Construction And Immunoscreening Of CDNA Library Of Tachyzoites Of Toxophasma Gondii ME49Strain

Toxoplasma gondii is an obligate intracellular protozoan parasite whichbelongs to the phylum Apicomplexa,subclass Coccidia. It infects a largevariety of domestic and wild animals including birds and human beings. Itcan cause Toxoplasmosis,which is an infectious disease with globaldistribution. T. gondii have widespread infection in the crowd. T. gondiiinfection can be acquired by ingesting raw or uncooked infected meat orthrough food, water or unwashed vegetables contaminated with oocystsexcreted by cats.Most people whose immune function is normal infect withT. gondii usually asymptomatic. When women infected T. gondii duringpregnancy (3to5weeks), the pathogens will through the placenta barrierspread to the fetus, lead to abortion,premature birth,dead fetus andcongenital toxoplasmosis. Congenital toxoplasmosis can lead tohydrocephalus,chorioretinitis,hepatosplenomegaly,thrombopenia,microcap-halus in fetal and baby.Nowadays, We have a couple of methods todiagnose toxoplasmosis,for example: microscopic examination directly,etiological examination, Animal inoculation, oocyst separation,Immunological inspection,indirect hemagglutination test(IHAT),enzymelinked immunosorbent assay(ELISA) etc.This experiment extract total RNA and construct a cDNA expresslibrary of tachyzoites of T. gondii ME49strain,then screening it usingserum of rat infected with tachyzoites.In order to search for the highimmunogenic antigens, to lay the foundation for express specific antigenprotein, to help to diagnose and prevent the toxoplasmosis in the earlyphage of the infection.Method：The strategy set up required two steps.The first involved theconstruction of complementary DNA libraries from tachyzoites of T. gondii ME49strain,then the tachyzoites of T. gondii were purified;the total RNA ofT. gondii was extracted using Trizol Reagent,then,we use the SMARTTMⅢ Oligonucleotide and long-distance PCR to generatefull-length,double-stranded cDNA. SMART(Switching Mechanism At the5’end of the RNA Template)technology ensures high representation offull-length cDNAs.After synthesizing and digesting the cDNAs with SfiⅠ,we enrich for large,full-length clones by size fractionation on alow-melting-point agarose gel.Finally,we excise cDNAs greater than1.0kband directionally clone them into our λTripleE×2phagemid,which isspecifically designed for efficient cloning of large inserts,finally packagedin vitro and amplified after tittering the packaging reaction,the titer of theunamplified library and the amplified library were determined.The second step involved immunoscreening in a prokaryotic system inorder to select the corresponding antigens.To do this,the expression librarywere screened with pooled rat immune serum collected6weekspost-infection.Positive plaques were cut out of the plates and the phageswere eluted in1ml SM buffer.The eluted phages were used in a secondaryscreening with50pfu per plate.After the secondary screening singleplaques were cut out and phages eluted in1ml SM buffer.The inserts fromthe positive phages were amplified by PCR,then the fragments weresubcloned to PMD-18T vector and subjected to sequencing.Results: The original cDNA library contained4×10~7pfu; the amplifiedlibrary have a titer of4.2×10~9pfu/ml;Primary screening of the T.gondiilibrary revealed40antigen positive plates.Subsequent secondary screeningidentified26positeve plaques.Conclusion: Screening phage express library by immunologicalmethods can screen the protective antigen genes that have the highresponse from the library,which is efficient method for searchingimmunogenic antigens. CELF and ROP8are candidate antigens fordiagnosis of T. gondii infection.