| Objective:In recent years our seminar found that the Lentinula edodes C91-3fermentationbroth, fermentation broth of total protein and its fractions protein not only had the invivo inhibition of tumor growth role but also had a significant role of in vitro directlykill the tumor cells. While, from the concentration of protein extract in a large numberof fermentation broth, stability can not fully meet the trials and future production needs.This experiment withdrew the total protein in the predecessor foundation from theLentinula edodes C91-3fermentation mycelium and carried on the separation purification,to obtain massively by the time highly effective had the anti-tumor active sole protein,and carry on the amino acid sequence appraisal to it, lay the foundation for the next stepgenetic engineering expression as well as the clinical practice.Methods：1. Fermentation mycelium protein gain Obtained the Lentinula edodes C91-3mycelium through the fermentation pot fermentation technology, using methods and soon refining, gentrifugalism obtained the fermentation mycelium protein to extract, againthrough the salting out, the dialysis, the freezing and drying, the gelatin filtrationchromatographic analysis carried on the separation purification to the fermentationmycelium thick protein, examined in various eluent according to the elution curve withthe SDS-PAGE electrophoresis the protein purity and the estimate protein molecularweight. To get a single protein component of the ingredients.2. Sole protein activeness examination Determined different density10μg/mL,30μg/mL, the100μg/mL sole protein with methyl azo thiazole blue colorimetric method (MTT) to the normal cell (human embryo liver, lung original generation of cell), thetumor cell (A549, Hca) affected24h,48h,72h cytotoxin activeness; Using flows thetype cell meter to examine this three density sole protein to A549, the Hcatumor cellaffected for24h, the induction tumor cell early time perished weakly with the laterperiod perished weakly and hindered to the A549cell cycle, to analyze its possibleanti-tumor mechanism; Induced the A549cell using the electron microscopeobservation sole protein to perish weakly the morphology change.3. Sole protein amino acid sequence appraisal Cutting after the SDS-PAGEelectrophoresis obtained sole protein banding, determined the sole protein peptidequality fingerprint with matrix assistance laser analysis ionization flight time massspectrum (MALDI-TOF-MS) to score (PMF) through to inquire the recognition waysole protein in the NCBI database to carry on the preliminary appraisal regarding this.Results：1.Using the homogenate,(NH4)2SO4salting out, dialysis, freeze-dried, we gotfermented mycelium crude proteins of C91-3. Eluted by the gel filtration chromatographyin8cm/h flow rate, the Microsoft Excel software drew the elution curve for us,whichcontained4elution peak.The results of SDS-PAGE electrophoresis demonstrated thatprotein at peak3was the single protein which molecular weight is about26kDa.2. MTT toxicity test showed that the single protein can obviously inhibit theactivity of the A549and Hca tumor cells in vitro. We used the single protein toinhibit the A549and Hcatumor cells at the concentration of10μg/mL,30μg/mL,100μg/mL and at the time of24h,48h,72h.The result showed us that the moreconcentration of single protein and the longer time of reaction we acted, the higherinhibition rates of A549and Hca tumor cells.The inhibition rate from a minimum of17.23%,11.25%increase to a maximum of76.21%,62.80%. There was statisticallysignificant differences between those groups(P <0.05); The inhibition rates of the twocells at the concentration of10μg/mL were both lower than the positive control DDP atthe concentration of5μg/mL. When A549cells at48h,72h and Hcacells at72h,therewere statistically significant differences between them (P <0.05).When the A549cellsat the concentration of30μg/mL,100μg/mL and the Hcacells at the concentration of100μg/mL,the inhibition rate was higher than control groups. There was statisticallysignificant differences between the control group and experiment group (P <0.05).Flowcytometric analysis (Annexin/PI decoration methods)showed that the single protein caninduce apoptosis of tumor cells.Different concentrations of a single protein used in A549and Hca tumor cells had different apoptosis rate.With the increase concentrationof protein, the A549and Hca tumor cells early apoptosis rate were3.62%,9.65%,19.71%,1.70%,4.91%,19.94%. There was statistically significant differences betweenthe different groups (P <0.05). Using Flow Cytometric Analysis(PI decoration method)to detect the different concentrations of the single protein acting on the A549cellscycle.The result showed that with the increase concentration of the single protein cellswe used, the G1phase cells were gradual increasing and the S phase cells weredecreasing,but the variation of G2/M phase cells is not obvious,and little changes in total.Compared with the control group,it is showed that the G1/S phase was retard. At theconcentration of30μg/mL used in A549cells for24h, TEM showed that the tumor cellsbegan shrinkage, the chromosome set of edges, intracytoplasmic had vacuolization andso on. MTT toxicity test verify that the protein have no significant inhibition in normalcells (human embryonic liver, lung primary cells).There was on statistically significantdifferences between the different concentration and time（P>0.05）.3.Using MALDI-TOF-MS to detect the PMF.There was no fitted protein with thePMF in NCBI database. Initially confirmed that it may be unknown protein.Conclusion:1. Using the homogenate to get fermented mycelium crude proteins of C91-3. Wegot4elution peak by salting out, dialysis and gel filtration chromatography. Protein inpeak3was the single protein which molecular weight is about26kDa.2. The experiments have proved that the growth of A549, HcaCell gweresignificantly inhibited by the single protein in vitro. With the longer time and thehigher concentration of single protein we used the action of inhibition was moreobvious.It was concentration and time dependent. Using Flow cytometric to analysecells apoptosis rate and cells cycle;And using the TEM to observe cell morphology.Theobservation of cell showed us that the Single protein can inhibit growth of tumorcells by inducing apoptosis and retarding G1/S phase.However, there was no significantinhibition in normal human embryonic cells.3.Detecting the single protein in NCBI database，There was no fitted protein inNCBI. Initially confirmed that it may be an unknown protein. |
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