Preliminary Isolation And Purification Of Effective Proteins From The Fermentative Liquor Of Lentinus Edodes C_91-3 And The Study Of Its Anti-tumor Mechanism

Objective:Our research team have confirmed that composite of crude protein from the Fermentative Liquor of Lentinus edodes C_91-3,named as LFP_91-3,have inhibition for growth of tumour cells in vivo and killing them in vitro directly.We carry out our work to surround that efficient and single protein should be separated.A member of our research team has purified a single protein through ion-exchange chromatography on DEAE-cellulose column:LFP_91-3-A.Then she has confirmed that the protein could conspicuously inhibit activities of tumour cells H22 and S₁₈₀.Its mechanism of antitumor could be related with inducing cell to apoptosis by analysis.At the base of these circumstances,we continue to separate LFP_91-3 out to other effective protein and keep on exploring better methods for separatation to get quantities of single protein with activity of anti-tumor easily.In this experiment,we separated composition of LFP_91-3 by Sephadex G-100. Furthermore,the activity determination of antitumor in vitro was maked for extracted protein,followed with preliminar study on mechanism and experiments of identification.For reducing composition of LFP_91-3 in order to separate active composition easily,this experiment also tried to prolong Fermentative time.Methods:(1) Extraction,separation and Preliminarily identification:at 4℃,centrifuge,salt-out,dialyze and concentrate the fermentation liquid. The final solution flow through Sephadex G-100 column(100×0.5 cm), determine concentration of every tub by Bradford method and draw the eluting curve.Group collected liquid according to the curve.The restraining tumor rate gotten by MTT method demonstrate that the third fraction of protein have the strongest effect of restraining tumor.Purity and molecular weight of protein for 23-26 tubes of the third fraction are determined by SDS-PAGE electrophoresis.Protein to polysaccharide ratios in finally separated protein is determined respectively by Bradford method and sulphuric acid-phenol method.(2)Functional determination:Make every fraction of collected liquid into three concentrations of protein.(20ug/ml,30ug/ml,40ug/ml).Respectively,determine their activity of refraining the growth of S₁₈₀ in vitro by the MTT methods,while it takes 24,48,72 hours as the time of effect.Adjust protein concentration of the third fraction of collected liquid to 5ug/ml,10ug/ml,15ug/ml,20ug/ml,30ug/ml, 40ug/ml,then determine respectively their activity of refraining the proliferation of S₁₈₀ in vitro by the MTT methods with just mentioned time of effect.(3)Mechanism analysis:Separated protein of three concentrations (20ug/ml,30ug/ml,40ug/ml)persist to function to S₁₈₀ cells for 24 hours. Stain above cells with Annexin/PI stain kit and analyze possible anti-tumor mechanism by flow cytometry and Cell Quest software.Observe above cells under TEM for Apoptosis of the cells.Results:(1)According to the elution curve(Fig 1),compounds was divided intoâ… ,â…¡,â…¢,â…£elution peaks(four fractions),It could draw a conclusion that the third fraction of collected liquid had the highest rate of restraining tumor.The results of SDS-PAGE electrophoresis demonstrated that protein in 23-26 tubes of the third fraction most was the one whose molecular weight is about 19kDa.Contents of other protein was extremely low.Protein to polysaccharide ratios in the separated protein was 3:1 according to determining results of Bradford method and Sulphuric acidphenol method.(2)Results of MTT methods demonstrated that the separated protein could restrain activity of S₁₈₀ cells significantly and its rate of restraining tumor ascended as concentration of proteins ascended with the same as incubating time was prolonged,The restraining tumor rate depended on concentration and time.Treated with 20ug/ml the separated protein after 48h,the S₁₈₀ cells'inhibition rates were 36.5%;Increasing concentration to 30ug/ml,the rates became 47.8%;After treated for 72 hours by 40ug/ml the separated protein,Sis0 cells'proliferations were significantly inhibited,the inhibition rates were up to76.7%.(3)Flow cytometric analysis(Annexin/PI decoration methods)showed the S₁₈₀ cells apoptosis rates 24 hours after treatment of the separated protein of the concentration 20ug/ml,30ug/ml, 40ug/ml,the early apoptosis rate was 0.83%,1.11%,1.42%,the late apoptosis rate was 0.33%,0.25%,0.25%;All apoptosis rates ofexperi-mental groups were different statistically compared with negative control and apoptosis rates ascended as concentration of the separated protein increased.It showed a correlation between dose and effect.The cell nuclei became progressively pyknotic and were extensively fragmented and some characteristic morphologic features of apoptosis were revealed by transmission electron microscopy.Conclusion:Four fractions of proteins were obtained from the fermentation liquid containing crude proteins by centrifuge,salt-out, dialyze,concentrate and gel permeation(Sephadex G-100) chromatography. The second and the third fraction had stronger inhibition to proliferation of S₁₈₀ cells in vitro.The separated protein in 23-26 tubes of the third fraction,which chiefly contained the protein of molecular weight of 19kDa approximately,had the strongest inhibition.Its anti-tumor mechanism could correlate with inducing tumor cells to appear apoptosis.The separated protein contained a quarter of polysaccharide.