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Preparation of human single chain Fv antibody against hepatitis C virus E2 protein and its identification in immunohistochemistry
Yan-Wei Zhong, Jun Cheng, Gang Wang, Shuang-Shuang Shi, Li Li, Ling-Xia Zhang, Ju-Mei Chen
Yan-Wei Zhong, Jun Cheng, Gang
Wang, Shuang-Shuang Shi, Li Li, Ling-Xia Zhang, Ju-Mei Chen,
Gene Therapy Research Center, Institute of Infectious Diseases, 302 Hospital of
PLA, 26 Fengtai Road, Beijing 100039, China
Supported by
the National Natural Science Foundation of China, No.39900130
Correspondence to:
Yan-Wei Zhong, Gene Therapy Research Center, Institute of Infectious
Diseases, 302 Hospital of PLA, 26 Fengtai Road, Beijing 100039, China. zyw@genetherapy.com.cn
Telephone:
+86-10-66933392 Fax: +86-10-63801283
Received 2002-01-28
Accepted 2002-03-05
Abstract
AIM: To screen human single chain Fv
antibody (scFv) against hepatitis C virus E2 antigen and identify its
application in immunohistochemistry.
METHODS:
The phage antibody library was panned by HCV E2 antigen, which was coated in
microtiter plate. After five rounds of biopanning,56 phage clones were
identified specific to HCV E2 antigen. The selected scFv clones were digested by
SfiI/NotI and DNA was sequenced. Then it was subcloned into the vector pCANTAB5E
for expression as E-tagged soluble scFv. The liver tissue sections from normal
person and patients with chronic hepatitis B and chronic hepatitis C were
immunostained with HCV E2 scFv antibody.
RESULTS: The data of scFv-E2 DNA
digestion and DNA sequencing showed that the scFv gene is composed of 750 bp.
ELISA and immunohistochemistry demonstrated that the human single chain Fv
antibody against hepatitis C E2 antigen has a specific binding character with
hepatitis virus E2 antigen and paraffin-embedded tissue, but did not react with
liver tissues from healthy persons or patients with chronic hepatitis B.
CONCLUSION: We have successfully
screened and identified HCV E2 scFv and the scFv could be used in the
immunostaining of liver tissue sections from patients with chronic hepatitis C.
Zhong YW, Cheng J, Wang G, Shi SS, Li L, Zhang LX, Chen JM. Preparation of human
single chain Fv antibody against hepatitis C virus E2 protein and its
identification in immunohistochemistry.World J Gastroenterol 2002;
8(5):863-867
INTRODUCTION
Hepatitis C virus (HCV) has been
identified as the major etiological agent of post-transfusion non-A non-B
hepatitis[1-10], responsible for most cases of non-A non-B hepatitis.
Hepatitis C is a disease of clinical importance because of its high infection
rate in blood donors and its persistence as chronic infections which may lead to
cirrhosis and hepatocellular carcinoma in the long term[11-26]. The
variability of the HCV genome has difficulties in serological detection and
vaccine design. Recent advance in phage technology offers a means of cloning
human anti-HCV antibodies of a defined specificity that may have potential
therapeutic use[27]. We now report the generation of phage display
antibody using phage antibody library. From this library we obtained specific
single-chain Fv antibody that recognizes the viral envelope protein E2, using
HCV E2 protein as the immobilized antigen and proceeding immunohistochemistry.
MATERIALS AND METHODS
Materials
Humanized scFv antibody phage
library in which the variable region coding gene of VL and VH were amplified by
polymerase chain reaction (PCR) with degenerate primers and connected with a
glycine linker [(Gly4Ser)3] was widely used in the screen and identification of
humanized scFv to various antigens[28-34]. The recombinant HCV E2
protein was purchased from Virostat Co, USA. Phage M13K07 was purchased from
Pharmacia Co., Sweden. Other reagents used in this experiment are all domestic
products of analytical grade.
Biopanning
The phage library was amplified in
37 ℃.
The host E. coli TG1was infected with phage M13K07 and incubated at 37 ℃
for 12 hours, the phage in the supernatant was harvested and concentrated by
PEG. Culture plate (Nunc) was coated with recombinant HCV E2 protein at the
concentration of 80 mg/L. The coating buffer was 0.05 mol/L NaHCO3,
pH 9.6. The plate was blocked with BSA at the concentration of 20 g/L for 2
hours and the concentrated phages were added to the well of the plate, incubated
at the room temperature for 90 min. The plates were washed 20 times with PBST
and PBS buffer respectively. The bound phage was eluted by the 0.1M of
triethylamine, and neutralized with 1M Tris buffer (pH 7.4). Recovered phages
were used to infect the host E. coli TG1 at the log phase growth and HCV
E2 protein-binding phages were amplified. The procedure of
absorption-elution-amplification was repeated 5 times.
Identification of phage clones
After 5 rounds of biopanning, 56
phage clones were selected randomly. The clones grew in 400 ml 2×TY-AMP-Glu at
37 ℃
overnight. The culture was transformed to another Eppendorf tube when its A600
nm reached 0.5. The culture was continued at
30 ℃
overnight after adding helper phage. ELISA for determining the supernatants was
repeated at least two times. The cross-reaction of the supernatants to the BSA
antigen was conducted. According to the ELISA results to the HCV E2 and BSA, one
clone with high reaction to HCV E2 and low reaction to BSA was selected.
Sequencing analysis
The plasmid DNA was prepared
using Wizard plus minipreps DNA Purification System (Promega Co., USA) and
sequenced using ABI automated DNA sequencing machine.
Expression of human HCV E2-scFv in E.coli
The selected HCV E2 scFv clone was
subcloned as SfiI/NotI fragments into the vector pCANTAB5E for expression as
E-tagged soluble scFv. DNA digestion and electrophoresis confirmed the
recombinant vector pCANTAB5E-E2-scFv. Competent E. coli XL1-Blue was
transformed with pCANTAB5E-E2-scFv and transformed XL1-Blue was induced by IPTG
for 20 h. The E. coli was harvested by centrifugation at 10 000rpm. The
culture supernatant was rendered for ELISA test according to the standard
procedure. In ELISA detection, Nunc plate was coated with 1mg/well of
recombinant HCV E2 antigen and blocked with 2 % bovine serum albumin (BSA) at 37
℃ for
2 h. The supernatants from induced and non-induced transformed E. coli were
added and incubated at 37 ℃
for 2 h. The plate was washed with PBS buffer, and 100 ml of HRP/anti-E Tag
1:4000 ratio diluted in PBS buffer containing 1 % BSA was added, and incubated
at 37 ℃
for 1 h. The substrate solution was added and A450nm value was measured.
Immunohistochemical identification of scFv in liver tissue
Paraffin-embedded liver tissue
slices were from patients with positive anti-HCV antibodies and HCV-RNA. After
deactivating endogenous hyperoxidase, these slices were submersed in the
methanol solution with 0.5 % H2O2 in the room temperature.
Fifty min later, they were washed with PBS buffer for 3 times,5 % BSA was added
and slices were stored overnight at 4 ℃.
Self-made scFv primary anti- HCV E2 single-chain antibodies were diluted at
1:100 ratio and added on to the slice. They were kept in the 37 ℃
incubator for 1 h, then 4 ℃
refrigerator overnight. HRP-sheep anti-M13 antibodies (diluted to 1:200)
solutions were dropped on to the tissue sections, incubated at 37℃
for 40 min. After washing 3 times with PBS buffer, DAB solutions (9 mg DAB, 13.5
ml Tris.cl, 1.5 ml CoCl2, 15 ml 30 % H2O2) were
dropped on to the tissue sections at room temperature. After ten minutes, the
slices were washed with PBS buffer for 3 times again, and 1 % heamatin solution
was used to stain the cell nucleus. Gradient ethanol was utilized to dehydrate
and dimethylbenzene to clear the sections, then neutral resin to envelope them.
The resultant slices were observed under microscope. The controls were set as
follows: 1) PBS buffer instead of anti- HCV E2 scFv; 2) HBsAg, HBcAg
double-positive liver tissue sections; and 3) Normal liver tissue sections.
RESULTS
Screening and identification phage clones
Using HCV E2 protein as
immobilized antigen, the humanized scFv phage library was biopanned. After 5
rounds of biopanning, 56 phage clones were selected randomly. ELISA and
cross-reaction of these clones to BSA confirmed their specificity to HCV E2.
Among the 56 phage clones, 16 showed good reactivity to the recombinant HCV E2
protein with high A value in the ELISA. In the cross-reaction screen, 6 among
the 16 showed low cross-reaction with BSA. The combined results indicated that 1
of the 6 showed the highest reaction to HCV E2 protein and lowest reaction to
BSA. One clone has been selected for further DNA digestion and sequence
analysis. The DNA sequence digestion was made by SfiI/NotI in Figure 1. Its
nucleic acid sequence and deduced amino acid sequence about HCV-E2-scFv fragment
are shown in Figure 2.
Figure 1 Restriction map of HCV E2-scFv
by SfiI/NotI digestion. A,B: HCV E2-scFv; C: DNA Marker
ATG GCC CAG GTG CAG CTG GTG CAG TCT GGG GCT GAG GTG AAG AAG CCT GGG GCC
M A Q
V Q
L V Q
S G A
E V K
K P G A
TCA GTG AAG GTT TCC TGC AAG GCT TCT GGA TAC
ACC TTC ACT AGC TAT GCT ATG
S V
K V S
C K A
S G
Y T
F T S
Y A M
CAT TGG GTG CGC CAG GCC CCC GGA CAA AGG CTT GAG TGG ATG GGA TGG ATC AAC
H W
V R Q
A P G
Q R L
E W M
G W I N
GCT GGC AAT GGT AAC ACA AAA TAT TCA CAG AAG TTC CAG GGC AGA GTC ACC ATT
A G
N G N
T K
Y S
Q K F
Q G R
V T I
ACC AGG GAC ACA TCC GCG AGC ACA GCC TAC ATG GAG CTG AGC AGC CTG AGA TCT
T R
D T S
A S T
A Y M
E L
S S L
R S
GAA GAC ACG GCC GTG TAT TAC TGT GCA AGA TCG AGT GGG CCG ATG CAT CGT GAG
E D
T A V
Y Y C
A R S
S G P
M H R E
TGG GGC CAA GGT ACC CTG GTC ACC GTG TCG AGA GGT GGA GGC GGT TCA GGC GGA
W G
Q G T
L V T
V S R
G G G
G S G G
GGT GGC TCT GGC GGT GGC GGA TCG TCT GAG CTG ACT CAG GAC CCT GCT GTG TCT
G G
S G G
G G S
S E
L T Q
D P A
V S
GTG GCC TTG GGA CAG ACA GTC AGG ATC ACA TGC CAA GGA GAC AGC CTC AGA AGC
V A
L G Q
T V
R I T
C Q G
D S L
R S
TAT TAT GCA AGC TGG TAC CAG CAG AAG CCA GGA CAG GCC CCT GTA CTT GTC ATC
Y Y
A S W
Y Q Q
K P G
Q A P
V L V I
TAT GGT AAA AAC AAC CGG CCC TCA GGG ATC CCA GAC CGA TTC TCT GGC TCC AGC
Y G
K N N
R P S
G I
P D R
F S G
S S
TCA GGA AAC ACA GCT TCC TTG ACC ATC ACT GGG GCT CAG GCG GAA GAT GAG GCT
S G
N T A
S L
T I
T G A
Q A E
D E A
GAC TAT TAC TGT AAC TCC CGG GAC AGC AGT GGT AAC CAT GTG GTA TTC GGC GGA
D Y
Y C N
S R D
S S G
N H V
V F G G
GGG ACC AAG CTG ACC GTC CTA GGT GCG GCC GCA GAA CAA AAA CTC ATC TCA GAA
G T
K L
T V L
G A A
A E
Q K
L I S E
GAG GAT CTG AAT GGG GCC GCA TAG
E D
L N G
A A *
Figure 2 Nucleic acid and deduced amino
acid sequences of scFv for HCV E2 protein GenBank accession number for this
sequence is AF317001
Figure 3 Absorbances of HCV-E2-scFv
binding to E2 antigen by ELISA. (a). supernant from induced XL1-blue transformed
with pCANTAB5E-E2-scFv; (b). posive control; (c). supernant from non-induced
XL1-blue transformed with pCANTAB5E-E2-scFv, d. negtive control
Expression of human HCV-E2-scFv in E. coli.
The expressed HCV-E2-scFv
antibody from E. coli XL1-blue transformed by pCANTAB5E and induced by
IPTG was confirmed by ELISA as shown in Figure3. The recombinant HCV E2 antigen
was taken as the positive control. The protein from induced and non-induced
E. coli XL1-blue transformed by expression vector was positive. But the
protein derived from the E. coli non-transformed by pCANTAB5E- HCV E2-
scFv was negative. These results indicated that the soluble form of human HCV
E2- scFv antibody has been successfully expressed in this procedure.
Immunostaining of HCV E2 antigen of liver tissue sections
The different sections from
liver tissues of healthy persons and patients with chronic hepatitis B or C were
immunostained. The positive immunostaining was seen only in the liver tissue
section of patients with chronic hepatitis C, but not in the liver tissue of
normal person and patients with chronic hepatitis B as seen in (Figure 4A). The
HCV E2 antigen was mainly located in the cytoplasm of the hepatocytes infected
by HCV virus (Figure 4B).
Figure 4
A. Immunohistochemistry of liver tissue from health person. B.
Immunohistochemistry of liver tissue from patients with chronic hepatitis C E2
antigen was detected in the cytoplasm of some liver cells
DISCUSSION
HCV is the etiological agent responsible
for most cases of non-A non-B hepatitis. It is a blood-related disease of
clinical importance since a large number of cases developed chronic infections
which may lead to cirrhosis and hepatocellular carcinoma. The variability of the
HCV genome has posed serious problems in serological detection of HCV and
vaccine design[27-38]. The possibility of using neutralizing
antibodies in passive immunization is also hampered by the existence of
genotypes and quasispecies[39-50]. To circumvent this, it is possible
to envisage the use of cross reactive antibodies with a broad reactivity
directed against functionally conserved surface domains common to various
genotypes. There has been in vitro and in vivo evidence that HCV
infection elicits a neutralizing antibody response in humans. The virus envelope
protein is the primary target for the host immune system. Despite the high
variability of the HCV envelope sequence, certain domains of biological
importance, e.g. ligands required in viral attachment to host cell receptors,
have to be preserved. Antibody specific for the envelope E2 protein has been
shown to be cross-reactive HCV E2 and to be able to block viral attachment to
cultured human fibroblast cells. However, attept to obtain human monoclonal anti
-HCV antibodies is an urgency.
Phage library is a powerful tool for the
selection of important and useful antibody specificities espeacially for getting
humanized scFv[51-53]. It has many advantages. First, it is the only
method to get specific antibody by passing the immunization step. It can mimic
the maturation procedure of human antibody in vivo, so it is possible to get
high affinity antibody from this selection. Second, the scFv with a low
molecular weight, can make it potentially applicable in the clinical diagnosis
and treatment of both infectious disease and cancer. Finally, it has no Fc
fragment, so the backgroud is very low.
The
phage technology offers a means of cloning human anti-HCV antibodies of a
defined specificity that may be potential for therapeutic and diagnostic use. In
this report, we have obtained anti-HCV E2 scFv by affinity selection and
purified recombinant hepatitis C virus E2 as coating antigen from synthetic
phage display antibody library. After 5 rounds of selection, 56 clones were
evaluated by enzyme-linked immunosorbent assay (ELISA). One phage clone was
selected from 56 clones according to the highest A450 nm value in the ELISA
identification and lowest cross-reaction to the bovine serum albumin (BSA). The
coding fragments of scFv were sequenced. The affinity and specificity of scFv
were evaluated by ELISA and immunohistochemistry. HCV E2-scFv DNA digestion and
sequence data showed that the scFv gene is composed of 750 bp. ELISA and
immunohistochemistry demonstrated that the human single chain Fv antibody
against HCV E2 antigen has a specific binding activity with hepatitis C virus E2
antigen. This study illustrated the feasibility of using antibody-engineering
technology with the phage display library that may be useful for future
therapeutic and detection purpose.
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Edited by Ma JY
