| Targeting 529bp repetitive sequence,ITS-1 and B1 gene has been developed for detecting Totoplasma gondii in swine blood samples which were highly conserved in RH strain of T. gondii were amplified with toch-down PCR, and cloned into PUM-T vector which was transformed into E. coli DH5α. The coefficient correlation (R2) of standard curve was 0.998 and efficiency was 96.724% and the solubility curve showed that the Tm value of 529bp, ITS-1 and B1 were 86.5±0.5℃,78.8±0.5℃ and 83.1±0.5℃. The specificity of triplex SYBR Green I real-time PCR showed that 529bp, ITS-1 and B1 of melt curve were 86.2℃,78.9℃ and 83.3℃, melt curve of the negative control have no amplification. The sensitivity of triplex SYBRGreen I real-time PCR showed that 529bp, ITS-1 and B1 were 173copies/μL,123copies/μL and 135 copies/uL respectively. The reproducibility of intra and inter-assay of Tm showed that the CV% of Tm were less than 0.3%.41.72%(131/314) of swine blood samples were detected by ELISA, and 53.44% (70/131) were tested by triplex SYBRGreen I real-time PCR (P<0.01). In conclusion, scientific evidence detecting for T. gondii was supplyed for more specific and sensitive method of triplex SYBRGreen I real-time PCR.In order to clone the truncated SAG2 gene of T. gondii and highly express it in E. coli, and then purify GST-SAG2t fusion protein which was established ELISA for detecting antibody against T. gondii in swine blood. A 438-bp DNA fragment encoding the truncated SAG2 without the highly hydrophobic signal peptide and C-terminus was amplified by PCR, subcloned into a prokaryotic expression vector PGEX-4T-3, then transformed in E. coli. The immunological activity of purified GST-SAG2t was analysed by western - blotting and ELISA. SDS-PAGE and western blotting analyzed purified recombinant SAG2t showed that the molecular mass of recombinant SAG2t was 41ku and recognized antibody in standard positive serum infected T. gondii. The recombinant protein was highly expressed in E. coli in condition of 25℃,0.5mmol/L and 4h. About 40mg soluble recombinant protein can be obtained every litre cultivation. Using target protein as the antigen, the optimum condition of ELISA was determined. The optimal dilution of GST-SAG2t for coating of plate was 2.0μg/mL, the optimal dilution of serum sample was 1:100, the threshold value of ELISA was 0.1780. The reproducibility of intra and inter-assay of rELISA showed that the CV% of OD450 value were 1%-9%. 90 swine serum samples were detected with this method and the kit respectively, It was found that the coincidence of ELISA were 97.14%. Truncated SAG2 fragment was highly expressed in E. coli, purified GST-SAG2t maintaining the immune activity of natural protein was developed ELISA that had good specificity for the detection of SAG2 antibody in serum, which provides an available technique for immune detection and serological survy of T. gondii. |
