title: Preparation and characterization of polyclonal antibody against Kaposi's sarcoma-associated herpesvirus lytic gene encoding RTA.
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.
authors.affiliation: Department of Oncology, Jiangsu Province Official Hospital, 65 Jiangsu Road, Nanjing, 210024, People's Republic of China.
authors.forename: Weifei
authors.initials: W
authors.lastname: Fan
authors.affiliation: Department of Clinical Laboratory, The Affiliated Nanjing First Hospital of Nanjing Medical University, Nanjing, 210029, People's Republic of China.
authors.forename: Qiao
authors.initials: Q
authors.lastname: Tang
authors.affiliation: Department of Microbiology, Nanjing Medical University, Nanjing, Jiangsu, 210029, People's Republic of China.
authors.forename: Chenyou
authors.initials: C
authors.lastname: Shen
authors.affiliation: Department of Microbiology, Nanjing Medical University, Nanjing, Jiangsu, 210029, People's Republic of China.
authors.forename: Di
authors.initials: D
authors.lastname: Qin
authors.affiliation: Department of Microbiology, Nanjing Medical University, Nanjing, Jiangsu, 210029, People's Republic of China.
authors.forename: Chun
authors.initials: C
authors.lastname: Lu
authors.affiliation: Department of Microbiology, Nanjing Medical University, Nanjing, Jiangsu, 210029, People's Republic of China. yanqin@njmu.edu.cn.
authors.forename: Qin
authors.initials: Q
authors.lastname: Yan
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology*; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006566:Herpesviridae Infections / Q000175:diagnosis / Q000821:virology*; D019288:Herpesvirus 8, Human / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D017874:Immediate-Early Proteins / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D011817:Rabbits; D015534:Trans-Activators / Q000032:analysis* / Q000235:genetics / Q000276:immunology
chemical_list: D000914:Antibodies, Viral; D017874:Immediate-Early Proteins; C417604:Rta protein, Human herpesvirus 8; D015534:Trans-Activators
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