Study On The Expression Purification And Biological Activities Of Latcripin-4-RCC1+ANK Domain Protein

Objective The aim of this study is to obtain the single-protein of Lentinus edodes with antitumor activity on the basis of the successful transcriptome of Lentinula edodes C91-3.The Lp-4 gene with RCC1 and ANK domains was cloned and expressed in the prokaryotic expression vector Rosetta-gami,and the activity of inhibiting the tumor growth was studied.Methods: 1.The total RNA of mycelia in the fermentation broth of Lentinula edodes C91-3 strain was obtained by Trizol method.The cDNA library was obtained by reverse transcription.The bioinformatics software was used to predict and compare the gene with the anti-tumor activity.And the full length of the Latcripin-4 gene was obtained by RACE.2.The Latcripin-4 domain fragment was obtained using PCR.After the expression vector pET32 a was digested,the Latcripin-4 gene fragment was ligated to the vector.The recombinant plasmid pET32a(+)-LP4 was transformed into the expression strain Ecoli.Rosetta-gami(DE3).After induction,the product was identified by SDS-PAGand Western-blot.3.LP4-RCC1 + ANK protein mostly in the form of inclusion bodies.After the bacteria were lysed,pets were collected.Separate the LP4-RCC1 + ANK protein with washing buffer containing detergent.The protein with high purity was obtained by the method of magnetic beads purification,and then refolded by dilution method.Concerntrate it by PEG.The concerntration was checked by BCA method.4.Identification of anti-tumor activity of LP4-RCC1 + ANK protein:The growth inhibition rate of LP4-RCC1 + ANK protein on HepG2 cells was determined by cck8 method.The ultrastructural changes of the cells were observed by transmission electron microscopy and Giemsa staining.The Apoptosis of HepG2 Cells were detected by Hoechst33258 Staining Method.At the same time,the toxic effect of LP4-RCC1 + ANK protein on human normal immortalized epidermal cells was detected by CCK8 method.Results: 1.The coding sequence of Latcripin-4 gene was obtained by RACE methodand the gene sequence of Latcripin-4 domain fragment was successfully obtained by PCR.The Latcripin-4 functional domain information was analyzedby bioinformatics software.The protein contains a plurality of chromosome-rich regulatory protein domains(RCC1)and anchor repeat sequence domains(ANK).2.Xhoi / BamHi double digestion showed that the Latcripin-4 domain fragment was successfully transferred into the expression plasmid.3.The expression of LP4-RCC1 + ANK protein was successfully induced and confirmed by SDS-PAG and Western-blot.4.The biological activity of the protein was checked by CCK8 method.And the result shows that,the protein has significant inhibitory activity on the growth of HepG2 cell line(p<0.05).Transmission electron microscopy,Giemsa staining and Hoechst33258 all showed that the HepG2 cells apoptosis.The results of CCK8 assay showed that LP4-RCC1 + ANK protein had no toxic effect on normal human immortalized epidermal cells.Conclution: 1.The full length of LP4 gene was obtained successfully.2.The recombinant expression plasmid has been constructed Successfully.3.The LP4-RCC1+ANK proteins were induced and purified successfully.4.The result of cck8 showed that LP4-RCC1 + ANK protein could inhibit the proliferation of HepG2 cells.The results of transmission electron microscopy,Giemsa staining and Hoechst33258 staining showed that LP4-RCC1 + ANK proteins can promote the apoptosis of HepG2 cells.5.TheLP4-RCC1+ANK proteins have no toxic effect on human normal immortalized epidermal cell line HACAT.