[cDNA cloning and expression in Escherichia coli of rice black-streaked dwarf virus segment 7]

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.