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Original Article



Expression of polyprotein and 3D polymerase protein in Sf9 cells and immunogenicity against enterovirus A71B5 (Thailand strain)

Nipatha Issaro, Aphisak Kongkaew, Akanitt Jittmittraphap, Pornsawan Leaungwutiwong, Wutigri Nimlamool, Mingkwan Na Takuathung.




Abstract
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Enterovirus 71 B5 subgenotype (EV-A71B5) is a primary human pathogen of hand, foot, and mouth disease. In this study, we aimed to prepare a novel recombinant protein polyprotein (P1) and 3D polymerase (3Dp°l) protein of EV-A71B5 and determine mice immunogenicity and neutralization activity against EV-A71 of the B5 subgenotype commonly found in Thailand. Using a dual promoter system and investigating its expression in Sf9 cells, we constructed a novel recombinant protein containing P1 and 3Dp°l protein. The purified P1-3Dp°l was observed by western blotting and transmission electron microscopy (TEM) to determine the particle size. Furthermore, we determined the immunogenicity and neutralization activity against EV-A71 of the B5 subgenotype using concatenation of Bagg and Albino (BALB/c) mice. The results revealed that P1-3Dp°l was expressed in Sf9 cells. We used TEM to visualize the particle size of P1-3Dp°l to be approximately 33 nm. P1-3Dp°l had the potential to elicit the production of immunoglobulin G and immunoglobulin M antibodies and the T helper type 1-dominant cytokine interferon-γ after immunizing BALB/c mice and inducing neutralization antibodies against EV-A71B5. Our results demonstrated that Sf9 cells successfully produced P1-3Dp°l, which can leverage immune efficacy in BALB/c mice and be used to develop vaccines against the EV-A71B5 strain prevalent in Thailand.

Key words: Enterovirus 71 subgenotype B5, polyprotein, 3D polymerase protein, transmission electron microscopy, T helper type 1-dominant cytokine interferon-γ






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