水稻瘤矮病毒S3和S8基因共表达杆状病毒转移载体构建及重组病毒的鉴定

Construction of baculovirus transfer vector for co-expression of Rice gall dwarf virus gene S3 and S8 and identification of recombinant baculovirus

为构建携带水稻瘤矮病毒(Rice gall dwarf virus,RGDV)主要内层衣壳蛋白S3基因和外层衣壳蛋白S8基因的重组杆状病毒,将目的基因(S3和S8)分别亚克隆到杆状病毒转移载体pFastBacDual多角体启动子(PH)和p10启动子的下游.经酶切和确证性序列测定,将其转化到DH10Bac感受态细胞中,获得重组杆粒rbpFBDS3-S8,采用脂质体转染法,将rbpF-BDS3-S8转染草地贪夜蛾(Spodoptera frugiperda)Sf9细胞包装病毒,PCR筛选鉴定重组病毒.结果表明:Sf9昆虫细胞被侵染72 h后,倒置显微镜下观察到细胞增大,培养液和细胞内出现颗粒状物质,部分细胞破裂甚至裂解,说明S3和S8基因已整合到重组杆状病毒基因组中,这为开展RGDV主要结构蛋白在昆虫细胞中的表达及其功能研究奠定了基础.

To construct a recombinant baculovirus co expressing the major core capsid protein gene S3 and outer capsid protein gene S8 of Rice gall dwarf virus （RGDV）, the target genes （S3 and S8 ） were subcloned into the downstream of PH promoter and pl0 promoter of the baculovirus transfer vector pFastBacDual, respectively. After transformation, pFBDS3 SS, which was identified with restriction enzyme digestion and conformed by sequence analysis, was introduced into the competent cells （Escherichia coli DH10Bac）, generating the recombinant bacmid rbpFBDS3 S8. Bacmid rbpFBDS3 S8 was transfected with Si9 （Spodopterafrugiper da） insect cells package virus by liposomal transfection method. The recombinant virus was identified by PCR. Results showed that an increased diameter, granular appearance and cells lysis, which were much different from the morphology of normal St9 cells were observed under fluorescence invert microscope, 72 h after infection. The gene S3 and S8 were integrated into genome of recombinant baculovirus, laying a foundation for the expression of the major structural protein gene in insect cells and the research of the function of gene being investigated.