摘要
为建立猪捷申病毒(PTV)抗体的间接ELISA检测方法,将PTV-8VP1基因克隆至原核表达载体pET-30a中,重组质粒转化大肠杆菌Rosetta表达重组的VP1蛋白。SDS-PAGE及Western-blot分析结果表明,表达的重组蛋白分子质量约为36.2ku,且能被PTV-8阳性血清特异性识别。以纯化的重组蛋白包被ELISA反应板,建立了检测PTV抗体的间接ELISA方法。特异性试验结果表明,重组VP1蛋白与猪繁殖与呼吸综合征病毒、猪伪狂犬病病毒、猪细小病毒、猪圆环病毒2型和猪瘟病毒阳性血清不发生反应。敏感性试验结果表明血清稀释至1∶800时仍检测为阳性。该方法的批内、批间变异系数均小于10%,表明具有较好的重复性。利用建立的方法对633份临床样品进行检测,阳性率是88.10%。选取58份临床样品与间接免疫荧光方法作比对试验,两者的符合率是94.80%。
To establish an indirect ELISA for the detection of antibodies against porcine teschovirus（PTV）,full sequence of gene encoding PTV-8 VP1 protein was cloned into pET-30a vector and the recombinant plasmid was transformed into Rosetta.The recombinant VP1 protein was analyzed by SDS-PAGE and Western-blot.Results indicated that the expressed protein was about 36.2 ku,and was able to specifi-cally react with PTV-8 positive sera.The indirect ELISA was established using the purified recombinant VP1 protein as coating antigen,and the detection limit of the method was 1∶800 of the PTV-8 positive serum dilution.The indirect ELISA established in the present study showed no cross reaction with positive serum of porcine reproductive and respiratory syndrome virus,pseudorabies virus,porcine parvovirus,porcine circovirus type 2,or classical swine fever virus.The coefficient of variation of inter-assay and intra-assay were less than 10%.A total of 633 swine serum samples were tested by the established assay and positive rate was 88.10%,and there was a 94.80% coincidence rate between the indirect ELISA and indirect fluorescence assay based on detection of 58 swine serum samples.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2012年第3期258-263,共6页
Chinese Veterinary Science
基金
国家自然科学基金资助项目(31001069
31172349
31172341)
兽医生物技术国家重点实验室项目(NKLVBP2001002)
中央级公益性科研院所基本科研业务费专项(2009ZJKJ01)
