Molecular cloning and sequencing of outer capsid gene of rice dwarf virus (RDV) and its expression in Escherichia coli

Lu RuiFang; Li Yi; Yang ChongLin; Yan Hua; Chen ZhangLiang

Acta Microbiologica Sinica 39(4): 305-314


Accession: 003502394

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The S2 full-length cDNA of rice dwarf virus (RDV) which encodes the viral outer capsid protein was cloned and its complete nucleotide sequence was determined. The results showed that S2 is 3512 bp in length with a large open reading frame which encodes a protein of 1116 amino acids. It shares 94.6% and 95,4% identity with RDV of Japanese H isolate in terms of nucleotide and amino acid sequences, respectively, and it also shows some homology with VP2 of rotavirus at the level of amino acid sequence. The search of deduced RDV S2 amino acid sequence in Blast network found that there were 4 leucine-rich motifs in P2 protein, and 10 amino acids within the hydrophilic region at the amino-terminus could form an alpha -helix. The predicted secondary structure of S2 cDNA suggested that a hairpin and a stem loop are present in the 5'-end within 50 bp, and a stem loop in the 3'-end within 50 bp. RDV S2 partial and full-length sequences were then cloned into expression vector pET-11d and pTrcHisC. SDS-PAGE and Western blot proved that amino- and carbon-termini of P2 were successfully expressed in E. coli.