healthmedicinet

Pasmids, strains and culture media

pMD20-T (Takara, Japan) was used for gene cloning. pET-21b (Novagen, USA) was employed
to construct expression vector. E. coli strain DH5? (Novagen, USA) was applied as the host for gene manipulation. E. coli strain BL21 (DE3; Novagen, USA) served as expression host for fusion protein. Luria-Bertani
(LB) medium was used for bacterial growth and protein expression.

Construction of the liver-targeting fusion interferon gene using E. coli preferred codon

To improve the expression level of IFN-CSP in E. coli, a new IFN-CSP coding sequence was designed. The optimized codons usage pattern of
E. coli genes was employed according to the codon usage pattern of E. coli (http://www.kazusa.or.jp/codon/). Based on the method of polymerase chain reaction (PCR)-based gene synthesis and
gene splicing by overlap extension, a modified three-step method 15], called “splicing by overlapping extension-PCR (SOE-PCR)”, was adopted to construct
the synthetic IFN-CSP gene. We designed 16 oligonucleotides encoding for the IFN-CSP
protein. The sense and antisense oligonucleotides with 19 bp complementary overlapping
sequence are presented in Table 1.

Table 1. Nucleotide sequences of oligonucleotides designed for assembly of IFN-CSP^*

Construction of expression plasmids IFN-CSP/pET-21b

The synthetic IFN-CSP gene fragments were cloned into the pMD20-T vector (Takara)
and transformed into E. coli DH5a according to the procedures described by the manufacturer. The generated recombinant
plasmids IFN-CSP/pMD20-T were digested and the inserts were cloned into Nde I/Xho I restriction sites of the expression vector pET-21b (Fig. 1a). The resulting expression plasmid IFN-CSP/pET-21b was finally transformed into
E. coli BL21 (DE3) for IFN-CSP expression.

Fig. 1. Schematic diagram of IFN-CSP gene in the expression vector IFN-CSP/pET-21b and expression of IFN-CSP protein in
E. coli BL21/pET-21b-IFN-CSP. a: A Schematic diagram of IFN-CSP/pET-21b (T7 pro, T7 promoter; T7 ter, T7 terminator).
b: SDS-PAGE analysis of protein expression. Lane M: Protein molecular weight marker,
Lane 1–2: Total proteins of E. coli BL21/pET-21b-IFN-CSP before and after induction, Lane 3–4: Supernatant and precipitation
after ultrasonication and centrifugation

Optimization of IFN-CSP expression

To improve the expression level of IFN-CSP, the induction conditions 16] like cultivation temperature, induction timing, inducer concentrations, induction
time were systematically examined by combining one-factor experiments with an orthogonal
test (L(9)(3)(4)). A fresh clone of recombinant E. coli BL21 with plasmid IFN-CSP/pET-21b grew in Luria-Bertani (LB) medium containing 100
?g/ml ampicillin. Different conditions of induction (temperatures: 17 °C, 22 °C, 27 °C,
32 °C, 37 °C, 42 °C; OD₆₀₀: 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 2.0; IPTG concentrations: 0.1, 0.2, 0.4, 0.6, 0.8,
1.0, 1.2, 1.5 mM; induction times: 1, 2, 4, 6, 8, 10, 12, 24 h) were conducted to
optimize the expression of heterologous protein in E. coli. To evaluate the expression level of IFN-CSP, the cells were harvested by centrifugation
at 10,000 rpm for 10 min. Samples were analyzed by 15 % sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE) and the percentage fraction of proteins was assessed
by densitometric using Gel-Pro analyzer Version 4.5 software. Total protein concentration
was determined by Bradford method and the concentration of IFN-CSP was calculated
according the percentage fraction and total protein concentration.

Purification, antigenicity and biological activity analysis

Cells were collected by centrifugation. After lysis by ultrasonication in an ice bath,
an improved seven-step process was conducted to obtain purified IFN-CSP based on previously
described methods 13], 15], 17]. Briefly, cellular pellet was separated from the cell lysate by centrifugation at
6000 g and 4 °C for 10 min. IB material was washed with 1 % Triton X-100 and 2 M urea
(containing 2 % deoxycholate), and then dissolved in 6 M guanidine hydrochloride (GuHCl)
(containing 2.5 mM DTT and 50 mM Tris-HCL buffer, pH 8.0). The solubilized IFN-CSP
was refolded by dialysis with the 50 mM Tris-HCl buffer systems (containing 0.2 mM
glutathione oxidized and 2 mM glutathione reduced), followed by changes of the buffer
containing decreasing GuHCl concentrations (4, 2 and 1 M) and finally with Tris-HCl
buffer (pH 7.4). The refolded IFN-CSP was purified by heparin affinity chromatography
according to the instruction of HiTrap affinity column (GE healthcare, USA). The IFN-CSP
was eluted with a linear gradient of 0.1–2 M NaCl. After dialysis against the Tris-HCl
buffer, the sample was further purified to remove endotoxin by polymyxin B column
(Bio-Rad, USA). After detecting the lipopolysaccharides (LPS) content with the chromogenic
limulus amoebocyte lysate assay (Associates of Cape Cod, USA), the purified IFN-CSP
was lyophilized and stored at -80 °C.

The antigenicity of the purified protein was characterized by western blot analysis.
We performed western blot with primary goat polyclonal anti-IFN ? antibody (1:200;
Santa Cruz Biotechnology, USA) and peroxidase-conjugated rabbit anti-goat IgG (1:2500;
Santa Cruz Biotechnology, USA) as previously described 13]. The purity of the purified protein was assessed by reverse phase high-performance
liquid chromatography (RP-HPLC) on a C18 column (250?×?4.6 mm, 5 ?m, and 300 Å, Agilent,
USA) in an analytical Alliance HPLC System (Waters, USA). The molecular weight of
purified IFN-CSP was characterized by mass spectrometry on a matrix-assisted laser
desorption/ionization (MALDI) mass spectrometer (Applied Biosystems, USA).

Biological activity of IFN-CSP was determined in a standard cytopathic effect inhibition
assay using vesicular stomatitis virus (VSV)/human amniotic cells (WISH) measure system
according to China Biologicals Requirements.

In vivo tissue distribution experiment

All animal experimental protocols were approved by the Guangdong Pharmaceutical University
Animal Care and Use Committee. Normal Balb/c mice (18–22 g) were purchased from the
Center for Experimental Animals of Guangdong Province (Guangzhou, China). Balb/c mice
(n?=?90) were injected via the tail vein with 9.01 ?g/kg (0.46 nmol/kg) of control
IFN ?2b or 10.09 ?g/kg (0.46 nmol/kg) of recombinant IFN-CSP. At 30, 60, 120, 240
and 480 min post injection, the mice were anesthetized, euthanized and then the liver,
heart, spleen, lung and kidney were excised, rinsed with saline, dried and weighed.
The concentration of IFN ?2b was determined by ELISA (Shanghai Senxiong Biotechnology
Limited Corporation.) according to the manufacturer.

In vivo anti-HBV experiment

Balb/c-HBV transgenic mice (18–22 g) were purchased from Infectious Disease Center
of No. 458 Hospital (Guangzhou, China) and kept under the pathogen free condition
in the facility of the Guangdong Pharmaceutical University. The Balb/c-HBV transgenic
mice were randomly assigned to either treatment or control groups (n?=?6). Six non-transgenic
Balb/c mice were used as normal control mice. Each mouse was intramuscular injected
with IFN-CSP (at dose of 10¹ U/g body weight, 10² U/g body weight, 10³ U/g body weight, respectively) once a day for 28 days. Sterile physiological saline
was used as control. IFN ?2b (10³ U/g body weight) was used as drug control. Mice were sacrificed at day 28. Serum
samples and livers were collected.

Measurement of serum HBsAg and HBV-DNA

Serum HBsAg was measured by enzyme-linked immunosorbent assay (ELISA) using a HBV
diagnostic kit (Shanghai Kehua Biotech Co. Ltd., China) following the manufacturer’s
recommendations. Serum HBV-DNA was measured using a commercially available real-time
fluorescent quantification PCR (FQ-PCR) diagnostic kit (Da-An Gene Co., Guangzhou,
China) according to the manufacturer’s instruction.

Immunofluorescence and western blot analysis for HBV core protein

Tissues were fixed in 4 % paraformaldehyde for 1 h, then in 30 % sucrose solution
overnight for cryoprotection. 10–12 ?m thick sections were cut using a Leica cryostat.
Immunohistological detection of HBcAg was performed on frozen sections using primary
rabbit polyclonal anti-HBcAg antibody (1:200; Abcam, England) and Alexa Fluor 488-conjugated
goat anti-rabbit IgG (1:200; Jackson ImmunoResearch, USA). Finally, the sections were
stained with 4?,6-diamidino-2-phenylindole (DAPI) for nuclear indication. Immunofluorescence
analyses were performed with a fluorescence microscope (Leica, German).

HBV core protein was also analyzed by a standard western blot procedure using primary
polyclonal anti-HBcAg antibody (1:200; Abcam, England), anti-actin monoclonal antibody
(1:1000; Santa Cruz Biotechnology, USA) and peroxidase-conjugated secondary antibodies.
The image was digitized using a scanner and signal was quantified using of Quantity
One software (Bio-Rad).

Statistical analysis

Each measurement was performed at least in triplicate. All data were expressed as
the mean?±?standard error of the mean (SEM). All statistical analyses were performed
by SPSS (version 13.0 for Windows) statistical software. Differences between mean
values were analyzed using One-Way Analysis of Variance (ANOVA). Statistical significance
was defined by a P value of less than 0.05.