Fan Weifei, Tang Qiao, Shen Chenyou, Qin Di, Lu Chun, Yan Qin
Department of Oncology, Jiangsu Province Official Hospital, 65 Jiangsu Road, Nanjing, 210024, People's Republic of China.
Department of Clinical Laboratory, The Affiliated Nanjing First Hospital of Nanjing Medical University, Nanjing, 210029, People's Republic of China.
Folia Microbiol (Praha). 2015 Nov;60(6):473-81. doi: 10.1007/s12223-015-0387-x. Epub 2015 Apr 2.
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.
复制与转录激活因子（RTA）是卡波西肉瘤相关疱疹病毒（KSHV）编码的一种关键裂解蛋白。为制备抗RTA的兔多克隆抗体，首先合成了KSHV RTA的三种抗原多肽。将RTA片段克隆到p3FlagBsd中构建重组质粒pRTA-Flag。用pRTA-Flag转染293T和EA.hy926细胞以获得RTA-Flag融合蛋白，并用抗Flag抗体进行检测。接下来，用钥孔血蓝蛋白偶联肽免疫新西兰白兔以产生抗RTA的多克隆抗体。进行酶联免疫吸附测定以鉴定多克隆抗体，抗RTA多克隆抗体的效价大于1:11,000。蛋白质印迹和免疫荧光分析表明，制备的抗体与RTA-Flag融合蛋白以及KSHV感染的原发性渗出性淋巴瘤细胞中的天然病毒蛋白发生特异性反应。总之，我们的工作成功构建了重组表达载体pRTA-Flag，并制备了抗RTA的多克隆抗体，这对于研究关键的KSHV裂解基因的生化和生物学功能具有重要价值。
