Expression and characterization of duck enteritis virus gI gene.

Abstract

At present, alphaherpesviruses gI gene and its encoding protein have been extensively studied. It is likely that gI protein and its homolog play similar roles in virions direct cell-to-cell spread of alphaherpesviruses. But, little is known about the characteristics of DEV gI gene. In this study, we expressed and presented the basic properties of the DEV gI protein.
The special 1221-bp fragment containing complete open reading frame(ORF) of duck enteritis virus(DEV) gI gene was extracted from plasmid pMD18-T-gI, and then cloned into prokaryotic expression vector pET-32a(+), resulting in pET-32a(+)-gI. After being confirmed by PCR, restriction endonuclease digestion and sequencing, pET-32a(+)-gI was transformed into E.coli BL21(DE3) competent cells for overexpression. DEV gI gene was successfully expressed by the addition of isopropyl-β-D-thiogalactopyranoside(IPTG). SDS-PAGE showed that the recombinant protein His6-tagged gI molecular weight was about 61 kDa. Subsequently, the expressed product was applied to generate specific antibody against gI protein. The specificity of the rabbit immuneserum was confirmed by its ability to react with the recombinant protein His6-tagged gI. In addition, real time-PCR was used to determine the the levels of the mRNA transcripts of gI gene, the results showed that the DEV gI gene was transcribed most abundantly during the late phase of infection. Furthermore, indirect immunofluorescence(IIF) was established to study the gI protein expression and localization in DEV-infected duck embryo fibroblasts (DEFs), the results confirmed that the protein was expressed and located in the cytoplasm of the infected cells, intensively.
The recombinant prokaryotic expression vector of DEV gI gene was constructed successfully. The gI protein was successfully expressed by E.coli BL21(DE3) and maintained its antigenicity very well. The basic information of the transcription and intracellular localization of gI gene were presented, that would be helpful to assess the possible role of DEV gI gene. The research will provide useful clues for further functional analysis of DEV gI gene.

Full text

Page 1

RESEARCHOpen Access
Expression and characterization of duck enteritis
virus gI gene
Lijuan Li1, Anchun Cheng1,2,3*, Mingshu Wang1,2*, Jun Xiang1, Xiaoyuan Yang1, Shunchuan Zhang1, Dekang Zhu2,3,
Renyong Jia1,2,3, Qihui Luo3, Yi Zhou3, Zhengli Chen3and Xiaoyue Chen2,3
Abstract
Background: At present, alphaherpesviruses gI gene and its encoding protein have been extensively studied. It is
likely that gI protein and its homolog play similar roles in virions direct cell-to-cell spread of alphaherpesviruses.
But, little is known about the characteristics of DEV gI gene. In this study, we expressed and presented the basic
properties of the DEV gI protein.
Results: The special 1221-bp fragment containing complete open reading frame(ORF) of duck enteritis virus(DEV)
gI gene was extracted from plasmid pMD18-T-gI, and then cloned into prokaryotic expression vector pET-32a(+),
resulting in pET-32a(+)-gI. After being confirmed by PCR, restriction endonuclease digestion and sequencing, pET-
32a(+)-gI was transformed into E.coli BL21(DE3) competent cells for overexpression. DEV gI gene was successfully
expressed by the addition of isopropyl-b-D-thiogalactopyranoside(IPTG). SDS-PAGE showed that the recombinant
protein His6-tagged gI molecular weight was about 61 kDa. Subsequently, the expressed product was applied to
generate specific antibody against gI protein. The specificity of the rabbit immuneserum was confirmed by its
ability to react with the recombinant protein His6-tagged gI. In addition, real time-PCR was used to determine the
the levels of the mRNA transcripts of gI gene, the results showed that the DEV gI gene was transcribed most
abundantly during the late phase of infection. Furthermore, indirect immunofluorescence(IIF) was established to
study the gI protein expression and localization in DEV-infected duck embryo fibroblasts (DEFs), the results
confirmed that the protein was expressed and located in the cytoplasm of the infected cells, intensively.
Conclusions: The recombinant prokaryotic expression vector of DEV gI gene was constructed successfully. The gI
protein was successfully expressed by E.coli BL21(DE3) and maintained its antigenicity very well. The basic
information of the transcription and intracellular localization of gI gene were presented, that would be helpful to
assess the possible role of DEV gI gene. The research will provide useful clues for further functional analysis of DEV
gI gene.
Background
Duck virus enteritis(DVE), also called duck plague, is an
acute and contagious herpesvirus infection of waterfowls
such as ducks, geese, and swans with high morbidity and
mortality [1]. The causative agent of DVE is duck enteri-
tis virus (DEV), which is a member of subfamily Alpha-
herpesvirinae of the family Herpesviridae, not assigned to
any genus according to the Eighth International Commit-
tee on Taxonomy of Viruses (ICTV) [2]. Like other her-
pesvirus, DEV establishes a lifelong infection, via a
quiescent state known as latency. The genome of DEV is
composed of a linear, double stranded DNA and the
G+C content is 64.3%, higher than any other reported
avian herpesvirus in the subfamily Alphaherpesvirinae
[3]. Recently, an increasing number of DEV genes, such
as UL5 [4], UL6 [5], UL22, UL23(TK) [6], UL24 [6,7],
UL25-UL30 [8], UL31-UL35 [9-11], UL38 [12], UL44(gC)
[13], UL46 [14], UL50(dUTPase) [15], UL51 [16], UL53
(gK) [17], US3-US5 [18,19], US8(gE) [20], US2 and US10
[21], have been identified. The DEV genomic library was
successfully constructed in our laboratory [22], and the
gI(Us7) gene(GenBank accession no.: EU035298) was iso-
lated and identified from DEV CHv strain [23].
* Correspondence: chenganchun@vip.163.com; mshwang@163.com
1Institute of Preventive Veterinary Medicine, Sichuan Agricultural University,
Wenjiang, Chengdu city, Sichuan, 611130, P.R.China
Full list of author information is available at the end of the article
Li et al. Virology Journal 2011, 8:241
http://www.virologyj.com/content/8/1/241
© 2011 Li et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

Page 2

The gI gene is located in unique short region (Us)
within the herpesviral genome, its homolog almost
existed in all alphaherpesvirus. The gI gene encoding
membrane protein glycoprotein I(gI) is conserved among
the alphaherpesviruses that have been sequenced. At pre-
sent, the most extensively studied on alphaherpesviruses
gI gene and its encoding protein are herpes simplex virus
type 1(HSV-1), varicella-zoster virus(VZV), and pseu-
dorabies virus(PRV). In all instances studied to date, the
glycoprotein I (gI) and glycoprotein E (gE) form a nonco-
valent complex gE/gI that are localized to the plasma
membrane, the virion envelope, and all internal mem-
branes (except for mitochondria) in infected cells [24].
Biological functions ascribed to gE/gI include cell-cell
spread, binding of antibody immunoglobulin G (IgG) Fc
receptor. Alphaherpesvirus gI protein played an impor-
tant role in virion sorting and promoting direct cell-to-
cell spread in polarized cells, but not enrty of extrcellular
virions [25]. Moreover, gI complexed with gE in HSV-1
[26], VZV [27] and PRV [28] to form Fc-receptor, partici-
pating in immune escape. Previous sequence analysis of
DEV CHv strain gI gene indicated that the ORF was
1116 bp in length and its primary translation product
was a polypeptide of 371 amino acids. The predicted pro-
tein possessed several characteristics of membrane glyco-
proteins and had a high degree of similarity to gI
homologs of other alphaherpesviruses [23]. Comparison
of predicted amino acid sequences to those of HSV-1,
VZV, and PRV homologs allowed the functions of DEV
gI protein to be putatively assigned. Nevertheless, little is
known about the characteristics of DEV gI gene.
In our study, the gI gene of DEV CHv-strain was extract
from recombinant plasmid pMD18-T-gI, in an effort to
elucidate the function of gI, we constructed a recombinant
plasmid pET-32a(+)-gI and successfully expressed the
DEV gI fused to His6 in a prokaryotic expression system.
We prepared polyclonal antiserum which allowed identify-
ing and characterizing the gI gene product of DEV. The
levels of the mRNA transcripts of gI were determined by a
real time-PCR method. In addition, the primary antibody
against the DEV gI recombinant protein was used for
intracellular localization by an indirect immunofluores-
cence assay(IIF). Taken together, the results indicate that
the gI gene was transcribed most abundantly during late
phase of infection, and the protein was expressed in DEV-
infected DEFs, principally locating in cytoplasm of the
infected cells. This work may provide a foundation for
further studies on the function of DEV gI gene.
Results
Identification of recombinant plasmid
The special 1221-bp fragment containing complete ORF
of DEV gI gene was cloned into pET-32a(+) vector,
resulting in construct pET-32a(+)-gI. For confirmation,
plasmid DNAs of constructs was verified by PCR analy-
sis(Figure 1A) and restriction enzyme digestion with
BamHI and XhoI(Figure 1B).
Expression and purification of recombinant protein
His6-tagged gI
The expression products collected at different culture peri-
ods were characterized by SDS-PAGE and Western blot-
ting. The results showed that there was a specific band
with a molecular weight of 61 kDa in crude cell extracts
(Figure 2A, lane 2), that is consistent with the calculated
molecular weight of the DEV gI protein. SDS-PAGE
revealed that the recombinant protein was expressed effi-
ciently and constantly in E.coli BL21(DE3) cells. The
expression level peaked 6 h after induction with 0.2 mM
IPTG. Based on the His6 tag present at its N-terminal end,
the recombinant gI was purified by Ni-NTA affinity chro-
matography (Figure 2A, lane 3). The purified protein was
identified by rabbit anti-DEV serum in Western blotting
(data not shown).
Preparation and specificity of anti-His6-tagged gI protein
antiserum
The rabbit anti-His6-tagged gI IgG, with 55 kDa and 25
kDa of the heavy chain and the light chain, was firstly
precipitated by ammonium sulfate precipitation (Figure
2B, lane 1) and then purified by High-Q anion exchange
chromatography (Figure 2B, lane 2). Western blotting
analysis showed that the purified His6-tagged gI was
recognized by the rabbit anti-His6-tagged gI IgG and
Figure 1 Identification of the recombination vector pET-32a
(+)-gI. A. PCR product of the fragment of DEV gI detected by 1%
agarose gel electrophoresis. Lane M, DNA marker(in bp); Lane 1, PCR
product of the DEV gI. B. DEV gI gene encoding DNA sequence was
cloned into pET-32a(+) prolaryotic expression vector as described in
materials and methods. The construct was digested with two
restriction enzymes. Lane M(in bp), DNA marker; Lane 1, BamHI and
XhoΙ generating two restriction fragments; Lane 2, BamHI
generating one restriction fragment.
Li et al. Virology Journal 2011, 8:241
http://www.virologyj.com/content/8/1/241
Page 2 of 9

Page 3

showed a specific band at 61 kDa, which is the expected
size of the fusion protein (Figure 2C, lane 1). No posi-
tive signal was observed when using the pre-immune
serum (Figure 2C, lane 2), indicating that the recombi-
nant protein induced an immunological response and
that the antiserum had a high level of specificity. Based
upon these results, this antiserum was deemed suitable
to characterize the structure, molecular mechanism and
functional involvement of the gI protein in the DEV life
cycle.
Determination of mRNA expression of gI in infected cells
In the real time-PCR(RT-PCR) analysis, the dissociation
curve of gI gene or b-actin gene showed a single peak at
expected temperature(data not shown), that indicated
specific amplification of those two genes. The standard
curve(Figure 3) for gI and corresponding internal control
b-actin gene obtained by RT-PCR using plasmid DNA as
template showed similar correlation coefficient and PCR
efficiency, it could be known that standard curve and the
established RT-PCR are excellent at performance [29].
The mRNA expression of gI gene at different times
after infection were determined by normalizing the cycle
threshold (CT) values with b-actin gene CTvalues, and
then the histogram reflecting the transcription tendency
of gI gene was constructed by iQ5 Optical System
Software(Figure 4). It cound be found that, the level of
mRNA was low in early phases of infection, presenting
slightly increased after 3 h p.i.. Subsequently, signal
intensity immediately increased after 12 h p.i., peaked at
48 h p.i., and then declined.
Intracellular localization of the gI protein in DEV-infected
cells
Intracellular distribution of DEV gI protein could be
visualized by IIF experiments utilizing rabbit immune
serum against expressed gI protein or pre-immune
serum. As shown in Figure 5, infected cells (Figure 5G-
R) showed a specific green fluorescent cytoplasmic
staining pattern, whereas essentially no signal was
detected in mock-infected cells(Figure 5A-C) or corre-
sponding preimmune serum (Figure 5D-F). The faint
fluorescence could be detected in the cytoplasm of
infected cells as early as 4 h p.i. (Figure 5G-I), and then
a strong fluorescence was found intensively distributed
in the cytoplasm and especially in the juxtanuclear
region at 12 h p.i.. A typical pattern of staining is shown
in Figure 5J-L. After that, following by a series of mor-
phological changes, the cytoplasm disintegration and
nuclear fragmentation in DEV-infected cells, fluores-
cence was gently dispersed at 36 h p.i.(Figure 5M-O)
and 48 h p.i. (Figure 5P-R).
Figure 2 SDS-PAGE of the purified fusion protein and rabbit anti-His6-tagged gI IgG and Western blotting analysis. A. Induction of the
His6-tagged gI fusion protien in E. coli. Recombinant plasmid pET-32a(+)-gI was transformed into bacteria. Lane M, molecular mass markers (in
kDa); lane 1, protein products of the uninduced recombinant bateria; lane 2, protein products of the induced recombinant bateria; lane3, purified
fusion protein His6-tagged gI. The arrowhead indicates the induced gI fusion protein; B. SDS-PAGE analysis of the purified rabbit anti-His6-
tagged gI IgG. Lane M, protein molecular weight marker(in kDa); lane 1, rabbit anti-His6-tagged gI IgG obtained by ammonium sulfate
precipitation; lane 2, rabbit anti-His6-tagged gI IgG obtained by High-Q anion exchange chromatography; C. Reactivity and specificity of the
purified gI antiserum analyzed by Western blotting. Proteins were separated by SDS-PAGE and transferred to PVDF membranes. Lane M, protein
molecular weight marker(in kDa); lane 1, the membranes were incubated with the gI antiserum; lane 2, the membranes were incubated with
preimmune rabbit serum. Arrowheads indicate the fusion protein His6-tagged gI.
Li et al. Virology Journal 2011, 8:241
http://www.virologyj.com/content/8/1/241
Page 3 of 9

Page 4

Figure 3 Establishment of the real time-PCR(RT-PCR) standard curve. A. The standard curve for gI gene obtained by SYBR Green RT-PCR
using recombinant plasmid pMD18-T-gI as template. B. The standard curve for b-actin gene obtained by SYBR Green RT-PCR using recombinant
plasmid pMD18-T-b-actin. Ten-fold dilutions of standard DNA were used, as indicated in the x-axis, whereas the corresponding cycle threshold
(CT) values are presented on the y-axis. Each dot represents the result of amplification of each dilution. The correlation coefficient and the slope
value of the regression curve were calculated and are indicated.
Figure 4 Determination of mRNA expression of gI in DEV-infected DEFs. The total RNA were harvested from uninfected(Neg) or DEV-
infected DEFs at different times p.i. (0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 36, 48, and 60 h), presented on the x-axis. After reverse transcription, the
cDNA was used as template for SYBR Green RT-PCR analysis, the data were normalized to the expression level of b-actin, as indicated in the y-
axis. Data were analyzed using the iQ5 optical system software (Bio-Rad)
Li et al. Virology Journal 2011, 8:241
http://www.virologyj.com/content/8/1/241
Page 4 of 9

Page 5

Discussion
Currently, gI gene has been studied extensively in
human and nonhuman herpesviruses [27,30-33]. As
mention in instruction, gI and gE formed a heterodimer
gE/gI in alphaherpesviruses, gE/gI can promote direct
cell-to-cell spread in polarized cells, but not entry of
extracellular virions. Given that gE/gI specifically func-
tions, this glycoprotein provides an excellent molecular
tool to study cell-to-cell spread [34]. According to the
previous report [23], a gene equivalent to the gI of other
alphaherpesviruses was identified and sequenced in DEV
CHv strain. The predicted amino acid sequence pos-
sesses several characteristics typical of membrane glyco-
proteins, including a N-terminal hydrophobic signal
sequence, C-terminal transmembrane and cytoplasmic
domains, and extra-cellular region containing three
potential N-linked glycosylation sites. Compared with
other alphaherpesviruses, DEV gI showed high identity
at the amino acid level. But the analysis of its expression
and characteristics have not been reported until now.
Experimental determination of the DEV gI gene expres-
sion and localization in infected cells has become
necessary.
The analysis of gene expression requires sensitive, pre-
cise, and reproducible measurement of specific mRNA
sequences. The methods used to quantify mRNA include
techniques based upon hybridization and real-time PCR
(RT-PCR), RT-PCR is becoming a common tool for
detecting and quantifying expression profiles of selected
genes [35]. SYBR Green I is the most frequently used
dsDNA-specific dye in RT-PCR today [36]. We have
developed a rapid real-time quantitative PCR method
using the icycler IQ Real-time PCR Detection System
coupled with SYBR Green chemistry, to evaluate the time
course of mRNA formation and decay of DEV gI gene.
Recently, relative quantitation has become the analytic
method of choice for many real-time PCR studies. In this
method a comparison within a sample is made with the
gene of interest to that of a control gene. Relative quanti-
tation relies on the assumption that the endogenous con-
trol gene does not vary under the experimental
conditions. Control genes that have been successfully
used include b-actin, GAPDH, 18S ribosomal RNA, His-
tone 3.3a, ubiquitin, and several others [29]. In our study,
to control for the variation in sample processing and in
reverse transcription reaction among samples, DEF b-
actin gene was amplified in parallel with the DEV gI
gene. The chosen control gene b-actin does not vary in
expression level among the samples of study.
Base on analyses of the HSV kinetics, both synthesis of
virus proteins and transcription of virus DNA were coor-
dinately regulated and sequentially ordered [37,38]. How-
ever, research on the expression kinetics of DEV genes has
been rare. Our study showed that the gI gene of DEV tran-
scription products appeared low level before 12 h p.i., then
increased acutely and reached a peak at 48 h p.i., declining
slowly thereafter, which owes the characterization of her-
pervirus late genes. Although gI gene of DEV was pre-
sumed as a late gene, its transcripts was keeping slightly
Figure 5 Intracellular location and distribution of DEV gI proein
analyzed by IIF. Mock-infected (A-C) and DEV-infected (D-R). DEFs
were fixed at different stages (4 h p.i., 12 h p.i., 36 h p.i., and 48 h p.i.)
as described in materials and methods. The samples were stained
with the gI antiserum (A-C, G-R) or preimmune serum (D-F), and
reacted with anti-rabbit IgG-conjugated FITC, and then counter-
stained with DAPI (blue is representative the cell nuclei). A, D, G, J, M,
and P show the FITC staining; and B, E, H, J, M, and Q show the DAPI
staining. C, F, I, L, O, and R are the merged images of FITC and DAPI
staining. The arrows indicate the DEV gI FITC fluorescence staining.
(Images were acquired by using 20× objective).
Li et al. Virology Journal 2011, 8:241
http://www.virologyj.com/content/8/1/241
Page 5 of 9