禽流感病毒同义NP蛋白的原核表达及其间接ELISA检测方法的建立

Establishment of indirect ELISA method with consensus NP protein of avian influenza viruses as coated antigen

为建立通用型禽流感病毒(AIV)快速检测方法,本研究将合成的同义NP(ConsNP)基因克隆于pET30a中,利用大肠杆菌系统进行原核表达,以纯化重组ConsN P蛋白作为包被抗原,建立了检测AIVNP抗体的间接ConsNP-ELISA方法,并优化了反应条件。结果表明:抗原包被量为1μg/孔;封闭剂为5%脱脂乳;一抗血清稀释度为1∶200;H RP标记的兔抗鸡IgG(酶标二抗)稀释度为1∶20 000;阴阳性临界值为:O D490nm值>0.239。该间接ELISA方法与其他亚型A IV阳性血清均呈阳性反应,与新城疫病毒,马力克病病毒,禽传染性囊病病毒阳性血清无交叉反应,具有良好的特异性和敏感性。本研究为进一步研制通用型的禽流感ConsN P-ELISA检测试剂盒奠定了基础。

To establish a rapid method for detection of antibody against avian influenza vires （AIV）, the consensus NP gene of AIV was synthesized based on NP genes of H5 subtype AIV and cloned into pET30a for expressing in E.coli. The recombinant consensus NP protein was expressed and purified to be used as coated antigen for development of the indirect ConsNP-ELISA method. The working conditions of ConsNP-ELISA were optimized as follows： coating with 1 μg/well of the consensus NP at 4℃ ovemight, blocking with 5% of skimmed milk, incubating for 1 hour at 37 ℃, detecting at 1：200 dilution serum with the 1：20,000 dilution of second antibody of rabbit anti-chicken HRP-IgG. The cut-off value was 0.239. This established method was reacted positively with all subtypes of AWs, but no cross-reaction with positive serum of Newcastle disease virus, Marek＇s disease virus and infectious bursal disease virus, which possessed a high specificity and sensibility. This study provided a solid basis for the development of a universal ConsNP-ELISA kit for detection of antibody against AWs.