长叶车前花叶病毒上海株(RMVsh)外壳蛋白基因的克隆、序列分析及原核表达

长叶车前花叶病毒上海分离株（RMVsh)是从上海郊区的青菜（Brassica chinensis)上分离鉴定的，以提纯的病毒为材料，SDS－酚法纯化的基因组RNA作为模板，通过RT－PCR方法克服了该病毒的外壳蛋白基因（CP），DNA序列测定结果表明，外壳蛋白基因全长474个碱基，编码157个氨基酸，将CP基因插入原核表达载体pBAD/His-C中，转化E.coli Top10后，诱导表达，经聚丙烯酰胺凝胶电泳分析呈－特异性的蛋白条带，Western blot检测表明，表达产物与RMVsh抗血清呈阳性反应，RMVsh CP基因的核苷酸序列及氨基酸序列与烟草花叶病毒属中其他能侵染十字花科作物的成员相比，同源率分别为83．5％－98．9％和87．9％－99．4％，并讨论了RMVsh的分类地位为烟草花叶病毒属侵染十字花科植物2个亚组中的第一亚组的代表。

Sequence Analysis of Coat Protein Gene of Ribgrass Mosaic Virus Shanghai Isolate and Expression in E. coli

An Shanghai isolate of ribgrass mosaic virus (RMVsh) was isolated from Brassica chinensis (Qingcai).Viral RNA extracted from virus preparation was used as template for cDNA synthesis of coat protein gene by RT PCR .PCR products were purified with agarose gel and cloned into pGEM T easy vector to construct the recombinant plasmid for nucleotide sequencing.The coat protein CP gene consists of 474 nt encoding 157 amino acids. The CP gene was inserted into pBAD/His C vector and expressed in E.coli Top10 strain. The product was identified by SDS PAGE and Western blot with RMVsh antiserum.The homology of nucleotide and predicted amino acid sequences of CP gene with those of other isolates of tobamovirus infecting crucifer were 83.5%-98.9% and 87.9%-99.4%,respectively. RMVsh as a representative in first sub group of Tobamovirus infecting cruciferae plants was discussed.