类泛素化蛋白NEDD8单克隆抗体制备

PREPARATION OF MONOCLONAL ANTIBODIES AGAINST NEDD8 PROTEIN

利用RT-PCR从A549细胞中扩增出类泛素化蛋白编码的nedd8基因,将其克隆至原核表达载体p ET-30a(+)中,测序验证后转化入BL21(DE3)进行IPTG诱导,表达获得NEDD8蛋白,经纯化后,免疫接种6周龄BALB/c小鼠。细胞融合后,用ELISA和Western blot方法筛选单克隆抗体细胞株。结果成功制备获得3株单克隆抗体。免疫特异性鉴定结果表明,所得到的3株单抗适用于Co-IP试验,可以特异性检测NEDD8修饰的Cullin-1底物。通过分段表达鉴定单抗针对的抗原表位可能在29RVEE32氨基酸区域,与泛素化蛋白(Ub)无交叉反应。本研究制备的特异性单抗为进一步鉴定NEDDylation修饰底物提供了良好的抗体工具。

The full-length fragment of nedd8 gene was amplified from A549 cells by RT-PCR and cloned into prokaryotic expression vector pET30 a(+).After sequencing and verification,it was transformed into BL21(DE3)and induced by IPTG.The results showed that the fusion protein was successfully expressed with a molecular mass of about 15 kDa.The expressed NEDD8 was purified and inoculated into 6-week-old BALB/c mice to prepare monoclonal antibodies(mAb).The hybridoma clones were screened and selected by Enzymelinked immunosorbent assay(ELISA)and Western blot analysis.Finally,three hybridoma cells were prepared.The mAbs could be successfully used in the co-immunoprecipitation assay(Co-IP)by checking NEDDylated Cullin-1.The epitope of NEDD8 mAbs was specially located at the 29 RVEE32 motif by identification with the truncated peptides.