新城疫病毒F蛋白在昆虫细胞中的表达及其融细胞作用

用RT-PCR方法扩增出新城疫病毒标准强毒株F48E8的F基因,并将其克隆到pGEM-T载体,命名为pGEM-NDF。鉴定正确后,以BamHI和XbaI双酶切将F基因从pGEM-NDF中释放出来,并插入到pFastBacI载体中,得到重组转移载体pFast-NDF。然后将该重组质粒转入含有穿梭质粒的感受态DH10Bac中,通过转座作用获得重组穿梭质粒reBacmid-NDF。再用reBacmid-NDF转染Sf9昆虫细胞,获得含有新城疫病毒F48E8株F基因的重组杆状病毒。间接免疫荧光和Western-blot分析结果表明F蛋白在昆虫细胞中获得表达,而且主要表达于细胞膜上,并使感染重组杆状病毒的昆虫细胞在96h发生融合作用。动物试验表明,表达的F蛋白能够产生中和抗体。本文的研究结果为F蛋白的进一步开发奠定了基础。

Expression and Characterization of the Fusion Protein of Newcastle Disease Virus Strain F48E8 in Insect Cells

The fusion protein gene of strain F48E8 of Newcastle disease virus was amplified with RT-PCR, and cloned into the pGEM-T vector and named pGEM-NDF. Then, F gene was released from pGEM-NDF by digestion with BamHI and XbaI, and ligated to baculovirus transfer vector, pFastBac1, which was digested with BamHI and XbaI. The ligation yielded a recombinant plasmid, pFast-NDF. The results of sequencing and PCR showed that F gene had been cloned into baculovirus genome correctly. After transfecting sf9 cells with pFast-NDF, the recombinant baculovirus was recovered from the cells. The results of IFA and Western-blot assay indicated that F gene was expressed well in sf9 cells. Furthermore, the F protein was present on the cell membrane, and sf9 cells were fused at 96 hours post-infection, which suggested that F protein had the activity of cell fusion. Animal tests showed that the fusion protein expressed in insect cells induced neutralizing antibody against NDV in mice. All these results provided foundation for the development of F protein.