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目的构建小鼠周脂素(prilipin,Plin)原核表达载体,表达周脂素蛋白并进行初步纯化,为研究周脂素的功能奠定基础。方法通过酶切从pMD18-Plin重组载体上酶切下周脂素目的基因,定向插入原核表达载体pET-32a(+)中,转化大肠杆菌BL21(DE3),IPTG诱导表达。表达产物经SDS-PAGE及Western blot鉴定后,进行纯化。结果构建的pET-32a-Plin载体能够表达周脂素重组蛋白,表达量约占菌体总蛋白的50%,经初步纯化后,纯度达95%以上。结论成功构建了周脂素原核表达载体,并在原核系统中表达了重组蛋白,纯化的蛋白纯度较高,可用于后续试验。
Objective To construct prokaryotic expression vector of mouse prilipin (Plin), express apolipoprotein and preliminarily purify it, which lays the foundation for the study on the function of perilipin. Methods The target gene was isolated from pMD18-Plin recombinant vector by restriction enzyme digestion and inserted into prokaryotic expression vector pET-32a (+). The recombinant plasmid was transformed into E. coli BL21 (DE3) and induced by IPTG. The expressed product was identified by SDS-PAGE and Western blot, then purified. Results The constructed pET-32a-Plin vector could express perifostine recombinant protein, which accounted for about 50% of the total bacterial proteins. After purification, the purity reached 95%. Conclusion The prokaryotic expression vector for periperin was successfully constructed and the recombinant protein was expressed in prokaryotic system. The purity of the purified protein was high and could be used in subsequent experiments.