| Lentinula edodes C91-3is an edible mushroom fungi isolated from BasidiomycetesUmbelliferae fungi. Currently, our group has confirmed Lentinula edodes C91-3fermentation broth crude extract protein can induce tumor cell apoptosis, which maybe related to the regulation of cell cycle. On this basis, Lentinula edodes C91-3bytranscriptome analysis initially screened17functional genes associated with apoptosis,using RACE technology to get their full-length gene sequences and the correspondingproteins sequences. This study is concerned the second gene of the17functional genes,named-latcripin-2gene (referred to as LP-2). At present, we have successfullyregistered the target protein sequence selected from transcriptome sequencing in theGenBank (Accession Number: KF668033).Objective: This study by the expression profile, screening will identify Lentinulaedodes C91-3of apoptosis-related protein the Latcripin-2in Ecoli. Rosetta-gami(DE3)recombinant expression, fermentation, and through its detection of biological activityto seek the most anti-tumor effect of functional proteins to Lentinula edodes C91-3. Atthe same time, the spatial structure of this protein analysis and comparison to clarifyinduced apoptotic pathway in tumor cells, and then be verified by experiment.①Tofind and identify Lentinula edodes C91-3apoptosis-related protein gene sequences andfunctional groups.②Construction of the prokaryotic expression vector to Lentinula edodes C91-3Latcripin-2apoptosis related proteins in Ecoli. Rosetta-gami(DE3)induced expression, while the expression products were identified.③Inducedexpression of Lentinula edodes C91-3apoptosis related protein the Latcripin-2study,investigated the induction of tumor cell apoptosis in human lung cancer cell line A549biologic functions of mechanism.Methods:①To test the anti-tumor protein in Lentinula edodes C91-3, weconducted a high-throughput transcriptome sequencing experiment to obtain Lentinulaedodes C91-3transcriptom sequences. Through a comprehensive analysis of theGenBank and Swiss-Model databases, we preliminarily screened the transcriptomesequence related apoptosis. According to the transcriptome sequence, we designedprimers and used the3’-Full RACE and5’-Full RACE method to produce thefull-length gene. According to the results of transcriptome sequencing, molecularbiology software analysis Latcripin-2protein physicochemical characterisation, aminoacid composition and secondary structure, and predict their functional domains mayexist in the tertiary structure of the homology modeling.②The latcripin-2gene wascloned into the prokaryotic expression vector pET32a(+) to construct prokaryoticexpression plasmid pET32a(+)-latcripin-2. Electricity conversion method was used totransform this plasmid into Ecoli. Rosetta-gami(DE3) and to inducible expression.Western blot analysis method was used to identify the expression products. Separatedand purified the target protein with affinity chromatography and decided the proteinconcentration by Bradford method.③Protein induced expression which has beenidentified of various concentrations was interacted in human lung cancer cell line A549,the inhibition rate of the target protein was determined by MTT, by flow cytometrytesting objective protein tumor cell apoptosis and cell cycle, by electron microscopymethod to detect the impact of various tumor cell morphology.Results:①Through the analysis of colonies PCR and gene sequence, we canassure that the fragment which inserted into the pET32a(+) is the latcripin-2genefragments. Through the analysis of the Latcripin-2protein domains, we can find thatthe protein tertiary structure is very special, which is Pkinase.②Results shows that protein expressed in Ecoli. Rosetta-gami(DE3) is the target protein by Western blot.We measure the concentration of protein by Bradford method. The linear equation isy=0.0299x+0.0123, R2=0.9921.③The reasult showed that different concentration ofprotein have different activities on the human lung cancer cell line A549growthinhibition. And the activities has a significant differences for control group (P <0.05).Flow cytometry results showed that this protein has the ability of inducing tumor cellsapoptosis. When the concentration of the protein in200μg/ml has the strongest actionof apoptosis. when the final concentration is100μg/ml,200μg/ml, the apoptosis rateof the tumor cells has a significant difference between the experimental group andcontrol group (P＜0.05).Conclusion:①Compared Transcriptomic sequence of Lentinula edodes C91-3through GenBank, we determine the apoptosis-related transcription sequence calledlatcripin-2. It is successfull for us to get latcripin-2gene through the method of3’-FullRACE,5’-Full RACE.②Recombinant plasmid pET32a(+)-latcripin-2has beensuccessfully constructed; The apoptosis-related protein Latcripin-2has beensuccessfully expressed in Ecoli. Rosetta-gami(DE3); the expression protein Latcripin-2was successfully purified by affinity chromatography.③Latcripin-2protein caninduced human lung cancer cells A549apoptosis. The specific mechanism needs to beelucidated in the furture. This experiment lays the foundation for further anti-tumormechanism study of Lentinula edodes C91-3. |
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