新肠道病毒71型病毒样颗粒的制备

Preparation of the EV71 VLPs

目的:研究新肠道病毒71型(Enterovirus 71,EV71)病毒样颗粒(Virus-like particles,VLPs)的制备方法。方法:依据昆虫细胞偏爱密码子优化EV71 P1和EV71 3CD基因,将二者分别克隆至供体质粒pFastBac Dual多角体蛋白启动子pPh和pP10上;构建重组pFastBac Dual EV71 P1-3CD质粒,并与Bacmid质粒转座后,获得重组杆状病毒表达质粒转染至Sf9细胞,应用PCR、免疫荧光、SDS-Page和Western blot方法鉴定EV71 VLPs表达情况,最后利用蔗糖密度梯度离心的方法纯化EV71 VLPs。结果:经昆虫密码子优化的EV71 P1和EV71 3CD基因,利用Bac-to-Bac系统成功组装了EV71 VLPs,纯化EV71VLPs的浓度为0.817 mg/ml,纯度>90%。结论:成功地构建了重组杆状病毒EV71 P1-3CD Bacmid表达质粒,并表达和纯化了EV71 VLPs,这为今后EV71 VLPs疫苗的研究奠定了基础。

Objective：The aim of the present study was to prepared EV71 VLPs,which was important for further studies in term of EV71 VLPs.Methods：Based on preferred codon optimization of insect cells,EV71 P1 and EV71 3CD genes were cloned into the donor vector pFastBac Dual under the polyhedrin promoter pPh and pP10,and the constructed donor plasmid pFastBac Dual-P1-3CD was transformed to E.coli DH10Bac.The obtained recombinant baculovirus plasmid Bacmid-P1-3CD was transfected into Sf9 cells to prepare recombinant baeulovirus and the expressed products were identified by PCR,IFA,SDS-Page and Western-Blot.Then the VLPs were purified by sucrose density gradient centrifugation.Results：The EV71 P1 and EV71 3CD genes insects codon optimized were assembled EV71 VLPs successfully by Bac-to-Bac system.Concentration of purified EV71 VLPs was 0.817 mg/ml,purity of 90%.Conclusion：Finally,the pFastBac Dual EV71 P1-3CD was constructed,and EV71 VLPs were expressed and purified successfully,which provide a valuable platform for the future EV71 VLPs vaccine research.