关键词: 牛, 牛传染性胸膜肺炎, 脂蛋白LppQ, 突变, ELISA

Abstract: 【Objective】Mycoplasma mycoides subsp mycoides SC (MmmSC) is the etiological agent of contagious bovine pleuropneumonia (CBPP). The N-terminal domain of the mature lipoprotein LppQ is hydrophobic, and it induces a strong, specific, early and persistent immune response in naturally and experimentally infected animals. People have good reason to believe N-terminal fragment of lipoprotein LppQ will become a kind of ideal CBPP diagnosis antigen.【Method】Mycoplasma-specific TGA (Trp) codons are utilized as stop codons in most other organisms. The lppQ N-terminal fragment from MmmSC HVRI X strain was mutated with a one-step overlapping extension PCR, and was expressed in E. coli. An indirect enzyme-linked immunosorbent assay was established by using recombination lipoprotein LppQ 【Result】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 42kD was purified by Ni-NTA His·Bind purification kit and the purity 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 μg·ml-1, and coated to microtiter 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). A total of 3 817 bovine serum samples were detected by indirect ELISA and CFT. 【Conclusion】The Kappa value is 0.63, which is middle or high agreemen between the two methods.

Key words: Bovine, Contagious bovine pleuropneumonia, Lipoprotein LppQ, Mutagenesis, Indirect enzyme-linked immunosorbent assay