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目的在真核生物酵母细胞中表达乙型肝炎病毒前S1蛋白反式激活基因5(PS1TP5)。方法以HepG2细胞来源的mRNA为模板,经RT-PCR法扩增PS1TP5基因,克隆到pGEM-T载体中,并测序鉴定,酶切回收后连接到酵母表达质粒pGBKT7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,进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和Western免疫印迹分析,以明确PS1TP5是否可在其中表达。结果成功扩增出PS1TP5,测序鉴定符合基因库报告序列,酶切回收的PS1TP5基因片段成功克隆入酵母表达载体pGBKT7,并转化入酵母细胞AH109中,Western免疫印迹显示该基因在酵母细胞中成功表达,表达产物相对分子质量为36950。结论成功构建了PS1TP5酵母表达载体,并在酵母细胞中表达。
Objective To express hepatitis B virus preS1 transactivator 5 (PS1TP5) in eukaryotic yeast cells. Methods PS1TP5 gene was amplified by RT-PCR from HepG2 cell-derived mRNA and cloned into pGEM-T vector. The recombinant plasmid was identified by sequencing and ligated into yeast expression plasmid pGBKT7 and transformed into yeast AH109 Positive colonies were screened on SD / -Trp medium and yeast proteins were extracted and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western immunoblot analysis to determine whether PS1TP5 was Can express in it. Results PS1TP5 was successfully amplified and sequenced. The PS1TP5 fragment recovered by enzyme digestion was successfully cloned into yeast expression vector pGBKT7 and transformed into yeast AH109. Western blotting showed that the PS1TP5 gene was successfully expressed in yeast cells , The relative molecular mass of the expressed product is 36950. Conclusion PS1TP5 yeast expression vector was successfully constructed and expressed in yeast cells.