The Effect Of ROP18on Apoptosis In Host Cell Infected By Toxoplasma Gondii

Objectives:To determine whether the rhoptry protein18has inhibiting effect on apoptosisin host cell infected by toxoplasma gondii and elucidate the mechanism of inhibitingapoptosis in host cell infected by toxoplasma gondii.Methods：The primers for amplifying ROP18gene of Toxoplasma gondii RH strain byPCR were designed using software and a Flag tag sequence was added upstream tothe initiator codon of ROP18gene. In order to facilitate subclone, two restrictionenzyme sites, BamHⅠand Hind Ⅲ, were added in5’ and3’of the construct.Genomic DNA was isolated from1x107parasite using isolation kit according to themanufacturer’s instruction. ROP18gene of Toxoplasma gondii RH strain wasamplified by PCR. Flag-ROP18was purified from agarose gel using kit accordingto manufacturer’s instruction.After digested with BamHⅠand Hind Ⅲ, Flag-ROP18was subsequently cloned into the same sites of pcDNA3.1(+) cleaved by the samerestriction enzymes resulting in expression plasmid pcDNA3.1(+)-Flag-ROP18. Theexpression plasmid pcDNA3.1(+)-Flag-ROP18was confirmed by DNA sequencingand subsequenctly transfected into RAW264.7cells with lipofectin. Expression ofROP18gene in RAW264.7cells was identified by Western blotting using an antibodyagainst Flag according standard protocol. RAW264.7cells and RAW264.7cellscontaining pcDNA3.1(+)-Flag-ROP18were treated with H2O2to induce the cellsto undergo apoptosis. The morphological markers for apoptosis in RAW264.7cellswere detected using a microscope under oil immersion. Cells induced apoptosis withH2O2were lysed with lysis buffer for20min on ice. Mitochondria and cytosolicfractions were separated by subcellular fractionation according to standard protocol and distribution of cytochrome c in mitochondrion and cytoplasm was detected bywestern blotting using an antibody against cytochrome c.At various times,DNA ofRAW264.7cells treated with H2O2was extracted and separated by size byelectrophoresis in an agarose gel and stained with ethidium bromide to detect thecleavage of nuclear DNA.Results：The DNA sequencing result showed that the expression plasmidpcDNA3.1(+)-Flag-ROP18was constructed correctly. Western blotting result showedthat the recombinant Flag-ROP18protein was expressed in RAW264.7cells. Afterinducing apoptosis using H2O2,Morphological markers for apoptosis in RAW264.7cells and RAW264.7cells containing pcDNA3.1(+)-Flag-ROP18were detectedusing microscope and stained in HE. The cells shrink and condence. The nuclearchromatin condense and the cells surface blebs and break up into apoptotic bodies.The western blotting result using an antibody against cytochrome c showed thatcytochrome c released from mitochrondria into the cytosol after inducing apoptisisusing H2O2. After cells were treated with H2O2,inducing the cells to undergoapoptosis, the cleavage of nuclear DNA into a characteristic ladder pattern offragments was detected in RAW264.7cells and RAW264.7cells containingpcDNA3.1(+)-Flag-ROP18. Three resultes above showed that there are nodifference in morphology, distribution of cytochrome C in Mitochondrion andCytoplasm and DNA fragmentation between RAW264.7cells and RAW264.7cellscontaining pcDNA3.1(+)-Flag-ROP18after inducing apoptosis using H2O2.Conclusions：Expression plasmid pcDNA3.1(+)-Flag-ROP18was constructed correctly and therecombinant Flag-ROP18protein was expressed in RAW264.7cells. ROP18has nodirect effect on RAW264.7cells apoptosis induced using H2O2.