【摘要】 目的 :对轮状病毒 SA1 1株 VP7基因进行修饰 ,使其表达后锚定于宿主细胞膜表面 ,同时构建一个可使目的蛋白分泌或跨膜表达的载体。方法 :用 PCR的方法把 VP7天然信号肽替换为流感病毒血凝素信号肽。然后 ,在基因下游添加流感病毒血凝素跨膜区编码序列 ,从而构建在宿主细胞表面表达的轮状病毒 SA1 1 VP7基因。利用遗传密码简并性在信号肽序列 3′端和跨膜区 5′端分别引入 Sty 和 Eco RV限制性内切酶识别位点 ,从而实现分泌 /跨膜基因修饰载体的构建。把野生型和跨膜型 VP7基因分别克隆入 pc DNA3哺乳动物细胞表达载体 ,转染 Hela细胞 ,得到稳定表达两种 VP7基因的细胞系。对外源基因在细胞染色体上的整合情况以及基因表达情况进行检测。结果 :酶切鉴定和 DNA序列分析表明膜锚定型 VP7基因和分泌 /跨膜基因修饰载体的构建正确。PCR和免疫荧光实验证明 ,VP7基因已经整合进入宿主细胞染色体 ,野生型和膜锚定型 VP7基因表达后分别定位于宿主细胞质和细胞膜。结论 :成功地构建了可在细胞膜表面锚定表达的 VP7基因 ,为进一步研究轮状病毒基因工程疫苗奠定了基础。新型分泌 /跨膜基因修饰载体为研究蛋白合成后加工和提高其免疫原性提供了有力的工具。更多还原

【Abstract】 Objective:To construct a rotavirus SA11 VP7 expressing at the cell surface;to construct a secrection/transmembrane gene modification vector;to generate Hela cells constantly expressing VP7 antigen in cytoplasm or cell surface.Methods:The signal sequence of SA11 VP7 was replaced by that of the influenza virus hemagglutinin (HA) by PCR.Then the coding sequence of membrane anchor and cytoplasmic domains also from the HA was added to the downstream of VP7 gene by in frame insertion.Recognition sites of restriction endonuclease StyⅠ and EcoRⅤ were introduced to the upstream and downstream of the VP7 sequence,respectively.Plasmid containing the novel VP7 gene,from which the VP7 sequence could be cut out,could be used to direct target proteins into secreting or cell surface anchoring pathway.The wild type and the modified form of VP7 genes were cloned into pcNA3 respectively.The recombinant plasmids were transfected into Hela cells and gene transfer was confirmed by G418 selection.Gene integrations to the host chromosome were tested by PCR.VP7 expression was investigated by ELISA and immunofluorescence assays which could also determine the localizations of expression products.Conclusion:SA11 VP7 was successfully expressed on the cell surface.This provided a foundation for further research on genetic engineering vaccine of rotavirus.The gene modification vector should be useful to modifying poorly immunogenic antigens expressed natively in the cytoplasm. 〔更多还原