Study On Sequencing And Detection And Expression Of SAG2 Gene Of Toxoplasma Gondii

Toxoplasma gondii was an obligate in tracellular parasite, causing Toxoplasmosis in avariety of mammals including animals and humans by meat. It was necessary to establish asensitive, specific PCR technique for the detection of Toxoplasma gondii infection in animal.Three aspects had been done in this paper.Firstly, Nested-PCR technology had been developed for detecting Toxoplasma gondii inmeat of animals. Two primers were designed on SAG2 sequence of Toxoplasma gondii.Fragment was amplified by this Nested-PCR procedure from Toxoplasma gondii genomic DNAsamples, however, could not be amplified from genomic DNA samples of ryptosporidium,E.tenella, Sarcocystis suihominis, E.bovis, Trypanosoma enansi. The results suggested that thismethod was specific for Toxoplasma gondii. Sensitivity of the PCR was at 10~2 Toxoplasmagondii genomic DNA level every gram meat, it demonstrated that sensitivity of the Nested-PCRwas higher. Toxoplasma gondii can be detected by this Nested-PCR. Total of 68 serum samplesoriginating from animal hospital and abattoirs were detected by PAPS and PCR. Results:16 outof 68 meat samples which tested by Nested-PCR were positive for Toxoplasma of toxoplasmosis,compared to 17out of 68 meat samples tested with PAPS. The data in this study demonstratedthat the Nested- PCR can be used for early diagnosis of toxoplasmosis in meat.Secondly, based on the SAG2 sequence ofToxoplasmosis gondii, a pair of full-length P22 primersare created. P22 gene were obtained from the genomic DNA of Toxoplasma Shanghai strains byPCR.The gel-purified fragments were inserted into pGEM T-Easy vector, the recombinantplasmids were transformed into JM109 competent cells for cloning and sequencing. The resultdemostrated that Toxoplasma shanghai SAG2 gene had high similar to the genes of RH strain.Thirdly, SAG2 gene that expressed the Toxoplasma gondii major surface antigen P22 wasamplified from SAG2-pGEM T-Easy vector. The fragment was inserted into T/A vector and tworestriction enzyme were selected for restriction digestion. The positive clone recombinantplasmid was obtained by QIA prep Spin Miniprep Kit. P22 gene was obtained by enzymedigestion and purified with the QIA quick Gel Extraction Kit. The purified gene were insertedinto prokaryotic plasmid PGEX-6p-1 with T4 DNA ligase. The recombinant plasmid wasidentified by PCR and enzyme digestion and gene sequencing,it was converted into E.coli BL21, then induced by 1mmol/L IPTG.. The expression product was analyzed by SDS-PAGE andWestern-blot.Result: Plasmid eP22-pGEX-6P-1 was recombinated successfully.The transformant(BL21 strain) was efficientiy expressed after being induced four hours.