Sequence analysis of DNA methyltransferase gene from Largemouth bass ulcerative syndrome virus and its expression in prokaryote.

In order to further reveal the characteristics of Largemouth bass ulcerative syndrome virus(LBUSV) isolated recently,and further study the group of LBUSV,DFV(Doctor fish virus) and LMBV(Largemouth bass virus),the full-length DNA methyltransferase(DNA MTase) gene was analyzed and expressed using prokaryotic system.A about 200 bp core fragment was amplified and sequenced,and the full-length of DNA MTase was identified by genome walking.The open reading frame(ORF) of DNA MTase gene was 663 bp encoding 220 amino acids(GenBank accession No.GU256634).Motif analysis indicated that LBUSV DNA MTase protein contained blocks Ⅰ,Ⅳ,Ⅵ and Ⅷ,and cofactor binding sites,substrate interacting sites and DNA binding sites were also found in LBUSV DNA MTase,and the proline-proline-cysteine tripeptide was proposed catalytic site.Additionally,the primers were designed according to DNA MTase ORF,and the PCR products were inserted in vector pBV220 and transformed to host Escherichia coli DH5α.After temperature inducement and SDS-PAGE analysis,the recombinant expression bacteria produced a special protein about 25 kD in molecular weight.The proportion of recombinant protein in total bacterial protein was about 30%.Characteristic analysis of DNA MTase gene showed that the predicted protein may play a role of DNA methyltransferase,and confirmed that the taxonomic status of LBUSV is genus Ranavirus of the family Iridoviridae.