Laboratory of General Surgery, The First Affiliated Hospital of Nanjing Medical University, Nanjing, P.R. China.
Int J Oncol. 2013 Jul;43(1):255-61. doi: 10.3892/ijo.2013.1919. Epub 2013 Apr 24.
Vasohibin-2 was recently identified as an important pro-angiogenesis factor in solid tumor and intracellular localization of its variants is important for elucidating the downstream mechanism(s) of its effects. Currently there are no reported antibodies affordable for intracellular localization. The aim of this study was to generate and characterize polyclonal antibodies against Vasohibin-2 and to determine the intracellular localization of Vasohibin-2. In this study, two polypeptides were synthesized and one prokaryotic Vasohibin-2 recombinant protein was custom-made. New Zealand rabbits were immunized with the polypeptide mixture and prokaryotic recombinant protein, respectively. The purified antibodies from the antiserum were validated by ELISA, western blotting (WB), immunofluorescence (IF), immunohistochemistry (IHC) and immunoprecipitation (IP). In order to determine intracellular localization, the cytoplasmic and nuclear proteins of the human liver cancer cell line HepG2 were isolated for the detection of Vasohibin-2 by western blotting. Vasohibin-2 cDNA, coding for 311 and 355 amino acid residues, fused with or without a DDK/V5 tag at the c-terminus, respectively, was cloned into the Lv-CMV-EGFP vector. Lentiviruses were successfully packaged. Vasohibin-2-overexpressing HepG2-VASH2 (355 amino acid residues) and HepG2-VASH2-V5 (311 amino acid residues fused with V5 tag at the c-terminus) human liver cancer cell lines were established. Approximately 1-2x106 HepG2, HepG2-VASH2 and HepG2-VASH2-V5 cells were injected subcutaneously into the flanks of BALB/c nude mice. Xenograft tumors were harvested for immunohistochemistry. HepG2 cells were transiently transfected with the Lv-CMV-EGFP vectors containing Vasohibin-2 cDNA (coding for 311/355 amino acid residues with a DDK tag at the c-terminal), followed by anti-DDK immunofluorescence. The antibodies obtained were able to detect human VASH2 successfully as applied in western blotting, IF, IHC and IP. Results from IF, IHC and WB (post cytoplasmic/nuclear protein isolation) showed a quite different intracellular localization of VASH2 protein. The VASH2 (with 355 amino acid residues) was located in the cytoplasm while VASH2 (with 311 amino acid residues) was located in the nucleus. The former was found to be a relatively low abundance protein. We successfully generated three rabbit anti-human Vasohibin-2 polyclonal antibodies which can be used for western blotting, IF, IP and IHC. These antibodies will provide a convenient tool for further studies on Vasohibin-2. This is the first study to report differences in the intracellular localization of the VASH2 protein and, hence, a new research direction on the study of VASH2.
血管生成素-2（Vasohibin-2）最近被确定为实体瘤中重要的促血管生成因子，其变体的细胞内定位对于阐明其作用的下游机制非常重要。目前，还没有报道可用于细胞内定位的抗体。本研究旨在生成和鉴定针对 Vasohibin-2 的多克隆抗体，并确定 Vasohibin-2 的细胞内定位。在这项研究中，合成了两个多肽，并定制了一个原核 Vasohibin-2 重组蛋白。新西兰兔分别用多肽混合物和原核重组蛋白免疫。从抗血清中纯化的抗体通过 ELISA、western blot（WB）、免疫荧光（IF）、免疫组织化学（IHC）和免疫沉淀（IP）进行验证。为了确定细胞内定位，从人肝癌细胞系 HepG2 中分离细胞质和核蛋白，通过 WB 检测 Vasohibin-2。Vasohibin-2 cDNA，编码 311 和 355 个氨基酸残基，分别在 C 末端融合或不融合 DDK/V5 标签，克隆到 Lv-CMV-EGFP 载体中。成功包装了慢病毒。建立了过表达 Vasohibin-2 的 HepG2-VASH2（355 个氨基酸残基）和 HepG2-VASH2-V5（311 个氨基酸残基，在 C 末端融合了 V5 标签）人肝癌细胞系。大约 1-2x106 HepG2、HepG2-VASH2 和 HepG2-VASH2-V5 细胞被皮下注射到 BALB/c 裸鼠的侧腹。异种移植物肿瘤被收获用于免疫组织化学。用含有 Vasohibin-2 cDNA（C 末端带有 DDK 标签，编码 311/355 个氨基酸）的 Lv-CMV-EGFP 载体瞬时转染 HepG2 细胞，然后进行抗 DDK 免疫荧光。获得的抗体能够成功地在 WB、IF、IHC 和 IP 中检测到人 VASH2。IF、IHC 和 WB（细胞质/核蛋白分离后）的结果显示 VASH2 蛋白的细胞内定位非常不同。VASH2（具有 355 个氨基酸残基）位于细胞质中，而 VASH2（具有 311 个氨基酸残基）位于细胞核中。前者被发现是一种相对低丰度的蛋白质。我们成功地生成了三种兔抗人 Vasohibin-2 多克隆抗体，可用于 WB、IF、IP 和 IHC。这些抗体将为进一步研究 Vasohibin-2 提供便利的工具。这是首次报道 VASH2 蛋白细胞内定位差异的研究，也是研究 VASH2 的一个新方向。
