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通过有机试剂抽提,CF-11纤维素柱层析,从感染水稻齿叶矮缩病毒菲律宾分离株(RiceRaggedStuntVirus,Philippineisolate,简称RRSV-P)的水稻植株中获取该病毒的全基因组,即获得从Segment1到Segment10(S1-S10)的10条双链RNA(dsRNA),然后设计合适的引物,用RT-PCR方法得到S9的cDNA并将其克隆到pUC119质粒上扩增,以双链测序法测定该cDNA的全序列。同时又将此cDNA克隆到大肠杆菌表达质粒pGEX-3X上,在大肠杆菌菌株DE3中用IPTG诱导表达,经超声波破菌、离心、Glutathione-sepharose4B亲和层析,得到纯化的分子量为64kD的融合蛋白。
The whole genome of the virus was obtained from rice plants infected with Rice Ragged Rats virus (Philippine isolate, RRSV-P) by organic reagent extraction and CF-11 cellulose column chromatography, (DsRNA) from Segment1 to Segment10 (S1-S10). Then, a suitable primer was designed. The cDNA of S9 was obtained by RT-PCR and cloned into pUC119 plasmid for amplification. The double-stranded sequencing The full sequence of this cDNA. At the same time, the cDNA was cloned into E. coli expression plasmid pGEX-3X and induced by IPTG in E. coli strain DE3. The recombinant plasmid was sonicated and centrifuged, and Glutathione-sepharose 4B affinity chromatography was performed to obtain a purified fusion protein with a molecular weight of 64 kD protein.