Expression And Cytotoxicity Analysis Of Bombyx Mori Bidensovirus NS1 Protein

Bombyx mori bidensovirus(Bm BDV) is a chronic virus and it can specifically infect Bombyx mori. Silkworm will survive about 7 to 12 days when it was infected with the virus, The symptoms of Bm BDV-infected silkworm are similar to that of silkworm infected with Bombyx mori desovirus. Bm BDV was originally named typeⅡ Bombyx mori desovirus. According to the specific genome organization, virus particles and DNA polymerase gene coding region contained in vial genome, 184 members of the International Committee on Taxonomy of Viruses voted to agree with the proposals to create a new genus, Bidensovirus, as well as a new family,Bidnaviridae, and Bombyx mori bidensovirus was designated as the type species of the Bidensovirus genus,.Bm BDV ns1 consisted of 951 nucleotides encoding a predicted 316-amino acid protein, which was identified to be a Non-structural protein involved in viral replication. The relative molecular weight of Bm BDV NS1 is 36 k Da and isoelectric point is 8.7. Previous study showed that the NS1 protein is a multi-functional protein,not only possessing ATPase and helicase activity, but also binding with the specific DNA sequences. Additionally, phosphorylation can regulate Bm BDV NS1 protein biological activity. To further study the effect of Bm BDV NS1 on silkworm development, silkworm were orally fed with purified Bm BDV NS1 or subcutaneously injected with Bm BDV NS1 in this study,.Recombinat plasmid for the expresson of Bm BDV NS1 fusion with GST was constructed, and optimizating translation initiation codon and a series of varied conditions were designed to study the optimal yield of target protein.. The purified target protein was subjected to MALDI-TOF-MS analysis, which was used to evaluate the toxicity on Bm N cells and silkworm. Purified Bm BDV NS1 was feed directly to silkworms(306 strains) and subcutaneously injected into the silkworms.Additionally, different concentrations of target protein were added into the culture medium of Bm N cells. The results showed that the mutations in TIR of Bm BDV ns1 have no effect on improving the efficiency of expression of Bm BDV ns1, and Bm BDV NS1 protein may be cytotoxic to cells and induces death of silkworm.Additionally, Bm BDV NS1 could reach its the highest yield at 20 ℃ compared with that of target protein in 16 ℃, 28 ℃ and 37 ℃.Moreover, previous studies showed that interactions between Bm BDV NS1 protein and Bm BDV DNA polymerase as well as silkworm serine protease Bm SP142.To further disclose the specific interaction sites, A series of truncations were expressed in E.coli purified from the lysates, which were respectively used to perform GST-pull down experiments in vitro. The results indicated that the interactions between Bm BDV NS1 and these truncations are weak. It was inferred that the truncations may affect the conformation of target protein or their interactions may be mediated by other proteins.In conlusion, these above results indicated that the low temperature induction at20 ℃can improve the yield of Bm BDV NS1 in soluble form, and the target protein was purified from lysate of E.coli successfully, and the purified Bm BDV NS1 can induce silkworm death and inhibit the proliferation of Bm N cells.