CAJ | 학술논문

FANCL是一个范可尼氏贫血新蛋白,它作为泛素E3连接酶催化FANCD2的单一泛素化,在修复DNA损伤、维持染色体稳定的FA途径中起着关键作用.胚胎期FANCL与小鼠原始生殖细胞增殖密切相关,成年睾丸中FANCL与几个生殖细胞特异性蛋白形成一个睾丸特异网络,可能参与影响精子的生成.采用RT-PCR方法从小鼠总RNA中扩增克隆FancL全长cDNA片段,构建表达质粒,在大肠杆菌中表达了6His-FANCL蛋白,用表达蛋白作为抗原免疫新西兰白兔制备了抗FANCL多抗血清.采用镍离子金属螯合柱纯化6His-FANCL蛋白后,通过与活性基团-NHS交联制备了FANCL抗原柱,亲和纯化了FANCL多抗.为了验证抗体活性和特异性,在HEK 293T细胞中瞬时表达了HA-FANCL融合蛋白,分别用HA单抗和纯化多抗进行Western印迹分析,结果表明获得了特异性的FANCL抗体.为了观察FANCL在组织中的表达谱,制备了多种小鼠组织匀浆蛋白,使用纯化的FANCL多抗进行Western印迹分析,在脑、心、肺、肝、脾、肾、睾丸、卵巢、子宫和肌肉组织中都检测到FANCL蛋白的表达,说明FANCL在小鼠组织中是广泛表达的,这与其是DNA修复复合物中的重要成员相一致.
FANCL시일개범가니씨빈혈신단백,타작위범소E3련접매최화FANCD2적단일범소화,재수복DNA손상、유지염색체은정적FA도경중기착관건작용.배태기FANCL여소서원시생식세포증식밀절상관,성년고환중FANCL여궤개생식세포특이성단백형성일개고환특이망락,가능삼여영향정자적생성.채용RT-PCR방법종소서총RNA중확증극륭FancL전장cDNA편단,구건표체질립,재대장간균중표체료6His-FANCL단백,용표체단백작위항원면역신서란백토제비료항FANCL다항혈청.채용얼리자금속오합주순화6His-FANCL단백후,통과여활성기단-NHS교련제비료FANCL항원주,친화순화료FANCL다항.위료험증항체활성화특이성,재HEK 293T세포중순시표체료HA-FANCL융합단백,분별용HA단항화순화다항진행Western인적분석,결과표명획득료특이성적FANCL항체.위료관찰FANCL재조직중적표체보,제비료다충소서조직균장단백,사용순화적FANCL다항진행Western인적분석,재뇌、심、폐、간、비、신、고환、란소、자궁화기육조직중도검측도FANCL단백적표체,설명FANCL재소서조직중시엄범표체적,저여기시DNA수복복합물중적중요성원상일치.
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 Ni2+-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.