人睾丸新基因SPAG4L的原核表达与纯化

Prokaryotic expression and purification of SPAG4L,a novel human testis gene

目的原核表达人睾丸生精相关基因SPAG4L,并对重组蛋白进行纯化,为下一步研究SPAG4L的生物学功能奠定基础。方法运用RT-PCR从人睾丸中扩增编码SPAG4L126-379的基因片段,并将PCR产物克隆至pUCm-T载体。用限制性内切酶EcoR I和Hind III消化后,将目的片段克隆至带有6个组氨酸标签的原核表达载体PQE30中。重组质粒PQE-30-SPAG4L经酶切和测序验证后,转化大肠杆菌JM15,IPTG诱导融合蛋白表达。SPAG4L融合蛋白以westernblot进行鉴定,最后用NI-NTA磁性琼脂糖珠进行纯化。结果成功构建了重组表达质粒PQE-30-SPAG4L,并能够在大肠杆菌JM15中诱导表达,经Western blot分析证实,IPTG诱导表达的是SPAG4L融合蛋白。建立小规模的SPAG4L融合蛋白表达、纯化系统,获得了带有6个组氨酸标签的纯化的SPAG4L融合蛋白。结论成功地对人睾丸生精相关基因SPAG4L进行了体外原核表达,获得了纯化的融合蛋白蛋白,为进一步研究该蛋白在精子发生中的生物功能奠定了基础。

Objective To express SPAG4L,a novel human testis gene in E.coli and purify it＇s fusion protein.Methods The fragment encoding SPAG4L126-379 was amplified by RT-PCR and the PCR products were cloned into PUCm-T vectors.After digestion by EcoR I and Hind III,the fragment was subcloned into PQE-30,a prokaryotic expression vector with 6×His tag.The recombinant plasmid PQE-30-SPAG4L was sequenced and transformed into E.coli M15.The expression of his-tagged fusion protein was induced by IPTG.The fusion protein was identified by Western blotting and purified using Ni-NTA magnetic agarose beads.Results The recombinant plasmid PQE-30-SPAG4L was constructed successfully and expressed in E.coli M15.The fusion protein SPAG4Lwith 6×his-tag was confirmed by Western blotting.The micro-scale purification system of 6×His-tagged SPAG4Lprotein was established and purified fusion protein was obtained.Conclusion The recombinant plasmid PQE-30-SPAG4L can be expressed in vitro and used for studying the biological function of SPAG4L in spermatogenesis.