新布尼亚病毒真核表达载体的构建及表达

目的 探究SFTSV-NSs基因的结构和功能以及病毒在感染细胞过程中与宿主的相互作用,通过构建SFTSV-NSs真核表达载体SFTSV-NSs-FLAG,并将其转染进293细胞内,为进一步研究SFTSV-NSs基因的结构和功能奠定基础。方法 利用RT-PCR 扩增SFTSV-NSs基因序列并测序,并将纯化得到的SFTSV-NSs cDNA 片段连接到pFLAG-CMV-3载体,通过EcoRI/BamHI双酶切鉴定重组质粒。将重组表达体pSFTSV-NSs-FLAG导入293细胞中,共聚焦显微镜下观察SFTSV-NSs在细胞中的定位、 Western Blot 检测SFTSV-NSs 蛋白在293细胞中的表达情况。结果 成功克隆测序SFTSV-NSs基因;成功构建SFTSV-NSs真核表达载体pSFTSV-NSs-FLAG,并将其转染到293细胞中。在293细胞中的表达检测结果显示共聚焦显微镜下观察重组体主要集中在细胞质中,并形成类包涵体样结构;且Western Blot 检测结果显示SFTSV-NSs 蛋白在293细胞中表达。结论 为进一步研究SFTSV-NSs基因的结构和功能以及病毒在感染细胞过程中与宿主的相互作用提供新思路。

Construction and expression of eukaryotic expression vector of SFTSV

NSs protein is a non-structural protein encoded by Bunya virus S RNA and plays an important role in the replication cycle of Severe fever with thrombocytopenia syndrome bunyavirus(SFTSV). Therefore it is possible to explore the structure and function of the SFTSV-NSs gene and the interaction of the virus with the host during infection of the cell. To construct SFTSV-NSs eukaryotic expression vector pSFTSV-NSs-FLAG and transfect it into 293 cells, which lays a foundation for further study of the structure and function of SFTSV-NSs gene. The SFTSV-NSs gene sequence was amplified by RT-PCR and sequenced. The purified SFTSV-NSs cDNA fragment was ligated into pFLAG-CMV-3 vector, and the recombinant plasmid was identified by EcoRI/BamHI double digestion. The recombinant expression pSFTSV-NSs-FLAG was introduced into 293 cells. The localization of SFTSV-NSs in cells was observed under a confocal microscope. The expression of SFTSV-NSs protein in 293 cells was detected by Western Blot.The SFTSV-NSs gene was successfully cloned and sequenced. The SFTSV-NSs eukaryotic expression pSFTSV-NSs-FLAG was successfully constructed and transfected into 293 cells. The expression of the expression in 293 cells showed that the recombinant group was mainly concentrated in the cytoplasm and formed a type of inclusion body-like structure a under confocal microscope. Western Blot analysis showed that SFTSV-NSs protein was expressed in 293 cells. This study provides a new way to further study the structure and function of the SFTSV-NSs gene and the interaction of the virus with the host in the process of infecting cells.