The Expression Of Circulating Antigen Of Toxoplasma Gondii And The Development Of The Lanthanide-labeled Fluorescent Microspheres Immunochromatographic Strip For The Serological Detection Of Toxoplasma Gondii Infection

Toxoplasma gondii,a worldwide zoonotic parasite,is an obligate intracellular protozoan.It can probably infect all warm-blooded vertebrates.Toxoplasma is a common cause of diseases in wildlife,livestock,and pets.Currently,lacking of effective drug treatment leads T.gondii great harm to the public health and animal husbandry.Circulating antigen(CAg)is excretion and pyrolysis products of T.gondii during the period of acute infection.CAg of T.gondii is a reliable indicator of early infection of this parasite.The main components of CAg in serum are excretory/secretory antigens(ESA).The ESA mainly include microneme protein(MIC),rhoptry protein(ROP)and dense granule protein(GRA).To develop the diagnostic method of T.gondii,truncated GRA1 and MIC4 fragments with strong antigenicity and high hydrophilicity were expressed in Escherichia coli.The truncated GRA1,mic4-1 and mic4-2 genes were amplified by RT-PCR and cloned into expression vector,then recombinant plasmids were expressed.Three soluble recombinant proteins GRA1,MIC4-1 and MIC4-2 were highly expressed when cultured at 28?,22?and 37?,respectively.The results of Western-blot demonstrated that recombinant proteins MIC4-1 and MIC4-2 were not recognized by Toxoplasma positive serum.But recombinant protein GRA1 reacted strongly with serum from dog infected with T.gondii RH strain and can be used as a serological diagnostic antigen.Subsequently,recombinant protein GRA1 was used as antigen to immunize BALB/c mice.Highly specific monoclonal antibodies were prepared by hybridoma technique.Two cell lines capable of stable and efficient secretion of specific antibodies were screened,and the titers of antibody were 6.4×10~4.The result of indirect immunofluorescence experiment(IFA)showed that the natural GRA1 protein of T.gondii could be recognized by the monoclonal antibody,suggesting that the monoclonal antibody could be used for the detection of T.gondii.The result of Western-blot also showed that the monoclonal antibody could recognize GRA1 present in ESA with a clear band at 24 kDa.So the monoclonal antibody could be used to diagnose of acute infection of T.gondii.To establish a detection method of serum antibody against T.gondii,this study used lanthanide-labeled fluorescent microspheres immunochromatographic strip(LFM-ICS).The recombinant protein and SPA were respectively microsprayed on the test line(T line)and the control line(C line).For this purpose,coupling conditions between microspheres and SPA,sample pretreatment method and the streak concentration of C line and T line were investigated to provide the optimum assay performance.The results demonstated that 10-60 min was the best detection time,and it could meet the requirements of rapid detection.The results showed that the method had good specificity.The detection limit of positive serum samples was 1:1600 dilution.The test results of serum sample suggested that the positive detection rates of LFM-ICS were 27.87%.The result of LFM-ICS has good consistency with ELISA and commercialized ELISA kit,and the positive coincidence rates were 93.33%and 85.71%respectively.The method of LFM-ICS had the characteristic of high sensitivity.In conclusion,this study significantly provided a powerful tool for the serological detection of T.gondii infection.