猪源粪肠球菌Ace蛋白间接ELISA方法的建立与应用

Development of indirect ELISA for detection of anti-Ace antibody for swine-originated Enterococcus faecalis

根据GenBank已发表粪肠球菌Ace基因(AF260879)的核苷酸序列设计1对引物,通过PCR扩增Ace基因保守序列A片段部分序列,将其克隆到pET-32a(+)裁体中,构建了原核表达载体pET-32a(+)-Ace,转化大肠杆菌BL21(DE3)后,成功表达并纯化了重组蛋白;以此重组蛋白作为抗原建立了检测粪肠球菌Ace抗体的间接ELISA方法。经反复试验,确定该间接ELISA的最佳反应条件为:抗原包被浓度为1mg/L,被检血清稀释度为1∶800,1%BSA为封闭液,血清和二抗的作用时间均为60min,底物TMB反应时间为15min,反应的阳性判定值为0.126。交叉试验、批内重复和批间重复试验证明,所建立的间接ELISA方法具有特异性高、重复性好的特点,可用于粪肠球菌Ace蛋白血清抗体的检测。

Based on the Ace gene sequence （NO. AF260879） of Enterococcus faecalis available in the GenBank, a pair of primers was designed for a partial sequence of fragment A gene of Ace. The amplified target sequence was then cloned into prokaryotic expression vector pET-32a（＋） and o- ver-expressed in E. coli BL21（DE3）. By using this recombinant protein as probe antigen,an indi- rect ELISA was established for detecting the anti-Ace antibodies. The optimal reaction conditions were determined as below：the antigen concentration is 1 mg/L; serum dilution 1 ： 800;1%BSA is blocking solution; serum and anti-porcine IgG incubation is 60 rain respectively; the substrate TMB reaction time is 15 min; the positive value was 0. 126. The cross-reaction tests, within- and between-batch reproducibility tests demonstrate that the indirect ELISA method established has a high specificity and good reproducibility, and is therefore promising for the serological detection of Enterococcus f aecalis.