摘要
目的构建重组高尔基体蛋白的原核表达质粒并在大肠杆菌中诱导表达,为进一步研发肝癌诊断试剂奠定基础。方法通过RT-PCR获得GP73目的片段,将其克隆入表达载体p ET-28a,经酶切及测序鉴定后,在BL21菌株中经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,用Western Blot鉴定。结果扩增的片段大小与预期一致;p ET-28a-GP73重组质粒通过酶切鉴定与DNA测序证实序列正确;诱导的蛋白用SDS-PAGE电泳和Western Blot检测,与预期相符可见一特异性条带。结论成功获得GP73目的基因并构建了p ET-28aGP73原核表达载体,实现了该融合蛋白的可溶性表达。
Objective To construct a recombinant prokaryotic expression plasmid containg Golgi protein 73 （ GP73 ） and express the protein in E. coli, and lays a foundation for an diagnostic method of hepatocellular carcinoma. Methods Amplify the target fragment encoding GP73 by RT - PCR and clone into prokaryotic expression vector pET - 28a. GP73 recombinant plasmid was induced by IPTG in E. coli BL21 after identifying by digestion and gene sequencing. The protein was detected by Western Blot. Results The length of the fragment amplified was same with expectation; pET- 28a - GP73 recombinant plasmid was confirmed by digestion and DNA sequencing;the rembinant GP73 protein was identified by SDS -PAGE and Western Blot, a special band was observed. Conclusion The prokaryotic expression plasmid was successfully constructed and expressed.
出处
《医药论坛杂志》
2015年第9期1-2,5,共3页
Journal of Medical Forum
基金
国家自然科学基金(No.81401970)
北京市佑安医院肝病艾滋病基金(No.BJYAH2013-1-005)
关键词
GP73
克隆
原核表达
肝癌
Golgi protein 73
Cloning
Prokaryotic expression
Hepatocellular carcinoma
