U.S. patent application number 11/609575 was filed with the patent office on 2007-07-12 for codon optimized recombinant dermaphagoides allergens.
This patent application is currently assigned to GlaxoSmithKline Biologicals, SA. Invention is credited to Alex Bollen, Paul Jacobs, Alain Jacquet, Marc Georges Francis Massaer.
Application Number | 20070161083 11/609575 |
Document ID | / |
Family ID | 9893457 |
Filed Date | 2007-07-12 |
United States Patent
Application |
20070161083 |
Kind Code |
A1 |
Bollen; Alex ; et
al. |
July 12, 2007 |
Codon Optimized Recombinant Dermaphagoides Allergens
Abstract
The present invention relates to codon optimised polynucleotides
which are efficiently expressed in mammalian cells and encode
insect proteins from Dermaphagoides dust mite. In particular, the
optimised codon polynucleotides encode a protein from
Dermaphagoides pteronyssinus, such as DerP1 or proDerP1. The
present invention also provides methods of preparing pharmaceutical
compositions comprising the expression of the codon optimised
polynucleotides, and vectors and transformed host cells comprising
them.
Inventors: |
Bollen; Alex; (Gosselies,
BE) ; Jacobs; Paul; (Brussels, BE) ; Jacquet;
Alain; (Gosselies, BE) ; Massaer; Marc Georges
Francis; (Brussels, BE) |
Correspondence
Address: |
GLAXOSMITHKLINE;Corporate Intellectual Property - UW2220
P.O. Box 1539
King of Prussia
PA
19406-0939
US
|
Assignee: |
GlaxoSmithKline Biologicals,
SA
|
Family ID: |
9893457 |
Appl. No.: |
11/609575 |
Filed: |
December 12, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10297563 |
Dec 9, 2002 |
7173117 |
|
|
PCT/EP01/06483 |
Jun 7, 2001 |
|
|
|
11609575 |
Dec 12, 2006 |
|
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Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/348; 530/350; 536/23.5 |
Current CPC
Class: |
C07K 14/43531 20130101;
A61P 37/08 20180101; A61K 38/00 20130101; A61P 11/06 20180101 |
Class at
Publication: |
435/069.1 ;
435/320.1; 435/348; 530/350; 536/023.5 |
International
Class: |
C07K 14/435 20060101
C07K014/435; C07H 21/04 20060101 C07H021/04; C12P 21/06 20060101
C12P021/06; C12N 5/06 20060101 C12N005/06 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 10, 2000 |
GB |
0014288.5 |
Claims
1. An isolated polynucleotide sequence which encodes a
Dermatophagoides mite protein, wherein the codon usage pattern of
the polynucleotide sequence resembles that of highly expressed
mammalian genes.
2. An isolated polynucleic acid sequence as claimed in claim 1,
wherein all of the amino acid types present in the protein are
optimised, such that the codons used to encode that amino acid are
used in the same frequency as the known mammalian frequency for
that amino acid.
3. An isolated polynucleic acid sequence as claimed in claim 1,
wherein the yield of protein encoded by the polynucleic acid when
expressed by an expression system is greater than 20% higher than
the amount of protein produced from the same expression system
using a non-optimised native gene for that Dermatophagoides
protein.
4. An isolated polynucleic acid sequence encoding a Dermaphagoides
protein, characterised in that the codons present in said
polynucleotide which are used to encode each amino acid are
selected to appear as set forth in the following table
TABLE-US-00006 Amino Frequency Acid Codon (percentage used) Ala GCG
17 GCA 13 GCT 17 GCC 53 Arg AGG 18 AGA 10 CGG 21 CGA 6 CGT 7 CGC 37
Asn AAT 22 AAC 78 Asp GAT 25 GAC 75 Cys TGT 32 TGC 68 Gln CAG 88
CAA 12 Glu GAG 75 GAA 25 Gly GGG 24 GGA 14 GGT 12 GGC 50 His CAT 21
CAC 79 Ile ATA 5 ATT 18 ATC 77 Leu TTG 6 TTA 2 CTG 58 CTA 3 CTT 5
CTC 26 Lys AAG 82 AAA 18 Phe TTT 20 TTC 80 Pro CCG 17 CCA 16 CCT 19
CCC 48 Ser AGT 10 AGC 34 TCG 9 TCA 5 TCT 13 TCC 28 Thr ACG 15 ACA
14 ACT 14 ACC 57 Tyr TAT 26 TAC 74 Val GTG 64 GTA 5 GTT 7 GTC
25
5. An isolated polynucleotide comprising SEQ ID NO: 15.
6. An isolated vector comprising the isolated polynucleotide of
claim 5.
7. An isolated host cell comprising the vector of claim 6.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 10/297,563 (Allowed) filed 9 Dec. 2002, which is a National
Stage Application filed under 35 U.S.C. .sctn.371 of PCT/EP01/06483
filed 7 Jun. 2001.
[0002] The present invention relates to codon optimised
polynucleotides which are efficiently expressed in mammalian cells
and encode insect proteins from Dermaphagoides dust mite. In
particular, the optimised codon polynucleotides encode a protein
from Dermaphagoides pteronyssinus, such as DerP1 or proDerP1. The
present invention also provides methods of preparing pharmaceutical
compositions comprising the expression of the codon optimised
polynucleotides, and vectors and transformed host cells comprising
them.
[0003] The allergens from the house dust mite Dermatophagoides have
long been recognised to be associated with allergic
hypersensitivity reactions such as asthma [1]. Amongst these
molecules, Der p 1 is an immunodominant allergen which elicits the
strongest IgE-mediated immune response [2,3]. The cysteine
proteinase activity of Der p 1 was shown to amplify its potent
allergenicity [4,5]. The Der p 1 encoding cDNA sequence reveals
that, like many mammalian and plant proteinases, Der p 1 is
synthesized as an inactive preproenzyme of 320 amino acid residues
which is subsequently processed into a 222-amino acid mature form
[6,7]. The maturation of ProDer p 1 is not known to date but it is
thought that the allergen is processed by the cleavage of the
80-residues proregion.
[0004] Mature Der p 1 was successfully purified from the whole
house dust mite culture but with weak overall yield [8].
Recombinant production of allergens represents an efficient way to
obtain defined materials with high yields for a variety of
experimental procedures such as immunological studies, diagnosis,
treatment of IgE-mediated allergic disorders by immunotherapy and
understanding structure-function relationships [9]. Previous
attempts of Der p 1 expression in bacteria and yeast indicated that
the allergen was poorly expressed and mainly under an insoluble
form [10-12]. Moreover, recombinant Der p 1 produced in bacteria
was shown to have weak IgE binding activity. The recombinant
protein expressed in yeast was recognized by specific IgE at,
however, a lower level than the natural protein.
[0005] Recombinant DerP1 allergens with reduced enzymatic activity
that are encoded by the native non-optimised Dermaphagoides gene
are described in WO 99/25823. Other recombinant Dermaphagoides
allergens include DerP1 (U.S. Pat. No. 6,077,518), DerPII (U.S.
Pat. No. 6,132,734), and DerFI and DerFII (U.S. Pat. No. 5,973,132;
U.S. Pat. No. 5,958,415; U.S. Pat. No. 5,876,722).
[0006] It is clearly desirable to enable the efficient expression
of recombinant Dermaphagoides allergens for use in the manufacture
of pharmaceuticals, vaccines or diagnostic assays. It is
furthermore desirable for the expression systems to produce
recombinant allergen at high levels that is also in the same
conformation and immunological properties as native Dermaphagoides
allergens.
[0007] The present invention achieves such advantages by providing
a polynucleotide sequence which encodes a Dermaphagoides protein,
wherein the codon usage pattern of the polynucleotide sequence is
altered to resemble that of highly expressed mammalian genes.
Accordingly, the cloning and expression of recProDer p 1 has been
achieved in Chinese Hamster Ovary cells (CHO) with high efficiency
and produces a product which displayed very similar IgE
reactivities to native purified DerP1.
[0008] The DNA code has 4 letters (A, T, C and G) and uses these to
spell three letter "codons" which represent the amino acids the
proteins encoded in an organism's genes. The linear sequence of
codons along the DNA molecule is translated into the linear
sequence of amino acids in the protein(s) encoded by those genes.
The code is highly degenerate, with 61 codons coding for the 20
natural amino acids and 3 codons representing "stop" signals. Thus,
most amino acids are coded for by more than one codon--in fact
several are coded for by four or more different codons.
[0009] Where more than one codon is available to code for a given
amino acid, it has been observed that the codon usage patterns of
organisms are highly non-random. Different species show a different
bias in their codon selection and, furthermore, utilization of
codons may be markedly different in a single species between genes
which are expressed at high and low levels. This bias is different
in viruses, plants, bacteria, insect and mammalian cells, and some
species show a stronger bias away from a random codon selection
than others. For example, humans and other mammals are less
strongly biased than certain bacteria or viruses. For these
reasons, there is a significant probability that a mammalian gene
expressed in E. coli or a viral gene expressed in mammalian cells
will have an inappropriate distribution of codons for efficient
expression. However, a gene with a codon usage pattern suitable for
E. coli expression may also be efficiently expressed in humans. It
is believed that the presence in a heterologous DNA sequence of
clusters of codons which are rarely observed in the host in which
expression is to occur, is predictive of low heterologous
expression levels in that host.
[0010] There are several examples where changing codons from those
which are rare in the host to those which are host-preferred
("codon optimization") has enhanced heterologous expression levels,
for example the BPV (bovine papilloma virus) late genes L1 and L2
have been codon optimised for mammalian codon usage patterns and
this has been shown to give increased expression levels over the
wild-type HPV sequences in mammalian (Cos-1) cell culture (Zhou et.
al. J. Virol 1999. 73, 4972-4982). In this work, every BPV codon
which occurred more than twice as frequently in BPV than in mammals
(ratio of usage >2), and most codons with a usage ratio of
>1.5 were conservatively replaced by the preferentially used
mammalian codon. In WO97/31115, WO97/48370 and WO98/34640 (Merck
& Co., Inc.) codon optimisation of HIV genes or segments
thereof has been shown to result in increased protein expression
and improved immunogenicity when the codon optimised sequences are
used as DNA vaccines in the host mammal for which the optimisation
was tailored. In this work, the sequences consist entirely of
optimised codons (except where this would introduce an undesired
restriction site, intron splice site etc.) because each viral codon
is conservatively replaced with the optimal codon for the intended
host.
LEGEND TO FIGURES
[0011] FIG. 1. Codon usage of ProDer p 1 and highly expressed human
(High) genes.
[0012] Codon usage of a synthetic ProDer p 1 gene (synthetic) after
optimisation of codon usage is also represented. Percentage
frequencies of individual codons are shown for each corresponding
amino acid. The most prevalent codon is shown in bold.
[0013] FIG. 2. PCR synthesis of ProDer p 1 cDNA.
[0014] A set of 14 mutually priming oligonucleotides were used for
PCR amplification of a synthetic ProDer p 1 cDNA. After one round
of amplification, amplified products were submitted to a second PCR
amplification using external primers (primers 1 and 14).
Oligonucleotides which served as PCR templates for the synthesis
are represented by solid bars. Unique restriction sites into the
synthetic Proder p 1 cDNA which were used for the cloning into the
eukaryotic pEE 14 expression vector are shown above. After each of
the two rounds of PCR amplification, electrophoresis on agarose gel
of the amplified fragments are also shown.
[0015] FIG. 3. Expression of synthetic and natural ProDer p 1 in
transient transfection assays.
[0016] Supernatants from COS cells transfected with plasmids
encoding natural (pNIV 4853) or synthetic ProDer p 1 (pNIV 4846)
were assayed for the presence of secreted recProDer p 1 in a Der p
1 ELISA. Supernatant from COS transfected with a plasmid without
insert was used as control.
[0017] FIG. 4. Purification of recProDer p 1.
[0018] Purified allergens were analyzed by SDS-PAGE and proteins
were detected by Coomassie blue staining (panel A), by
immunoblotting with rabbit polyclonal serum raised against Der p 1
peptide 245-267 (panel B). Lane 1: purified recProDer p 1. Lane 2:
purified Der p 1
[0019] FIG. 5. Carbohydrate analysis of recProDer p 1.
[0020] Glycosylations of purified allergens were analysed by lectin
staining with Galanthus nivalis agglutinin (GNA, Lane 1, 2), Datura
stramonium agglutinin (DSA, Lane 3, 4) and Maackia amurensis
agglutinin (MAA, Lane 5, 6). Lane 1, 3, 5: purified Der p 1. Lane
2, 4, 6: purified recProDer p 1
[0021] FIG. 6. Immune recognition of recProDer p 1 by monoclonal
antibodies directed to Der p 1.
[0022] Reactivity of Der p 1 (.circle-solid.) and recProDer p 1
(.box-solid.) towards monoclonal antibodies was assayed in a
two-site ELISA. Both allergens were used at the same concentration
which was determined in a total protein assay (MicroBCA,
Pierce).
[0023] FIG. 7. Correlation between the IgE reactivity of recProDer
p 1 and Der p 1.
[0024] Immunoplates were coated with 500 ng/well of purified Der p
1 or recProDer p 1 and incubated with 95 sera (diluted 1:8)
radioallergosorbent positive to D. pteronyssinus. Bound IgE was
quantitated by incubation with mouse anti-human IgE and alkaline
phosphatase-labelled anti-mouse IgG antibodies, followed by an
enzymatic assay. Results are expressed as OD.sub.410 nm values.
[0025] FIG. 8. Histamine release activity of recProDer p 1
[0026] Basophils isolated from the peripheral blood of one allergic
donor were stimulated with serial dilutions of natural Der p 1
(.lamda.) or recProDer p 1 (.nu.). The histamine released from
cells was measured by ELISA. The total amount of histamine in
basophils was quantified after cell disruption with the detergent
IGEPAL CA-630. Results are shown as the ratio of released histamine
by allergens to total histamine.
SUMMARY OF THE INVENTION
[0027] According to a first aspect, the present invention provides
a polynucleotide sequence which encodes a Dermatophagoides mite
protein, wherein the codon usage pattern of the polynucleotide
sequence resembles that of highly expressed mammalian genes.
[0028] Preferably the polynucleotide sequence is a DNA sequence.
Desirably the codon usage pattern of the polynucleotide sequence is
typical of highly expressed human genes. Preferably the house dust
mite protein in all of the following aspects of the present
invention is derived from Dermatophagoides pteronyssinus or
Dermatophagoides farinae. Most preferably, the Dermatophagoides
pteronyssinus protein is DerP1 or ProDerP1 or DerP2.
[0029] Accordingly there is provided in a first aspect of the
present invention, a synthetic gene comprising a plurality of
codons together encoding a Dermatophagoides protein; wherein the
selection of the possible codons used for encoding the recombinant
insect protein amino acid sequence has been changed to closely
mimic the optimised mammalian codon usage, such that the frequency
of codon usage in the synthetic gene is substantially the same as a
mammalian gene which encodes the same protein.
[0030] Preferably in this first aspect, all of the amino acid types
present in the protein are optimised such that the codons used to
encode that amino acid are used in the same frequency as the known
mammalian frequency for that amino acid. In addition, in preferred
optimised codon synthetic genes are used in expression systems that
have a protein yield which is greater than 20% higher, and more
preferably greater than 50% and most preferably more than 100%
higher in yield than the amount of protein produced from the same
expression system using a non-optimised native gene for that
Dermatophagoides protein.
[0031] Alternatively, in a second aspect of the present invention
there is provided an isolated nucleic acid molecule encoding an
Dermaphagoides protein, characterised in that the codons present in
said polynucleotide which are used to encode each amino acid are
selected to appear in substantially the same frequency as set forth
in table 1. TABLE-US-00001 TABLE 1 Codon usage frequency in
mammalian cells Amino Frequency Acid Codon (percentage used) Ala
GCG 17 GCA 13 GCT 17 GCC 53 Arg AGG 18 AGA 10 CGG 21 CGA 6 CGT 7
CGC 37 Asn AAT 22 AAC 78 Asp GAT 25 GAC 75 Cys TGT 32 TGC 68 Gln
CAG 88 CAA 12 Glu GAG 75 GAA 25 Gly GGG 24 GGA 14 GGT 12 GGC 50 His
CAT 21 CAC 79 Ile ATA 5 ATT 18 ATC 77 Leu TTG 6 TTA 2 CTG 58 CTA 3
CTT 5 CTC 26 Lys AAG 82 AAA 18 Phe TTT 20 TTC 80 Pro CCG 17 CCA 16
CCT 19 CCC 48 Ser AGT 10 AGC 34 TCG 9 TCA 5 TCT 13 TCC 28 Thr ACG
15 ACA 14 ACT 14 ACC 57 Tyr TAT 26 TAC 74 Val GTG 64 GTA 5 GTT 7
GTC 25
[0032] In this context, the meaning of "substantially" is intended
to mean that the percentage usage of a particular codon is the
figure as appearing in the table .+-.20%, more preferably .+-.15%,
more preferably .+-.10%, and ideally .+-.5%.
[0033] Alternatively, in a third aspect of the present invention
there is provided, a synthetic gene comprising a plurality of
codons together encoding a Dermatophagoides; protein, characterised
in that each type of amino acid type has a .chi..sup.2 value which
is not significantly different, at a confidence interval of between
80-99%, to the corresponding .chi..sup.2 value of that same amino
acid type as found in a theoretical mammalian gene; said
.chi..sup.2 value being calculated using the following formula:
.chi. k 2 = ( x ij - x j / n ) 2 ( x j / n ) ##EQU1##
[0034] wherein x.sub.ij is the number of codons of type j in
sequence i, n is the total number of codons for a particular amino
acid k in the sequence, and x.sub.j is the total number of codons
of type j in the 2 sequences. The degrees of freedom of the
variable is equal to the number of different possible codons minus
1.
[0035] Along these same lines, the present invention can also be
expressed as providing a synthetic gene comprising a plurality of
codons together encoding a Dermatophagoides protein; characterised
in that between 60-100% of the different types of amino acids
present in the synthetic gene are optimised, characterised in that
an amino acid type is considered to be optimised if its .chi..sup.2
value in the synthetic gene is less that the Limit .chi..sup.2
value for significance (5%), for that particular amino acid as
defined in the following table: TABLE-US-00002 Amino Acid Limit
.chi..sup.2 value for significance (5%) Ala 7.81 Cys 3.84 Asp 3.84
Glu 3.84 Phe 3.84 Gly 7.81 His 3.84 Ile 5.99 Lys 3.84 Leu 11.1 Asn
3.84 Pro 7.81 Gln 3.84 Arg 11.1 Ser 11.1 Thr 7.81 Val 7.81 Tyr
3.84
said .chi..sup.2 value being calculated using the following
formula: .chi. k 2 = ( x ij - x j / n ) 2 ( x j / n ) ##EQU2##
[0036] wherein x.sub.ij is the number of codons of type j in
sequence i, n is the total number of codons for a particular amino
acid k in the sequence, and x.sub.j is the total number of codons
of type j in the 2 sequences. The degrees of freedom of the
variable is equal to the number of different possible codons minus
1. Preferably, more than 70% of the amino acids are optimised, more
preferably more than 80% are optimised and most preferably greater
than 90% of the codons are optimised.
[0037] Surprisingly such optimised Dermatophagoides genes express
very well in mammalian cells such as CHO cells, but also express
very well in yeast cells despite the different codon usage of
yeast.
[0038] The present invention also provides an expression vector is
provided which comprises, and is capable of directing the
expression of, a polynucleotide sequence according to the first to
third aspects of the invention, encoding a Dermatophagoides amino
acid sequence wherein the codon usage pattern of the polynucleotide
sequence is typical of highly expressed mammalian genes, preferably
highly expressed human genes. The vector may be suitable for
driving expression of heterologous DNA in bacterial insect or
mammalian cells, particularly human cells.
[0039] Host cells comprising a polynucleotide sequence according to
the first aspect of the invention, or an expression vector
according the second aspect, is provided. The host cell may be
bacterial, e.g. E. coli; mammalian, e.g. human; or may be an insect
cell. Mammalian cells comprising a vector according to the present
invention may be cultured cells transfected in vitro or may be
transfected in vivo by administration of the vector to the
mammal.
[0040] Pharmaceutical compositions comprising a recombinant
Dermatophagoides protein expressed by the polynucleotides of the
present invention, or the codon optimised polynucleotide sequences
are also provided.
[0041] Preferably the pharmaceutical compositions comprises a DNA
vector according to the second aspect of the present invention. In
preferred embodiments the composition comprises a plurality of
particles, preferably gold particles, coated with DNA comprising a
vector encoding a polynucleotide sequence which encodes a
Dermatophagoides amino acid sequence, wherein the codon usage
pattern of the polynucleotide sequence is typical of highly
expressed mammalian genes, particularly human genes. In alternative
embodiments, the composition comprises a pharmaceutically
acceptable excipient and a DNA vector according to the second
aspect of the present invention. The composition may also include
an adjuvant.
[0042] In a further aspect, the present invention provides a method
of making a pharmaceutical composition including the step of
altering the codon usage pattern of a wild-type Dermatophagoides
nucleotide sequence, or creating a polynucleotide sequence
synthetically, to produce a sequence having a codon usage pattern
typical of highly expressed mammalian genes and encoding a
wild-type Dermatophagoides amino acid sequence or a mutated
Dermatophagoides amino acid sequence comprising the wild-type
sequence with amino acid changes sufficient to inactivate one or
more of the natural functions of the polypeptide. The method
further comprising the expression of the synthetic polynucleotide
sequence in a mammalian host cell, purification of the expressed
recombinant protein, and formulation with pharmaceutically
acceptable excipients.
[0043] Methods of preparing a vaccine are provided when the
pharmaceutically acceptable excipients comprises an adjuvant.
Adjuvants are well known in the art (Vaccine Design--The Subunit
and Adjuvant Approach, 1995, Pharmaceutical Biotechnology, Volume
6, Eds. Powell, M. F., and Newman, M. J., Plenum Press, New York
and London, ISBN 0-306-44867-X).
[0044] Codon usage patterns for mammals, including humans can be
found in the literature (see e.g. Nakamura et. al. Nucleic Acids
Research 1996, 24:214-215).
[0045] The polynucleotides according to the invention have utility
in the production by expression of the encoded proteins, which
expression may take place in vitro, in vivo or ex vivo. The
nucleotides may therefore be involved in recombinant protein
synthesis, for example to increase yields, or indeed may find use
as therapeutic agents in their own right, utilised in DNA
vaccination techniques. Where the polynucleotides of the present
invention are used in the production of the encoded proteins in
vitro or ex vivo, cells, for example in cell culture, will be
modified to include the polynucleotide to be expressed. Such cells
include transient, or preferably stable mammalian cell lines.
Particular examples of cells which may be modified by insertion of
vectors encoding for a polypeptide according to the invention
include mammalian HEK293T, CHO, HeLa, 293 and COS cells. Preferably
the cell line selected will be one which is not only stable, but
also allows for mature glycosylation and cell surface expression of
a polypeptide. Expression may be achieved in transformed oocytes. A
polypeptide may be expressed from a polynucleotide of the present
invention, in cells of a transgenic non-human animal, preferably a
mouse. A transgenic non-human animal expressing a polypeptide from
a polynucleotide of the invention is included within the scope of
the invention.
[0046] Where the polynucleotides of the present invention find use
as therapeutic agents, e.g. in DNA vaccination, the nucleic acid
will be administered to the mammal e.g. human to be vaccinated. The
nucleic acid, such as RNA or DNA, preferably DNA, is provided in
the form of a vector, such as those described above, which may be
expressed in the cells of the mammal. The polynucleotides may be
administered by any available technique. For example, the nucleic
acid may be introduced by needle injection, preferably
intradermally, subcutaneously or intramuscularly. Alternatively,
the nucleic acid may be delivered directly into the skin using a
nucleic acid delivery device such as particle-mediated DNA delivery
(PMDD). In this method, inert particles (such as gold beads) are
coated with a nucleic acid, and are accelerated at speeds
sufficient to enable them to penetrate a surface of a recipient
(e.g. skin), for example by means of discharge under high pressure
from a projecting device. (Particles coated with a nucleic acid
molecule of the present invention are within the scope of the
present invention, as are delivery devices loaded with such
particles).
[0047] Suitable techniques for introducing the naked polynucleotide
or vector into a patient include topical application with an
appropriate vehicle. The nucleic acid may be administered topically
to the skin, or to mucosal surfaces for example by intranasal,
oral, intravaginal or intrarectal administration. The naked
polynucleotide or vector may be present together with a
pharmaceutically acceptable excipient, such as phosphate buffered
saline (PBS). DNA uptake may be further facilitated by use of
facilitating agents such as bupivacaine, either separately or
included in the DNA formulation. Other methods of administering the
nucleic acid directly to a recipient include ultrasound, electrical
stimulation, electroporation and microseeding which is described in
U.S. Pat. No. 5,697,901.
[0048] Uptake of nucleic acid constructs may be enhanced by several
known transfection techniques, for example those including the use
of transfection agents. Examples of these agents includes cationic
agents, for example, calcium phosphate and DEAE-Dextran and
lipofectants, for example, lipofectam and transfectam. The dosage
of the nucleic acid to be administered can be altered. Typically
the nucleic acid is administered in an amount in the range of 1 pg
to 1 mg, preferably 1 pg to 10 .mu.g nucleic acid for particle
mediated gene delivery and 10 .mu.g to 1 mg for other routes.
[0049] A nucleic acid sequence of the present invention may also be
administered by means of specialised delivery vectors useful in
gene therapy. Gene therapy approaches are discussed for example by
Verme et al, Nature 1997, 389:239-242. Both viral and non-viral
vector systems can be used. Viral based systems include retroviral,
lentiviral, adenoviral, adeno-associated viral, herpes viral,
Canarypox and vaccinia-viral based systems. Non-viral based systems
include direct administration of nucleic acids, microsphere
encapsulation technology (poly(lactide-co-glycolide) and,
liposome-based systems. Viral and non-viral delivery systems may be
combined where it is desirable to provide booster injections after
an initial vaccination, for example an initial "prime" DNA
vaccination using a non-viral vector such as a plasmid followed by
one or more "boost" vaccinations using a viral vector or non-viral
based system.
[0050] A nucleic acid sequence of the present invention may also be
administered by means of transformed cells. Such cells include
cells harvested from a subject. The naked polynucleotide or vector
of the present invention can be introduced into such cells in vitro
and the transformed cells can later be returned to the subject. The
polynucleotide of the invention may integrate into nucleic acid
already present in a cell by homologous recombination events. A
transformed cell may, if desired, be grown up in vitro and one or
more of the resultant cells may be used in the present invention.
Cells can be provided at an appropriate site in a patient by known
surgical or microsurgical techniques (e.g. grafting,
micro-injection, etc.)
[0051] The pharmaceutical compositions of the present invention may
include adjuvant compounds, or other substances which may serve to
increase the immune response induced by the protein which is
encoded by the DNA. These may be encoded by the DNA, either
separately from or as a fusion with the antigen, or may be included
as non-DNA elements of the formulation. Examples of adjuvant-type
substances which may be included in the formulations of the present
invention include ubiquitin, lysosomal associated membrane protein
(LAMP), hepatitis B virus core antigen, FLT3-ligand (a cytokine
important in the generation of professional antigen presenting
cells, particularly dentritic cells) and other cytokines such as
IFN-.gamma. and GMCSF.
[0052] Examples of other mite allergens that may be codon optimised
according to the methods of the present invention are DerF3 and
DP15. DerF3 is a serine protease from Dermatophagoides farinae
(accession D63858NID/g1311456). DP15 is major allergen p Dp
15=glutathione S-transferase homolog from Dermatophagoides
pteronyssinus (accession S75286/g807137).
[0053] The codon usage pattern of DerF3 and DP15 are shown in the
following table: TABLE-US-00003 .chi..sup.2 value of .chi..sup.2
value of native Limit .chi..sup.2 value for native DerF3 DP15
significance (5%) Ala 10.6 3.4 7.81 Cys 4.7 0.5 3.84 Asp 17.5 8.0
3.84 Glu 11.5 8.2 3.84 Phe 4 4.7 3.84 Gly 25.7 11.3 7.81 His 9.6
3.0 3.84 Ile 16.0 8.9 5.99 Lys 14.6 10.2 3.84 Leu 22.4 12.7 11.1
Asn 9.9 15.2 3.84 Pro 9.4 6.3 7.81 Gln 16.1 10.9 3.84 Arg 12 13.7
11.1 Ser 21.1 4.3 11.1 Thr 7.3 3.0 7.81 Val 19 5.9 7.81 Tyr 9.2
12.7 3.84
Values in bold are statistically significant (amino acids that are
not codon optimised)
[0054] Optimised genes may be designed using a Visual Basic program
called Calcgene, written by R. S. Hale and G Thompson (Protein
Expression and Purification Vol. 12 pp. 185-188 (1998)). For each
amino acid residue in the original sequence, a codon was assigned
based on the probability of it appearing in highly expressed
mammalian or human genes. Details of the program, which works under
Microsoft Windows 3.1, can be obtained from the authors. In this
article, certain rare codons were excluded from the optimisation
process to obviate the possibility of generating clusters of rare
codons together which would otherwise prejudice the efficient
expression of the gene. In the context of this invention,
therefore, either the man skilled in the art can visually check the
sequence of the polynucleotide to verify that no clusters of rare
codons were present in the optimised gene, or alternatively, one or
more rare codons may be excluded from the optimisation process.
REFERENCES
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[0075] 21. Hauser H: Heterologous expression of genes in mammalian
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[0076] 22. Holm L, Nucleic Acids Res 1986; 14:3075-3087. [0077] 23.
Haas et al., Current Biology 1996; 6:315-324. [0078] 24. Zhou et
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12:417-422. [0081] 27. Kleber-Janke and Becker, Prot Expr Purif
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XVIIth International Congress of Allergology and Clinical
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30:1582-1590.
[0086] The present invention is exemplified but not limited to the
following examples.
EXAMPLE 1, EXPRESSION OF RecProDerP1 IN COS AND CHO CELLS
Construction of ProDer p 1 Synthetic Gene
[0087] A "humanized" ProDer p 1 gene was synthesized using a set of
14 partially overlapping oligonucleotides. These primers were
designed, based on the codon usage of highly expressed human genes,
and produced by an 394 DNA/RNA Applied Biosystem synthetizer The
degenerately encoded amino acids were not encoded by the most
prevalent codons but taking the frequencies of the individual
codons into account. For example, histidine residue is encoded by
CAC or CAT with a respective frequency of 79% and 21% in highly
expressed human genes. Consequently, we attempted to follow the
same codon frequency instead of selecting only the CAC codon for
each histidine residue in the synthetic ProDer p 1. The native Der
p 1 signal sequence was exchanged with the highly efficient leader
peptide of the VZV glycoprotein E (gE) to facilitate secretion. The
oligonucleotides were the following: TABLE-US-00004
.sup.5'GAAGCTTCGGGCGAATTGCGTGGTTTTAAGTGACT SEQ ID NO. 1
ATATTCGAGGGTCGCCTGTAATATGGGGACAGTTAAT
AAACCTGTGGGTGGGGGTATTGATGGGGTTCGGAATT ATCACG.sup.3' (oligo 1,
coding); .sup.5'GAAGGCTTTCTTGTATTCCTCGAAGGTCTTAATGG SEQ ID NO. 2
AGCTCGGCCGTGCTCTGACCGGATTCGTTATACGC A
AGGTACCCGTGATAATTCCGAACCC.sup.3' (oligo 2, non coding);
.sup.5'GGAATACAAGAAAGCCTTCAACAAGAGCTATGCCA SEQ ID NO. 3
CCTTCGAGGACGAGGAGGCCGCGCGCAAGAACTTCCT
GGAAAGCGTGAAATACGTGCAGAGC.sup.3' (oligo 3, coding);
.sup.5'GTCTTAAGGTGTTCGAAAGCCTCGGCGCTCATCAG SEQ ID NO. 4
GAACCGGTTCTTGAACTCGTCTAAAGACAGGTCGGAC
AGGTGATTTATAGCCCCGCCGTTGCTCTGCACGTATT TCAC.sup.3' (oligo 4, non
coding); .sup.5'CTTTCGAACACCTTAAGACCCAGTTTGATCTCAAC SEQ ID NO. 5
GCGGAGACCAACGCCTGCAGTATCAACGG CAATGCC CCCGCTGAGATTGATCTGCGCC.sup.3'
(oligo 5, coding); .sup.5'GACTCTGTCGCGGCCACGCCTGAAAAGGCCCAACA SEQ
ID NO. 6 CCGACCCGCAGCCGCCTTGCATGCGGATGGGAGTCAC
GGTCCTCATCTGGCGCAGATCAATCTCAG.sup.3' (oligo 6, non coding);
.sup.5'GTGGCCGCGACAGAGTCGGCATACCTCGCGTATCG SEQ ID NO. 7
GAATCAGAGCCTGGACCTCGCTGAGCAGGAGCTCGTT GACTGCGCCTCCCAAC ACGG.sup.3'
(oligo 7, coding); .sup.5'GCTACGTATCGGTAATAGCTTTCCTGCACGACGCC SEQ
ID NO. 8 ATTATGCTGGATGTATTCGATACCTCTGGGAATCGTA
TCCCCATGACATCCGTGTTGGGAGGCGC.sup.3' (oligo 8, non coding);
.sup.5'GCTATTACCGATACGTAGCTAGGGAGCAGTCCTGC SEQ ID NO. 9
CGCCGTCCTAACGCACAGCGCTTCG GCATTTCCAAT TATTGCCAGATCTACC.sup.3'
(oligo 9, coding); .sup.5'CCTTGATTCCGATGATGACAGCGATGGCGCTGTGC SEQ
ID NO. 10 GTCTGCGCCAGGGCCTCCCTGATCTTGTTGGCATTAG
GGGGGTAGATCTGGCAATAATTG.sup.3' (oligo 10, non coding);
.sup.5'GTCATCATCGGAATCAAGGATCTGGACGCATTCCG SEQ ID NO. 11
GCACTATGACGGGCGCACAATCATCCAGCGCGACAAC GGATATCAGCCAAACTACC.sup.3'
(oligo 11, coding); .sup.5'GTAGTCCACCCCCTGGGCGTTCGAGTAACCCACGA SEQ
ID NO. 12 TGTTGACCGCGTGGTAGTTTGGCTGATATCC.sup.3' (oligo 12, non
coding); .sup.5'CCAGGGGGTGGACTACTGGATCGTGAGAAACAGTT SEQ ID NO. 13
GGGACACTAACTGGGGCGACAACGGCTACGGCTACTT CGCCGCCAAC.sup.3' (oligo 13,
coding); .sup.5'GCTCTAGACTCGAGGGATCCTTACAGGATCACCAC SEQ ID NO. 14
GTACGGGTACTCCTCGATCATCATCAGGTCGATGTTG GCGGCGAAGTAGC.sup.3' (oligo
14, non coding).
[0088] The oligonucleotides were incubated together for the
amplification of a synthetic ProDer p 1 gene in a PCR reaction.
Typically, PCR was conducted using High Fidelity Polymerase
(Boehringer) with the following conditions: 30 cycles, denaturation
at 94.degree. C. for 30 s, annealing at 50.degree. C. for 30 s and
elongation at 72.degree. C. for 30 s. The generated products were
amplified using the 3' and 5' terminal primers (oligo 1 and 14) in
the same conditions. The resulting 1080 bp fragment was cloned into
a pCRII-TOPO cloning vector (Invitrogen). The resulting plasmid
pNIV4845 was used to transform the E. coli strain TOP 10
(Invitrogen).
[0089] The sequences of the natural and codon-optimised genes,
together with the encoded amino acid sequence are listed below and
are SEQ ID NO.s 15, 16, and 17 respectively. The sequences encode
the full ProDerP1, and the nucleic acid or amino acid which is
underlined indicates the start of the mature DerP1 sequence
resulting from the cleavage of the Pro region. TABLE-US-00005 SEQ
ID NO. 15, Natural gene
CGTCCATCATCGATCAAAACTTTTGAAGAATACAAAAAAGCCTTCAACAA
AAGTTATGCTACCTTCGAAGATGAAGAAGCTGCCCGTAAAAACTTTTTGG
AATCAGTAAAATATGTTCAATCAAATGGAGGTGCCATCAACCATTTGTCC
GATTTGTCGTTGGATGAATTCAAAAACCGATTTTTGATGAGTGCAGAAGC
TTTTGAACACCTCAAAACTCAATTCGATTTGAATGCTGAAACTAACGCCT
GCAGTATCAATGGAAATGCTCCAGCTGAAATCGATTTGCGACAAATGCGA
ACTGTCACTCCCATTCGTATGCAAGGAGGCTGTGGTTCATGTTGGGCTTT
CTCTGGTGTTGCCGCAACTGAATCAGCTTATTTGGCTTACCGTAATCAAT
CATTGGATCTTGCTGAACAAGAATTAGTCGATTGTGCTTCCCAACACGGT
TGTCATGGTGATACCATTCCACGTGGTATTGAATACATCCAACATAATGG
TGTCGTCCAAGAAAGCTACTATCGATACGTTGCACGAGAACAATCATGCC
GACGACCAAATGCACAACGTTTCGGTATCTCAAACTATTGCCAAATTTAC
CCACCAAATGTAAACAAAATTCGTGAAGCTTTGGCTCAAACCCACAGCGC
TATTGCCGTCATTATTGGCATCAAAGATTTAGACGCATTCCGTCATTATG
ATGGCCGAACAATCATTCAACGCGATAATGGTTACCAACCAAACTATCAC
GCTGTCAACATTGTTGGTTACAGTAACGCACAAGGTGTCGATTATTGGAT
CGTACGAAACAGTTGGGATACCAATTGGGGTGATAATGGTTACGGTTATT
TTGCTGCCAACATCGATTTGATGATGATTGAAGAATATCCATATGTTGTC ATTCTCTAA SEQ ID
NO 16, Synthetic gene
CGGCCGAGCTCCATTAAGACCTTCGAGGAATACAAGAAAGCCTTCAACAA
GAGCTATGCCACCTTCGAGGACGAGGAGGCCGCGCGCAAGAACTTCCTGG
AAAGCGTGAAATACGTGCAGAGCAACGGCGGGGCTATAAATCACCTGTCC
GACCTGTCTTTAGACGAGTTCAAGAACCGGTTCCTGATGAGCGCCGAGGC
TTTCGAACACCTTAAGACCCAGTTTGATCTCAACGCGGAGACCAACGCCT
GCAGTATCAACGGCAATGCCCCCGCTGAGATTGATCTGCGCCAGATGAGG
ACCGTGACTCCCATCCGCATGCAAGGCGGCTGCGGGTCTTGTTGGGCCTT
TTCAGGCGTGGCCGCGACAGAGTCGGCATACCTCGCGTATCGGAATCAGA
GCCTGGACCTCGCTGAGCAGGAGCTCGTTGACTGCGCCTCCCAACACGGA
TGTCATGGGGATACGATTCCCAGAGGTATCGAATACATCCAGCATAATGG
CGTCGTGCAGGAAAGCTATTACCGATACGTAGCTAGGGAGCAGTCCTGCC
GCCGTCCTAACGCACAGCGCTTCGGCATTTCCAATTATTGCCAGATCTAC
CCCCCTAATGCCAACAAGATCAGGGAGGCCCTGGCGCAGACGCACAGCGC
CATCGCTGTCATCATCGGAATCAAGGATCTGGACGCATTCCGGCACTATG
ACGGGCGCACAATCATCCAGCGCGACAACGGATATCAGCCAAACTACCAC
GCGGTCAACATCGTGGGTTACTCGAACGCCCAGGGGGTGGACTACTGGAT
CGTGAGAAACAGTTGGGACACTAACTGGGGCGACAACGGCTACGGCTACT
TCGCCGCCAACATCGACCTGATGATGATCGAGGAGTACCCGTACGTGGTG ATCCTGTAA SEQ ID
NO. 17, protein sequence
RPSSIKTFEEYKKAFNKSYATFEDEEAARKNFLESVKYVQSNGGAINHLS
DLSLDEFKNRFLMSAEAFEHLKTQFDLNAETNACSINGNAPAEIDLRQMR
TVTPIRMQGGCGSCWAFSGVAATESAYLAYRNQSLDLAEQELVDCASQHG
CHGDTIPRGIEYIQHNGVVQESYYRYVAREQSCRRPNAQRFGISNYCQIY
PPNANKIREALAQTHSAIAVIIGIKDLDAFRHYDGRTIIQRDNGYQPNYH
AVNIVGYSNAQGVDYWIVRNSWDTNWGDNGYGYFAANIDLMMIEEYPYVV IL
Construction of Humanized ProDer p 1 Expression Vector.
[0090] As the sequencing of eight bacterial clones demonstrated
some mutations in the synthetic ProDer p 1 gene, the plasmid for
stable expression was generated starting from four ProDer p 1 DNA
fragments derived from bacterial clones carrying pNIV4845. Clones
n.degree.5 and n.degree.20 were respectively submitted to double
digestions by HindIII-BssHII and SphI-BglII, to isolate the 228 bp
HindIII-BssHII and 272 bp SphI-BglII ProDer p 1 DNA fragments.
Clone n.degree.7 was restricted with BssHII-SphI and BglII-XbaI to
generate the 239 bp BssHII-SphI and 329 bp BglII-XbaI ProDer p 1
DNA fragments. These fragments were inserted into the HindIII-XbaI
cut pEE14 expression vector (Celltech) [16] to give the final
plasmid pNIV4846. The correct recombinants were confirmed by DNA
sequencing.
Transient Transfections and Selection of RecProDer p 1-Producing
Stable CHO-K1 Lines.
[0091] To determine the expression levels of recProDer p 1, COS
cells (ATCC) were transiently transfected with 10 .mu.g of pNIV4846
or pNIV4853, a plasmid carrying authentic ProDer p 1 gene, by
calcium phosphate coprecipitation. For stable recProDer p 1
expression, CHO-K1 cells (ATCC) were transfected with pNIV4846
plasmid by lipofection. After a 3-weeks 25 .mu.M
methionylsulphoximin (MSX, Sigma) selection, one round of gene
amplification was carried out with 100 .mu.M MSX.
Expression of the Recombinant Allergen in CHO Cells.
[0092] The best producing recombinant CHO-K1 clone was cultured in
cell factories in GMEM medium (Invitrogen) supplemented with 2%
fetal calf serum (Gibco). Spent culture medium was harvested every
72 h and stored at -20.degree. C. until purification.
Purification of Natural Der p 1 from Natural Mite Whole Body
Extracts.
[0093] Purification of natural Der p 1 from whole mite culture was
performed as previously described [13]. Briefly, D. pteronyssinus
extracts were submitted to (NH.sub.4).sub.2SO.sub.4 precipitation
to 60% saturation. The precipitate, collected by
ultracentrifugation and resuspended in PBS containing
(NH.sub.4).sub.2SO.sub.4 1M, was applied onto a Resource Phenyl
column (Pharmacia) equilibrated in PBS containing
(NH.sub.4).sub.2SO.sub.4 1M. Der p 1 was eluted from the column
with water. After the pH and conductivity adjustments of the Der p
1-enriched fractions, the pool was applied onto a Q sepharose fast
flow column (Pharmacia) equilibrated in 20 mM Tris-HCl pH 9. Der p
1 was eluted by addition of 200 mM NaCl in the starting buffer. The
Der p 1 purification was achieved by a gel filtration
chromatography onto a superdex-75 column (Pharmacia) equilibrated
in PBS pH 7.3. Purified Der p 1 was concentrated and stored at
-20.degree. C.
Purification of RecProDer p 1 from CHO Spent Culture Medium.
[0094] CHO spent culture medium was diluted two times with water
and the pH was adjusted to 7.2. The modified supernatant was loaded
onto a Q sepharose fast flow column (5.times.10 cm, Pharmacia)
equilibrated in 20 mM Tris-HCl pH 7.2 which is coupled to a
hydroxyapatite column (2.6.times.15 cm, Bio-Rad) conditioned in the
same buffer. The flow-through containing recProDer p 1 of both
columns was adjusted to pH 9 and applied onto a Q sepharose fast
flow column (1.6.times.10 cm) equilibrated in 20 mM Tris-HCl pH 9.
The column was washed with the starting buffer and with the same
buffer supplemented with 100 mM NaCl. ProDer p 1 was eluted by a
linear NaCl gradient (100-300 mM, 15 column volumes). The recProDer
p 1-enriched fractions were pooled and concentrated by
ultrafiltration onto a Filtron membrane (Omega serie, cut-off: 10
kD). The recProDer p 1 purification was achieved by a gel
filtration chromatography onto a superdex-75 column (1.times.30 cm,
Pharmacia) equilibrated in PBS pH 7.3. Purified recProDer p 1 was
concentrated and stored at -20.degree. C.
SDS PAGE and Western Blot Analysis
[0095] Proteins were analyzed by SDS-PAGE on 12.5% polyacrylamide
gels. After electrophoresis, proteins were transferred onto
nitrocellulose membranes using a semi-dry transblot system
(Bio-Rad). Membranes were saturated for 30 min with 0.5% Instagel
(PB Gelatins) in TBS-T (50 mM Tris HCl pH 7.5, 150 mM NaCl, 0.1%
Tween 80) and incubated with rabbit polyclonal serum raised against
Der p 1 peptide 245-267 diluted in blocking solution (1:5000)
(Kindly provided by Dr Pestel, Institut Pasteur de Lille, France)
[17]. Immunoreactive materials were detected using alkaline
phosphatase-conjugated goat anti-rabbit antibodies (Promega,
1:7500) and 5-bromo,4-chloro,3-indolylphosphate (BCIP,
Boehringer)/nitroblue tetrazolium (NBT, Sigma) as substrates.
Glycan Analysis
[0096] Carbohydrate analysis was carried out with the Glycan
Differenciation Kit (Boehringer) using the following lectins:
Galanthus nivalis agglutinin (GNA), Sambucus nigra agglutinin
(SNA), Maackia amurensis agglutinin (MAA), Peanut agglutinin (PNA)
and Datura stramonium agglutinin (DSA). Briefly, purified proteins
were transferred from SDS-PAGE onto nitrocellulose membranes.
Membranes were incubated with the different lectins conjugated to
digoxigenin. Complexes were detected with anti-digoxigenin
antibodies conjugated to alkaline phosphatase.
Enzymatic Assays
[0097] Enzymatic assays were performed in 50 mM Tris-HCl pH 7,
containing 1 mM EDTA and 20 mM L-cysteine at 25.degree. C. in a
total volume of 1 ml. Hydrolysis of
Cbz-Phe-Arg-7-amino-4-methylcoumarin (Cbz-Phe-Arg-AMC) and
Boc-Gln-Ala-Arg-7-amino-4-methylcoumarin (Boc-Gln-Ala-Arg-AMC)
(Sigma) (both substrates at a final concentration of 100 .mu.M) was
monitored using a SLM 8000 spectrofluorimeter with
.lamda..sub.ex=380 nm and .lamda..sub.em=460 nm. Assays were
started by addition of cysteine activated allergen to a final
concentration of 100 nM. Before any assay, purified Der p 1 or
recProDer p 1 was incubated with a mixture of aprotinin- and
p-aminobenzamidine-agarose resins (Sigma) to remove any putative
trace of serine protease activity.
Protein Determination
[0098] Total protein concentration was determined by the
bicinchoninic acid procedure (MicroBCA, Pierce) with bovine serum
albumin as standard.
Der p 1 ELISA
[0099] Der p 1 or recProDer p 1 was detected with an ELISA kit
using Der p 1 specific monoclonal antibodies 5H8 and 4C1 (Indoor
Biotechnologies). The Der p 1 standard (UVA 93/03) used in the
assay was at a concentration of 2.5 .mu.g/ml.
IgE-Binding Activity.
[0100] Immunoplates were coated overnight with Der p 1 or recProDer
p 1 (500 ng/well) at 4.degree. C. Plates were then washed 5 times
with 100 .mu.l per well of TBS-Tween buffer (50 mM Tris-HCl pH 7.5,
150 mM NaCl, 0.1% Tween 80) and saturated for 1 hr at 37.degree. C.
with 150 .mu.l of the same buffer supplemented with 1% BSA (Sigma).
Sera from allergic patients to D. pteronyssinus and diluted at 1/8
were then incubated for 1 hr at 37.degree. C. Out of the 95 sera
used in the experiments, 16 sera ranged in their specific anti-D.
pteronyssinus IgE values (RAST assays) from 58.1 kU/L to 99 kU/L
and 79 above the upper cut-off value of 100 kU/L. Plates were
washed 5 times with TBS-Tween buffer and the allergen-IgE complexes
were detected after incubation with a mouse anti-human IgE antibody
(Southern Biotechnology Associates) and a goat anti-mouse IgG
antibody coupled to alkaline phosphatase (dilution 1/7500 in
TBS-Tween buffer, Promega). The enzymatic activity was measured
using the p-nitrophenylphosphate substrate (Sigma) dissolved in
diethanolamine buffer (pH 9.8). OD.sub.410 nm was measured in a
Biorad Novapath ELISA reader.
[0101] For IgE inhibition assays, plates were coated with Der p 1
or recProDer p 1 at the same concentration (0.12 .mu.M). A pool of
20 human sera from allergic patients (RAST value>100 kU/L) was
preincubated overnight at 4.degree. C. with various concentrations
(3.6-0.002 .mu.M) of Der p 1 or recProDer p 1 as inhibitors and
added on ELISA plates. IgE-binding was detected as described
above.
Histamine Release
[0102] The histamine release was assayed using leukocytes from the
peripheral heparinized blood of an allergic donor and by the
Histamine-ELISA kit (Immunotech). Basophils were incubated with
serial dilutions of recProDer p 1 or Der p 1 for 30 min at
37.degree. C. The total amount of histamine in basophils was
quantified after cell disruption with the detergent IGEPAL CA-630
(Sigma).
RESULTS
Synthesis of Humanized ProDer p 1 Gene.
[0103] The codon prevalence of ProDer p 1 gene displayed many
divergences compared with that used for highly expressed human
genes (FIG. 1). In consequence, oligonucleotides were designed for
the construction of a synthetic ProDer p 1 gene to optimise the
allergen expression in mammalian cells. As shown in FIG. 1, the
final codon frequency in the synthetic ProDer p 1 gene was very
similar to that used in highly expressed mammalian genes.
[0104] The synthetic ProDer p 1 was assembled from mutually priming
oligonucleotides that were subsequently amplified by PCR (FIG. 2).
After one round of PCR, amplified products displayed a molecular
weight ranging from 3000 to 300 bp. A subsequent amplification with
primers complementary to the 5' end of VZV gE leader peptide and to
the 3' end of synthetic ProDer p 1 gene led to a 1072 bp fragment
of excepted size. The amplified fragment was cloned into the pCRII
cloning vector. Sequence analysis of recombinant clones revealed
the presence of point mutations and deletions in the synthetic
ProDer p 1 gene. Finally, the correct coding cassette was obtained
after ligation of 4 different fragments isolated from 3 independent
bacterial clones, and inserted in the mammalian expression vector
pEE14 to give the final plasmid pNIV4846.
Transient and Stable Expression of RecProDer p 1
[0105] To compare the expression efficiency of the synthetic ProDer
p 1 construct with the original sequence, COS cells were
transfected with pNIV4846 and pNIV4853, a pEE14-derived plasmid
carrying authentic ProDer p 1 cDNA. The recProDer p 1 expression
level of the supernatants was estimated by an ELISA assay, using
two anti-Der p 1 monoclonal antibodies. As shown in FIG. 3, the
expression vector carrying the synthetic cDNA directed the
recProDer p 1 synthesis more efficiently than the same vector
containing the authentic gene. The expression level of humanized
ProDer p 1 gene was enhanced up to 450 ng/ml/72 h which represents
a 6-fold increase compared with the reference construction (75
ng/ml/72 h). As expected, no recProDer p 1 expression was detected
using COS cells transfected by a control vector without any
insert.
[0106] CHO-K1 cells were transfected with pNIV4846 and clones
resistant to 25 .mu.M MSX were selected. The recProDer p 1 level
assayed by ELISA indicated that three independent clones secreted
recProDer p 1 up to 11 .mu.g/ml/72 h. Addition of sodium butyrate,
a molecule previously reported to enhance expression level of
recombinant proteins in culture medium [18], did not influence the
recProDer p 1 synthesis. A further amplification of 25 .mu.M
MSX-resistant clones up to 100 .mu.M MSX increased expression,
raising 26 to 34 .mu.g/ml/72 h recProDer p 1 in culture medium. The
clone n.degree.1 was used for recProDer p 1 large-scale production
in cell factories and purification. Spent culture medium was
collected every 72 h and up to 9 harvests were performed. In these
conditions, the best recProDer p 1 expression level raised 15
.mu.g/ml in the culture medium before purification.
Purification of RecProDer p 1.
[0107] Purification of recProDer p 1 was achieved by a combination
of three chromatographic steps, using anion-exchange,
hydroxyapatite and gel filtration media. The final purification
yield was about 6 mg of recProDer p 1 per litre of culture medium
with a recovery close to 40%. On SDS PAGE, purified recProDer p 1
migrated as three immunoreactive species: two major bands with a
respective molecular weight of 41 and 36 kD and one minor band of
38 kD (FIG. 4). This result indicated that the propeptide cleavage,
to yield mature Der p 1, did not occur during the expression and
purification steps, as natural Der p 1 migrated on SDS PAGE as a 29
kD band. The purity of the product was higher than 90%.
Biochemical Characterization of ProDer p 1
[0108] All the recProDer p 1 species were submitted to an
amino-terminal amino acid sequencing. The N-terminal sequence of 41
and 38 kDa species were identical and started at residue
Arg.sub.19. The sequence was identified as Arg-Pro-Ser-Ser-Ile,
which corresponds to the N-terminal sequence of the Der p 1
propeptide and indicates that cleavage of VZV gE signal peptide
proceeded efficiently. Surprisingly, the N-terminal sequence of the
36 kDa band started at residue Ala.sub.38 (the obtained sequence
was Ala-Thr-Phe-Glu-Asp) showing that, for the 36 kDa molecule, an
internal cleavage of the prosequence occurred between Tyr.sub.37
and Ala.sub.38. Carbohydrate analysis of recProDer p 1 was
performed by glycan recognition with several specific lectins.
Among the five lectins used, only the GNA lectin reacted with the
36 kDa recProDer p 1, pointing to the presence of terminal mannose
residues on this molecule, either as high-mannose N-glycan chains
or as exposed mannose in hybrid chains (FIG. 5). The 38 and 41 kDa
bands were recognized respectively by the DSA and MAA lectins,
showing that the 38 kDa molecule carried terminal galactose linked
.beta.(1-4) to N-acetyl-glucosamine in N-glycan chains whereas the
carbohydrate structure of the upper band was terminated by sialic
acid linked .alpha.(2-3) to galactose. As previously showed [13],
Der p 1 did not react with any lectin confirming that Der p 1 is
not glycosylated.
[0109] The enzymatic activity of recProDer p 1 was measured using
Cbz-Phe-Arg-AMC and Boc-Gln-Ala-Arg-AMC as substrates [19,20]. As
expected, because of the presence of the Pro region, RecProDer p 1
was totally inactive in our assays. In the same experimental
conditions, fluorogenic molecules were fully degraded within 4 min
by natural Der p 1 used at the same molarity.
IgG- and IgE-Reactivities of RecProDer p 1
[0110] RecProDer p 1 was tested in ELISA assays to determine
whether the recombinant allergen displayed reactivities similar to
those of Der p 1 towards specific anti-Der p 1 IgG and
anti-Dermatophagoides pteronyssinus IgE. As shown in FIG. 6,
equimolar concentrations of both allergens reacted similarly with
two Der p 1 specific monoclonal and conformational antibodies,
suggesting that recProDer p 1 displayed the overall structure of
the natural allergen. The IgE reactivity of recProDer p 1 and Der p
1 was compared in a direct ELISA wherein immunoplates were directly
coated with Der p 1 or recProDer p 1. A set of 95 human sera with
positive radioimmunosorbent tests to D. pteronyssinus extract was
used at dilution 1:8. IgE titer determinations clearly showed a
close correlation of IgE reactivity with both allergens, indicating
that recProDer p 1 has very similar IgE binding characteristics
compared with Der p 1 (R.sup.2=0.8171, P<0.0001) (FIG. 7).
Histamine Releasing Activity of RecProDer p 1
[0111] To compare the allergenic activity of natural Der p 1 and
recProDer p 1, basophils from one allergic patient were challenged
in vitro with various concentrations of both allergens and the
released histamine was measured. Natural Der p 1 was able to induce
histamine release from basophils even at a concentration of 1
ng/ml. By contrast, recProDer p 1 could only release histamine at
1000-fold higher concentration (FIG. 8). From this result,
recProDer p 1 was shown to be less allergenic that the natural Der
p 1.
EXAMPLE 2, EXPRESSION OF RecProDerP1 IN Pichia pastoris
Construction of ProDer p 1 Expression Vector
[0112] The ProDer p 1 coding cassette from pNIV4846 (full-length
1-302aa ProDer p 1 cDNA with optimised mammalian codon usage) was
amplified by PCR using the following primers:
5'ACTGACAGGCCTCGGCCGAGCTCCATTAA3' (SEQ ID NO. 18) (StuI restriction
site in bold, forward) and 5'CAGTCACCTAGGTCTAGACTCGAGGGGAT3' (SEQ
ID NO. 19) (AvrII restriction site in bold, reverse). The amplified
fragment was cloned into the pCR2.1 TOPO cloning vector. The
correct ProDer p 1 cassette was verified by DNA sequencing.
Recombinant TOPO vector was digested with StuI-AvrII to generate a
918 bp fragment which was introduced into the pPIC9K expression
vector restricted with SnaBI-AvrII. The resulting plasmid,
pNIV4878, contains the ProDer p 1 cassette downstream to the S.
cerevisae .alpha. factor
Site-Directed Mutagenesis
[0113] Expression plasmid for the production of unglycosylated
ProDer p 1 (N52Q, mature Der p 1 numbering) was derived from
pNIV4878 by overlap extension PCR using a set of four primers. The
following primers: 5'GGCTTTCGAACACCTTAAGACCCAG3' (SEQ ID NO. 20)
(primer 1, AflII restriction site in bold, forward) and
5'GCTCCCTAGCTACGTA TCGGTAATAGC3' (SEQ ID NO. 21) (primer 2, SnaBI
restriction site in bold, reverse) were used to amplify a 317 bp
fragment encoding the ProDer p 1 amino acid sequence 71-176. The
following primers 5'CCTCGCGTATCGGCAACAGAGCCTGGACC3' (SEQ ID NO. 22)
(primer 3, mutation N52Q in bold, forward) and 5'GGTCCAGGCTCT
GTTGCCGATACGCGAGG3' (SEQ ID NO. 23) (primer 4, mutation N52Q in
bold, reverse) were used to introduce mutation N52Q in the ProDer p
1 sequence.
[0114] The mutated 317 bp AflII-SnaBI fragment was generated by a
three-step process. In PCR n.degree.1, primers 1 and 4 were mixed
with pNIV4878 to produce a 200 bp fragment. In PCR n.degree.2,
primers 2 and 3 were mixed with pNIV4878 to produce a 140 bp. The
two PCR products were purified onto agarose gel and used as
templates for a third round of PCR to obtain a .about.340 bp
fragment. This purified fragment was cloned into the pCR2.1 TOPO
cloning vector. The mutation was verified by DNA sequencing.
Recombinant TOPO vector was digested with AflII-SnaBI to generate a
317 bp fragment which was ligated into the similarly digested
pNIV4878. The resulting plasmid, pNIV4883, contains the ProDer p 1
N52Q downstream to the S. cerevisae .alpha. factor
[0115] To obtain unglycosylated variants of ProDer p 1 carrying
mutations of Der p 1 cysteine residues at position 4, 31 or 65
(mature Der p 1 numbering), overlap extension PCR using the same
set of primers were performed with plasmids pNIV4873, pNIV4875 and
pNIV4874. The resulting plasmids pNIV4884, 4885 and 4886 encode
respectively ProDer p 1 N52Q C4R, N52Q C31R and N52Q C65R.
Transformation of P. pastoris
[0116] Plasmid pNIV4878 was introduced into P. pastoris using the
spheroplast transformation method. Transformants were selected for
histidinol deshydrogenase (His+) prototrophy. The screening of His+
transformants for geneticin (G418) resistance was performed by
plating clones on agar containing increasing concentrations of
G418.
Production of ProDer p 1 by Recombinant Yeast
[0117] G418 resistant clones were grown at 30.degree. C. in BMG
medium to an OD.sub.600 nm of 2-6. Cells were collected by
centrifugation and resuspended to an OD.sub.600 nm of 1 in 100 ml
of BMG medium. Proder p 1 expression was induced by daily addition
of methanol 0.5% for 6 days. The supernatant was collected by
centrifugation and stored at -20.degree. C. until purification.
Purification of ProDer p 1 from Yeast Culture Supernatant
[0118] Supernatants were diluted 10 times with water and, after pH
adjustment to 9, directly loaded onto a Q sepharose column
equilibrated in 20 mM Tris-HCl pH 9. The column was washed with the
starting buffer. Protein elutions proceeded by step-wise increasing
NaCl concentration in the buffer. The ProDer p 1-enriched fractions
were pooled and concentrated by ultrafiltration onto a Filtron
membrane (Omega serie, cut-off: 10 kD). The ProDer p 1 purification
was achieved by a gel filtration chromatography onto a superdex-75
column (1.times.30 cm, Pharmacia) equilibrated in PBS pH 7.3.
Purified ProDer p 1 was concentrated and stored at -20.degree. C.
Surprisingly, given the fact that yeast codon usage is
significantly different from the human profile, this humanized
ProDerP1 expressed very well in this system with a high yield of
protein.
Discussion
[0119] The inability to obtain large amounts of Der p 1, the major
allergen from D. pteronyssinus is a major obstacle for the
development of biochemical and immunological studies. Indeed, whole
mite culture is cost effective, the growth rate is slow and the
purification yield of native Der p 1 is relatively low, about 1 mg
Der p 1 being purified from 1 gram of whole mite culture in our
experimental conditions. Moreover, previous attempts of Der p 1
expression in bacteria and yeast indicated that the allergen was
poorly expressed and mainly under an insoluble form [10-12].
[0120] The present study clearly reports that production of
recProDer p 1 in mammalian cells is very low indicating that the
presence of prosequence is not sufficient to induce high-level
recProDer p 1 expression.
[0121] The codon prevalence of the Proder p 1 gene was different
from that most frequently used in highly expressed human genes. To
assess the importance of an appropriate codon usage for the
recProDer p 1 expression in CHO cells, we decided to engineer a
synthetic ProDer p 1 gene based on the mammalian prevalent codons.
Our results clearly demonstrate that codon optimisation is
beneficial to induce high-level expression of recProDer p 1 in
mammalian cells.
[0122] In summary, codon usage optimisation can induces high-level
expression of recProDer p 1, an allergen difficult to produce in
CHO cells. This strategy could also be applicable for expression of
other allergens and could be extrapolate to other expression
systems. Synthetic genes with appropriate codons could thus provide
new tools for allergy diagnosis and specific immunotherapy.
[0123] RecProDer p 1, immobilized on solid phases, could substitute
natural Der p 1 in diagnostic test for the detection of specific
IgE. Considering the reduced recProDer p 1 anaphylactogenic
potential, this recombinant allergen could be used in the future as
alternative reagents for immunotherapy to replace the commonly used
allergen extracts.
Sequence CWU 1
1
23 1 114 DNA Artificial Sequence Codon Optimised Dermaphagoides
gene 1 gaagcttcgg gcgaattgcg tggttttaag tgactatatt cgagggtcgc
ctgtaatatg 60 gggacagtta ataaacctgt ggtgggggta ttgatggggt
tcggaattat cacg 114 2 96 DNA Artificial Sequence Codon Optimised
Dermaphagoides gene 2 gaaggctttc ttgtattcct cgaaggtctt aatggagctc
ggccgtgctc tgaccggatt 60 cgttatacgc aaggtacccg tgataattcc gaaccc 96
3 97 DNA Artificial Sequence Codon Optimised Dermaphagoides gene 3
ggaatacaag aaagccttca acaagagcta tgccaccttc gaggacgagg aggccgcgcg
60 caagaacttc ctggaaagcg tgaaatacgt gcagagc 97 4 113 DNA Artificial
Sequence Codon Optimised Dermaphagoides gene 4 gtcttaaggt
gttcgaaagc ctcggcgctc atcaggaacc ggttcttgaa ctcgtctaaa 60
gacaggtcgg acaggtgatt tatagccccg ccgttgctct gcacgtattt cac 113 5 93
DNA Artificial Sequence Codon Optimised Dermaphagoides gene 5
ctttcgaaca ccttaagacc cagtttgatc tcaacgcgga gaccaacgcc tgcagtatca
60 acggcaatgc ccccgctgag attgatctgc gcc 93 6 100 DNA Artificial
Sequence Codon Optimised Dermaphagoides gene 6 gactctgtcg
cggccacgcc tgaaaaggcc caacaagacc cgcagccgcc ttgcatgcgg 60
atgggagtca cggtcctcat ctggcgcaga tcaatctcag 100 7 92 DNA Artificial
Sequence Codon Optimised Dermaphagoides gene 7 gtggccgcga
cagagtcggc atacctcgcg tatcggaatc agagcctgga cctcgctgag 60
caggagctcg ttgactgcgc ctcccaacac gg 92 8 100 DNA Artificial
Sequence Codon Optimised Dermaphagoides gene 8 gctacgtatc
ggtaatagct ttcctgcacg acgccattat gctggatgta ttcgatacct 60
ctgggaatcg tatccccatg acatccgtgt tgggaggcgc 100 9 87 DNA Artificial
Sequence Codon Optimised Dermaphagoides gene 9 gctattaccg
atacgtagct agggagcagt cctgccgccg tcctaacgca cagcgcttcg 60
gcatttccaa ttattgccag atctacc 87 10 95 DNA Artificial Sequence
Codon Optimised Dermaphagoides gene 10 ccttgattcc gatgatgaca
gcgatggcgc tgtgcgtctg cgccagggcc tccctgatct 60 tgttggcatt
aggggggtag atctggcaat aattg 95 11 91 DNA Artificial Sequence Codon
Optimised Dermaphagoides gene 11 gtcatcatcg gaatcaagga tctggacgca
ttccggcact atgacgggcg cacaatcatc 60 cagcgcgaca acggatatca
gccaaactac c 91 12 66 DNA Artificial Sequence Codon Optimised
Dermaphagoides gene 12 gtagtccacc ccctgggcgt tcgagtaacc cacgatgttg
accgcgtggt agtttggctg 60 atatcc 66 13 82 DNA Artificial Sequence
Codon Optimised Dermaphagoides gene 13 ccagggggtg gactactgga
tcgtgagaaa cagttgggac actaactggg gcgacaacgg 60 ctacggctac
ttcgccgcca ac 82 14 85 DNA Artificial Sequence Codon Optimised
Dermaphagoides gene 14 gctctagact cgagggatcc ttacaggatc accacgtacg
ggtactcctc gatcatcatc 60 aggtcgatgt tggcggcgaa gtagc 85 15 909 DNA
Dermaphagoides pteronyssinus 15 cgtccatcat cgatcaaaac ttttgaagaa
tacaaaaaag ccttcaacaa aagttatgct 60 accttcgaag atgaagaagc
tgcccgtaaa aactttttgg aatcagtaaa atatgttcaa 120 tcaaatggag
gtgccatcaa ccatttgtcc gatttgtcgt tggatgaatt caaaaaccga 180
tttttgatga gtgcagaagc ttttgaacac ctcaaaactc aattcgattt gaatgctgaa
240 actaacgcct gcagtatcaa tggaaatgct ccagctgaaa tcgatttgcg
acaaatgcga 300 actgtcactc ccattcgtat gcaaggaggc tgtggttcat
gttgggcttt ctctggtgtt 360 gccgcaactg aatcagctta tttggcttac
cgtaatcaat cattggatct tgctgaacaa 420 gaattagtcg attgtgcttc
ccaacacggt tgtcatggtg ataccattcc acgtggtatt 480 gaatacatcc
aacataatgg tgtcgtccaa gaaagctact atcgatacgt tgcacgagaa 540
caatcatgcc gacgaccaaa tgcacaacgt ttcggtatct caaactattg ccaaatttac
600 ccaccaaatg taaacaaaat tcgtgaagct ttggctcaaa cccacagcgc
tattgccgtc 660 attattggca tcaaagattt agacgcattc cgtcattatg
atggccgaac aatcattcaa 720 cgcgataatg gttaccaacc aaactatcac
gctgtcaaca ttgttggtta cagtaacgca 780 caaggtgtcg attattggat
cgtacgaaac agttgggata ccaattgggg tgataatggt 840 tacggttatt
ttgctgccaa catcgatttg atgatgattg aagaatatcc atatgttgtc 900
attctctaa 909 16 1511 DNA Artificial Sequence CDS (1)...(909) Codon
Optimised Dermaphagoides gene misc_feature 79, 160, 225, 230, 239,
308, 384, 391, 449, 459, 464, 527, 548, 697, 699, 711, 775, 852,
857, 865, 940, 955, 959, 1018, 1026, 1034, 1039, 1101, 1194, 1250,
1257, 1261, 1273, 1333, 1337, 1356, 1416, 1425, 1443 n = A,T,C or G
Codon Optimised Dermaphagoides gene 16 cgg ccg agc tcc att aag acc
ttc gag gaa tac aag aaa gcc ttc aac 48 Arg Pro Ser Ser Ile Lys Thr
Phe Glu Glu Tyr Lys Lys Ala Phe Asn 1 5 10 15 arg rsr sry sth rhg
ugu tyr ysy saa has naa gag cta tgc cac ctt 96 Xaa Xaa Xaa Xaa Xaa
Cys Xaa Xaa Xaa Xaa Xaa Glu Leu Cys His Leu 20 25 30 cga gga cga
gga ggc cgc gcg caa gaa ctt cys srt yra ath rhg uas 144 Arg Gly Arg
Gly Gly Arg Ala Gln Glu Leu Xaa Xaa Xaa Ile Xaa Xaa 35 40 45 gug
uaa aaa rgy sas nhc tgg aaa gcg tga aat acg tgc aga gca acg 192 Val
* Lys Xaa Xaa Xaa Trp Lys Ala * Asn Thr Cys Arg Ala Thr 50 55 60
gcg ggg cta taa atc acu gus rva yst yrv agn sra sng ygy aaa snh 240
Ala Gly Leu * Ile Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Lys Xaa 65 70
75 sct gtc cga cct gtc ttt aga cga gtt caa gaa ccg gtt cct gat gag
288 Xaa Val Arg Pro Val Phe Arg Arg Val Gln Glu Pro Val Pro Asp Glu
80 85 90 cus ras usr uas guh ysa sna rgh umt srg ccg agg ctt tcg
aac acc 336 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Pro Arg Leu Ser
Asn Thr 95 100 105 tta aga ccc agt ttg atc tca acg cgg aga agu aah
guh suy sth rgn 384 Leu Arg Pro Ser Leu Ile Ser Thr Arg Arg Ser Xaa
Xaa Xaa Xaa Xaa 110 115 120 125 has uas naa gua cca acg cct gca gta
tca acg gca atg ccc ccg ctg 432 Xaa Xaa Xaa Val Pro Thr Pro Ala Val
Ser Thr Ala Met Pro Pro Leu 130 135 140 aga ttg atc tgt hra sna acy
ssr asn gya sna ara agu asu cgc cag 480 Arg Leu Ile Cys Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Ser Xaa Arg Gln 145 150 155 atg agg acc gtg act
ccc atc cgc atg caa ggc ggc tgc ggg arg gnm 528 Met Arg Thr Val Thr
Pro Ile Arg Met Gln Gly Gly Cys Gly Xaa Xaa 160 165 170 tar gth rva
thr rar gmt gng ygy cys gyt ctt gtt ggg cct ttt cag 576 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Val Gly Pro Phe Gln 175 180 185 gcg
tgg ccg cga cag agt cgg cat acc tcs rcy str aah srg yva aaa 624 Ala
Trp Pro Arg Gln Ser Arg His Thr Xaa Xaa Xaa Xaa Xaa Xaa Lys 190 195
200 205 ath rgu sra aty rug cgt atc gga atc aga gcc tgg acc tcg ctg
agc 672 Ile Xaa Xaa Xaa Xaa Arg Ile Gly Ile Arg Ala Trp Thr Ser Leu
Ser 210 215 220 agg agc tcg ttg aca aty rar gas ngn sru asu aag ugn
guu vaa stg 720 Arg Ser Ser Leu Thr Xaa Xaa Xaa Xaa Xaa Xaa Lys Xaa
Val Xaa Xaa 225 230 235 cgc ctc cca aca cgg atg tca tgg gga tac gat
tcc cag agg tat ccy 768 Arg Leu Pro Thr Arg Met Ser Trp Gly Tyr Asp
Ser Gln Arg Tyr Xaa 240 245 250 saa srg nhs gyc ysh sgy ast hrr arg
gyg aat aca tcc agc ata atg 816 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Asn Thr Ser Ser Ile Met 255 260 265 gcg tcg tgc agg aaa gct att
acc gat acg uty rgn hsa sng yva vag 864 Ala Ser Cys Arg Lys Ala Ile
Thr Asp Thr Xaa Xaa Xaa Xaa Xaa Xaa 270 275 280 285 ngu srt yrt yra
rgt yrg tag cta ggg agc agt cct gcc gcc gtc 909 Xaa Xaa Xaa Xaa Xaa
Xaa * Leu Gly Ser Ser Pro Ala Ala Val 290 295 ctaacgcaca gcgcttcggc
vaaaarggug nsrcysarga rgrasnaagn arghgyattt 969 ccaattattg
ccagatctac ccccctaatg ccaacaagat caggsrasnt yrcysgntyr 1029
rrasnaaasn ysarggaggc cctggcgcag acgcacagcg ccatcgctgt catcatcgga
1089 atcguaauaa gnthrhssra aaavagyaag gatctggacg cattccggca
ctatgacggg 1149 cgcacaatca tccagysasu asaaharghs tyrasgyarg
thrgncgcga caacggatat 1209 cagccaaact accacgcggt caacatcgtg
ggtargasas ngytyrgnra sntyrhsaav 1269 aasnvagyta ctcgaacgcc
cagggggtgg actactggat cgtgagaaac agttggtyrs 1329 rasnaagngy
vaastyrtrv aargasnsrt rgacactaac tggggcgaca acggctacgg 1389
ctacttcgcc gccaacatca sthrasntrg yasasngyty rgytyrhaaa aasngacctg
1449 atgatgatcg aggagtaccc gtacgtggtg atcctgtaaa sumtmtgugu
tyrrtyrvav 1509 au 1511 17 299 PRT Artificial Sequence VARIANT 17,
18, 19, 20, 21, 23, 24, 25, 26, 27, 43, 44, 45, 47, 48, 51, 52, 53,
68, 69, 70, 71, 72, 73, 74, 75, 77, 78, 94, 95, 96, 97, 98, 99,
100, 101, 102, 103, 121, 122, 123, 124, 125, 126, 127, 128, 146,
147, 148, 149, 150, 151, 152 Xaa = Any Amino Acid VARIANT 153, 155,
172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 199,
200, 201, 202, 203, 204, 207, 208, 209, 210, 227, 228, 229, 230,
231, 232, 234, 236, 237, 253, 254, 255, 256, 257, 258, 259, 260,
261, 262, 263, 280, 281, 282 Xaa = Any Amino Acid VARIANT 283, 284,
285, 286, 287, 288, 289, 290, 291 Xaa = Any Amino Acid Codon
Optimised Dermaphagoides gene 17 Arg Pro Ser Ser Ile Lys Thr Phe
Glu Glu Tyr Lys Lys Ala Phe Asn 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Cys
Xaa Xaa Xaa Xaa Xaa Glu Leu Cys His Leu 20 25 30 Arg Gly Arg Gly
Gly Arg Ala Gln Glu Leu Xaa Xaa Xaa Ile Xaa Xaa 35 40 45 Val Lys
Xaa Xaa Xaa Trp Lys Ala Asn Thr Cys Arg Ala Thr Ala Gly 50 55 60
Leu Ile Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Lys Xaa Xaa Val Arg 65
70 75 80 Pro Val Phe Arg Arg Val Gln Glu Pro Val Pro Asp Glu Xaa
Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Pro Arg Leu Ser Asn
Thr Leu Arg Pro 100 105 110 Ser Leu Ile Ser Thr Arg Arg Ser Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 Val Pro Thr Pro Ala Val Ser Thr
Ala Met Pro Pro Leu Arg Leu Ile 130 135 140 Cys Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Ser Xaa Arg Gln Met Arg Thr 145 150 155 160 Val Thr Pro
Ile Arg Met Gln Gly Gly Cys Gly Xaa Xaa Xaa Xaa Xaa 165 170 175 Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Leu Val Gly Pro Phe Gln Ala Trp Pro 180 185
190 Arg Gln Ser Arg His Thr Xaa Xaa Xaa Xaa Xaa Xaa Lys Ile Xaa Xaa
195 200 205 Xaa Xaa Arg Ile Gly Ile Arg Ala Trp Thr Ser Leu Ser Arg
Ser Ser 210 215 220 Leu Thr Xaa Xaa Xaa Xaa Xaa Xaa Lys Xaa Val Xaa
Xaa Arg Leu Pro 225 230 235 240 Thr Arg Met Ser Trp Gly Tyr Asp Ser
Gln Arg Tyr Xaa Xaa Xaa Xaa 245 250 255 Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Asn Thr Ser Ser Ile Met Ala Ser Cys 260 265 270 Arg Lys Ala Ile Thr
Asp Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 275 280 285 Xaa Xaa Xaa
Leu Gly Ser Ser Pro Ala Ala Val 290 295 18 29 DNA Artificial
Sequence rge ProDer p 1 18 actgacaggc ctcggccgag ctccattaa 29 19 29
DNA Artificial Sequence ProDer p 1 19 cagtcaccta ggtctagact
cgaggggat 29 20 25 DNA Artificial Sequence ProDer p 1 20 ggctttcgaa
caccttaaga cccag 25 21 27 DNA Artificial Sequence ProDer p 1 21
gctccctagc tacgtatcgg taatagc 27 22 29 DNA Artificial Sequence
ProDer p 1 22 cctcgcgtat cggcaacaga gcctggacc 29 23 29 DNA
Artificial Sequence ProDer p 1 23 ggtccaggct ctgttgccga tacgcgagg
29
* * * * *