人NEDD8 原核表达及兔高免血清制备

摘要: 本研究根据基因比对，将合成的nedd8 基因( HN8) 酶切克隆入pCold-TF 原核表达载体中，命名为pCold-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 kDa。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。

Prokaryotic Expression and Antibody Preparation of Human NEDD8

Abstract: In the study，human nedd8 gene ( HN8) was synthesized and subcloned into pMD19-T simple vector．After sequencing，HN8 gene was transferred into the prokaryotic expression vector，pCold-TF． The final construct is designated as pCold-HN8． The plasmid was transformed into E． coli BL21 ( DE3) competent cells． Induced with Isopropyl β-D-1-thiogalactopyranoside ( IPTG) ，the bacteria were grown in LB broth at 16℃ for 24 hours． The products were sonicated and then purified by His-bind purification kit． The purified protein was used to immunize three rabbits to prepare polyclonal antibody． The results showed that the fused protein were soluble expressed with a size of 64 kDa by SDS-PAGE analysis． Both indirect immunofluorescence and Western blotting assay，it confirmed the polyclonal antibody could recognize the HN8 protein． This antibody provides a useful tool for a further study of the neddylation modification by human NEDD8．