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Easily purified baculovirus/insect-system-expressed recombinant hepatitis B virus surface antigen fused to the N- or C-terminus of polyhedrin

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Abstract

Baculoviruses are circular double-stranded DNA viruses that infect insects and are widely used as the baculoviral expression vectors (BEVs), which provide a eukaryotic milieu for heterologous expression. The most frequently used vector is based on Autographa californica multiple nucleopolyhedrovirus (AcMNPV). However, purification of recombinant proteins produced using BEVs is laborious, time-consuming, and often expensive. Numerous strategies have been explored to facilitate purification of heterologous proteins, such as fusion with occlusion body (OBs)-forming proteins like polyhedrin (Polh). Baculoviruses produce OBs in the late stages of infection to protect the virion in the cellular environment, and the main protein responsible for OB formation is Polh. In this study, we investigated the effect of fusing the gene that encodes the surface antigen (S-HBsAg) of hepatitis B virus (HBV) to either the N- or C-terminus of the AcMNPV Polh. The production of recombinant viruses and recombinant proteins was confirmed, and the ability to form chimeric S-HBsAg-containing OBs was accessed by light and scanning electron microscopy of infected cells. The fusion was found to affect the shape and size of the OBs when compared to wild-type OBs, with the N-terminal fusion producing less-amorphous OBs than the C-terminal construct. In addition, the N-terminal construct gave higher levels of expression than the C-terminal construct. Quantitative and qualitative immunoassays with human serum or plasma antibodies against HBsAg showed that the two forms of the antigen reacted differently. Although both reacted with the antibody, the N-terminal fusion protein reacted with more sensitivity (2.27-fold) and is therefore more suitable for quantitative assays than the C-terminal version. In summary, the BEVs represents a promising tool for the production of reagents for the diagnosis of HBV infection.

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Funding

This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, grant number 428799/2018-3 and 305468/2019-7), Coordenação de Aperfeiçoamento de Pessoal de nível Superior (CAPES/PROEX, grant number 23038.004173/2019-93) and Fundação de Apoio à Pesquisa do Distrito Federal (FAPDF, grant number 193.001532/2016).

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Authors and Affiliations

  1. Laboratory of Baculovirus, Cell Biology Department, University of Brasilia, Brasília, DF, 70910-900, Brazil

    Leonardo A. Silva, Brenda R. Camargo, Ana Carolina Araújo, Bergmann M. Ribeiro & Daniel M. P. Ardisson-Araújo

  2. Laboratory of Insect Virology, Cell Biology Department, University of Brasília, Brasília, DF, 70910900, Brazil

    Taylice Leonel Batista & Daniel M. P. Ardisson-Araújo

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  1. Leonardo A. Silva
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  2. Brenda R. Camargo
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  3. Ana Carolina Araújo
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  5. Bergmann M. Ribeiro
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  6. Daniel M. P. Ardisson-Araújo
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Correspondence to Bergmann M. Ribeiro or Daniel M. P. Ardisson-Araújo.

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705_2021_5305_MOESM1_ESM.tif

Supplementary file1 Supplementary Fig. S1 Carboxy- (CT) and amino-terminal (NT) versions of S-HBsAg fused separately to AcMNPV polyhedrin. (A) Schematic representation of the modified polh gene cloned into pFastBac1 (pFB1) to generate pFB1-S-HBsAg (CT) and pFB1-S-HBsAg (NT). (B) Tn5B cells at 72 h after infection with the respective second-passage virus stock at an MOI of 5. The photographs show cells infected with vAc-S-HBsAg (CT) and vAc-S-HBsAg (NT). The insets show details of infected cells with an apparently amorphous mass accumulating in the infected cell. (TIF 14671 KB)

705_2021_5305_MOESM2_ESM.tif

Supplementary file2 Supplementary Fig. S2 PCR analysis of the S-HBsAg gene fused to 6xHis-Polh (NT) and Polh-6xHis (CT) after sequential passages in a Trichoplusia ni-derived cell line (Tn5B). PCR was carried out using virus genomic DNA recovered from the infected cell supernatant. M, marker 1 kbp; 1, first passage of vAc-S-HBsAg (NT); 2, fourth passage of vAc-S-HBsAg (NT); 3, first passage of vAc-S-HBsAg (CT); 4, fourth passage of vAc-S-HBsAg (CT); +, positive control (S-HBsAg gene). The red arrow indicates the expected size of the amplicon (742 bp). (TIF 2136 KB)

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Silva, L.A., Camargo, B.R., Araújo, A.C. et al. Easily purified baculovirus/insect-system-expressed recombinant hepatitis B virus surface antigen fused to the N- or C-terminus of polyhedrin. Arch Virol 167, 345–354 (2022). https://doi.org/10.1007/s00705-021-05305-6

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