The Characteristics And Antitumor Activity Of Parasporins From Lysinibacillus Sphaericus X050

The soil sample was collected from the mountain of yuelu in changsha, and was screened a bacillus, which was detected the antitumor activity, and combined with the morphology observation and physiological, biochemical characteristics, 16 s rRNA genes Blast homologous sequence alignment analysis to determine a novel Lysinibacillus sphaericus. The Lysinibacillus sphaericus had parasporal crystal and highly toxicity to tumour cell, then was named Lysinibacillus sphaericus BsX050, hereinafter referred to as BsX050(CCTCC NO: 2014160 M).The fermentation growth curve of Bs X050 strain was determined and the supernatant of different periods was collected to test the antitumor activity. The results showed that the fermentation supernatant of BsX050 strain had significance toxicity against B16 cells and HeLa after entering stationary phase. Precipited with 60%ammonium sulfate, the antitumor spectrum of bold protein was determined. The results showed that the fermentation supernatant of BsX050 strain had a highly toxicity against B16, 4T1, Hep-3B, HT29,MCF-7 or HeLa and had a broad spectrum of anti-tumor activity.Further, Separation and purification condition of antitumor activity in fermented liquid was preliminary optimized by AKTA and UHPLC.Parasporal crystal of BsX050 strain was extracted and observed by electron microscope. The results showed that the parasporal crystal presented the circular or elliptic, then its structure was loose and compactness was bad. Parasporins was Surface protein slpC OS and its molecular weigh was 125 kDa by SDS-PAGE analysis and in-gel digestion. Analyzed the influence on parasporal crystal production of Zn2+ showed parasporal crystal production was improved when the concentration of Zn2+ was 10-6 mol/L and parasporal crystal productionwas inhibited when the concentration of Zn2+ was 10-3 mol/L. Then the solubility and stability of parasporins was analyzed, the results found that a large number of parasporins was dissolved out when 50 mM Na2CO3 without beta mercaptoethanol at pH 12.5. The volume ratio of trypsin and parasporins was 1/10 and parasporins was hydrolyzed completely at 37 ℃ for 3 h. The morphology of 4T1 and Hep-3B change significantly was observed after cells were treated with actived parasporins for 24 h by inverted microscope and scanning electron microscopy(sem), however, the morphology of HUVEC was unchanged. MTT assay found actived parasporins had a highly toxicity for 4T1 and Hep-3B and inhibited the proliferation of Hep-3B and 4T1 in a dose-dependent manner and time-dependent manner, but had no obvious effect on cell proliferation. Submicroscopic structure of 4T1 and Hep-3B was impaired, nucleus shrinked and diminished, tubulin decreased and rounded up on the nucleus, then cell rounded,mitochondria fluorescence intensity weaken, but HUVEC was not obvious damaged by confocal observed. 4T1 and Hep-3B treated with parasporins was detected DNA Ladder by gel electrophoresis. Flow cytometry showed parasporins induced 4T1 apoptosis, and cell was blocked in G0/G1 phase. Western blotting found that Bax protein and PARP shear protein expression up-regulation and Bcl-2 protein expression down- regulation. The above results show that parasporins had a highly toxicity to 4T1 and Hep-3B and impaired submicroscopic structure of cell, induced 4T1 cell apoptosis and cell was blocked in G0/G1 phase, but had no toxicity to HUVEC obviously. This provided Strain resources and laied theory foundation to the application of parasporins from Lysinibacillus sphaericus as well as the research on mechanism of antitumor.