Extract
Note: Please read the complete
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Introduction
Accurate allergy diagnosis and efficient immunotherapy
protocols are strongly dependent on the use of
standardized extracts or well-characterized allergen
mixtures[1-3]. However, standardization of specific allergens from natural
allergen extracts is a huge task[4] and it is almost impossible
to carry out standardization on a large scale for day-to-day
clinical practice. Recombinant allergens that have similar
structures, functions and immunoreactivity to their native
counterparts could be used to overcome this problem, given
that they can be produced in large quantities, leading to
more specific, effective and safer immunotherapy
treatment[5].
Currently, gene cloning for novel allergens is mainly
based on the construction of a cDNA library, which is
time-consuming and expensive[6-9]. Other methods for isolating
unknown allergens involve using the polymerase chain
reaction (PCR) with either degenerate primers consisting of a
pool of primers containing most or all of the possible
nucleotide sequences encoding a conserved amino acid
motif[10], or consensus primers consisting of a single primer
containing the most common nucleotide at each codon position
within the motif[11]. Although these strategies have been
successful in isolating closely related sequences, they have
generally failed when sequences are more distantly
related[11]. Many programs and algorithms have been developed for
sequence comparisons of multiple members of protein
families[12-15]. By using alignment techniques, the first amino
acid sequence that emerges, that is, the sequence aligned
uppermost in each large and dense cluster in the output tree
produced, has provided the most information about amino
acid diversity in the cluster. In the current study, we refer to
these sequences as "core sequences", and we designed the
degenerate primers based on the core sequences and their
cognates. Under special PCR conditions, we expected that
the degenerate primers could match all associated sequences
in the abovementioned cluster, and thus could be used for
cloning new allergen gene members in a distantly related
species. We will hereafter refer to these primers as
"pan-degenerate primers". We expected that the process
involving the pan-degenerate primers would be more efficient and
less expensive than the cDNA library technique, and would
have a higher success rate than an ordinary PCR method.
Humulus scandens (Lour) Merr (Moraceae: Cannaboideae;
Lücao in Chinese) is a weed that grows in most provinces of
China. The plant blossoms in summer and bears a large
quantity of pollen. Short ragweed (Ambrosia
artemisiifolia L; Compositae) is one of the most invasive weeds in the
world. It was introduced to China in 1930s, and is now
distributed from the Yantze River to the 3 northeastern prov
inces of China. Nanning, Wuhan, Nanchang-Jiujiang and
Shenyang-Tieling-Dandong are the 4 areas in which the weed
is most pervasive in China. Pollen from H scandens
and A artemisiifolia have become the main source of pollen
allergens in this country. However, little is known of the actual
allergens in the pollen of the weeds. In this study, we report
on the cloning, expression and characterization of 6 pollen
allergen genes from the 2 weeds. Following the non-fusion
expression and purification of the 5 encoded proteins, 2
representative proteins of the 5, provisionally named rAmb a
8(D03) and rHum s 1(LCM9), were immunologically
characterized in detail.
Materials and methods
Sample preparations Wild plants of H
scandens and A artemisiifolia were harvested in Wuhan. Flowers were
bagged, and the pollen collected from each plant was mixed
with other pollen collected from the same plant. All pollen
collected was immediately submerged in liquid nitrogen
until use. Twenty-five patients with A
artemisiifolia pollen allergy and 10 patients with
H scandens pollen allergy (as assessed by a positive skin prick test reaction to
A artemisi-ifolia and a serum level of A artemisiifolia/H scandens
specific IgE higher than 3.5 kUA/L) and 13 healthy volunteers
were recruited. Blood samples were taken from all
participants in the Allergy Clinic, Shenyang North Hospital. All
the samples were tested by using the UniCAP Specific IgE
Fluoroenzymeimmunoassay (Pharmacia Diagnostics AB,
Uppsala, Sweden). After centrifugation at
500×g, 4 °C, for 10 min, serum from each individual was collected and stored at
-70 °C until use. The study was approved by the Ethics
Committee of Shenyang North Hospital, Shenyang, China,
and written informed consent was obtained from all
participants.
Primer design The 478 pollen allergens and their related
sequences were retrieved by keyword searches in the
SWISS-PROT and TrEMBL websites (http://www.expasy.org) and
were multiple-aligned online by using Clustal W
software[16]. For alignment, the default values were used to obtain the
output tree in a phylogram pattern, with the absolute
distances labeled. The uppermost allergen sequence in each
large and dense cluster in the output tree was taken as the
core sequence. There were several of these core sequences
found in the output phylogenetic trees. Based on the core
sequences, allergen sequences with high homology (with
BLAST E values £10-10) were selected for further analysis to
acquire conservative gene domains. Pan-degenerate primers,
which we expected to be successful in all species, were thus
designed according to cDNA sequences of the
conservative domains chosen.
RT-PCR analysis Frozen pollen samples were ground in
a mortar filled with liquid nitrogen, and the total RNA was
extracted by using an Rneasy Maxi Kit (Qiagen Valencia,
CA, USA) according to the manufacturer¡¯s instructions.
After quality and concentration assessments, the RNA was
either immediately used for RT-PCR analysis or stored at
-80 ºC until use.
After reverse transcription (2 µg total RNA per sample)
was performed by using the ProtoScript First Strand cDNA
Synthesis Kit (New England Biolabs, Beverly, MA, USA)
according to the manufacturer¡¯s instructions, PCR was
carried out by using 50 ng of the reverse transcription product
in 25 µl of the reaction mixture containing 1.25 U HotstarTaq
DNA polymerase (Qiagen), 1× PCR buffer (Qiagen), 0.2
mmol/L dNTP, and 0.4 µmol/L of each primer. Thermal cycling was
performed in a PTC-200 Peltier Thermal Cycler (MJ Research,
Watertown, MA, USA) as follows: after an initial step at
95 °C for 15 min to activate the DNA polymerase, 9
touchdown cycles were carried out at 94 °C for 45 s,
68 °C for 30 s and 72 °C for 30 s, with the annealing temperature
decreasing by 1 ºC per cycle. This was followed by 26 gradient
cycles at 94 ºC (45 s)¡ú55
ºC¡ú61 ºC (30
s)¡ú72 ºC (30 s). After purification with a
QIAquick PCR Purification Kit (Qiagen), the PCR products were cloned into the pGEM-T
easy vector (Promega, Madison, WI, USA). The inserted
plasmids were transformed into JM109 (Promega). Positive
clones were confirmed first by colony PCR with the former
PCR primers, and then by sequencing. DNA sequencing
was carried out by the BioAsia Biotechnology Co (Shanghai,
China) by using the dideoxy chain-terminating method.
Full-length cDNA synthesis and sequencing
The clones derived from the degenerate primers did not cover the
full-length coding region. Gene-specific primers were therefore
synthesized (Table 1), and combined using the RACE
process[17] with RA3, a 3¡¯ reverse primer from the GeneRacer Kit
(Invitrogen Corporation, Carlsbad, CA, USA). The PCR
conditions were similar to those for RT-PCR, with a slight
modification in the denaturing temperature and extension time.
PCR products were also cloned into the pGEM-T easy vector,
and positive clones were confirmed by colony PCR and
sequencing. Full-length cDNAs, acquired by deduction of
the truncated gene and its 3¡¯ end, were translated into amino
acid sequences, with their isoelectric point and molecular
weight calculated (http://www.expasy.org). cDNA and
protein homologues, retrieved by using the BLAST program in
the NCBI website (http://www.ncbi.nlm.nih.gov/), were
aligned by using the CLUSTAL W (1.82)
program[16]. Phylogenetic trees were subsequently generated using MEGA2
software[18].
Northern blot Northern blots were performed according
to the membrane manufacturer¡¯s instructions. Briefly, a total
of 20 mg RNA was loaded on to a formaldehyde-containing
gel. After running the gels at 40 V for 3 h, the RNA bands
were electrically transferred onto Nytran supercharge
nylon transfer membrane (Schleicher & Schull BioScience, New
Hampshire, USA). After UV cross-linking treatment, the
membrane was stained with methylene blue [0.02%
[w/v] methylene blue in 0.3 mol/L sodium acetate(pH 5.5)] to investigate
the transferring efficiency. Following destaining, BrightStar
Psoralen-Biotin (Ambion Inc, Autin, TX, USA)-labeled probe
D03 was applied for hybridization in ULTRhyb hybridization
buffer (Ambion Inc). The membrane was washed twice with
a buffer containing 2× SSC (0.3 mol/L NaCl, 0.03 mol/L
sodium citrate) and 0.1% sodium dodecylsulfate before the
probes were detected by using the Phototope Star
Detection Kit (New England Biolabs). Finally, the filter was
exposed to Fuji Medical X-ray film at room temperature for
5-8 min.
Protein expression and immunoblotting To enable
non-fusion protein expression, the S-protein tag and His-tag were
removed from the pET-44 vector and the allergen-coding
regions were decorated by introducing restriction sites (5¡¯
NdeI and 3¡¯ PstI) by PCR using the corresponding primers (Table
1). Subsequent to double restriction digestion and
purification, the PCR products were ligated with the pET-44
EK/LIC vector (Novagen, Madison, WI, USA). After
transformation of the ligation products into NovaBlue Singles
cells (Novagen), positive clones and in-frame insertions were
confirmed, initially by colony PCR with the primer SProt and
3¡¯ primers related to each clone, and then by sequencing
with the S-Tag 18-mer primer or ColiDown primer (Novagen).
Plasmid DNA was extracted with the QIAprep Miniprep Kit
(Qiagen), and transformed into E coli RosettaBlue (DE3) cells
(Novagen) for expression, following the manufacturer¡¯s
instructions. Cells were grown in 1 L Luria-Bertani
(LB)-medium until the OD600 value reached 0.6. Target protein
expression was induced by the addition of
isopropyl-D-thio-galactopyranoside (IPTG) to 1 mmol/L for 4 h at 30 °C before
harvest. Finally, the recombinant proteins were purified by
affinity chromatography using poly-(L)-proline as the solid
phase. The bacterium lysate was applied to the column,
followed by washing with 1 mol/L urea and elution with 6
mol/L urea. The elution product was dialyzed stepwise
against decreasing amounts of urea (6, 4, 2 mol/L, no urea) in
phosphate-buffered saline (PBS; 1.8 mmol/L potassium
phosphate, pH 7.4, 0.137 mol/L NaCl) and then dialyzed
against double-distilled water, followed by freeze-drying.
Purified rAmb a 8(D03) and rHum s(LCM9) were selected
as representatives for immunoblotting. Fifteen micrograms
of protein were loaded onto the 15% SDS-PAGE gel, and
were then electrically transferred to a polyvinylidene
difluoride (PVDF) membrane (Amersco, Solon, OH, USA).
Following blocking the membrane overnight with a 4%
bovine serum albumin solution at 4 ºC, allergic sera (diluted 1:7)
were added to each membrane strip for 2 h before the
addition of peroxidase conjugated goat anti-human IgE
(Sigma-Aldrich, Saint Louis, MO, USA). The blotted band was
finally visualized with a diaminobenzidine (DAB)
chromogenic substrate solution (Amersco).
N-terminal end sequencing Sequencing of the
N-terminal end of recombinant proteins was performed by using the
Applied Biosystems Procise 491 Sequencer from Applied
Biosystems, according to the manufacturer¡¯s instructions.
Results
Homology between different allergen genes The 478
pollen allergens obtained from the SWISS-PROT and TrEMBL websites were clustered into 8 large groups, which
consisted of a number of subgroups. Further searching with
the core sequences from each subgroup demonstrated that
these core sequences matched a large quantity of closely
related homologues. For instance, Q9XF40, the first core
sequence in group 8, was an apple pollen allergen with
extensive homology with other pollen allergens from different
species (Figure 1), such as Poa pratensis, Lolium
perenne, Dactylis glomerata, Hordeum vulgare, Holcus
lanatus (e values <2e-25). The conservative domains in the Q9XF40
cDNA sequence (1-20 bp, 343-369 bp) as resolved by
multiple sequence alignment of Q9XF40 and its cognates were
taken as the Sg1p5/Sg1p3 primer sequence (Table 1). Other
degenerate primers were designed following the same
principle.
Full-length cDNA generation By combining the
gradient PCR with touchdown PCR, 3 gene fragments were
obtained from each weed species, and were subsequently
inserted into the pGEM-T vector. Sequence analysis showed
that the inserts, 5 of 363 bp and 1 of 369 bp, were
homologous to the known allergen gene profilin from different
species with up to 90% (5 clones except for D03) and 79% (for
D03) identity, but with ~30 bp truncated at the 3¡¯ ends,
suggesting a further requirement of the RACE process for the
full-length cDNA. After cloning of the amplificants from the
specific PCR with primer pairs PC11/RA3, PM9/RA3,
PD10/RA3 and PD03/RA3, a total of 29 positive RACE clones were
detected. Subsequent sequencing reactions demonstrated
that they all contained the initiation and stop codons, primer
sequences and poly (A) tails, reflecting the mRNA source of
the clones. Northern blotting with a clone D03 probe further
confirmed that the cDNA cloned was derived from the pollen
mRNA (Figure 2). Bridging the 3¡¯ end and the corresponding
fragment genes produced 6 novel full-length coding genes,
among which LCS13, LCM20 and LCM9 were from H
scandens, whereas A0418, D106 and D03 were from
A artemisiifolia. They are all available in GenBank under
accession Nos AY268422-AY268427.
Sequence analysis Sequence analysis revealed that there
were several altered nucleotides in the cDNA sequences
compared with the degenerate primers used in the
fragment-gene cloning process (data not shown), which means that
the degeneracy was effectively expanded. The predicted
amino acid sequences of the 6 genes were acidic proteins (pI
of approximately 5.0) with molecular weights of approximately
14 kDa. Multiple sequence alignment demonstrated that
these 6 novel sequences shared a high homology, up to
83%. Moreover, cDNA and protein sequence searches with
Blastn and Blastx programs in GenBank showed that the new
genes were homologous to profilin genes from more than 10
plant families including Rosaceae, Euphorbiaceae,
Phaseo-leae, Liliaceae, Poaceae, Urticaceae, Apiaceae, Oleaceae,
Sola-naceae, Brassicaceae, and Compositae. Alignment of the
cognates derived from this search produced a phylogenetic
tree, which showed that the 6 novel genes cloned were clearly
separated into 3 subgroups, but with high homology (Figure 3).
Of the 6 clones, A0418, D106 and LCM9, 3 closely related
clones that encoded 131-amino acid residues, exhibited high
homology with apple pollen allergen Q9XF41 (Mal d 4;
GenBank accession number: AF129427). LCS13 and LCM20,
2 clones from H scandens, had only differences in the cDNA
non-coding regions and therefore encoded the same protein,
which had 80% and 85% identity with Q8L5D8 from date
palm pollen and Q9XF40 from apple pollen, respectively.
Clone D03 encoded 133 amino acid residues, and had an
identity of 79% and 72% with O81982 (Hela
2)[19] and CAD12862[20], 2 profilin proteins from common sunflower
and mugwort. Hel a 2 and D03, along with the timothy grass
profilins O24282, P35079, O24650, were clustered into one
subgroup (Figure 3). Furthermore, D03 was quite different
from the other 5 clones in 4 regions of its amino acid
sequence, which represents an approximately 90% difference
between D03 and other profilins (Figure 4).
Protein expression and purification, and immunoblotting
After detecting the correct reading frame of the target
protein gene in the expression vector, the in-frame plasmids
were then successfully transformed into E coli
RosettaBlue (DE3) cells. Following induction and further incubation at
30 ºC for 6 h, the recombinant non-fusion proteins were
visualized using SDS-PAGE, and had an apparent molecular mass
of ~14 kDa (Figure 5A). The N-terminal sequence of 10 amino
acids of rAmb a 8(D03) confirmed that the recombinant
protein was the same as that deduced from the cDNA sequence.
The yield of purified protein was approximately 8 mg per L of
cultured bacteria.
IgE antibodies from 6 of 8 sera (75%) from the
A artemisi-ifolia allergic patients reacted with recombinant protein rAmb
a 8(D03) in the immunoblot experiments. The bands occurred
at approximately 14 kDa (Figure 5B). Other recombinant
proteins were also very efficiently expressed and purified in
non-fusion form (Figure 5C, 5D). As a representative from
H scandens, the immunoblotting profile of rHum s 1(LCM9) is
shown in Figure 5E. Interestingly, rHum s 1(LCM9) not only
reacted with the sera from 5 patients with H
scandens allergy, but also reacted with sera from patients with
A artemisiifolia allergy, which suggests that cross-reactivity occurred for
rAmb a 8(D03) and rHum s 1(LCM9).
Discussion
In the past, most allergen genes have been cloned by
cDNA library screening[7-9], and hundreds of pollen allergen
genes from more than 170 species are available in online
databases, which can be sorted into several groups. This
means that homologous genes exist in many related or
unrelated species, which provided support for the current method.
In this study, we designed pan-degenerate primers and
succeeded in cloning pollen allergen cDNAs from different
weeds, thus showing that there is a way to clone
homologous genes from distantly related species or little-studied
species. Furthermore, by using a combination of the
gradient PCR process and touchdown steps, the genes with some
divergence in their primer regions could be obtained in the
weed genome; that is, the degeneracy of the primers was
expanded. The method could therefore be used to clone the
same allergens in other species or to clone other kinds of
allergen genes.
It has been argued that allergenicity cannot be
rationalized on the basis of either overall folding pattern or
biological function, and thus any protein should be regarded as a
potential allergen[19]. According to alignment studies, the
proteins coded by our new clones had more than 60%
identity over the full-length peptide with the known allergens,
suggesting that the proteins would be allergenic. Thus, D03
and LCM9, the most different variants of the 6 clones
obtained, were expressed as recombinant proteins. Through
efficient expression in a pET-44 system, ideal solubility of all
the recombinant proteins was obtained, which facilitated
protein purification through affinity chromatography, and
would ensure efficiency in commercial production.
The molecular basis of cross-reactivity has been of
primary concern in numerous
publications[19-21]. The relationship between sequence similarity, as obtained by pair-wise
alignment, and structural or functional properties has been
the goal of much research[22-24]. Recent studies have
confirmed a widely accepted rule-of-thumb that 30% or 35%
identity over aligned regions suffices for structural or functional
deduction[25,26]. A remote homologue
study[27] demonstrated that allergens from diverse sources have a common
structural motif, namely a groove located inside an
a-b motif, which could potentially serve as a ligand-binding site, thus
leading to the stimulation of the T cell helper type 2 (Th2)
response and a subsequent bias towards the synthesis of
IgE. This result expanded cross-reactivity to between
microbes and higher plants. In the present study, the clones
were different from each other in their cDNA or protein
sequence, suggesting open pollination and/or large genetic
variation in the weeds. This was also consistent with other
studies on ragweed and timothy grass sequence
polymorphism[28,29]. However, multiple sequence analysis
demonstrated that the newly cloned genes had up to 83%
homology with each other and 60%-90% identity with previously
described allergen profilins from latex, food, and pollens from
various organisms, which can produce extensive cross-reactivity[30,31]. This result demonstrated that proteins from
H scandens and A artemisiifolia would have cross-reactivity.
In a previous study we also inferred that the proteins coded
by clone D106 would have cross-reactive properties for food-
and/or pollen-sensitive allergic
individuals[32,33]. All of these results imply that the newly cloned genes would function
the same way as pan-allergen profilins. Based on the issues
described herein and the results obtained in the current
study, we assume that cross-reactivity would occur so widely
that atopic individuals would be surrounded by a dynamic
network of allergens, and would be predisposed towards
allergic diseases.
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