摘要
目的原核表达并纯化高尔基体糖蛋白-73(GolgiProtein73,GP73)。方法以人肝癌细胞系HepG2总RNA为模板,经RT-PCR扩增GP73基因,克隆至原核表达载体pET-21a(+)-TRX中,转化大肠杆菌BL21(DE3),IPTG诱导表达。His-tag磁珠纯化重组蛋白GP73,SDS-PAGE鉴定。结果克隆目的基因的序列正确,未发生碱基突变;重组表达质粒pET21a(+)-TRX-GP73经双酶切鉴定构建正确;表达的重组蛋白相对分子量为80 kD,与预期相符。结论本实验成功在大肠杆菌BL21(DE3)中表达并纯化了GP73重组蛋白,为后续研究奠定了基础。
Objective To prokaryotically express and purify Golgi protein 73（GP73）. Methods We amplified GP73 gene from human hepatoma cell HepG2 by RT-PCR and cloned it into the prokaryotic expression vector pET21a（＋）-TRX, then transformed it into Escherichia coli BL21 （DE3）. The expression of GP73 protein was induced by isopropylβ-D-1-thiogalactopyranoside （IPTG） and purified by His-tag magnetic beads. SDS-PAGE was used to identify GP73. Results Gene GP73 was cloned from HepG2 without any mutation. The recombinant plasmid pET21a（＋）-TRX-GP73 was con-structed correctly and the recombinant protein GP73 was expressed successfully with an expected molecular weight of 80KD. Conclusion We successfully expressed and purified the recombinant protein GP73 in Escherichia coli BL21 （DE3） and layed a foundation for following study.
出处
《肿瘤药学》
CAS
2014年第3期172-175,共4页
Anti-Tumor Pharmacy
基金
横向课题(11430101001261)
