摘要
目的:为进一步探讨乙型肝炎病毒(HBV)X蛋白(HBxAg)的功能,在真核生物酵母细胞中表达X基因。 方法:用多聚酶链反应(PCR)的方法以HBV ayw亚型全序质粒pCP10为模板扩增X基因,克隆到pGEM-T载体中扩增并测序鉴定,EcoRI和PstI双酶切后回收连接到酵母表达载体pGBKT-7中并转化酵母AH109,色氨酸缺陷型培养基(SD/-Trp)上筛选阳性菌落,提取酵母蛋白质,进行十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和Western免疫印迹分析,以明确HaxAg是否在其中表达。 结果:成功地扩增出HBx基因,测序鉴定符合Gene Bank报告序列;酶切回收的HBx基因成功地克隆入酵母表达载体pGBKT-7并转化入酵母细胞AH109中,Western免疫印迹显示X基因在酵母细胞中表达,表达产物在胞内存在,相对M_r为35000左右。 结论:成功地构建了HBxAg在酵母表达载体,并在酵母细胞中表达。
AIM:To study the exact function of HBx protein so as to investigate the gene expression of HBxAg in yeast.METHODS: PCR was used to amplify the gene of HBx from the plasmid pCP10 containing the whole fragment of ayw subtype of HBV and the PCR product was cloned into pGEM-T vector and was evaluated by sequencing. The gene of HBx was cut from pGEM-T vector and cloned into yeast expression plasmid pGBKT7, and was transformed into yeast cell AH 109. The yeast protein was isolated, and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting.RESULTS: HBx gene was successfully amplified and identified by DNA sequensing. The digested fragment was cloned into pGBKT7 vector and transformed into yeast cell AH109. The SDS-PAGE and Western blotting assay showed that the relative molecular weight of the expressed product was about 35000 Da, and HBx protein existed in yeast cells.CONCLUSIONS: HBx was successfully expressed in yeast system.
出处
《世界华人消化杂志》
CAS
2002年第1期15-18,共4页
World Chinese Journal of Digestology
