鸡传染性法氏囊病血清抗体间接ELISA检测方法的建立
Development of an Indirect ELISA for the Detection of Antibodies against Infectious Bursal Disease Virus in Chickens
  
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中文摘要:
      为建立可检测鸡传染性法氏囊病血清抗体的间接ELISA检测方法,本研究经RT-PCR扩增IBDV VP2(1nt-708nt)基因片段,并应用pET28a载体进行原核表达,亲和层析纯化重组蛋白作为包被抗原,建立检测IBDV血清抗体的间接ELISA方法。应用所建方法和IDEXX试剂盒,对采自不同地区的156份鸡血清样品进行平行检测和比较。结果表明,RT-PCR扩增获得708bp的VP2基因片段;含重组表达质粒pET28a-VP2的大肠杆菌BL21经IPTG诱导后,表达了约27kDa的VP2重组蛋白,表达量约占菌体蛋白的30.1%;建立的间接ELISA检测方法与IDEXX试剂盒比较,其特异性、敏感性和符合率分别达到90.24%、95.65%和94.23%。由此可见,本研究所建立的间接ELISA方法敏感、特异,适用于IBDV血清抗体的检测。
英文摘要:
      In order to develop an indirect ELISA for the detection of antibodies against IBDV, VP2 gene(1nt~708nt) was amplified by RT-PCR and expressed using the pET-28a vector in Escherichia coli BL21. The recombinant protein was purified through Ni-chelating affinity chromatography and based on it,an indirect ELISA was developed. To verify the performance of this assay, 156 clinical serum samples were detected by the assay and IDEXX ELISA kit in parallel. The results showed that VP2 gene was successfully amplified by RT-PCR and expressed in E.coli BL21 with IPTG induction. Compared to IDEXX ELISA kit, the specificity,sensitivity,and coincidence rate of the developed indirect ELISA were 90.24%,95.65% and 94.23% respectively.The results demonstrated the assay was sensitive and specific,and could be used to detect antibodies against IBDV in chicken sera.
作者单位
张华,南文龙,巩明霞,张悦勇,彭大新,陈义平  
中文关键词:  传染性法氏囊病  VP2  原核表达  间接ELISA
英文关键词:IBDV  VP2  prokaryotic expression  indirect ELISA
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DOI:
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