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目的 :利用酵母Pichiapastoris真核蛋白表在系统 ,表达HPV16 (新疆株 )E6 (HPV16XJE6 )蛋白。方法 :根据HPV16XJE6基因序列设计引物 ,并分别在 5′引物和 3′引物中引入了EcoRI和XbaI酶切位点 ,经PCR扩增后与pMD18-T载体相连 ,再将HPV16XJE6从T载体上切下并克隆至穿梭质粒pGAPZαA上 ,获得的重组穿梭质粒pGAPZαA -E6经线性化后 ,采用LiCl法将重组穿梭质粒转入酵母细胞内 ,Zeocin+筛选鉴定 ,经小瓶发酵后 ,取上清作SDSPAGE检测。结果 :HPV16XJE6成功地在酵母真核表达系统获得表达 ,表达产物的分子量为 2 0kD ,为深入研究HPV16XJE6蛋白功能奠定了理论基础。
OBJECTIVE: To systematically express HPV16 (HPV16XJE6) protein of HPV16 (Xinjiang strain) using the yeast Pichia pastoris eukaryotic protein expression system. Methods: The primers were designed according to the HPV16XJE6 gene sequence. The EcoRI and XbaI restriction sites were introduced into the 5 ’and 3’ primers respectively. The PCR products were ligated to the pMD18-T vector and then the HPV16XJE6 was excised from the T vector And cloned into the shuttle plasmid pGAPZαA. The recombinant shuttle plasmid pGAPZαA-E6 obtained after linearization was transformed into yeast cells by LiCl method and identified by Zeocin +. After being vial-fermented, the supernatant was subjected to SDSPAGE . Results: HPV16XJE6 was successfully expressed in yeast eukaryotic expression system. The molecular weight of HPV16XJE6 was 20kD, which laid the theoretical foundation for further study on the function of HPV16XJE6 protein.