【摘要】 为建立一种快速检测猪圆环病毒2型(PCV2)抗体的方法,本研究通过基因合成PCV2的Rep编码序列,将其克隆至pET-28a(+)并在E.coli中表达,表达的重组Rep蛋白经Ni2+亲和纯化。采用纯化的目的蛋白作为包被抗原,建立检测PCV2 Rep蛋白抗体的间接ELISA方法。结果表明最佳抗原包被浓度为200 ng/孔,血清和二抗最佳反应时间分别为1 h和30 min。该方法具有良好的特异性和敏感性,与韩国金诺公司试剂盒的符合率为81.4%,表明建立的ELISA方法可以用于PCV2的流行病学检测。更多还原

【Abstract】 To develop a method for porcine circovirus 2(PCV2) detection,the gene coding the Rep of PCV2 was synthesized and cloned into pET-28a(+),which was expressed in Escherichia coli.The recombinant Rep protein was purified by nickel affinity chromatography and used as the coating antigen to establish an indirect ELISA as a fast method to detect PCV2 antibody.The reaction conditions were optimized which included 200 ng of recombinant Rep as coating antigen for per well,and an hour and a half incubation for sample serum and secondary antibody,respectively.This recombinant Rep-based indirect ELISA was both specific and sensitive,and showed an accordance rate of 81.4% with the South Korean company kit for detection of PCV2 antibody,indicating it might be favorable in epidemiologic test of PCV2.更多还原