Generation and characterization of rabbit polyclonal antibodies against Vasohibin-2 for determination of its intracellular localization
Generation and characterization of rabbit polyclonal antibodies against Vasohibin-2 for determination of its intracellular localization
复制标题
兔抗 Vasohibin-2 多克隆抗体的生成和表征，用于确定其细胞内定位
DOI：
10.3892/ijo.2013.1919
复制
发表时间：
2013-07-01
影响因子：
5.2
通讯作者：
Gao, Wentao
中科院分区：
文献类型：
--
作者：
Sun, Jie;Tu, Min;Gao, Wentao
Vasohibin-2是近年来发现的一种重要的促血管生成因子，其变异体的细胞内定位对于阐明其作用的下游机制具有重要意义。目前还没有报道可用于细胞内定位的抗体。本研究的目的是产生和表征针对Vasohibin-2的多克隆抗体，并确定Vasohibin-2的细胞内定位。本研究合成了两个多肽，并定制了一个原核表达的Vasohibin-2重组蛋白。分别用该多肽混合物和原核重组蛋白免疫新西兰大白兔。用ELISA、免疫印迹（WB）、免疫荧光（IF）、免疫组化（MC）和免疫沉淀（IP）对纯化的抗血清进行鉴定。为了确定细胞内定位，分离人肝癌细胞系HepG 2的胞质和核蛋白，用于通过蛋白质印迹法检测Vasohibin-2。将编码311和355个氨基酸残基的Vasohibin-2 cDNA克隆到Lv-CMV-EGFP载体中，所述Vasohibin-2 cDNA在C-末端分别与DDK/V5标签融合或不与DDK/V5标签融合。慢病毒被成功包装。建立了Vasohibin-2过表达的HepG 2-VASH 2（355个氨基酸残基）和HepG 2-VASH 2-V5（311个氨基酸残基，在c-末端与V5标签融合）人肝癌细胞系。将大约1- 2x 10（6）个HepG 2、HepG 2-VASH 2和HepG 2-VASH 2-V5细胞皮下注射到BALB/c裸鼠的侧腹。收获异种移植肿瘤用于免疫组织化学。用含有Vasohibin-2 cDNA（编码311/355个氨基酸残基，在C-末端具有DDK标签）的Lv-CMV-EGFP载体瞬时转染HepG 2细胞，随后进行抗DDK免疫荧光。所获得的抗体能够成功地用于Western印迹、IF、IHC和IP检测人VASH 2。IF、IHC和WB（胞质/核蛋白分离后）的结果显示VASH 2蛋白的细胞内定位完全不同。VASH 2（含355个氨基酸残基）定位于细胞质，而VASH 2（含311个氨基酸残基）定位于细胞核。发现前者是相对低丰度的蛋白质。成功制备了3株兔抗人Vasohibin-2多克隆抗体，可用于免疫印迹、免疫荧光、免疫沉淀和免疫组化。这些抗体将为进一步研究Vasohibin-2提供方便的工具。这是第一个研究报告的差异在细胞内定位的VASH 2蛋白，因此，一个新的研究方向的研究VASH 2。
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 poly peptides 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 (MC) 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 1-2x10(6) 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.