[Expression and activity identification of APOBEC3A-HBc fusion protein]

Objective To construct the expression vector of the fusion protein of apolipoprotain B mRNA editing enzyme catalytic polypeptide-like 3A (APOBEC3A) and hepatitis B virus core proteins (HBc) with different sequences, and identify its expression, intracellular localization and cytosine deamination activity. Methods The APOBEC3A gene and the coding sequence of HBc and four kinds of truncated HBc containing C-termimal domain were amplified by PCR. The APOBEC3A with full-length HBc antigen or four kinds of truncated HBc were cloned into a eukaryotic expression vector pcDNA3.0 by In-Fusion method, and confirmed by DNA sequencing. The recombinants were then transfected into HEK293T cells. The expression and localization of the fusion proteins were detected by Western blotting and immunofluorescence cytochemistry. The cytosine deamination activity was analyzed by electrophoresis on a urea denaturing polyacrylamide gel. Results Five kinds of fusion protein were expressed in HEK293T cells successfully, and the expression of the vectors containing the truncated HBc were higher than that of the APOBEC3A-HBc vector containing the full-length HBc. The fusion protein of APOBEC3A-HBc was mainly expressed in the cytoplasm of HEK293T cells, and the fusion proteins of APOBEC3A-HBc144S, APOBEC3A-HBc144E, APOBEC3A-HBc144AAA, APOBEC3A-HBc144A were expressed mainly in the nuclei of HEK293T cells. The cytosine deamination activity of the fusion proteins containing the truncated HBc was higher than that of the fusion protein APOBEC3A-HBc. Conclusion The fusion protein expression vectors of APOBEC3A and HBc with different lengths and sequences have been constructed successfully. The expressing ability, intracellular localization and cytosine deamination activity are obviously different between the fusion protein APOBEC3A-HBc and the fusion proteins containing the truncated HBc.