Protein Expression and Purification 188 (2021) 105965

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Protein Expression and Purification

journal homepage: www.elsevier.com/locate/yprep

Expression of HCV genotype-4 core antigen in prokaryotic E. coli system for

diagnosis of HCV infection in Egypt
Eman M. Saleh a, *, Abdullah E. Gouda b, Amina M. Medhat a, Hend O. Ahmed b, Mohamed
A. Shemis b
a
Biochemistry Department, Faculty of Science, Ain Shams University, Cairo, Egypt
b
Biochemistry and Molecular Biology Department, Theodor Bilharz Research Institute, Giza, Egypt

A R T I C L E I N F O A B S T R A C T

Keywords: Background: Egypt has a high prevalence of hepatitis C virus (HCV) infection with 92.5% of genotype-4.
HCV Aim: This study aimed to clone and express the core gene of HCV genotype-4 for using it to develop a highly
Core protein sensitive, specific, and cost-effective diagnostic assay for detecting HCV infection.
Inclusion bodies
Methods: Using synthetic HCV genotype-4 core gene, pET15b as E. coli expression vector, and 1 mM lactose as
Refolding
inducer, the HCV core protein (MW 17 kDa) was expressed in the form of inclusion bodies (IBs) that was purified
ELISA
and solubilized using 8 M guanidinium HCl. The recombinant core protein was in vitro refolded by a rapid
dilution method for further purification using weak cation exchange liquid chromatography. The immunoge­
nicity of the purified protein was tested by ELISA using 129 serum samples.
Results: The recombinant core protein was successfully expressed and purified. The results also showed that the
in-house anti-HCV core assay is accurate, specific (~96.6%), and highly sensitive (~100%) in accordance with the
commercial ELISA kit.
Conclusion: The sensitivity, specificity, and reproducibility of the developed assay were high and promising to be
used as a screening assay for detecting HCV infection.

1. Introduction Poland, and Northwest Ethiopia. In participating HCWs, a lower prev­

The Egyptian Demographic Health Surveys (EDHS) estimated that
hepatitis C infection is a major public burden in Egypt. EDHS measured
antibody prevalence among the adult population aged 15–59 years in
2009 and it was found to be 14.7% [1], 10.0% in 2015 [2], and 11.9% in
a 2018 meta-analysis [3]. These percentages were significantly higher
than the global levels estimated by WHO (1.4%) [4]. After the con­
duction of several national programs to manage HCV infection, Waked
et al. reported an overall prevalence rate of 4.6% for HCV antibody
positivity and 3.5% for HCV viremia among the screened cohort
(included approximately 50 million Egyptians) during 2018–2019 [5].
In a recent study including 500 patients admitted to the internal medi­
cine and surgery Ain Shams hospitals in Cairo, Egypt, in addition to 50
health care workers (HCWs), Anwar et al. found a 19.80% prevalence of
HCV antibodies among patients, which is considered a high rate
compared to available estimates that are typically less than 5% among
hospital patients from other countries as USA, Belgium, England,
alence of 8.0% was found, although this is again higher than estimates
from Poland, Mexico, Syria, and Pakistan [6].
The widespread of HCV infection among the Egyptian population is
considered the leading cause of liver disease, hepatocellular carcinoma
(HCC), and the primary signs for liver transplantation [7,8]. Moreover,
HCV disease expands the threat of type 2 diabetes mellitus by increasing
insulin resistance and is related to various additional hepatic in­
dications. Also, HCV increases the hazard of circulatory and kidney
diseases (e.g., lymphoproliferative disorders and
membrane-proliferative glomerulonephritis with or without cry­
oglobulinemia) [9], renal failure, and cancers of the esophagus, pros­
tate, and thyroid that all-cause mortality [10,11]. In Egypt, the most
common HCV genotype is genotype-4, representing 92.5% of all HCV
cases, and genotyping of HCV assumes a vital role in the treatment of
HCV [12,13].
The diagnosis of HCV infection is usually based on two categories of
laboratory tests: the indirect tests or serologic assays, which can detect

* Corresponding author.
E-mail address: e_ahmed@sci.asu.edu.eg (E.M. Saleh).

https://doi.org/10.1016/j.pep.2021.105965
Received 22 April 2021; Received in revised form 21 August 2021; Accepted 24 August 2021
Available online 27 August 2021
1046-5928/© 2021 Elsevier Inc. All rights reserved.
E.M. Saleh et al.
specific antibody to HCV (anti-HCV), and direct tests, which can detect,
measure, or differentiate the components of HCV viral particles, such as
HCV RNA and core antigen. Both direct and indirect virological assays
have an essential role in diagnosing infection, choosing the type of
treatment, and estimating the virological response to therapy [14,15].
With the advancement in direct-acting antivirals (DAAs) for HCV,
the expectation of removing viral hepatitis has turned into a genuine
probability. The Egyptian National Committee for the Control of Viral
Hepatitis does its best to supply Egyptian HCV patients with DAAs.
Egypt approved a strategy that denotes a model of consideration for
helping other countries with a high HCV predominance rate in their
fight against HCV [16]. Regardless of how the rate of HCV infection is
decreasing, HCV infections’ clinical and economic impacts are estimated
to develop extensively [17].
The core protein (191 aa) is responsible for packaging viral RNA and
virion budding. This protein is configured into three domains that
include the N-terminal (D1, ~120 aa), the C-terminal (D2, ~50 aa), and
the last 21 aa that serves as a signal sequence for targeting E1 (D3).
Besides being an important target in developing preventive and thera­
peutic strategies against HCV infection, the core antigen is a crucial
element for diagnostic application. The HCV core antigen is a highly
antigenic protein that induces a cellular and humoral-specific response.
It plays a significant role in the pathogenesis of HCV viral infection and
HCC [18]. Antibodies against HCV core protein epitopes (residues 7–21,
31–45, 49–63, 99–113) were detected in the serum of HCV patients.
These conserved epitope clusters were thought to be the most immu­
nogenic among the HCV proteins and could elicit a more pronounced
humoral response than the other HCV antigens in acute, chronic,
resolved, or progressive chronic hepatitis C (CHC) [19,20].
Austria and Wu [21] reported that the presence of cryptogenic
occult HCV infection carrying antibodies of IgG class directed against an
immunodominant epitope of HCV core protein facilitates the detection
of silent occult HCV infection. In a cohort of over 140 examined patients,
it was found that 40% were positive for the IgG anti-HCV core antibody,
including 10% of individuals who were antibody non-reactive at the
time of the first sample testing by ELISA kit based on different HCV
antigen. This finding is rather interesting given that the assay was only
evaluated antibodies against a single N-terminal epitope (aa 5–19) of the
core protein.
Fig. 1. The pMA-T cloning vector map with the core protein insert. CP: core protein.
2
Protein Expression and Purification 188 (2021) 105965
This study aimed to clone and express HCV genotype-4 core gene in
the pET15b expression vector and study the immunoreactivity of the
purified recombinant protein for developing an assay for serological
diagnosis of HCV genotype-4.
2. Materials and methods
2.1. Amplification of the core gene of HCV genotype-4
HCV core synthetic gene encoding the first 140 aa of the HCV core
protein genotype-4 was synthesized after retrieving the HCV core gene
(Gene ID: AF029298.1, DQ418789.1) from the National Center for
Biotechnology Information (NCBI) server (www.ncbi.nlm.nih.gov) fol­
lowed by multiple alignments using both NCBI nucleotide blast and
cluster Omega (https://www.ebi.ac.uk/Tools/msa/clustalo) online
programs to select the most conserved region in HCV core gene. The
multiple alignments resulted in 96.18%–100% nucleotide identity with
the different isolates and subtypes of HCV genotype-4. The HCV core
gene was synthesized and inserted into the pMA-T cloning vector
(Construct No: 17ADOWAC) by Invitrogen (Thermo Fisher Scientific
Inc., Germany), as shown in Fig. 1. The core gene primers specific to
HCV genotype-4 were also synthesized by Invitrogen (Thermo Fisher
Scientific Inc., Germany) (Table 1).
According to the pET15b expression vector’s multiple cloning sites
(Cat No: 69257, Novagen Co, USA), the HCV core gene-specific forward
(F) and reverse (R) primers were designed to contain restriction sites
NdeI and BamHI, respectively (Fig. 2). The designed primers were
analyzed using an IDT-sequence analyzer, an online primer program (htt
Table 1
Primer sequences for the amplification of HCV genotype-4 core gene (first 140
aa).
Primer Sequence Restriction Amplified gene
site product size
core-F 5′ GGGAATTCCAT ATG AGC ACG NdeI (~441 bp)
AAT CCT AAA C3′
core-R 5′ GGATCCGCG CCA CGA GCG BamHI
GGA TGT ATC3′
F, forward primer; R, reverse primer.
E.M. Saleh et al.
Fig. 2. The pET15b expression vector map with the T7 promoter and lacO.
p://www.idtdna.com) to calculate primers’ melting temperature (Tm)
and GC content tested for self-complementary, hairpin loop, and primer
dimer formation.
Different annealing temperatures 58 ◦ C, 55 ◦ C, 53 ◦ C, 50 ◦ C, and
48 ◦ C were tested, and the optimum annealing temperature was found to
be 48 ◦ C. PCR program was designed as the following: one cycle 94 ◦ C
for 3 min and 30 cycles at 94 ◦ C for 45 s, 48 ◦ C for 45 s, and 72 ◦ C for 45 s
(T100 PCR thermal cycler, Bio-Rad, Singapore).
2.2. Molecular subcloning of core gene into pET15b expression vector
To obtain the core gene from pMA-T cloning vector for subcloning
into pET15b expression vector, the digestion of pMA-T and pET15b
vectors was performed using NdeI (Cat No: FD0583) and BamHI (Cat No:
FD0054) restriction enzymes (Thermo Fisher Scientific Inc., Germany).
The digested products were identified using 2% agarose gel
electrophoresis.
The digested core gene and pET15b products were purified from the
agarose gel using GeneJET gel extraction kit (Cat No: k0691, Fermentas,
Thermo Fisher Scientific Inc., Germany). The HCV core gene was ligated
to the linearized pET15b expression vector at molar ratio 3:1 using T4
DNA ligase (Cat No: 1522407, Thermo Fisher Scientific Inc., Germany)
[22]. To express the recombinant core protein, chemically Rosetta 2
(DE3) competent cells (Cat No: 70954, Novagen Co, USA) were trans­
formed with 1 μl of the recombinant pET15b/core gene vector using
heat-shock protocol [23]. To confirm the successful ligation into a
pET-15b vector, PCR using T7 universal and reverse primers of core gene
was done at the annealing temperature of 55 ◦ C to confirm the
right-oriented ligation of the cloned gene.
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Protein Expression and Purification 188 (2021) 105965
2.3. Expression of HCV genotype-4 recombinant core antigen
A single colony of Rosetta 2 (DE3) harboring pET15/core gene was
inoculated into 10 ml medium supplemented with ampicillin (50 μg/ml)
and chloramphenicol (35 μg/ml) and incubated overnight at 37 ◦ C with
shaking at 225 rpm. The overnight culture was diluted at 1:100 dilution
in sterile LB medium containing antibiotics and incubated at 37 ◦ C with
shaking to achieve the exponential phase, i.e., optical density (OD600nm
= 0.6). To induce the expression of the target gene, 1 mM lactose was
added to the culture having OD600nm = 0.6 and incubated for 3 h at 37 ◦ C
for the maximum production of the core antigen. The cells were then
harvested by centrifugation at 6000 rpm at 4 ◦ C for 5 min, and the cells
were resuspended in 1 M Tris/HCl pH 6.8 for testing core antigen
expression using SDS-PAGE [24].
SDS-PAGE analysis was performed using 15% Tris-glycine acryl­
amide gel. The culture samples were prepared by mixing 25 μl of 2×
loading dye with 25 μl of each cell suspension. The solutions were mixed
well and heated at 100 ◦ C for 5 min. In each lane of the gel, 3 μl of the
dye-sample mixture was loaded, and the electrophoresis was performed
in an SDS Tris-glycine buffer at 100 V constant until the dye reached the
bottom of the gel. The protein bands were stained using the Coomassie-
staining technique [25].
2.4. Inclusion bodies purification, solubilization, and in vitro protein
refolding
As E. coli is a prokaryotic system, it can produce foreign proteins as
insoluble aggregates called inclusion bodies (IBs). To purify IBs, cells
were usually disrupted by lysozyme treatment followed by homogeni­
zation and centrifugation at 6000 rpm, 4 ◦ C for 15 min. The pellets were
washed several times with 0.1 M Tris/HCl buffer (pH 8) containing low
concentrations of chaotropic agents (0.5 M Guanidinium hydrochloride;
E.M. Saleh et al.
GdmCl) and detergents (1% Triton X-100). After purification, 100 mg of
the pellets were dissolved in 5 ml solubilization buffer [8 M GdmCl, 0.1
M Tris/HCl (pH 8), 100 mM dithiothreitol (DTT), and 1 mM EDTA] and
incubated for 2 h at 25 ◦ C. The pH of the solution was lowered to a value
between pH 3 and 4 by drop-wise addition of HCl and incubated for 10
min at 25 ◦ C, followed by centrifugation at 12,000 rpm for 15 min at 4 ◦ C
to remove debris [26]. The supernatant containing solubilized IBs was
quantified at 280 nm using Nanodrop 2000c spectrophotometer
(Thermo Fisher Scientific Inc., USA). The solubilized IBs were diluted in
a ratio of approximately 1:100 (v/v) into the pre-cooled refolding buffer
(10 ◦ C) (0.1 M Tris HCl; pH 9, 2 M L-Arg/HCl, and 1 mM EDTA) under
rapid mixing. After 12 h, centrifugation was performed at 10,000 rpm,
4 ◦ C for 20 min to precipitate the unfolded protein.
2.5. Purification of recombinant core antigen by liquid chromatography
Depending on the core antigen isoelectric point (pI), which was 11.4
using IPC-Isoelectric Point Calculation of Proteins (http://isoelectric.
org/index.html), the refolded recombinant core antigen protein was
purified using a weak cation CM sepharose fast flow column (GE
Healthcare Life Sciences, Barcelona, Spain). A volume of 20 ml of
refolded core antigen protein was centrifuged at 5000 rpm for 20 min at
4 ◦ C using 20 ml vivaspin (MWCO 10000) (GE Healthcare Life Sciences,
Barcelona, Spain). The sample was concentrated to about 2 ml, four
sample volumes of 20 mM sodium phosphate buffer (pH 7) were added,
and centrifugation was repeated to obtain a volume of 2 ml concentrated
refolded protein. The sample was centrifuged at 4 ◦ C for 10 min before
being applied to the column. Automated purification was accomplished
using AKTA Purifier 100 FPLC system (GE Healthcare Life Sciences,
Sweden). The isolated peaks were tested using 15% SDS-PAGE. The
concentration of the total purified protein was quantified at 280 nm
using a Nanodrop 2000c spectrophotometer (Thermo Fisher Scientific
Inc., USA).
2.6. Immunodetection of recombinant HCV core antigen activity using
ELISA
The HCV core genotype-4 (Cat No: A58287, Antibodies Co, NY, USA)
was used as a standard control to generate a standard curve for deter­
mining the concentration of the expressed core protein. The recombi­
nant purified core antigen was diluted to a final concentration of 3 μg/
ml by a coating buffer (0.33% Na2CO3 and 0.6% NaHCO3), pH 9.6. The
wells of a PVC microtiter plate were coated with 50 μl core antigen
dilution. The plate was incubated for 2 h at room temperature. The
coating solution was then removed, and the plate was washed twice with
200 μl washing buffer (1× PBS with 0.05% Tween 20). A volume of 200
μl of blocking buffer (1.5% bovine serum albumin) was added to each
well, and the plate was incubated for 2 h at room temperature. After
removing the blocking buffer, the plate was washed twice using a
washing buffer, then 100 μl of human anti-hepatitis C core protein (0.5
μg/ml) as a primary antibody (Cat No: ab123076, Abcam, MA, USA) was
added to each well, and the plate was incubated for 2 h at room tem­
perature. After removing the primary antibody, the plate was washed
twice using a washing buffer, then 100 μl of anti-human IgG horseradish
peroxidase (1:10000) as a secondary antibody (Cat No: B109026-02,
Foresight, ACON Laboratories Inc., USA) was added to each well, and
the plate was incubated for 2 h at room temperature. After removing the
secondary antibody, the plate was washed twice using a washing buffer
followed by 100 μl of substrate solution (0.1% o-phenylenediamine
dihydrochloride with 50 μl of 20% H2O2). The plate was incubated in the
dark for 30 min, and then the reaction was stopped by 50 μl of 3 M HCl.
The plate was read at 490 nm [27].
2.7. Validation of the developed in-house anti-HCV core assay
For the validation of ELISA analysis, 135 archived serum samples
4
Protein Expression and Purification 188 (2021) 105965
divided into two groups were enrolled in this study: 75 archived serum
samples were collected from patients with HCV infection along with 60
HCV-negative control samples. All samples were collected from
Biochemistry and Molecular Biology Laboratory, Theodor Bilharz
Research Institute (TBRI).
Samples were tested by real-time PCR technique using Abbott real-
time HCV amplification reagent pack (Cat No: 1N30, Abbott Molecu­
lar Inc., USA). The positive samples were genotyped using modified
multiplexed nested PCR [12]. The HCV genotype-4 positive and nega­
tive samples were re-tested by ELISA using both the in-house anti-HCV
core assay and the commercial kit (Foresight, HCV antibody EIA test kit
(Cat No: B010401-03, ACON Laboratories Inc., USA).
The study was approved by the ethics committee from TBRI ac­
cording to the institutional committee to protect human subjects and
adopted by the 18th world medical assembly, Helsinki, Finland, with
approval no. FWA00010609.
2.8. Statistical analysis
The lower detection limit (LOD) of the in-house anti-HCV core assay
was expressed according to the formula: mean of blank + (3*SD) of
blank, meanwhile, the lower quantification limit (LOQ) was calculated
according to the formula: mean of blank + (10*SD) of blank, where SD is
the standard deviation of the reagent blank (lowest concentration) [28].
The GraphPad Prism v.7.01 paired t-test was used to analyze the in-
house anti-HCV core assay versus the commercial ELISA kit. The
GraphPad Prism v.7.01 ROC curve was plotted to calculate the speci­
ficity and sensitivity for both kits.
3. Results
3.1. Amplification of core gene of HCV
PCR amplification products were detected on 2% agarose gel, and a
band at a molecular size of ~441 bp was obtained, as shown in Fig. 3.
3.2. Molecular subcloning of core gene into pET15b expression vector
Digestion of pMA-T plasmid with NdeI and BamHI restriction en­
zymes was conducted to confirm the presence of the target HCV
genotype-4 core gene. In Fig. 4, two bands were obtained indicating the
successful cloning of HCV genotype-4 core gene into the pMA-T vector.
Fig. 3. 2% agarose gel electrophoresis showing the PCR products of HCV core
gene.
Lane 1: 100–1000 bp GeneRuler DNA ladder (Cat No: SM0241, Thermo Fisher
Scientific Inc., Germany), lane 2: negative control, lanes 3 and 4: PCR prod­
ucts of HCV core gene with expected size ~441 bp.
E.M. Saleh et al.
(Lanes 2 and 3). Lanes 4 and 5 represent the undigested and digested
pET15b, respectively.
3.3. Construction of expression plasmid and bacterial transformation
Two methods examined the positive clones: PCR using T7 universal
primer and core gene reverse primer and automatic bidirectional using
core gene-specific primers. The PCR product showed a right orientation
of the core gene into the pET15b expression vector as shown in Fig. 5
compared to the PCR product of core gene-specific forward and reverse
primers.
3.4. Expression of recombinant core antigen
Induction of recombinant core antigen was identified in 15% SDS-
PAGE as a sharp protein band of MW ~17 kDa. The protein was
detected in the pellet lysate of induced clones; in contrast, no protein
band was found in the non-induced bacteria, as shown in Fig. 6.
Fig. 5. 2% agarose gel electrophoresis of PCR amplification products.
Lane 1: 100–1000 bp GeneRuler DNA ladder (Cat No: SM0241, Thermo Fisher
Scientific Inc., Germany), lane 2: PCR using core gene-specific primers with a
band of ~441 bp, lane 3: PCR using T7 universal primer and core gene reverse
primer with a band of ~550 bp.
5
Protein Expression and Purification 188 (2021) 105965
Fig. 4. 2% agarose gel electrophoresis
showing digested pMA-T and pET15b vec­
tors with NdeI and BamHI restriction en­
zymes.
Lane 1: 100–1000 bp GeneRuler DNA ladder
(Cat No: SM0241, Thermo Fisher Scientific
Inc., Germany), lane 2: undigested recom­
binant pMA-T plasmid, lane 3: digestion
products showed successful digestion
providing a pMA-T plasmid of ~2377 bp and
an insert of ~441 bp, lane 4: undigested
pET15b vector, lane 5: digested pET15b
vector.
Fig. 6. 15% SDS-PAGE of the lysate of induced Rosetta 2 (DE3) cells.
Lane 1: pre-stained low MW chromatin protein ladder (Cat No: PR0602,
Vivantis, Malaysia). A band of ~17 kDa was detected in lanes 3, 4, and 5,
whereas it was not detected in lane 2.
3.5. Purification of recombinant core antigen by liquid chromatography
A weak cation exchange chromatography was performed using the
CM sepharose column at different pH below the isoelectric point (pI) of
the core antigen. The concentration of total refolded protein loaded on
the column was 2 mg/ml at 280 nm (Table 2). The protein was eluted
with a stepwise gradient elution of 1 M NaCl in 20 mM sodium phos­
phate buffer pH 7. As shown in the chromatogram (Fig. 7a), a sharp peak
of the core antigen protein was detected at 0.5 M NaCl elution buffer at a
retention time of 12.3 min. The concentration of the total purified
protein was 1.25 mg/ml at 280 nm. A 15% SDS-PAGE of purified frac­
tion from weak cation exchange column was shown in Fig. 7b. The size
of the detected band was ~17 kDa.
3.6. Immunodetection analysis and validation of the in-house anti-HCV
core assay
The immunogenicity of purified recombinant core antigen from the
CM sepharose column was evaluated using the ELISA technique. Positive
and negative results were differentiated based on the cutoff value (for
commercial ELISA kit), which was calculated by submitting the negative
control result with 0.145 and numerically calculated to be 0.149 ac­
cording to the instructions of the manufacturer and based on the lower
detection limit (LOD) for in-house anti-HCV core assay and the anti-
E.M. Saleh et al. Protein Expression and Purification 188 (2021) 105965

Table 2
The progress of purification and yields of the obtained protein at each purification step.
Sample Purification step Fraction Volume (ml) Ag concentration (mg/ml) Ag (mass/mg) Yield (%)

Crude IBs purification Total 5 (ml) ≥20 (mg/ml) ≥100 (mg) 100%
Refolded core – Total 20 (ml) ≥0.2 (mg/ml) ≥4 (mg) –
Refolded core Cross filtration Total 2 (ml) ≥2 (mg/ml) ≥4 (mg) –
Concentrated refolded core CM sepharose 1 (0.5 M NaCl elution) 1 (ml) loaded 2 (mg/ml) 2 (mg) 100%
1 (ml) collected 1.25 (mg/ml) 1.25 (mg) 62.5%

Fig. 7. (a) Core antigen purification onto CM sepharose column using AKTA Purifier 100 FPLC system (GE Healthcare Life Sciences, Sweden). The Y-axis represents
protein absorbance UV280 in the milli-absorbance unit (mAU). The X-axis represents time in minutes (min). The smooth curve characterizes UV280 absorbance. The
dotted curve demonstrates the stepwise gradient increasing in salt concentration (0–100% of 1 M NaCl).
(b) 15% SDS-PAGE of purified fraction from weak cation exchange column.
Lane 1: pre-stained low MW chromatin protein ladder (Cat No: PR0602, Vivantis, Malaysia). Lane 2: collected peak from CM sepharose column at a retention time of
12.3 min. The size of a band detected was ~17 kDa.

human IgG conjugate that was calculated to be 0.114 (1.6 ng/ml). The
limit of quantification (LOQ) was also determined to be 0.157 (2.2 ng/
ml) (Fig. 8). In this study, the tested 135 serum samples were classified
according to the quantitative real-time PCR technique results into two
groups: HCV-positive and HCV-negative. A total of 69 (92%) of the 75
positive samples were genotype-4 and selected for re-testing by both the
in-house anti-HCV core assay and the commercial assay along with 60
negative control samples. These positive samples were of different viral
loads ranging from 225 IU to 6,934,590 IU.
Of the 129 selected sera, 69 were positive by real-time PCR, 68 by in-
house anti-HCV core assay, and 66 by the commercial assay. The
remaining 60 sera were negative by real-time PCR, 59 were confirmed
by in-house anti-HCV core assay, and 56 were confirmed by the com­
mercial assay. Comparative analysis of the selected sera by the two as­
says is summarized in Table 3.
The paired t-test data of the in-house anti-HCV core assay versus the
commercial assay was statistically highly significant with a P-value <
0.001 and a low 95% confidence interval for both the positive and
negative samples. The results also showed that the in-house anti-HCV
core assay is specific, accurate, and highly sensitive in accordance with

Fig. 8. Calibration curve using standard core protein and ELISA method.

6
E.M. Saleh et al.
Table 3
Comparative analysis of the commercial ELISA kit as a reference standard and the in-house anti-HCV core assay.
Cut-off Sensitivity Specificity PPV
Commercial ELISA kit 0.149 100.0% 100.0% 100.0%
In-house anti-HCV core assay 0.114 100.0% 96.6% 97.2%
Where; PPV: Positive predictive value, NPV: Negative predictive value, AUC: Area under curve.
the commercial ELISA kit. The area under the curve (AUC) was calcu­
lated according to the ROC curve of paired t-test data for both the in-
house anti-HCV core assay and the commercial ELISA kit (Fig. 9).
Also, plotting of both positive and negative samples showed higher
reproducibility of in-house anti-HCV core assay compared with the
commercial ELISA kit, as shown in Fig. 10a and b.
4. Discussion
HCV infection is a major public health burden in Egypt, with the
highest prevalence rate in the world. Detection and treatment of this
disease in the early stage are critical [17]. HCV screening begins with
blood investigation to detect antibodies against the HCV, using EIA or
CIA because nucleic acid amplification techniques (NATs) are costly and
prone to contamination. Besides, rapid tests with inaccurate, deceptive
results are not preferred to screen HCV at blood collection centers of
developing countries like Egypt [15,29].
Different serological kits are used to screen HCV in Egypt, but these
assays are not prepared from the antigens representing local HCV
strains; hence, a good reason for the screened sera may give false-
negative results. Also, Egypt is a developing country, which increases
the need for national HCV screening kits. To the best of our knowledge,
no methods had been developed so far in Egypt based on the existent
HCV genotypes or the virus isolates.
In this study, the core antigen gene of HCV genotype-4 encoding the
first 148 aa (partial protein) was cloned, characterized, expressed, and
purified, following that the serological reactivity of the purified re­
combinant core protein was tested using ELISA. The results showed a
promising, more effective, and high-level yield of the expressed protein.
The purification of recombinant HCV core protein in the present study
depended upon the protein’s pI (pI = 11.4). Using a pH lower than the pI
value, the recombinant HCV core protein was positively charged and
easily attached to its counter ions on the weak cation exchange column
(CM sepharose). Thereafter, washing of column for removing undesired
Fig. 9. ROC curve of paired t-test for (a) the in-house anti-HCV core assay versus (b) the commercial ELISA kit.
7
Protein Expression and Purification 188 (2021) 105965
NPV Accuracy AUC (95%C.I) p-value
Lower bound Upper bound
100.0% 100% 100.0% 100.0% 100.0% 0.001**
100.0% 98.4% 99.8% 100.0% 100.0% 0.001**
proteins followed by elution of the recombinant HCV core protein was
carried out at retention time 12.3 min. The concentration of purified
active protein was 1.25 mg/ml, which represents 62.5% of the total
protein injected into the column (2 mg/ml). This high-yield concentra­
tion was useful in detecting anti-HCV antibodies in human sera on a
large scale and paved the way to develop an in-house anti-HCV assay for
HCV genotype-4 in Egypt.
The concentration of HCV genotype-4 core antigen was first deter­
mined according to a calibration line generated using a standard core
protein and ELISA technique. A total number of 129 archived serum
samples detected for HCV infection using the real-time PCR technique
were re-tested for anti-HCV antibodies by both in-house anti-HCV assay
and commercial ELISA kits to evaluate both sensitivity and specificity of
the developed method. The in-house anti-HCV core assay detected 68 of
69 total HCV-positive samples and 59 of total 60 HCV-negative samples.
The in-house anti-HCV assay results versus the commercial ELISA kit
were analyzed using the paired t-test ‘GraphPad Prism version 7.01’. The
results were statistically significant with a P-value < 0.001 and a low
95% confidence interval for both positive and negative samples. Also,
plotting of both positive and negative samples showed higher repro­
ducibility of in-house anti-HCV core assay compared to the commercial
ELISA kit.
The present study indicates that a partial core protein with the major
epitopes showed high reactivity; however, another former study
demonstrated that the full-length core protein has better reactivity than
truncated core antigen [30].
A Pakistani study [31] reported the expression of HCV genotype-3a
whole core protein in E. coli and purification of the expressed protein
using GST sepharose fast flow column (an affinity column chromatog­
raphy). A small amount of purified protein was used in ELISA to detect
antibodies in HCV-infected human sera, and the ELISA results agreed
with this study in being sensitive, specific, and of higher reproducibility
in accordance with the commercial kit. Another report from Iran [32]
described the expression of HCV genotype-3a whole core antigen in
E.M. Saleh et al.
Fig. 10. (a) The reproducibility of the tested positive samples for both the in-house anti-HCV core assay and the commercial kit. (b) The reproducibility of the tested
negative samples for both the in-house anti-HCV core assay and the commercial kit.
E. coli, but IBs containing the core protein without purification were
used in the dot blot assay instead of ELISA to capture the antibodies in
HCV-infected human sera. Also, cloning and expression of the antigenic
regions of core and E2 genes from local HCV genotype-3a from Lahore
were performed. Both HCV core and E2 inactive proteins were purified
with a good yield using Ni-NTA affinity chromatography and were tested
using ELISA and immunoblot assay. Similar results to the present study
were obtained with increased sensitivity and specificity of both the
ELISA and immunoblot assay compared to the commercial ELISA kit
[33].
Alternatively, Mirnurollahi et al. [34] reported that the reverse
staining method (an imidazole-SDS-Zn reverse staining method that
purified the proteins using dialysis membrane) was preferable to Ni-NTA
affinity chromatography under native conditions for purifying the core
and core-E1-E2 recombinant HCV proteins because Ni-NTA affinity
chromatography could not purify completely the recombinant proteins
compared to reverse staining method.
The evaluation of the in-house assay was focused on the functional
characteristics, such as ease of handling, specificity, and sensitivity on a
group of well-characterized samples obtained from geographically
diverse regions of Egypt and its suitability for manipulation in small
laboratories, i.e., blood collection centers. A common drawback of this
in-house anti-HCV core assay based on the ELISA technique is that it may
not detect HCV infection in the sample taken during the period between
actual infection and production of antibodies.
Although there are many commercial assays for the diagnosis of
HCV, several assays for detecting the core antigen of HCV by different
techniques such as ELISA have been developed. These assays were used
as alternatives to NAT to be used in resource-limited settings, where
molecular laboratory tests are either unavailable or not widely
employed due to their cost issues. The major limitation of the HCV core
assay is its lower sensitivity limiting its utility. Moreover, most of these
kits are not sensitive and lack specificity for HCV genotype-4. Alterna­
tively, ELISA-based assays require less technical equipment and are less
expensive than molecular techniques. Hence, the current target focused
on designing highly efficient ELISA diagnostic assay with high speci­
ficity and sensitivity to maximize efficiency by minimizing the cost.
Hence, the present in-house anti-HCV core ELISA is convenient, sensi­
tive, and cost-effective for screening HCV genotype-4 infection in Egypt
and other developing countries. This outcome strengthens the effec­
tiveness of the current in-house anti-HCV core ELISA to hinder the
process of the HCV-induced HCC.
8
Protein Expression and Purification 188 (2021) 105965
5. Conclusion
A high level of the expressed recombinant HCV genotype-4 core
antigen was obtained through the present study and used as capturing
antigen in an ELISA with high sensitivity, specificity, and reproduc­
ibility. The obtained recombinant protein can be considered for in-house
screening of HCV infection.
CRediT authorship contribution statement
Eman M. Saleh: Conceptualization, Software, Investigation, Formal
analysis, Data curation, Writing – original draft, Writing – review &
editing, Visualization, Supervision. Abdullah E. Gouda: Methodology,
Software, Validation, Investigation, Formal analysis, Resources, Data
curation, Writing – original draft, Writing – review & editing, Visuali­
zation. Amina M. Medhat: Conceptualization, Visualization, Investi­
gation, Formal analysis, Data curation, Writing – original draft, Writing
– review & editing, Visualization, Supervision. Hend O. Ahmed: Soft­
ware, Validation, Formal analysis, Resources, Writing – original draft.
Mohamed A. Shemis: Conceptualization, Methodology, Software,
Validation, Formal analysis, Resources, Data curation, Writing – original
draft, Writing – review & editing, Visualization, Supervision, Project
administration, Funding acquisition.
Declaration of competing interest
The authors declare no conflict of interest.
Acknowledgment
This work was partially supported by the ASRT-project no. EGY/
FR10-06.
References
[1] F. El-Zanaty, A. Way, Egypt Demographic and Health Survey 2008, Minist. Heal,
2009. San Fr. http://www.measuredhs.com. (Accessed 19 August 2021). accessed
[2] Ministry of Health and Population [Egypt], El-Zanaty and Associates [Egypt], and
ICF International, Egypt Health Issues Survey 2015, 2015, p. 234.
[3] S.P. Kouyoumjian, H. Chemaitelly, L.J. Abu-Raddad, Characterizing hepatitis C
virus epidemiology in Egypt: systematic reviews, meta-analyses, and meta-
regressions, Sci. Rep. 8 (2018), https://doi.org/10.1038/s41598-017-17936-4.
[4] World Health Organization, Global Hepatitis Report 2017, World Health
Organization, Who, 2017.
[5] I. Waked, G. Esmat, A. Elsharkawy, M. El-Serafy, W. Abdel-Razek, R. Ghalab,
G. Elshishiney, A. Salah, S. Abdel Megid, K. Kabil, M.H. El-Sayed, H. Dabbous, Y. El
Shazly, M. Abo Sliman, K. Abou Hashem, S. Abdel Gawad, N. El Nahas, A. El Sobky,
S. El Sonbaty, H. El Tabakh, E. Emad, H. Gemeah, A. Hashem, M. Hassany,
E.M. Saleh et al.
N. Hefnawy, A.N. Hemida, A. Khadary, K. Labib, F. Mahmoud, S. Mamoun,
T. Marei, S. Mekky, A. Meshref, A. Othman, O. Ragab, E. Ramadan, A. Rehan,
T. Saad, R. Saeed, M. Sharshar, H. Shawky, M. Shawky, W. Shehata, H. Soror,
M. Taha, M. Talha, A. Tealaab, M. Zein, A. Hashish, A. Cordie, Y. Omar, E. Kamal,
I. Ammar, M. AbdAlla, W. El Akel, W. Doss, H. Zaid, Screening and treatment
program to eliminate hepatitis C in Egypt, N. Engl. J. Med. 382 (2020), https://doi.
org/10.1056/nejmsr1912628.
[6] W.A. Anwar, M. El Gaafary, S.A. Girgis, M. Rafik, W.M. Hussein, D. Sos, I.
M. Mossad, A. Fontanet, L. Temime, Hepatitis C virus infection and risk factors
among patients and health-care workers of Ain Shams University hospitals, Cairo,
Egypt, PLoS One 16 (2021), https://doi.org/10.1371/journal.pone.0246836.
[7] A.R.N. Zekri, A.S.E.D. Youssef, Y.M. Bakr, R.M. Gabr, O.S. Ahmed, M.H. Elberry, A.
M. Mayla, M. Abouelhoda, A.A. Bahnassy, Early detection of hepatocellular
carcinoma co-occurring with hepatitis C virus infection: a mathematical model,
World J. Gastroenterol. (2016), https://doi.org/10.3748/wjg.v22.i16.4168.
[8] Y.E.E. Abo-Amer, S. Abd-Elsalam, H. Eldosoky, A.K. Elshenawy, S. Awny,
W. Elagawy, M. El Abgeegy, H.F. Elsergany, H. Elashry, M.S. Negm, Declining
prevalence of hepatitis C virus among university students in one of the main
governorates in Egypt, Infect. Drug Resist. 11 (2018) 2435–2441, https://doi.org/
10.2147/IDR.S183462.
[9] A. El Ray, T. Asselah, R. Moucari, M. El Ghannam, A.A. Taha, M.A. Saber, M. Akl,
R. Atta, M. Shemis, A.S. Radwan, A. Ghali, V. Paradis, P. Marcellin, Insulin
resistance: a major factor associated with significant liver fibrosis in Egyptian
patients with genotype 4 chronic hepatitis C, Eur. J. Gastroenterol. Hepatol.
(2013), https://doi.org/10.1097/MEG.0b013e32835c9f69.
[10] P. Cacoub, L. Gragnani, C. Comarmond, A.L. Zignego, Extrahepatic manifestations
of chronic hepatitis C virus infection, Dig. Liver Dis. 46 (2014) S165–S173, https://
doi.org/10.1016/j.dld.2014.10.005.
[11] Z. Mariño, A. Darnell, S. Lens, V. Sapena, A. Díaz, E. Belmonte, C. Perelló, J.
L. Calleja, M. Varela, M. Rodriguez, C. Rodriguez de Lope, S. Llerena, X. Torras,
A. Gallego, M. Sala, R.M. Morillas, B. Minguez, J. Llaneras, S. Coll, J.A. Carrion,
M. Iñarrairaegui, B. Sangro, R. Vilana, M. Sole, C. Ayuso, J. Ríos, X. Forns, J. Bruix,
M. Reig, Time association between hepatitis C therapy and hepatocellular
carcinoma emergence in cirrhosis: relevance of non-characterized nodules,
J. Hepatol. 70 (2019) 874–884, https://doi.org/10.1016/j.jhep.2019.01.005.
[12] M.A. Shemis, D.M. El-Abd, D.I. Ramadan, M.I. El-Sayed, B.S. Guirgis, M.A. Saber,
H.M. El-Said Azzazy, Evaluation of multiplex nested polymerase chain reaction for
routine hepatitis C virus genotyping in Egyptian patients, Hepat. Mon. (2012),
https://doi.org/10.5812/hepatmon.6012.
[13] D. Omran, M. Alboraie, R.A. Zayed, M.N. Wifi, M. Naguib, M. Eltabbakh,
M. Abdellah, A.F. Sherief, S. Maklad, H.H. Eldemellawy, O.K. Saad, D.M. Khamiss,
M. El Kassas, Towards hepatitis C virus elimination: Egyptian experience,
achievements and limitations, World J. Gastroenterol. 24 (2018) 4330–4340,
https://doi.org/10.3748/wjg.v24.i38.4330.
[14] E. Gupta, M. Bajpai, A. Choudhary, Hepatitis C virus: screening, diagnosis, and
interpretation of laboratory assays, Asian J. Transfus. Sci. 8 (2014) 19–25, https://
doi.org/10.4103/0973-6247.126683.
[15] A. Do, N.S. Reau, Chronic viral hepatitis: current management and future
directions, Hepatol. Commun. 4 (2020) 329–341, https://doi.org/10.1002/
hep4.1480.
[16] W. Doss, G. Shiha, M. Hassany, R. Soliman, R. Fouad, M. Khairy, W. Samir,
R. Hammad, K. Kersey, D. Jiang, B. Doehle, S.J. Knox, B. Massetto, J.
G. McHutchison, G. Esmat, Sofosbuvir plus ribavirin for treating Egyptian patients
with hepatitis C genotype 4, J. Hepatol. (2015), https://doi.org/10.1016/j.
jhep.2015.04.023.
[17] A. Gomaa, N. Allam, A. Elsharkawy, M. El Kassas, I. Waked, Hepatitis C infection in
Egypt: prevalence, impact and management strategies [Corrigendum], Hepat. Med.
Evid. Res. (2017), https://doi.org/10.2147/hmer.s143866.
Protein Expression and Purification 188 (2021) 105965
[18] K. Yasui, T. Wakita, K. Tsukiyama-Kohara, S.I. Funahashi, M. Ichikawa, T. Kajita,
D. Moradpour, J.R. Wands, M. Kohara, The native form and maturation process of
hepatitis C virus core protein, J. Virol. (1998).
[19] E. Torbati, M. Bandehpour, P. Pakzad, N. Mosaffa, B. Kazemi, The evaluation of
Hepatitis C virus core antigen in immunized Balb/C mice, Hepat. Mon. 12 (2012)
71–77, https://doi.org/10.5812/hepatmon.6141.
[20] S. Hekmat, S.D. Siadat, M.R. Aghasadeghi, S.M. Sadat, G. Bahramali, M.M. Aslani,
M. Mahdavi, S. Shahbazi, From in-silico immunogenicity verification to in vitro
expression of recombinant Core-NS3 fusion protein of HCV, Bratislava Med. J. 118
(2017) 189–195, https://doi.org/10.4149/BLL_2017_038.
[21] A. Austria, G.Y. Wu, Occult hepatitis C virus infection: a review, J. Clin. Transl.
Hepatol. 6 (2018) 155–160, https://doi.org/10.14218/JCTH.2017.00053.
[22] M.L. Poulin, I.-M. Chiu, Direct cloning of λgt11 cDNA inserts into a plasmid vector,
in: Protoc. Gene Anal., Humana Press, New Jersey, 1994, pp. 9–18, https://doi.
org/10.1385/0-89603-258-2:9.
[23] M. Rahimzadeh, M. Sadeghizadeh, F. Najafi, S. Arab, H. Mobasheri, Impact of heat
shock step on bacterial transformation efficiency, Mol. Biol. Res. Commun. 5
(2016) 257–261.
[24] H. Okasha, S.M. Nasr, S. Samir, Recombinant expression of Cec-B peptide in
Escherichia coli with a significant anticancer effect on hepatocellular carcinoma,
Curr. Pharmaceut. Biotechnol. 22 (2021) 1235–1245, https://doi.org/10.2174/
1389201022666210104121709.
[25] B. Kazemi, M. Bandehpour, N. Seyed, M. Roozbehi, N. Mosaffa, Cloning and
expression of hepatitis C virus core protein in pGemex-1 expression vector, Arch.
Iran. Med. 11 (2008) 173–178.
[26] A. Singh, V. Upadhyay, A.K. Panda, Solubilization and refolding of inclusion body
proteins, Methods Mol. Biol. 1258 (2015) 283–291, https://doi.org/10.1007/978-
1-4939-2205-5_15.
[27] M. Seki, Y. Honda, J. Kondo, K. Fukuda, K. Ohta, jiro Sugimoto, E. Yamada,
Effective production of the hepatitis C virus core antigen having high purity in
Escherichia coli, J. Biotechnol. (1995), https://doi.org/10.1016/0168-1656(94)
00134-X.
[28] A.H.B. Wu, T.A. Onigbinde, S.S. Wong, K.G. Johnson, Evaluation of full-scanning
gc/ion trap ms analysis of nida drugs-of-abuse urine testing in urine, J. Anal.
Toxicol. 16 (1992) 202–206, https://doi.org/10.1093/jat/16.3.202.
[29] A.M. Ibrahim, N. Gamal, M. Abo-El-Azaem, A. Mohamed, A. Abd-El, A. Ghaith,
S. Hassan, H. Ahmed, M. Mohamed, A. Zaki, Evaluation of Some Available HCV
Antibody Detection Tests (ELISA, Chemiluminescence, Immune Assay) and RT-PCR
Assay in the Diagnosis of Hepatitis C Virus Infection, 2018. https://ejhm.journals.
ekb.eg/article_10167.html.
[30] T. Xiang, Z. Jiang, J. Zheng, C. Lo, H. Tsou, G. Ren, J. Zhang, A. Huang, G. Lai,
A novel double antibody sandwich-lateral flow immunoassay for the rapid and
simple detection of hepatitis C virus, Int. J. Mol. Med. 30 (2012) 1041–1047,
https://doi.org/10.3892/ijmm.2012.1121.
[31] M.Z. Yousaf, M. Idrees, Z. Saleem, I.U. Rehman, M. Ali, Expression of core antigen
of HCV genotype 3a and its evaluation as screening agent for HCV infection in
Pakistan, Virol. J. (2011), https://doi.org/10.1186/1743-422X-8-364.
[32] B. Kazemi, M. Bandehpour, N. Seyed, M. Roozbehi, N. Mosaffa, Cloning and
expression of hepatitis C virus core protein in pGemex-1 expression vector, Arch.
Iran. Med. (2008).
[33] A. Ali, M. Nisar, M. Idrees, S. Rafique, M. Iqbal, Expression of Hepatitis C Virus
Core and E2 antigenic recombinant proteins and their use for development of
diagnostic assays, Int. J. Infect. Dis. 34 (2015) e84–e89, https://doi.org/10.1016/j.
ijid.2015.03.010.
[34] S.M. Mirnurollahi, A. Bolhassani, S. Irani, N. Davoudi, Expression and purification
of HCV core and core-E1E2 proteins in different bacterial strains, Iran. J.
Biotechnol. 13 (2015) 57–62, https://doi.org/10.15171/ijb.1249.