HBV-Core-rekombinantes Antigen, Delta

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.

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 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).