【摘要】 本研究以高致病性猪繁殖与呼吸综合征病毒(HPPRRSV)重组NSP2蛋白为包被抗原,建立了检测PRRSV抗体的间接ELISA方法。通过RT-PCR技术,从PRRSV SCMS08株中,成功扩增出NSP2的主要抗原区域。将其克隆到pET-32a(+),成功构建原核表达载体,并转化宿主菌(E.coli)Rosetta 2(DE3)。以镍柱纯化表达蛋白,SDS-PAGE检测证实重组蛋白获得了高效表达,Western blot检测表明所表达蛋白能够被抗组氨酸标签单克隆抗体和PRRSV阳性血清识别。以此蛋白包被酶标板,初步建立了PRRSV NSP2-ELISA诊断方法。优化的NSP2-ELISA的最佳抗原包被浓度为1.7μg·mL-1,血清的最佳稀释度为1∶40。用该方法检测185份血清样品,与IDEXX公司ELISA试剂盒检测结果符合率为90.8%。建立的间接ELISA方法敏感性高、特异性强,可用于猪繁殖与呼吸综合征的常规诊断及流行病学调查。更多还原

【Abstract】 The present experiment was performed with the objective of establishing an indirect ELISA for detection of PRRSV with the purified NSP2 fusion protein as coating antigen. The major antigen region of NSP2 was successfully amplified by RT-PCR from a highly pathogenic PRRSV (SCMS08 strain), and inserted into pET-32a(+) vector. The recombinant plasmid was transformed into E. coli Rosetta 2 (DE3) and induced with IPTG. The fusion protein was treated with Ni-chelated chromatography under denaturing conditions. With the purified fusion protein as coating antigen, an ELISA for detection of PRRSV was established. SDS-PAGE showed that the fusion protein was expressed at high level in Rosetta 2. Western blot analyses showed that the expressed protein was recognized specifically by anti-His monoclonal antibody as well as PRRSV positive serum. In the optimized NSP2-ELISA, the fusion protein was coated at 1.7 μg·mL-1 and swine serum samples were diluted at 1∶40. About 185 serum samples were detected by the method and IDEXX-ELISA kit, respectively. The agreement ratio between the two methods reached at 90.8%. The results indicated that NSP2-ELISA was specific, sensitive and suitable for routine diagnosis of PRRS and also for epidemiological surveys.更多还原