A recombinant multiepitope protein for hepatitis B diagnosis

Abstract

Hepatitis B is a liver inflammation caused by hepatitis B virus (HBV) and can be diagnosed in clinical stage by hepatitis B core antibody from IgM class (anti-HBcIgM). Hepatitis B core antibody from IgG class (Anti-HBcIgG) appears quickly after IgM, reaching high titers in chronic hepatitis, and remains even after cure. Since hepatitis B core antibody (anti-HBc) is the first antibody identified and sometimes the only marker detected during the course of infection, it can be used both to indicate HBV acute infection (anti-HBc-IgM) and to identify individuals who have come into contact with the virus (anti-HBc-IgG). In this work we propose a recombinant hepatitis B core multiepitope antigen (rMEHB) to be used for diagnosis of hepatitis B. For this purpose, a synthetic gene coding for rMEHB was designed and cloned into vector pET21a with a 6xHis tag at the C-terminal. Time course induction in E. coli showed an induced protein with an apparent molecular mass of ~21 kDa. Protein purification was performed by a single step with affinity chromatography Ni-NTA. Circular dichroism spectroscopy indicated rMEHB as a thermal stable protein at pH 7.0 and 8.0. In these conditions rMEHB was successfully used to perform an enzyme linked immuno sorbent assay (ELISA) with positive and negative sera.

Figure 1

Nucleotide and predicted amino acid sequences of rMEHB.

Figure 2

Time course rMEHB expression in E. coli BL21 (λDE3) and analysis by SDS-PAGE12%. (a) Lane M, unstained protein molecular mass marker (Fermentas Life Sciences); Lane 1, uninduced control; Lanes 2–4, after 0.5, 1.5, and 2.5 hours of induction, respectively. (b) Western blotting analysis of the purified rMEHB. The monoclonal antibody, antipolyhistidine conjugated alkaline phosphatase, was diluted 1 : 1000 in PBS.

Figure 3

Analysis of rMEHBby SDS-PAGE 12% after purification procedure. Fractions were collected after Ni-NTA chromatography. Lane M, molecular mass markers (Weight Standard, Broad Range, BioRad). Lane 1, flow-through; Lanes 2–4, wash steps; Lanes 5–8 purified rMEHB after elution with 500 mM imidazole.

Figure 4

Far-UV CD spectra of rMEHB (0.08 mg·mL⁻¹) in 2 mM MOPS buffer at 25°C. Solid and dashed lines indicate the CD spectra of MEHB at pH 7.0 and 8.0, respectively.

Figure 5

Heat-induced unfolding of rMEHB by circular dichroism (CD). (a) Far UV CD spectra in 2 mM MOPS buffer pH 7.0 and (b) pH 8.0, as a function of temperature. Molar ellipticities [θ] were measured from 260 to 195 nm at temperature rising from 25°C to 95°C. The arrows indicate decreased dichroic signal at 200 nm with increased temperature. (c) Temperature induced unfolding curves of rMEHB in 2 mM MOPS buffer pH 7.0 (––) and pH 8.0 (—) monitored at 200 nm.

Figure 6

Enzyme linked immuno sorbent assay (ELISA) using rMEHB and monoclonal antibody Anti-HB, present on the plate surface of a commercial kit for hepatitis B diagnosis. Several concentrations of rMEHB were used as following: 50 ng, 500 ng, 1 μg, 10 μg, and 15 μg. PBS was used as negative control. Antipolyhistidine conjugated alkaline phosphatase was the second antibody and the substrate was pNPP.

Figure 7

Immunological assay of rMEHB. The commercial antigen was replaced by rMEHB for the competitive test. NS: commercial negative control serum. PS: commercial positive control serum. Antipolyhistidine conjugated alkaline phosphatase was the second antibody and the substrate was pNPP.