Prokaryotic expression and polyclonal antibody production of transactivator Tas for potential application in detection of human foamy virus infection

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-dthiogalactoside. 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.

Prototype foamy virus elicits complete autophagy involving the ER stress-related UPR pathway

“…Anti-phospho-JNK/JNK, anti-phospho-ERK1/2pY204, anti-phospho-IRE1, anti-phospho-ATF6 and anti-β-actin polyclonal Abs were purchased from Abcam. Antibody against PFV Gag was kindly provided by Professor Li Zhi, and anti-Tas was produced by immunizing mouse with prokaryotic expressed Tas and purified according to standard procedures [43]. HRP-conjugated goat anti-mouse or HRP-conjugated goat anti-rabbit secondary antibodies were from PM BIOPRIMACY.…”

ZNF219, a novel transcriptional repressor, inhibits transcription of the prototype foamy virus by interacting with the viral LTR promoter

“…Antibody against Tas protein of PFV was stored in our laboratory. Anti-Tas was produced by immunizing mouse with prokaryotic expressed Tas and purified according to standard procedures ( Qiu et al., 2012 ). HRP-conjugated goat anti-rabbit or goat anti-mouse secondary antibodies were purchased from Proteintech (Wuhan, China).…”