GST-Gankyrin融合蛋白的原核表达及抗体制备

Expression of Gankyrin-GST fusion protein in E.coli and preparation of its specific polyclonal antibody

目的:为进一步研究在肝癌上过表达的癌相关基因Gankyrin的功能,进行GSTGankyrin融合蛋白表达载体的构建、原核表达、及抗体制备.方法:采用逆转录PCR(RTPCR)法从人肝癌细胞系hepG2中扩增GankyrincDNA编码区,并将其重组于谷胱甘肽硫转移酶(GST)融合蛋白表达质粒pGEX4T2中,经酶切、序列鉴定分析后,用该重组质粒转化大肠杆菌DH5α,经IPTG诱导获得表达,并将Gankyrin蛋白免疫家兔,经过Westernblot检测抗Gankyrin抗体的产生.结果:成功构建了Gankyrin原核表达载体,并在大肠杆菌DH5α中获得表达,Westernblot检测证实获得了抗Gankyrin抗体.结论:成功表达了Gankyrin蛋白并制备了其特异性抗体.

AIM: To express Gankyrin in E. coli and to prepare antibody against it. METHODS: The core fragment of Gankyrin was cloned into plasmid pGEX-4T-2 containing glutathione s-transferase (GST) fusion protein gene. Following the restriction enzyme digestion analysis and sequencing, pGEX-4T-Gankyrin was transformed into E. coli DH-5α. GST-Gankyrin fusion protein was expressed under IPTG induction and the Gankyrin protein was used to immunize New Zealand rabbits. The specific antibody against Gankyrin was identified by ELISA and Western blot. RESULTS: GST-Gankyrin fusion protein was expressed in E.coli and its specific polyclonal antibody was obtained. CONCLUSION: The successful expression of GST-Gankyrin fusion protein in E.coli and the preparation of its specific polyclonal antibody will facilitate the study of the biological functions of Gankyrin.