Development of an Indirect Enzyme-Linked Immunosorbent Assay for Seromonitoring Contagious Bovine Pleuropneumonia Using Recombinant Lipoprotein LppQ of Mycoplasma mycoides subsp mycoides SC as Antigen

Mycoplasma mycoides subsp mycoides SC (MmmSC) is the etiological agent of contagious bovine pleuropneumonia(CBPP). The lipoprotein LppQ encoded by lppQ gene is specific to MmmSC and is found in the type strain and in fieldstrains isolated in Europe, Africa, and Australia, as well as in vaccine strains. No serological cross-reactions wereobserved with the related mycoplasmas of the Mycoplasma mycoides cluster. The N-terminal domain of the maturelipoprotein LppQ is hydrophilic, and it induces a strong, specific, early, and persistent immune response in naturally andexperimentally infected animals. Mycoplasma-specific TGA (Trp) codons are utilized as stop codons in most otherorganisms. The lppQ N-terminal fragment from MmmSC HVRI Ⅹ strain, the Chinese strain for CF antigen production,was mutated with one-step overlapping extension PCR. Sequence analysis confirmed the successful mutation from A toG in codon 198 in the lppQ gene. The fragment containing the mutation site was subcloned into the pET32a expressionvector. The recombinant protein with molecular weight of 42 kDa was purified using the Ni-NTA His·Bind purificationkit, with a purity of up to 95%. Western blot indicated that the standard positive serum of CBPP could react with therecombinant protein. The purified protein was diluted to 0.35 μg mL-1, and coated to microtiter enzyme-linkedimmunosorbent assay (ELISA) plates. Indirect ELISA reaction conditions were optimized. The value of P/N wasdetermined to be 4.8 (0.934/0.193), the sensitivity to be 95.8% (46/48), and the specificity to be 98.9% (161/163). 3 817cattle serum samples from three different provinces were detected by the indirect ELISA and CFT. The Kappa value is0.63, which is middle or high agreement between the two methods.