水稻黑条矮缩病毒第七号片段的cDNA克隆及其在大肠杆菌中的表达

cDNA Cloning and Expression in Escherichia coli of Rice Black-streaked Dwarf Virus Segment 7

运用RT PCR技术 ,根据玉米粗缩病毒S6的末端序列设计引物 ,从玉米粗缩病感病材料中克隆得到一条 2 2kb长的cDNA片段 ,序列分析表明 ,该片段全长 2 1 93bp ,含两个开放阅读框 ,分别编码 41 0kD和 36 3kD的多肽。序列分析结果表明 ,该cDNA片段及其编码产物与水稻黑条矮缩病毒S7的cDNA序列及编码产物存在最高的相似性 ,推测该cDNA片段为水稻黑条矮缩病毒的S7片段 ,而不是玉米粗缩病毒的S6。分别将该片段的两个阅读框克隆到原核表达载体pET2 1 d及pGEX KG中 ,经IPTG诱导后 ,两种蛋白均得到了高效的表达。表达产物回收后制备并得到了高效价的抗血清。

Using primers designed from the terminal sequences of maize rough dwarf virus S6, a 2.2 kb cDNA fragment was amplified by RT-PCR from maize plants showing maize rough dwarf disease. Sequence analysis shows that the full length of this cDNA is 2193bp. It contains two open reading frames that encoded two polypeptides with molecular weight of 41.0kD and 36.3kD, respectively. Results of multi-sequences alignment suggest that, this cDNA sequence has significant similarity to rice black-streaked dwarf virus S7, much higher than to MRDV S6. The ORFs were cloned into expression vectors, pET21-d (ORF1) or pGEX-KG(ORF2), respectively, and then transformed to BL21(DE3)-gold. After induction with IPTG, both proteins were highly expressed. The recombinant proteins were purified and high titer antisera of these two proteins were prepared.