本研究根据基因比对,将合成的nedd8基因(HN8)酶切克隆入p Cold-TF原核表达载体中,命名为p Cold-HN8,转化到感受态宿主菌BL21(DE3)中,加入不同浓度异丙基硫代半乳糖苷(IPTG)于16℃中诱导表达24 h,超声波裂解,通过His-bind纯化试剂盒纯化,分别进行SDS-PAGE检测,结果 HN8蛋白获得可溶性表达,融合蛋白大小为64 k Da。用纯化的蛋白免疫新西兰兔,制备多克隆抗体,经Western Blot和间接免疫荧光试验证实,兔抗HN8蛋白的血清能够特异性识别HN8蛋白。这为进一步研究HN8蛋白在Neddylation通路中的修饰作用奠定了基础。 In this study, we cloned the synthesized nedd8 gene (HN8) into the p Cold-TF prokaryotic expression vector according to the gene alignment and named it p Cold-HN8 and transformed it into competent host strain BL21 (DE3) Isopropylthiogalactopyranoside (IPTG) was induced at 16 ℃ for 24 h and was lysed by ultrasonic wave and purified by His-bind purification kit. SDS-PAGE analysis showed that IPTG protein was soluble and the size of fusion protein was 64 k Da. The purified rabbit polyclonal antibody was used to immunize New Zealand rabbits. Western Blot and indirect immunofluorescence assay confirmed that the rabbit anti - HN8 protein could specifically recognize HN8 protein. This laid the foundation for the further study of the modification of HN8 protein in the Neddylation pathway.