小鼠附睾分泌蛋白Spink12的原核表达与纯化

目的原核表达带有His标签的小鼠附睾蛋白Spink12,对表达产物进行鉴定和纯化,以研究Spink12的免疫避孕效果。方法提取小鼠附睾组织总RNA,RT-PCR扩增不含信号肽的Spink12蛋白编码序列,以NdeⅠ和EcoRⅠ酶切后,将其连入原核表达载体pET28(a);经酶切和测序鉴定正确的重组质粒pET28(a)-Spink12,转化大肠埃希菌BL21感受态细胞。融合蛋白经IPTG诱导表达,产物经SDS-PAGE电泳和Western blotting免疫印迹鉴定。用Ni-NTA在非变性条件下利用不同的咪唑梯度浓度纯化融合蛋白6×His-Spink12。结果酶切和测序结果显示成功构建出原核表达载体pET28(a)-Spink12。重组质粒转化大肠埃希菌BL21并经IPTG诱导,SDS-PAGE电泳显示表达出约9kD的融合蛋白6×His-Spink12,与理论分子量相符。融合蛋白的表达量占菌体蛋白总量的23.5%。用抗His单克隆抗体进行Western blotting免疫印迹鉴定,检测到同样大小的条带。经纯化获得较高纯度的融合蛋白6×His-Spink12,纯度达91.1%。结论成功表达并纯化了融合蛋白6×...

Prokaryotic expression and purification of Spink12, a mouse epididymal secretary protein

Objective To study the effect of immunocontraception of Spink12, the fusion protein 6×His-Spink12 was induced to express in E: coli BL21, and purified.Methods The total mouse epididymal RNA was extracted, and the Spink 12 protein coding exon without signal peptide sequence was amplified by RT-PCR, digested by restrictive enzyme Nde Ⅰ and EcoR Ⅰ, and then cloned into the prokaryofic expression vector pET28 (a) which contained the 6×His tag in N terminal .The recombinant plasmid pET28a-Spink12, after being confirmed by enzyme digestion and sequencing, was transformed into the competent cells of E.coli BL21.The fusion protein 6×His-Spink12 was expressed by IPTG induction and analyzed by SDS-PAGE and Western blotting.The products were purified by Ni-NTA under native condition with different imidazole gradients and characterized by Western blotting with anti-His monoclonal antibody.Results The results of digestion and sequencing indicated that the recombinant prokaryotic expression vector pET28a-Spink12 was successfully constructed.After E.coli BL21 was transformed with pET28a-Spink12 and induced with IPTG, the recombinant target protein of about 9 kDa was obtained by SDS-PAGE analysis, which was coincident with the theoretical value.The fusion protein was of 23.5% of the total E.coli cell proteins.The Western blotting analysis with anti- His monoclonal antibody showed that the band was fusion protein 6 × His-Spink12.After purified with Ni-NTA, a higher quality of fusion protein 6×His-Spink12 was obtained in a purity of 91.1%.Conclusions The fusion protein 6×His-Spink12 has been successfully expressed in E.coli BL21 and purified.It may provide a base for the study of the effect of Spink12 on immunocontraception.