Study On The Expression,Purification And Antioxidant Activity Of Mycoplasma Penetrans Ferritin Protein

BackgroundMycoplasma penetrans is an AIDS-associated mycoplasma that is named for adhering to and penetrating eukaryotic cells.M.penetrans has been reported potentially facilitate the malignant transformation.The human immune system can produce large amounts of reactive oxygen species?ROS?to kill pathogens.Therefore,M.penetrans inevitably face the oxidative stress during adhesion and entry into the host.Antioxidant mechanism is extremely important for the survival of M.penetrans and its pathogenic function,however,it remains unclear so far.ObjectiveIn this study,we constructed a prokaryotic expression plasmid pET15b-Ferritin which express the M.penetrans Ferritin protein.The Ferritin protein was expressed and purified by affinity chromatography.The antioxidant activity of Ferritin protein was detected,and the immunogenicity of Ferritin was determined,which will provides an experimental basis for knowing the pathogenicity of M.penetrans.Methods1.The binary and tertiary structures and other biological characteristics of Ferritin were analyzed through bioinformatics methods.2.The M.penetrans Ferritin gene was synthesized,which could encode the full-length Ferritin protein in E.coli.The specific primers were designed according to the sequences of Ferritin gene and were used to amplify the Ferritin gene.Ferritin gene was double digested by NdeI and BamHI and the product was recovered.The recovered product was then ligated to pET15b to gain the recombinant plasmid pET15b-Ferritin which was transformed into the E.coli BL21.3.The fused Ferritin protein was induced by IPTG and was purified by using Ni²⁺affinity.After the endotoxin was removed,the concentration of protein was determined by BCA Method.4.Ferrous oxidase activity of Ferritin protein was determined by spectrophotometric analysis.The antioxidant activity of Ferritin was determined through in vitro experiment.5.The growth of E.coli expressing Ferritin and control E.coli after H₂O₂ treatment were determinated.6.The antiserum was obtained from mice immuned with Ferritin protein and the antibody titer was determined.Results1:The full-length Mpe Ferritin gene is 540 bp and encodes 180amino acids.Mpe Ferritin protein localizes at the cytoplasm without a signal peptide.It has an?-helix of 71.5%,a?-turn of 6.7%,an extended chain of 5.0%,and a random coil of 16.8%.The tertiary structure of Ferritin protein was a 24-mer which composed of 24 homologous identical subunits.Ferritin protein has multiple B cell epitopes.2.The recombinant plasmid pET15b-Ferritin encoding the 6*His fused recombinant Ferritiin protein was obtained,which was transformed into the E.coli to obtain the recombinant bacteria BL21/pET15b-Ferritin.The 22.9 kDa recombinant Ferritin was induced by IPTG and the protein with high purity was harvested.3.Ferritin protein can rapidly catalyze Fe²⁺into Fe³⁺and has ferrous oxidase activity;the semi-inhibitory concentration(IC₅₀)of Ferritin protein hydroxyl radical scavenging is 0.51 mg/mL,which is significantly lower than the IC50 value of BSA protein?0.80 mg/mL?4.After H₂O₂ treatment,the E.coli transformed with pET15b-Ferritin had a higher survival rate than the control strain transformed with pET15b empty vector.5.Immunization of mice with Ferritin protein stimulates mice to secrete high titers of antibodies.Conclusion:1.Mpe Ferritin has ferrous oxidase activity and antioxidant function,which can improve the ability of E.coli to resist H₂O₂ stress,indicating that Mpe Ferritin protein has antioxidant function.2.Ferritin protein can induce high titer of antibodies in mice.