| Toxoplasmosis is a zoonotic protozoal disease in humans and other warm-bloodanimals, caused by Toxoplasma gondii which is an obligate intracellular parasite. It iswidely popular all over the world. Not only is it caused a serious threat to human health,but also has a great economic losses of animal husbandry. The diagnostic method oftoxoplasmosis is mainly etiology and immunological methods, with the poor sensitivityand specificity. Therefore, for control toxoplasmosis, it is a great significance to establish asimple, rapid, sensitivity and specificity diagnostic method of Toxoplasmosis.Unfortunately, there has not been safe and effective drug developed to prevent or curetoxoplasmosis yet. Researching the protective immunity of parasite protein and developingan effective vaccine are new means against T. gondii infection. ROP18involved in theinvasion of host cell, and mediated immune evasion of T.gondii, has been shown to be animportant virulence factor. Some researchers have already cloned and expressed ROP18,and analyzed the reactionogenicity of it. But there are only few reports about its immuneprotection.Firstly, the conserved sequence was obtained by analysising the clone sequence of529bp PCR product which is from swine in AnHui province. Secondly, a pair of LAMPamplification primers was designed by Primer Explorer4.0, a program which can designLAMP sepecific primers online, being analysed and screened by Oligo6.0and PrimerPremier5.Then, a LAMP amplification reaction was estabished, using pMD-19T-529bprecombinant plasmid as the template, and, following, the reaction system and conditionswere optimized by orthogonal test. Lastly, the sensitivity, specificity and reproducibility ofthe method established were estimated, and compared with the PCR method. Results: The529bp sequence of T.gondii from swine was amplified, and we found its main mutationregion was31to74and100to141bases. The LAMP detection method of T.gondii wasestablished successfully after the LAMP amplification primers were designed. The reactiontime was33min when added a ring primers. This LAMP reaction has good specificity, itcan not amplify ultra-pure water and the DNA of pigs, Cryptosporidium and Coccidium;and high sensitivity, it can amplify1.0×103copies/mL which is10times of the PCRmethod.The ROP18gene of T.gondii was amplified, cloned and sequenced, then prokaryotic expression plasmid pET-32a-ROP18and eukaryotic expression plasmid pcDNA 3.1-ROP18-Flag was constructed, which were expressed in E.coil Rosseta and BHK-21cells respectively. The prokaryotic expression product was identified by SDS-PAGE, which form of expression was analyzed, and the induced conditions were optimized. The recombinant protein was purified by Ni-NTA, and which reactogenicitywas detected by Western-blot. The product of eukaryotic expression indentified by indirect immunofluorescence assay (IFA), and its reactogenicity was also detection by Western-blot. The Kunming mice were immunized with recombinant plasmid andrecombinant proteins emulsified with adjuvant respectively, while a blank control group were immunized with nothing and PBS control group were immunized with PBS.10d after the third immunization all of the mice were attacked by3000tachyzoites. Results: The ROP18gene sequences were cloned, and the prokaryotic expression plasmid pET-32a-ROP18and eukaryotic expression plasmid pcDNA3.1-ROP18-Flag were constructed, which were expressed in E.coil Rosseta and BHK-21cells. Theprokaryotic expression plasmid was expressed with the form of inclusionbody, which was2.3mg/mL after purified. In IFA, the cells transfected with recombinant plasmid be observed with green fluorescence, while the control cells can not. Both of the two products have well antigenicity identified by Western-blot. Indirect ELISA showed that recombinant proteins and recombinant plasmid can induce the anti-Toxoplasma antibodies in mice. The average survival time of mice in recombinant proteins group is9.5days, and9.7days in recombinant plasmid group, which are significant difference with control group, but no significant difference between the two groups, and also between in the other control group.We successfully established a rapid, specific, sensitive Toxoplasma gondii LAMP detection method, which has important significance to rapid and accurate diagnostic method of Toxoplasmosis. We also found that ROP18of T.gondii can induce anti-Toxoplasma immune protection, which provides a theoretical basis for the researchof the anti-Toxoplasma vaccine. |
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