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Preliminary Isolation and Purification of Effective Proteins from the Fermentative Liquor of Lentinus Edodes C91-3 and the Study of Its Anti-tumor Mechanism

Author: WuYongHong
Tutor: HuangMin
School: Dalian Medical University
Course: Pathogen Biology
Keywords: Fermentation broth Mushrooms Protein Gel filtration chromatography Apoptosis
CLC: R285.5
Type: Master's thesis
Year: 2009
Downloads: 91
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Abstract


Objective: This research group has been confirmed by experiment mushrooms C the fermentation broth 91-3 strain mycelium crude protein mixture ingredients LFP 91-3 in vivo inhibition of tumor growth in vitro direct anti-tumor activity. We focus on separation and purification of LFP 91-3 the efficient protein component Expand work by ion exchange chromatography separation to obtain a single protein component the LFP 91-3-A < / sub>, and confirmed in vitro significantly inhibited H 22 , the S 180 tumor cell growth. Analysis of its anti-tumor mechanism may be associated with the induction of apoptosis. On this basis, we continue to separate purified LFP 91-3 other active protein ingredient and continue to explore better separation and purification methods, making it easy to get a more effective active protein. This experiment was carried out by gel filtration chromatography on LFP 91-3 component separation, purification, and protein extraction for the determination of the in vitro anti-tumor activity, a preliminary study the mechanism and identification experiment. Thereby facilitating separation and purification of the active ingredient for the decrease in the crude protein component, the present experiment also attempt to lengthen the fermentation time. Method: (1) Separation and Purification: shiitake C 91-3 mycelia fermentation broth at 4 ° C the crude protein by salting out, dialysis, Sephadex G-100 gel filtration chromatography separation. Determination of protein concentration in the liquid collected in each tube, and so draw the elution curve with Coomassie blue method. (2) functional assay and preliminary identification: tetrazolium blue method (MTT) were measured each elution peak collect fluid in the protein concentration 20ug/ml, 30ug/ml 40ug/ml on S < Sub> 180 tumor cells in vitro growth inhibitory activity, duration of action is 24 hours, 48 ??hours and 72 hours. Each tube of the maximum inhibitory rate of the elution peak collected liquid were done silver staining of SDS-PAGE electrophoresis, and the purity and the molecular weight of the protein in the eluate of each tube was measured. Take electrophoretic bands a relatively single the Feng Jian number of tube collector were combined, concentrated proteins were extracted after-oriented experiments, dubbed concentration (5ug/ml, 10ug/ml 15ug/ml, 20ug/ml 30ug / ml, 40ug/m1), were determined by MTT assay the the S 180 tumor cell growth inhibitory activity in vitro, the role of time above. Measured using the Coomassie blue method and sulfuric acid - phenol given sugar method to extract the proportion of protein in the protein and sugar. (3) Mechanism: different concentrations of (20ug/ml, 30ug/ml, 40ug/ml) to extract protein S 180 tumor cells for 24 hours, the induction of tumor cell flow cytometry early apoptosis rate and late apoptotic rate in order to analyze the possible anti-tumor mechanism; TEM morphological observation by the role of the protein S 180 tumor cell apoptosis. (4) used in this experiment fermentation broth fermentation time is 3-4 days longer time than the previous group members. Results: the LFP of (1) mushrooms C 91-3 mycelium fermentation broth crude protein 91-3 by salting out, dialysis, Sephadex G-100 gel layer analysis, elution curve plotted using Microsoft Excel software, available I - IV 4 elution peak inhibition rate of each peak collected fluid was determined by the MTT assay, a third elution peak the collected liquid inhibitory rate highest. SDS-PAGE electrophoresis showed that the top of the third elution 23-26 collected liquid having a molecular weight of about 19kDa protein, very low molecular weight protein content. Coomassie blue method and sulfuric acid - phenol method shows that the protein in the protein and sugar ratio of about 3:1. (2) MTT assay showed protein in vitro significantly inhibited the S 180 tumor cell activity, the inhibition rate will be increased with the increase in protein concentration and the extension of time, concentration and time dependence. Extract protein interaction 20ug/ml 48 hours, S 180 cell activity inhibition rate 36.5%; improve concentration 30ug/ml proteins were extracted for 48 hours, the S 180 cells inhibition was 47.8%: When the concentration of 40ug/ml to 72 hours, to extend the duration of action on the S 180 cells activity inhibition rate of 76.7%. (3) double staining of Annexin / PI staining, flow cytometry determination of S 180 tumor cell apoptosis rate results show that the extracted protein can be induced in vitro S 180 cells apoptosis, different concentrations of extract protein (20ug/ml, 30ug/ml, 40ug/ml) the S 180 cells 24 hours the S 180 cells apoptosis rate (early late) were 1.38%, 1.59%, 2.17%, compared to 0.71% with the negative control has a significant difference. Tumor cell apoptosis rate increased with increasing concentration of extract protein in a dose - response relationship. Transmission electron microscope by extracting protein role of S 180 cells, the visible cell shrinkage, nuclear condensation and chromatin concentrated set of edges typical apoptotic morphology also apoptotic bodies. (4) The product obtained in the fermentation time after prolonged compared with the previous, the protein components in a decrease in molecular weight also decreased. Conclusion: the LFP of mushrooms C 91-3 mycelium fermentation broth crude protein 91-3 by salting out, dialysis, Sephadex G-100 gel filtration chromatography the four groups, wherein the first group and the third group of the protein in vitro the S 180 cells having a strong inhibitory effect on the growth of The third group of the strongest inhibition, in the group with a molecular weight of about 19kDa protein elution peak at the top of 23-26 collected liquid, and contains a quarter of the sugar component, suggesting that the glycoprotein. Of the extracted protein the S 180 cell growth has a strong inhibitory effect, its mechanism may be associated with the induction of apoptosis of tumor cells.

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