蓝氏贾第鞭毛虫末端结合蛋白1基因的克隆与原核表达

Cloning and prokaryotic expression of Giardia lamblia eb1 gene

目的克隆并原核表达C2株蓝氏贾第鞭毛虫(Giardia lambia,简称贾第虫)末端结合蛋白1(End-binding protein 1,geb1)基因,获得重组gEB1蛋白。方法由于geb 1基因无内含子,我们以C2株贾第虫基因组为模板,以国际标准株WB株geb 1基因序列为参考序列,设计引物克隆geb 1基因,经NcoⅠ和XhoⅠ双酶切与原核表达载体pET-28α(+)连接,转化感受态E.coli TOP10,经筛选和测序验证后导入大肠杆菌E.coli Rosetta(DE3),异丙基-β-D-硫代半乳糖(IPTG)诱导gEB1蛋白表达,产物经SDS-PAGE和Western blot进行检测和验证。结果成功构建了表达C2株贾第虫geb1基因的原核表达载体pET-28α(+)-gEB1,转化入大肠杆菌E.coli Rosetta(DE3),重组菌株经0.1 mmol/L IPTG,30℃低温诱导5 h,SDS-PAGE和Western blot显示,在相对分子量约29 KDa的位置出现目的蛋白条带,与理论值一致。结论用大肠杆菌成功表达了gEB1蛋白,为gEB1蛋白的功能研究和抗体制备提供了材料。

Objective To clone and express the C2 strain of Giardia lambia End-binding protein 1(gEB1)in E.coli.Methods Recombinant gEB1 protein was obtained,on which basis the primer was designed to clone gEB1 gene using C2 strain of Giardia lamblia as template and sequence reference to gEB1 gene by international standard WB strain The PCR product was cloned into prokaryotic expression vector pE-28α(+)with restriction enzymes Nco I and Xho I.The recombinant vector pET-28a(+)-gEB1 was transformed into E.coli host strain Rosetta(DE3),then the fusion protein was expressed by IPTG induction at 30℃for 5 h.All products were tested and confirmed by SDS-PAGE and Western blot.Results The prokaryotic expression vector was successfully constructed,and the EB1 was highly expressed in E.coli Rosetta(DE3).SDS-PAGE and Western blot showed that the expressed product was about 29 kDa that was consistent with the defined value.Conclusion gEB1 gene has been successfully cloned and expressed in E.coli Rosetta(DE3),the recombinant protein can be used for further functional study and antibody preparation.