Expression Cloning, Purification And The Study Of The Role Of Apoptosis And Nucleic Acids Of Latcripin-5

Lentinula edodes C91-3 strain by my research group for many years screened a medicinal fungus. The experimental group after a pair of Lentinula edodes C91-3strains years transcriptome sequencing and functional analysis,, thus initial screening17 genes associated with cell activity. We use RACE(rapid amplification of c DNA ends) technique to obtain the full-length gene sequences of all genes and their protein sequences corresponding to the screening. This study is concerned that all the functions of genes selected by the commander of Article 5 of the gene, so which was named latcripin-5 gene(referred to as LP-5). We used latcripin-5 gene by bioinformatics than to arrive at the gene nucleic acid degradation effect.ObjectiveIn this study, mainly through building Lentinula edodes C91-3 strain p ET32a(+)-latcripin-5 of prokaryotic expression vector, and prokaryotic expression vector induced expression in E. coli of Ecoli R. Biological activities induced by the expression product of the detection, to determine the nucleic acid to cells having Lentinula edodes occur degradation and biological activity of the functional protein.(1) Find and determine Latcripin-5 gene sequence and functional proteins through specific software.(2) The recombinant expression vector in the original Latcripin-5gene into E. coli carried Ecoli.R induced, and induced expression of product identification and function of biological activity, as its mechanism of action of nucleicacid cells do well prepared.Methodspart I(1) Total RNA was extracted from the selected strains Lentinula edodes C91-3mycelium, the laboratory results based on transcriptome sequencing data obtained by RACE technique to obtain the full-length latcripin-5 gene, bioinformatics software physical and chemical properties Latcripin-5 protein, amino acid composition were analyzed, and Latcripin-5 functional domain proteins to predict.(2) The domain gene latcripin-5 of Endonuclease-NS cloned into prokaryotic expression vector p ET32a(+),and thus construct p ET32a(+)- latcripin-5 this prokaryotic expression plasmid.(3)Prokaryotic expression of this recombinant plasmid was transformed into E. coli Ecoli.R and induce its expression, the induced expression of the product was subjected to SDS- polyacrylamide gel electrophoresis(SDS-PAGE) electrophoresis and Western blot methods were identified.(4)Nickel affinity chromatography column to separate the purified recombinantly-expressed Latcripin-5 protein.part II(1) The resulting purified Latcripin-5 protein refolding and desalination and concentrated, and the concentrated protein concentration was measured.(2) Protein and human genomic DNA obtained by the action of refolded and concentrated, with the digestion of the resulting protein refolding was detected activity exists.(3) The active role of the identified proteins in the presence of different concentrations of A549 human lung cancer cells and chick embryo cells with MTT(Methyl Thiazoly Ltetra Zolium, tetrazolium blue) The method for determining the inhibition of protein Latcripin-5 cells.(4) 33258 staining and flow cytometry were used to detect the role Latcripin-5 protein on apoptosis and cell cycle.The role of electron microscopy was used to detect Latcripin-5 protein on the cell micro-structures.Resultspart I(1) Experimental methods utilizing double digestion of determining inserted into p ET32a(+) of the gene fragment for the gene fragment latcripin-5. By Latcripin-5 protein domain analysis, proved domain Latcripin-5 protein is a non-enzyme(Endonuclease-NS) within the restriction endonuclease domain.(2) Use the results of SDS-PAGE and Western blot showed that the protein of E. coli was Ecoli. R expressed as Latcripin-5 protein.(3) SDS-PAGE electrophoresis can be seen by nickel affinity chromatography to the purification of the target protein.part II(1) SDS-PAGE electrophoresis and Western blot analysis showed that the protein refolding was desalted and concentrated after.(2) We measured the expression of the protein concentration using Bradford method, to obtain the linear equation for the standard protein y = 0.0537 x + 0.0021, R2 = 0.9950.(3) Results were digested with recombinantly expressed protein obtained by the method revealed that the protein of the nucleic acid having a concentration gradient, temperature gradient dependence.(4)The results obtained using the MTT assay showed obvious differences in concentrations of Latcripin-5 protein on tumor cell growth inhibition with the control group, and different concentrations of this protein in chick embryo cell growth inhibition comparison with the control group had the same differences.(5) The results obtained by flow cytometry showed that this protein can induce apoptosis, Latcripin-5protein concentration for inducing apoptosis when 200 μg / ml strongest; this protein on the cell cycle results show in Latcripin-5 protein role 200μg / ml of protein after major role in the G1 phase of the cell’s arrest, G2 phase unchanged, decrease in S phase.(6) 33258 staining showed apoptotic nuclei significant shrinkage phenomenon.(7) Electron microscopy to detect the results of the micro-structures on tumor cells showed cell nuclei occurred significant degradation phenomena, cell becomes large,reducing the concentration of cytoplasm.Conclusion(1) The whole transcriptome sequencing was carried out genetic strains of Lentinula edodes C91-3 comparison, according to the preliminary results of gene function annotation, the laboratory successful full-length latcripin-5 gene.(2) The experimental group was successfully constructed p ET32a(+)- recombinant plasmid of latcripin-5;Recombinant expression Latcripin-5 protein was successfully expressed inE. coli Ecoli. R; Latcripin-5 protein by nickel affinity chromatography column was purified success.(3) Latcripin-5 protein in the human genome with a nucleic acid digestion role, while the role of cellular nucleic acid with a restriction enzyme and its specific mechanism needs further study.