U.S. patent application number 10/727619 was filed with the patent office on 2004-12-23 for gene prv-1 and its use.
Invention is credited to Pahl, Heike.
Application Number | 20040259110 10/727619 |
Document ID | / |
Family ID | 7885492 |
Filed Date | 2004-12-23 |
United States Patent
Application |
20040259110 |
Kind Code |
A1 |
Pahl, Heike |
December 23, 2004 |
Gene PRV-1 and its use
Abstract
This document describes a nucleotide sequence which encodes the
PRV-1 protein, and essentially comprises the sequence ID No. 1, and
also a process for detecting this gene and the polypeptide encoded
by this gene.
Inventors: |
Pahl, Heike; (Freiburg,
DE) |
Correspondence
Address: |
MILLEN, WHITE, ZELANO & BRANIGAN, P.C.
2200 CLARENDON BLVD.
SUITE 1400
ARLINGTON
VA
22201
US
|
Family ID: |
7885492 |
Appl. No.: |
10/727619 |
Filed: |
December 5, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10727619 |
Dec 5, 2003 |
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09830189 |
Aug 6, 2001 |
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6686153 |
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09830189 |
Aug 6, 2001 |
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PCT/EP99/07238 |
Sep 30, 1999 |
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Current U.S.
Class: |
435/6.17 ;
435/320.1; 435/325; 435/69.1; 530/395; 536/23.5 |
Current CPC
Class: |
A61P 29/00 20180101;
A61P 9/12 20180101; A61P 35/02 20180101; A61P 25/04 20180101; A61K
38/00 20130101; A61P 9/00 20180101; A61P 7/00 20180101; A61P 7/06
20180101; A61P 19/06 20180101; A61P 17/04 20180101; A61P 7/02
20180101; A61P 35/00 20180101; A61P 43/00 20180101; C07K 14/475
20130101; A61P 31/04 20180101; A61P 13/12 20180101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/320.1; 435/325; 530/395; 536/023.5 |
International
Class: |
C12Q 001/68; C07H
021/04; C07K 014/47 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 23, 1998 |
DE |
198 49 044.5 |
Claims
1. An isolated polypeptide, comprising one of the following amino
acid sequences: amino acids 1-437 of SEQ. ID NO.: 2; amino acids
1-409 of SEQ. ID NO.: 2; amino acids 22-437 of SEQ. ID NO.: 2;
amino acids 22-409 of SEQ. ID NO.: 2; or a fragment thereof
containing at least 50 amino acids.
2. (Cancelled).
3. (Cancelled).
4. An isolated polynucleotide, comprising one of the following
nucleotide sequences: nucleotides 1-1600 of SEQ. ID NO.: 1;
nucleotides 36-1346 of SEQ. ID NO.: 1; nucleotides 36-1262 of SEQ.
ID NO.: 1; nucleotides 39-1346 of SEQ. ID NO.: 1; nucleotides
39-1262 of SEQ. ID NO.: 1; nucleotides 99-1346 of SEQ. ID NO.: 1;
or nucleotides 99-1262 of SEQ. ID NO.: 1.
5. (Cancelled).
6. (Cancelled).
7. (Cancelled).
8. (Cancelled).
9. Antibody against a polypeptide of claim 1, or an epitome
thereof.
10. Antibody according to claim 9, characterized in that it is a
monoclonal antibody.
11. Process for detecting polycythaemia vera, characterized in that
the polypeptide according to claim 1 is reacted, in an immunoassay,
with one or more antibody(ies) being monoclonal.
12. Process according to claim 11, characterized in that the
antibody employed is a polyclonal or monoclonal antibody.
13. Process for detecting polycythaemia vera, characterized in that
the PRV-1 polynucleotide is detected using an RT-PCR method or a
blotting method.
14. Drug for treating polycythaemia vera, characterized in that, in
addition to customary excipients, it comprises polyclonal or
monoclonal antibodies according to claim 10.
15. Drug, comprising a polypeptide according to claim 1 and at
least one pharmaceutically tolerated excipient.
16. Drug, comprising a polynucleotide according to claim 4 and at
least one pharmaceutically tolerated excipient.
17. Use of a polypeptide according to claim 1 as a growth
factor.
18. Use of polypeptide according to claim 1 for producing a drug
for treating pancytopenias and pancytopathies in the bone marrow
and in the circulation.
19. Use of a polynucelotide according to claim 4 for producing a
drug for treating pancytopenias and pancytopathies in the bone
marrow and in the circulation.
20. Use of a polypeptide according to claim 1 for treating and/or
multiplying endogenous cells and/or established cell lines ex vivo
or in vitro.
21. Kit for detecting polycythaemia vera, comprising at least one
polynucleotide according to claim 4, or a fragment thereof.
22. Kit for detecting disturbances of the haematopoietic system,
comprising at least one polynucleotide according to claim 4, or a
fragment thereof.
23. Kit for detecting the PRV-1 protein according to claim 21,
characterized in that it is an ELISA test kit.
24. Kit for detecting polycythaemia vera, comprising at least one
polypeptide according to claim 1.
25. Kit for detecting polycythaemia vera, comprising at least one
antibody according to claim 9.
26. Kit for detecting polycythaemia vera, comprising at least one
antibody according to claim 10.
27. Kit for detecting disturbances of the haematopoietic system,
comprising at least one polypeptide according to claim 1.
28. Kit for detecting disturbances of the haematopoietic system,
comprising at least one antibody according to claim 9.
29. Kit for detecting disturbances of the haematopoietic system,
comprising at least one antibody according to claim 10.
Description
[0001] The invention relates to a nucleotide sequence which encodes
the PRV-1 gene, to recombinant DNA which contains this nucleotide
sequence, to vectors which contain the recombinant DNA and to cells
which are transformed with these vectors, and also to a PRV-1
polypeptide, to antibodies against this polypeptide, to a process
for detecting the PRV-1 polypeptide and to drugs which comprise the
PRV-1 polypeptide or antibodies which are directed against the
PRV-1 polypeptide.
[0002] Polycythaemia rubra vera (erythraemia), also termed
polycythaemia vera or p. vera, is a malignant haematological
disease in which there is an increased formation of erythroid,
granulocytic and megakaryocytic cells. The disease is of clonal
origin and arises as a result of the mutation of a single
haematopoietic precursor cell. In Germany, the incidence of p. vera
is from 4 to 6 per million inhabitants. If left untreated, the
disease leads to death within 18 months. Treatment by means of
blood-letting or chemotherapy extends the average survival time to
more than 13 years.
[0003] P. vera is diagnosed by means of clinical criteria. The
clinical picture includes headaches, pruritus, splenomegaly in two
thirds of the patients, bleeding or thromboses, hypertension in a
third of the patients, gout, which is brought about by an increase
in the production of uric acid, and, in some cases, septic ulcers.
The most important laboratory finding is an increase in the values
for haemoglobin, haematocrit, erythrocyte count and total
erythrocyte volume, and also a neutrophilic granulocytosis or
thrombocytosis in many cases. Since, on the one hand, most of the
criteria are rather diffuse and, on the other hand, not all the
patients fulfil these criteria, it is frequently difficult to
distinguish p. vera from other myeloproliferative diseases, such as
chronic granulocytic leukaemia or essential thrombocytosis, and
thereby confirm the diagnosis. To date, the molecular cause of p.
vera is completely unknown. Since, however, p. vera takes a severe
course if it is not treated, accurate diagnosis is important.
[0004] An object of the invention was therefore to find the
molecular cause of polycythaemia rubra vera and to create the
possibility of diagnosing it.
[0005] This object was achieved by isolating a gene which is
expressed specifically in association with p. vera and not in
healthy control individuals. This gene is designated the PRV-1 gene
(polycythaemia rubra Vera).
[0006] A similar nucleotide sequence is disclosed in International
application WO 98/50552.
[0007] One part of the subject-matter of the invention therefore
relates to a polynucleotide which encodes the PRV-1 gene and
essentially comprises the sequence ID No. 1. The polynucleotides of
the present invention can be single-stranded or double-stranded DNA
or RNA. If they are RNA, it is then clear to the skilled person
that "U" nucleotides are present in place of "T" nucleotides.
"Polynucleotide" is understood as meaning nucleic acids which
contain 15 or more nucleotides.
[0008] The nucleotide sequence according to the invention is
depicted in FIG. 1. The invention therefore relates to a
polynucleotide which corresponds to the sequence shown in FIG. 1
and also to a polynucleotide whose nucleotide sequence exhibits
minor differences. Within the meaning of the present application,
minor differences are understood as meaning those sequences in
which a few, preferably not more than 50 and particularly
preferably not more than 25, nucleotides can be exchanged, with,
however, the function of the gene encoded by the nucleotide
sequence being unaffected. The skilled person is familiar with the
fact that a base triplet encoding an amino acid can be replaced
with another triplet which encodes the same amino acid. In addition
to this, regions which are of less importance can be deleted and/or
mutated to a minor extent. In a particular embodiment, the
polynucleotide comprises nucleotides 36 to 1346 of sequence No. 1,
that is the coding region of the PRV-1 gene. Another embodiment
comprises nucleotides 36 to 1262 of sequence No. 1. This region
presumably encodes the active region of the PRV-1 polypeptide.
Finally, the polynucleotide of the invention can also comprise
nucleotides 39 to 1346 or 39 to 1262 of sequence No. 1, such that
the codon which encodes the starting methionine is not present. A
preferred embodiment is a polynucleotide which comprises
nucleotides 99-1346or 99 to 1262 of sequence No. 1. This results in
the codons at the 5' end which encode the signal peptide of the
PRV-1 polypeptide not being present.
[0009] The polynucleotide according to the invention can also be a
fragment of the PRV-1 gene. As a rule, the fragment possesses more
than 100 nucleotides, preferably, however, more than 300
nucleotides. The fragments can also be used as primers or as
probes, in particular for PCR; in this case, the fragments can be
truncated to fit the purpose. Usually, primers have a length of
between 10 and 30 nucleotides and probes have a length of between
15 and 50 nucleotides.
[0010] The PRV-1 gene is an endogenous gene whose expression in
healthy individuals is, however, restricted to only a few organs.
Normally, it is expressed in the main in the haematopoietic organs,
i.e. in bone marrow and foetal liver; and weakly expressed in the
spleen, but not expressed in heart, muscle, pancreas or kidney. In
patients who are suffering from p. vera, this gene is very strongly
overexpressed in the haematopoietic cells, in particular.
[0011] The PRV-1 gene encodes a protein which exhibits the protein
sequence shown in FIG. 2. The signal peptide, which is present in
the protein sequence of all surface molecules and normally removed
when the protein is processed, is divided off by a hyphen. The
protein has the sequence ID No. 2. Another aspect of the invention
is consequently an essentially pure polypeptide having the sequence
No. 2 or a polypeptide having the sequence No. 2 but lacking the
signal peptide (i.e. amino acids 22 to 437 of sequence No. 2).
Other embodiments encompass amino acids 1 to 409 or 22 to 409 of
sequence No. 2 (what is probably the active region of the
protein).
[0012] With regard to biological activity, the polypeptide
according to the invention is preferably glycosylated; it is most
preferably N-glycosylated. It can then be glycosylated at at least
one of the amino acids Asn-46, Asn-189 and Asn-382 of the PRV-1
polypeptide (the amino acid numbers refer to the sequence No. 2).
The invention also encompasses fragments of the polypeptides
according to the invention which are N-glycosylated. The fragments
are at least 50 amino acids in length, preferably at least 100
amino acids and most preferably at least 150 amino acids. In
another embodiment, a polypeptide can be O-glycosylated.
[0013] It is clear to the skilled person that particular amino
acids can be replaced with other amino acids without impairing the
biological activity of the protein. Such modified forms of the
polypeptides according to the invention are also part of the
subject-matter of the invention. The amino acid replacements are
those which do not have a negative effect on the biological
activity of the protein. The skilled person can make use of well
known rules for selecting the replacements.
[0014] Depending on the method of preparation, the PRV-1
polypeptide can, for example, possess a glycosyl
phosphatidylinositol anchor. This is then bonded to the amino acids
which correspond to amino acids 407 to 409 in sequence ID No. 2. A
GPI anchor is used to anchor a protein by means of a lipid on the
outside of the cell membrane. However, for reasons which have not
so far been conclusively elucidated, it is frequently observed that
GPI-linked proteins are also released into the medium. This is
referred to as "shedding". To date, it has not been clarified
whether this is a specific process, i.e. such proteins are cleaved
from the membrane by enzymes in a controlled manner, or whether it
represents a non-specific loss of the anchor. It is consequently
very probable that PRV-1 is to be found both on the cell membrane
and extracellularly. The secreted form, which is not
membrane-bound, is probably more important for the effect of the
polypeptide as a growth factor since, as a growth factor, this form
is able to diffuse and reach other cells.
[0015] It is clear to the skilled person that he can influence the
attachment of the protein to the cell membrane by manipulating
these amino acids. This particularly concerns the preparation of
defined DNA constructs which are intended for expressing the PRV-1
polypeptide or fragments of this polypeptide. The codons which
encode these amino acids can be mutated or deleted.
[0016] The gene encodes a surface receptor of the uPAR/Ly6 family.
This receptor family can transduce mitogenic signals, i.e. signals
which stimulate cell division. It is therefore assumed that
overexpression of the PRV-1 gene, inter alia on the granulocytes of
p. vera patients, contributes to hyperproliferation of these
cells.
[0017] It has been found that PRV-1 is not expressed on
granulocytes in healthy individuals or in patients suffering from
other myeloproliferative diseases, e.g. suffering from chronic
granulocytic leukaemia, acute granulocytic leukaemia or essential
thrombocytosis.
[0018] In order to be able to use the polypeptide encoded by the
PRV-1 gene for analyses and detection methods, it is expediently
generated from recombinant DNA, with the recombinant DNA preferably
comprising the nucleotide sequence ID No. 1 or at least the coding
region of the PRV-1 gene, that is nucleotides 36 to 1346 of
sequence ID No. 1, at least, however, nucleotides 39 to 1262,
functionally linked to a promoter. However, the recombinant DNA can
also comprise only a fragment of sequence No. 1.
[0019] The invention furthermore relates to a vector which contains
the recombinant DNA for the PRV-1 polypeptide, or a fragment
thereof, and to a host cell which is transfected or transformed
with this vector. The host cells may be prokaryotic, for example
bacteria such as E. coli. However, the polypeptides which are
expressed are then not glycosylated. Preference is therefore given
to eukaryotic host cells, which are able to glycosylate the
expressed protein post-transitionally and modify it in other ways.
Examples of eukaryotic host cells are insect cells, such as Sf9
cells, for expression following infection with recombinant
baculoviruses, and mammalian cells, such as COS cells, CHO cells
and HeLa cells. These examples are not exhaustive. It is also
possible to use yeast cells as host cells. It is clear to the
skilled person that the glycosylation pattern can differ depending
on the host cell. The biological activity of the expression product
can therefore also vary. Particular preference is given to host
cells which glycosylate the expression product in such a way that
the biological activity of the protein is retained.
[0020] The PRV-1 polypeptide which is isolated from granulocytes or
produced recombinantly can be employed both for diagnosing
polycythaemia vera and for treating the disease.
[0021] One therapeutic possibility is that of "antisense therapy".
This method employs an "antisense" RNA molecule, that is an RNA
which is complementary to the PRV RNA. Since the PRV-1 RNA has the
sequence 5'-AAAAGCAGAAAGAGATTACCAGCC-3' (seq. ID No. 3) at its
beginning, the requisite antisense RNA directed against this
sequence would possess the following nucleotide sequence:
5'-GGCTGGTAATCTCTTTCTGCTTTT-3' (seq. ID No. 4). This antisense RNA
is incorporated into a vector and introduced into the p. vera
cells. This RNA is introduced, for example, by means of
transfection, with the vector used for the transfection preferably
being configured such that it is introduced specifically into the
p. vera cells. Expression of the antisense RNA results in it no
longer being possible for the PRV-1 mRNA to be translated into a
polypeptide. Cells which have been treated in this way do not then
form any PRV-1 protein.
[0022] The invention therefore also relates to a process for
detecting p. vera which is characterized in that the PRV-1
polypeptide, or an epitope thereof, is detected and the extent of
the expression is determined.
[0023] Overexpression of this receptor on mature cells outside of
the bone marrow, e.g. on granulocytes, is a strong indication of
the presence of the disease p. vera. This overexpression is
expediently detected by means of an immunoassay using antibodies
which are directed against the PRV-1 receptor. Suitable test
methods are the known immunoassay variants which make use of PRV-1
polypeptide-specific antibodies together with other labelled
antibodies which can be immobilized or in solution. The labelling
can be effected in a manner known per se, for example using
radioactive isotopes, by means of fluorescence or luminescence,
using enzymes, by means of colour-forming reactions or using other
groups which are suitable for the determination. These variants are
known to the skilled person and do not require any more detailed
explanation here. According to the invention, ELISA tests are
particularly preferred.
[0024] The antibodies which are required for specifically detecting
the PRV-1 receptor can likewise be prepared in a manner which is
known per se. Both monoclonal and polyclonal antibodies are
suitable, with preference being given to using monoclonal
antibodies.
[0025] Peptides which are derived from the protein can also be used
for preparing antibodies. Within the context of the present
invention, success was achieved using the peptides having the
sequences:
1 a) KVSDLPRQWTPKN (amino acids [seq. ID No.5] 34 to 46), and b)
SAREKRDVQPPASQH (amino acids [seq. ID No.6] 391 to 405).
[0026] The polyclonal antibodies are normally produced by
immunizing a suitable host (rabbit) with the PRV-1 polypeptide,
where appropriate bound to an immunological support (adjuvant), and
eliciting an immune response. Monoclonal antibodies can be
generated in a manner known per se using the hybridoma technique.
The antibodies can be purified by means of affinity purification.
The preparation and purification of antibodies are described, for
example, in "Antibodies: A Laboratory Manual" by Harlow and Lane,
Cold Spring Harbor Laboratory Press.
[0027] Furthermore, such polyclonal or monoclonal antibodies which
are directed against PRV-1 can also be used for treating the
disease.
[0028] In another embodiment, the PRV-1 receptor can be detected
using an RT-PCR method. For this, RNA is first of all isolated from
the PRV-1-overexpressing cells, which are as a rule granulocytes. A
reverse transcription is then performed in a manner known per se
using an RT primer. The RT primer is preferably a primer which has
the following nucleotide sequence. (SEQ ID No. 7)
2 ATTAGGTTATGAGGTCAGAGGGAGGTT.
[0029] In this way, the specific PRV-1 RNA is transformed into DNA.
This DNA is then amplified in a PCR reaction in a manner known per
se. The following two primers are preferably employed for the
amplification cycles:
3 As the sense primer GCAGAAAGAGATTACCAGCCACAGACGG. (SEQ ID No.8)
As the antisense primer GAATCGTGGGGGTAATAGAGTTAGCAGG. (SEQ ID
No.9)
[0030] The skilled person is readily able to use the disclosed
sequence to find other primers which are also suitable.
[0031] Since the RNA is used as the starting material for this
method, the PCR signal is only positive when the PRV-1 gene is also
expressed. As explained above, this is only the case when the
patient is suffering from p. vera. PRV is not expressed in
granulocytes of healthy patients. Consequently, the absence of any
RT-PCR signal indicates that no p. vera is present.
[0032] In another alternative, it is also possible to use a
blotting method, preferably a Northern Blot, for diagnosing p.
vera. For such a method, the RNA is isolated from granulocytes and
then examined for the expression of PRV-1 using a blotting method,
for example Northern blotting. The cDNA sequence of SEQ ID No. 1,
or a segment of the sequence, can be used as the probe.
Hybridization then only occurs if the granulocytes are derived from
a patient suffering from p. vera since only then is there any
expression on the granulocytes. The absence of hybridization
indicates that the individual from whom the granulocytes are
derived does not have p. vera.
[0033] It is also possible to use a fragment of the gene for the
Northern blot hybridization. Such a fragment is normally more than
100 bases in length, preferably more than 300 bases in length.
Alternatively, various different fragments of the gene, which can
be used as probes in the Northern blot, can be prepared by
digesting the gene with restriction endonucleases. If the fragments
are derived from the cDNA, they are then present as double strands
which have to be separated into the single strands for the
hybridization. Suitable examples are the Bam HI-PstI fragment from
base pair 420 to base pair 831, or the PstI-PstI fragment from base
pair 831 to base pair 1900.
[0034] PRV-1 mRNA, and consequently the expression of PRV-1, can
also be detected by first of all reverse-transcribing the mRNA in
an RT-PCR reaction and then amplifying the cDNA; the amplified DNA
is then detected with a probe in a hybridization method.
[0035] In the case of a positive diagnosis, the disease has to be
treated since it otherwise leads to death within a relatively short
period of time. For this treatment, it is possible to use specific
antibodies which are directed against PRV-1 and to which cytotoxic
components can be bonded, where appropriate.
[0036] The invention therefore furthermore relates to a drug which,
in addition to the customary excipients, comprises antibodies which
are directed against the PRV-1 receptor.
[0037] Since the PRV-1 receptor is overexpressed in p. vera, many
antibodies are bound on the surface of the affected granulocytes
when they come into contact with the anti-PRV-1 antibody. The
binding of many antibodies to these cells stimulates the
immunological cells to destroy these granulocytes. In this way, it
is possible to eliminate the p. vera cells specifically.
[0038] Surprisingly, it has also been found that the PRV-1
polypeptide exhibits haematopoietic activity. The PRV-1 polypeptide
is able to stimulate certain haematopoietic precursor cells to form
erythroid colonies. It is particularly the N-glycosylated PRV-1
polypeptides which display this function. The polypeptides
according to the invention which are preferred are therefore the
N-glycosylated PRV-1 polypeptides, and fragments thereof, which
display the growth factor activity.
[0039] Another aspect of the invention is therefore a drug which,
in addition to a pharmaceutically tolerated excipient, comprises
the PRV-1 polypeptide or a biologically active fragment thereof.
The PRV-1 polypeptide is preferably glycosylated PRV-1 polypeptide
and, even more preferably, N-glycosylated PRV-1 polypeptide or a
biologically active fragment thereof. The invention also relates to
drugs which comprise at least one polynucleotide according to the
invention.
[0040] The present invention furthermore relates to the use of
PRV-1 polypeptide, or a biologically active fragment thereof, as a
growth factor in vivo and ex vivo. The PRV-1 polypeptide, or a
biologically active fragment thereof, can be used for treating all
pancytopenias and pancytopathies in the bone marrow and in the
circulation (change in the cellular constituents of the peripheral
blood and bone marrow). The polypeptides of the present invention
can, for example, be used for treating anaemias in the case of
kidney failure, chemotherapy or whole body radiation, for treating
neutropenias and thrombocytopenias during chemotherapy or whole
body radiation, for the ex-vivo treatment of peripheral or bone
marrow stem cells for expansion (multiplication) and retransfusion
into the patients, and for treating sepsis, systemic inflammatory
response syndrome (SIRS) or regional inflammatory reactions. The
polypeptides of the present invention, or drugs which comprise
them, can be administered in a wide variety of ways. The forms of
administration comprise intravenous, intramuscular, subcutaneous,
intraperitoneal, oral, transdermal and transmucosal
administration.
[0041] The polynucleotides according to the invention can also be
used for treating pancytopenias and pancytopathies. In this case,
the aim is to express a PRV-1 polypeptide, or a functional fragment
thereof, in cells of the affected patient. Gene therapy methods are
first and foremost used in this connection. Cells can be isolated
from the patient and transfected with a polynucleotide according to
the invention (ex-vivo manipulation), after which they are then
returned to the patient. It is also possible to conceive of methods
in which the polynucleotides according to the invention gain access
into the target cells by means of viral transfer. Expression of the
inserted nucleic acids then leads to haematopoietic activity.
[0042] The invention also relates to kits for detecting either
polycythaemia vera or disturbances of the haematopoietic system.
These kits comprise a polynucleotide according to the invention
and/or a polypeptide according to the invention and/or one or more
antibodies according to the invention. In addition to this, the kit
can also comprise a container or compositions which are suitable
for implementing detection reactions. Examples of such compositions
are buffer solutions, reagents for blocking membranes,
hybridization solutions, secondary antibodies, substrate solutions
for detection reactions, etc. The kit is preferably used for
implementing PCR reactions, Northern blots, Southern blots, Western
blots and ELISA, RIA or similar reactions.
[0043] The following examples are given in explanation.
EXAMPLE 1
[0044] Characterizing the PRV gene
[0045] The following experiments were carried out in order to
characterize the gene:
[0046] the following protocol was used to isolate granulocytes from
stored blood or from blood obtained by bleeding p. vera
patients:
[0047] an equal volume of 3% dextran solution in 0.9% NaCl was
added to the blood and the mixture was left to stand at room
temperature (RT) for 20 minutes.
[0048] The mixture separated into two phases. The upper,
light-coloured phase was removed and centrifuged for 10 minutes at
1800 g and at RT.
[0049] The supernatant was discarded and the cell pellet was
resuspended in the same volume of 0.9% NaCl.
[0050] In each case 35 ml of the cells in NaCl were layered on 15
ml of Ficoll-Hypaque.
[0051] The cells on the Ficoll-Hypaque were then centrifuged for 60
minutes at 1800 g and at RT without using the brake.
[0052] A cell pellet and two layers with an interphase were
formed.
[0053] The layers and interphase were aspirated off and the cell
pellet was resuspended for 30 seconds in 10 ml of ice-cold 0.2%
NaCl, and 10 ml of ice-cold 1.6% NaCl were added immediately after
30 seconds.
[0054] The cells were centrifuged down for 10 minutes at 1800 g and
at RT.
[0055] They were then washed once in 10 ml of PBS and centrifuged
down.
[0056] The cell pellet contained 95-99%-pure granulocytes.
[0057] RNA was isolated from these cells using standard
methods.
[0058] 10 mg of this RNA were examined for the expression of PRV-1
in a Northern blot. The entire cDNA sequence shown in SEQ ID No. 1
was used as a probe.
[0059] This experiment was performed on 19 p. vera patients and 21
control samples of stored blood. The PRV-1 probe was found to
hybridize strongly in the case of the p. vera patients. No
hybridization was observed in healthy control samples.
EXAMPLE 2
[0060] PRV-1 Possesses Growth Factor Activity
[0061] Embryos were removed from a pregnant mouse 13.5 days after
fertilization. The foetal livers were removed. The cells contained
in them were stained using antibodies and enriched for particular
cells, and depleted for other cell types, by means of column
chromatography. This results in a cell mixture which is enriched
for certain haematopoietic precursor cells (colony forming
units-erythroid, CFU-E). Thus, while in all approximately 2% of the
foetal liver consists of CFU-E, 30-40% of the enriched cells
consist of CFU-E.
[0062] These CFU-Es were transfected using a retrovirus. To do
this, a packaging cell line, designated 293-T, was itself
transfected 48 hours previously. 293-T cells are an established
human embryonic kidney cell line. 293-T cells are stably
transfected with several genes from a retrovirus. If these 293-T
cells are now transfected with two plasmids, termed pOS and pKAT,
the 293-T cells then produce a retrovirus which is able to infect
murine foetal liver cells. If the 293-T cells are transfected with
an empty pOS vector and pKAT, a wild-type retrovirus, which only
expresses retroviral proteins, is then produced. On the other hand,
cloning a human gene, e.g. PRV-1, into the pOS vector results in
the production of a retrovirus which expresses this protein when it
has infected cells. The 293-T cells secrete the retrovirus into the
cell culture medium.
[0063] After two days, the cell culture medium from the transfected
293-T cells which contains the retrovirus is harvested and filtered
once through, a 0.45 .mu.m filter. In order to transfect the foetal
liver cells, these latter cells are mixed with the filtered cell
culture medium, which contains the retrovirus, and centrifuged for
2 hours at 1800 rpm and 20.degree. C. in the added presence of
Polybren. The transfected foetal liver cells were then cultured in
a medium (Methocult, from Cell Systems) which contains, in addition
to the usual salts and amino acids, foetal calf serum, 0.0001-0.4
IU of erythropoeitin (EPO)/ml and methyl cellulose 5 (0.8%). The
CFU-Es require EPO in order to form haematopoietic colonies. The
methyl cellulose solidifies the medium in the form of a jelly,
thereby fixing individual cells in this jelly so that, in contrast
to being in a liquid medium, they cannot move. It is therefore
possible to observe whether a haematopoietic colony is or is not
formed from a single cell. CFU-Es form erythroid colonies, that is
colonies which contain red blood cells and their precursor
cells.
[0064] After three days, a count is taken of the number of
haematopoietic colonies which have developed. Various mixtures are
compared. The mixtures were not all examined in each experiment;
mixtures 1-3 are very similar controls and each of them can be
compared 20 individually with mixture 4.
[0065] Mixture 1: Cells which were not transfected with a
retrovirus;
[0066] Mixture 2: Cells which were transfected with an empty pOS
vector;
[0067] Mixture 3: Cells which were transfected with a "green
fluorescent protein" (GFP), a protein which is not
haematopoietically active.
[0068] Mixture 4: Cells which were transfected with pOS-PRV-1
(vector+gene according to the invention).
4TABLE 1 The table lists the results obtained from three
experiments which were performed as described. The figures in each
case indicate the number of colonies. Mixture 4 Mixture 2 Mixture 3
PRV-1 Mixture 1 empty vector GFP (pOS- untransfected (pOS)
(pOS-GEP) PRV-1) Experiment 1 116 156 80 326 Experiment 2 271 273
410 Experiment 3 120 131 291
[0069] The experiments demonstrate that CFU-Es which were
transfected with PRV-1 form very many more colonies (up to three
times as many) than do the various control CFU-Es. This result
indicates that PRV-1 is a growth factor for CFU-E.
EXAMPLE 3
[0070] Solubility of the PRV-1 Growth Factor
[0071] A further experiment was carried out in order to investigate
whether PRV-1 is a soluble growth factor or whether cell-cell
contact is required. It is not only a retrovirus which is produced
by the packaging cell line 293-T after it has been transfected with
the pOS and pKAT vectors. In addition, the 293-T cells also
synthesize the protein encoded by the gene cloned in pOS, i.e.
PRV-1 in the present case. If the gene product is a soluble
protein, it is secreted into the medium which surrounds the
packaging cell line 293-T. If the 293-T cells are transfected only
with the pOS vector, without pKAT, no retroviruses are then formed.
The cell culture medium then only contains the soluble protein
produced by the cells. Medium which is derived from
pOS-PRV-1-transfected cells, and which does not contain any
retrovirus, is mixed with CFU-Es and the whole is plated out in the
methyl cellulose medium; the resulting colonies are then
counted.
[0072] The following results were obtained:
5TABLE 2 Solubility of PRV-1. The figures in each case indicate the
number of colonies. Mixture 4 Mixture 2 Mixture 3 PRV-1 Mixture 1
empty vector GFP (pOS- untransfected (pOS) (pOS-GFP) PRV-1)
Experiment 4 137 187 557
[0073] In this experiment, too, CFU-Es which were treated with
PRV-1-containing medium formed very many more haematopoietic
colonies than did control cells. It can be concluded from this
result that PRV-1 is a soluble growth factor.
EXAMPLE 4
[0074] The Growth Factor PRV-1 is N-glycosylated
[0075] Granulocytes were isolated from a patient suffering from p.
vera, and protein extracts were prepared from these cells using a
standard protocol. These protein extracts were treated in
accordance with the protocol for the "N-Glycosidase F
Deglycosylation Kit" supplied by Boehringer Mannheim. In detail,
this means that a "denaturation buffer" was added to the protein
extracts and the mixtures were heated at 95.degree. C. for 3
minutes, after which they were treated either with "reaction
buffer" or with "reaction buffer" plus N-glycosidase. Each mixture
was incubated overnight at 37.degree. C. and the proteins were
analysed on a PAGE gel electrophoresis followed by a Western blot.
The PRV-1 protein was detected with an antibody directed against a
protein having the amino acid sequence ID No. 5. The results show
that while PRV-1 protein purified from granulocytes is 60-65 kDa in
size, it is only 40 kDa in size after having been digested with
N-glycosidase. This clearly proves that PRV-1 is glycosylated on
asparagine residues (asparagine=N).
Sequence CWU 1
1
9 1 1600 DNA Homo sapiens 1 aaaagcagaa agagattacc agccacagac
gggtcatgag cgcggtatta ctgctggccc 60 tcctggggtt catcctccca
ctgccaggag tgcaggcgct gctctgccag tttgggacag 120 ttcagcatgt
gtggaaggtg tccgacctgc cccggcaatg gacccctaag aacaccagct 180
gcgacagcgg cttggggtgc caggacacgt tgatgctcat tgagagcgga ccccaagtga
240 gcctggtgct ctccaagggc tgcacggagg ccaaggacca ggagccccgc
gtcactgagc 300 accggatggg ccccggcctc tccctgatct cctacacctt
cgtgtgccgc caggaggact 360 tctgcaacaa cctcgttaac tccctcccgc
tttgggcccc acagccccca gcagacccag 420 gatccttgag gtgcccagtc
tgcttgtcta tggaaggctg tctggagggg acaacagaag 480 agatctgccc
caaggggacc acacactgtt atgatggcct cctcaggctc aggggaggag 540
gcatcttctc caatctgaga gtccagggat gcatgcccca gccaggttgc aacctgctca
600 atgggacaca ggaaattggg cccgtgggta tgactgagaa ctgcaatagg
aaagattttc 660 tgacctgtca tcgggggacc accattatga cacacggaaa
cttggctcaa gaacccactg 720 attggaccac atcgaatacc gagatgtgcg
aggtggggca ggtgtgtcag gagacgctgc 780 tgctcataga tgtaggactc
acatcaaccc tggtggggac aaaaggctgc agcactgttg 840 gggctcaaaa
ttcccagaag accaccatcc actcagcccc tcctggggtg cttgtggcct 900
cctataccca cttctgctcc tcggacctgt gcaatagtgc cagcagcagc agcgttctgc
960 tgaactccct ccctcctcaa gctgcccctg tcccaggaga ccggcagtgt
cctacctgtg 1020 tgcagcccct tggaacctgt tcaagtggct ccccccgaat
gacctgcccc aggggcgcca 1080 ctcattgtta tgatgggtac attcatctct
caggaggtgg gctgtccacc aaaatgagca 1140 ttcagggctg cgtggcccaa
ccttccagct tcttgttgaa ccacaccaga caaatcggga 1200 tcttctctgc
gcgtgagaag cgtgatgtgc agcctcctgc ctctcagcat gagggaggtg 1260
gggctgaggg cctggagtct ctcacttggg gggtggggct ggcactggcc ccagcgctgt
1320 ggtggggagt ggtttgccct tcctgctaac tctattaccc ccacgattct
tcaccgctgc 1380 tgaccaccca cactcaacct ccctctgacc tcataaccta
atggccttgg acaccagatt 1440 ctttcccatt ctgtccatga atcatcttcc
ccacacacaa tcattcatat ctactcacct 1500 aacagcaaca ctggggagag
cctggagcat ccggacttgc cctatgggag aggggacgct 1560 ggaggagtgg
ctgcatgtat ctgataatac agaccctgtc 1600 2 437 PRT Homo sapiens 2 Met
Ser Ala Val Leu Leu Leu Ala Leu Leu Gly Phe Ile Leu Pro Leu 1 5 10
15 Pro Gly Val Gln Ala Leu Leu Cys Gln Phe Gly Thr Val Gln His Val
20 25 30 Trp Lys Val Ser Asp Leu Pro Arg Gln Trp Thr Pro Lys Asn
Thr Ser 35 40 45 Cys Asp Ser Gly Leu Gly Cys Gln Asp Thr Leu Met
Leu Ile Glu Ser 50 55 60 Gly Pro Gln Val Ser Leu Val Leu Ser Lys
Gly Cys Thr Glu Ala Lys 65 70 75 80 Asp Gln Glu Pro Arg Val Thr Glu
His Arg Met Gly Pro Gly Leu Ser 85 90 95 Leu Ile Ser Tyr Thr Phe
Val Cys Arg Gln Glu Asp Phe Cys Asn Asn 100 105 110 Leu Val Asn Ser
Leu Pro Leu Trp Ala Pro Gln Pro Pro Ala Asp Pro 115 120 125 Gly Ser
Leu Arg Cys Pro Val Cys Leu Ser Met Glu Gly Cys Leu Glu 130 135 140
Gly Thr Thr Glu Glu Ile Cys Pro Lys Gly Thr Thr His Cys Tyr Asp 145
150 155 160 Gly Leu Leu Arg Leu Arg Gly Gly Gly Ile Phe Ser Asn Leu
Arg Val 165 170 175 Gln Gly Cys Met Pro Gln Pro Gly Cys Asn Leu Leu
Asn Gly Thr Gln 180 185 190 Glu Ile Gly Pro Val Gly Met Thr Glu Asn
Cys Asn Arg Lys Asp Phe 195 200 205 Leu Thr Cys His Arg Gly Thr Thr
Ile Met Thr His Gly Asn Leu Ala 210 215 220 Gln Glu Pro Thr Asp Trp
Thr Thr Ser Asn Thr Glu Met Cys Glu Val 225 230 235 240 Gly Gln Val
Cys Gln Glu Thr Leu Leu Leu Ile Asp Val Gly Leu Thr 245 250 255 Ser
Thr Leu Val Gly Thr Lys Gly Cys Ser Thr Val Gly Ala Gln Asn 260 265
270 Ser Gln Lys Thr Thr Ile His Ser Ala Pro Pro Gly Val Leu Val Ala
275 280 285 Ser Tyr Thr His Phe Cys Ser Ser Asp Leu Cys Asn Ser Ala
Ser Ser 290 295 300 Ser Ser Val Leu Leu Asn Ser Leu Pro Pro Gln Ala
Ala Pro Val Pro 305 310 315 320 Gly Asp Arg Gln Cys Pro Thr Cys Val
Gln Pro Leu Gly Thr Cys Ser 325 330 335 Ser Gly Ser Pro Arg Met Thr
Cys Pro Arg Gly Ala Thr His Cys Tyr 340 345 350 Asp Gly Tyr Ile His
Leu Ser Gly Gly Gly Leu Ser Thr Lys Met Ser 355 360 365 Ile Gln Gly
Cys Val Ala Gln Pro Ser Ser Phe Leu Leu Asn His Thr 370 375 380 Arg
Gln Ile Gly Ile Phe Ser Ala Arg Glu Lys Arg Asp Val Gln Pro 385 390
395 400 Pro Ala Ser Gln His Glu Gly Gly Gly Ala Glu Gly Leu Glu Ser
Leu 405 410 415 Thr Trp Gly Val Gly Leu Ala Leu Ala Pro Ala Leu Trp
Trp Gly Val 420 425 430 Val Cys Pro Ser Cys 435 3 24 DNA Homo
sapiens 3 aaaagcagaa agagattacc agcc 24 4 24 DNA Homo sapiens 4
ggctggtaat ctctttctgc tttt 24 5 13 PRT Homo sapiens 5 Lys Val Ser
Asp Leu Pro Arg Gln Trp Thr Pro Lys Asn 1 5 10 6 15 PRT Homo
sapiens 6 Ser Ala Arg Glu Lys Arg Asp Val Gln Pro Pro Ala Ser Gln
His 1 5 10 15 7 27 DNA Artificial Sequence Description of
Artificial Sequence Primer 7 attaggttat gaggtcagag ggaggtt 27 8 28
DNA Artificial Sequence Description of Artificial Sequence Primer 8
gcagaaagag attaccagcc acagacgg 28 9 28 DNA Artificial Sequence
Description of Artificial Sequence Primer 9 gaatcgtggg ggtaatagag
ttagcagg 28
* * * * *