Cloning And Sequence Analysis Of Toxoplasma Gondii RON5 Partial Gene And Its Expression Patterns In Different Developmental Stages

Toxoplasma gondii,an obligate intracellular parasitic protozoan,is one of the food-borne and opportunistic agents that can essentially infect all warm-blooded vertebrate,including humans.The investigations indicated that one-third of population was infected with T.gondii in the world.However,there is no safe and effective vaccines and drugs to ragainst it up to now.The rhoptry only is a secretory subcellular organism to Apicomplexa protozoan,which can be divided into rhoptry protein(ROPs)and rhoptry neck protein(RONs)according to the location of the secreted protein in rhoptry.12 RONs have been found and confirmed including TgRON1,TgRON2,TgRON3,TgRON4,TgRON5,TgRON6,TgRON8,TgRON9 and TgRON10.Many studies have been shown that the rhoptry neck protein is closely associated with the mechanism of invasion of T.gondii,and it also is one of the virulence factors of this parasitic protozoa.Experiment 1: To investigate the different transcription and expression of rhoptry neck protein 5(RON5)gene in differential developmental stages,real-time quantitative PCR was applied to determined it.Genomic DNA of 9 T.gondii isolates from different hosts was used as template to amplify the target gene,and the obtained products were sequenced,and the homology analysis and phylogenetic tree also was made.The results showed that the RON5 gene could not effectively distinguish different genotypes of T.gondii,so it couldn’t be used as genetic marker.In antigenity,it demonstrated that RON5 protein has 28 α-helices,45 β-folds,9 β-rotations,18 random coils and 16 B cell epitopes containing 14 hydrophilic regions.It indicated the soluble protein RON5 has the better immunogenicity,which could be used as a vaccine candidate molecules against Toxoplasma.Experiment 2: In the study,T.gondii Type II PRU strain was selected as the research objectives to analyze the differential expression of TgRON5 gene at different developmental stages,including tachyzoite,bradyzoite,non-sporulated oocyst and sporulated oocyst,as well as TgACT1 gene was used as the reference gene.Specific primers were designed according to the sequence of aim and reference gene,then the real-time fluorescence relative quantitative PCR(qRT-PCR)was made to examine the variety of the TgRON5 gene expression.According to the reported 3’-terminal coding sequence of RON5 gene from GenBank,the specific primers were designed and the whole reading fragment of actin(ACT1)coding-resign gene of T.gondii was involved,then the amplification genes were transformed into prokaryotic expression vectors to construct pET30a-RON5 C and pET30a-ACT1,the recombinant proteins were expressed by IPTG induction and purified by immuno-magnet separation.New Zealand rabbits were immunized with the obtained recombinant proteins to prepare advance the polyclonal antibody,after the polyclonal antibody were purified by immuno-magnet separation,it was identified by SDS-PAGE and Western blotting technique;Finally,Western-blot was used to detect and testify the relative expression of RON5 protein at different developmental stages of T.gondii in this study.The mRNA expression result demonstrated that TgRON5 gene could be expressed in all developmental stages,but it was the highest expression in the sporulated oocysts,following as the non-sporulated oocysts,tachyzoite and bradyzoite.It suggested that the expression level of TgRON5 gene in T.gondii is various at its different developmental stages;Furthermore,the aim genes of RON5 and ACT1 were successfully cloned,and these recombinant plasmids could be expressed by IPTG induction,their molecular size respectively was about 61 kDa and 53 kDa according to the expected;After immunization,the polyclonal antibodies could be extracted from serum,their molecular weight respectively was about 53 kDa and 22 kDa according to the expected.It could be found that T.gondii RON5 protein could be expressed in all developmental stages,but the sporulated oocysts was higher than that of others.Over all,the TgRON5 gene is highly conserved,it can not regard as the gene marker to distinguish gene-type of T.gondii.Additionally,both the mRNA and protein of TgRON5 could be expressed in all developmental stages.It can be suggestted that the RON5 may be one of the key molercular of T.gondii involves in invading into host cell,and its protein expression is related to its invasiveness.