坦布苏病毒MM1775株反向遗传操作系统的建立

ESTABLISHMENT OF REVERSE GENETICS SYSTEM FOR TEMBUSU VIRUS STRAIN MM1775

本研究将坦布苏病毒MM1775株全基因组分3段克隆,获得3个重组质粒,利用融合PCR方法扩增得到5'端含有T 7启动子的病毒全长c DNA,经体外转录得到感染性的RNA,将RNA转染DF-1细胞,48 h后出现细胞病变。当细胞病变达到90%,将细胞上清接种DF-1细胞,72 h后进行间接免疫荧光(indirect immunofulorescence,IFA)和RT-PCR鉴定,结果表明拯救病毒成功。序列测定结果显示,拯救毒的全基因组序列与亲本毒完全一致,表明MM1775反向遗传操作系统构建成功,为进一步研究坦布苏病毒致病性等相关分子机制奠定了基础。

The c DNA of Tembusu virus strain MM1775, were inserted segmentally into 3 plasmids, by PCR amplification, cloning and sub-cloning methods. The full-length cDNA, with T7 promoter sequence added at 5 ＇ ends, was amplified by infusion-PCR using the recombinant plasmids as templates and transfected in vitro to produce the virus RNA, which was then transfected into DF-1 cells to rescue virus. Detectable cytopathic effect（CPE） was found at 48 hours post-transfection. To test the rescued virus, fresh DF-1 cell were infected with the supernatant of transfected-cell culture when 90% transfected-cells showed CPEs. Specific green fluorescence could be detected in the newly infected cells by indirect immuno fluorescence（IFA） and specic RT-PCR products could be amplified from the supernatants at 72 hours post-infection, suggesting the success of MM1775 rescue. Sequence analysis confirmed the rescued virus have no unexpected nucleotide change comparing with its parental virus. The establishment of the reverse genetics system of MM1775 settled the foundation for further study on the molecular basis of the pathogenicity of Tembusu virus.