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[目的]利用杆状病毒表达系统在昆虫细胞中表达水稻条纹病毒(Rice stripe virus,RSV)RNA聚合酶(RdRp)基因。[方法]RT-PCR扩增RdRp基因功能区片段,克隆于转移载体p Fast Bac HTb上,转化到大肠杆菌DH10Bac中,得到重组杆状病毒表达载体Bacmid-rp。提取Bacmid-rp DNA,转染Sf9细胞获得重组病毒v Ac-rp。将重组病毒v Ac-rp感染Sf9细胞使目的蛋白在细胞内表达,并利用亲和层析纯化,SDS-PAGE检测RdRp的表达情况。将纯化的RdRp蛋白免疫家兔制备多克隆抗体,Western blot分析RdRp的免疫原性。[结果]RSV RdRp基因功能区能够在Sf9细胞中表达,蛋白分子量约为45k Da,与预测大小一致,且可与RdRp抗血清发生特异性反应。[结论]在真核细胞中成功表达了RSV RdRp,并得到了纯化的RdRp和其多克隆抗体。
[Objective] The baculovirus expression system was used to express Rice stripe virus (RSV) RNA polymerase (RdRp) gene in insect cells. [Method] The functional fragment of RdRp gene was amplified by RT-PCR, cloned into p Fast Bac HTb and transformed into E. coli DH10Bac to obtain Bacmid-rp recombinant baculovirus expression vector. Bacmid-rp DNA was extracted and transfected into Sf9 cells to obtain recombinant virus v Ac-rp. The recombinant protein v Ac-rp was transfected into Sf9 cells to express the target protein in cells. The recombinant protein was purified by affinity chromatography and the expression of RdRp was detected by SDS-PAGE. The purified RdRp protein was immunized in rabbits to prepare polyclonal antibodies, and the immunogenicity of RdRp was analyzed by Western blot. [Result] The RSV RdRp gene functional region could be expressed in Sf9 cells. The molecular weight of RSR was about 45 kDa, consistent with the predicted size, and could specifically react with RdRp antisera. [Conclusion] RSV RdRp was successfully expressed in eukaryotic cells, and purified RdRp and its polyclonal antibody were obtained.