小鼠附睾分泌的防御素Defb48的原核表达及纯化

Prokaryotic Expression and Purification of Mouse Epididymial Specific β-Defensin 48

目的:获取小鼠附睾分泌的防御素Defb48的基因并原核表达、纯化,为研究其功能奠定基础。方法:提取小鼠附睾组织的总RNA,用RT-PCR技术扩增出不含信号肽的Defb48编码序列,将2段Defb48编码序列串联克隆至原核表达载体pET28(a)中,经酶切和测序鉴定正确的重组质粒转化大肠杆菌Rosetta(DE3),IPTG诱导表达重组蛋白;用镍柱进行重组蛋白的纯化,然后进行SDS-PAGE和Western印迹分析鉴定。结果:获得实验所需小鼠附睾分泌的防御素Defb48的基因序列,测序结果与已发表的基因序列一致;在大肠杆菌Rosetta(DE3)中表达了His-Defb48融合蛋白,经Western印迹分析,在相对分子质量18 000处有特异的蛋白条带,镍柱纯化后获得纯度较好的融合蛋白。结论:克隆了小鼠附睾分泌的防御素Defb48基因,并在大肠杆菌Rosetta(DE3)中表达纯化出融合蛋白,为制备其多克隆抗体,进而进一步研究Defb48的功能奠定了基础。

Objective： To clone mouse epididymial specific β-defensin 48（Defb48） cDNA fragment,and to ex-press fusion protein His-Defb48 in E.coli Rosetta（DE3） and purify it.Methods： Two cDNA fragments of mouse epididymial specific Defb48,with the difference of presence of stop codon,were amplified from mouse epididymal RNA by RT-PCR respectively,and they were ligated into the vector pET-28（a） in turn.The resulting expression plasmid consists of the vector pET-28（a） and the tandem cDNA fragment without the signal peptide coding sequence.The recombinant plasmid was verified by sequencing and then transformed into Rosetta（DE3）.The recombinant protein was expressed under the induction of IPTG,and then further purified by using Ni-NTA and analyzed by SDS-PAGE and Western blotting.Results： The targeted mouse epididymial specific Defb48 gene was cloned,which was consistent with those early reported.The recombinant protein His-Defb48 about 18 kD was expressed in E.coli Rosetta（DE3）,which was verified by Western blotting.After purified with Ni-NTA,high quality recombinant protein was obtained.Conclusion： Mouse epididymial specific β-defensin 48 had been successfully cloned and efficiently expressed in E.coli Rosetta（DE3）,and the fusion protein had been highly purified.Our work paved ways for antibody preparation and functional study of Defb48 in sperm maturation.