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http://www.abbs.info e-mail:[email protected] ISSN 0582-9879 ACTA BIOCHIMICA et BIOPHYSICA SINICA 2002, 34(4): 400-404 CN 31-1300/Q |
Expression
and Characterization of Hepatitis C Virus E2 Glycoprotein Fused to Hepatitis B
Virus preS1(21-47)
Fragment in CHO Cells
(
State Key Laboratory of Molecular Biology, Institute of Biochemistry and
Cell Biology,
Shanghai
Institutes for Biological Sciences, the Chinese Academy of Sciences, Shanghai 200031,
China )
HCV
has a positive-sense, single
stranded RNA genome of about 9.6 kb encoding a polyprotein precursor of about 3
000 amino acids by a single large open reading frame and is further processed
into various precursors and mature viral proteins[2, 3]. Three structural proteins
located at the N-terminal of the polyprotein are core, E1 and E2. The core protein is thought
to constitute the viral capsid with E1 and E2 being the virus envelope
proteins. HCV E2 glycoprotein has drawn much attention in vaccine
development, and host humoral and
cellular immune responses against this protein were elicited (e.g. references
[4-7]).
Previous work indicated that E2 expressed in mammalian system has advantage in
inducing neutralizing antibodies compared with E2 expressed in
prokaryotic, yeast or insect
expression system[8],
it made the CHO system a preferred system for expression of E2 protein.
As the preparation of full-length HCV envelope proteins from the cell lysate
was a laborious and high-cost procedure[8] due to the highly
hydrophobic character of the proteins,
secreted E2 without C-terminal hydrophobic region is preferred for
purification[9].
To
favor secretory expression of E2 protein,
the gene fragment encoding a C-terminally truncated form of E2 was
inserted into the secretory expression vector pSecTagB in this study. In
addition, to facilitate the
detection and purification of expressed E2 protein, a DNA fragment encoding a 27 amino acids fragment of
hepatitis B virus (HBV) preS1,
which contains the hepatocyte binding site of HBV[10], was incorporated at the N terminus of
HCV E2. A stable recombinant CHO cell line carrying above fusion gene was
established, which expresses an E2
protein fused to preS1 of HBV. The expression products were further
characterized.
1.1
Gene and antibodies
HCV
E2 gene (gene type 1b, GenBank
accession # D10934, in plasmid
pUC18/E) was kindly provided by Professor WANG Yu[11] (Peking
University, Beijing). Rabbit
anti-HCV E2 polyclonal antibodies RE2116 raised against E.coli
derived HCV E2 peptide was produced in our lab[12], HBV preS1 peptide (21-47)
specific monoclonal antibody 125E11,
peroxidase conjugated 125E11 and 125E11-agarose were generated as
described[13].
1.2
Construction of plasmid pSecTag2B/sS1E2
The
HCV E2 sequence encoding amino acids 384-661
of the HCV polyprotein amplified from pUC18/E was cloned into pSecTagB
(Invitrogen), then a sequence
encoding an HBV preS1 polypeptide (21-47
aa) amplified from preS1 expression vector pcDNA3/SS1[14] was
incorporated at the N-terminus of E2. A factor Xa cleavage site was introduced
between preS1 epitope and HCV E2. A schematic diagram of the plasmid construct
is shown in Fig.1. The insertion of the sequence encoding preS1-tagged E2 was
confirmed by DNA sequencing.
Fig.1 Schematic maps of part of the HCV cDNA
and pSecTagB/sS1E2
The fusion genes were expressed under
control of the promoter PCMV. Ig kappa chain secretion
signal, Xa cleavage site, HBV preS1 (21-47)
coding sequence and HCV E2 (384-661)
coding sequence are indicated by arrowheads. Numbers refer to amino acids of
the HCV polyprotein.
1.3
Construction of stable cell line CHO/sS1E2
C400
strain of CHO cells, kindly gifted
from Dr. WU Jia-Rui in the same institute, were cultured in Ham's F12 medium supplemented with 10% FCS.
The cells were transfected with pSecTagB/sS1E2 by the standard calcium
phosphate method. After 24 hours,
transfectants were selected with Ham's F12 containing 400 mg/L Zeocin
(Invitrogen). A Zeocin-resistant clone named CHO/sS1E2 was expanded for further
studies. Regents for cell culture were from Gibco BRL unless stated otherwise.
1.4
Immunofluorescence analysis (IFA)
Indirect
immunofluorescence analysis was carried out as described[15]. Both Zeocin-resistant
CHO cells and CHO control cells were freshly cultured for 16 h in parallel. The
cells were then fixed in ice-cold CMA solution (chloroform∶methanol∶acetone=1∶2∶1), incubated with anti-preS1 monoclonal
antibody 125E11 1000-fold diluted in 1% BSA (prepared in PBS) followed by
incubation with FITC conjugated goat-anti-mouse IgG (Santa Cruz
Biotechnology, Inc.). Fluorescence
was viewed with fluorescence microscope (Olympus).
1.5
Enrichment of the expressed preS1-E2 protein with 125E11-Sepharose
108
CHO/sS1E2 cells were harvested and washed twice with PBS, then lysed in 1 ml 1%NP-40 (prepared in
PBS). The clarified lysate was allowed to bind to the 125E11 conjugated onto
Sepharose. The resin was then washed with PBS and bound protein was eluted with
0.1 mol/L glycine-HCl, pH 2.7. The
eluant was neutralized with 1 mol/L Tris buffer (pH 8.5) and subjected for
Western blotting analysis with RE2116. CHO cells treated equally was served as
control.
1.6
Western blot
Protein
samples were separated by 10% SDS-PAGE gels and transferred onto nitrocellulose
membranes. Blots were reacted with peroxidase conjugated 125E11 (1∶2
000) or RE2116 (1∶1
000) followed by HRP-conjugated swine anti-rabbit IgG (Dako, 1∶1
000). Bands were visualized by the ECL system (Amersham Phamacia Biotech).
1.7
Deglycosylation analysis
To
analyze the glycosylation of the expression products, cell pellets were boiled in denaturing buffer provided by
the manufacturer and then digested with PNGase (NEB) F or Endo H (NEB) for 2
hours at 37 ℃.
To
analyze the secreted expression products,
CHO/sS1E2 and CHO cells were cultured in serum free F12/OptiMEM(1∶1)
for 12 hours. The culture medium was precipitated by equal volume of ice-cold
ethanol after clarified by centrifugation at 20 000 g, 4 ℃
for 30 min. The precipitant was then resuspended with small volume of PBS and
subjected to further analysis.
2.1
Construction of stable cell line CHO/sS1E2 which expresses the fusion
antigen preS1-E2
Recombinant
plasmid pSecTagB/sS1E2 carrying target DNA fragment encoding fusion genes of
HBV preS1(21-47)
and HCV E2 under the control of CMV promoter were constructed as
described in Materials and Methods. Besides enzymatic analysis and DNA sequencing,
the correct construction of this plasmid was confirmed by in vitro
transcription/translation, where protein band of about 35 kD, as expected from
the polypeptide backbone of the fusion antigen, were detected (data not shown).
This
plasmid DNA was then transfected into CHO cells, a Zeocin-resistant CHO cell
line (CHO/sS1E2) carrying preS1-tagged E2 gene was established. Expression of
preS1-E2 was analyzed by immunofluorescence staining using anti-preS1 mAb
125E11. Perinuclear fluorescence was observed, consistent with ER-localization
of HCV E2 [Fig.2(A)]. In contrast,
no fluorescence was seen with the control CHO cell line [Fig.2(B)].
Expression products were further analyzed by Western blot. A specific protein
band of about 50 kD was detected with the anti-E2 polyclonal antibodies (Fig.3,
lane 2). A specific band of the same size was also detected with monoclonal
anti-preS1 antibody 125E11 (Fig.3, lane 4), suggesting that the fusion double
antigen be properly expressed.
Fig.2 Immunofluorescence assay of preS1-E2
fusion antigen expressed in CHO/sS1E2 cells
CHO/sS1E2
(A) and CHO (B) cells were analyzed using monoclonal anti-preS1 antibody
125E11. Magnification was 400×.
Fig.3 Western blot analysis of the cell lysate
CHO cells (lane 1, 3) and CHO/sS1E2 cells (lane 2, 4) were directly lysed in SDS-PAGE
loading buffer, and detected with
anti-E2 rabbit serum RE2116 (lane 1, 2) or anti-preS1 125E11 (lane
3, 4), respectively.
2.2
Fusion antigen preS1-E2 is N-glycosylated and secretable
Post-translational
modification of HCV envelope proteins was considered closely associated with
the protective immunogenicity[8], therefore the glycan type of the expressed fusion antigen
was analyzed with the treatment of glycosidases. CHO/sS1E2 cells and CHO cells
were lysed with the denaturing buffer and digested with PNGase F and Endo
H, respectively, before Western blotting analysis with
peroxidase conjugated 125E11. Multiple bands representing differently processed
fusion antigen were detected before deglycosylation treatment [Fig.4（A）, lane 3 and 7], which were sensitive to both
glycosidases. After digestion,
those multiple bands of about 50 kD co-migrated to about 35 kD [Fig.4（A）, lane 4 and 8], in consistent with the peptide backbone
of the expressed fusion antigen. PNGase F hydrolyzes all types of N-glycan
chains from glycopeptides and glycoproteins unless they carry α-1-3
linked core fucose residues present in insect and plant glycoproteins[16]
while Endo H cleaves only high mannose structures and hybrid structures on N-linked
oligosaccharides of glycoproteins[17]. The results thus demonstrated
that the cell associated expression products were mostly high-mannose-type
glyco-sylated.
The
expressed products were also secretable. The bands specifically reacted with
125E11 could be detected in the culture medium [Fig.4（B）, lane 3 and 7]. The secreted expression
products were also analyzed with PNGase F and Endo H, respectively. Expression products of about 60 kD were
migrated to about 35 kD after deglycosylation with PNGase F [Fig.4（B）, lane 8]. However, this band was resistant to Endo H
[Fig.4（B）, lane 4], indicating further modifications of Golgi apparatus was
happened upon secretion of the fusion antigen.
Fig.4 Deglycosylation analysis of the
expressed products
Cell lysates (A) and proteins precipitated
from serum free culture medium (B) were analyzed by Western blot analysis with
peroxidase conjugated 125E11 after N-glycosidase treatment. 1, 2, 5, 6, samples prepared from cultured CHO cell;
3, 4, 7, 8, samples prepared from cultured CHO/sS1E2
cell; 1, 3, samples incubated with Endo H digestion
buffer; 2, 4, samples digested with Endo H; 5, 7, samples incubated with PNGase F digestion buffer; 6, 8, samples digested with PNGase F.
2.3
Fusion antigen preS1-E2 could be enriched with anti-preS1 immunoaffinity
resin
The
immunoaffinity resin of monoclonal anti-preS1 antibody coupled Sepharose
(125E11-Sepharose) was successfully used to purify a preS1(21-47)
tagged protein[13]. Here we assessed if 125E11-Sepharose could also be
useful in purification of the preS1 tagged E2 protein. Cell lysates were
prepared and allowed to bind to 125E11-Sepharose. From the Western blot
analysis with RE2116 (Fig.5),
it was shown that the E2 specific bands were absorbed efficiently by
this resin. The absorbed fusion antigen could be eluted with low pH buffer.
Comparing with the pre-binding sample,
unspecific bands were disappeared from the eluant, suggesting that preS1-E2 was able to be
enriched and purified with the immunoaffinity resin.
Fig.5 125E11-Sepharose binding analysis of
the expressed preS1E2 fusion antigen
CHO (lane 1, 3, 5) and
CHO/sS1E2 (lane 2, 4, 6) cells were lysed and allowed to bind
to pre-balanced 125E11-Sepharose,
washed and eluted as described in “Materials
and Methods”.
Pre-binding (lane 1, 2), flow-through (lane 3, 4) and eluant (lane 5, 6) were analyzed by Western blot with RE2116.
3
Discussion
We
report here the construction of a stable CHO cell CHO/sS1E2 which expresses
C-terminally truncated HCV E2 fused to HBV preS1(21-47).
Upon characterization of the expression products, it was found that both cell-associated form and secreted
form of the preS1-tagged E2 protein,
which reacted specifically to both HCV E2 specific and HBV preS1
specific antibodies, were
expressed with this cell line. The inclusion of the HBV preS1(21-47)
peptide at the N-terminal of HCV E2 protein allowed for efficient detection of
the expression products. More importantly, the preS1 peptide tag facilitated the purification of the
fusion antigen. The expressed fusion antigen could specifically bind to and be
eluted from the 125E11-Sepharose resin in a primary experiment, suggesting that the anti-preS1 affinity
chromatography could be used in large-scale preparation of the fusion antigen
under non-denaturing conditions.
In
this study, we analyzed the glycan types of both cell associated and secreted
expression products. The cell-associated form was found to be high-mannose-type
glycosylated, while the secreted form complex-type glycosylated. This is
different from the processing of HCV E1, where both cell-associated and
secreted forms of E1 were found to be high-mannose-type glycosylated[19].
It was reported that differently processed HCV envelope proteins have different
ability in eliciting protective immunity[8]. Also E2 targeted to
cell surface was shown to be more immunogenic than ER-retained E2, and the surface expressed E2 was
properly folded[18]. However,
less glycosylated ER-retained E2 was shown to be the optimal E2 antigen
to elicit antibody responses with neutralization-of-binding (NOB) activity[20].
As the CHO/sS1E2 could efficiently express both cell-associated and secreted
forms of E2 fused to preS1 peptide,
which are respectively high-mannose-type glycosylated or complex-type
glycosylated, this cell line would
be useful to compare these two forms of E2 antigen. No matter which form is
better in diagnosing HCV patient or eliciting protective antibodies, this cell line would be very useful for
future research.
The
transmission pathway of HCV is similar to that of HBV, which is still a serious infectious
pathogen of liver diseases, and
HCV infection is frequently associated with HBV infection[21, 22].
Hence, a vaccine capable of
eliciting protection against both virus would be very useful in protecting
people under high-risk of infection. The preS1 epitope we used is the major HBV
receptor binding site for hepatocytes[23]. Anti-serum against
preS1(21-47)
synthetic peptide could block the attachment of HBV to hepatocyte and was
virus-neutralizing. The expressed fusion antigen displayed both HBV preS1 and
HCV E2 antigenicity, suggesting
its possibility to be used in further study of immunological properties of HCV
E2 glycoprotein and in the study of recombinant HBV/HCV vaccine.
Acknowledgements The authors thank Dr. WU
Jia-Rui for kindly providing CHO C400 cell line, Ms. KONG Yu-Ying for help in cell culture, Ms PING Bei-Fang for help in
preparation of 125E11 and its derivatives, TANG Ke for help in immuno-fluorescence assay.
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Received:
January 30, 2002
Accepted: February 25, 2002
This
work was supported by the State 863 High Technology R&D Project of China
(No.2001AA215171)
*Corresponding
authors:Tel, 86-21-64374430-5326;Fax, 86-21-64338357; e-mail,
[email protected]