It is estimated that around 240 million people are chronic hepatitis B surface antigen (HBsAg) carriers, of whom around 15–20 million are also infected with hepatitis delta virus (HDV) . The HDV virion comprises an RNA genome, a single HDV-encoded antigen (HDAg), and a lipoprotein envelope provided by HBsAg . HDAg includes two isoforms, small HDAg (S-HDAg) and large HDAg (L-HDAg).
These two isoforms share the same core sequence, but L-HDAg is extended an additional 19 amino acids at the carboxyl terminus of S-HDAg.
S-HDAg could be a candidate for human vaccine development. Protection induced by immunization of adjuvanted S-HDAg (p24) was assessed in HDV-challenged woodchucks by measuring humoral and T-cell mediated responses to HDAg . In another study, a DNA vaccine expressing S-HDAg produced a higher titer of anti-HDV antibodies than one expressing L-HDAg . However, efforts to characterize and evaluate the immunological properties of S-HDAg have been limited due to the lack of suitable methods to efficiently express and purify S-HDAg. In this work, we present a brief procedure for the expression and recognition of S-HDAg in Pichia pastoris culture medium.
methods
PCR amplification of the S – HDAg gene
Two primers, HDAg-F: 5′-GC TCTAGA TTTGGGAATCCCTGGTTTCC-3′ and HDAg-R: 5′-GC GGTACC ATGAGCCGGTCCGAATCG-3′ ( XbaI and KpnI sites underlined, respectively) were used to amplify the S – HDAg Gen. The volume of the PCR reaction was 50 µl, including: 1 × Phusion buffer, 0.2 mM dNTP (NEB, N0446S), 0.5 mM each primer (IDT) and 5 ng pHDV3 plasmid as template, 1 U Phusion high-fidelity DNA polymerase (NEB , M0530S). The PCR reaction was performed using the following program: 98°C for 30 s; 30 cycles (98 °C for 10 s, 55 °C for 30 s, 72 °C for 30 s) and final extension at 72 °C for 5 min. The PCR product was purified by electrophoresis using a 1% (w/ v) Agarose gels (BioBasic, D0012) analyzed and visualized by Red-Safe Solution (iNtRON, 21141) on a blue LED illuminator. The desired ~590 bp DNA band was excised from the gel and purified using the QIAquick Gel Extraction Kit (Qiagen, 28706) according to the manufacturer’s instructions.
Cloning of the S – HDAg gene into pPICZαA
Enzymatic digestion and ligation
The purified S – HDAg gene and the vector pPICZαA (TFS, V19520) were digested with XbaI and KpnI (NEB, R0145S and R0142S, respectively) and purified using the QIAquick PCR Purification Kit (Qiagen, 28106) according to the manufacturer’s instructions. The digested HDAg gene was ligated into the linearized vector pPICZαA using T4 DNA ligase (NEB, M0202S). The reaction was carried out in a volume of 20 μl including 2 μl 10× Rapid Ligation Buffer, 8 μl DNA (~100 ng), 1 μl 5 U/μl T4 DNA ligase and incubated at 22° C. for 2 h.
Transformation and screening of E. coli
10 μl of the ligation mixture were transformed into competent E. coli DH5α cells by heat shock at 42° C. for 30 s. The cells were then recovered by adding 500 µl of liquid LB medium and incubating at 37°C for 1 hour, and then plated on LB plates supplemented with 25 µg/ml Zeocin (TFS, R25001). After incubation at 37°C overnight, ten colonies were cultured in 3 ml of liquid LB medium supplemented with 25 µg/ml Zeocin at 37°C overnight. The recombinant plasmids were isolated from the cell pellets using a GeneJET plasmid miniprep kit (TFS, K0503) according to the manufacturer’s instructions and using XbaI and KpnI to screen for positive plasmids expressing the S – carry HDAg gene, digested.
sequencing and analysis
To confirm positive clones, purified plasmid was used for nucleotide sequencing. 5 µl of the eluted plasmid was subjected to cycle sequencing with 1.0 µl of the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit (ABI) using 0.5 µl of 5’AOX-F:5′-GACTGGTTCCAATTGACAAGC-3′ on the AOX1 – Promoter and 3 subjected ‚AOX1-R:5′-GCAAATGGCATTCTGACATCC-3‘ to the AOX1 terminator. Consensus sequences were generated by aligning both sequenced strands after validation with DNAstar software V7.
Expression of recombinant S-HDAg in P. pastoris
plasmid preparation
A positive colony of E. coli was cultured at 37°C overnight in 50 ml of liquid LB medium supplemented with 25 µg/ml Zeocin. The recombinant plasmid (pPICAαA-S-HDAg) was then isolated with the GeneElute plasmid midiprep kit (Sigma-Aldrich, NA0200) according to the manufacturer’s instructions and linearized using PmeI (NEB, R0560S). The linearized plasmid was then separated on a 1.5% agarose gel and purified by Wizard SVGel and PCR Clean-Up System (Promega, A9281) according to the manufacturer’s instructions.
Transformation and screening of yeast
transformation
5 µg of the linearized recombinant vector was transferred into 50 µl of P. pastoris X33 or SMD1163 competent cells using a Gene Pulser electroporator (Bio-Rad) at 1800 V (25 µF, 600 Ω) in a 10 mm gap electroporator cuvette transformed. After addition of 1 ml of 1 M ice-cold sorbitol, the cells were harvested at 30°C for 2 hours. 100 µl of the transformation mixture was then plated onto YDPS plates supplemented with 100, 500 and 1000 µg/ml Zeocin and then incubated for 2-3 days at 30°C until colonies appeared.
Induction in miniature
For expression screening, 24 colonies of each parental strain, X33 or SMD1163, were cultured at 30°C in 2.5 ml liquid BMGY medium without Zeocin in a Micro-24 plate (Corning) at A 600 µm 15-20. The cells were centrifuged and transferred to 10 ml BMMY induction medium. Cells and culture media were harvested every 24 h after induction. 1% methanol was added every 24 h after induction.
Secreted protein preparation
The cultures were centrifuged at 5000 rpm for 3 min and 20 µl of the supernatant was taken for immunoblot analysis.
Intracellular protein preparation
Cell pellets were used to determine total intracellular protein. 1 ml breakup buffer (50 mM Na 2 HPO 4 , 50 mM NaH 2 PO 4 , 2.0 mM EDTA, pH 7.4, 100 mM NaCl and 5% glycerol; pH 7.4), 2.0 µl protease inhibitor (Calbiochem) and 200 mg glass beads were added to the cell pellets. The cells were then lysed by disruption at 50 Hz for 3 min in a Tissue Lyser LT (Qiagen). The supernatant was transferred to a 1.5 ml Eppendorf and centrifuged at 13,000 rpm for 15 min. The cell lysate was used for immunoblot analysis.
immunoblot
To detect the HDAg-His-tag fusion protein, immunoblotting was used to detect the His 6 -tag fused to the HDAg protein in the supernatant (culture medium) or intracellular protein (cell lysate). 20 µl (10 µg) of each sample and 5 µl withProtomarker prestained protein ladder (National Diagnostics) (10-225 kDa) were loaded onto a 12.5% SDS gel and run in 1x Tris/Glycine/SDS (GeneFlow) 100 V for 1 h. The SDS gel was transferred to a nitrocellulose membrane (Whatman, 09-301-111), in 5% milk in 1x PBS buffer and blocked with primary antibody (6x His monoclonal antibody (Serotec) at a dilution of 1:5000 ) incubated 1 h at room temperature). After washing with 1x PBST, the membrane was incubated with secondary antibody against mouse IgG conjugated to HRP (Sigma, A0545) at a dilution of 1:5000 for 1 h. After washing with 1x PBST, protein bands were detected on the membrane using EZ-ECL chemiluminescent solution (Geneflow,
Nickel affinity purification
Recombinant protein was purified using a His-Trap column. Total secreted protein from 300 ml culture broth was dialysed against binding buffer (300mM NaCl, 10mM imidazole, 50mM NaH 2 PO 4 , pH 8.0; Sigma-Aldrich, 56750) which was also used as binding and equilibration solution.
HBV core recombinant antigen, Delta
00121-V-01mg
Virogen
0,1 mg
321 EUR
HBV core recombinant antigen, Delta
00121-V-1000ug
Virogen
1000 ug
1539 EUR
HBV core recombinant antigen
00120-V-01mg
Virogen
0,1 mg
321 EUR
HBV core recombinant antigen
00120-V-1000ug
Virogen
1000 ug
1539 EUR
Recombinant HBV Core Antigen
VAng-Wyb8585-inquire
Creative Biolabs
inquire
Ask for price
Recombinant HBV Core Antigen (aa 1-183)
VAng-0501Lsx-inquire
Creative Biolabs
inquire
Ask for price
Recombinant HBV Core Antigen (aa 1–183)
VAng-Wyb8583-inquire
Creative Biolabs
inquire
Ask for price
Recombinant HBV Core Antigen (aa 1–144)
VAng-Wyb8584-inquire
Creative Biolabs
inquire
Ask for price
QuickTiter HBV Core Antigen ELISA Kit
VPK-150
Cell Biolabs
96 assays
1096.8 EUR
QuickTiter HBV Core Antigen ELISA Kit
VPK-150-5
Cell Biolabs
5 x 96 assays
4507.2 EUR
Recombinant HBV Core Protein
VAng-Lsx04579-1mg
Creative Biolabs
1 mg
2683.2 EUR
HBV Core Antigen (aa 1 - 186) [His]
DAG1452
Creative Diagnostics
100 µg
774 EUR
HBV Core Antibody
20-abx137186
Abbexa
Recombinant Hepatitis B HBV core delta Protein, Untagged, E.coli-100ug
QP12131-100ug
EnQuireBio
100ug
261.6 EUR
Recombinant Hepatitis B HBV core delta Protein, Untagged, E.coli-1mg
QP12131-1mg
EnQuireBio
1mg
1513.2 EUR
Recombinant Hepatitis B HBV core delta Protein, Untagged, E.coli-500ug
QP12131-500ug
EnQuireBio
500ug
795.6 EUR
HBV core (107 - 115)
5-01268
CHI Scientific
4 x 5mg
Ask for price
Virus Hepatitis B Virus 320 Core Antigen (HBV 320 Core Ag) Protein
abx670003-50ug
Abbexa
50 ug
493.2 EUR
HBV surface recombinant antigen HBsAg antigen
00123-V-01mg
Virogen
0,1 mg
321 EUR
HBV surface recombinant antigen HBsAg antigen
00123-V-1000ug
Virogen
1000 ug
1539 EUR
HBV surface recombinant antigen HBsAg antigen
00124-V-01mg
Virogen
0,1 mg
321 EUR
HBV surface recombinant antigen HBsAg antigen
00124-V-1000ug
Virogen
1000 ug
1539 EUR
HBV Core Antigen (aa 1 - 149)(subtype ayr ) [His]
DAG2293
Creative Diagnostics
100 µg
868.8 EUR
HBV core protein (128-140)
5-01270
CHI Scientific
4 x 1mg
Ask for price
HBV core (107 115) Peptide
20-abx266079
Abbexa
HBV surface recombinant antigen HBsAg
00122-V-01mg
Virogen
0,1 mg
321 EUR
HBV surface recombinant antigen HBsAg
00122-V-1000ug
Virogen
1000 ug
1539 EUR
HCV core recombinant antigen
00115-V-01mg
Virogen
0,1 mg
321 EUR
HCV core recombinant antigen
00115-V-1000ug
Virogen
1000 ug
1539 EUR
HCV core recombinant antigen
00152-V-01mg
Virogen
0,1 mg
321 EUR
HCV core recombinant antigen
00152-V-1000ug
Virogen
1000 ug
1539 EUR
Recombinant HCV Core Antigen
VAng-Lsx04584-1mg
Creative Biolabs
1 mg
2023.2 EUR
Recombinant HBV Core Protein (aa 1-186) [His]
VAng-Lsx0176-inquire
Creative Biolabs
inquire
Ask for price
Recombinant HBV Surface Antigen (Subtype ay)
VAng-Wyb8577-inquire
Creative Biolabs
inquire
Ask for price
HBV antigen HBsAg Antibody
abx140205-01mg
Abbexa
0.1 mg
393.6 EUR
HBV antigen HBsAg Antibody
abx140206-01mg
Abbexa
0.1 mg
393.6 EUR
Recombinant Hepatitis B Virus Core Delta
7-07417
CHI Scientific
100µg
Ask for price
Recombinant Hepatitis B Virus Core Delta
7-07418
CHI Scientific
500µg
Ask for price
Recombinant Hepatitis B Virus Core Delta
7-07419
CHI Scientific
1000µg
Ask for price
HCV core recombinant antigen, Rhodamine
00115-V-R-01mg
Virogen
0,1 mg
321 EUR
HCV core recombinant antigen, Rhodamine
00115-V-R-1000ug
Virogen
1000 ug
1539 EUR
Recombinant Hepatitis B HBV core Protein, Untagged, E.coli-100ug
QP12130-100ug
EnQuireBio
100ug
261.6 EUR
Recombinant Hepatitis B HBV core Protein, Untagged, E.coli-1mg
QP12130-1mg
EnQuireBio
1mg
1513.2 EUR
Recombinant Hepatitis B HBV core Protein, Untagged, E.coli-500ug
QP12130-500ug
EnQuireBio
500ug
795.6 EUR
Recombinant HBV e Antigen (aa 1-160)
VAng-0502Lsx-inquire
Creative Biolabs
inquire
Ask for price
Recombinant HBV Surface Antigen (Subtype adw) (Yeast)
VAng-Wyb8578-inquire
Creative Biolabs
inquire
Ask for price
Recombinant HBV Surface Antigen (Subtype adr) (Yeast)
VAng-Wyb8580-inquire
Creative Biolabs
inquire
Ask for price
HBV Surface Antigen (Subtype ay)
VAng-0496Lsx-1mg
Creative Biolabs
1 mg
1912.8 EUR
Human Anti-HBV surface Antigen Antibody,Anti-HBV ELISA
YLA1996HU-48T
Shanghai YL Biotech
48T
435 EUR
Human Anti-HBV surface Antigen Antibody,Anti-HBV ELISA
YLA1996HU-96T
Shanghai YL Biotech
96T
562.5 EUR
Recombinant Feline Immunodeficiency Virus Core Antigen
DAG552
Creative Diagnostics
100ug
1138.8 EUR
HCV core recombinant antigen, Biotin conjugate
00115-V-B-01mg
Virogen
0,1 mg
321 EUR
HCV core recombinant antigen, Biotin conjugate
00115-V-B-1000ug
Virogen
1000 ug
1539 EUR
HCV core recombinant antigen, FITC conjugate
00115-V-F-01mg
Virogen
0,1 mg
321 EUR
HCV core recombinant antigen, FITC conjugate
00115-V-F-1000ug
Virogen
1000 ug
1539 EUR
HCV core 2-119aa recombinant antigen
00150-V-01mg
Virogen
0,1 mg
321 EUR
HCV core 2-119aa recombinant antigen
00150-V-1000ug
Virogen
1000 ug
1539 EUR
HIV 1 p24 core recombinant antigen
00177-V-01mg
Virogen
0,1 mg
321 EUR
HIV 1 p24 core recombinant antigen
00177-V-1000ug
Virogen
1000 ug
1539 EUR
HTLV-I p24 core recombinant antigen
00204-V-01mg
Virogen
0,1 mg
321 EUR
HTLV-I p24 core recombinant antigen
00204-V-1000ug
Virogen
1000 ug
1539 EUR
Hepatitis C virus core recombinant antigen
8802
Virostat
100 ug
477.6 EUR
Hepatitis B virus core recombinant antigen
8914
Virostat
100 ug
673.36 EUR
Hepatitis C virus core recombinant antigen
8924
Virostat
100 ug
412.36 EUR
Hepatitis B virus core recombinant antigen
8927
Virostat
100 ug
412.36 EUR
Hepatitis B virus core recombinant antigen
8929
Virostat
100 ug
477.6 EUR
Feline Immunodef. virus core recombinant antigen
8963
Virostat
100 ug
673.36 EUR
Recombinant HBV Core Protein (aa 1-149) (Subtype ayr) [His]
VAng-Lsx0179-100g
Creative Biolabs
100 µg
712.8 EUR
Recombinant HBV Core Protein (aa 1-149) (Subtype ayr) [His]
VAng-Lsx0179-1mg
Creative Biolabs
1 mg
3682.8 EUR
HCV Core 24 Antigen
00151-V-01mg
Virogen
0,1 mg
321 EUR
HCV Core 24 Antigen
00151-V-1000ug
Virogen
1000 ug
1539 EUR
HCV Core Antigen Antibody
abx021744-1mg
Abbexa
1 mg
2389.2 EUR
HCV Core Antigen Antibody
abx021825-1mg
Abbexa
1 mg
1387.2 EUR
HCV Core Antigen Antibody
abx021826-1mg
Abbexa
1 mg
1387.2 EUR
HCV Core Antigen Antibody
abx021827-1mg
Abbexa
1 mg
1554 EUR
HCV Core Antigen Antibody
abx021828-1mg
Abbexa
1 mg
1387.2 EUR
HCV Core Antigen Antibody
abx021829-1mg
Abbexa
1 mg
1387.2 EUR
HCV Core Antigen Antibody
abx021835-100ug
Abbexa
100 ug
718.8 EUR
HCV Core Antigen Antibody
abx021836-100ug
Abbexa
100 ug
718.8 EUR
HCV Core Antigen Antibody
abx021837-100ug
Abbexa
100 ug
727.2 EUR
HCV Core Antigen Antibody
abx021838-100ug
Abbexa
100 ug
777.6 EUR
HCV Core Antigen Antibody
abx021841-100ug
Abbexa
100 ug
678 EUR
HCV Core Antigen Antibody
abx023013-1ml
Abbexa
1 ml
844.8 EUR
Recombinant HBV Surface Antigen (Subtype ad) (Saccharomyces cerevisiae)
VAng-0497Lsx-100g
Creative Biolabs
100 µg
1447.2 EUR
Recombinant HBV Surface Antigen (Subtype adr) (CHO Cells)
VAng-0498Lsx-500g
Creative Biolabs
500 µg
4018.8 EUR
Recombinant HBV Surface Antigen (Subtype ayw) (S Region)
VAng-0499Lsx-inquire
Creative Biolabs
inquire
Ask for price
Recombinant HBV e Antigen [GST] (1 mg/mL)
VAng-0503Lsx-inquire
Creative Biolabs
inquire
Ask for price
Recombinant HBV Surface Antigen (Subtype ad) (Human Plasma)
VAng-Wyb8581-inquire
Creative Biolabs
inquire
Ask for price
Recombinant HBV e Antigen [GST] (6.0 mg/mL)
VAng-Wyb8586-inquire
Creative Biolabs
inquire
Ask for price
Recombinant HBV Surface Antigen (Subtype ad) (Insect Cell)
VAng-Lsx04580-1mg
Creative Biolabs
1 mg
637.2 EUR
Hepatitis B Virus Core delta Protein
20-abx260228
Abbexa
Hepatitis C virus core protein recombinant antigen
9007
Virostat
100 ug
679.87 EUR
Recombinant HCV Core Antigen (aa 2-192)
VAng-0512Lsx-inquire
Creative Biolabs
inquire
Ask for price
Recombinant HBV Protein
VAng-Lsx0175-1mg
Creative Biolabs
1 mg
4164 EUR
Recombinant HBV Surface Antigen (Subtype adw) (aa 1-227)
VAng-0500Lsx-100g
Creative Biolabs
100 µg
1710 EUR
Recombinant HBV Surface Antigen (Subtype adw) (aa 1-227)
VAng-0500Lsx-500g
Creative Biolabs
500 µg
7318.8 EUR
HCV type 4 Core Antigen
DAG3322
Creative Diagnostics
1 mg
972 EUR
HCV Core Antigen Antibody (Biotin)
abx021833-100ug
Abbexa
100 ug
811.2 EUR
HCV Core Antigen Antibody (FITC)
abx021834-100ug
Abbexa
100 ug
811.2 EUR
HCV Core Antigen Antibody (Biotin)
abx021839-100ug
Abbexa
100 ug
718.8 EUR
A 5 mL His-Trap HP column (GE Healthcare) was equilibrated with 5 column volumes of binding buffer. Total dialyzed protein (5 mg) was loaded onto the column at a flow rate of 1 mL/min for 50 min. The column was washed with 5 column volumes of binding buffer followed by 5 column volumes of wash buffer (300mM NaCl, 30mM imidazole, 50mM NaH 2 PO 4 , pH 8.0). The protein was eluted with elution buffer (300mM NaCl, 250mM imidazole, 50mM NaH 2 ) PO 4 , pH 8.0) at a flow rate of 1 ml/min for 20 min. Each 1 ml fraction was analyzed by SDS-PAGE and visualized using a silver staining kit (Sigma-Aldrich). The protein concentration of the eluted fractions was quantified using a Bradford kit (BioBasic).
Leave a Comment