Expression and serodiagnostic efficacy of a novel echinococcosis-specific recombinant fusion antigen rAgB8/1-Em18-Eg95

Abstract

Objective The echinococcosis-specific antigens Eg95 and AgB8/1 and the Em18 gene sequences were fused and expressed as the novel recombinant antigens rAgB8/1-Em18-Eg95 (T3) and rEm18-Eg95 (T2), used for the diagnosis of hydatid disease, prepared into an enzyme-linked immunosorbent reaction (ELISA) kit, and evaluated for their serological diagnostic value. Method The genes sequences were obtained from the GenBank database, and the T3 and T2 gene sequences were optimized and transformed into the pET-22b (+) BL21(DE3) prokaryotic expression system. Expression was induced, and the bacteria were disrupted. After obtaining the target recombinant protein, an ELISA was used to evaluate the serological diagnostic effect of the recombinant antigens. Results T3 and T2 recombinant gene sequences were successfully constructed. The relative molecular weight of the T3 protein was 88.1 kDa. The prokaryotic expression of T3 was higher in the supernatant, which was consistent with the predicted molecular weight. The purified concentration was 1.5 mg/mL, and the purity was 80%. The relative molecular weight of T2 protein was 79.9 kDa, the total protein concentration was 0.5 mg/mL, and the purity was less than 50%. The overall coincidence rate of T2 protein was low, and it was impossible to distinguish between negative and positive sera. The T3 antigen was coated at 1.0 µg/mL, the cutoff value was 0.5271, and the serum dilution ratio was 1:400. A T3 ELISA 96T kit was constructed to detect the serum of 272 clinically and pathologically confirmed cases. The sensitivity of T3 was 93.75%, and the specificity was 93.75%. The Youden coefficient was 77.08%, the parasite cross-reaction was 30%. Satisfactorily, the Pearson correlation coefficient between the T3 OD value and lesion diameter was 0.707, showing a strong correlation. Conclusions T3 exhibits good antigenicity, and the prepared ELISA diagnostic kit reached the laboratory diagnostic level of commercial kits. T3 can distinguish human cystic echinococcosis (CE) and alveolar echinococcosis (AE) more significantly and predict the diameter of lesions according to the OD value, which provides practical value for drug or surgical efficacy.