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Preparation and characterization of polyclonal antibody against Kaposi’s sarcoma-associated herpesvirus lytic gene encoding RTA

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Abstract

Replication and transcription activator (RTA) is a critical lytic protein encoded by Kaposi’s sarcoma-associated herpesvirus (KSHV). To prepare rabbit polyclonal antibody against RTA, three antigenic polypeptides of KSHV RTA were initially synthesized. The fragment of RTA was cloned into p3FlagBsd to construct the recombinant plasmid, pRTA-Flag. 293 T and EA.hy926 cells were transfected with pRTA-Flag to obtain RTA-Flag fusion protein, which was detected using anti-Flag antibody. Next, New Zealand white rabbits were immunized with keyhole limpet hemocyanin-conjugated peptides to generate polyclonal antibodies against RTA. Enzyme-linked immunosorbent assays were performed to characterize the polyclonal antibodies, and the titers of the polyclonal antibodies against RTA were greater than 1:11,000. Western blotting and immunofluorescence assay revealed that the prepared antibody reacted specifically with the RTA-Flag fusion protein as well as the native viral protein in KSHV-infected primary effusion lymphoma cells. Collectively, our work successfully constructed the recombinant expression vector, pRTA-Flag, and prepared the polyclonal antibody against RTA, which was valuable for investigating the biochemical and biological functions of the critical KSHV lytic gene.

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Acknowledgments

We are grateful to Dr. Shou-Jiang Gao (University of Southern California) for generously providing plasmid. This work was supported by grants from the Natural Science Youth Foundation of Jiangsu Province (BK20140908) and Science Development Foundation of Nanjing Medical University (2013NJMU007).

Conflict of interest disclosure

The authors declare no competing financial interest.

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Authors and Affiliations

  1. Department of Oncology, Jiangsu Province Official Hospital, 65 Jiangsu Road, Nanjing, 210024, People’s Republic of China

    Weifei Fan

  2. Department of Clinical Laboratory, The Affiliated Nanjing First Hospital of Nanjing Medical University, Nanjing, 210029, People’s Republic of China

    Qiao Tang

  3. Department of Microbiology, Nanjing Medical University, Nanjing, Jiangsu, 210029, People’s Republic of China

    Chenyou Shen, Di Qin, Chun Lu & Qin Yan

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  1. Weifei Fan
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  2. Qiao Tang
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  4. Di Qin
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  5. Chun Lu
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  6. Qin Yan
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Correspondence to Qin Yan.

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Qin Yan holds a PhD, Nanjing Medical University.

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Fan, W., Tang, Q., Shen, C. et al. Preparation and characterization of polyclonal antibody against Kaposi’s sarcoma-associated herpesvirus lytic gene encoding RTA. Folia Microbiol 60, 473–481 (2015). https://doi.org/10.1007/s12223-015-0387-x

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  • DOI: https://doi.org/10.1007/s12223-015-0387-x

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