| Background: Throughout the world, cancer is one of the most serious diseasesthreatening the health of human beings, due to the complexity of cancer pathogenesisand effective treatment difficult. Although modern medical research has made greatprogress, but still the majority of tumors can be cured. The genetic and environmental factorslead to the occurrence of malignant tumors. Oxidative stress-induced DNA damage andits interference with the signal transduction pathway play an important role in thedevelopment of tumors.The vast majority of complex life forms are dependent on oxygen to survive, but itis a highly reactive oxygen molecules, biological decomposition of oxygen to produceexperience, including hydrogen peroxide (H2O2), free radicals such as hydroxyl radical(OH), superoxide anion (O2-), and lipid peroxide active oxygen (reactive oxygenspecies, ROS), these metabolites may do harm to nucleic acids, lipids and proteinstemporary or permanent directly or indirectly, and damage to the organism itself. Manyliving cells requires the enzymatic oxidation reaction, the oxidation reaction is ahydrogen proton or electron transfer between molecules, such metabolic reactions required life process is the main source of energy.Oxidation-antioxidative imbalance status cause the occurrence of many diseases,such as cancer, neurological diseases, atherosclerosis, acute respiratory distresssyndrome. Based on its own protection, the body needs various types of anti-oxidants tomaintain balance oxidative metabolism, such as glutathione, vitamin C, vitamin E andsome enzymes, including catalase (catalase, CAT), superoxide dismutase (superoxidedismutase, SOD), glutathione peroxidase (glutathione peroxidase, GPX) and so on.Antioxidant enzymes may protect cells from oxidative stress, disorders associated withtumor activity are closely related to the expression of antioxidant enzymes, which is animportant factor of tumor development.Natural antioxidants in medicine, food and other fields have broad applicationprospects, mushrooms are medicinal and edible fungus, antioxidants extracted frommushrooms have certain theoretical and practical value.Our preliminary experimentsproved that the Lentinula edodes C91-3fermentation broth has a good anti-tumor andantibacterial effect. Lentinula edodes C91-3fermentation broth contained a variety ofprotein components. From2005, this group of Lentinula edodes C91-3fermentation brothprotein (LFP91-3) was studied with the initial separation and in vitro anti-tumorexperiments. The results showed that the protein composition had good anti-tumoreffect, can induce tumor cell apoptosis. These properties were conducive to be a goodclinical drug. In recent years, to seek a new drug with little side effects and strongfunction and to improve the long-term survival of cancer patients is an urgent task toresolve.The foreign scholars’ results showed that the Lentinula edodes fungus extractionhas the inhibitory action to many kinds of tumor cell. Our previous study also found thatthe hybrid protein extracted from fungi LFP91-3can effectively inhibit tumor growth,directly kill tumor cells, and enhance the immunity of multiple anti-tumor effects.While, the composition of this protein complex contains a lot of low concentrations ofprotein components in a broad PH range and the molecular weight range. Therefore, thein-depth study on the protein composition of this group to continue making good clinical anti-tumor drugs is the current problem that we need to solve. So, we carried outthe study of Lentinula edodes C91-3expression profile detecting, transcriptomesequencing and expression, trying to find the most useful anti-tumor proteins.Currently, we have successfully selected the target protein sequences wereregistered in GenBank (GenBank Accession Number: KF682440), and namedLatcripin-3.The protein is a medicinal fungi in addition to other anti-tumorpolysaccharide outside the main pharmacological components. At present the report ofanti-tumor clinical trials of fungi protein or application were rare, there are widerdevelopment space and a larger tap potential in antitumor fungal protein. With thecontinuous further study of the antitumor activity of fungal protein, we believe that thedevelopment of anti-tumor fungal protein will become an increasing concern andattention to the people.Objective: This study by the expression profile, screening will identify Lentinulaedodes C91-3of anti-oxidant activity, anti-tumor effect and apoptosis-related protein theLatcripin-3in E.coli Rosetta-gami (DE3) strain recombinant expression, fermentation,and through its detection of biological activity to seek the most anti-tumor effect offunctional proteins to Lentinula edodes C91-3. At the same time, the spatial structure ofthis protein analysis and comparison to clarify induced apoptotic pathway in tumor cells,and then be verified by experiment.①To find and identify Lentinula edodes C91-3anti-oxidate, anti-tumor protein apoptosis-related protein gene sequences;physicochemical properties, analysis of protein secondary structure prediction of thespatial conformation and functional groups.②Construction of the pET32a-Latcripin-3expression vector Lentinula edodess C91-3strain of anti-oxidation, anti-tumor proteinLatcripin-3expression in strain Rosetta-gami (DE3), while the expression productswere identified.③Purification of the target protein Latcripin-3, A549lung cancer cellsto study its impact on human biological functions, explore the mechanism of inductionof tumor cell apoptosis.Methods:①To test the anti-oxidation and anti-tumor protein in Lentinula edodes C91-3,we conducted a high-throughput transcriptome sequencing experiment to obtain Lentinula edodes C91-3transcriptom sequences. Through a comprehensive analysis ofthe GenBank and Swiss-Model databases, we preliminarily screened the transcriptomesequence correlated to apoptosis. Total RNA was extracted from the Lentinula edodesmycelium strain C91-3. According to the transcriptome sequence, we designed primersand used the3’-Full RACE and5’-Full RACE method to produce the full-length gene.According to the results of transcriptome sequencing, molecular biology softwareanalysis Latcripin-3protein physicochemical properties, amino acid composition andsecondary structure, and predict their functional domains may exist in the tertiarystructure of the homology modeling.②the Latcripin-3gene was cloned into the E.coliRosetta-gami (DE3)expression vector pET32a (+) to construct prokaryotic expressionplasmid pET32a (+)-Latcripin-3. Use hot conversion methods to transform this plasmidinto E.coli Rosetta-gami (DE3) and to express the target protein. Western blot analysismethod and mass spectrometry identified the expression products. With affinitychromatography separation and purification methods of the objective protein anddetermination of protein concentration.③Protein which has been identified of variousconcentrations of induced expression was interacted in human lung cancer cell lineA549, the inhibition rate of the target protein was determined by MTT, by flowcytometry testing objective protein tumor cell apoptosis and cell cycle, by electronmicroscopy method to detect the impact of various tumor cell morphology.Results:①By double digestion product DNA electrophoresis and gene sequence, wecan assure that the fragment which inserted into the pET32a (+) is the Latcripin-3genefragments. Through the analysis of the Latcripin-3protein domains, we can find that theprotein tertiary structure is very special, which having a plurality of consecutive α helix,forming a pocket-like spatial structure. The protein has a peroxidase superfamilydomain in upstream and a DUF3415domain in downstream. Two domains contribute tothe bioactivity of the protein.②Western blot showed that protein expression for thepurpose of containing histidine-tagged proteins. The process of induced expression thetarget protein is considered to production after3hours complete after adding IPTG, thetotal expression amount of the production keep relatively constant with time. By affinity chromatography, the successful purification of the target protein, protein electrophoresisshowed a single band object. After urea gradient dialysis, restore the spatial structure ofthe protein, and the use of centrifugal ultrafiltration protein concentrate. By LTQ massspectrometry to verify the biological activity of the protein. We use the Bradfordmethod to measure the concentration of protein by Nanjing KGI BCA proteinconcentration kit. To detect the expression of the protein concentration was determined0.736mg/mL. The standard curve was y=38.845x-2.5791, R2=0.9958.③Antioxidantexperiments protein Latcripin-3on DPPH scavenging was increased in a concentrationdependent manner compared to ascorbic acid, which was used as the positiveantioxidant control in this investigation. In low concentration protein Latcripin-3andascorbic acid, the bioactivity of scavenging DPPH has no statistical significant(P>0.05).④MTT assay showed that the different roles of the time (24hours,48hours), a different protein concentrations in A549cells (ATCC CCL185) growthinhibition (OD495) compared with control group differences were statisticallysignificant (P <0.05).7.5μg/mL,15μg/mL,30μg/mL and60μg/mL proteinshowed significant inhibition rate concentration-dependent and time-dependent manners.⑤Apoptotic function by flow cytometry showed that this protein in a finalconcentration of30μg/mL,60μg/mLwith the ability of early apoptosis of A549cells,induction of apoptosis compared with control group differences were statisticallysignificant (P <0.05), at a concentration of60μg/mL role in the induction of apoptosisstrongest when.⑥Flow cycle showed that with Latcripin-3protein concentrationincreases, G0/G1phase proportion has decreased; Proportion of G2/M phase wassignificantly decreased, and in Latcripin-3protein concentration of60μg/mL, thepercentage of G2/M phase turn to0(P <0.05). S phase was significantly higherproportion of obvious trend when Latcripin-3protein concentration of30μg/ml and60μg/mlS period there was a significant difference (P <0.05), and more stagnant growthin S phase.⑦Electron microscopy showed that cells Latcripin-3protein on apoptosisin the state after the presentation of apoptotic cells smaller size, cytoplasmicconcentration, increased cytoplasmic vacuoles, chromatin condensation, and edge set or broken into irregular lumps swelling of mitochondria.Conclusion:①Compared transcriptomic sequence of Lentinula edodes C91-3strainstranscriptome obtained for comparison analysis, screening is likely to determine thetranscriptional activity of antioxidant enzymes sequences Latcripin-3, primers weredesigned based on this sequence, with the3’-Full RACE (Rapid Amplificatioon ofcDNA Ends),5’-Full RACE method was successfully obtained Latcripin-3full-lengthgene, by the analysis of physical and chemical properties of this protein secondarystructure prediction of the spatial conformation and functional groups. Latcripin-3protein has two functional domains: a peroxidase domain and an unknown DUF3415domain with its biological function may play a role in common by these two domains.②The recombinant plasmid pET32a (+)-Latcripin-3, and successfully expressed in E.coli Rosetta-gammi (DE3) host, protein Latcripin-3was purified by affinitychromatography success for further research Latcripin-3the biological function of thefoundation.③Lentinula edodes C91-3strain of transcription histone Latcripin-3with afree radical scavenging antioxidant activity; Latcripin-3protein in A549human lungcancer cells induce apoptosis in early and make A549lung cancer cells in the S phasearrest, inhibition of the tumor cell growth. Specific biological mechanism remains to beelucidated. This experiment further revealed Lentinula edodes C91-3strain of anti-tumormechanism of the foundation. |
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