Construction of a novel liver-targeting fusion interferon by incorporation of a Plasmodium region I-plus peptide

Abstract

Interferon alpha (IFN α) exerts a multiplicity of biological actions including antiviral, immunomodulatory, and antiproliferative effects. Administration of IFN α is the current treatment for chronic hepatitis B; however, therapy outcome has not been completely satisfactory. The systemic effects of IFN α may account for its low in vivo biological activity and multiple adverse events. The purpose of this study was to design a novel liver-targeting fusion interferon (IFN-CSP) by fusing IFN α2b with a Plasmodium region I-plus peptide, thus targeting the drug specifically to the liver. The DNA sequence encoding IFN-CSP was constructed using improved splicing by overlapping extension-PCR method, and then cloned into the pET-21b vector for protein expression in E. coli BL21 (DE3). The recombinant protein was expressed as a His-tagged protein and purified using a combination of Ni affinity and HiTrap affinity chromatography at a purity of over 95%. The final yield of biologically active IFN-CSP was up to 270 mg/L culture. The purified recombinant protein showed anti-HBV activity and liver-targeting potentiality in vitro. These data suggests that the novel fusion interferon IFN-CSP may be an excellent candidate as a liver-targeting anti-HBV agent.

Figure 1

Schematic diagram of the gene synthesis method. The target DNA is dissected into oligos of between 29 and 58 nt long. Each four adjacent primers were mixed in a separate tube; after the first PCR, fragments overlap adjacent ones by up to 175 bp. In the overlap extension PCR step, the terminal fragments can be easily extended to full length.

Figure 2

Schematic representation of expression vector IFN-CSP/pET-21b.

Figure 3

Construction of fusion gene IFN-CSP and screening of recombinant plasmids containing fusion gene. (a) Fusion gene IFN-CSP was constructed by SOE-PCR. Lane M: DNA molecular weight marker. Lane 1–4: first PCR was performed for fusion gene assembly using eight pairs of oligonucleotides with different concentrations as templates to give PCR products ranging 132–175 bp in size. Lane 5: target amplification fragment of fusion gene IFN-CSP. (b) Screening of recombinant plasmids containing IFN-CSP gene by colony PCR and restriction endonuclease analysis. Lane M: DNA molecular weight marker, Lane 1: PCR products of recombinant plasmids, and Lane 2: Recombinant plasmids digested with Nde I/Xho I.

Figure 4

Analysis of fusion protein by SDS-PAGE and Western blot. (a) Expression and purification of IFN-CSP. Lane M: protein molecular weight marker. Lanes 1 and 2: total proteins of E. coli BL21/pET-21b-IFN-CSP before and after induction. Lanes 3 and 4: supernatant and precipitation after ultrasonication and centrifugation. Lane 5: purified IFN-CSP using Ni affinity chromatography. Lane 6: Purified IFN-CSP using HiTrap affinity chromatography. (b) IFN-CSP was analyzed by SDS-PAGE, transferred to PVDF membrane, and detected by goat polyclonal anti-human IFN α antibody. Lane M: protein molecular weight marker. Lanes 1 and 2: total proteins of E. coli BL21/pET-21b-IFN-CSP before and after induction.

Figure 5

RP-HPLC analysis of purified fusion protein IFN-CSP with a C18 column.

Figure 6

Inhibition of HBV DNA replication by IFN α2b and IFN-CSP in HepG 2.2.15 cells. The HBV DNA in supernatants of the HepG 2.2.15 cells was harvested and analyzed by real-time PCR. The copies of the HBV DNA from the HepG 2.2.15 cells were calculated based on their Ct value and the standard curves. Data shown represent the mean values (±S.D.) based on three independent experiments. **P < 0.01 as compared with the control group.

Figure 7

Fluorescence photomicrographs of tissue from an adult BALB/c mouse after being incubated with IFN-CSP solution. (a) Heart. (b) Liver. (c) Spleen. (d) Lung. (e) Kidney. 1: green fluorescent labeling of IFN-CSP stained by anti-IFN antibody. 2: blue nuclear stained with DAPI. 3: merged images of 1 and 2. Note the distinct green fluorescent labeling of IFN-CSP in liver, but not in heart, spleen, lung, or kidney. Bar, 100 μm, is the same for all photomicrographs. Arrows indicate two examples of sinusoidal capillary profiles.