College of Basic Medical Science, Tianjin Medical University, 22 Qixiangtai Road, Tianjin 300070, People's Republic of China.
Appl Biochem Biotechnol. 2010 Feb;160(4):1217-26. doi: 10.1007/s12010-009-8604-1. Epub 2009 Mar 31.
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 approximately 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.
卡波氏肉瘤相关疱疹病毒（KSHV）是卡波氏肉瘤的病原体，卡波氏肉瘤是未经治疗的 HIV-1 感染者中最常见的肿瘤，也是几种 B 细胞疾病的病原体。KSHV 感染经历裂解和潜伏阶段，从潜伏到裂解复制的转换由病毒复制和转录激活剂（RTA）控制。RTA 由 691 个氨基酸组成，包含一个 N 端 DNA 结合域和一个 C 端激活域。在本研究中，生成并评估了针对 RTA 的多克隆抗体。RTA 的 C 端区域（E482 到 D691）在大肠杆菌中表达，通过亲和层析纯化，并用于在 BALB/c 小鼠中产生多克隆抗体。获得了高亲和力的抗血清，该抗血清在 ELISA 中的稀释度为 1:13500，在 Western blot 分析中的稀释度为 1:20000 时，均可成功检测到抗原。该抗体可特异性识别在大肠杆菌和哺乳动物细胞中表达的全长 RTA。此外，在 TPA 诱导的 BCBL-1 细胞中，该抗体可在各种条件下检测到内源性 RTA。这些结果表明，该抗体对于研究 RTA 的生化特性和生物学功能具有重要价值。
