Prokaryotic Expression of the Toxoplasma gondii SAG2 Gene in Pet Cats and Establishment of an Indirect ELISA Method
Prokaryotic Expression of the Toxoplasma gondii SAG2 Gene in Pet Cats and Establishment of an Indirect ELISA Method
Liyun Chang1, Aiju Liu2, Jianshuang Zhang3, Yingbin Chen1 and Zhiyong Liu4*
ABSTRACT
This study aimed to establish an indirect enzyme-linked immunosorbent assay (ELISA) method based on the SAG2 gene of Toxoplasma gondii (T. gondii) to improve the detection of toxoplasmosis in pet cats. Escherichia coli BL21 (DE3) was transformed with a prokaryotic expression vector, pET-21a-SAG2, which was constructed and induced to express a recombinant protein (SAG2), identified using SDS-PAGE and Western blot analysis. The purified protein was then used as a coating antigen to establish an indirect ELISA method for detecting the T. gondii antibody in pet cats, whose reaction conditions were optimized. The SAG2 gene was successfully cloned into a pET-21a (+) prokaryotic expression vector, and the recombinant plasmid pET-21a-SAG2 was obtained. The size of the recombinant protein was approximately 19 kDa, and it was expressed mainly in the form of an inclusion body. The optimal reaction conditions were as follows: antigen-coating concentration, 0.6 μg·mL-1; serum dilution, 1:200; 60-min serum action time, 1:2000 working dilution of the enzyme-labeled secondary antibody; and 5% skimmed milk used as a blocking solution. No cross-reactivity was observed with the positive serum of Eperythrozoon, Trichinella spiralis (T. spiralis) and Hydatid cysts. The coefficients of the inter- and intraassay variations in the repeatability tests were lower than 10%. The established method and the indirect hemagglutination (IHA) test were used to detect 50 clinical samples at the same time. The positive coincidence rate of the two was 94.11%. Therefore, the newly established indirect ELISA method had high sensitivity, specificity, stability, and intra- and interbatch repeatability. It could be used for the diagnosis and epidemiological investigation of toxoplasmosis in cats, thus laying a good foundation for preparing ELISA kits for clinical detection.
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