水稻齿叶矮缩病毒Segment 9 dsRNA的序列分析及其在大肠杆菌DE3中的表达  

SEQUENCE ASSAY AND EXPRESSION IN E.COLI DE3 OF SEGMENT 9 dsRNA OF PHILIPPINE ISOLATE OF RICE RAGGED STUNTVIRUS

作　　者：卢雄斌[1] 周国瑛[1] 吴建华[1] 龚祖埙[1] 

机构地区：[1]中国科学院上海生物化学研究所

出　　处：《病毒学报》1997年第1期68-74,共7页Chinese Journal of Virology

基　　金：美国洛氏基金会(Rockfeler Foundation)经费资助

摘　　要：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。Ten genomic dsRNA (S1-S10) of Philippine isolate of rice ragged stunt virus (RRSV) were extracted and purified with organic reagents and CF-11 cellulose column.Based on the known sequences of two other isolates,RRSV-T (Thailand) and RRSV-I(India),we designed two suitable primers with EcoRI and BamHI cleavage sites and then the complementary cDNA of RRSV-P S9 was obtained by RT-PCR method.S9 cDNA of RRSV-P was incorporated into the bacterial expression vector pGEX-3X which produced a fusion protein with molecular weight about 64 kD.The result of Western blot experiment showed that the fusion protein was able to react strongly with antibodies raised against the purified RRSV-P particles.

关 键 词：植物病毒 水稻 齿叶矮缩病毒 Segment9 序列分析 

分 类 号：S432.41[农业科学—植物病理学]