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Generation and Application of Polyclonal Antibody Against Replication and Transcription Activator of Kaposi’s Sarcoma-Associated Herpesvirus

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Abstract

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi’s sarcoma, the most common neoplasm in untreated HIV-1-infected individuals, and several B cell disorders. KSHV infection goes through lytic and latent phases, and the switch from latency to lytic replication is governed by viral replication and transcription activator (RTA). RTA consists of 691 amino acids, containing an N-terminal DNA-binding and a C-terminal activation domain. In the present study, polyclonal antibody against RTA was generated and evaluated. The C-terminal region of RTA (E482~D691) was expressed in Escherichia coli, purified by affinity chromatography, and utilized to raise polyclonal antibody in BALB/c mice. High-affinity antisera were obtained, which successfully detected the antigen at a dilution of 1:13,500 for ELISA and 1:20,000 for Western blot analysis. The antibody can specifically recognize full-length RTA expressed in both E. coli and mammalian cells. Furthermore, endogenous RTA can be detected with the antibody in TPA-induced BCBL-1 cells under various conditions. These results suggested that the antibody is valuable for the investigation of biochemical properties and biological functions of RTA.

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Acknowledgements

This work was supported by grants from the National 973 Program of China (No. 2005CB522903) and the National Program of Major Scientific Projects (2008ZX1001-1010) to JW, the National Natural Science Foundation of China (No. 30570083 and 30870129 to JW, and No. 30500024 to YW), and NIH grant (RO1 TW007294) to JW. We thank Prof. Charles Wood (University of Nebraska-Lincoln) for the pCMV-Tag50 expression plasmid, Lingling Zu and Yuangen Wang for technical assistance.

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Authors and Affiliations

  1. College of Basic Medical Science, Tianjin Medical University, 22 Qixiangtai Road, Tianjin, 300070, People’s Republic of China

    Yu Qin & Xiaomian Li

  2. TEDA School of Biological Sciences and Biotechnology, Nankai University, Key Laboratory of Molecular Microbiology and Technology of the Ministry of Education, and Tianjin Key Laboratory of Microbial Functional Genomics, 23 Hongda Street, TEDA, Tianjin, 300457, People’s Republic of China

    Zhao Liu, Tianzheng Zhang, Ying Wang & Jinzhong Wang

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  1. Yu Qin
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  2. Zhao Liu
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Correspondence to Xiaomian Li or Jinzhong Wang.

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Qin, Y., Liu, Z., Zhang, T. et al. Generation and Application of Polyclonal Antibody Against Replication and Transcription Activator of Kaposi’s Sarcoma-Associated Herpesvirus. Appl Biochem Biotechnol 160, 1217–1226 (2010). https://doi.org/10.1007/s12010-009-8604-1

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