Zhao Qing-Guo, Zhou Yu, Zhu Heng-Qi, Lu Bai-Song, Huang Pei-Tang
Beijing Institute of Biotechnology, Beijing 100071, China.
Yi Chuan Xue Bao. 2006 Jan;33(1):49-55. doi: 10.1016/S0379-4172(06)60008-5.
Fanconi anemia complementation group L (FANCL) is a novel Fanconi anemia protein, which mono-ubiquitinates FANCD2 as a ubiquitin E3 ligase, and plays a crucial role in DNA damage repair and chromosome stability maintenance. FANCL is involved in the proliferation of primordial germ cells (PGC) in early embryonic stages, and may play a role in the development of germ cells by forming a novel testis-specific network with testis-specific proteins in the adult testis. FancL cDNA sequence was cloned by RT-PCR from mouse testis total RNA, and expressed in E. coli BL21(DE3). Rabbit FANCL polyclonal antiserum was generated using the recombinant protein as the antigen. To prepare an antigen column for affinity purification of FANCL-specific antibody, recombinant His-tagged FANCL was purified by Ni(2+)-charged HiTrap Chelating HP column and coupled to an NHS-activated HiTrap column. To confirm the activity and specificity of the FANCL antibody, we constructed plasmid pCMV-HA/FANCL to transfect HEK 293T cells. Transiently expressed HA-FANCL fusion protein was analyzed by immunoblotting with both the FANCL antibody and HA monoclonal antibody. The antibody was used in Western blotting to check the expression of FANCL protein in mouse tissues. We found wide expression of FANCL in brain, muscle, heart, lung, liver, spleen, kidney, testis, ovary and uterus, indicating the functional importance of this novel protein.
范可尼贫血互补组L（FANCL）是一种新型的范可尼贫血蛋白，作为泛素E3连接酶对FANCD2进行单泛素化，在DNA损伤修复和维持染色体稳定性中起关键作用。FANCL参与早期胚胎阶段原始生殖细胞（PGC）的增殖，并且在成年睾丸中可能通过与睾丸特异性蛋白形成新型的睾丸特异性网络而在生殖细胞发育中发挥作用。通过RT-PCR从小鼠睾丸总RNA中克隆FancL cDNA序列，并在大肠杆菌BL21(DE3)中表达。以重组蛋白为抗原制备兔FANCL多克隆抗血清。为制备用于亲和纯化FANCL特异性抗体的抗原柱，用镍离子（Ni(2+)）负载的HiTrap Chelating HP柱纯化重组His标签的FANCL，并将其偶联到NHS活化的HiTrap柱上。为了确认FANCL抗体的活性和特异性，我们构建了质粒pCMV-HA/FANCL转染HEK 293T细胞。用FANCL抗体和HA单克隆抗体通过免疫印迹分析瞬时表达的HA-FANCL融合蛋白。该抗体用于蛋白质印迹法检测小鼠组织中FANCL蛋白的表达。我们发现FANCL在脑、肌肉、心脏、肺、肝脏、脾脏、肾脏、睾丸、卵巢和子宫中广泛表达，表明这种新型蛋白具有重要的功能。
