Abstract
The human foamy virus (HFV) may pose a risk to humans as an infectious agent. The HFV transactivator Tas plays a critical role as a regulatory factor in viral replication, and potently activates gene expression via the 5' LTRs (long terminal repeats) of HFV. However the anti-Tas antibody currently available can rarely be applied in the investigation and diagnosis of HFV because of its high valence. To better understand the possible role of Tas as a potential candidate for the diagnosis and understanding of the infectious mechanism of HFV, the cloning, expression, and purification of Tas are described in this report. Additionally, the preparation of a polyclonal antibody directed against Tas is described. The Tas gene was amplified from wild type HFV (pHSRV13) by PCR, subcloned into the pGEX-4T-1 expression plasmid, and then introduced into Escherichia coli BL21 (DE3) cells. To make the GST-Tas fusion protein, the recombinant expression plasmid pGEX-4T-1-Tas was induced using isopropyl-β-d-thiogalactoside. Optimal levels of GST-Tas were obtained by optimizing the concentration of IPTG and the induction time. After purification, GST-Tas was used to immunize mice, after which a standard protocol was used to acquire antiserum. Western blot analysis indicated that the prepared antiserum reacted specifically to the recombinant protein when expressed in eukaryotic cells, demonstrating good antigenicity of the antiserum. The HFV Tas protein showed low homology when compared to the foamy virus Tas protein in other species, suggesting that the HFV Tas polyclonal antibody may be specific to HFV Tas. The present study sheds light on the mechanism of the transactivator function and immunogenicity of Tas. Information from this study could be used to develop antibody or antigen detection assays for the clinical detection of the HFV pathogen. Key words: HFV, Tas, polyclonal antibody, expression, western blot.
Highlights
The foamy virus belongs to the spumavirus genus of retroviruses, and is endemic in most humans, monkeys, and other mammals
The human foamy virus (HFV) Tas protein showed low homology when compared to the foamy virus Tas protein in other species, suggesting that the HFV Tas polyclonal antibody may be specific to HFV Tas
The Tas gene, encoding the transactivator of spumavirus, which was amplified by PCR from the pHSRV13 plasmid and inserted into the expression vector pGEX-4T-1
Summary
The foamy virus belongs to the spumavirus genus of retroviruses, and is endemic in most humans, monkeys, and other mammals (such as cats and cattle). Human foamy virus (HFV) was isolated in 1971 from a. Wide tissue tropism commonly found in mammals. The virus can infect cells from many vertebrate species and can infect diverse tissue types (Achong et al, 1971). The infection has not been associated with a specific disease, transgenic mice carrying the bel ( called Tas) region of HFV displayed a progressive degenerative disease of the central nervous system and the striated muscle (Bothe et al, 1991), which suggested the pathogenic potential of HFV in humans. Concern about an HFV outbreak is increasing and prevention should be stressed
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