小鼠精囊自身抗原的原核表达和纯化

Prokaryotic Expression and Purification of Mouse Seminal Vesicle Autoantigen

目的:在原核细胞中表达小鼠精囊自身抗原(SVA),并对表达产物进行鉴定和纯化。方法:提取小鼠附睾组织总RNA,RT-PCR获得SVA的cDNA,设计并合成特异引物序列,进一步扩增出不含信号肽的SVA编码序列,连入原核表达载体pET28a中,经酶切和测序鉴定正确的重组质粒转化大肠杆菌Rosetta(DE3)感受态细胞,IPTG诱导表达,Western印迹分析表达产物His-SVA,采用Ni-NTA纯化融合蛋白His-SVA。结果:原核表达获得融合6个组氨酸的SVA,用抗His单克隆抗体进行Western印迹鉴定,检测到相对分子质量约18×103的目的蛋白,与理论值一致;经Ni-NTA纯化获得较高纯度的His-SVA融合蛋白。结论:获得了在大肠杆菌中表达的小鼠附睾蛋白SVA,为后续研究其对小鼠生殖的影响奠定了基础。

Objective： To prokaryotic express and purify mouse seminal vesicle autoantigen（SVA）. Methods： The gene of mouse SVA was cloned from mouse epididymal RNA by RT-PCR. The cDNA without signal peptide sequence was cloned into pET28a vector. The recombinant plasmid was proved correct by sequencing and enzymes cutting, then was transformed into E.coli Rosetta（DE3）. The fusion protein His-SVA was expressed by IPTG induction, purified by Ni-NTA and characterized by Western blot. Results： The Western blot analysis with anti-His mono-antibody showed that the molecular weight of the fusion protein expressed in E.coli Rosetta（DE3） is 18 kD, which is coincident with theoretical value. After purified with Ni-NTA, high quality fusion protein His-SVA was obtained. Conclusion： The fusion protein His-SVA was expressed in E.coli Rosetta（DE3）. It provided a base for studying the effect of SVA on mouse fertility.