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.

摘要

M. mycoides subsp. mycoides SC (MmmSC) is the aetiological agent of contagious bovine pleuropneumonia (CBPP). The lipoprotein LppQ encoded by the lppQ gene is specific to MmmSC and is found in the type strain and in field strains isolated in Europe, Africa and Australia, as well as in vaccine strains. No serological cross-reactions were observed with the related mycoplasmas of the M. mycoides cluster. The N-terminal domain of the mature lipoprotein LppQ is hydrophilic and it induces a strong, specific, early and persistent immune response in naturally and experimentally infected animals. Mycoplasma-specific TGA (Trp) codons are utilized as stop codons in most other organisms. The lppQ N-terminal fragment from MmmSC HVRI X strain, the Chinese strain for CF antigen production, was mutated with one-step overlapping extension polymerase chain reaction (PCR). Sequence analysis confirmed the successful mutation from A to G in codon 198 in the lppQ gene. The fragment containing the mutation site was subcloned into the pET32a expression vector. The recombinant protein with molecular weight of 42 kDa was purified using the Ni-NTA His-Bind purification kit, with a purity of up to 95. Western blot indicated that the standard positive serum of CBPP could react with the recombinant protein. The purified protein was diluted to 0.35 micro g/ml and coated to microtitre enzyme-linked immunosorbent assay (ELISA) plates. Indirect ELISA reaction conditions were optimized. The value of P/N was determined 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). 3817 cattle serum samples from three different provinces were detected by the indirect ELISA and CFT. The Kappa value is 0.63, which is middle or high agreement between the two methods..