homogenizer (Digital Homogenizer; manufactured by Inouchi, Co.),
while adding thereto 35 ml of 5.5 M GTC solution (containing 324.5
g of guanidine isothiocyanate, 3.7 g of sodium citrate, and 3.3 g
of Sarkosyl in 500 ml) and 492 .mu.l of 2-mercaptoethanol, and the
homogenate in suspension was passed four times through an injection
needle of gauge 18 arranged on a 50-ml injection cylinder.
[0164] The suspension was then transferred into a 15-ml centrifuge
tube, for centrifugation at 6,000 rpm at 20.degree. C. for 10
minutes, to recover the supernatant.
[0165] The supernatant was then overlaid in 16-ml portions on a
CsTFA preparative solution [a mixture solution of CsTFA solution
(100 ml) manufactured by Pharmacia, Co., 82.06 ml of 0.25 M EDTA
solution (pH 7.0), and 23.09 ml of H.sub.2O] in a 40-ml polyallomer
tube for ultracentrifugation, and the tube was then
ultra-centrifuged under conditions of 25,000 rpm and 18.degree. C.
for 25 hours.
[0166] After discarding the supernatant, the tube was cut at the
position of about 1.5 cm from the bottom of the tube, and the
resulting precipitate was dissolved in 0.6 ml of 4M GTC solution [a
mixture solution of 5.5M GTC solution (4 ml), 1.5 ml of H.sub.2O,
and 56 .mu.l of 2-mercaptoethanol].
[0167] The dissolved solution was centrifuged at 14,000 rpm for 15
seconds, to recover the supernatant.
[0168] After adding 15 .mu.l of 1M sodium acetate and 0.45 ml of
ethanol to the supernatant and thereby suspending the precipitate,
the resulting suspension was centrifuged to recover the
precipitate.
[0169] The precipitate was rinsed in 70% ethanol, suspended in 1 ml
of TE buffer [10 mM Tris-HCl (pH 8.0), 1 mM EDTA-NaOH (pH 8.0)],
and centrifuged at 14,000 rpm for 15 seconds, to recover the
supernatant.
[0170] Adding a 2.5-fold volume of 70% ethanol to the supernatant,
followed by centrifugation, the resulting precipitate was
recovered.
[0171] The precipitate was rinsed in 70% ethanol and was then
dissolved in 500 .mu.l of TE buffer.
[0172] Through the procedure, the whole RNA was recovered from the
kidneys from the rats with activity induction and the rats with no
activity induction, which was calculated as 639 .mu.g and 918
.mu.g, respectively, on the basis of the absorbance at 260 nm.
[0173] The whole RNA solution (150 .mu.l) derived from the rats
with activity induction was effected with thermal treatment at
65.degree. C. for 5 minutes, which was immediately cooled in
ice.
[0174] To the solution were added 0.5 ml of 5M NaCl and 0.15 g
oligo dT cellulose (manufactured by Collaborative Research, Co.,
Type 3) equilibrated with TE/NaCl [10 mM Tris-HCl (pH 7.5), 500 mM
NaCl], to adsorb the whole RNA onto the cellulose.
[0175] The cellulose was packed in a column, through which the
TE/NaCl solution was passed for washing the column, followed by
elution of mRNA with TE solution of 0.5 ml, to fractionate and
collect the eluate in 200 .mu.l-fractions.
[0176] From the individual fractionated solutions, 2 .mu.l portions
were sampled, followed by addition of 1 .mu.g/ml ethidium bromide
(20 .mu.l), to detect luminescent sampled solutions under
ultraviolet irradiation.
[0177] Ethanol was added to the fractionated solutions
corresponding to the luminescent sampled solutions, to recover
precipitates.
[0178] The precipitates were rinsed in 80% ethanol and suspended in
TE buffer.
[0179] Through the procedures, mRNA of 14.3 .mu.g was recovered
from the kidneys of the rats with activity induction.
Example 3
[0180] Preparation of cDNA Library
[0181] By using ZAP-cDNA synthesis kit (#200400) manufactured by
Stratagene Co., a cDNA library was constructed according to the
instruction manual attached to the kit.
[0182] By using 4 .mu.g of mRNA derived from the rats with activity
induction as prepared in Example 2, fist strand DNA was synthesized
through reverse-transcriptase reaction, and after RNase reaction,
second strand DNA was synthesized with DNA polymerase I.
[0183] Under high temperature conditions, PfuDNA polymerase
reaction was effected to make the termini of the cDNA to blunt
end.
[0184] By ligating an EcoRI adapter fragment to the cDNA for
phosphorylation and digesting the resulting cDNA with XhoI, a cDNA
fragment with EcoRI-XhoI cleavage sites on both the termini was
prepared.
[0185] The cDNA fragment was inserted into the EcoRI-XhoI site of
.lambda. ZAP II, and by subsequent packaging with Giga pack Gold
Packaging Kit (manufactured by Stratagene, Co.) and infection by
using Escherichia coli host XL1-Blue, MRF' strain and helper phage
VCS257, a cDNA library was constructed.
Example 4
[0186] Selection of a Clone Harbouring mRNA Molecule Specifically
Expressed in the Kidneys of the Rats with Induced Activity
[0187] The amino acid sequences of the rat-derived hydroxylase of
the position 25 of vitamin D.sub.3 and the hydroxylase of the
position 24 thereof were previously reported, and among the regions
well preserved in these vitamin D.sub.3hydroxylases of the family
P450, the partial amino acid sequences of the adrenodoxin binding
region (region A) essential for the enzyme activity and of the hem
binding region (region H) were selected, and on the basis of the
DNA sequences were designed a sense primer and an antisense primer
for PCR amplification of the gene in the regions.
[0188] More specifically, DNA comprising the nucleotide sequence
represented by SEQ ID No. 7 corresponding to the region A was used
as the sense primer; and DNA comprising the nucleotide sequence
represented by SEQ ID NO. 8 corresponding to the region H was used
as the antisense primer.
[0189] By using the ZAP-cDNA synthesis kit (#200400) manufactured
by Stratagene Co. and 4 .mu.g of the mRNA derived from the rats
having activity induction, first strand DNA was synthesized with a
primer random hexamer.
[0190] By using the first strand DNA as the template, the DNA
comprising the nucleotide sequence represented by SEQ ID NO. 7 as
the sense primer and the DNA comprising the nucleotide sequence
represented by SEQ ID NO. 8 as the antisense primer and by
utilizing RT-PCR kit manufactured by Stratagene, Co., PCR was
effected.
[0191] By using DNA Thermal Cycler 480 manufactured by Perkin
Elmer, Co., PCR was effected at 35 cycles, each cycle composed of
94.degree. C. for 30 seconds, 42.degree. C. for one minute and
72.degree. C. for one minute.
[0192] The reaction product was analyzed by agarose gel
electrophoresis, and a 255-bp amplification fragment (AH fragment)
was observed. By using a DNA purification kit (manufactured by Bio
Rad, Co.), the fragment was extracted from agarose, which was then
inserted into pCRII vector (manufactured by Invitrogen, Co.).
[0193] From the whole RNAs derived from the rats induced with
25-hydroxyvitamin D.sub.3-1.alpha.-hydroxylase activity and the
non-induced rats were prepared poly(A) .sup.+RNAs, which were then
subject individually to agarose electrophoresis, to transfer the
electrophoresed mRNAs onto membrane filters in a conventional
manner.
[0194] By using these membrane filters, Northern hybridization was
effected by using the amplified AH fragment as the probe.
[0195] The amplified AH fragment was hybridized only when the
membrane filter prepared from the mRNA derived from the rats with
activity induction was used.
[0196] The AH fragment had nucleotide sequences corresponding to
the regions A and H.
[0197] By using the AH fragment and 3' RACE system kit manufactured
by BRL, Co., a PCR amplified fragment containing the 3' noncoding
region of the DNA encoding the 25-hydroxyvitamin
D.sub.3-1.alpha.-hydroxylase was recovered by the following
method.
[0198] By using the Oligo dT/AUAP primer attached to the 3' RACE
system kit manufactured by BRL, Co. and 4 .mu.g of the mRNA from
the rats with activity induction as recovered in Example 2, cDNA
was synthesized.
[0199] The cDNA was used as a template.
[0200] Based on the sequence of the AH fragment amplified above,
DNA comprising the nucleotide sequence represented by SEQ ID NO. 9
was synthesized and used as a sense primer.
[0201] The AUAP primer attached to the 3' RACE system kit
manufactured by BRL, Co. was used as an antisense primer.
[0202] By using the template, the sense primer and the antisense
primer, PCR was effected at 35 cycles, each cycle composed of
94.degree. C. for one minute, 55.degree. C. for one minute and
72.degree. C. for 2 minutes.
[0203] The reaction product was analyzed by agarose gel
electrophoresis, and an amplified fragment of 1.3 kb (A3 fragment)
was observed. By using a DNA purification kit (manufactured by Bio
Rad, Co.), the fragment was extracted from agarose, which was then
inserted into pCRII vector.
[0204] In the same manner as for the AH fragment, the A3 fragment
was specifically hybridized with the mRNA from the rats with
activity induction.
[0205] The A3 fragment contained almost whole length of the AH
fragment.
Example 5
[0206] Recovery of DNA encoding 25-hydroxyvitamin
D.sub.3-1.alpha.-hydroxy- lase.
[0207] The cDNA phage library prepared in Example 3 was spread on
an agar medium and cultivated to a final concentration of 10,000 to
20,000 plaques per one petri dish.
[0208] HybondN.sup.+ membrane (manufactured by Amersham, Co.) was
placed on each of the petri dishes with the plaques formed thereon,
to transfer the plaque DNA onto the membrane. Two transcription
membranes were prepared per one petri dish.
[0209] The transcription membranes were subject to alkali treatment
(immersion in 1.5 M NaCl and 0.5 M NaOH) and SDS treatment
(immersion in 2.times.SSC and 0.1% SDS solution), rinsed and dried,
and then, the resulting membranes with plaque DNA immobilized
thereon were used as blotting membranes for the following
hybridization.
[0210] By using DIG labeling kit.(#1 175 033; manufactured by
Boehringer Mannheim, Co.) and 2 ng each of the AH fragment and A3
fragment as templates, PCR was effected, to recover DIG labeled AH
fragment or A3 fragment.
[0211] PCR was effected under conditions of 30 cycles, each cycle
of a process of 94.degree. C. for one minute, 50.degree. C. for one
minute and 72.degree. C. for one minute.
[0212] The resulting DIG labeled AH fragment and DIG labeled A3
fragment were used as the following probes.
[0213] The blotting membranes prepared above were immersed in a
hybridization solution [5.times.SSC, 0.1% Sarkosyl, 0.02% SDS, 1%
hybridization blocking solution (manufactured by Boehringer
Mannheim, Co.)] at 60.degree. C. for 5 hours, followed by addition
of thermally treated DIG labeled probe (10 .mu.l/10
ml-hybridization solution), for overnight hybridization at
65.degree. C.
[0214] After hybridization, the membranes were subject to rinsing
(rinsing twice in 2.times.SSC and 0.1% SDS at room temperature for
5 minutes, rinsing twice in 0.1.times.SSC and 0.1% SDS at
60.degree. C. for 15 minutes), blocking [effected by using
1.times.blocking solution (manufactured by Boehringer Mannheim,
Co.), 0.1M maleic acid, 0.15M NaCl, pH 7.5], reaction with AP
labeled anti-DIG antibody (effected according to the protocol by
Boehringer Mannheim, Co.), and alkali treatment [0.1M Tris-HCl (pH
9.5), 0.1M NaCl and 50 mM MgCl.sub.2], and by using thereafter DIG
luminescence detection kit (#1 363 514; manufactured by Boehringer
Mannheim, Co.), plaques hybridizable with the probes were screened
on an X-ray film.
[0215] By using firstly the DIG labeled AH fragment as the DIG
labeled probe to select plaques hybridizable with the fragment and
by subsequently using the DIG labeled A3 fragment, plaques
hybridizable with the fragment were selected from the plaques
described above.
[0216] The plaques selected at each stage were again inoculated on
petri dishes, and then, it was confirmed that these were
hybridizable. By PCR using both the primers of the region A and
AUAP, additionally, it was confirmed that the plaques had the
nucleotide sequence of the A3 fragment.
[0217] After screening of 35 petri dishes in total, finally, four
plaques (Nos. 221, 522, 411, 111) were selected.
[0218] From individual plaque clones was extracted DNA, which was
then ligated to pBluescript vector by using rapid excision kit
(#211204; manufactured by Stratagene, Co.), and subsequently, the
nucleotide sequence of DNA inserted into the clone was analyzed by
using M13 primer.
[0219] By the analysis with the clone No. 221, DNA comprising a
nucleotide sequence of 2469 bp was observed, as represented by SEQ
ID No. 5.
[0220] An open reading frame (referred to as ORF hereinafter)
encoding 501 amino acids was observed in the DNA, in which amino
acid sequences believed as the hem binding region and adrenodoxin
binding region in common with the P450 family protein were
present.
Example 6
[0221] Expression of Isolated 25-hydroxyvitamin
D.sub.3-1.alpha.-hydroxyla- se Gene in Animal Cells
[0222] From the clone No. 221 described in Example 5 was prepared a
plasmid, which was subsequently digested with HindIII and XbaI.
Expression vector pcDNA3 (manufactured by Invitrogen, Co.) for
animal cells was similarly digested with HindIII and XbaI.
[0223] The cleavage fragments recovered above were individually
subject to agarose electrophoresis, which were thereby separated
and extracted.
[0224] The resulting DNA fragments from the vector and the inserted
gene fragment were ligated together, by using a DNA ligation
kit(manufactured by TaKaRa Brewery), to recover a ligated
plasmid.
[0225] By using the plasmid, Escherichia coli strain DH5.alpha. was
transformed, and thereafter, an ampicillin resistant strain was
selected, from which the plasmid was extracted according to a known
method.
[0226] Based on the analysis of the plasmid by restriction
cleavage, it was confirmed that the plasmid inserted the objective
gene. The plasmid was named pCMD3R.
[0227] By electroporation [Potter et. al., Proc. Natl. Acad. Sci.
USA, 81, 716 (1984)], pCMD3R was introduced into an animal cell, to
be expressed therein as follows. COS7 cell was cultivated in a DMEM
culture medium (manufactured by GIBCO BRL, Co.) supplemented with
10% FCS (fetal calf serum) in a petri dish for 2 days.
[0228] After cultivation, the cells were peeled off from the petri
dish by trypsin treatment, and the cells were rinsed in PBS and
then suspended in 0.5 ml of KPBS (137 mM KCl 2.7 mM NaCl, 8.1 mM
Na.sub.2HPO.sub.41.5 mM NaH.sub.2PO.sub.4 4 mM MgCl.sub.2), to a
final concentration of 2 to 6.0.times.10.sup.6/ml.
[0229] The suspension and 15 .mu.g of pCMD3R plasmid were mixed
together in a pulser cuvette (manufactured by BIO-RAD, Co.) with a
groove width of 0.4 cm, and the resulting mixture was then applied
to an electroporation system Gene pulser (manufactured by BIO-RAD,
Co.) for pulse loading under conditions of 960 .mu.F and 0.22 kV,
to introduce the DNA into the cell.
[0230] The DNA introduced cell was suspended in 10 ml of DMEM
culture medium containing 10% FCS, for cultivation in a 5% CO.sub.2
incubator at 37.degree. C. for 48 to 72 hours.
[0231] By discarding the culture in the petri dish and rinsing the
cell twice in PBS, the cell was scraped off with a scraper,
followed by centrifugation to collect the cell.
Example 7
[0232] Recovery of human-derived 25-hydroxyvitamin
D.sub.3-1.alpha.-hydrox- ylase gene
[0233] From 1.2 g of tissue resected from human kidney cancer, the
whole RNA (750 .mu.g) was recovered according to the method
described in Example 2, and 9.5 .mu.g of mRNA was recovered from
the whole RNA.
[0234] By using 5 .mu.g of the mRNA, a human cDNA library was
constructed by the method described in Example 3.
[0235] According to the method described in Example 5, DNA encoding
the human derived 25-hydroxyvitamin D.sub.3-1.alpha.-hydroxylase
was recovered.
[0236] The whole length of the rat vitamin D.sub.3hydroxylase gene
of 2469 bp as isolated in Example 5 was DIG labeled according to
the method described in Example 5, which was then used as a
probe.
[0237] Hybridization was effected overnight in a hybridization
solution containing formamide at 40% under a condition of
42.degree. C.
[0238] Through the hybridization, four clones were selected.
[0239] According to the method described in Example 5, DNA was
extracted from these clones, to analyze the nucleotide sequence of
the DNA inserted into the clones.
[0240] The DNA had the nucleotide sequence represented by SEQ ID
NO. 6. In the DNA fragment was observed ORF encoding a peptide of
508 amino acids.
[0241] The peptide had an amino acid sequence in common with the
rat-derived 25-hydroxyvitamin D.sub.3-1.alpha.-hydroxylase in terms
of 413 amino acid residues, and contained amino acid sequences
possibly corresponding to the hem binding region and adrenodoxin
binding region, commonly observed in the P450 family protein.
[0242] Additionally, the DNA sequence included a sequence of 1724
residues, which is the same as the sequence derived from rats, and
therefore, it was indicated that the DNA had high homology.
Example 8
[0243] Expression of Rat-derived Vitamin
D.sub.3-1.alpha.-hydroxylase Gene and Assay of the Activity
[0244] According to the method of Example 6, gene expression
plasmid carrying the rat-derived vitamin
D.sub.3-1.alpha.-hydroxylase gene, namely pCMD3R, was introduced
into COS-7 cell by electroporation.
[0245] The gene-introduced cells of 5.times.10.sup.5 in number were
cultivated in 10 ml of a DMEM culture medium containing 10% FCS for
24 hours, and then, the culture medium was exchanged to a DMEM
culture medium (8 ml) containing 1% FCS, followed by addition of
[26, 27-.sup.3H]-25-hydroxyvitamin D.sub.3(manufactured by
Amersham, Co.) at 2000 Bq/3 .mu.l-methanol solution, and then, the
resulting mixture was cultivated for 24 hours.
[0246] After cultivation, vitamin D.sub.3 metabolites were
extracted from the culture supernatant and the cells by the Bligh
& Dyer's method [Can. J. Biochem., 37, 911 (1959)]. More
specifically, the culture was transferred into a 50-ml centrifuge
tube equipped with a screw cap, while 10 ml methanol was added into
the petri dish, to scrape the cells with a scraper, and the cells
were then transferred into the centrifuge tube. Methanol (10 ml)
was again added into the petri dish, to suspend the cells remaining
in the petri dish, and the resulting suspension was thoroughly
transferred into the centrifuge tube.
[0247] Chloroform (10 ml) was added into the centrifuge tube for
thorough mixing, followed by further addition of 10 ml of
chloroform and subsequent complete re-mixing, and the resulting
tube was left to stand to separate a chloroform layer from an
aqueous layer.
[0248] The chloroform extract solution in the separated chloroform
layer was placed in another centrifuge tube, followed by further
addition of 10 ml of chloroform to the remaining aqueous layer, for
mixing and extraction in the same manner, and the resulting
chloroform extract solution was combined together with the
previously recovered chloroform extract solution.
[0249] Distilled water was added into the chloroform extract
solution to a final total volume of 60 ml, followed by addition of
two drops of saturated sodium chloride solution and subsequent
sufficient mixing.
[0250] The mixture solution was centrifuged, to separate the
chloroform layer from the aqueous layer.
[0251] The resulting chloroform layer fraction was concentrated in
nitrogen gas stream to recover the residue.
[0252] The residue was dissolved in 400 .mu.l of a mixture solution
iso-propanol/methanol/n-hexane=6:6:88.
[0253] With HPLC system 880 PU manufactured by JASCO, Co. with TSK
silica gel 150 column (4.6.times.250 mm; manufactured by Toso, Co.)
arranged thereon, the resulting solution was subject to analysis
under conditions such that the mixture solution
iso-propanol/methanol/n-hexane=6:6:88 was used as the mobile phase
at a flow rate of 1 ml/minute. On comparison with the elution time
of a standard substance, vitamin D.sub.3 metabolites were
identified.
[0254] Similarly, vitamin D.sub.3 metabolites were identified by
using a vector pcDNA3 which does not carry the gene of the present
invention.
[0255] The results are shown in FIG. 1.
[0256] "A" represents the analytical results of metabolites in the
cells introduced with pcMD3R; and "B" represents the analytical
results of metabolites in the cells introduced with pcDNA3. Because
1.alpha., 25-hydroxyvitamin D.sub.3 was detected only in the cells
introduced with pcMD3R carrying the gene of the present invention,
it was indicated that only the cells had 25-hydroxyvitamin
D.sub.3-1.alpha.-hydroxylase activity, which further indicates that
the gene of the present invention encodes 25-hydroxyvitamin
D.sub.3-1.alpha.-hydroxylase.
INDUSTRIAL APPLICABILITY
[0257] In accordance with the present invention, the following can
be provided; a polypeptide having 25-hydroxyvitamin
D.sub.3-1.alpha.-hydroxy- lase activity, being useful for the
prevention, diagnosis and therapeutic treatment of adult diseases
such as osteoporosis induced by the decrease of active type vitamin
D.sub.3, DNA encoding the polypeptide, a recombinant DNA prepared
by inserting the DNA in a vector, a transformant carrying the
recombinant DNA, a method for preparing 25-hydroxyvitamin
D.sub.3-1.alpha.-hydroxylase by using the transformant, a method
for preparing 1.alpha., 25-dihydroxyvitamin D.sub.3 by using the
polypeptide having 25-hydroxyvitamin D.sub.3-1.alpha.-hydroxylase
activity, and an antibody recognizing the polypeptide.
Sequence CWU 1
1
9 1 501 PRT Rat 1 Met Thr Gln Ala Val Lys Leu Ala Ser Arg Val Phe
His Arg Val Gln 1 5 10 15 Leu Pro Ser Gln Leu Gly Ser Asp Ser Val
Leu Arg Ser Leu Ser Asp 20 25 30 Ile Pro Gly Pro Ser Thr Pro Ser
Phe Leu Ala Glu Leu Phe Cys Lys 35 40 45 Gly Gly Leu Ser Arg Leu
His Glu Leu Gln Val His Gly Ala Ala Arg 50 55 60 Tyr Gly Pro Ile
Trp Ser Gly Ser Phe Gly Thr Leu Arg Thr Val Tyr 65 70 75 80 Val Ala
Asp Pro Ala Leu Val Glu Gln Leu Leu Arg Gln Glu Ser His 85 90 95
Cys Pro Glu Arg Cys Ser Phe Ser Ser Trp Ser Glu His Arg Arg Arg 100
105 110 His Gln Arg Ala Cys Gly Leu Leu Thr Ala Asp Gly Glu Glu Trp
Gln 115 120 125 Arg Leu Arg Ser Leu Leu Ala Pro Leu Leu Leu Arg Pro
Gln Ala Ala 130 135 140 Ala Gly Tyr Ala Gly Thr Leu Asp Ser Val Val
Ser Asp Leu Val Arg 145 150 155 160 Arg Leu Arg Arg Gln Arg Gly Arg
Gly Ser Gly Leu Pro Asp Leu Val 165 170 175 Leu Asp Val Ala Gly Glu
Phe Tyr Lys Phe Gly Leu Glu Gly Ile Gly 180 185 190 Ala Val Leu Leu
Gly Ser Arg Leu Gly Cys Leu Glu Ala Glu Val Pro 195 200 205 Pro Asp
Thr Glu Thr Phe Ile Glu Ala Val Gly Ser Val Phe Val Ser 210 215 220
Thr Leu Leu Thr Met Ala Met Pro Ser Trp Leu His Arg Leu Ile Pro 225
230 235 240 Gly Pro Trp Ala Arg Leu Cys Arg Asp Trp Asp Gln Met Phe
Ala Phe 245 250 255 Ala Gln Lys His Val Glu Gln Arg Glu Gly Glu Ala
Ala Val Arg Asn 260 265 270 Gln Gly Lys Pro Glu Glu Asp Leu Pro Thr
Gly His His Leu Thr His 275 280 285 Phe Leu Phe Arg Glu Lys Val Ser
Val Gln Ser Ile Val Gly Asn Val 290 295 300 Thr Glu Leu Leu Leu Ala
Gly Val Asp Thr Val Ser Asn Thr Leu Ser 305 310 315 320 Trp Ala Leu
Tyr Glu Leu Ser Arg His Pro Glu Val Gln Ser Ala Leu 325 330 335 His
Ser Glu Ile Thr Gly Ala Val Asn Pro Gly Ser Tyr Ala His Leu 340 345
350 Gln Ala Thr Ala Leu Ser Gln Leu Pro Leu Leu Lys Ala Val Ile Lys
355 360 365 Glu Val Leu Arg Leu Tyr Pro Val Val Pro Gly Asn Ser Arg
Val Pro 370 375 380 Asp Arg Asp Ile Cys Val Gly Asn Tyr Val Ile Pro
Gln Asp Thr Leu 385 390 395 400 Val Ser Leu Cys His Tyr Ala Thr Ser
Arg Asp Pro Ala Gln Phe Arg 405 410 415 Glu Pro Asn Ser Phe Asn Pro
Ala Arg Trp Leu Gly Glu Gly Pro Ala 420 425 430 Pro His Pro Phe Ala
Ser Leu Pro Phe Gly Phe Gly Lys Arg Ser Cys 435 440 445 Ile Gly Arg
Arg Leu Ala Glu Leu Glu Leu Gln Met Ala Leu Ala Gln 450 455 460 Ile
Leu Thr His Phe Glu Val Leu Pro Glu Pro Gly Ala Leu Pro Val 465 470
475 480 Lys Pro Met Thr Arg Thr Val Leu Val Pro Glu Arg Ser Ile His
Leu 485 490 495 Gln Phe Val Asp Arg 500 2 508 PRT human 2 Met Thr
Gln Thr Leu Lys Tyr Ala Ser Arg Val Phe His Arg Val Arg 1 5 10 15
Trp Ala Pro Glu Leu Gly Ala Ser Leu Gly Tyr Arg Glu Tyr His Ser 20
25 30 Ala Arg Arg Ser Leu Ala Asp Ile Pro Gly Pro Ser Thr Pro Ser
Phe 35 40 45 Leu Ala Glu Leu Phe Cys Lys Gly Gly Leu Ser Arg Leu
His Glu Leu 50 55 60 Gln Val Gln Gly Ala Ala His Phe Gly Pro Val
Trp Leu Ala Ser Phe 65 70 75 80 Gly Thr Val Arg Thr Val Tyr Val Ala
Ala Pro Ala Leu Val Glu Glu 85 90 95 Leu Leu Arg Gln Glu Gly Pro
Arg Pro Glu Arg Cys Ser Phe Ser Pro 100 105 110 Trp Thr Glu His Arg
Arg Cys Arg Gln Arg Ala Cys Gly Leu Leu Thr 115 120 125 Ala Glu Gly
Glu Glu Trp Gln Arg Leu Arg Ser Leu Leu Ala Pro Leu 130 135 140 Leu
Leu Arg Pro Gln Ala Ala Ala Arg Tyr Ala Gly Thr Leu Asn Asn 145 150
155 160 Val Val Cys Asp Leu Val Arg Arg Leu Arg Arg Gln Arg Gly Arg
Gly 165 170 175 Thr Gly Pro Pro Ala Leu Val Arg Asp Val Ala Gly Glu
Phe Tyr Lys 180 185 190 Phe Gly Leu Glu Gly Ile Ala Ala Val Leu Leu
Gly Ser Arg Leu Gly 195 200 205 Cys Leu Glu Ala Gln Val Pro Pro Asp
Thr Glu Thr Phe Ile Arg Ala 210 215 220 Val Gly Ser Val Phe Val Ser
Thr Leu Leu Thr Met Ala Met Pro His 225 230 235 240 Trp Leu Arg His
Leu Val Pro Gly Pro Trp Gly Arg Leu Cys Arg Asp 245 250 255 Trp Asp
Gln Met Phe Ala Phe Ala Gln Arg His Val Glu Arg Arg Glu 260 265 270
Ala Glu Ala Ala Met Arg Asn Gly Gly Gln Pro Glu Lys Asp Leu Glu 275
280 285 Ser Gly Ala His Leu Thr His Phe Leu Phe Arg Glu Glu Leu Pro
Ala 290 295 300 Gln Ser Ile Leu Gly Asn Val Thr Glu Leu Leu Leu Ala
Gly Val Asp 305 310 315 320 Thr Val Ser Asn Thr Leu Ser Trp Ala Leu
Tyr Glu Leu Ser Arg His 325 330 335 Pro Glu Val Gln Thr Ala Leu His
Ser Glu Ile Thr Ala Ala Leu Ser 340 345 350 Pro Gly Ser Ser Ala Tyr
Pro Ser Ala Thr Val Leu Ser Gln Leu Pro 355 360 365 Leu Leu Lys Ala
Val Val Lys Glu Val Leu Arg Leu Tyr Pro Val Val 370 375 380 Pro Gly
Asn Ser Arg Val Pro Asp Lys Asp Ile His Val Gly Asp Tyr 385 390 395
400 Ile Ile Pro Lys Asn Thr Leu Val Thr Leu Cys His Tyr Ala Thr Ser
405 410 415 Arg Asp Pro Ala Gln Phe Pro Glu Pro Asn Ser Phe Arg Pro
Ala Arg 420 425 430 Trp Leu Gly Glu Gly Pro Thr Pro His Pro Phe Ala
Ser Leu Pro Phe 435 440 445 Gly Phe Gly Lys Arg Ser Cys Met Gly Arg
Arg Leu Ala Glu Leu Glu 450 455 460 Leu Gln Met Ala Leu Ala Gln Ile
Leu Thr His Phe Glu Val Gln Pro 465 470 475 480 Glu Pro Gly Ala Ala
Pro Val Arg Pro Lys Thr Arg Thr Val Leu Val 485 490 495 Pro Glu Arg
Ser Ile Asn Leu Gln Phe Leu Asp Arg 500 505 3 1503 DNA Rat CDS
(1)..(1503) 3 atg acc cag gca gtc aag ctc gcc tcc aga gtc ttc cat
cga gtc caa 48 Met Thr Gln Ala Val Lys Leu Ala Ser Arg Val Phe His
Arg Val Gln 1 5 10 15 ctg cct tct cag ctg ggc agt gac tcg gtt ctc
cgg agt tta tct gat 96 Leu Pro Ser Gln Leu Gly Ser Asp Ser Val Leu
Arg Ser Leu Ser Asp 20 25 30 atc cct ggg ccc tct aca cct agc ttc
ctg gct gaa ctc ttc tgc aaa 144 Ile Pro Gly Pro Ser Thr Pro Ser Phe
Leu Ala Glu Leu Phe Cys Lys 35 40 45 ggg ggg ctg tcc agg cta cat
gaa ctg cag gtg cat ggc gct gcg cgg 192 Gly Gly Leu Ser Arg Leu His
Glu Leu Gln Val His Gly Ala Ala Arg 50 55 60 tac ggg cca ata tgg
tcc ggc agc ttc ggg aca ctt cgc aca gtt tat 240 Tyr Gly Pro Ile Trp
Ser Gly Ser Phe Gly Thr Leu Arg Thr Val Tyr 65 70 75 80 gtg gcc gac
cct gca ctt gta gag cag ctc ctg cga caa gaa agt cat 288 Val Ala Asp
Pro Ala Leu Val Glu Gln Leu Leu Arg Gln Glu Ser His 85 90 95 tgt
cca gag cgc tgt agt ttc tca tct tgg tca gag cac cgt cgc cgc 336 Cys
Pro Glu Arg Cys Ser Phe Ser Ser Trp Ser Glu His Arg Arg Arg 100 105
110 cac cag cgg gct tgc ggg ttg cta acg gcg gat ggt gaa gaa tgg cag
384 His Gln Arg Ala Cys Gly Leu Leu Thr Ala Asp Gly Glu Glu Trp Gln
115 120 125 agg ctc cga agt ctc ctg gcc ccg cta ctc ctc cga cct caa
gca gcc 432 Arg Leu Arg Ser Leu Leu Ala Pro Leu Leu Leu Arg Pro Gln
Ala Ala 130 135 140 gcc ggc tat gct gga act ctg gac agc gtg gtc agt
gac ctc gtg cga 480 Ala Gly Tyr Ala Gly Thr Leu Asp Ser Val Val Ser
Asp Leu Val Arg 145 150 155 160 cga cta agg cgc cag cgg gga cgt ggc
tct ggg cta ccg gac cta gtt 528 Arg Leu Arg Arg Gln Arg Gly Arg Gly
Ser Gly Leu Pro Asp Leu Val 165 170 175 ctg gac gtg gcg gga gag ttt
tac aaa ttt ggc cta gaa ggc ata ggc 576 Leu Asp Val Ala Gly Glu Phe
Tyr Lys Phe Gly Leu Glu Gly Ile Gly 180 185 190 gcg gtg ctg ctg gga
tcg cgc ctg ggc tgc ctg gag gct gaa gtt cct 624 Ala Val Leu Leu Gly
Ser Arg Leu Gly Cys Leu Glu Ala Glu Val Pro 195 200 205 ccc gac aca
gaa acc ttc att gag gcc gtg ggc tcg gtg ttt gtg tct 672 Pro Asp Thr
Glu Thr Phe Ile Glu Ala Val Gly Ser Val Phe Val Ser 210 215 220 aca
ctc ttg acc atg gca atg ccc agt tgg ctg cac cgc ctt ata ccc 720 Thr
Leu Leu Thr Met Ala Met Pro Ser Trp Leu His Arg Leu Ile Pro 225 230
235 240 gga ccc tgg gcc cgc ctc tgc aga gac tgg gat cag atg ttt gcc
ttt 768 Gly Pro Trp Ala Arg Leu Cys Arg Asp Trp Asp Gln Met Phe Ala
Phe 245 250 255 gcc cag aag cac gtg gag cag cgc gaa ggc gaa gct gcc
gtg agg aac 816 Ala Gln Lys His Val Glu Gln Arg Glu Gly Glu Ala Ala
Val Arg Asn 260 265 270 cag gga aag cct gag gag gat ttg cca acg ggg
cat cac tta acc cac 864 Gln Gly Lys Pro Glu Glu Asp Leu Pro Thr Gly
His His Leu Thr His 275 280 285 ttc ctt ttt cgg gaa aag gtg tct gtc
cag tcc ata gtg gga aat gtg 912 Phe Leu Phe Arg Glu Lys Val Ser Val
Gln Ser Ile Val Gly Asn Val 290 295 300 aca gag cta cta ctg gct gga
gtg gac acg gta tcc aat acg ctc tcc 960 Thr Glu Leu Leu Leu Ala Gly
Val Asp Thr Val Ser Asn Thr Leu Ser 305 310 315 320 tgg gca ctc tat
gag ctc tcc cgg cac ccg gaa gtc cag tct gca ctc 1008 Trp Ala Leu
Tyr Glu Leu Ser Arg His Pro Glu Val Gln Ser Ala Leu 325 330 335 cac
tct gag atc aca ggc gct gtg aac cct ggc tcc tat gcc cac ctc 1056
His Ser Glu Ile Thr Gly Ala Val Asn Pro Gly Ser Tyr Ala His Leu 340
345 350 caa gcc act gct ctg tcc cag cta ccc ctg cta aag gct gtg atc
aaa 1104 Gln Ala Thr Ala Leu Ser Gln Leu Pro Leu Leu Lys Ala Val
Ile Lys 355 360 365 gaa gtg ttg aga ttg tac cct gtg gta cct ggg aac
tcc cgt gtc cca 1152 Glu Val Leu Arg Leu Tyr Pro Val Val Pro Gly
Asn Ser Arg Val Pro 370 375 380 gac aga gac atc tgt gta gga aac tat
gtt att ccc caa gat aca ctg 1200 Asp Arg Asp Ile Cys Val Gly Asn
Tyr Val Ile Pro Gln Asp Thr Leu 385 390 395 400 gtt tcc ctc tgt cac
tat gcc act tca agg gac ccc gcc cag ttt cgg 1248 Val Ser Leu Cys
His Tyr Ala Thr Ser Arg Asp Pro Ala Gln Phe Arg 405 410 415 gaa ccc
aac tct ttt aat cca gct cga tgg ctt gga gag ggt cca gcc 1296 Glu
Pro Asn Ser Phe Asn Pro Ala Arg Trp Leu Gly Glu Gly Pro Ala 420 425
430 ccc cac cca ttt gca tct ctt cct ttt ggc ttt ggc aaa cga agt tgc
1344 Pro His Pro Phe Ala Ser Leu Pro Phe Gly Phe Gly Lys Arg Ser
Cys 435 440 445 ata ggg aga cgc ttg gca gag ctc gag cta caa atg gcg
ttg gcc cag 1392 Ile Gly Arg Arg Leu Ala Glu Leu Glu Leu Gln Met
Ala Leu Ala Gln 450 455 460 atc ttg acc cat ttt gag gtg ctg cct gag
cca ggt gct ctt cca gtc 1440 Ile Leu Thr His Phe Glu Val Leu Pro
Glu Pro Gly Ala Leu Pro Val 465 470 475 480 aaa ccc atg acc cgg act
gtc ctg gta cct gag agg agc atc cat ctc 1488 Lys Pro Met Thr Arg
Thr Val Leu Val Pro Glu Arg Ser Ile His Leu 485 490 495 cag ttt gta
gac aga 1503 Gln Phe Val Asp Arg 500 4 1524 DNA human CDS
(1)..(1524) 4 atg acc cag acc ctc aag tac gcc tcc aga gtg ttc cat
cgc gtc cgc 48 Met Thr Gln Thr Leu Lys Tyr Ala Ser Arg Val Phe His
Arg Val Arg 1 5 10 15 tgg gcg ccc gag ttg ggc gcc tcc cta ggc tac
cga gag tac cac tca 96 Trp Ala Pro Glu Leu Gly Ala Ser Leu Gly Tyr
Arg Glu Tyr His Ser 20 25 30 gca cgc cgg agc ttg gca gac atc cca
ggc ccc tct acg ccc agc ttt 144 Ala Arg Arg Ser Leu Ala Asp Ile Pro
Gly Pro Ser Thr Pro Ser Phe 35 40 45 ctg gcc gaa ctt ttc tgc aag
ggg ggg ctg tcg agg cta cac gag ctg 192 Leu Ala Glu Leu Phe Cys Lys
Gly Gly Leu Ser Arg Leu His Glu Leu 50 55 60 cag gtg cag ggc gcc
gcg cac ttc ggg ccg gtg tgg cta gcc agc ttt 240 Gln Val Gln Gly Ala
Ala His Phe Gly Pro Val Trp Leu Ala Ser Phe 65 70 75 80 ggg aca gtg
cgc acc gtg tac gtg gct gcc cct gca ctc gtc gag gag 288 Gly Thr Val
Arg Thr Val Tyr Val Ala Ala Pro Ala Leu Val Glu Glu 85 90 95 ctg
ctg cga cag gag gga ccc cgg ccc gag cgc tgc agc ttc tcg ccc 336 Leu
Leu Arg Gln Glu Gly Pro Arg Pro Glu Arg Cys Ser Phe Ser Pro 100 105
110 tgg acg gag cac cgc cgc tgc cgc cag cgg gct tgc gga ctg ctc act
384 Trp Thr Glu His Arg Arg Cys Arg Gln Arg Ala Cys Gly Leu Leu Thr
115 120 125 gcg gaa ggc gaa gaa tgg caa agg ctc cgc agt ctc ctg gcc
ccg ctc 432 Ala Glu Gly Glu Glu Trp Gln Arg Leu Arg Ser Leu Leu Ala
Pro Leu 130 135 140 ctc ctc cgg cct caa gcg gcc gcc cgc tac gcc gga
acc ctg aac aac 480 Leu Leu Arg Pro Gln Ala Ala Ala Arg Tyr Ala Gly
Thr Leu Asn Asn 145 150 155 160 gta gtc tgc gac ctt gtg cgg cgt ctg
agg cgc cag cgg gga cgt ggc 528 Val Val Cys Asp Leu Val Arg Arg Leu
Arg Arg Gln Arg Gly Arg Gly 165 170 175 acg ggg ccg ccc gcc ctg gtt
cgg gac gtg gcg ggg gaa ttt tac aag 576 Thr Gly Pro Pro Ala Leu Val
Arg Asp Val Ala Gly Glu Phe Tyr Lys 180 185 190 ttc gga ctg gaa ggc
atc gcc gcg gtt ctg ctc ggc tcg cgc ttg ggc 624 Phe Gly Leu Glu Gly
Ile Ala Ala Val Leu Leu Gly Ser Arg Leu Gly 195 200 205 tgc ctg gag
gct caa gtg cca ccc gac acg gag acc ttc atc cgc gct 672 Cys Leu Glu
Ala Gln Val Pro Pro Asp Thr Glu Thr Phe Ile Arg Ala 210 215 220 gtg
ggc tcg gtg ttt gtg tcc acg ctg ttg acc atg gcg atg ccc cac 720 Val
Gly Ser Val Phe Val Ser Thr Leu Leu Thr Met Ala Met Pro His 225 230
235 240 tgg ctg cgc cac ctt gtg cct ggg ccc tgg ggc cgc ctc tgc cga
gac 768 Trp Leu Arg His Leu Val Pro Gly Pro Trp Gly Arg Leu Cys Arg
Asp 245 250 255 tgg gac cag atg ttt gca ttt gct cag agg cac gtg gag
cgg cga gag 816 Trp Asp Gln Met Phe Ala Phe Ala Gln Arg His Val Glu
Arg Arg Glu 260 265 270 gca gag gca gcc atg agg aac gga gga cag ccc
gag aag gac ctg gag 864 Ala Glu Ala Ala Met Arg Asn Gly Gly Gln Pro
Glu Lys Asp Leu Glu 275 280 285 tct ggg gcg cac ctg acc cac ttc ctg
ttc cgg gaa gag ttg cct gcc 912 Ser Gly Ala His Leu Thr His Phe Leu
Phe Arg Glu Glu Leu Pro Ala 290 295 300 cag tcc atc ctg gga aat gtg
aca gag ttg cta ttg gcg gga gtg gac 960 Gln Ser Ile Leu Gly Asn Val
Thr Glu Leu Leu Leu Ala Gly Val Asp 305 310 315 320 acg gtg tcc aac
acg ctc tct tgg gct ctg tat gag ctc tcc cgg cac 1008 Thr Val Ser
Asn Thr Leu Ser Trp Ala Leu Tyr Glu Leu Ser Arg His 325 330 335 ccc
gaa gtc cag aca gca ctc cac tca gag atc aca gct gcc ctg agc 1056
Pro Glu Val Gln Thr Ala Leu His Ser Glu Ile Thr Ala Ala Leu Ser 340
345 350 cct ggc tcc agt gcc tac ccc tca gcc act gtt ctg tcc cag ctg
ccc 1104 Pro Gly Ser Ser Ala Tyr Pro Ser Ala Thr Val Leu Ser Gln
Leu Pro 355 360 365 ctg ctg aag gcg gtg gtc aag gaa gtg cta aga ctg
tac cct gtg gta 1152 Leu Leu Lys Ala Val Val Lys Glu Val Leu Arg
Leu Tyr Pro Val Val 370
375 380 cct gga aat tct cgt gtc cca gac aaa gac att cat gtg ggt gac
tat 1200 Pro Gly Asn Ser Arg Val Pro Asp Lys Asp Ile His Val Gly
Asp Tyr 385 390 395 400 att atc ccc aaa aat acg ctg gtc act ctg tgt
cac tat gcc act tca 1248 Ile Ile Pro Lys Asn Thr Leu Val Thr Leu
Cys His Tyr Ala Thr Ser 405 410 415 agg gac cct gcc cag ttc cca gag
cca aat tct ttt cgt cca gct cgc 1296 Arg Asp Pro Ala Gln Phe Pro
Glu Pro Asn Ser Phe Arg Pro Ala Arg 420 425 430 tgg ctg ggg gag ggt
ccc acc ccc cac cca ttt gca tct ctt ccc ttt 1344 Trp Leu Gly Glu
Gly Pro Thr Pro His Pro Phe Ala Ser Leu Pro Phe 435 440 445 ggc ttt
ggc aag cgc agc tgt atg ggg aga cgc ctg gca gag ctt gaa 1392 Gly
Phe Gly Lys Arg Ser Cys Met Gly Arg Arg Leu Ala Glu Leu Glu 450 455
460 ttg caa atg gct ttg gcc cag atc cta aca cat ttt gag gtg cag cct
1440 Leu Gln Met Ala Leu Ala Gln Ile Leu Thr His Phe Glu Val Gln
Pro 465 470 475 480 gag cca ggt gcg gcc cca gtt aga ccc aag acc cgg
act gtc ctg gta 1488 Glu Pro Gly Ala Ala Pro Val Arg Pro Lys Thr
Arg Thr Val Leu Val 485 490 495 cct gaa agg agc atc aac cta cag ttt
ttg gac aga 1524 Pro Glu Arg Ser Ile Asn Leu Gln Phe Leu Asp Arg
500 505 5 2469 DNA Rat CDS (24)..(1526) 5 gagcagactc ctcaaacaca aac
atg acc cag gca gtc aag ctc gcc tcc aga 53 Met Thr Gln Ala Val Lys
Leu Ala Ser Arg 1 5 10 gtc ttc cat cga gtc caa ctg cct tct cag ctg
ggc agt gac tcg gtt 101 Val Phe His Arg Val Gln Leu Pro Ser Gln Leu
Gly Ser Asp Ser Val 15 20 25 ctc cgg agt tta tct gat atc cct ggg
ccc tct aca cct agc ttc ctg 149 Leu Arg Ser Leu Ser Asp Ile Pro Gly
Pro Ser Thr Pro Ser Phe Leu 30 35 40 gct gaa ctc ttc tgc aaa ggg
ggg ctg tcc agg cta cat gaa ctg cag 197 Ala Glu Leu Phe Cys Lys Gly
Gly Leu Ser Arg Leu His Glu Leu Gln 45 50 55 gtg cat ggc gct gcg
cgg tac ggg cca ata tgg tcc ggc agc ttc ggg 245 Val His Gly Ala Ala
Arg Tyr Gly Pro Ile Trp Ser Gly Ser Phe Gly 60 65 70 aca ctt cgc
aca gtt tat gtg gcc gac cct gca ctt gta gag cag ctc 293 Thr Leu Arg
Thr Val Tyr Val Ala Asp Pro Ala Leu Val Glu Gln Leu 75 80 85 90 ctg
cga caa gaa agt cat tgt cca gag cgc tgt agt ttc tca tct tgg 341 Leu
Arg Gln Glu Ser His Cys Pro Glu Arg Cys Ser Phe Ser Ser Trp 95 100
105 tca gag cac cgt cgc cgc cac cag cgg gct tgc ggg ttg cta acg gcg
389 Ser Glu His Arg Arg Arg His Gln Arg Ala Cys Gly Leu Leu Thr Ala
110 115 120 gat ggt gaa gaa tgg cag agg ctc cga agt ctc ctg gcc ccg
cta ctc 437 Asp Gly Glu Glu Trp Gln Arg Leu Arg Ser Leu Leu Ala Pro
Leu Leu 125 130 135 ctc cga cct caa gca gcc gcc ggc tat gct gga act
ctg gac agc gtg 485 Leu Arg Pro Gln Ala Ala Ala Gly Tyr Ala Gly Thr
Leu Asp Ser Val 140 145 150 gtc agt gac ctc gtg cga cga cta agg cgc
cag cgg gga cgt ggc tct 533 Val Ser Asp Leu Val Arg Arg Leu Arg Arg
Gln Arg Gly Arg Gly Ser 155 160 165 170 ggg cta ccg gac cta gtt ctg
gac gtg gcg gga gag ttt tac aaa ttt 581 Gly Leu Pro Asp Leu Val Leu
Asp Val Ala Gly Glu Phe Tyr Lys Phe 175 180 185 ggc cta gaa ggc ata
ggc gcg gtg ctg ctg gga tcg cgc ctg ggc tgc 629 Gly Leu Glu Gly Ile
Gly Ala Val Leu Leu Gly Ser Arg Leu Gly Cys 190 195 200 ctg gag gct
gaa gtt cct ccc gac aca gaa acc ttc att gag gcc gtg 677 Leu Glu Ala
Glu Val Pro Pro Asp Thr Glu Thr Phe Ile Glu Ala Val 205 210 215 ggc
tcg gtg ttt gtg tct aca ctc ttg acc atg gca atg ccc agt tgg 725 Gly
Ser Val Phe Val Ser Thr Leu Leu Thr Met Ala Met Pro Ser Trp 220 225
230 ctg cac cgc ctt ata ccc gga ccc tgg gcc cgc ctc tgc aga gac tgg
773 Leu His Arg Leu Ile Pro Gly Pro Trp Ala Arg Leu Cys Arg Asp Trp
235 240 245 250 gat cag atg ttt gcc ttt gcc cag aag cac gtg gag cag
cgc gaa ggc 821 Asp Gln Met Phe Ala Phe Ala Gln Lys His Val Glu Gln
Arg Glu Gly 255 260 265 gaa gct gcc gtg agg aac cag gga aag cct gag
gag gat ttg cca acg 869 Glu Ala Ala Val Arg Asn Gln Gly Lys Pro Glu
Glu Asp Leu Pro Thr 270 275 280 ggg cat cac tta acc cac ttc ctt ttt
cgg gaa aag gtg tct gtc cag 917 Gly His His Leu Thr His Phe Leu Phe
Arg Glu Lys Val Ser Val Gln 285 290 295 tcc ata gtg gga aat gtg aca
gag cta cta ctg gct gga gtg gac acg 965 Ser Ile Val Gly Asn Val Thr
Glu Leu Leu Leu Ala Gly Val Asp Thr 300 305 310 gta tcc aat acg ctc
tcc tgg gca ctc tat gag ctc tcc cgg cac ccg 1013 Val Ser Asn Thr
Leu Ser Trp Ala Leu Tyr Glu Leu Ser Arg His Pro 315 320 325 330 gaa
gtc cag tct gca ctc cac tct gag atc aca ggc gct gtg aac cct 1061
Glu Val Gln Ser Ala Leu His Ser Glu Ile Thr Gly Ala Val Asn Pro 335
340 345 ggc tcc tat gcc cac ctc caa gcc act gct ctg tcc cag cta ccc
ctg 1109 Gly Ser Tyr Ala His Leu Gln Ala Thr Ala Leu Ser Gln Leu
Pro Leu 350 355 360 cta aag gct gtg atc aaa gaa gtg ttg aga ttg tac
cct gtg gta cct 1157 Leu Lys Ala Val Ile Lys Glu Val Leu Arg Leu
Tyr Pro Val Val Pro 365 370 375 ggg aac tcc cgt gtc cca gac aga gac
atc tgt gta gga aac tat gtt 1205 Gly Asn Ser Arg Val Pro Asp Arg
Asp Ile Cys Val Gly Asn Tyr Val 380 385 390 att ccc caa gat aca ctg
gtt tcc ctc tgt cac tat gcc act tca agg 1253 Ile Pro Gln Asp Thr
Leu Val Ser Leu Cys His Tyr Ala Thr Ser Arg 395 400 405 410 gac ccc
gcc cag ttt cgg gaa ccc aac tct ttt aat cca gct cga tgg 1301 Asp
Pro Ala Gln Phe Arg Glu Pro Asn Ser Phe Asn Pro Ala Arg Trp 415 420
425 ctt gga gag ggt cca gcc ccc cac cca ttt gca tct ctt cct ttt ggc
1349 Leu Gly Glu Gly Pro Ala Pro His Pro Phe Ala Ser Leu Pro Phe
Gly 430 435 440 ttt ggc aaa cga agt tgc ata ggg aga cgc ttg gca gag
ctc gag cta 1397 Phe Gly Lys Arg Ser Cys Ile Gly Arg Arg Leu Ala
Glu Leu Glu Leu 445 450 455 caa atg gcg ttg gcc cag atc ttg acc cat
ttt gag gtg ctg cct gag 1445 Gln Met Ala Leu Ala Gln Ile Leu Thr
His Phe Glu Val Leu Pro Glu 460 465 470 cca ggt gct ctt cca gtc aaa
ccc atg acc cgg act gtc ctg gta cct 1493 Pro Gly Ala Leu Pro Val
Lys Pro Met Thr Arg Thr Val Leu Val Pro 475 480 485 490 gag agg agc
atc cat ctc cag ttt gta gac aga tagtcctgtg gaaggcagct 1546 Glu Arg
Ser Ile His Leu Gln Phe Val Asp Arg 495 500 gtcatcatct ctctccagac
tggatttttc ttactatgca caagaggcac actctccctc 1606 gaggcctgtc
tgtctgagca aacttcagga agcaggcccg ggcctatctg tgcttgacct 1666
gactcagcag gtaccacaga accaggatcc tttctcctgc tcagtacctc tcctgatcat
1726 tcctcaagat ccaaagcctt cagattttaa cacatcctta aagggccaac
tcgggggtta 1786 actaacagcc ccaggcagcc tgggcaggga tcccccactg
atccttccat gcttacagtg 1846 ttcactgaca gctgtctaag catccattgc
agcacaaact aagtgactgt gcacctggtc 1906 tgcacctggt ctgcacctgg
ttgcgtctct gcctgaccat gtgagctctt tgagaagagt 1966 gatgactact
gggcttttag ctcttttcct ttttgggaca cagtcttgct attgtactcc 2026
atgctgtcct tgaacccaca agccctcacc tcaccttccc aagtgttggg ttacggacat
2086 tagctatggc ttccagcttt attagtcttt ctatctcctg ccatggtcta
tccccggcta 2146 tttgatacta tatattctca gattgaatct ggaccatgtg
gtagaaggga tgaccactca 2206 ccaggctcta cccaccactt tatcttaatc
ttttctctag gaaagtgaat ctctccttgc 2266 cttacagcat tttaaagctc
cccttggctg ttctgctctt tagccactct aaagtggatc 2326 cactctactt
ctcaccaccc atctttctgc accccagcct gtctttttat atttaaaaaa 2386
ttgtatttat tatgttttca aataaaatgt ttactccttg aaaaaaaaaa aaaaaaaaaa
2446 aaaaaaaaaa aaaaaaaaaa aaa 2469 6 2469 DNA human CDS
(122)..(1645) 6 aggagggatt ggctgaggag cttggagagg gggcgtcatc
acctcaccca aaggttaaat 60 aggggttgag atatgatgct caggagaagc
gctttctttc gcgagcaccc tgaaccagac 120 c atg acc cag acc ctc aag tac
gcc tcc aga gtg ttc cat cgc gtc cgc 169 Met Thr Gln Thr Leu Lys Tyr
Ala Ser Arg Val Phe His Arg Val Arg 1 5 10 15 tgg gcg ccc gag ttg
ggc gcc tcc cta ggc tac cga gag tac cac tca 217 Trp Ala Pro Glu Leu
Gly Ala Ser Leu Gly Tyr Arg Glu Tyr His Ser 20 25 30 gca cgc cgg
agc ttg gca gac atc cca ggc ccc tct acg ccc agc ttt 265 Ala Arg Arg
Ser Leu Ala Asp Ile Pro Gly Pro Ser Thr Pro Ser Phe 35 40 45 ctg
gcc gaa ctt ttc tgc aag ggg ggg ctg tcg agg cta cac gag ctg 313 Leu
Ala Glu Leu Phe Cys Lys Gly Gly Leu Ser Arg Leu His Glu Leu 50 55
60 cag gtg cag ggc gcc gcg cac ttc ggg ccg gtg tgg cta gcc agc ttt
361 Gln Val Gln Gly Ala Ala His Phe Gly Pro Val Trp Leu Ala Ser Phe
65 70 75 80 ggg aca gtg cgc acc gtg tac gtg gct gcc cct gca ctc gtc
gag gag 409 Gly Thr Val Arg Thr Val Tyr Val Ala Ala Pro Ala Leu Val
Glu Glu 85 90 95 ctg ctg cga cag gag gga ccc cgg ccc gag cgc tgc
agc ttc tcg ccc 457 Leu Leu Arg Gln Glu Gly Pro Arg Pro Glu Arg Cys
Ser Phe Ser Pro 100 105 110 tgg acg gag cac cgc cgc tgc cgc cag cgg
gct tgc gga ctg ctc act 505 Trp Thr Glu His Arg Arg Cys Arg Gln Arg
Ala Cys Gly Leu Leu Thr 115 120 125 gcg gaa ggc gaa gaa tgg caa agg
ctc cgc agt ctc ctg gcc ccg ctc 553 Ala Glu Gly Glu Glu Trp Gln Arg
Leu Arg Ser Leu Leu Ala Pro Leu 130 135 140 ctc ctc cgg cct caa gcg
gcc gcc cgc tac gcc gga acc ctg aac aac 601 Leu Leu Arg Pro Gln Ala
Ala Ala Arg Tyr Ala Gly Thr Leu Asn Asn 145 150 155 160 gta gtc tgc
gac ctt gtg cgg cgt ctg agg cgc cag cgg gga cgt ggc 649 Val Val Cys
Asp Leu Val Arg Arg Leu Arg Arg Gln Arg Gly Arg Gly 165 170 175 acg
ggg ccg ccc gcc ctg gtt cgg gac gtg gcg ggg gaa ttt tac aag 697 Thr
Gly Pro Pro Ala Leu Val Arg Asp Val Ala Gly Glu Phe Tyr Lys 180 185
190 ttc gga ctg gaa ggc atc gcc gcg gtt ctg ctc ggc tcg cgc ttg ggc
745 Phe Gly Leu Glu Gly Ile Ala Ala Val Leu Leu Gly Ser Arg Leu Gly
195 200 205 tgc ctg gag gct caa gtg cca ccc gac acg gag acc ttc atc
cgc gct 793 Cys Leu Glu Ala Gln Val Pro Pro Asp Thr Glu Thr Phe Ile
Arg Ala 210 215 220 gtg ggc tcg gtg ttt gtg tcc acg ctg ttg acc atg
gcg atg ccc cac 841 Val Gly Ser Val Phe Val Ser Thr Leu Leu Thr Met
Ala Met Pro His 225 230 235 240 tgg ctg cgc cac ctt gtg cct ggg ccc
tgg ggc cgc ctc tgc cga gac 889 Trp Leu Arg His Leu Val Pro Gly Pro
Trp Gly Arg Leu Cys Arg Asp 245 250 255 tgg gac cag atg ttt gca ttt
gct cag agg cac gtg gag cgg cga gag 937 Trp Asp Gln Met Phe Ala Phe
Ala Gln Arg His Val Glu Arg Arg Glu 260 265 270 gca gag gca gcc atg
agg aac gga gga cag ccc gag aag gac ctg gag 985 Ala Glu Ala Ala Met
Arg Asn Gly Gly Gln Pro Glu Lys Asp Leu Glu 275 280 285 tct ggg gcg
cac ctg acc cac ttc ctg ttc cgg gaa gag ttg cct gcc 1033 Ser Gly
Ala His Leu Thr His Phe Leu Phe Arg Glu Glu Leu Pro Ala 290 295 300
cag tcc atc ctg gga aat gtg aca gag ttg cta ttg gcg gga gtg gac
1081 Gln Ser Ile Leu Gly Asn Val Thr Glu Leu Leu Leu Ala Gly Val
Asp 305 310 315 320 acg gtg tcc aac acg ctc tct tgg gct ctg tat gag
ctc tcc cgg cac 1129 Thr Val Ser Asn Thr Leu Ser Trp Ala Leu Tyr
Glu Leu Ser Arg His 325 330 335 ccc gaa gtc cag aca gca ctc cac tca
gag atc aca gct gcc ctg agc 1177 Pro Glu Val Gln Thr Ala Leu His
Ser Glu Ile Thr Ala Ala Leu Ser 340 345 350 cct ggc tcc agt gcc tac
ccc tca gcc act gtt ctg tcc cag ctg ccc 1225 Pro Gly Ser Ser Ala
Tyr Pro Ser Ala Thr Val Leu Ser Gln Leu Pro 355 360 365 ctg ctg aag
gcg gtg gtc aag gaa gtg cta aga ctg tac cct gtg gta 1273 Leu Leu
Lys Ala Val Val Lys Glu Val Leu Arg Leu Tyr Pro Val Val 370 375 380
cct gga aat tct cgt gtc cca gac aaa gac att cat gtg ggt gac tat
1321 Pro Gly Asn Ser Arg Val Pro Asp Lys Asp Ile His Val Gly Asp
Tyr 385 390 395 400 att atc ccc aaa aat acg ctg gtc act ctg tgt cac
tat gcc act tca 1369 Ile Ile Pro Lys Asn Thr Leu Val Thr Leu Cys
His Tyr Ala Thr Ser 405 410 415 agg gac cct gcc cag ttc cca gag cca
aat tct ttt cgt cca gct cgc 1417 Arg Asp Pro Ala Gln Phe Pro Glu
Pro Asn Ser Phe Arg Pro Ala Arg 420 425 430 tgg ctg ggg gag ggt ccc
acc ccc cac cca ttt gca tct ctt ccc ttt 1465 Trp Leu Gly Glu Gly
Pro Thr Pro His Pro Phe Ala Ser Leu Pro Phe 435 440 445 ggc ttt ggc
aag cgc agc tgt atg ggg aga cgc ctg gca gag ctt gaa 1513 Gly Phe
Gly Lys Arg Ser Cys Met Gly Arg Arg Leu Ala Glu Leu Glu 450 455 460
ttg caa atg gct ttg gcc cag atc cta aca cat ttt gag gtg cag cct
1561 Leu Gln Met Ala Leu Ala Gln Ile Leu Thr His Phe Glu Val Gln
Pro 465 470 475 480 gag cca ggt gcg gcc cca gtt aga ccc aag acc cgg
act gtc ctg gta 1609 Glu Pro Gly Ala Ala Pro Val Arg Pro Lys Thr
Arg Thr Val Leu Val 485 490 495 cct gaa agg agc atc aac cta cag ttt
ttg gac aga tagtcccatg 1655 Pro Glu Arg Ser Ile Asn Leu Gln Phe Leu
Asp Arg 500 505 gaaagagact gtcatcatca ccctttcatt catcataggg
ataagatttt ttgtaggcac 1715 aagaccaagg tatacatctt cccctaatgc
ctatctgacc aaactggata gaaccaccat 1775 agtgaagtgt gaggcggccc
tgaccaatgt gtgaagtatg cacttggcct gactcaggaa 1835 gccaggtgag
aaaaccatgg tctctctgct tgcttggccc ttctgatcat gtatgcatcc 1895
cccaaggatg aaatcagatt ttaactaata atgctggatg gcctgaggaa agattcaact
1955 gcctctcttt ttgggctttc atagtgttca ttgatgctgc tggctaagca
tttatcaaag 2015 cataagctca gtaactgtgc atctggtctg tacctggttg
gtccttcgtc tttgcatgta 2075 agctctttga gaggaagggt gaagccttat
ttgtttttta tgtcccctgc cagggcctgt 2135 ctctgactag gtgtcaccat
acacattctt agattgaatc tgaaccatgt ggcagaaggg 2195 ataagcagct
tacttagtag gctctgtcta cccccttcct tctttgtctt gcccctagga 2255
aggtgaatct gccctagcct ggtttacggt ttcttataac tctcctttgc tctctggcca
2315 ctattaagtg ggtttgcccc atcacttagt tctcaggcag agacatcttt
gggcctgtcc 2375 ctgcccaggc ctctggcttt ttatattgaa aatttttaaa
tattcacaaa ttttagaata 2435 aatcaaatat tccattaaaa aaaaaaaaaa aaaa
2469 7 23 DNA synthetic DNA 7 ctsctsaarg chgtsatyaa rga 23 8 22 DNA
synthetic DNA 8 ckcttbccra abccraargg va 22 9 25 DNA synthetic DNA
9 aaggcagtga ttaaggaagt gttga 25
* * * * *