Preliminary Study On Hydralysin-2 Of Bacillus Bombyseptieus

Bacillus bombyseptieus is a kind of pathogen of silkworm, Bombyx mori. Bb has a circular plasmid with the molecular weight of 578 kb, which is similar with Bacillus thuringiensis, the same genus of Bb. The 384 th gene on the plasmid of Bb named hydralysin-2(GI:61249318), is similar with hydralysin from the green hydra(Chlorohydra Viridissima) which has selective toxicity to insects and parasporin-2 from Bacillus thuringiensis which recognizes and kills cancer cells. Based on the sequence similarity, there is two questions to be investigated in the present thesis. Firstly, is hydralysin-2 specifically insecticidal or cytolytic to cancer cells? Secondly, dose hydralysin-2 play a role in the pathogenesis of Bb to Bombyx mori? The main contents and results are as follows.1. The q RT-PCR results showed that hydralysin-2 transcription was upregulated when Bb infected silkworm compared with cultivate Bb. It suggested hydralysin-2 might be a virulence factor of Bb.2. SUMO protein was used as a fusion partner for the production of hydrlysin-2 in E. coli BL21(DE3). Hydrslyin-2 was purified using the combination of NI-IDA affinity chromatography,digestion by the SUMO protease and Sephadex G-25. The purified protein was about 28 k Da as determined on SDS-PAGE gel. MALDI-TOF/TOF-MS analysis confirmed that this protein was hydralysin-2.3. hydralysin-2 affected the Bm-12 cell proliferation by CCK8 assay.Pore-forming activity of hydralysin-2 to Bm-12 cell was detected by cytoplasmic LDH assay. Futhermore, scanning electron microscopy showed hydralysin-2 caused shrinkage, cracking on the Bm-12 cell sureface and the microvilli decreasd obviously. All the above data revealed that hydralysin-2 had toxic effect on Bm-12 cells.4. The toxicity of hydralysin-2 on silkworm in vivo was determined by injection or feeding to silkworm larvae. The results indicated that hydralysin-2 was lethal to silkworm larvae by injection. However, no mortality of larvae but the retarded growth effect on larvae was observed by feeding.5.The hydralysin-2 had no cytotoxic on Hep G2 cells by CCK8 assay. Its inhibition effect on cancer cells is still to be tested on other cancer cells.In summary, hydralysin-2 was lethal to the silkworm, showing hydralysin-2 was an virulence factor of Bb. More research work is needed to explore if hydralysin-2 is lethal to the lepidopteran pests or if it is worthwhile to be developed as a novel insecticide.