Analysis Of Partial DNA Sequences Of Bombyx Mori Densovirus (Zhenjiang Strain) And Expression Of Its Major Structural Protein (vp) Gene In Escherichia Coli

An attempt to identify the relationship between Bombyx mori Densovirus (BmDNV) Zhenjiang strain and BmDNV Yamanashi strain, partial DNA sequences of BmDNV (Zhenjiang strain) were determined and compared with the corresponding region of BmDNV (Yamanashi strain). The major viral structural protein (vp) gene was expressed by Escherichia coli (E. coli) expression system and examined by Western-blotting assay.I. Based on nucleotide sequences of BmDNV (Yamanashi strain) VD1 segment, oiigonuclotide primers was designed. Employing the designed-primers, a fragment of nucleotide sequences about 1.5 Kb, which is speculated to be the major structural protein (vp) gene, was amplified from the genomic DNA of BmDNV (Zhenjiang strain) by polymerase chain reaction (PCR).Sequenced and analyzed with Dna-star software, the nucleotide sequence of the cloned structural protein (vp) gene and its deduced amino acid sequence are highly homologous with its homologue in BmDNV Yamanashi strain, with the identity of 98.1% and 98.6% respectively. Compared with Yamanashi strain, 29 bases and 7 amino acids of the vp gene were substituted in Zhenjiang strain, respectively. No reading frame shift mutant was observed. The newly determined sequence has been submitted to GeneBank and the accession number is AY 236978.2. Based on nucleotide sequences of BmDNV (Yamanashi strain) VD2 segment, oiigonuclotide primers toward between two open reading frames (ORFs) on DNA of the virus was designed. Another fragment of nucleotide sequences about 1.0 Kb was generated from BmDNV (Zhenjiang strain) by polymerase chain reaction (PCR).Sequenced and analyzed with Dna-star software, the similarity between the corresponding region of Zhenjiang strain and Yamanashi strain was 98.5%. 15 nucleotide mutants, including 3 gaps, were found in the segment.3. On-line searching with Blast software in NCBI data base, no homologous sequences were found except DNA sequence of Densovirus Yamanashi strain.4. The major structural protein (vp) gene of BmDNV (Zhenjiang strain) was inserted into between the EcoRI and BamHI sites of the multiple cloning sites (MCS) of the expression plasmid pET-28a under the inducible promoter LacZ. Transformed and expressed in E.coli BL21(DE3), a 53KD protein, identical to the predicted molecular mass, was demonstrated by SDS-PAGE analysis. The product can react with polyclonal antibody of virus specifically by Western-blotting assay. This indicated that thecloned gene was viral structural protein gene.5. The above results indicate that the genome of BmDNV Zhenjiang strain composes of two DNA segments as BmDNV Yamanashi strain does. The two BmDNVs are highly homologous in nucleotide sequences (about 98%) and no other homologous sequence was found in NCBI data base. These results reveal that the two BmDNVs are different isolate of the same virus.