The Preliminary Study On Bioactivity And Isolation Of Protein Produced By DNN6Endophyte From Laminaria Japonica Aresch

In order to develop the use of marine biological resources, we have previously confirmedstrong broad-spectrum antimicrobial activity of the proteins of symbiotic bacteria DNN6fromthe brown seaweed laminaria japonica Aresch. This paper made a preliminary study to exploreits anti-tumor cell activity, hemolytic activity, cardiovascular activity and acute toxicityevaluation, and preliminary isolation of protein components. The conclusions of the paper are asfollows:(1) With MTT assay, obvious inhibition of DNN6proteins against four different lines of tumorcells (K562, HepG-2, MCF-7and MDA-MB-231) and human endothelial cells (HuvEC). TheIC50were58、126、126、127and115μg/ml respectively.(2) Hemolytic determination of DNN6proteins, the calculated HU50is3.6μg/ml. In vivohemolytic activity was determined in mice (180mg/kg, i.v.), however, no significant hemolysisby DNN6proteins was observed. Different ratios of mouse blood serum and BSA were addedinto the erythrocyte suspensions, hemolysis was significantly blocked. Osmotic protection ofhemolytic activity in the treatment series with PEG, when the DNN6proteins were added to a0.45%erythrocyte suspension containing PEG2000or PEG8000at a final concentration of25mM, the hemolysis was blocked obviously. The levels of MDA increased depended on the dosesof DNN6proteins. Confirmed the pore formation is one of the mechanisms of non-specific celldamage. We also found lipid peroxidation in erythrocyte hemolysis effect was an important rolein DNN6protein damage(3) Activity Evaluation of cardiovascular of DNN6proteins in the whole animal and on theisolated heart experiments found that the DNN6proteins has function of strong heart, longerduration of action, can be used as an indicator of active substances screened for furtherexperiments.(4) Acute toxicity test in mice, the mice showed malaise, scrunching, slowness, sluggishnessand horripilation within5min. The toxic manifestations of the mice obviously depended on thedoses of DNN6proteins and its recovery time were consistent with the severity of toxicmanifestations, even the time of death. We only observed slight effects on the serum biochemicalindexes by DNN6proteins. Obvious damage in heart and kidney organ. Other organs also havedifferent degrees of damage. The non-specific cytotoxicity and inhibition of hemoglobin to carry oxygen work together, leading to a variety of serious toxic symptoms in vivo, even death inmice.·(5) Protein Purification of the active ingredient, DNN6protein bands of different molecularweights successfully separated by gel filtration chromatography, but the gel filtrationchromatography can not successfully separated the active ingredient; strong anion exchangecolumn can be successfully separated DNN6protein according isoelectric point of differentproteins. The major antibacterial activity protein molecular weights include the protein of55KDand36KD.