摘要
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。
Abstract
Objective To structure the prokaryotic expression vector of mouse prilipin gene, was expressed in prokaryotic cells and purify the expressed product. Methods By enzyme digestion from pMD18-Plin recombinant enzyme cut the prilipin gene.The target gene was recovered and cloned into expression vector pET-32a(+), and the constructed recombinant plasmid pET-32a-Plin was transformed to E.coli BL21(E3) for expression under induction of IPTG. The expressed product was identified by SDS-PAGE and Western blot, then purified by His label chromatography. Results The prokaryotic expression vector of mouse prilipin gene can express prilipin. The expressed recombinant prilipin, with a relative molecular mass of about 57 kDa, contained about 50% of total somatic protein. It showed good reactogenicity and reached a purity of more than 95% after purification. Conclusion The prokaryotic expression vector of mouse prilipin gene was successfully structured and expressed in prokaryotic cells, and the expressed product reached a high purity, which laid a foundation of study on the function of prilipin.
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维普
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