猪巨细胞病毒gB基因的原核表达与间接ELISA检测方法的建立

摘要

猪巨细胞病毒(PCMV)是引起新生仔猪死亡、鼻炎、肺炎和生长速度减缓的重要病原之一,我国尚无有效的诊断方法.为建立检测PCMV的ELISA方法,本研究以重组PCMV囊膜糖蛋白B(gB蛋白)为包被抗原,优化反应条件:抗原包被量0.4 μg/mL,待检血清稀释度1∶100,37℃包被2h后4℃过夜,5％脱脂乳37℃封闭2h,二抗1∶10 000稀释,37℃作用2h,抗体临界值为S/P≥0.354判为阳性,S/P≤0.295判为阴性,介于二者之间为可疑,建立了PCMV抗体间接ELISA检测方法.与猪瘟、猪伪狂犬病、猪附红细胞体病、副猪嗜血杆菌及猪圆环病毒2型等血清抗体无交叉反应,批间和批内重复性较好,对江苏省259份仔猪血清样品进行检测,抗体阳性率达56.76％,从而为PCMV检测和流行病学调查提供了有效方法.%Porcine cytomegalovirus (PCMV) was one of the agents causing fetal and piglet deaths, runting, rhinitis, and pneumonia, as well as poor live weight gain. To establish the diagnostic method, an indirect ELSA was established for detection of the antibody against this virus by using recombinant gB protein as coating antigen expressed in E.coli. The optimized reaction conditions were including: coating with 0.4 μg/mL antigen of PCMV gB at 37℃ for 2 hour and then overnight at 4℃, blocking with 5% skim milk at 37℃ for 2 hours, incubating with the secondary antibody diluted in 1:10,000 at 37℃ for 0.5 hour and judging with the cutoff value of S/P ≥0.354. The assay was reproducibility and specific to PCMV antibody, but no reactions with the positive serum to swine fever, pseudorabies, porcine reproductive and respiratory syndrome, porcine circovirus type 2 and Haemophilus parasuis. Furthermore, a total 259 clinical serum samples collected from pig farms in Jiangsu province were detected and the positive rate was 56.76%. These results indicated that this method could be used for PCMV epidemiological surveys and diagnosis of PCMV infections in pigs.