【摘要】 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。更多还原

【Abstract】 The paper reported that HPV16 XJ E6 proteins were experessed in Pichia pastoris eukaryote expression system.The 5’ primer and 3’ primer were designed according to HPV16 XJ E6 gene with EcoR I & Xba I.After amplified with PCR,HPV16 XJ E6 gene was cloned into pMD18-T vector.Then the HPV16 XJ E6 gene was cloned into shuttle plasmid pGAPZ α A by cleaved from T-vector.The recombinant plasmid were linearized and transformed into pichia pastoris and screened positive clone with zeocin.At last,the supernatant of medium was analyzed by SDS-PAGE after fermentation.The results indicated that the HPV16 XJ E6 protein has been expressed successfully in pichia pastoris eukaryote expression system,and the MW of HPV16 XJ E6 protein is 20 kD.This could lead to further investigating the function of HPV16 protein development.更多还原