表达SFTSV Gn蛋白的重组狂犬病病毒的构建、鉴定与免疫反应

Construction, identification and immunoreactivity of recombinant rabies virus expressing Gn protein of severe fever with thrombocytopenia syndrome virus

目的构建表达发热伴血小板减少综合征病毒(SFTSV) Gn蛋白的重组狂犬病病毒,并研究重组病毒的免疫反应。方法将SFTSV的保护性抗原Gn蛋白基因插入至狂犬病病毒(RABV) SAD株基因组的假基因区,通过反向遗传技术拯救获得重组病毒SAD-Gn株。对SAD-Gn进行生物学特性鉴定,并将SAD-Gn株免疫小鼠,测其血清中和抗体效价。结果通过酶切、RT-PCR和荧光抗体染色鉴定,证明SAD-Gn株基因组中含有Gn基因,且能正确表达Gn蛋白。该重组病毒细胞适应性好,滴度可达3. 3×108FFU/ml。免疫小鼠后,可同时诱导产生高效价的抗RABV和SFTSV的中和抗体。结论成功构建了重组病毒SAD-Gn,并诱导小鼠产生针对RABV和SFTSV的中和抗体,为进一步研制预防狂犬病和发热伴血小板减少综合征的二联疫苗奠定了基础。

Objective To construct a recombinant rabies virus（RABV） live vector strain expressing the Gn protein of severe fever with thrombocytopenia syndrome virus （SFI＇SV） and prevent these two zoonotic diseases. Methods The Gn protein gene was inserted into the Pseudogene region of Street Alabama Dufferin（SAD） strain and the rescued recombinant virus was immunized in BALB/c mice to detect serum neutralizing antibody titers. Results The recombinant RABV,named SAD-Gn and expressing Gn protein, was screened via reverse transcription-polymerase chain reaction （RT-PCR） , indirect imnmnofluorescent assay（IFA） and restriction endonuclease digestion. The titer of SAD-Gn strain remained at 3.3 × 108 FFU/ml. SAD-Gn showed high neutralizing antibody titers against both BABV and SFTSV by immunized mice. Conclusion A recombinant SAD-Gn strain is obtained in high titer,which induces high levels of neutralizing antibody titers in mice.