2H9-L intrabody found in E coli and silkworm pupae can bind to HCV core variants

Wayne Kuznar, for MDLinx | June 27, 2018

The intrabody 2H9-L, which functions as an anti-hepatitis C virus (HCV) inhibitor in human hepatic cells, has the ability to bind to HCV core protein variants. The finding of HCV core-intrabody interfaces may permit a novel strategy to design and generate chemical drugs with antiviral activities, according to Japanese researchers, who published their results in Science Direct.

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Silkworm pupae and HCV

Researchers observed 2H9-L intrabody expression in Escherichia coli and silkworm pupae, and demonstrated its ability to bind to all HCV core protein variants.

HCV core protein plays a key role in viral particle formation and is involved in viral pathogenesis. Intrabodies, which are expressed intracellularly, can block the function of a protein in cells by directly binding to intracellular antigens.

Previously, several single-domain intrabodies against the HCV core protein were generated from the anti-core monoclonal antibodies. One specific intrabody, 2H9-L, was found to function as an inhibitor of both HCV production and the host cell-signaling pathway activated by HCV.

In the current study, the 2H9-L intrabody was expressed in Escherichia coli and silkworm pupae and was purified. Several forms of the viral core protein were also bacterially expressed and purified, and their binding ability to 2H9-L was evaluated.

Enzyme-linked immunosorbent assay (ELISA) confirmed the binding of both intrabodies to all of the HCV core protein variants. Higher absorbance was observed when using each HCV core protein variant than when using bovine serum albumin as a negative control, “which indicates that both intrabodies have an ability to bind HCV core protein variants,” the investigators wrote in Protein Expression and Purification. Binding of both intrabodies to the HCV core protein variant C191 appeared to be weaker than their binding to other variants.

Immunoprecipitation demonstrated that the intrabodies that were purified from both E. coli and silkworm pupae bound to all of the HCV core protein variants. As with ELISA, binding of both intrabodies to C191 was weaker than their binding to the other HCV core protein variants.

“In this study, the intrabody 2H9-L…was expressed in E coli and silkworm pupae and was successfully purified as a soluble form, which has binding activity to the HCV core protein in vitro,” the researchers concluded. “Solubility is advantageous for an intrabody, which functions intracellularly to bind the HCV core protein during HCV amplification.”

Additionally, the authors concluded that, since these purified intrabodies can be used in biochemical analyses of the inhibitory mechanism of HCV propagation and as protein interference reagents, they may provide a potential pathway to developing new types of antiviral treatments.

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