Cloning And Expression Of Nucleopolyhedrovirus ORF75 Gene To Bombyx Mori

With primers designed according to sequence(L33180) published on GenBank and Bombyx mori nucleopolyhedrovirus, a DNA fragment of BmNPV gene was obtained using PCR amplification and was further cloned into pMD18-T vector. The mature protein open reading frame of the DNA was obtained. The fragment was digested by two restriction enzyme BamH I / Xho I and was joined with pGEX-4T-2 plasmid which was digested by BamH I / Xho I , too. The pGEX/Bm75 was obtained, then pGEX/Bm75 was transformed into BL21. By inducing with IPTG, the GST-Bm75 fusion protein was checked by SDS-PAGE (SDS polyacrylamide gel electrophoresis) and Western bloting. The molecular weight of fusion protein was about 57kDa with a GST tag which provide a convenient way for seperation and purification of this protein.By bioinformatics analysis of BmNPV ORF75 gene, we identified that Bm75 gene was between 70485bp å’Œ 71264bp, molecular weight is 30.8kDa, theoretical pI is 7.67, formula is C₁₄₀₉H₂₁₅₇N₃₅₁O₃₉₀S₁₉, estimated half-life is beyond 10 hours in E.coli, total number of negatively charged residues (Asp+Glu) is 10.5%, total number of positively charged residues (Arg+Lys) is 10.8%, the instability index is computed to be 42.67 and the protein is unstable. By scaning Prosite data, we found 4 kinds of protein modified sites.The blast indicate Bm75 protein only exits in Lepidoptera virus.The phylogenetic tree showed the amino acid of Bm75 and Ac92 are just the same, the amino acid of Bm75 and Ro89 have high homology.