| Toxoplasmosis is an important zoonotic parasitic disease caused by Toxoplasma gondii, which can cause human and animal abortion, malformation or stillbirth and make serious harm to public health and safety and Livestock production. Cats are the final host, play an important role in the spread of Toxoplasma gondii. Therefore, a rapid and accurate detection of Toxoplasma gondii has great significance for human health. Toxoplasma gondii dense granule proteins(GRAs) are secretory proteins, wherein GRA1 is related to the Signal Transduction of host cell infected with T. gondii tachyzoites, and GRA7 is a potential vaccine candidate molecules which can be secreted during various periods in Toxoplasma gondii. It has been proved that GRA1 and GRA7 have good immunogenicity as diagnostic antigens, which can be the genetic markers of serological diagnosis in Toxoplasmosis. In this study, we use the prokaryotic expression of Toxoplasma gondii dense granule protein GRA1, GRA7 to establish GRA1-ELISA and GRA7-ELISA detection methods.The study using prokaryotic expression vector pET-28a-GRA1 and pET-28a-GRA7 that were constructed in our laboratory transformed into Escherichia coli,the high expression was induced by IPTG, after analyzed by SDS-PAGE, the molecular mass of the recombinant GRA1 and GRA7 protein were 25 kDa and 27 kDa. Expression products analized by Western blotting shows an strong immune response immunogenicity. The expression protein was purified by Ni-NTA, Protein purity percentage was as high as 98% the recombinant GRA1, GRA7 protein concentration is about 200μg/ml and 600μg/ml.Using the purified recombinant protein GRA1 and GRA7 as a diagnostic antigen established the GRA1-ELISA and GRA7-ELISA diagnostic methods. Establishing the optimal antigen coating concentration is 5μg/ml and 5μg/ml, serum dilution is 1:64, the HRP-labeled anti-rabbit IgG concentration of secondary antibodies is 1:20000. Description of the method with high sensitivity and good reproducibility. Application of this method to detect Trichinella, suis, hydatid and cats Toxoplasma positive serum, the results shows the GRA1-ELISA and GRA7-ELISA methods can detect other parasitic diseases with no cross-reactivity and good specificity.Established GRA1-ELISA and GRA7-ELISA detection method which used MAT/IFA test results as the standard was compared and evaluated, GRA1-ELISA detection rate of 10.8% false positive and false negative rate of 4.1%, while GRA7-ELISA detection of false positives rate of 7.5%, the false negative rate of 1.4%, indicating that the rate of false-positive diagnosis GRA7 for antigen detection of Toxoplasmosis and false negative rate is low. There were no significant differences among part of the test(P> 0.05) by McNemar chi-square, the consistency in part by Kappa test, GRA1: Kappa = 0.83, the consistent accuracy rate of results were 94.6%, GRA7: Kappa=0.92, the consistent accuracy rate of results were 97.2%, it showed that GRA7-ELISA has a higher concordance rate.ROC curve analysis of the stability of a better GRA7-ELISA was established. GRA1-ELISA the cut-off value of 0.11, the sensitivity was 83.6%, a specificity of 87%; GRA7-ELISA the cut-off value of 0.26, the sensitivity was 89.7, a specificity of 92.5%. The study showed that GRA1 established as a diagnostic antigen GRA1-ELISA method to detect ineffective, low sensitivity and specificity, weak stability. Therefore, GRA7 was a diagnostic antigen of quality for the detection of Toxoplasmosis in cats, GRA7-ELISA detection method which was established with high sensitivity and specificity, strong stability.GRA7-ELISA diagnostic method which was established in this study solved the problem that Toxoplasmosis is not easy to diagnose accurately and quickly,had an great significant in the clinical diagnosis of Toxoplasmosis in cats, disease prevention, screening high-quality TgGRA7 diagnostic antigen can provide the basis for the rapid diagnosis of Toxoplasmosis in cats and the development of kits. |
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