小鼠附睾分泌蛋白Fam12b的原核表达与纯化

Prokaryotic expression and purification of novel protein Fam12 in mouse epididymis

本研究旨在克隆小鼠附睾基因fam12b,构建pET28(a)-fam12b-fam12b原核表达重组质粒,从大肠杆菌Rosetta(DE3)中获得融合蛋白。应用RT-PCR方法从小鼠附睾中扩增出fam12编码序列,串联克隆至原核表达载体pET28(a),转化大肠杆菌Rosetta(DE3)细胞,诱导表达,Ni-NTA纯化融合蛋白。结果显示,获得小鼠附睾基因fam12b,纯化后的融合蛋白经过SDS-PAGE和Western印记分析鉴定,在相对分子质量31kD处有特异的条带,并获得高纯度融合蛋白。本研究成功克隆了小鼠附睾基因fam12b,并从Rosetta(DE3)中获得高纯度融合蛋白,为进一步研究Fam12b的功能创造了条件。

To prokaryotic express and purify Fam12b,a mouse epididymial secretory protein.We constructed the expression plasmid consisting of prokaryotic vector pET-28（a） and fam12b double copy gene.Two fam12b fragments excluding the signal peptide sequence were amplified from mouse epididymal RNAs by RT-PCR,one had stop codon while the other didn′t.The recombinant plasmid was verified by sequencing and then transformed into E.coli Rosetta（DE3）.The recombinant protein was expressed under the induction of IPTG,then further purified by using Ni-NTA and analyzed by SDS-PAGE and Western blot.The results showed that the sequence of cloned fam12b gene was identical with those early reported.The fusion protein was verified by SDS-PAGE and Western-blot,showing its molecular was around 31 kD.Then the fusion protein was highly purified.The results indicated that the mouse epididymis gene fam12b had been successfully closed and efficiently expressesd in Rosetta（DE3）,and fusion protein had been highly purified,our work paved ways for antibody preparation and functional study of Fam12b in sperm maturation.