s0">2.3. Primer Design and PC R Amplificatio n of the
cpsE Gene
The full-length of cpsE gene in this study (GenBank accession
no.HQ888682) consists of 450 base pairs. Primers with restric-
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168
tion sites Bam HI and Hind III (underlined), respectively. Pri-
mers: upstream 5'
CGGGATCCATGAAAATTTGTCTGGTTGG3’
downstream 5'
CCAAGCTT
Bacterial isolates from infected tilapia were initially identified
as GBS by conventional phenotypic characteristics (Gram-
positive cocci, β-haemolytic, catalase negative, API 20 STREP
system, Slidex Strepto-kit). Slidex Strepto-kit was positive for
group B and API 20 STREP gave profile number 3663414,
which co r r es ponded to excellent matches to GBS.
3.2. Bioinformatics Prediction
The analysis results obtained by the software DNAStar indi-
cated that there were nine distinct antigenic domains and six
surface probability domains. This program also showed a mea-
surable amount of beta strands with few turn regions and ran-
dom coil. Prediction of SignalP 3.0 Server confirmed the ab-
sence of signal peptide in CpsE sequence. Program SOSUI
Server indicated that amino acids from 80-102aa in the cpsE
gene may be part of a transmembrane region in the CpsE pro-
tein sequence .Results indicate that this region (80-102aa) is
likely anchored to the bacterial wall. And possibility of insolu-
bility when over-expressed in E.coli was 64.1%.
3.3. Plasmids Con struction
Coning plasmid pMD19-t/cpsE was confirmed by double re-
striction enzymes digestion (Bam HI and Hind III) (Figure 1)
and sequencing analysis. Correct fragment was fused with
pET-32a (+) to form the expression plasmid pET-32a (+)/cpsE.
The initial transformation was performed with competent
DH5αcells for screening. The positive clones were identified by
PCR amplification and double restriction enzymes digestion
(Figure 2).
TTAAAAAATTCCTCCTAAATT3’).
Amplification was done at 94 for 5 min and then at 94
for 1 min, 55for 1 min, and 72 for 40 s for a total of 30
cycles, followed by 72 for 10 m in.
2.4. Construction of Cloning Plasmid and Expre ssion
Plasmids
The target fragment was ligated into cloning vector pMD19-T
to construct recombinant cloning plasmid pMD19-T/cpsE.
Competent E.coli strain DH5α cells were used for host cloning
construction.
The pET-32a (+) vector and the recombinant cloning plas-
mids were both digested with Bam HI and Hind III and were
both spliced with T4 ligase (Takara, Japan) to construct the
recombinant prokaryotic expression plasmid pET-32a(+)/ cpsE.
The ligated product was transformed into competent DH5a cells .
Selection of positive clones was done by PCR amplification,
double enzymes digestion and sequence analysis. The positive
clones were transformed into expression hosts of BL21 (DE3)
cells.
2.5. Expression and O ptimization o f Expression
Conditions
Transformants were inoculated into 5 ml of culture medium in
test tubes and grown overnight at 37with constant agitation
(220 rpm) until they reached the exponential phase (approx-
imately OD600≈ 0.50.6 measured by absorbance at 600nm) and
were subsequently induced with IPTG. Preliminary tests were
performed with 1mM IPTG induction at 37 °C for 4 h and for
the optimization of mature CpsE expression. Optimization of
the expression conditions were performed at different tempera-
tures (30, 34, and 37), for different induction periods
(1h, 2h, 3h, 4h, 5h, 6h, 7 h and overnight) and with different
IPTG concentrations (0.2mM, 0.4mM. 0.6mM, 0.8mM, 1.0mM,
1.2mM, 1.5mM and 2.0mM).
2.6. Purification of the His6-tagged Recombinant
Protein
The His6-tagged recombinant protein was subjected to Bio-
RadTM metal affinity chromatography for purification. Under
denatured state, CpsE-containing fractions were pooled and
dialyzed against PBS buffer (10mM Na2 HPO 4 1.8mM KH 2
PO4, pH 7.4, 140mM NaCl, and 2.7mM KCl) with step-wise
lower con centration s of urea (6M, 4M, 2M and 0M ) for a total
time of 96h. The purified recombinant protein was stored at 4
for use within 1 week or at –70 for longer time.
3. Results
3.1. GBS Strain Isolate Confirming
Figure1. Characterization of the recombinant plasmid pMD19-
T/cpsE by restriction digestion and PCR-based amplification.M1:
DNA marker of 7000bp; lane 1:pMD19-T/cpsE digested with Bam
HI; lane 2:pMD19-T/cpsE dig e s t e d wi t h Bam H I an d Hind III; la ne
3:PCR amplification product of cpsE (ORF450bp); M2:DNA
m arker of 200 0b p
Fi gur e 2. Characterization of the recombinant plasmid
pET-32a(+)/cp sE by restriction digestion and PCR-based amplifi-
cation. M1:DNA marker of 200bp;lane 1:PCR amplification prod-
uct of cpsE ; lane 2:pET-32a(+)/cpsE digested with Bam HI and
Hind III; lane 3: pET-32a(+)/c p sE digested with Bam HI; lane 4:
pET-32a(+)digested with Bam HI and Hind III; lane 5: pET-32a(+)
digested with Bam HI.
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169
3.4. Expression of the Reco mbinant Protein
The protein expression was observed in expression host BL21
(DE3). A distinct band of approximately 36kDa of molecular
wei g h t , which corresponded to the expected size of the His6-
tagged CpsE fusion protein, was observed (Figure 3 a). Distinct
or correct band was not detected in both un-induced sample and
the pET-32a (+) vector culture in BL21 (DE3) cells (Figure
3(a)).The target protein was mainly expressed in the insoluble
fraction, in the form of inclusion bodies.
3.5. Optimization of Cul ture Conditions
We examined effects of the induction temperature, induction
periods and IPTG concentration on the CpsE expression level.
The expression level was analyzed by SDS-PAGE.The recom-
binant protein expression was best in BL21 (DE3) cells with
0.2mM IPTG induction (Figure 3b) at 3 7(Figure 3a) for 3h
(Figure 3c) after the cult ure r eached an OD600 of 0.6. Therefore,
this condition was used throughout the paper for the induction
of CpsE expression.
3.6. Purification and Immunogenicity Analysis of the
Reco mbinant Prote in
The CpsE was mainly eluted from Ni-NTA column using
250mM imidazole (Data not shown). A clear band corres-
ponded to a molecular mass of about 36kDa was observed on
the SDS-PAGE gel staining with Coomassie brilliant blue
(Figure 4). From a 1-L culture, 1.62mg of purified CpsE was
obtained. (4g of wet weight cell pellets were obtained from 1L
of bacterial culture in confirmed expression conditions in our
study) The purification profile is summarized in Table 1.
4. Discussion
GBS is among a select number of significant human and animal
pathogenic bacteria expressing polysaccharide. CPS confer
virulence on these organisms by interfering with the mechan-
isms of host immune recognition[27] .The striking conservation
of cpsE gene among GBS of different serotypes has suggested
that the product of this gene has function that is independent of
the repeating unit structure of the associated polysaccharide. To
our knowledge, there are no reports on characterization of CpsE
in fish.
Studies report that CpsE is expressed in heterologous expres-
sion systems in E.coli [18].I n this work, cpsE from GBS name-
ly serotype Ia, isolated from clinical tilapia, was cloned for
expression at high levels in E.coli with the intention of detect-
ing immunogenicity and further characterization of this protein
in fish.
The result obtained for CpsE solubility indicated that the
mature protein (without signal peptide), with the transmem-
brane region and a theoretic relative high possibility of insolu-
bility when over-expressed in E .coli, is mainly retained in the
insoluble fraction. This result corroborates the bioinformatics
prediction that overall the recombinant protein presents a hy-
drophobic ch aracter.
(a) (b)
(c)
Figure 3. (a) His 6 -tagged CpsE expression in BL21(DE3) at differe nt temperature conditions on a 12.5%SDS-PAGE gel.The sample are di-
luted five folds relative to the standard preparation for the gel(25μl of the sample buffer for each 1mL of cell pellet at Abs600=0.6); (b)
SDS-PAGE analysis for optimization of the concentration of IPTG for His6-tagged CpsE expression; (c) His6 -tagged CpsE expression for
differe nt induction period o n a 12.5%SDS-PAGE gel.It is able to compare the expression levels among lanes for an equal amount of sample
was lo a d ed in to ea ch lane.
X. FU ET AL.
Copyright © 2012 SciRes. ENG
170
Fi gur e 4. Purified CpsE on a 12.5%SDS-PAGE gel eluted from
Ni-NTA column using 250mM imidazole. Expression of all lanes
ar e a cco mplished at optimal co nditions (at 37 for 1h with 0.2mM
IPTG induction).
Table 1. Purification of cpse protein from 1l culture.
Step Total
proteina
(mg)
CpsE
(mg)
Protein
concentration
μg/mlb
Purity
(%)c
Cru de ext ractd 67.5 15.48 1,214 23
Immobilized metal affinity
chromatography 1.90 1.62 480 85
aTotal protein was isolated from 1liter of culture medium in optimal expression
condition; b Protein concentration was estimated by the Bradford method using
BSA as standard; c The purity was determined as the amount of the stain asso-
ciated with the CpsE band as a fraction of the stain associated with all the bands
on the SDS-PAGE[28]; d The pellets containing the insoluble fraction (crude
extract) obta ine d from 1liter of culture.
To date, there are no reports in literature that ap proached the
expression optimization allowing the development of a process
to produce recombinant CpsE in E.coli. Our work is able to
present conditions that are optimal for production of recombi-
nant protein. Distribution of B cell epitopes is largely related to
the secondary structures of the protein because regions of turn
and coil can easily change in shape that benefits combination of
protein with antibodies. Hydrophilic regions of the protein
would serve as likely B cell epitopes. Prediction results of hy-
drophilicity character and transmembrane region indicate that
there may have certain B cell epitopes in the CpsE protein.
Comprehensive analysis of turn regions, coil regions, surface
probability regions and hydrophilic regions presented here
suggest that the most possible sites for B cell epitopes are
Phe52-Asn57, Gly7-Gly11, Ser83-Gly84 and Asp112-Pro 114.
This speculate is able to locate B cell epitopes, which could
help to find sp ecific antigen and furth er benefit development of
subunit vaccin e.
5. Acknowledgements
This work was supported by grant from Program for Chang-
jiang Scholars and Innovative Research Teamin in University
(PCSIRT) (Grant no. IRT0848 ); Science and Technology
project of Sichuan Province (No.08ZA082). Kaiyu Wang is the
corresponding author. Fish Disease Research Center & Key
Laboratory of Animal Disease and Human Health of Sichuan
Province, College of Veterinary Medicine of Sichuan Agri-
cultural University, 46# Xinkang Road, Yucheng District, Yaan
62501 4, Si c h ua n Pr ovince of Chi na .
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