Study On Bombyx Mori Nuclear Polyhedrosis Virus (BmNPV) Ubiquitin And Purification Of Bombyx Mori Ubiquitin

Ubiquitin(UB) is a small polypeptide of 76 amino acids, which highly conserved in eukaryotes from insects to humans and consist in cytoplasm and nucleolus. UB degradated cytoplasmic and nucleolus proteins efficiently, particularly and selectively via a ATP -dependent Ubiquitin-proteasome pathway (UPP). UB has many functions, including helping formation of ribosome and inner-engulfment of cell. At present, insect viruses are the only viruses known to encode UB.We cloned the fragment of UB gene-39 K from the genome of BmNPV-ZJ. Sequencing and bioinformatics analyses indicated that UB-39K gene consisted two integrated ORF, BmVUB and BmV39K. The sequence of UB was as same as BmNPV-T3's, while it was quite different with other insect viruses'. The nucleotide acids sequences difference between 39K and BmNPV-T3 was only three while the homology was very low between 39K and insect pox virus. The phylogenetic trees using BmVUB and BmV39k sequences was consistent to that based on the other amino acids sequences of the baculovirus. The second structure of BmUVB and BmV39K included a-helix and random coil. The modification rate of BmV39K was higher than BmUVB by SUMO, which had certain effects on BmUVB and BmV39K.In this paper, we constructed two kinds of prokaryotic expressing vector, pETBmVUB and pGEXBmVUB. The expression level was 50% and 35% of total protein respectively, which were all soluble. We obtained the antibody against fused protein (His) 6-BmVUB purified by affinity chromatography. And the effect of antibody refined with deposited and chromatography, was up to 3×10^-5. Western Blotting analysis of antibody specification was successful.The phagocyte 12 peptides pool was constructed by affinity selection, using fused protein(His)₆-BmVUB as target. We identified positive phagocyte clone with ELISA, analysed its DNA sequences and homology comparition and detected the effect of phagocyte containing fused peptides on the multiplication of BmN cell with MTT. The results showed that the 20 phagocyte clones, which was selected at random after three times selection, could combine with BmVUB with ELISA analysis. 10 clones were selected, sequenced and deduced its amino acid sequence, which could bedivided into 5 types of amino acid sequence, containing at least three hydrophobic amino acids. The further identification indicated that free BmVUB could block the combination of 10 positive phagocyte clone with solid BmVUB. The difference of effect of five kinds of phagocyte combined peptides on growth and multiplication of BmN cell was significant. Especially, the peptide VAPHHAYAPMRT was concentration dependent, which meaned that it stimulated growth of cell intensively with low concentration while inhibited growth of cell strongly with high concentration. The result could provide technical assistance to design peptide drug against BmVUB.We identified two kinds of DNA binding protein combined with UB sequence with non-radioactive Southwestern Blotting from nucleus protein using a modified protocol, whose molecular weight was 66 KDa and 70 KDa respectively. The combination characteristics were specific and could be competitively inhibited by BmVUB.We isolated and purified the fused protein with six histidine using prokaryotic expressing system through a series steps, including affinity chromatogram, gelatin chromatogram and reverse-phase HPLC. By analysing with mass spectrum, the amino acids sequence of every fragment is homologous with known UB sequence. We concluded that the expression product was BmVUB.This research provided experimental evidence for the development of peptide drugs against UB and virus UB further.