Study On The Cloning Expression, Purification And Biological Activities Of Transcript Protein Latcripin-9 From Lentinula Edodes C_91-3

Lentinula edodes C_91-3 is one fungus which has great application value and has been studied for years.It has been confirmed that the Lentinula edodes C_91-3 fermentation broth can induce the apoptosis of some tumor cells.Hereby,some genes that can induce the apoptosis of tumor cells has been screened and named.The full-length genes and the corresponding amino acid sequences were obtained by the RACE methods and the sequence blast.Here,the research object of this experiment is the Latcripin-9 gene of Lentinula edodes C_91-3.Objective: Obtained the Latcripin-9 gene sequence,functional domain fragment and the corresponding amino acid sequence by bioinformatics methods.To harvest more useful information,the secondary structure and tertiary structure were predicted by the bioinformatics software.The prokaryotic expression vector pET32a（+）-Latcripin-9 was constructed,the recombinant Latcripin-9 protein was expressed in E.coli Rosetta-gami（DE3）and identified after inducing by IPTG.The bioactivities and functions were predicted by the bioinformatics methods.The antioxidant activity was checked by different methods in vitro.The tumor cells were treated by the recombinant protein to study the effect on tumor cell biological function preliminarily.These researches may lay a foundation for the further study of the bioactivity and the potential functions of Latcripin-9 protein.Methods: 1.Extracted the total RNA from the mycelium of Lentinula edodes C_91-3.Complementary DNA library（cDNA library）was obtained by reverse transcription.According to the result of reverse transcription,sequencing the transcriptome by the high-throughput sequencing,the target sequence was obtained by blasting through the bioinformatics software.3’-Full RACE 、5’-Full RACE were adopt to obtain the Latcripin-9 full-length gene.The functional domain sequence of Latcripin-9 gene was acquired from NCBI and Pfam database.The amino acid composition,physical and chemical properties,even the bioactivities were acquired through the prediction of bioinformatics.2.The recombinant plasmid pET32a（+）-Latcripin-9 was constructed by cloning the functional domain fragment of Latcripin-9 gene to the prokaryotic expression vector pET32a（+）,then transforming the recombinant plasmid pET32a（+）-Latcripin-9 to E.coli Rosetta-gami（DE3）,the Latcripin-9 protein was induced by IPTG and identified by SDS-PAGE and Western Blotting methods.Affinity chromatography method was used for the purification of the Latcripin-9 protein,the purified protein was refolding by urea gradient dialysis method,BCA method was used to measuring the concentration of the Latcripin-9 protein.3.The circular dichroism spectra method was used to identify the refolding Latcripin-9 protein.4.The antioxidant activity of Latcripin-9 protein was checked by three different methods,including DPPH,FRAP,and ABTS methods.5.MTT method was used to observe the growth situation of human lung cancer cell A549 after the cell was treated with Latcripin-9 protein,the normal cells（Chicken Embryo Fibroblast cell and Vero cell）were used to detect whether the Latcripin-9 protein is toxic to normal cells at the same time.The transmission electron microscopy was used to observe the morphology impact of human tumor cells（lung cancer cell A549 and laryngeal cancer cell Hep-2）after being treated with Latcripin-9 protein.The influence of cells apoptosis situation of human lung cancer cell A549 was detected by flow cytometry.Results: 1.The full-length sequence of Latcripin-9 gene was acquired by RACE methods,the functional domain of Latcripin-9 gene was obtained by the PCR method.The functional domain is Dye-decolorizing peroxidase structural domain after predicted by bioinformatics.2.The result of double enzymes（the two restriction endonuclease enzymes EcoRⅠand XhoⅠ）digestion showed that the functional domain sequence was inserted into the prokaryotic expression vector pET32a（+）successfully.The expression of the Latcripin-9 protein was identified by SDS-PAGE and Western Blotting methods.The purified Latcripin-9 protein was harvested by affinity chromatography method.The concentration of target protein was measured by BCA method,the standard linear equation of the standard protein is y = 0.0462 x + 0.0108,R2 = 0.9986.The concentration of Latcripin-9 protein was calculated with the equation is 1.671mg/m L.3.The results of three common antioxidant activity assays in vitro showed that Latcripin-9 protein has significant antioxidant activity,but weaker than VC.4.MTT assay showed that Latcripin-9 protein is nontoxic for Chicken Embryo Fibroblast cell and Vero cell,but the protein can inhibit the growth of human lung cancer cell A549 significantly,and showed concentration-dependent and time-dependent as well,the inhibition rate approaches 30% when treated with Latcripin-9 protein（the concentration is 100 μg/m L）for 12 h.The cellular structure of cancer cells observed by TEM showed that Latcripin-9 protein can lead to autophagy of human lung cancer cell A549,induce apoptosis of laryngeal cancer cell Hep-2.The result of flow cytometry assay showed that target protein has killing effect of human lung cancer cell A549,and it can induce apoptosis on A549 cell.Conclusion: 1.Through the bioinformatics analysis of transcription sequence of the lentinula edodes C_91-3 strain,the Latcripin-9 gene with peroxidase activity was screened out,and the corresponding full-length gene sequences were obtained by RACE method.2.The recombinant plasmid pET32a（+）-Latcripin-9 was obtained by gene recombination technology successfully,the plasmid was transformed into E.coli Rosetta-gami（DE3）by heat transfer method.Latcripin-9 protein was purified by affinity chromatography,and the Latcripin-9 protein was successfully refolding by urea gradient dialysis.3.In vitro antioxidant activity study showed that Latcripin-9 protein has antioxidant activity,which lay a foundation for further study of its application in antioxidation.4.The MTT results showed the Latcripin-9 protein has antitumor activity,while it is nontoxic for normal Chicken Embryo Fibroblast cell and Vero cell,Latcripin-9 protein can inhibit the growth of both Hep-2 cell and A549 cell,also lay a foundation for further studies on the antitumor mechanism and biological functions of Latcripin-9 protein.