摘要
目的原核表达轴突再生抑制蛋白Nogo-66,并对其进行鉴定。方法采用聚合酶链反应方法从含Nogo-66编码序列的质粒DNA中扩增出Nogo-66基因开放阅读框,构建原核表达载体,转化大肠杆菌,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,裂解细菌抽提蛋白,表达产物进行聚丙烯酰胺凝胶电泳(SDS-PAGE)、Western免疫印迹鉴定。结果测序鉴定Nogo-66开放阅读框成功插入pET-46-EK/LIC质粒;重组质粒在大肠杆菌中成功表达大鼠Nogo-66蛋白,SDS-PAGE测定其相对分子质量(Mr)约7000,WesternBlot结果证实表达产物融合有聚组氨酸标签。结论应用原核表达系统成功表达了Nogo-66蛋白,为下一步制备Nogo-66蛋白疫苗及疫苗功能研究打下了基础。
Objective To express and identify rat Nogo-66 peptide in E.coli . Methods The open reading frame of rat Nogo-66 was amplified by PCR using DNA vaccine as plate, then it was inserted into PET-46-EK/LIC vector. Recombinant plasmid was identified by colony PCR and transformed into BL21 (DE3) pLysS, and then induced by IPTG in 37 ℃ incubation. The expression product was analyzed by SDS-PAGE and Western blotting. Results The recombinant plasmid was constructed successfully, and then verified by sequencing. The expressed proteins in E.coli mainly exist in supernatant. The Nogo-66 fusion protein of M _r 7 000 was characterized by 6×his tag. Conclusion Rat Nogo-66 protein is successfully expressed in E.coli system, which paves a way for preparation of vaccine for Nogo-66 and for related function research.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2005年第3期256-258,共3页
Immunological Journal
基金
重庆市科委自然科学基金资助项目(CSTC
2004BB5032)
