阴道毛滴虫RRas基因的cDNA克隆和原核表达

Cloning and Expression of Trichomonas vaginalis RRas cDNA

目的克隆和表达阴道毛滴虫RRas(TvRRas)基因,以便进一步探讨其组织定位及功能。方法应用PCR方法从构建的阴道毛滴虫cDNA表达文库中扩增TvRRas基因片段,克隆至pGEM-T载体,经PCR、酶切和测序鉴定后亚克隆至原核表达载体pET-41a,转化大肠埃希菌(E.coli)BL21,经异丙基硫代--βD-半乳糖苷(IPTG)诱导表达,镍-硝基三乙酸(Ni-NTA)柱纯化表达产物后用SDS-聚丙稀酰胺凝胶电泳(SDS-PAGE)进行分析鉴定。结果从阴道毛滴虫cDNA表达文库中扩增的TvRRas基因片段长522bp,酶切及DNA测序证实pET-41a/TvRRas重组质粒构建正确,表达融合蛋白分子量约为45000u。结论成功构建重组原核表达质粒pET-41a/TvRRas,并能在大肠埃希菌内表达,所表达的融合蛋白分子量大小与预期一致。

Objective To clone the Trichomonas vaginalis RRas （TvRRas） cDNA and express the TvRRas as a fusion protein in E.coli. Method A eDNA fragment of TvRRas was amplified by PCR, and cloned into pGEM- T easy vector. The recombinant plasmid pGEM-T/TvRRas was digested with restriction endonuclease BamH Ⅰ and Pst Ⅰ . The fragment was then subeloned into the expression vector pET-41a. E.coli BL21 cells were transformed by the recombinant plasmid. The expression of the recombinant protein was induced by IPTG, and the fusion protein was purified with Ni-NTA agarose column and analyzed by SDS-PAGE. Result A 522 base pairs TvRRas eDNA fragment was PCR amplified, cloned into pGEM-T easy vector and then was used to construct the recombinant plasmid pET- 41a/TvRRas. The fusion protein （about 45 000 u） was expressed and purified from E.coli. Conclusion The recombinant plasmid pET-41a/TvRRas have been constructed successfully and the fusion protein can be expressed in E.coli BL21 cells.