汉坦病毒S基因真核表达载体的构建及表达

目的 将编码汉坦病毒76-118株核蛋白的S基因片段克隆到真核表达载体DTARGET TM，构建含汉坦病毒核蛋白S基因的重组真核表达质粒pTARGET-hans，并在体外进行瞬间表达。方法 采用PCR产物直接克隆方法，将核蛋白S基因插入到真核表达载体pTARGE TM中；酶切鉴定；用电穿孔法将重组质粒转染入Vero-E6细胞；用间接免疫荧光法检测其表达产物。结果 酶切鉴定证实S基因已被正确地插入pTARGET TM中；在部分转染重组质粒的Vem-E6细胞胞质内观察到中等强度的特异性荧光。结论 重组的真核表达载体pTARGET-hans已被成功地构建并可在体外表达．为下一步基因免疫打下基础。

Construction and expression of eukaryotic expression vector containing Hantaan virus S gene

Objective Hantaan virus S gene encoding Nucleocapsid protein was cloned into the eukaryotic expressing plasmid pTARGET TM to get the recombinant plasmid pTARGET-hans,then to express it in vitro.Methods Polymerase chain reaction(PCR) was used to clone S gene segment and its product was inserted into eukaryotic expression vector pTARGET TM ;then the recombinant expression vector pTARGET-hans was identified by restriction enzyme analysis and transfected into Vero-E6 cells by electroporation.The transient expression of Hantaan virus nucleocapsid protein in Vero-E6 cells was detected by indirect immunofluorescence assay(IFA).Results Restriction enzyme analysis demonstrated that S gene was successfully inserted into pTARGET TM and IFA result showed that green fluorescence was observed in some transfected Vero-E6 cells.Conclusion The eukaryotic expression vector pTARGET-hans was successfully constructed and expressed in vitro,which would lay a foundation for further researching S gene segment immunization against Hantaan Virus.