人Eppin蛋白在大肠杆菌中的可溶性表达

Soluble expression of human Eppin protein in Escherichia coli

目的构建人类附睾蛋白酶抑制剂Eppin蛋白原核表达质粒,诱导、表达与纯化后获得到可溶重组蛋白,并检测其免疫学活性。方法将目的基因克隆到表达载体pMALc,转化大肠杆菌表达菌株Rossetta2-OrigamiB(DE3),用异丙基-β-D硫代半乳糖苷(IPTG)诱导表达。通过Ni-NTA亲和层析柱及Superdex 200分子筛层析进行纯化后,使用Thrombin酶切,并对目标蛋白进行质谱及Western印迹鉴定。结果成功构建pMALc-Eppin重组质粒,诱导后在上清中检测到Mr57 000大小的可溶MBP-Eppin融合蛋白。目标蛋白纯化与酶切后仍可溶,SDS-PAGE分析显示重组Eppin蛋白相对分子质量约15 000。肽指纹图谱分析证实目标蛋白的序列与人Eppin蛋白吻合,Western印迹显示其具有抗原性。结论经过优化,Eppin蛋白可在原核系统中以可溶形式表达,为以后研究该蛋白的生物活性、避孕免疫机制及制备避孕疫苗奠定坚实的基础。

Human Epididymal protease inhibitor （Eppin） may be a potential candidate as a male contraceptive vaccine. In this study, we aimed to produce soluble Eppin in bacterial expression system, establishing a solid basis for vaccine design. We subcloned the Eppin gene into expression vector pMALc, and then transformed into Escherichia coli host strain Rossetta2-OrigamiB （DE3） for expression trial. The recombinant MBP-Eppin fusion protein was purified using affinity and size-exclusion chromatography, and later digested with thrombin. SDS-PAGE showed that the molecular weight of the fusion protein is 57 KD; Eppin was still soluble after MBP-tag removal; peptide fingerprinting spectrometry demonstrated that the expressed product was the authentic Eppin protein and Western blot confirmed its antigenic activity. Our experimental results reported here definitely benefit functional studies on the biological activity of Eppin and contraceptive vaccine design.