Prokaryotic expression of the fusion protein α-β2-ε of Clostridium perfringens and establishment of an indirect ELISA method

Abstract:

[Background] Clostridium perfringens is the main pathogen causing sudden death syndrome in cattle, seriously endangering the cattle industry. Enzyme-linked immunosorbent assay (ELISA) is an important tool for diagnosis of this disease and antibody detection. [Objective] We expressed the recombinant α-β2-ε fusion protein and used this protein as the target antigen to establish an indirect ELISA method for antibody detection, aiming to provide technical support for the clinical detection of C. perfringens in yaks and epidemiological investigation. [Methods] The bioinformatic tools EditSeq and Protean and the Jameson-Wolf method were employed to analyze the antigenic indices of α, β2, and ε proteins of C. perfringens from cattle. The peptides with higher antigenic indices were captured, ligated, optimized for codons, and then cloned into the pET-32a(+) vector to construct the recombinant expression plasmid pET-32α-β2-ε, which was transformed into BL21(DE3) competent cells. The cells were cultured and induced for protein expression, and the expressed protein was purified and identified by SDS-PAGE and Western blotting. With the recombinant α-β2-ε fusion protein as the coating antigen, we optimized the reaction conditions by the checkerboard assay and single factor tests to establish an indirect ELISA method. The cut-off was determined by the receiver operating characteristic (ROC) curve, and the specificity, sensitivity, reproducibility, and clinical efficacy of the established method were evaluated. [Results] The peptides with high antigenic indices of α (aa 241–403), β2 (aa 161–355), and ε (aa 154–331) toxins were selected for fusion expression, and the fusion protein was determined as correctly expressed by SDS-PAGE and Western blotting. The optimal ELISA conditions for antibody detection were as follows: incubation with the coating antigen (5 μg/mL) at 4 ℃ overnight or 37 ℃ for 60 min; incubation with 5% skimmed milk at 37 ℃ for 1 h; incubation with the serum (dilution at 1:150) at 37 ℃ for 30 min; incubation with secondary antibody (1:2 000) at 37 ℃ for 30 min; color development for 10 min with protection from light. The cut-off of detection was 0.470 2. The established method demonstrated high specificity because of no cross-reactivity with positive sera of Mannheimia haemolytica, Salmonella, Escherichia coli, and Staphylococcus aureus. It showcased high sensitivity, with the maximum dilution of detectable serum being 1:3 200. The coefficients of variation within and between batches were less than 10%, which indicated high reproducibility of the established method. The detection for 277 yak serum samples from Ganzi Tibetan Autonomous Prefecture, Sichuan Province with the established method showed that the antibody positivity rate of C. perfringens was 89.89%, which was basically in agreement with the results obtained by the commercial ELISA kit for the detection of C. perfringens α-toxin. [Conclusion] We successfully constructed the recombinant α-β2-ε fusion protein and established an indirect ELISA method for the detection of C. perfringens.

Key words:Clostridium perfringens;α-toxin;β2-toxin;ε-toxin;fusion expression;indirect ELISA