发热伴血小板减少综合征布尼亚病毒结构和非结构蛋白表达研究

发热伴血小板减少综合征布尼亚病毒(SFTSV)是我国2010年新发现的新布尼亚病毒,可导致人类严重发热伴血小板减少综合征。SFTS新布尼亚病毒全基因组已解析,但病毒分子生物学结构蛋白特征及功能尚需更多研究。本文通过蔗糖密度梯度离心确定发热伴血小板减少综合征布尼亚病毒(HB29株)病毒颗粒的沉降密度及超离纯化条件,得出该病毒颗粒在蔗糖中的沉降密度为1.135g/mL。利用PCR方法扩增SFTSV病毒株HB29株病毒RNA聚合酶(RdRp)、糖蛋白前体蛋白(M)、包膜糖蛋白(Gn)、包膜糖蛋白(Gc)、核蛋白(NP)及非结构蛋白(NSs)的编码区基因片段,分别克隆入真核表达载体pcDNA5/FRT或VR1012,在293T细胞上获得上述基因表达。通过SDS-PAGE分析纯化病毒颗粒和重组蛋白,并通过免疫印迹(Western blotting)和间接免疫荧光(IFA)确定蛋白活性和分子量。本研究结果将有利于对新布尼亚病毒分子生物学特征的认识,为后期研究提供基础。

Expression of structural and non-structural proteins of severe fever with thrombocytopenia syndrome bunyavirus

Severe fever with thrombocytopenia syndrome bunyavirus (SFTSV) is a novel phlebovirus, causing a life-threatening illness associated with the symptoms of severe fever and thrombocytopenia syndrome. The sequence and structure of the genome have already been illustrated in previous study. However, the characteristics and function of the structure and non-structure proteins is still unclear. In this study, we identified the density of the purified SFTSV virions as 1.135 g/mL in sucrose solution. Using RT-PCR method, we amplified the full coding sequence of RNA dependent RNA polymerase(RdRp), glycoprotein precursor (M), glycoprotein n (Gn), glycoprotein c (Gc), nuclear protein (NP) and non structural protein (NSs) of SFTSV (strain HB29). Respectively inserted the target genes into eukaryotic expression vector pcDNA5/FRT or VR1012, the target protein in 293T cell were successfully expressed. By analyzing the SFTSV virions in SDS-PAGE and using recombinant viral proteins with SFTS patients sera in Western blotting and Immunofluorescent assay, the molecule weight of structure and non-structure proteins of SFTSV were defined. The study provides the first step to understand the molecular characteristics of SFTSV.