U.S. patent application number 13/748017 was filed with the patent office on 2013-09-12 for viral expression plasmids for production of proteins, antibodies, enzymes, virus-like particles and for use in cell-based assays.
This patent application is currently assigned to ICOSAGEN CELL FACTORY OU. The applicant listed for this patent is ICOSAGEN CELL FACTORY OU. Invention is credited to Aare ABROI, Jelizaveta GEIMANEN, Kadri JANIKSON, Anne KALLING, Tiiu MANDEL, Toomas SILLA, Ingrid TAGEN, Radi TEGOVA, Urve TOOTS, Andres TOVER, Ene USTAV, Mart USTAV.
Application Number | 20130236958 13/748017 |
Document ID | / |
Family ID | 43780826 |
Filed Date | 2013-09-12 |
United States Patent
Application |
20130236958 |
Kind Code |
A1 |
SILLA; Toomas ; et
al. |
September 12, 2013 |
Viral expression plasmids for production of proteins, antibodies,
enzymes, virus-like particles and for use in cell-based assays
Abstract
This disclosure shows that the EBV FR-element comprised of EBNA1
multimeric binding sites can provide the stable maintenance
replication function to the mouse polyomavirus (PyV) core origin
plasmids in the presence of BPV-1 E2 protein and PyV large
T-antigen (LT).
Inventors: |
SILLA; Toomas; (Tartu,
EE) ; TAGEN; Ingrid; (Tartu, EE) ; GEIMANEN;
Jelizaveta; (Tartu, EE) ; JANIKSON; Kadri;
(Tartu, EE) ; ABROI; Aare; (Tartu, EE) ;
USTAV; Ene; (Tartu, EE) ; USTAV; Mart; (Tartu,
EE) ; MANDEL; Tiiu; (Tartu, EE) ; TOOTS;
Urve; (Tartu, EE) ; TOVER; Andres; (Tartu,
EE) ; KALLING; Anne; (Tartu, EE) ; TEGOVA;
Radi; (Tartu, EE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ICOSAGEN CELL FACTORY OU |
Tartu |
|
EE |
|
|
Assignee: |
ICOSAGEN CELL FACTORY OU
Tartu
EE
|
Family ID: |
43780826 |
Appl. No.: |
13/748017 |
Filed: |
January 23, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12803831 |
Jul 7, 2010 |
8377653 |
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13748017 |
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11351809 |
Feb 10, 2006 |
7790446 |
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12803831 |
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60652390 |
Feb 11, 2005 |
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Current U.S.
Class: |
435/354 ;
435/320.1; 435/352; 435/366 |
Current CPC
Class: |
C12N 2740/16023
20130101; C12N 2800/108 20130101; C12N 7/00 20130101; C12N
2710/16222 20130101; A61K 48/00 20130101; C12N 15/85 20130101; C12N
15/86 20130101; C07K 14/005 20130101; C12N 2820/60 20130101; C12N
2710/16244 20130101; C12N 2800/40 20130101; C12N 2710/22044
20130101; C12N 2710/22022 20130101; C12N 2820/10 20130101 |
Class at
Publication: |
435/354 ;
435/320.1; 435/352; 435/366 |
International
Class: |
C12N 15/85 20060101
C12N015/85 |
Claims
1. An expression system to provide extended episomal replication of
hybrid plasmid in eukaryotic cell lines, said system comprising: a)
a vector comprising a polyoma virus core origin according to SEQ ID
NO:2, an FR element of EBV, a selection marker, and a gene of
interest operably linked within an expression cassette expressing
the gene in a eukaryotic cell; and b) a compatible mammalian cell
line, where the vector is expressed, said cell line constitutively
expressing EBV EBNA1 protein and PyV LT protein either in presence
or in absence of selective pressure.
2. The system according to claim 1, wherein the FR element
comprises at least 21 EBNA1 binding sites.
3. The system according to claim 2, wherein the EBNA 1 binding
sites are according to sequences selected from the group consisting
of SEQ ID NOs: 3, 4, and 6.
4. The system according to claim 3, wherein the FR element is
according to SEQ ID NO:1.
5. The system according to claim 1, wherein the cell line is of
mouse, hamster or human origin.
6. The system according to claim 1, wherein the gene of interest
encodes a protein for pharmaceutical use.
7. The system according to claim 6, wherein the protein is a
monoclonal antibody.
8. The system according to claim 6, wherein the protein is virus
envelope or capside protein.
9. The system according to claim 6, where the protein is fused to
an epitope-tag.
10. The system according to claim 9, where the epitope tag has an
amino acid sequence selected from the group consisting of SEQ ID
NO:20, 21, 22 and 23.
11. An expression plasmid for use in a mammalian cell expression
system, said vector comprising: a) a polyoma virus core origin, b)
an FR element of Eppstein Barr Virus (EBV); c) a selection marker;
and d) an expression cassette for expression a gene of interest in
a eukaryotic cell.
12. The expression plasmid of claim 11, where the polyoma virus
core origin is according to SEQ ID NO:2 and the FR element is
according to SEQ ID NO:1.
13. The expression plasmid of claim 11, wherein the expression
cassette comprises a multicloning site for insertion of the gene of
interest, and a promoter.
14. The expression plasmid of claim 13, wherein the promoter is
selected from the group consisting of Cytomegalovirus immediate
early (CMV)-promoter, EF1.alpha.; EF1.alpha.-HTLV, heIF 4a,
.beta.-actine, and Rous Sarcoma Virus proviral Long Termina Repeat
(RSV-LTR)-promoter.
15. The expression plasmid of claim 13, wherein the gene of
interest encodes a protein for pharmaceutical use.
16. The expression plasmid of claim 15, wherein the protein is a
monoclonal antibody.
17. The expression plasmid of claim 15, wherein the protein is
virus envelope or capside protein.
18. The expression plasmid of claim 15, where the protein is fused
to an epitope-tag.
19. The expression plasmid of claim 18, where the epitope tag has
an amino acid sequence selected from the group consisting of SEQ ID
NO:20,21,22, and 23.
20. A cell lien comprising the expression plasmid of claim 11.
Description
PRIORITY
[0001] This application is continuation application of non
provisional application Ser. No. 12/803,831 which was filed on Jul.
7, 2010 and claimed priority of non provisional patent application
Ser. No. 11/351,809 which was filed on Feb. 10, 2006 and claimed
priority of provisional application 60/652,390 filed on Feb. 11,
2005, all of which are incorporated herein by reference in their
entirety.
SEQUENCE LISTING
[0002] This application contains sequence data provided on a
computer readable diskette and as a paper version. The paper
version of the sequence data is identical to the data provided on
the diskette.
TECHNICAL FIELD OF THE INVENTION
[0003] The present invention relates to production of proteins,
antibodies, enzymes and virus like particles in novel engineered
animal and human cell lines using eukaryotic hybrid replication
origin carrying extrachromosomally replicating expression plasmids.
The present invention also relates to short term production of the
proteins of interest for research purposes lasting up to 14 days.
The present invention also relates to production of the proteins of
interest using stable long term extrachromosomal replication of
hybrid expression plasmid in engineered eukaryotic cell lines in
large quantities in bioreactors or fermentors. The present
invention also relates to use of long term extrachromosomal
replication of the hybrid expression plasmids in engineered animal
and human cells lines for providing expression of the proteins at
physiological level in the form of epitope-tagged proteins for the
purpose designing cell-based assays for identification of the
active drug candidates using High Content Analysis.
BACKGROUND OF THE INVENTION
[0004] Several eukaryotic DNA viruses maintain their genomes as
extrachromosomal multicopy nuclear episomes in proliferating host
cells. Such episomal maintenance is characteristic of latent
infection of the Bovine papillomavirus type 1 (BPV-1), Epstein-Barr
Virus (EBV) as well as for Kaposi sarcoma associated Human
herpesvirus type 8 (HHV8). The latency of the viral genome in
dividing cell population requires activity of the viral genome at
the two phases of the cell cycle: the viral genome replication
during the S phase and proper segregation and partitioning of the
replicated genomes into daughter cells during the host cell
mitosis. For BPV-1 and two members of the gammaherpesvirus
family--EBV and HHV8 an effective segregation of viral genomes into
daughter cells and nuclear retention during mitosis is mediated
through a single viral protein serving as a molecular linker, which
attaches viral genomes to the host mitotic chromosomes. This linker
protein is a viral regulatory protein E2 for BPV-1, viral
transactivator EBNA1 for EBV and viral transcription repressor
LANA1 for HHV8.
[0005] For initiation of the BPV-1 DNA replication in vivo, minimal
origin region in cis and two viral proteins--E1 and E2, in trans,
are absolutely essential. However, the minimal origin (MO) is not
sufficient for stable extrachromosomal replication in dividing
cells. An additional element, the Minichromosome Maintenance
Element (MME) ensures the long-term episomal persistence of the
genome in the presence of viral E1 and the E2 proteins in the
dividing cells. In the BPV-1 genome, total of 17 E2 protein binding
sites with different affinities to E2 can be identified: 12 of
these are locating in the noncoding upstream regulatory region
(URR). We have shown that the minichromosome maintenance element
(MME) activity can be provided by the subset of the E2 binding
sites. The function of multimeric E2 protein binding sites in the
stable maintenance of the BPV-1 genomes is to provide the anchoring
function for the E2 protein, which therefore tethers MME containing
plasmids to mitotic chromosomes. This linkage between the BPV-1
genome and host chromatin ensures also that the viral genome is
targeted to the nucleus when the nuclear membrane is reassembled
during mitosis. In the case of EBV, the stable maintenance of
replicated genomes is achieved due to the EBNA1 protein and
FR-element, which is comprised of multimeric EBNA1 protein binding
sites.
[0006] We have shown that the BPV1 E2 protein dependent MME (Abroi
et al. 2004) and EBV EBNA1 dependent FR (Mannik, Janikson and
Ustav, unpublished) segregation/partitioning activities function
independently from replication of the plasmids.
[0007] Transfection or infection of permissive cells with
polyomavirus genome or replicator results in amplificational
replication leading to cell death due to the over-replication. The
mechanism of the BPV-1 origin based episomal replication is more
complex and controlled. On one hand the first amplificational
replication step, resembling in many aspects polyomavirus lytic
over-replication is crucial for establishment of the stable
episomal replication of the papillomavirus DNA. Such replication
leads to increase of expression level of the viral proteins and
copy-number of the viral genome. Increase of the E1 protein
concentration, however, over certain limit induces the "onion-skin"
type replication of the BPV-1 origin and generation of the
replication intermediates having tendency for high frequency of DNA
rearrangements and integration of the fragments of the viral DNA
into chromosomal DNA. To maintain the stability and intactness of
the viral genome, virus has to apply certain mechanism to assure
proper balance between initiation and elongation of replication
fork as well as segregation/partitioning of the viral plasmids
during cell division
[0008] Therapeutic protein production in small and large scale is
important field of development in pharmaceutical industry, because
proteins produced in animal cells have proper processing,
post-translational modification and therefore have adequate
activity for treatment of the physiological condition. In general,
for research purposes, the transient expression systems are used.
The expression plasmids equipped with strong promoter driving
expression of gene of interest is transfected into the appropriate
cells using either chemical transfection (like Lipofectamine 2000)
or physical transfection, like electroporation. Transfection could
be carried out in small scale, resulting in small amount of
produced protein or transfected in large scale (up to 100 liters of
cell suspension) allowing harvesting expressed protein in large
quantity. Problem with large scale transfections is high cost for
expensive transfection agent, large quantity of the expression
plasmid, and large cost for maintaining large quantity of the cell
culture. In these cases the transient transfection has been
optimized for 293 HEK cells. However, therapeutic proteins for
human use are mostly produced in Chinese Hamster Ovary (CHO cells),
which have been proved to be safe and effective for production of
therapeutic proteins. This is achieved by generating CHO stable
super-producer cell lines isolated as result of screening and
several subclonings. These steps are time and money consuming and
therefore impractical for research applications. Use of CHO cells
has turned out to be difficult in transient production assays due
to the poor transfection and production capability.
[0009] In order to overcome the shortage of CHO cells in production
of therapeutic proteins in transient transfections, use of episomal
expression vectors is one of the possibilities enhancing expression
plasmid copy-number and maintaining it in the cells for extended
time for enlarging fraction of cell producing therapeutic protein
as well as improving yield of the protein production.
[0010] The stable episomal maintenance systems described earlier
(U.S. Pat. No. 6,479,279) were provided with homologous replication
origins. Characteristic for these systems is for example a high
mutation frequency, especially recombination. Furthermore, the
system does not maintain stably replicating episomes in cells
because part of the cells lose their plasmid in every generations.
This deficiency of the system can be compensated by application of
continuous antibiotic selection pressure on cells in order to
eliminate the plasmid-negative cells from the culture. This fact
creates serious limits for the system to be used for example in
protein production.
[0011] U.S. patent application Ser. No. 10/938,864 (Kunaparaju)
provides a heterologous system, which is capable of stable episomal
replication lasting a couple of weeks. Kunaparaju uses two
functional replicons in the expression plasmid--one dependent on
wtPyV replication origin and Large T-antigen and second, oriP and
EBNA1. Limitations in this system is use of wild type polyomavirus
origin equipped with enhancer which we have shown to initiate
over-replication and generating too high copy-number of the plasmid
leading to death of the cells. Cell adaptation to too high
copy-number will include rearrangement of the plasmid leading to
genetic instability and therefore incompatibility with the
requirements for therapeutic protein production. Second limitation
is use of complete oriP, which is comprised of two elements--Dyad
Symmetry Element (DUE), which is eukaryotic origin of replication
functioning as a result of cellular replication factors and Family
of Repeats (FR), which is serving as cis-sequence for EBNA1
dependent segregation/partitioning of the plasmid. It means that
Kunaparaju et al. invention uses two viral eukaryotic origins of
replication, which may fire independently of each other and
generate a conflict between initiation of DNA replication. The
present disclosure provides improvements over the problems
encountering prior systems.
SUMMARY OF THE INVENTION
[0012] The present disclosure provides an extended episomal
maintenance system with heterologous replication origin, stable
episomal replication lasting longer than with any prior art method,
cell viability staying high for longer than with any other known
method.
[0013] The present disclosure provides a system where plasmids
containing core polyoma virus origin (not containing enhancer
function) partition into daughter cells. The system according to
this disclosure provides a hybrid system where a unique
configuration of polyoma virus origin (core polyomavirus origin not
containing enhancer function) is stable for initiation of
replication by the large T antigen and is segregated due to the
action of helper protein EBNA1 from Epstein-Barr virus acting on
multimeric EBNA1 binding sites fused to the PyV enhancerless
replication origin during the mitosis.
[0014] An advantage of the present system is that high copy-number
extrachromsomal state of chimeric origins is maintained without
rearrangement. The present invention further provides a possibility
to combine vectors with polyoma virus origin and papilloma origin
into a single cell, thereby enabling expression of more than one
different recombinant proteins or RNAs in one cell. A further
advantage of the current system is that it provides stable episomal
maintenance in the cell that lasts up to several months as opposed
to all previous systems, which provide maintenance of maximally a
few weeks.
[0015] In U.S. patent application Ser. No. 11/351,809 we showed how
the stable maintenance can be provided with a hybrid plasmid
containing the PyV core origin, and MME element containing at least
5 E2-binding sites. We also showed that the extended stable
maintenance can be provided also by replacing the enhancer with
EBNA1-binding sites of EBV. Here we describe in more details the
expression system comprising PyV core origin, and FR element of EBV
in various applications.
[0016] The problem that we aim to solve is that production of gene
products in animal cell systems is costly and time consuming. This
problem has been solved in the present disclosure by providing
data, which show that transient expression of gene products in the
engineered cells is possible due to the high efficiency of delivery
of the expression plasmids. Since the genes of interest are
replicated and maintained outside the chromatin in the nucleus, the
replicating expression plasmids do not have positional effect of
the host cell DNA on expression of the gene of interest like it has
been shown for the stable cell lines carrying integrated expression
cassettes. The extrachromosomal plasmids go to the progeny in cell
division and segregation, maintain transcriptional activity and
with the method of the current disclosure, the expression of the
gene products can be continued for months. This enables generation
of the production of the proteins of interest using cell population
with homologous copy-number of extrachromosomal expression plasmids
without additional subcloning. Use of the expression system
according to this disclosure allows production of therapeutic
proteins in research setting using the transient mode of
expression. Additional benefit of the system disclosed here is that
we can generate cell banks and maintain these in liquid nitrogen,
which allows repeated production of the protein of interest.
Further, we can use the same cell banks for large scale production
of the therapeutic proteins in cost and time effective way.
[0017] Development of stable expression in cell lines takes usually
several months or even years. The system that we describe here is
enabling generation of production cell culture in transient format
as well as stable long-term culture and is much faster and
therefore useful and novel. Further more, transient systems so far
known do not have capability of maintaining expression plasmid in
the cell and therefore they have very limited half-life; i.e.
maximally up to 7 days. In addition, those systems may need
construction of recombinant viruses, which makes the systems
expensive and very time consuming. Our system provides a marked
improvement to the existing art; the system according to this
disclosure provides a transient expression system that maintains
the expression levels for several weeks and even up to several
months.
[0018] The present disclosure provides a possibility to develop
stable cell lines when the vector according to this disclosure
contains a selection marker and the cells are cultivated on a
medium containing the selective agent. The present disclosure also
provides a possibility to express gene products in a cell line for
shorter time when the vector does not contain a selection marker.
However, even without using selection pressure the current system
provides stable maintenance that lasts longer than with any other
comparable system previously known.
[0019] The present disclosure further enables development of a
multi-replicon expression system, where more than one gene products
are expressed from different replicons and the replicons are
locating in same cell. Such a mechanism is useful for example to
express different subunits of antibodies or enzyme subunits in one
cell or to study interactions of macromolecules expressed in the
cell.
[0020] An object of the present disclosure is to provide a
mechanism to extended episomal maintenance of polyoma virus core
origin.
[0021] Another object of the present disclosure is to provide a
mechanism to extended episomal maintenance of polyoma virus core
origin plasmids without selective pressure for use in transient
production of the proteins.
[0022] Yet another object of the present disclosure is to provide
constructs in conjunction with the segregations/partitioning
elements from EBV.
[0023] A further object of the present disclosure is to provide
cell lines capable of supporting the replication and episomal
maintenance of hybrid plasmids.
[0024] A still further object of the present disclosure is to
provide a transient system for extended (up to 14 days without
selection) expression of gene products in eukaryotic cells allowing
expansion of the volume of the production culture an therefore
output of the protein of interest.
[0025] An even further object of the present disclosure is to
provide cell lines harboring more than one different vector and
thereby providing expression of more than one different genes of
interest.
[0026] Yet a further object of the present disclosure is to provide
a transient system for long lasting production therapeutic,
prophylactic or endotoxine free gene products for diagnostic and
other applications in eukaryotic cells.
[0027] Another object of the present disclosure is to provide a
transient system for long lasting production of RNA or proteins in
eukaryotic cells. The cells can be cultivated and gene products can
be expressed in small and large scale; from laboratory flasks and
Petri dishes up to big fermentors.
[0028] Yet another object of the present disclosure is to provide a
system for extended production of proteins, antibodies, enzymes and
virus-like particles.
[0029] In patent application Ser. No. 11/351,809, we showed that
the BPV-1 E2 protein dependent MME comprising E2 multimeric binding
sites can provide extended maintenance replication function to the
mouse polyomavirus (PyV) core origin plasmids in the presence of
BPV-1 E2 protein and PyV large T-antigen (LT), but fail to do so
for the complete PyV origin. In mouse fibroblast cell-lines
expressing PyV LT and BPV-1 E2 (COP5/E2), the plasmids carrying PyV
core origin linked to at least five multimeric E2 protein binding
sites show the capacity for long term episomal replication, which
can be monitored for more than 5 months (under selective
conditions). Overall structural integrity as well as the intactness
of domains of BPV-1 E2 are required for efficient episomal
maintenance.
[0030] Our data showed clearly that the large T antigen dependent
replication function of the polyomavirus and extended maintenance
functions of the BPV-1 are compatible in certain configurations.
Further quantitative analysis of the loss of the episomal plasmids
carrying hybrid origin showed that MME dependent plasmids are lost
with the frequency of 6% per generation.
[0031] Similar hybrid origins comprising the EBV FR-element and
polyomavirus replication origin were constructed and studied in the
cell lines expressing EBNA1 and polyomavirus large T antigen (LT).
Our data suggest convincingly that segregation/partitioning
functions of the BPV-1 and EBV can effectively be used for extended
episomal maintenance of the polyomavirus core origin.
BRIEF DESCRIPTION OF THE FIGURES
[0032] FIG. 1. Schematic representation of mouse polyoma virus core
origin of replication and FR (Family of repeats) element. Here, FR
element (SEQ ID NO: 1) contains 21 possible EBNA-1 binding
sequences. DNA fragments containing EBNA-1 binding sequences are
aligned.
[0033] FIG. 2. Schematic representation of new generation
expression plasmid.
1. Replication and maintenance in QMCF cells: Maintenance
element--(Epstein-Barr Virus Family of Repeats (FR) (SEQ ID NO:1)
of Bovine Papilloma type I Minichormosome Maintenance element (MME)
SEQ ID NO:7); PyV core origin of replication--murine polyomavirus
origin of replication without enhancer (SEQ ID NO:2); 2. Antibiotic
selection: SV40 pr--SV 40 promoter (SEQ ID NO:17) controlling
expression of Neo resistance gene; Neo/Km--Neomycin/Kanamycin
resistance marker (SEQ ID NO:18) for selection of plasmid
containing cells in bacteria and eukaryotic cells; 3. Replication
in bacteria: pMMB origin of replication (SEQ ID NO:25); 4.
Expression cassette: Promoter (CMV (SEQ ID NO:13), hEF1.alpha.-HTLV
(SEQ ID NO:14), hEF1alpha (SEQ ID NO:8), RSV-LTR (SEQ ID 016),
heIF4A (SEQW ID NO:9) or .beta.-actin (SEQ ID NO:12)); Intron--hEF1
alpha intron (SEQ ID NO:19) in 5' position from gene of interest
cDNA; MCS--multicloning sequence; polyA--SV40 polyadenylation
sequence (SEQ ID NO:15) (regulating expression of Neo/Km and gene
of interest simultaneously).
[0034] FIGS. 3 A and B. Strength of different promoters used in
pGMCF and pQMME plasmids. Gaussia luciferase gene was used as a
reporter and data is represented as relative promoter strength
compared to strongest CMV promoter-3' intron containing vector
(pQMCF-1/pQMME-1). A. Promoter comparison in different pQMCF
vectors. B. Promoter comparison in different pQMCF vectors. Also
comparison with old version pQMCF-CMV-0.1 vector is resented in
comparison to pQMCF-1.
[0035] FIG. 4. Growth of the CDNF-expressing CHOEBNALT85 cell
culture. 48 h after transfection G418 (700 .mu.g/ml) was added. 12
days after transfection expression cell bank was generated. From
day 13 to 21 the production phase was performed. Temperature was
reduced to 30.degree. C., additional nutrients were added to the
medium. The viability of the cell culture was more than 85% during
antibiotic selection and production. At day 21 when viability of
the culture starts to decline, supernatant of the culture was
clarified by centrifugation and filtered through 0.45 .mu.m
filter.
[0036] FIG. 5. Analysis of expression of the CDNF by CHOEBNALT85
cells using Western Blot 48 hours after transfection. Lane 1.
PageRuler Prestained Protein Ladder (#SM0671, lot: 00036958,
Fermentas); lane 2 --17 .mu.L of the supernatant from cells
transfected with 1 .mu.g of the CDNF expression vector; lane 3--17
.mu.L of supernatant from the cells transfected with 5 .mu.g of the
CDNF expression vector; lane 4-17 .mu.L supernatant from the
untransfected CHOEBNALT85 cells; lane 5-17 .mu.L of the
untransfected CHOEBNALT85 cell lysate; lane 6-17 .mu.L of the cell
lysate transfected with 1 .mu.g of the CDNF expression vector; lane
7-17 .mu.L of the cell lysate transfected with 5 .mu.g of the CDNF
expression vector.
[0037] FIG. 6. Southern-Blot analysis of CDNF production plasmids.
Lines 1-12 pQMCF-1-CDNF (CDNF expressed under control of CMV
promoter). Lines 13-21 pQMCF-2-CDNF (CDNF under control of
hEF1.alpha.-HTLV promoter). I and II designated cells transfected
and grown from production cell bank, respectively. Line 1 and Line
12 designate time point 48 hours after transfection. Lines 2, 6, 13
and 16 designate time point at the beginning of production. All
other lines--different time points during CDNF production
phase.
[0038] FIG. 7. Growth of the bovine DNaseI-expressing CHOEBNALT85
cell culture. 48 h after transfection G418 (700 .mu.g/ml) was
added. 12 days after transfection expression cell bank was
generated. From day 13 to 21 the production phase was performed.
Temperature was reduced to 30.degree. C., additional nutrients were
added to the medium. The viability of the cell culture was more
than 85% during antibiotic selection and production. At day 17 when
viability of the culture starts to decline, supernatant of the
culture was clarified by centrifugation and filtered through 0.45
.mu.m filter.
[0039] FIG. 8. Production of Bovine DNaseI in CHOEBNALT85 Cells
after Selection of Plasmid-Positive Cells
1. CHOEBNALT85 [pQMCF-CMV-0.2-DNaseI] culture supernatant (14
.mu.l) 2. CHOEBNALT85 [pQMCF-CMV-0.2-DNaseI] culture supernatant (7
.mu.l) 3. CHOEBNALT85 [pQMCF-CMV-0.2-DNaseI] culture supernatant
(3.5 .mu.l) 4. CHOEBNALT85 [pQMCF-CMV-0.2-DNaseI] culture
supernatant (1.25 .mu.l) 5. "Ambion" DNaseI (0.25 .mu.g) 6.
"Ambion" DNaseI (0.125 .mu.g) 7. "Ambion" DNaseI (0.0625 .mu.g)
[0040] FIG. 9. Production of Bovine DNaseI by CHOEBNALT85 Cells
Started from Newly Transfected Cells or from Production Cell
Nank.
Line 1. Protein marker (#SM0671, lot: 00052778, Fermentas); Line 2.
CHOEBNALT85 [pQMCF-1-DNaseI] culture supernatant before production
phase, Line 3. CHOEBNALT85 [pQMCF-1-DNaseI] culture supernatant,
4.sup.th day of production Line 4. CHOEBNALT85 [pQMCF-1-DNaseI]
culture supernatant, last day of production; Line 5. CHOEBNALT85
[pQMCF-1-DNaseI] culture supernatant, cells were taken from
production cell bank (last day of production).
[0041] FIG. 10. Southern-Blot analysis of bovine DNaseI production
plasmids. Lines 1-4 Old version pQMCF vector during production
phase; Lines 5-9 pQMCF-1-DNaseI (CMV promoter containing vector),
Lines 10-14 pQMCF-2-DNaseI (hEF1-HTLV promoter containing vector),
Lines 15-21 pQMCF-3-DNaseI (heIF4a promoter-containing vector),
Lines 22-26 pQMCF-5-DNaseI (RSV-LTR containing vector).), Lines
27-31 pQMCF-6-DNaseI (hEF1.alpha.containing vector). Lines 5, 10,
15 22 and 27 exhibit time point 48 h after transfection. All other
lines represent samples taken before production phase or during
production phase. Line 32--control; Line 33 DNA size marker.
[0042] FIG. 11. Analysis of ETAR-translocation assay. Adherent
CHOEBNALT cells were stably transfected with ETAR-EGFP (SEQ ID
NO:24) bearing QMCF plasmid. After 4 weeks of G418 selection cells
were treated with 300 nM of endothelin-1. Non-endotheline-1 treated
cells; 2: ETAR-EGFP internalization in after endothelin-1
treatment. Green--GFP signals from ETAR-EGFP fusion protein;
red--cytosol and nucleoli (Nikon Eclipse TE-2000-U; 60.times.).
[0043] FIG. 12. Detection of ETAR-5E11tag fusion protein in
adherent U2OSEBNALTD3 cells. Green--ETAR-5E11tag fusion protein;
red--cytosol and nucleoli (ArrayScan VTi 40.times.).
[0044] FIG. 13. Schematic representation of single-expression (A)
and double-expression (B) cassette containing vector for expression
of Virus Like Particles (VLP-s).sub.1. Replication and maintenance
in QMCF cells: Maintenance element--(Epstein-Barr Virus Family of
Repeats (FR)); PyV core origin--murine polyomavirus origin of
replication without enhancer element; 2. Antibiotic selection: SV40
pr--SV 40 promoter controlling expression of Neo resistance gene;
Neo/Km--Neomycin/Kanamycin resistance marker for selection of
plasmid containing cells in bacteria and eukaryotic cells; 3.
Replication in bacteria: pMMB origin of replication; 4. Expression
cassette: Promoters (CMV for gag protein expression, RSV-LTR or
hEF1.alpha.-HTLV for protein of interest expression);
Intron--hEF1.alpha. intron (Intron 1 and intron 2) in 5' position
from gene of interest, bgh intron (SEQ ID NO:26) (Intron 3) in 3'
position of gag protein expression gene; Gag--HIV-1 or MLV gag
protein; 2A--Foot-and-mouth disease virus (FMDV) 2A peptide (SEQ ID
NO:27); ETAR--G-protein coupled receptor (example protein);
polyA--SV40 polyadenylation sequence (PolyA and PolyA2) regulating
expression of Neo/Km and gene of interest simultaneously, polyA
1--bgh polyA regulating expression of gag protein.
[0045] FIG. 14. Production of ETAR-pseudotyped HIV-gag based VLP-s
using 293EBNALT75 cell line. All samples were ultracentrifuged for
VLP purification. Lines 1 and 2 ETAR is detected by BPV E2 tag
antibody, lines 5-6 detected using HIV-gag specific antibody. Line
1. Protein marker (#SM0671, lot: 00052778, Fermentas). Line 2.
Supernatant of 293EBNALT75 expressing ETAR. Line 3. Supernatant of
293EBNALT75 expressing ETAR and HIV-gag. Line 4. Protein marker
(#SM0671, lot: 00052778, Fermentas). Line 5. Supernatant of
293EBNALT75 expressing ETAR. Line 6. Supernatant of 293EBNALT75
expressing HIV-gag.
[0046] FIG. 15. Schematic representation of single- and
double-expression cassette containing vector for expression of
monoclonal antibodies 1. Replication and maintenance in QMCF cells:
Maintenance element--(Epstein-Barr Virus Family of Repeats (FR));
PyV core origin--murine polyomavirus origin of replication without
enhancer element; 2. Antibiotic selection: SV40 pr--SV 40 promoter
controlling expression of Neo resistance gene;
Neo/Km--Neomycin/Kanamycin resistance marker for selection of
plasmid containing cells in bacteria and eukaryotic cells; 3.
Replication in bacteria: pMMB origin of replication; 4. Expression
cassette: Promoters (CMV, RSV-LTR or hEF1.alpha.-HTLV for
expression of antibody heavy and light chain is used);
Intron--hEF1.alpha.intron and CMV intron in 5' position from gene
of interest) 2A--Foot-and-mouth disease virus (FMDV) 2A peptide;
IgG HC and IgG LC--coding regions for expression of antibodu heavy-
and light chains; polyA--SV40 polyadenylation sequence regulating
expression of Neo/Km and gene of interest simultaneously or bgh
polyA is used for regulation of expression of antibody light- or
heavy chain.
[0047] FIG. 16. Growth of the monoclonal antibody-expressing
CHOEBNALT85 cell culture. 48 h after transfection G418 (700
.mu.g/ml) was added. 9 days after transfection expression cell bank
was generated. From day 10 to 16 the production phase was
performed. Temperature was reduced to 30.degree. C., additional
nutrients were added to the medium. The viability of the cell
culture was more than 85% during antibiotic selection and
production. At day 16 when viability of the culture starts to
decline, supernatant of the culture was clarified by centrifugation
and filtered through 0.45 .mu.m filter.
[0048] FIG. 17. Western-Blot analysis of partially humanized
(chimeric) tyrosinase A antibody expressed by CHOEBNALT85 cells.
Line 1. Protein marker (#SM0671, lot: 00052778, Fermentas); Line 2.
Non-transfected CHOEBNALT85 cell growth medium; Line 3. Supernatant
of CHOEBNALT85 cell line expressing recombinant monoclonal
partially humanized (chimeric) tyrosinase A antibody; Line 4.
Positive control (ImmunoPure Human IgG Whole Molecule 31154,
Pierce).
[0049] FIG. 18. Western-Blot analysis of partially humanized
(chimeric) tyrosinase A antibody expressed by CHOEBNALT85 cells.
Line 1. Supernatant of CHOEBNALT85 cell line expressing recombinant
monoclonal partially humanized (chimeric) tyrosinase A antibody,
time point before production phase; Lines 2 and 3. Supernatant of
CHOEBNALT85 cell line expressing recombinant monoclonal partially
humanized (chimeric) tyrosinase A antibody, time point during
production phase; Line 4. Supernatant of CHOEBNALT85 cell line
expressing recombinant monoclonal partially humanized (chimeric)
tyrosinase A antibody, time point at the end of production
phase.
[0050] FIG. 19. Southern-Blot analysis of pFRG-EFFP and
pFRG-EGFP-FR (without FR element). Lines 1-4 pFRG-EGFP; Lines 5-8
pFRG-EGFP-FR (without FR element). Time points of analysis in
figure: Line 1 and 5. 48 hours after transfection; Lines 2 and 6 14
days after transfection; Lines 3 and 7 21 days after transfection;
Lines 4 and 8 22 days after transfection.
[0051] FIG. 20. Schematic representation of PyV hybrid origin
constructs used in flow cytometry analysis (A). Time course of
long-term EGFP (B) or short-term d1EGFP (C,D) expression in the
presence or absence of G418 selection for various cell lines.
COP5E2/PuroMMEG (B); COP5E2/PuroMMEG* (C); COP5EBNA1/PuroFRG* cell
line (D).
[0052] FIG. 21A-F is a schematic illustration of the novel vectors
according to this disclosure. The vectors are called pQMCF1-pQMCF6
A. pQMCF1 contains CMV promoter-driven expression cassette. B.
pQMCF2 contains EF1.alpha.-HTLV promoter-driven expression
cassette. C. pQMCF-3 contains heIF4a promoter-driven expression
cassette D. pQMCF-4 contains .beta.-actine promoter-driven
expression cassette. E. pQMCF-5 contains RSV-LTR promoter-driven
expression cassette. F. pQMCF-6 contains hEF1.alpha.promoter-driven
expression cassette.pQMCF-6 Single-cutting restriction sites are
shown in map and the sequence.
[0053] FIG. 22 A-F is a schematic illustration of the novel vectors
according to this disclosure. The vectors are called pQMME 1-6. A.
pQMME-1 contains CMV promoter-driven expression cassette. B.
pQMME-2 contains hEF1.alpha.-HTLV promoter-driven expression
cassette. C. pQMME-3 contains heIF4a promoter-driven expression
cassette. D. pQMME-4 contains human .beta.-actin promoter-driven
expression cassette. E. pQMME-5 contains RSV-LTR promoter-driven
expression cassette. F. pQMME-6 contains RSV-LTR promoter-driven
expression cassette. Single-cutting restriction sites are shown in
the maps.
[0054] FIG. 23 A-B is a schematic illustration of the novel VLP
expression vectors according to this disclosure. A.
pQMCF-VLP-srcgag1 vector contains two expression cassettes: CMV
promoter-driven expression cassette for srcgag protein (cDNA
sequence according to SEQ ID NO:26) and hEFLalpha.-HTLV
promoter-driven expression cassette for ETAR expression. B.
pQMCF-VLP-hangag1 vector contains two expression cassettes: CMV
promoter-driven expression cassette for srcgag protein and
hEF1.alpha.-HTLV promoter-driven expression cassette for ETAR
expression. Single-cutting restriction sites are shown in the maps
and the sequence.
[0055] FIG. 24 is a schematic illustration of the novel monoclonal
antibodies expression vectors. A. Map of partially humanized
(chimeric) tyrosinase A antibody expression vector (two expression
cassettes: CMV promoter-driven antibody light chain expression
cassette and RSV-LTR promoter-driven expression cassette for
antibody heavy chain expression cassette). Single-cutting
restriction sites are shown in the map. Expression cassettes of
antibody light- and heavy chain are locating in same direction. B.
Map of partially humanized (chimeric) tyrosinase A antibody
expression vector (two expression cassettes: CMV promoter-driven
antibody light chain expression cassette and RSV-LTR
promoter-driven expression cassette for antibody heavy chain
expression cassette). Single-cutting restriction sites are shown in
the map. Expression cassettes of antibody light- and heavy chain
are locating in opposite direction.
[0056] FIG. 25 is Southern-Blot analysis of pQMME-1-EGFP and in
U2OSEBNALTE2 adherent cell line. Lines 1-3 and 4-6 exhibit results
of plasmid stability in two different U2OSEBNALTE2 cell lines.
Lines 1 and 4 show results of time points 48 hours after
transfection. Lines 2 and 6 show results of time points 32 days
after transfection. Lines 3 and 6 show results of time points 52
days after transfection.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0057] In this disclosure the following terms are used as defined
below:
[0058] "Papillomavirus" refers to a member of the papilloma family
of viruses, including but not limited to bovine papillomavirus
(BPV) and human papillomavirus (HPV).
[0059] "Polyomavirus" refers to a member of polyoma family of
viruses, including but not limited to mouse polyomavirus (PyV).
[0060] "Polyomavirus core origin" refers to a minimal cis-sequence
within a polyomavirus that is necessary for initiation of DNA
synthesis. The PyV core origin is essentially according to SEQ ID
NO: 2. The core origin of PyV is located at nucleotides
5232-5297/1-88 in total 154 bp (5232 and 88 included) in sequence
PLY2CG (Genebank accession number J02288). The polyomavirus core
origin is also referred as PyV core origin or as minimal core
origin.
[0061] The Minimum origin (MO) of BPV1 is defined as described in
U.S. Pat. No. 6,479,279.
[0062] FR element refers to Epstein-Ban virus family of repeats. It
comprises at least 16 EBNA1-binding sites. SEQ ID NO:1 gives
nucleotide sequence of one alternative synthetic FR-element. In
this element 21 EBNA binding sites were used. The EBNA 1-binding
sites in the FR-element do not need to be similar to each other.
The EBNA1-binding sites may be according to any one of SEQ ID NO:
3-6: (SEQ ID NO: 3 GGGTATCATATGCTGACT; SEQ ID NO: 4:
GGGTATCATATGCTGACT; SEQ ID NO: 5 GGATAGCATATGCTACCC; SEQ ID NO:6:
GGATAGCATATACTACCC) In the vectors according to this disclosure the
EBNA-1 binding sites are separated by spacers.
[0063] "EBNA1" refers to viral transactivator for EBV and is
encoded by nt 7421-8043 in EBV sequence with Genbank accession
number V01555.
[0064] "E1" refers to the protein encoded by nt 849-2663 of BPV
subtype 1, or to nt 932-2779 of HPV of subtype 11, or to equivalent
E1 proteins of other papillomaviruses, or to functional fragments
or mutants of a papillomavirus E1 protein, i.e. fragments or
mutants of E1 which possess the replication properties of E1.
[0065] "E2" refers to the protein encoded by nt 2594-3837 of BPV
subtype 1; or to nt 2723-3823 of HPV subtype 11, or to equivalent
E2 proteins of other papillomaviruses, or to functional fragments
or mutants of a papillomavirus E2 protein, i.e. fragments or
mutants of E2 which possess the replicating properties of E2.
[0066] "Minichromosomal maintenance element" (MME) refers to a
region of the papilloma viral genome to which viral or human
proteins essential for papilloma viral replication bind, which
region according to this invention is essential for stable episomal
maintenance of core origin in a host cell. Preferably, the MME is a
sequence containing multiple binding sites for E2. According to
this disclosure the MME contains at least 5 E2 binding sites. The
sequential binding sites which constitute the MME need not be
identical in sequence, but must be able to bind E2. SEQ ID NO:7 is
one preferred example of MME.
[0067] "E2 binding site" (E2BS) refers to the minimum sequence of
papillomavirus double-stranded DNA to which the E2 protein binds.
E2 binding site may be of BPV or of HPV. The affinities of the E2
binding sites vary and according to this disclosure E2 binding site
means a high affinity binding site. The E2 binding site may be
according to SEQ ID NO: 4, preferably it is according to SEQ ID NO:
5. It may also be according to SEQ ID NO:9. In the vectors
according to this disclosure the repetitive E2 binding sites are
separated by spacers.
[0068] "Heterologous replication origin" refers to a system where
the replication origin locates in a vector containing MME or
FR-element of another virus species.
[0069] "A host cell" which is stably transformed according to the
disclosure is a eukaryotic cell and preferably a mammalian cell,
most preferably a human, mouse or hamster cell. The cell may be
derived from any tissue. The host cell may be derived from CHO
(hamster), COP (mouse) or human cell lines HEK293 or U2OS:
CHOEBNALT85 is a cell line derived from CHO and expressing EBV
EBNA1-protein and PyV-LT protein. 293EBNALT75 is a cell line
derived from 293 and expressing EBV EBNA1-protein and PyV
LT-protein. U2OSEBNALTD3 is a cell line derived form U2OS and
expressing EBV EBNA1-protein and PyV LT-protein.
[0070] "A gene of interest" refers to a gene encoding a gene
product of interest such as a protein or RNA of interest.
[0071] "A gene product" refers to a product of the gene of
interest. The product may be an expression product on RNA level or
it can as well be an expressed protein or peptide. The gene
products may be used for example as therapeutic or prophylactic
purposes. The gene products may be endotoxine free products for
diagnostic purpose. These uses are exemplary only and one skilled
in the art would realize that there are other purposes as well
according to this disclosure.
[0072] "Helper protein" refers to various viral proteins including
viral regulatory proteins E1, E2, EBNA1, and LT.
[0073] "Extended episomal replication": refers to long term (during
20-30 cell generations) replication and maintenance of the
expression plasmids and expression of protein of interest after
transfection into the engineered cell without selective
pressure
[0074] We describe here a mechanism of extended replication of
chimeric origins. We have developed PyV origin based constructs in
conjunction with the segregation/partitioning elements from the EBV
and the cell lines capable of supporting the replication and
episomal maintenance of these plasmids. Polyomaviruses exhibit
replication patterns that are uncoupled from the regulatory
mechanisms of the host cell, so that each viral genome replicates
many times within each cell cycle to the maximal level. The
complete polyomavirus origin (wild type origin) includes
transcriptional and replicational enhancer sequences, which dictate
the origin activity and the efficiency of replication in specific
cells by determining the availability of the replication factors
and nucleotides. Papillomavirus origin replication control is
similar to polyomavirus replication in the first, amplificational
phase of the replication. However, in the latent replication phase
copy number control mechanism is applied, which assures the
controlled initiation of replication of the episomal viral genome
in the latent replication phase. Epstein-Barr virus (EBV) uses
entirely cellular replication machinery for initiation of the
latent origin oriP replication, which strictly replicates once per
cell cycle. Although the BPV-1 and polyomaviruses use the host
replication machinery for viral genome replication, the initiation
of replication is achieved by viral factors, while for stable
maintenance with the EBV entirely cellular initiation and
elongation machinery is used. The polyomavirus replicational
enhancer can be exchanged with binding sites for different factors
such as c-Jun and Ga14, without loosing its ability to promote
replication (Guo et al., 1996).
[0075] The inventive step in this disclosure includes the finding
that substitution of the wild-type PyV enhancer with at least 16
synthetic EBNA1 binding sites for the EBV protein, can replace
replication enhancer function and makes it dependent on E2 protein.
Similarly as in the previous disclosure (U.S. application Ser. No.
11/351,809) where addition of five or ten E2 binding sites to PyV
wt origin did not cause additional replication activation, addition
of the EBNA 1 binding sites to a PyV wt origin did not cause
additional replication activation. Therefore, the viral origin
seems to achieve in a host cell a maximum activity when a strong
enhancer is present and after that point enhancement of replication
is not possible, even if additional enhancer elements are added.
This may be because of limitation of cellular factors or saturation
of the nucleus with the active genetic elements. Accordingly, we
observed many dead cells after transfection with PyV wt origin
constructs.
[0076] It is known that EBNA1 protein of the EBV is necessary and
sufficient for linking of the FR containing plasmids to the
chromatin. The novelty of this disclosure includes the finding that
FR functions outside of its natural replication origin and provides
extended maintenance function only for constructs, which contain
PyV core origin. In the case of wt PyV origin very strong transient
replication was observed, however, it was impossible to rescue
stable episomal replication of these plasmids, even after
antibiotic selection for origin constructs. It is important to note
that stably maintained constructs were in episomal state, no
integration to host chromosomes was detected.
[0077] According to the present disclosure, EBV EBNA1
protein-dependent FR-element can provide extended maintenance
functions to the PyV core origin plasmids in the presence of viral
trans-factors. We have used stable replication assay and flow
cytometric EGFP reporter expression assay for the analysis of the
kinetics of the extended maintenance of the episomes. In the case
of the BPV-1 and PyV, the origin of replication is fired several
times during their amplificational replication in host cell S-phase
and even during the stable replication of the BPV-1 the origin is
not restricted to precisely once in each cell cycle. At the same
time the EBV latent origin oriP replicates strictly once per cell
cycle, the same way as chromosomal DNA. The present disclosure
suggests that the extended maintenance of the episomes provided by
the function of MME or FR-element, is not connected to the mode of
replication of the episome. FR-element can provide an extended
maintenance function to both types of origins--in its natural
context within EBV latent origin oriP and in our hybrid replicon
together with PyV core origin (SEQ ID NO:2).
[0078] The present disclosure also shows that the replication
function is not connected to the stable maintenance function of the
virus--replication origins of different viruses can be combined
with different stable maintenance elements without the loss of
either function. It has been shown previously that the cellular
receptors of BPV-1 E2 protein and EBV EBNA1 protein, which link the
episomes to mitotic host chromatin and therefore provide the stable
maintenance function, are different. The present disclosure
suggests that the different localization of the episome on mitotic
chromosomes does not interfere with the replication of PyV core
origin.
The Rate of Loss of Episomal Plasmids is Lower than in Control
Plasmids
[0079] We have analyzed the episomal maintenance of the pMMEG,
pMMEG* and pFRG* plasmids (Materials and Methods) in cells cultured
without geneticine selection. These plasmids contained PyV minimal
core origin (SEQ ID NO: 2) and either BPV-1 Minichromosome
maintenance element (MME) or EBV FR-element. The viral
trans-factors (either PyV LT and BPV1 E2 or PyV LT and EBV EBNA1
protein) were stably expressed in the cell line. For the analysis
of the plasmid loss we measured the expression of the reporter gene
EGFP (or d1EGFP) with flow cytometry. In the case of plasmids
containing the PyV minimal core origin and BPV-1 MME the rate of
episomal loss was .about.6% per cell division in the absence of
geneticine selection. For plasmids containing PyV minimal origin
and EBV FR-element, the rate of episomal loss was faster
(.about.13%), but compared to the 22-30% rate of loss of the
control plasmids (pEGFP-C1 and pd1EGFP-N1), which contained neither
replication origin nor segregation element, this rate is still
significantly lower. The rate of loss of plasmids containing PyV
minimal core origin and FR-element (pFRG*) is also different from
the previously published results of the rate of loss of several
replicating plasmids that contained FR-element as stable
maintenance factor, where the rate of loss was 2.1-7.8%
(Wade-Martins et al., 1999) but it is very similar to the 15% rate
of loss previously estimated for oriP containing plasmids (Hung et
al., 2001). We have verified the requirement of FR-element in
long-term experiment. For that we have deleted FR element from the
pFRG-EGFP plasmid. Plasmids with or without FR element were
electroporated into CHOEBNALT85 cells. Samples for Southern-Blot
analysis were taken from different time-points (48 hours, 14, 21
and 22 days after transfection). As shown in FIG. 19, lines 1-4
plasmid (pFRG-EGFP) could be detected from all time points
observed. At the same time plasmid (pFRG-EGFP-FR without FR element
could be detected only 48 hours after transfection. In all other
time points no plasmid could be detected within CHOEBNALT85
cells.
[0080] The examples presented below are meant to be descriptive and
by no mean limiting the various embodiments of the present
invention.
Materials and Methods Used in the Examples
[0081] Plasmids
[0082] For constructing hybrid replicons (FIG. 1), containing PyV
origin (core origin), we used vector pUC19 as the basic backbone
where we cloned 1, 5 or 10 head-to-tail copies of high-affinity E2
binding site 9. PyV wt and core origin were amplified by PCR from
vectors pmu1046/CAT and pmu1047/CAT using primers Py4963
(5'-AGGGAGCTACTCCTGATG-3'') (SEQ ID NO:10) and Py174
(--CTACCACCACTCCGACTT-3'') (SEQ ID NO:11). Amplified PyV origin
fragments were digested with enzymes EheI and BelI and inserted
between BamHI and HincII sites of pUC19 vector containing different
number of BPV-1 E2 binding sites. In the vector the E2 binding
sites exists together with spacers.
[0083] For constructing hybrid replicons (FIG. 1), containing PyV
origin (core origin), we used vector pUC19 as the basic backbone
and FR element originated from EBV genome (Strain 95-8; GenBank:
V01555.2). FR element was cloned into the pUC19 containing Py core
origin (SEQ ID NO: 2). Sequence of FR element used in plasmids
comprised at least 16 EBNA1-binding sites separated by spacer
sequences. FIG. 1 shows and FR element containing 21 EBNA1 binding
sites and having a sequence according to SEQ ID NO: 1 For
construction of new generation pQMCF plasmids, modified pFRG vector
(pFRG-shorty-SV40 pA) without expression cassette for protein of
interest was used. Plasmids containing different promoters (CMV,
hEF1.alpha., heIF4a, RSV-LTR, .beta.-actine and hEF1.alpha.-HTLV)
and hEF1.alpha. intron in 5' position from protein of interest were
constructed in pUC19 cloning vector. DNA fragments containing
different promoters with intron sequence were cloned into
pFRG-shorty-SV40pA which contains also SV40 polyA sequence (FIG.
2). SV40 PolyA sequence regulating expression of Neomycine
resistance and gene of interest simultaneously. After that
multi-cloning sequence containing sites for single-cutting enzymes
for cloning of genes of interest were added into the vectors (Table
1).
[0084] For construction of new generation pQMCF plasmids, modified
pFRG vector (pFRG-shorty-SV40 pA) without expression cassette for
protein of interest was used. Plasmids containing different
promoters (CMV, hEF1.alpha., heIF4a, RSV-LTR, .beta.-actine and
hEF1.alpha.-HTLV) and hEF1.alpha. intron in 5' position from
protein of interest were constructed in pUC19 cloning vector. DNA
fragments containing different promoters with intron sequence were
cloned into pFRG-shorty-SV40pA which contains also SV40 polyA
sequence (FIG. 2). SV40 PolyA sequence regulating expression of
Neomycine resistance and gene of interest simultaneously. After
that multi-cloning sequence containing sites for single-cutting
enzymes for cloning of genes of interest were added into the
vectors (Table 1).
TABLE-US-00001 TABLE 1 pQMCF and pQMME vectores and promoters used
for expessin of protein Plasmid Promoter MCS pQMCF-1 CMV
Intron-Pfl23II-SfaAI-NheI-Bsp1407I-SgsI- Bsp119I-BoxI-polyA pQMCF-2
hEF1.alpha.-HTLV Intron-Pfl23II-SfaAI-NheI-Bsp1407I-SgsI-
Bsp119I-BoxI-polyA pQMCF-3 heIF4a
Intron-Pfl23II-SfaAI-NheI-Bsp1407I-SgsI- Bsp119I-BoxI-polyA pQMCF-4
h.beta.-actin Intron-Pfl23II-SfaAI-Bsp1407I-SgsI-
Bsp119I-BoxI-EcoRI-polyA pQMCF-5 RSV-LTR
Intron-Pfl23II-SfaAI-NheI-Bsp1407I-SgsI- Bsp119I-BoxI-polyA pQMCF-6
hEF1.alpha. Intron-Pfl23II-SfaAI-NheI-Bsp1407I-SgsI-
Bsp119I-BoxI-EcoRI-polyA
[0085] For construction of antibody- or VLP-expressing vectors
another expression cassette containing CMV, hEF1.alpha.-HTLV or
RSV-LTR promoter for expression of antibody light- or heavy chain
or gag protein was added into the pQMCF-1 plasmid.
[0086] Construction of cell lines. For construction of cell lines,
which express BPV-1 wt E2 protein and its mutant forms E39A and
R68A, the vector pBabePuro was linearized using enzyme SalI and was
ligated with equal amount of E2 expression vectors (pCGE2,
pCGE2/R68, pCGE2/E39), which were linearized with XhoI
endonuclease. 1 .mu.g of ligated hybrid plasmids was electroporated
into COP5 cell line. COP5 cell line is derived from mouse C127
cells (ATCC CRL-1804) and described in Tyndall et al. 1981.
Electroporation experiments were preformed with a Bio-Rad Gene
Pulser with capacitance and voltage settings of 975 .mu.F and 220
V. For selection puromycin (2 .mu.g/ml) was added. The expression
of the proteins was analyzed by Western blot.
[0087] A cell line which expresses wt E2 and carries neomycine
selection cassette was constructed by the same protocol described
above, using vector pBabeNeo instead of pBabePuro.
[0088] A cell line expressing PyV T-antigens and EBV EBNA1 protein
was generated as a result of transfection of the NotI linearized
plasmid pBabePuro/EBNA1 (EBNA1 coding sequence inserted into
EcorI/SalI sites in pBabePuro vector) into COP5 cell line and
selection for puromycin (2 .mu.g/ml). The expression of the
proteins was analyzed by Western blot. The cell line was named
COP5EBNA1/Puro.
[0089] Sometimes cell line CHO4.15 was used. This cell line is
derived from CHO-K1 cell line (ATCC CCL 61) and described in Ustav
1993. CHO, HEK293 and U2OS derived cell lines expressing EBV EBNA1,
PyV LT, BPV E2 were constructed using the same method as used for
construction of COP derived cell lines.
[0090] Cells and transfection. COP5 cells (Tyndall et al., 1981)
and its derivatives COP5E2/Puro, COP5E2/Neo COP5R68/Puro,
COP5E39/Puro, COP5EBNA1/Puro expressing polyomavirus T-antigens and
BPV-1 wt E2 or its mutant forms or EBNA1 were grown in Iscove's
modified Dulbecco's medium ("Difco") supplemented with 10% fetal
calf serum. For selection G418 (500 .mu.g/ml) or puromycin (2
.mu.g/ml) were added, depending on selection marker.
Electroporation experiments were performed with a Bio-Rad Gene
Pulser with capacitance and voltage settings of 975 .mu.F and 220
V, respectively.
[0091] COP5E2/Puro cells transfected with neomycin-constructs were
selected with G418 at 500 .mu.g/ml. COP5E2/Neo cells co-transfected
with pBabePuro (Morgenstern, J. P., and H. Land. 1990) were
selected with puromycin at 2 .mu.g/ml. After transfection with
plasmids carrying geneticine resistance marker and GFP coding
sequence, COP5EBNA1/Puro cell line was grown in IMDM medium
containing 500 .mu.g/ml G418 (medium contained no puromycin).
[0092] The CHOEBNALT 85 cells are adapted to serum-free suspension
culture in 1:1 mixture of CD CHO and 293 SFMII medium supplemented
with L-Glutamine, HT Supplement and puromycin (20 .mu.g/ml). CD CHO
Medium (Invitrogen Cat. No. 10743-029) supplemented with 8 mM
Lglutamine and 20 m1/1 HT Supplement and.cndot. 293 SFM II Medium
(Invitrogen Cat. No. 11686-029) supplemented with 4 mM
Lglutamine.
[0093] Electroporation were performed with a Bio-Rad Gene Pulser
with capacitance and voltage settings of 975. F and 230 V.
6.times.10.sup.6 CHOEBNALT 85 cells were transfected with pQMCF
plasmids. 48 hours after transfection G418 is added at final
concentration 700 .mu.g/ml.
[0094] The 293EBNALT 75 cells are adapted to serum-free suspension
culture in 1:1 mixture of Pro293s-CDM and 293 SFMII medium
supplemented with L-Glutamine and puromycin. To prepare a 293
medium for 293EBNALT 75 cells mix in equal amounts: Pro293s-CDM
(BioWhittaker.TM. Cat. No. 12002-026) and 293 SFM II Medium
(Invitrogen Cat. No. 11686-029), supplemented with 4 mM L-Glutamine
and puromycin to the final concentration of 0.8 .mu.g/mL. For
electroporation 4106 viable cells were taken. Electroporation
settings are 975 .mu.F and 150V. 48 hours after transfection G418
is added at final concentration 10 .mu.g/ml.
[0095] The U2OSEBNALTD3 cells are grown in DMEM high Glucose (4.5
g/L) with Sodium Pyruvate and Lglutamine (PAA E15-843 or
equivalent); 10% Foetal Bovine Serum (FBS)(PAA E15-151 or
equivalent) in the presence of Puromycin (2 .mu.g/mL). For
transfection 3.5 10.sup.6 cells from dish containing cells growing
in logarithmic growth phase with 70-80% confluency were used.
Electroporation settings are 250V; 975 .mu..F.
[0096] Southern Blot Analysis
[0097] Total DNA was extracted from cells following standard
protocol. Extraction of low-molecular-weight DNA from cells as well
as analysis of origin constructs levels in both low molecular
weight and total DNA preparation were performed as described
previously (Ustav and Stenlund, 1991; Piirsoo and Ustav 1996).
Specific probes were labeled with [.sup.32P]dCTP by
random-hexamer-primed synthesis using DecaLabel kit (Fermentas,
Lithuania). Hybridizing species were visualized by autoradiography.
Radioactive signals on the blots were quantified on PhosphorImager
using ImageQuant software (Molecular Dynamics, Amersham
Biosciences, UK).
[0098] Immunoprecipitation
[0099] Cells (1.5.times.10.sup.7) were lysed with ice-cold 1%
sodium dodecyl sulfate (SDS)-phosphate-buffered saline on ice,
collected in a 15--mil tissue culture tube, and sonicated. From
this step an aliquot for the Bradford assay was taken. SDS was
diluted to 0.1% by adding ice-cold radioimmunoprecipitation assay
(RIPA) buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 0.5%
dexycholate, 0.1 mM dithiotreitol (DDT), 0.5 mM
phenylmethylsulfonyl fluoride, protease inhibitors). The insoluble
fraction was sedimented by centrifugation at 5,000.times.g for 15
min. The soluble fraction was transferred to a new tube and
incubated with 5H4, 3E8, 1'E4 and 3F12 antibodies over night at 4
C. Then protein G-Sepharose (Amersham Biosciences) was added and
incubated for 1 h. Sepharose beads where washed three times with
RIPA buffer and resuspended in SDS loading buffer and subjected to
immunoblotting analysis with horseradish peroxidase-conjugated eE11
(subclone of MAb 3F12(antibody (Quattromed AS, Tartu, Estonia).
[0100] Immunoblotting
[0101] Total protein from the same number of cells lysed in
standard loading buffer supplemented with 100 mM DDT was separated
by electrophoresis on 8% polyacrylamide-SDS gel and transferred to
Immobilon-P membrane (Millipore, USA). Antibody 1E4 (Kurg et al.,
1999) was used to detect E2 proteins. Antibodies BM3167 and BM1083
(DPC Biermann) was used to detect EBNA1 protein.
Peroxydase-conjugated goat-anti mouse antibody and the enhanced
chemoluminescence detection kit (Amersham Biosciences) were used
for subsequent developing of the blot, using a standard protocol
provided by the supplier.
[0102] The plasmid rescue assay was performed for detection of the
episomal state of the plasmid as described previously in Mannik et
al 2003. Two micrograms of uncut genomic DNA was electrotransformed
in to Escherichia coli strain DH10B. The electorcompetent cells
were prepared and the transformations were performed using a Pulser
apparatus and 2-mm electroporation cuvette (Bio-Rad Laboratoires,
Hercules, Calif.) according to the manufacturer's instructions. The
cells were recovered by centrifugation and were grown on medium
containing ampiclillin at 100 .mu.g/ml. Plasmid DNA from single
colonies was purified and analyzed using restriction
endonucleases.
[0103] Flow Cytometry Analysis
[0104] EGFP expression was analysed by flow cytometry using
Becton-Dickinson FACSCalibur flow cytometer with associated
CellQuest software. 100 000-200 000 signals were analysed from each
sample. The threshold for autofluorescence was set to 99% of the
signals from the mock-transfected control cells. All the signals
above the threshold were considered to correspond to EGFP-positive
cells. For calculating the episomal rates of loss in the FIG. 8,
EGFP expression data was analyzed on days 0 and 12 (pEGFP-C1,
pd1EGFP-N1), on days 0 and 55 for pMMEG, on days 0 and 37 for
pMMEG* and on days 0 and 30 for pFRG*. For this calculation first
order rate-of-loss model was used: rate of loss .lamda.=(-1/t)(ln
N.sub.t/N.sub.0). N.sub.o is the percentage of the green cells at
the beginning of the experiment of non-selective conditions and
N.sub.t is the percentage of the green cells after t
generations.
[0105] Expression of Luciferase Analysis
[0106] The expression analysis was done in CHO4.15 E2 cell line
with plasmids carrying different regulatory elements and
recombinant EGFP-luciferase gene. The cells were electroporated
with the equimolar amounts of the EGFP-luciferase vectors. For
negative control the cells were transfected with carrier DNA only.
In different time-point, the cells were washed with PBS and lyzed
with appropriate amount of 1*CCLR ragent (Promega). Luciferase
activities in the samples were measured using Luciferase Assay
System kit (Promega) and plate reading luminometer (Tecan).
Different dilutions of the samples in 1*CCLR buffer were used for
verifying that all measurements are done at linear range. For
normalisation of the activities of to the total protein in the
samples, these were diluted 4 times with water. Thereafter, BCA
assay kit (Pierce) was used for measurements.
Western-Blot Analysis
[0107] Total protein from the same number of cells or supernatant
of equal number of cells were taken for analysis. Cells were lysed
in standard loading buffer (Laemmli buffer) supplemented with 100
mM DDT. Supernatant of the cells was mixed with Laemmli buffer with
or without DDT. Samples were separated by electrophoresis on 8 to
12% polyacrylamide-SDS gel and transferred to Immobilon-P membrane
(Millipore, USA). Antibody 1E4 (Kurg et al., 1999) was used to
detect E2 proteins. Antibodies BM3167 and BM1083 (DPC Biermann) was
used to detect EBNA1 protein. Peroxydase-conjugated goat-anti mouse
antibody and the enhanced chemoluminescence detection kit (Amersham
Biosciences) were used for subsequent developing of the blot, using
a standard protocol provided by the supplier. For detection of
DNaseI, CDNF or ETAR protein expression appropriate antibodies were
used and for visualization of expression alkaline-phosphatase
conjugated secondary antibodies were used.
Example 1
PyV Hybrid Origin Constructs Including PyV Core Origin and EBV FR
Element and Construction of Plasmids for Extended Episomal
Maintenance
[0108] FIG. 1 schematically shows mouse polyoma virus core origin
of replication (SEQ ID NO:2) and FR (Family of repeats) element.
Here, FR element (SEQ ID NO: 1) contains 21 possible EBNA-1 binding
sequences. Table 1 above and FIG. 21 show the novel QMCF-plasmids
according to this disclosure. pQMCF1 contains CMV promoter-driven
expression cassette. pQMCF2 contains EF1.alpha.-HTLV
promoter-driven expression cassette. EF1.alpha.-HTLV promoter
GenBank J04617.12 and HTLV leader sequence GenBank AB513134).
pQMCF-3 contains heIF4a promoter-driven expression cassette.
(heIF4a GenBank J04617.1) pQMCF-4 contains .beta.-actine
promoter-driven expression cassette. pQMCF-5 contains RSV-LTR
promoter-driven expression cassette.
Example 2
Comparison of BPV-1 MME and EBV FR Element in Providing
Segregation/Partitioning Function to the PyV Core Origin
Plasmids
[0109] Maintenance of plasmids containing PyV core origin, MME or
FR, selection marker (geneticine resistance) and green fluorescent
protein marker (either long half-life EGFP or short half-life
d1EGFP) was analyzed by flow cytometry. The flow cytometry analysis
was conducted in the COP5EBNA1/Puro cell line with plasmid
containing PyV core origin, FR-element, selection marker
(geneticine resistance) and green fluorescent protein marker (short
half-life d1EGFP) (pFRG*). In this case the replication function of
the plasmid is provided by PyV core origin and LT protein and the
segregation/partitioning function is provided by FR-element and
EBNA1 protein of the EBV. The results are similar to the flow
cytomery analysis with plasmids pMMEG and pMMEG* in COP5E2/Puro
cell line. Transfected cells were grown in continuous culture in
the presence or absence of geneticine for up to 75 days. Selection
of the transfected COP5EBNA1/PuroFRG* for geneticine resulted in
the cell culture, which had approximately 40% d1EGFP positive cells
(FIG. 20 D). When the geneticine selection was removed the
percentage of d1EGFP-positive cells decreased from 40% to 1% in 30
days. When the geneticine selection on COP5E2/PuropFRG* cell line
was restored at this point, the proportion of EGFP expressing cells
increased back to the initial level (FIG. 20D). These results show
that episomal persistence of the plasmid occurs with certain
efficiency, which is different from 100%. Clearly also, EBNA1/FR
and E2/MME elements confer comparablee segregation/partitioning
funcitions for the PyV core origin reporter plasmids in the cell
models.
[0110] To exclude the possibility that the loss of EGFP
fluorescence is due to inactivation of the promoter of EGFP, we
also analyzed the DNA content in the cells. After removal of
geneticin selection total DNA was extracted from cells and digested
with MluI (linearizes pMMEG* and pFRG* plsamids) and DpnI. Equal
amonts of total DNA were then anlayzed using Southern blotting with
a radiactively labelled probe against the pMMEG* or pFRG* plasmid.
The loss of the episomal plasmid DNA from the cells grown without
Geneticin selection correlates with the flow cytometry analysis
(results not shwon). On the other hand, these results indicate that
EGFP fluorescence was indeed measured from plasmids which exists in
the episomal state. In the cse of pasmid integration the
hybridization signals remained constant.
Example 3
Production of Recombinant Proteins and Generation of Cell-Based
Assay (CBA) Test-System for Development Drug Candidates
[0111] We have constructed set of new expression plasmids
containing polyoma virus (PyV) core origin (SEQ ID NO: 2) in
combination with Family of Repeats (FR) of Epstein-Barr Virus, an
element for extrachromosomal genome maintenance of Epstein-Barr
virus (EBV) or MME (minichromosome maintenance element) from Bovine
Papilloma virus (BPV-1) in proliferating cells. (construction of
the plasmids is described above and in previous patent application
Ser. No. 11/351,809 which is incorporated herein by reference; and
plasmids are shown in FIG. 22). Such hybrid origin uses mouse
polyomavirus (PyV) Large-T antigen for initiation of plasmid
replication during S-phase of the cell cycle and EBV EBNA-1 or BPV
E2 protein for segregation/partitioning of the extrachromosomal
plasmid into the daughter cells during cell division. We engineered
six different promoters--four human originated cellular promoters
(EF1.alpha.; EF1.alpha.-HTLV; heIF 4a; .beta.-actine) and two viral
promoters Cytomegalovirus immediate early (CMV) and Rous Sarcoma
Virus proviral Long Terminal Repeat (RSV-LTR) driven expression
cassettes in new expression vectors (FIG. 21). Different strength
of selected promoters allows modulation of the protein of interest
expression depending on purpose of use of expression system. In all
cases the expression cassettes were equipped with hEF Lalpha.
intron (GenBank J04617.1) locating at 5' position from gene of
interest coding sequence. SV40 polyadenylation (polyA) sequence
regulating expression of Neo/Km and gene of interest simultaneously
is used in protein of interest expression cassettes. New generation
plasmids are suitable for expression of recombinant proteins or
target-proteins for generation of CBA-s. Cell lines for expression
of recombinant proteins and for generation of cell based assays are
different. For protein expression modified suspension cell lines
based on CHO or HEK293 are used. For generation of CBA-modified
CHO, HEK293, U2OS adherent cell lines are suitable.
A. Testing of pQMCF and pQMME Expression Vectors Using Gaussia
Luciferase as Test System in Transient Expression of Reporter
Gene
[0112] To characterize new expression vectors we use gaussia
luciferase as reporter system. For analysis of pQMCF vectors
suspension CHOEBNALT85 and 293EBNALT75 cell line was used, for
analysis of pQMME vectors CHOmLTE2 63 cell line was used. In FIG.
3A, results of gaussia luciferase activities using different
vectors in suspension CHOEBNALT85 and 293EBNALT75 is shown. In FIG.
3B, results of gaussia luciferase activities using different
vectors in suspension CHOmLTE2 is shown.
[0113] Activity of luciferase was measured 24 hours after
transfection. As shown in FIG. 2A, CMV promoter in pQMCF-1 is
approximately two times higher compared to our initial vector
pQMCF-CMV-0.1 (modified plasmid pFRG). Also in pQMME-1 CMV promoter
exhibits highest activity compared to all other promoters. All
other promoters in pQMCF show two to four times lower activity
compared to pQMCF-1 vector (FIG. 3). In pQMME expression vectors
hEF1.alpha., heIF4a and RSV-LTR promoters are approximately
two-times weaker than CMV promoter. Other two promoters
(.beta.-actin and hEF1.alpha.) exhibit 10-15 times lower activity
compared to CMV promoter. Differences in promoter strength in QMCF
cell lines makes these useful for expression of those proteins
which high-level expression is toxic to the cells and allows
modulate protein of interest expression level.
[0114] Activity of luciferase was measured 24 hours after
transfection. As shown in FIG. 3, CMV promoter in pQMCF-1 is
approximately two times higher when compared to another
pQMCF-CMV-0.1 vector containing different configuration of
expression cassette (CMV-promoter and 3' intron). All other
promoters show two to four times lower activity as compared to
pQMCF-1 vector (FIG. 3) which makes these promoters useful for
expression of proteins which are toxic to the cells in
high-levels.
B. Use of the Novel Constructs, Vectors and Cell Lines of this
Disclosure for Expression of Human Neurotrophic Factors e.g.
Expression of Human CDNF
[0115] cDNA encoding human CDNF protein (UniProtKB/Swiss-Prot
Q49AHO) was cloned into all new pQMCF vectors (pQMCF-1 to pQMCF-6,
vectors are shown in FIG. 21) and expressed in CHOEBNALT85 cell
line. In FIG. 4, growth of the CHOEBNALT85 cells expressing CDNF
[pQMCF-1-CDNF] is shown.
[0116] The expression of the CDNF was analyzed in the cells as well
as in culture media after transient transfection using
electroporation of the QMCF vector encoding CDNF. Transfection
efficiency of the CHOEBNALT85 suspension cells was around 85%. As
shown on FIG. 5, CDNF is expressed effectively in cells carrying
the leader peptide as well as processed CDNF. At the same time only
processed CDNF can be found in the culture media, indicating that
only processed form of the CDNF is secreted out of the cells.
Approximately 9-12 days after transfection production cell bank
could be generated (1 10.sup.7 cells/vial). Human CDNF expression
by cells taken from expression cell bank is comparable to the
expression started by newly transfected CHOEBNALT85 cells.
Stability of pQMCF Expression Vectors
[0117] We have seen that one important reason for plasmid
instability during production phase is the dependence of the
protein of interest on the expression level. The stress generated
due to the over-expression of the protein depends strongly on the
physiological effect of the protein and level of expression. We
have analyzed by Southern-Blot method the intactness of the input
plasmid in CHOEBNALT85 suspension cell line 48 h after
transfection, after selection, and before production phase and
during production. As shown in FIG. 5 in the CDNF expression vector
(pQMCF-2-CDNF) containing EF1.alpha.-HTLV promoter (HTLV leader
sequence--GeneBank AB513134; hEF1. alpha. promoter--GenBank
J04617.1) exhibits the least plasmid rearrangements and reduction
of the plasmid copy-number as compared to CMV promoter driven
vector (pQMCF-1-CDNF) both during the expression of CDNF originated
from transfection and production cell bank (for more detailed
information is provided in FIG. 6 legend).
C. Expression of Recombinant Bovine DNaseI Using the Novel
Constructs Vectors and Cell Lines of this Disclosure
[0118] cDNA of Bovine DNaseI protein was cloned into the different
pQMCF expression vectors (pQMCF-1 to pQMCF-6, see FIG. 21). For
production of CDNF 1 .mu.g of plasmid DNA (expression vector) was
used for transfection of the 4 10.sup.6 CHOEBNALT85 suspension
cells. 48 hours after transfection 700 .mu.g/ml of G418 is added to
the growth medium. Duration of G418 selection depends on
transfection efficiency and rate of toxicity of protein of interest
to the appropriate cell line. In most cases 75-80% transfection
efficiency is achieved with CHOEBNALT85 suspension cells. After
G418 selection production phase (7 days) is performed. Production
phase is started with 610.sup.6 cells/ml, temperature is shifted to
30.degree. C., feed is added to the cell culture (FIG. 7). After
production phase supernatant is clarified and frozen prior the use.
Similarly to CDNF production, there was no significant difference
between bovine DNaseI production started from transfected cells or
from production cell bank (data not shown).
[0119] For selection of plasmid-containing cells 48 h after
transfection G418 (700 .mu.g/ml) was added and cells were grown
additionally 10 days. 4 vials (1.times.107) cells were frozen as
expression cell bank. For production of bovine DNaseI in 200 ml
volume 1 vial from expression cell bank was taken and grown 6 days
to reach to the 200 ml volume (4.times.10.sup.6 cells/ml). After
7-days of production phase the viability of culture was 83%
containing 8 10.sup.6 cells/ml. Supernatant of the cells was
clarified, frozen down and analyzed for DNaseI expression level
using semi-quantitative Western-Blot method (FIG. 8).
[0120] Bovine DNaseI expression by cells taken from expression cell
bank is comparable to the expression started by newly transfected
CHOEBNALT85 cells (FIG. 9).
[0121] Stability of pQMCF expression vectors. We have analyzed by
Southern-Blot analysis the stability of plasmid in CHOEBNALT85
suspension cell line 48 h after transfection, before production
phase and during production. As shown in FIG. 10, new generation
pQMCF vectors are much more stable (FIG. 10, Lines 5-26) as
compared to pFRG-type plasmid (FIG. 10, Lines 1-4).
Example 4
Examples of Cell-Based Assays (CBA) Generated by QMCF Plasmids and
Cell Lines
[0122] We developed adherent cell lines based on U205, CHO and
HEK293 stably expressing Mouse Polyomavirus (PyV) Large-T antigen
for initiation of plasmid replication during S-phase of the cell
cycle and Epstein-Barr Virus (EBV) EBNA-1 protein for
segregation/partitioning of the extrachromosomal plasmid into the
daughter cells during cell division. Adherent, monolayer growth of
cell culture allows use of these cell lines as cell-based assay
test-systems. New generation pQMCF expression plasmids (pQMCF-1 to
pQMCF-6, as shown in FIG. 21) are suitable for this approach. cDNA
of target protein is inserted into the pQMCF expression vector,
adherent QMCF cell lines are transfected by electroporation,
plasmid-containing cells are selected by G418 and protein
expression or translocation is detected. For detection target
proteins are fused with EGFP* (in the case of target-protein-EGFP
fusion systems) or by N- or C-terminal epitope-tag sequences.
Appropriate fluorescent monoclonal anti-epiotpe-tag antibodies are
used for protein localization visualization. We have developed 4
different epitope-tags, which allow detecting more than one
target-protein localization within same cell-based assay
simultaneously. Detection of more than one target-protein within
same cell gives good possibility to reconstruct signal-pathways and
investigate possible side effects of drug candidate.
[0123] Epitope-tag sequences used for target-protein detection:
TABLE-US-00002 (SEQ ID NO: 20) 6G5 - TVKAKLLSVE (SEQ ID NO: 21)
5E11 - SSTSSDFRDR (SEQ ID NO: 22) HIV-1 p24 - TPQDLNTMLNTVGGH (SEQ
ID NO: 23) 9A2.1 - LSSKAVNHIRSVWKDLLEDT
A. Endothelin a Receptor (ETAR-EGFP) Translocation Assay.
[0124] Adherent CHOEBNALT cells were transfected with expression
plasmid containing ETAR-EGFP fusion protein under the control of
CMV promoter. 800 ng of expression plasmid was transfected into 4
10.sup.6 CHOEBNALT adherent cells. 48 hours after transfection 400
.mu.g/ml of G418 is added to the growth medium. Duration of G418
selection depends on transfection efficiency and rate of toxicity
of target protein into the appropriate cell line. After G418
selection cells were treated with 300 nM endothelin (ETAR
internalization effector) and visualized by fluorescent microscopy
(FIG. 11).
B. Endothelin a Receptor (ETAR-5E11 Tag) Detection Using
Fluorescent Dye Labelled Antibody Directed Against Epitope Tag.
[0125] Adherent U2OSEBNALTD3 cells were transfected with expression
ETAR-5E11tag-fusion plasmid. 800 ng of expression plasmid was
transfected into 4.times.10.sup.6 CHOEBNALT adherent cells. 48
hours after transfection cells were fixed and permeabilized and IF
(immunofluorescence) analysis was performed using Thermo Scientific
DyLight488 fluorescent dye conjugated anti 5E11 epitope-tag
antibody (FIG. 12).
Example 5
Expression of Virus Like Particles (VLP-s)
[0126] Expression of viral envelope or capside proteins results
self-assembly of virus like particles (VLP-s). VLPs could be
produced in different cell systems, including mammalian, insect,
yeast and plant cells. One good possibility to produce membrane
proteins in functional and correct conformational manner is
expression of these proteins in composition of virus like particles
(VLP-s). VLP-s could be used for different approaches e.g. vaccine
development, investigation of receptor functions and also for
expression of different membrane proteins.
[0127] We have constructed expression vectors for production of
membrane-bound proteins eg. ion-channels, receptors, viral
glycoproteins in composition of gag protein-based VLP-s using new
generation pQMCF vectors (FIG. 21,23) and the mammalian cell lines
as described in this application. All expression vectors for
production of VLP-s carry maintenance, replication and antibiotic
selection elements as described above.
[0128] Two different types of expression vectors are constructed:
single- or two-expression cassettes-containing vectors. (FIGS. 12A
and B respectively). Also, two different types of gag proteins,
HIV-1 and MLV (murine leukemia virus) gag proteins are used for
formation of VLP-s.
[0129] As it is shown in FIG. 9, strong CMV promoter is used for
expression of gag protein or in the case of single expression
cassette-containing vector also protein of interest expression. Gag
and protein of interest expression is divided in single-expression
cassette vector by foot-and-mouth disease virus (FMDV) 2A peptide
(FIG. 10A). As expression of ion channels and receptor proteins is
sometimes toxic to the cells, we use two "weaker" RSV-LTR or
hEF1.alpha.-HTLV promoters for protein of interest expression.
A. Expression of Endotheline A Receptor (ETAR) in HIV-1 gag
Protein-Based VLP-s
[0130] Production of VLP-s by Use of Novel Constructs, Vectors and
Cell Lines of this Disclosure
[0131] We found in our experiments that for production of VLP-s
HEK293-based cell line 293EBNALT75 gives more stable VLP expression
than CHOEBNALT85 cell line. For production of VLP-s 1 .mu.g of
plasmidial DNA (expression plasmid) for transfection of the
4.times.10.sup.6 293EBNALT75 cells. 48 hours after transfection 10
.mu.g/ml (293EBNALT75) G418 is added to the growth medium. Duration
of G418 selection depends on transfection efficiency and rate of
toxicity of protein of interest to the appropriate cell line. In
most cases 30-50% transfection efficiency is achieved with
293EBNALT75 cells. After G418 selection production phase (3-5 days)
is performed. Production phase is started with 6.times.10.sup.6
cells/ml, temperature is shifted to 30.degree. C. After production
phase supernatant is clarified and VLP-s are purified by
ultracentrifugation or precipitation and gel-filtration.
[0132] In FIG. 14, production of ETAR-pseudotyped HIV-gag based
VLP-s is shown. ETAR protein (P25101) is tagged by 5E11 epitope tag
(U.S. Pat. No. 7,189,540) and appropriate antibody was used for
detection of protein expression. VLP-s are produced by 293EBNALT75
cell line in small, 20 ml medium volume. Compared to cells
expressing only ETAR protein (Line 2), type II ETAR-pseudotyped
VLP-s are formed efficiently (Line 3, FIG. 14).
Example 6
Production of Recombinant Monoclonal Antibodies
[0133] All expression vectors for production of monoclonal
antibodies carry maintenance, replication and antibiotic selection
elements as described above. Antibody expression vectors contain
two separate expression cassettes (FIG. 24). All DNA elements
(promoters, introns, polyA sequences) in expression cassettes were
chosen as different as possible.
[0134] Two different types of antibody expression vectors are
constructed: single- or two-expression cassettes-containing
vectors. (FIGS. 15 A and B respectively).
A. Expression of Partially Humanized (Chimeric) Tyrosinase a
Antibodies
[0135] We have validated methods for generation of production
system for recombinant murine- or partially humanized (chimeric)
tyrosinase A antibodies. cDNA-s encoding variable regions of IgG1
antibody light- or heavy chains were generated from appropriate
hybridoma culture and recombinant IgG1 antibody-expressing vectors
were generated fusing antibody variable regions-encoding DNA
fragments to the antibody constant regions. Recombinant antibody
expression plasmids were transfected into the CHOEBNALT85 cells and
antibodies were produced, purified and in vitro antigene-binding
affinity was measured in comparison with tyrosinase A antibody
expressed and purified from hybridoma culture.
[0136] For production of partially humanized (chimeric) tyrosinase
A antibody 1 .mu.g of plasmid DNA (expression vector) was used for
transfection of the 4.times.10.sup.6 CHOEBNALT85 cells. 48 hours
after transfection 700 .mu.g/ml of G418 is added to the growth
medium. Duration of G418 selection depends on transfection
efficiency and rate of toxicity of protein of interest to the
appropriate cell line. In most cases 75-80% transfection efficiency
is achieved with CHOEBNALT85 cells. After G418 selection production
phase (6 days) is performed. Production phase is started with
6.times.10.sup.6 cells/ml, temperature is shifted to 30.degree. C.,
feed is added to the cell culture (FIG. 16). After production phase
supernatant is clarified and frozen prior usage.
[0137] In FIG. 18 expression of partially humanized (chimeric)
tyrosinase A antibody was verified by Western-Blot analysis. In
FIG. 16 expression of partially humanized (chimeric) tyrosinase A
antibody was analyzed from different time points of growth. As
shown in FIG. 18, significantly larger amount of monoclonal
antibody is detected at the end of production (FIG. 18, Line
4).
Example 7
Stability of pQMME in U2OSEBNALTE2 Cell Lines
[0138] Southern-Blot Analysis of pQMME Plasmid in U2OSEBNALTE2 Cell
Lines
[0139] We have analyzed stability of plasmid pQMME (FIG. 22) in
U2OSEBNALTE2 cell line. For construction of U2OSEBNALTE2 cell line
BPV E2 protein-expressing concatamer is inserted into U2OSEBNALTD3
cell line. EGFP-expression plasmid pQMME-1-EGFP is transferred into
the U2OSEBNALTE2 cell line and cells are grown under the G418
selection constantly passaging for more than 8 weeks. Samples for
plasmid analyses were taken 48 hours, 32 days and 52 days after
transfection. As shown in FIG. 25, plasmid remains stable during
all experiment.
REFERENCES
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bovine papillomavirus type 1 E2 protein. Journal of Virology
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Long-term episomal maintenance of bovine papillomavirus type 1
plasmids is determined by attachment to host chromosomes, which is
mediated by the viral E2 protein and its binding sites. Journal of
Virology 73:4404-12. [0142] 3. Kurg, R., J. Parik, E. Juronen, T.
Sedman, A. Abroi, I. Liiv, U. Langel, and M. Ustay. 1999. Effect of
bovine papillomavirus E2 protein-specific monoclonal antibodies on
papillomavirus DNA replication. Journal of Virology 73:4670-7.
[0143] 4. Mannik, A., M. Piirsoo, K. Nordstrom, E. Ustav, B.
Vennstrom, and M. Ustay. 2003. Effective generation of transgenic
mice by Bovine papillomavirus type 1 based self-replicating plasmid
that is maintained as extrachromosomal genetic element in three
generations of animals. Plasmid 49:193-204. [0144] 5. Morgenstern,
J. P., and H. Land. 1990. Advanced mammalian gene transfer: high
titre retroviral vectors with multiple drug selection markers and a
complementary helper-free packaging cell line. Nucleic Acids
Research 18:3587-96. [0145] 6. Nilsson, M., M. Forsberg, Z. Y. You,
G. Westin, and G. Magnusson. 1991. Enhancer effect of bovine
papillomavirus E2 protein in replication of polyomavirus DNA.
Nucleic Acids Research 19:7061-5. [0146] 7. Piirsoo, M., E. Ustav,
T. Mandel, A. Stenlund, and M. Ustay. 1996. Cis and trans
requirements for stable episomal maintenance of the BPV-1
replicator. EMBO Journal 15:1-11. [0147] 8. Tyndall, C., G. La
Mantia, C. M. Thacker, J. Favaloro, and R. Kamen. 1981. A region of
the polyoma virus genome between the replication origin and late
protein coding sequences is required in cis for both early gene
expression and viral DNA replication. Nucleic Acids Research
9:6231-50. [0148] 9. Ustav E, Ustav M, Szymanski P, Stenlund A.
1993 The bovine papillomavirus origin of replication requires a
binding site for the E2 transcriptional activator. Proc. Natl.
Acad. Sci. USA 90 (3): 898-902 [0149] 10. Ustav, M., and A.
Stenlund. 1991. Transient replication of BPV-1 requires two viral
polypeptides encoded by the E1 and E2 open reading frames. EMBO
Journal 10:449-57. [0150] 11. Wade-Martins, R., J. Frampton, and M.
R. James. 1999. Long-term stability of large insert genomic DNA
episomal shuttle vectors in human cells. Nucleic [0151] 12. Guo, Z.
S., and M. L. DePamphilis. 1992. Specific transcription factors
stimulate simian virus 40 and polyomavirus origins of DNA
replication. Mol Cell Biol 12:2514-24. Acids Research 27:1674-82.
[0152] 13. Hung, S.C., M. S. Kang, and E. Kieff. 2001. Maintenance
of Epstein-Barr virus (EBV) oriP-based episomes requires
EBV-encoded nuclear antigen-1 chromosome-binding domains, which can
be replaced by high-mobility group-I or histone H1. Proceedings of
the National Academy of Sciences of the United States of America
98:1865-70.
Sequence CWU 1
1
271622DNAartificial sequencechemically synthesized 1gggtatcata
tgctgactgt atacgcatga ggatagcata tgctacccgg atacagatta 60ggatagcata
tactacccag atatagatta ggatagcata tgctacccag atatagatta
120ggatagccta tgctacccag atataaatta ggatagcata tactacccag
atatagatta 180ggatagcata tgctacccag atatagatta ggatagccta
tgctacccag atatagatta 240ggatagcata tgctacccag atatagatta
ggatagcata tgctatccag atatttgggt 300agtatatgct acccagatat
aaattaggat agcatatact accctaatct ctattaggat 360agcatatgct
acccggatac agattaggat agcatatact acccagatat agattaggat
420agcatatgct acccagatat agattaggat agcctatgct acccagatat
aaattaggat 480agcatatact acccagatat agattaggat agcatatgct
acccagatat agattaggat 540agcctatgct acccagatat agattaggat
agcatatgct atccagatat ttgggtagta 600tatgctaccc atggcaacat ta
6222212DNAartificial sequencechemically synthesized 2gccagtcctg
ttttgacaag ttgcctctgg aagcctctac aatgcctctc ttctttttct 60ccagagtaag
cggaggccag gggcccccgg cctctgctta atactaaaaa aaacagctgt
120tgtcatagta atgattgggt ggaaacattc taggcagatc tagtcgggag
gaaaattact 180gtgttggagg ccctccgcca tcttctgaag ct
212319DNAartificial sequencechemically synthesized 3cgggtatcat
atgctgact 19419DNAartificial sequencechemically synthesized
4aggatagcat atgctaccc 19519DNAartificial sequencechemically
synthesized 5aggatagcat atactaccc 19619DNAartificial
sequencechemically synthesized 6aggatagcct atgctaccc
197206DNAartificial sequencechemically synthesized 7ctgtaccgtt
gccggtcgga tctgtaccgt tgccggtcgg atctgtaccg ttgccggtcg 60gatctgtacc
gttgccggtc ggatctgtac cgttgccggt cggatctgta ccgttgccgg
120tcggatctgt accgttgccg gtcggatctg taccgttgcc ggtcggatct
gtaccgttgc 180cggtcggatc tgtaccgttg ccggtc 2068244DNAHomo sapiens
8gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg
60gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg
120atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat
ataagtgcag 180tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc
cagaacacag ctgaagcttc 240gagg 2449528DNAHomo sapiens 9ctagatgatt
tccttcatcc ctggcacacg tccaggcagt gtcgaatcca tctctgctac 60aggggaaaac
aaataacatt tgagtccagt ggagaccggg agcagaagta aagggaagtg
120ataaccccca gagcccggaa gcctctggag gctgagacct cgcccccctt
gcgtgatagg 180gcctacggag ccacatgacc aaggcactgt cgcctccgca
cgtgtgagag tgcagggccc 240caagatggct gccaggcctc gaggcctgac
tcttctatgt cacttccgta ccggcgagaa 300aggcgggccc tccagccaat
gaggctgcgg ggcgggcctt caccttgata ggcactcgag 360ttatccaatg
gtgcctgcgg gccggagcga ctaggaacta acgtcatgcc gagttgctga
420gcgccggcag gcggggccgg ggcggccaaa ccaatgcgat ggccggggcg
gagtcgggcg 480ctctataagt tgtcgatagg cgggcactcc gccctagttt ctaaggaa
5281018DNAartificial sequencechemically synthesized 10agggagctac
tcctgatg 181118DNAartificial sequencechemically synthesized
11ctaccaccac tccgactt 18121528DNAHomo sapiens 12ctagagttcc
atgtccttat atggactcat ctttgcctat tgcgacacac actcaatgaa 60cacctactac
gcgctgcaaa gagccccgca ggcctgaggt gcccccacct caccactctt
120cctatttttg tgtaaaaatc cagcttcttg tcaccacctc caaggagggg
gaggaggagg 180aaggcaggtt cctctaggct gagccgaatg cccctctgtg
gtcccacgcc actgatcgct 240gcatgcccac cacctgggta cacacagtct
gtgattcccg gagcagaacg gaccctgccc 300acccggtctt gtgtgctact
cagtggacag acccaaggca agaaagggtg acaaggacag 360ggtcttccca
ggctggcttt gagttcctag caccgccccg cccccaatcc tctgtggcac
420atggagtctt ggtccccaga gtcccccagc ggcctccaga tggtctggga
gggcagttca 480gctgtggctg cgcatagcag acatacaacg gacggtgggc
ccagacccag gctgtgtaga 540cccagccccc ccgccccgca gtgcctaggt
cacccactaa cgccccaggc ctggtcttgg 600ctgggcgtga ctgttaccct
caaaagcagg cagctccagg gtaaaaggtg ccctgccctg 660tagagcccac
cttccttccc agggctgcgg ctgggtaggt ttgtagcctt catcacgggc
720cacctccagc cactggaccg ctggcccctg ccctgtcctg gggagtgtgg
tcctgcgact 780tctaagtggc cgcaagccac ctgactcccc caacaccaca
ctctacctct caagcccagg 840tctctcccta gtgacccacc cagcacattt
agctagctga gccccacagc cagaggtcct 900caggccctgc tttcagggca
gttgctctga agtcggcaag ggggagtgac tgcctggcca 960ctccatgccc
tccaagagct ccttctgcag gagcgtacag aacccagggc cctggcaccc
1020gtgcagaccc tggcccaccc cacctgggcg ctcagtgccc aagagatgtc
cacacctagg 1080atgtcccgcg gtgggtgggg ggcccgagag acgggcaggc
cgggggcagg cctggccatg 1140cggggccgaa ccgggcactg cccagcgtgg
ggcgcggggg ccacggcgcg cgcccccagc 1200ccccgggccc agcaccccaa
ggcggccaac gccaaaactc tccctcctcc tcttcctcaa 1260tctcgctctc
gctctttttt tttttcgcaa aaggagggga gagggggtaa aaaaatgctg
1320cactgtgcgg cgaagccggt gagtgagcgg cgcggggcca atcagcgtgc
gccgttccga 1380aagttgcctt ttatggctcg agcggccgcg gcggcgccct
ataaaaccca gcggcgcgac 1440gcgccaccac cgccgagacc gcgtccgccc
cgcgagcaca gagcctcgcc tttgccgatc 1500cgccgcccgt ccacacccgc cgaccggt
152813606DNAHuman cytomeglovirus 13ggctggatcg gtcccggtgt cttctatgga
ggtcaaaaca gcgtggatgg cgtctccagg 60cgatctgacg gttcactaaa cgagctctgc
ttatatagac ctcccaccgt acacgcctac 120cgcccatttg cgtcaatggg
gcggagttgt tacgacattt tggaaagtcc cgttgatttt 180ggtgccaaaa
caaactccca ttgacgtcaa tggggtggag acttggaaat ccccgtgagt
240caaaccgcta tccacgccca ttgatgtact gccaaaaccg catcaccatg
gtaatagcga 300tgactaatac gtagatgtac tgccaagtag gaaagtccca
taaggtcatg tactgggcat 360aatgccaggc gggccattta ccgtcattga
cgtcaatagg gggcgtactt ggcatatgat 420acacttgatg tactgccaag
tgggcagttt accgtaaata ctccacccat tgacgtcaat 480ggaaagtccc
tattggcgtt actatgggaa catacgtcat tattgacgtc aatgggcggg
540ggtcgttggg cggtcagcca ggcgggccat ttaccgtaag ttatgtaacg
gggcgagctc 600gaattc 60614532DNAartificial sequencechemically
synthesized 14gctccggtgc ccgtcagtgg gcagagcgca catcgcccac
agtccccgag aagttggggg 60gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg
cggggtaaac tgggaaagtg 120atgtcgtgta ctggctccgc ctttttcccg
agggtggggg agaaccgtat ataagtgcag 180tagtcgccgt gaacgttctt
tttcgcaacg ggtttgccgc cagaacacag ctgaagcttc 240gaggggctcg
catctctcct tcacgcgccc gccgccctac ctgaggccgc catccacgcc
300ggttgagtcg cgttctgccg cctcccgcct gtggtgcctc ctgaactgcg
tccgccgtct 360aggtaagttt aaagctcagg tcgagaccgg gcctttgtcc
ggcgctccct tggagcctac 420ctagactcag ccggctctcc acgctttgcc
tgaccctgct tgctcaactc tacgtctttg 480tttcgttttc tgttctgcgc
cgttacagat ccaagctgtg accggcgcct ac 53215269DNAsimian virus
15aattaacatt taaatgatct accacatttg tagaggtttt acttgcttta aaaaacctcc
60cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta
120ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
aataaagcaa 180tagcatcaca aatttcacaa ataaagcatt tttttcactg
cattctagtt gtggtttgtc 240caaactcatc aatgtatctt atcatgtcg
26916527DNARous sarcoma virus 16ctgctccctg cttgtgtgtt ggaggtcgct
gagtagtgcg cgagcaaaat ttaagctaca 60acaaggcaag gcttgaccga caattgcatg
aagaatctgc ttagggttag gcgttttgcg 120ctgcttcgcg atgtacgggc
cagatatacg cgtatctgag gggactaggg tgtgtttagg 180cgaaaagcgg
ggcttcggtt gtacgcggtt aggagtcccc tcaggatata gtagtttcgc
240ttttgcatag ggagggggaa atgtagtctt atgcaatact cttgtagtct
tgcaacatgg 300taacgatgag ttagcaacat gccttacaag gagagaaaaa
gcaccgtgca tgccgattgg 360tggaagtaag gtggtacgat cgtgccttat
taggaaggca acagacgggt ctgacatgga 420ttggacgaac cactgaattc
cgcattgcag agatattgta tttaagtgcc tagctcgata 480caataaacgc
catttgacca ttcaccacat tggtgtgcac ctccaag 52717330DNAsimian virus
17aggcctccaa aaaagcctcc tcactacttc tggaatagct cagaggccga ggcggcctcg
60gcctctgcat aaataaaaaa aattagtcag ccatggggcg gagaatgggc ggaactgggc
120ggagttaggg gcgggatggg cggagttagg ggcgggacta tggttgctga
ctaattgaga 180tgcatgcttt gcatacttct gcctgctggg gagcctgggg
actttccaca cctggttgct 240gactaattga gatgcatgct ttgcatactt
ctgcctgctg gggagcctgg ggactttcca 300caccctaact gacacacatt
ccacagggtc 33018795DNAartificial sequencechemically synthesized
18atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
60ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
120gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct
gaatgaactg 180caggacgagg cagcgcggct atcgtggctg gccacgacgg
gcgttccttg cgcagctgtg 240ctcgacgttg tcactgaagc gggaagggac
tggctgctat tgggcaaagt gccggggcag 300gatctcctgt catctcacct
tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 360cggcggctgc
atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
420atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga
tctggacgaa 480gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc
tcaaggcgcg catgcccgac 540ggcgaggatc tcgtcgtgac ccatggcgat
gcctgcttgc cgaatatcat ggtggaaaat 600ggccgctttt ctggattcat
cgactgtggc cggctgggtg tggcggaccg ctatcaggac 660atagcgttgg
ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
720ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta
tcgccttctt 780gacgagttct tctga 79519943DNAhomo sapiens 19ctgaaatgga
agaaaaaaac tttgaaccac tgtctgaggc ttgagaatga accaagatcc 60aaactcaaaa
agggcaaatt ccaaggagaa ttacatcaag tgccaagctg gcctaacttc
120agtctccacc cactcagtgt ggggaaactc catcgcataa aacccctccc
cccaacctaa 180agacgacgta ctccaaaagc tcgagaacta atcgaggtgc
ctggacggcg cccggtactc 240cgtggagtca catgaagcga cggctgagga
cggaaaggcc cttttccttt gtgtgggtga 300ctcacccgcc cgctctcccg
agcgccgcgt cctccatttt gagctccctg cagcagggcc 360gggaagcggc
catctttccg ctcacgcaac tggtgccgac cgggccagcc ttgccgccca
420gggcggggcg atacacggcg gcgcgaggcc aggcaccaga gcaggccggc
cagcttgaga 480ctacccccgt ccgattctcg gtggccgcgc tcgcaggccc
cgcctcgccg aacatgtgcg 540ctgggacgca cgggccccgt cgccgcccgc
ggccccaaaa accgaaatac cagtgtgcag 600atcttggccc gcatttacaa
gactatcttg ccagaaaaaa agcgtcgcag caggtcatca 660aaaattttaa
atggctagag acttatcgaa agcagcgaga caggcgcgaa ggtgccacca
720gattcgcacg cggcggcccc agcgcccaag ccaggcctca actcaagcac
gaggcgaagg 780ggctccttaa gcgcaaggcc tcgaactctc ccacccactt
ccaacccgaa gctcgggatc 840aagaatcacg tactgcagcc aggggcgtgg
aagtaattca aggcacgcaa gggccataac 900ccgtaaagag gccaggcccg
cgggaaccac acacggcact tac 9432010PRTartificial sequencechemically
synthesized 20Thr Val Lys Ala Lys Leu Leu Ser Val Glu 1 5 10
2110PRTartificial sequencechemically synthesized 21Ser Ser Thr Ser
Ser Asp Phe Arg Asp Arg 1 5 10 2215PRTartificial sequencechemically
synthesized 22Thr Pro Gln Asp Leu Asn Thr Met Leu Asn Thr Val Gly
Gly His 1 5 10 15 2320PRTartificial sequencechemcially synthesized
23Leu Ser Ser Lys Ala Val Asn His Ile Arg Ser Val Trp Lys Ser Leu 1
5 10 15 Leu Glu Asp Thr 20 242025DNAartificial sequencechemcially
synthesized 24ttacttgtac agctcgtcca tgccgagagt gatcccggcg
gcggtcacga actccagcag 60gaccatgtga tcgcgcttct cgttggggtc tttgctcagg
gcggactggg tgctcaggta 120gtggttgtcg ggcagcagca cggggccgtc
gccgatgggg gtgttctgct ggtagtggtc 180ggcgagctgc acgctgccgt
cctcgatgtt gtggcggatc ttgaagttca ccttgatgcc 240gttcttctgc
ttgtcggcca tgatatagac gttgtggctg ttgtagttgt actccagctt
300gtgccccagg atgttgccgt cctccttgaa gtcgatgccc ttcagctcga
tgcggttcac 360cagggtgtcg ccctcgaact tcacctcggc gcgggtcttg
tagttgccgt cgtccttgaa 420gaagatggtg cgctcctgga cgtagccttc
gggcatggcg gacttgaaga agtcgtgctg 480cttcatgtgg tcggggtagc
ggctgaagca ctgcacgccg taggtcaggg tggtcacgag 540ggtgggccag
ggcacgggca gcttgccggt ggtgcagatg aacttcaggg tcagcttgcc
600gtaggtggca tcgccctcgc cctcgccgga cacgctgaac ttgtggccgt
ttacgtcgcc 660gtccagctcg accaggatgg gcaccacccc ggtgaacagc
tcctcgccct tgctcaccat 720tttgtcgtca tcgtctttgt agtcgttcat
gctgtcctta tggctgctcc ggtctgtgtt 780gtggttgttt tgatcgtggt
tcttccactg gatgcttgtt ccgttcatgg ggaccgaggt 840catcagactt
ttggactggt aacagcagca gcagaggcat gactggaaac aatttttaaa
900tttcttgctc acaaaataca gagctatggg gtttatacat gaattcatgg
ttgccaagtt 960aataccgatg taatccatga gcagtaagaa actaagtaat
tcacatcggt tcttgtccat 1020ctcgttatac acagttttct tcaatatacg
gcttaaatga agagggaacc agcaaagagc 1080aaaaattaca accaagcaga
aaactgtttt tgccacttct cgacgctgct taagatgttc 1140actgagggca
attctcaagc tgccattcct tctgttcaac atctcacaag tcatgagggt
1200gtagaagatc gcagtgcaca ccaagggcat acagaaatag aacccgaaga
gccaccagtc 1260ctttacatct tggtagaact ccatgaattt tgatgtggca
ttgagcatac aggttttatg 1320ctgttcaccc ctatattcaa agggtaccat
gacgaagcca atcgcttcag gaatggccag 1380gataaaggac aggatccaga
tggagacaat ttcaatggca gttaccaaag gaatcccaat 1440tccctgaaca
cgactccagg aggcaactgc tctgtacctg tcaacactaa gagcgcagag
1500gttgaggacg gtgatcccca ccgaggactt ctgcaaaaag gggaacagct
tgcaaagaaa 1560tacgccaaag tcattgtgat caaaaggcca gcgcccagcc
agcagcttaa atacattgat 1620agggagatca atgaccacat agataaggtc
tccaagggca agactggcta tcagcgcgtt 1680ggggccattc ctcatacatt
tgttctggta aatgatcctg agcagagttg cattccccac 1740cattcccacg
atgaaaatag tacaagatat cacagtgtta atgtatttga aagctgaagt
1800aattttagtc tgctgtgggc aatagttgtg cattgagcca ttgctgggta
ggaccaaatt 1860agtgggttga tgagtggtaa ccaggaagct gagctctgtg
ccacgaaaag tggtgaaatc 1920atccacatga ttgcttagat ttgtgctgta
tctctcagga ttatcactga ttacacatcc 1980aaccagtgcc agccaaaagg
atgccctgag gcaaagggtt tccat 202525584DNAartificial
sequencechemcially synthesized 25gcgttgctgg cgtttttcca taggctccgc
ccccctgacg agcatcacaa aaatcgacgc 60tcaagtcaga ggtggcgaaa cccgacagga
ctataaagat accaggcgtt tccccctgga 120agctccctcg tgcgctctcc
tgttccgacc ctgccgctta ccggatacct gtccgccttt 180ctcccttcgg
gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
240taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
cgaccgctgc 300gccttatccg gtaactatcg tcttgagtcc aacccggtaa
gacacgactt atcgccactg 360gcagcagcca ctggtaacag gattagcaga
gcgaggtatg taggcggtgc tacagagttc 420ttgaagtggt ggcctaacta
cggctacact agaagaacag tatttggtat ctgcgctctg 480ctgaagccag
ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
540gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcg 584261433DNAHIV
26atgggatcct ccaagagtaa gcccaaggac cacatcgtgt gggccagccg cgagctggag
60cgcttcgccg tgaaccccgg cctgctcgag accagcgaag gctgccgcca gatcatgggc
120cagctccagc ccagcctcca gaccggcagc gaggagctgc gcagcctgta
caacaccgtg 180gccaccctgt actgcgtgca ccagaagatc gaggtgaagg
acaccaagga ggccctggac 240aaggtggagg aggagcagaa caacagcaag
aagaaggccc agcaggaggc cgccgacgcc 300ggcaaccgca accaagtcag
ccagaactac cccatcgtgc agaacctgca gggccagatg 360gtgcaccagg
ccatcagccc ccgcaccctg aacgcctggg tgaaggtggt ggaggagaag
420gccttcagcc ccgaggtgat ccccatgttc agcgccctga gcgagggcgc
taccccccag 480gacctgaaca ccatgctgaa caccgtgggc ggccaccagg
ccgccatgca gatgctgaag 540gagaccatca acgaggaggc cgccgagtgg
gaccgcctgc accccgtgca cgccgggccc 600atcgcccccg gccagatgcg
cgagccccgc ggcagcgaca tcgccggcac caccagcacc 660ctccaggagc
agatcggctg gatgaccaac aaccccccca tccccgtggg cgagatctac
720aagcgctgga tcatcctggg cctgaacaag atcgtccgca tgtacagccc
caccagcatc 780ctggacatca agcagggccc caaggagccc ttccgcgact
acgtggaccg cttctacaag 840accctgcgcg ccgagcaggc cacccaggag
gtgaagaact ggatgaccga gaccctgctg 900gtgcagaacg ccaaccccga
ctgcaagacc atcctcaagg ccctgggacc cgccgccacc 960ctggaggaga
tgatgaccgc ctgccaaggc gtgggaggcc caggccacaa ggccagagtg
1020ctggccgagg ccatgagcca ggtgaccggc agcgctgcca tcatgatgca
gagaggcaac 1080ttcagaaacc agagaaagac cgtgaagtgc ttcaactgcg
gcaaggaggg acacatcgcc 1140agaaactgca gagctcccag aaagaagggc
tgctggaagt gcggaaagga gggacaccag 1200atgaaggact gcaccgagag
acaggccaac ttcctgggca agatctggcc cagccacaag 1260ggcagacccg
gcaacttcct gcagagcaga cccgagccca ccgctcctcc cgaggagagc
1320ttcagattcg gcgaggccac cgctcctagc cagaagcagg agcccatcga
caaggagctg 1380tacccactgg ccagcctgaa gagcctgttc ggcagcgacc
caagcagcca gat 14332772DNAFoot and mouth disease virus 27gcaccggtga
aacagacttt gaattttgac cttctcaagt tggcgggaga cgtggagtcc 60aaccctgggc
cc 72
* * * * *